IMAGING SYNAPSE

STRUCTURE AND
FUNCTION
EDITED BY : George J. Augustine and Marc Fivaz
PUBLISHED IN : Frontiers in Synaptic Neuroscience
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ISBN 978-2-88945-175-3
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Frontiers in Synaptic Neuroscience 1 May 2017  |  Imaging Synapse Structure and Function
 IMAGING SYNAPSE STRUCTURE
AND FUNCTION
Topic Editors:
George J. Augustine, Lee Kong Chian School of Medicine, Nanyang Technological University,
Singapore
Marc Fivaz, Duke-NUS Medical School, Singapore

Two complementary super-resolution imaging techniques reveal interactions of single protein

molecules with an individual synapse. The scaffold molecule PSD-95 was mapped using live-cell
PALM, and the positions of those molecules are plotted color-coded by the density of their neighbors.
Trajectories of a probe transmembrane protein were simultaneously measured, using UPAINT
technology, and are shown in pink.
Image by Tuo Peter Li and Thomas Blanpied; see article by these authors in this eBook

Development of new imaging technologies in recent years has transformed neuroscience in

profound ways. Following on the heels of the revolution based on the Green Fluorescent Protein,
refined genetically-encoded fluorescent reporters and genetic targeting strategies now enable
optical recording of synaptic transmission in defined neuronal populations at speeds approach-
ing the enviable temporal resolution of electrophysiology. Super-resolution light microscopy

Frontiers in Synaptic Neuroscience 2 May 2017  |  Imaging Synapse Structure and Function
 permits observation of synapses and their molecular machinery at sub-diffraction resolution. At
the ultrastructural level, automated forms of electron microscopy, improvements in specimen
fixation methods, and recent efforts to correlate data from light and electron micrographs now
make the reconstruction of functional neural circuits a reality. Finally, the use of optogenetic
actuators, such as channelrhodopsins, allows precise temporal and spatial manipulation of neu-
ronal activity and is revealing profound insights into the organization of neural circuits and
their roles in behavior.

This research topic highlights recent advances in both light and electron microscopy, with a
specific focus on approaches that combine innovations from several different fields to obtain
novel information about synapse structure and function. We are confident that this collection
of articles - three original research papers, six reviews, one methods paper and one perspective
article - will enable neuroscientists to achieve the next generation of experiments aimed at
cracking the neural code.

Citation: Augustine, G. J., Fivaz, M., eds. (2017). Imaging Synapse Structure and Function.
Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-175-3

Frontiers in Synaptic Neuroscience 3 May 2017  |  Imaging Synapse Structure and Function
 Table of Contents

05 Editorial: Imaging Synapse Structure and Function

George J. Augustine and Marc Fivaz

Section 1: Imaging presynaptic signaling

07 Stimulation of Synaptic Vesicle Exocytosis by the Mental Disease Gene DISC1
is Mediated by N-Type Voltage-Gated Calcium Channels
Willcyn Tang, Jervis Vermal Thevathasan, Qingshu Lin, Kim Buay Lim,
Keisuke Kuroda, Kozo Kaibuchi, Marcel Bilger, Tuck Wah Soong and Marc Fivaz
21 Visualizing Presynaptic Calcium Dynamics and Vesicle Fusion with a Single
Genetically Encoded Reporter at Individual Synapses
Rachel E. Jackson and Juan Burrone
33 Flash-and-Freeze: Coordinating Optogenetic Stimulation with Rapid Freezing to
Visualize Membrane Dynamics at Synapses with Millisecond Resolution
Shigeki Watanabe

Section 2: Imaging postsynaptic signaling

43 The Postsynaptic Density: There Is More than Meets the Eye
Ayse Dosemeci, Richard J. Weinberg, Thomas S. Reese and Jung-Hwa Tao-Cheng
51 Optogenetic Monitoring of Synaptic Activity with Genetically Encoded Voltage
Indicators
Ryuichi Nakajima, Arong Jung, Bong-June Yoon and Bradley J. Baker
60 Control of Transmembrane Protein Diffusion within the Postsynaptic Density
Assessed by Simultaneous Single-Molecule Tracking and Localization Microscopy
Tuo P. Li and Thomas A. Blanpied
74 The Emergence of NMDA Receptor Metabotropic Function: Insights from Imaging
Kim Dore, Jonathan Aow and Roberto Malinow

Section 3: General synapse imaging technologies

83 Correlative Light Electron Microscopy: Connecting Synaptic Structure and
Function
Isabell Begemann and Milos Galic
95 Advanced Fluorescence Protein-Based Synapse-Detectors
Hojin Lee, Won Chan Oh, Jihye Seong and Jinhyun Kim
107 Organelle-Specific Sensors for Monitoring Ca2+ Dynamics in Neurons
Seok-Kyu Kwon, Yusuke Hirabayashi and Franck Polleux
116 Understanding Synaptogenesis and Functional Connectome in C. elegans by
Imaging Technology
Jung-Hwa Hong and Mikyoung Park

Frontiers in Synaptic Neuroscience 4 May 2017  |  Imaging Synapse Structure and Function
 EDITORIAL
published: 15 December 2016
doi: 10.3389/fnsyn.2016.00036

Editorial: Imaging Synapse Structure

and Function
George J. Augustine 1 and Marc Fivaz 2*
1
Lee Kong Chiang School of Medicine, Nanyang Technological University, Singapore, Singapore, 2 Program in Neuroscience
and Behavioral Disorders, Duke-NUS Medical School, Singapore, Singapore

Keywords: synapse, optical microscopy, electron microscopy, circuits, genetically-encoded biosensors,

optogenetics

Editorial on the Research Topic

Imaging Synapse Structure and Function

These are the glory days for imaging synapse structure and function. Thanks to recent advances
in both optical and electron microscopy, it is now possible to image individual synapses with
unprecedented spatial and temporal resolution. The parallel development of a wide range
of genetically-encoded synaptic reporters enables all-optical recording of synaptic activity in
genetically-defined neuronal populations. These engineering breakthroughs allow neuroscientists
to interrogate the brain in ways that were inconceivable just a few years ago. The ability to image
synaptic structure and activity in large functional circuits is beginning to yield key insights into
how the brain stores, processes, and computes information. This research topic consists of eleven
articles (methods, primary research papers, and reviews) that provide an overview of the latest
developments in synapse imaging. Rather than attempting an exhaustive list of synaptic reporters
and microscopy techniques, our collection emphasizes approaches that merge technical advances
from diverse areas to extract a rich palette of novel information from individual synapses.
Watanabe presents a method that combines optogenetics and rapid freezing (Flash-and-Freeze)
to visualize the synaptic vesicle (SV) cycle at the ultrastructural level with millisecond resolution
Watanabe. This revolutionary approach revealed ultra-fast endocytosis of SVs at central synapses
and neuromuscular junctions (Watanabe et al., 2013a,b) and promises to uncover many new
kinetic aspects of synapse dynamics. Begemann and Galic review recent efforts to image neuronal
preparations with both light and electron microscopy, with a series of hybrid techniques referred
Edited and reviewed by: to as Correlative Light Electron Microscopy (CLEM). Jackson and Burrone describe the first
Per Jesper Sjöström, genetically-encoded fluorescent reporter (sypHy-RGECO) that enables concurrent monitoring
McGill University, Canada of calcium dynamics and SV fusion. sypHy-RGECO will undoubtedly be a powerful means of
*Correspondence: examining calcium triggering of SV exocytosis at the level of individual presynaptic boutons. Using
Marc Fivaz similar probes, Tang et al. show that the mental disease gene DISC1 (Disrupted-In-Schizophrenia-
[email protected] 1) accelerates SV exocytosis by facilitating calcium influx through N-type voltage-gated Ca2+
channels. Calcium transients at synapses are also shaped by both mobilization and sequestration
Received: 19 October 2016
of calcium by intracellular stores. Kwon et al. report on the latest advances in organelle-specific
Accepted: 22 November 2016
calcium sensors and review the contribution of the endoplasmic reticulum and mitochondria to
Published: 15 December 2016
calcium dynamics and synaptic transmission/plasticity.
Citation:
Until recently, one major impediment to imaging of synaptic activity has been our inability to
Augustine GJ and Fivaz M (2016)
Editorial: Imaging Synapse Structure
directly measure membrane potential with adequate signal/noise ratio. This is rapidly changing
and Function. with the recent improvement of a wide range of genetically-encoded voltage indicators (GEVIs)
Front. Synaptic Neurosci. 8:36. that now are capable of monitoring both single action potentials and even subthreshold
doi: 10.3389/fnsyn.2016.00036 synaptic potentials, both in vitro and in vivo Nakajima et al. Three papers describe recent

Frontiers in Synaptic Neuroscience | www.frontiersin.org December 2016 | Volume 8 | Article 36 | 5

Augustine and Fivaz Editorial: Imaging Synapse Structure and Function

advances on the localization, dynamics and function of Overall, we hope that the fine collection of papers contained
postsynaptic receptors and scaffolds. Using a combination within this research topic highlights a useful synapse imaging
of single-molecule tracking (uPAINT) and photoactivated toolkit for the neuroscience community. The next big challenge
localization microscopy (PALM), Li and Blanpied assess in brain imaging will be to scale up these synaptic measurements
the diffusion properties of membrane proteins within the to large ensembles of neurons to comprehend how circuits
postsynaptic density (PSD). The same authors recently used compute. This will require synaptic reporters that operate in
localization microscopy to demonstrate the existence of a synaptically-relevant time scale (milliseconds), along with
transsynaptic nanocolumns that align the neurotransmitter improved genetic targeting strategies, further advances in
release machinery to postsynaptic receptors (Tang et al., 2016a). automated high-speed microscopy, and refined bioinformatics
Dosemeci et al. review a series of EM studies that reveal the tools for analysis of the resulting large datasets.
presence of a dense lamina—the “pallium”—just beneath the
core layer of the PSD, and discuss how translocation of signaling AUTHOR CONTRIBUTIONS
proteins and scaffolds in and out of the pallium may shape
activity-induced changes in dendritic spines. In keeping with the All authors listed, have made substantial, direct and intellectual
theme of postsynaptic signaling, Dore et al. discuss evidence for contribution to the work, and approved it for publication.
metabotropic functions of NMDARs, based on time-resolved
FRET and other imaging approaches. Finally, at the level of FUNDING
synaptic circuits, two reviews describe the use of genetically-
encoded synaptic labels to trace neural circuits in a variety of This work was supported by Singapore Ministry of Education
different model systems, ranging from C. elegans to mammals grants MOE2015-T1-001-069 and MOE2015-T2-2-095 to GA
(Hong and Park; Lee et al.). and grant MOE2013-T2-1-053 to MF.

REFERENCES Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
Tang, A. H., Chen, H., Li, T. P., Metzbower, S. R., Macgillavry, H. D., and Blanpied, be construed as a potential conflict of interest.
T. A. (2016a). A trans-synaptic nanocolumn aligns neurotransmitter release to
receptors. Nature 536, 210–214. doi: 10.1038/nature19058 Copyright © 2016 Augustine and Fivaz. This is an open-access article distributed
Watanabe, S., Liu, Q., Davis, M. W., Hollopeter, G., Thomas, N., Jorgensen, N. B., under the terms of the Creative Commons Attribution License (CC BY). The
et al. (2013a). Ultrafast endocytosis at Caenorhabditis elegans neuromuscular use, distribution or reproduction in other forums is permitted, provided the
junctions. Elife 2:e00723. doi: 10.7554/eLife.00723 original author(s) or licensor are credited and that the original publication in
Watanabe, S., Rost, B. R., Camacho-Perez, M., Davis, M. W., Söhl-Kielczynski, this journal is cited, in accordance with accepted academic practice. No use,
B., Rosenmund, C., et al. (2013b). Ultrafast endocytosis at mouse hippocampal distribution or reproduction is permitted which does not comply with these
synapses. Nature 504, 242–247. doi: 10.1038/nature12809 terms.

Frontiers in Synaptic Neuroscience | www.frontiersin.org December 2016 | Volume 8 | Article 36 | 6

 ORIGINAL RESEARCH
published: 14 June 2016
doi: 10.3389/fnsyn.2016.00015

Stimulation of Synaptic Vesicle

Exocytosis by the Mental Disease
Gene DISC1 is Mediated by N-Type
Voltage-Gated Calcium Channels
Willcyn Tang 1† , Jervis Vermal Thevathasan 1† , Qingshu Lin 2 , Kim Buay Lim 1 ,
Keisuke Kuroda 3 , Kozo Kaibuchi 3 , Marcel Bilger 4 , Tuck Wah Soong 2 and Marc Fivaz 1,2 *
1
DUKE-NUS Medical School, Program in Neuroscience and Behavioral Disorders, Singapore, Singapore, 2 Department
of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore, 3 Department
of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya, Japan , 4 DUKE-NUS Medical School,
Program in Health Services and Systems Research, Singapore, Singapore

Lesions and mutations of the DISC1 (Disrupted-in-schizophrenia-1) gene have

been linked to major depression, schizophrenia, bipolar disorder and autism,
but the influence of DISC1 on synaptic transmission remains poorly understood.
Using two independent genetic approaches—RNAi and a DISC1 KO mouse—we
examined the impact of DISC1 on the synaptic vesicle (SV) cycle by population
imaging of the synaptic tracer vGpH in hippocampal neurons. DISC1 loss-of-
Edited by: function resulted in a marked decrease in SV exocytic rates during neuronal
Stéphane Martin,
stimulation and was associated with reduced Ca2+ transients at nerve terminals.
IPMC CNRS UMR7275 - University
of Nice Sophia antipolis, France Impaired SV release was efficiently rescued by elevation of extracellular Ca2+ ,
Reviewed by: hinting at a link between DISC1 and voltage-gated Ca2+ channels. Accordingly,
Timothy A. Ryan, blockade of N-type Cav2.2 channels mimics and occludes the effect of
Weill Medical College of Cornell
University, USA DISC1 inactivation on SV exocytosis, and overexpression of DISC1 in a
Yann Humeau, heterologous system increases Cav2.2 currents. Collectively, these results show that
Centre National de la Recherche
Scientifique (CNRS), France DISC1-dependent enhancement of SV exocytosis is mediated by Cav2.2 and point
*Correspondence: to aberrant glutamate release as a probable endophenotype of major psychiatric
Marc Fivaz disorders.
[email protected]
†
These authors have contributed Keywords: DISC1, hippocampus, glutamate, psychiatric disorders, schizophrenia, neurotransmitter, synaptic
vesicle release
equally to this work.

Received: 01 February 2016

Accepted: 31 May 2016
INTRODUCTION
Published: 14 June 2016
Genome-wide association studies and exome sequencing efforts have led to the identification
Citation:
Tang W, Thevathasan JV, Lin Q,
of hundreds of variants associated with psychiatric disorders (International Schizophrenia
Lim KB, Kuroda K, Kaibuchi K, et al., 2009; Moskvina et al., 2009; Glessner et al., 2010; Fromer et al., 2014; Schizophrenia
Bilger M, Soong TW and Fivaz M Working Group of the Psychiatric Genomics, 2014), confirming the complex polygenic
(2016) Stimulation of Synaptic Vesicle nature of these diseases. These genomic data also revealed a significant overlap in genes
Exocytosis by the Mental Disease or gene networks associated with distinct mental illnesses (Cross-Disorder Group of the
Gene DISC1 is Mediated by N-Type
Voltage-Gated Calcium Channels.
Psychiatric Genomics et al., 2013), suggesting a common genetic and perhaps circuitry
Front. Synaptic Neurosci. 8:15. basis for major psychiatric disorders. However, the synaptic and circuitry defects underlying
doi: 10.3389/fnsyn.2016.00015 these disorders remain poorly defined, hindering the development of therapeutic solutions.

Frontiers in Synaptic Neuroscience | www.frontiersin.org June 2016 | Volume 8 | Article 15 | 7

Tang et al. DISC1 Enhances Synaptic Vesicle Cycling

DISC1 is the prototypical example of a gene associated in large synapse populations. We show that DISC1 elevates
with several major psychiatric disorders. It was discovered synaptic Ca2+ signals and boosts SV exocytosis at glutamatergic
in a Scottish family at the site of a balanced chromosomal terminals. Our results further indicate that N-type voltage-gated
translocation that strongly segregates with major depression, Ca2+ channels (VGCCs) mediate the stimulatory effect of DISC1
schizophrenia and bipolar disorder (St Clair et al., 1990; on SV release. These findings identify a central role of DISC1
Millar et al., 2000, 2001). The high penetrance (∼70%) of in neurotransmitter release and provide new insights on the
this translocation for mental illness supports a causal link biological basis of synaptic dysfunction in major psychiatric
between this genetic lesion and major psychiatric conditions disorders.
(Chubb et al., 2008). DISC1 variants (haplotypes, single
nucleotide polymorphisms and copy number variations) MATERIALS AND METHODS
have since been independently associated with depression,
schizophrenia, bipolar disorders and autism spectrum DNA, shRNA Constructs, Lentiviruses and
disorders (Ekelund et al., 2001, 2004; Sachs et al., 2005;
Kilpinen et al., 2008). Thus, DISC1 is a major susceptibility
Antibodies
The pCAGGs vGlut1-pHluorin (vGpH) and pCAGGs
factor for mental illness and a relevant genetic entry point to
Synaptophysin-GCaMP3 (SyGC3) constructs were gifts from
identify core endophenotypes implicated in neuropsychiatric
R. Edwards (UCSF; Voglmaier et al., 2006) and S. Voglmaier
disorders.
(UCSF; Li et al., 2011). The pFUGW (Addgene #14883) shRNA-
The translocation breakpoint in this Scottish family is
expressing lentiviral vector was modified to express mCherry
located in the C-terminal portion of DISC1 and results in
(pFUmChW). The shRNA targeting sequences are as follows:
overall reduced expression of the full-length transcript and
(1) Scramble 50 GGAGCAGACGCTGAATTAC30 (Kamiya
protein (Millar et al., 2005), suggesting that haploinsufficiency
et al., 2005); (2) DISC1-E 50 GGCTACATGAGAAGCACAG30
is the main mechanism by which this chromosomal alteration
(exon 2; Duan et al., 2007); and (3) DISC1-A 50 GGAAGG
confers risk to disease. Alternatively, it has been proposed
GCTAGAGATGTTC30 (exon 9) designed with Block-it shRNA
that a C-terminal truncated form of DISC1 is expressed
from Invitrogen. pFUGW scramble shRNA was a gift from
from the translocated allele and may be pathogenic (Hikida
A. Sawa (Johns Hopkins). The DISC1 shRNAs constructs
et al., 2007; Pletnikov et al., 2008), although expression of the
were cloned by introducing double-stranded DNA oligos
truncated DISC1 protein in translocation carriers remains to
into lentiviral vector pll3.7 (Addgene #11795) using the
be demonstrated. Consistent with a disease mechanism based
HpaI and XhoI sites. DNA fragments containing the U6
on DISC1 loss-of-function, DISC1 expression is also attenuated
promoter and shRNAs were then PCR amplified and cloned
in human induced pluripotent stem (iPS) cells derived from
into pFUmChW using the PacI site. The human DISC1 gene
members of a family with a DISC1 frame-shift mutation that
L variant (NCBI Refseq NM018662.2) was PCR amplified
co-segregates with major psychiatric disorders (Wen et al.,
from pCMV6-XL5 DISC1-tGFP (Origene) and cloned into
2014).
pIRES2-DsRed-Express (Clontech) using NheI and SmaI sites.
The identification of a large DISC1 interactome consisting
All constructs were sequenced before use. Lentiviral particles
of proteins belonging to different ontologic families suggests
expressing pFUmChW shRNAs were prepared as described in
broad functions of DISC1 in nerve cells. Accordingly, DISC1
Tiscornia et al. (2006). The DNA constructs used for whole-
has been implicated in multiple aspects of neuronal and brain
cell patch clamp recording are as follows: Cav2.1 (generated
development, including neurogenesis (Clapcote et al., 2007;
in T. W. Soong’s lab), Cav2.2 (Addgene #26568), GFP-β2a
Shen et al., 2008; Mao et al., 2009; Singh et al., 2010; Lee
and α2 δ1 (kindly provided by T. Snutch, UBC). The rabbit
et al., 2011), neuronal migration (Kamiya et al., 2005; Duan
polyclonal Abs against Cav2.1 (#ACC-001) and Cav2.2 (#ACC-
et al., 2007; Kubo et al., 2010; Steinecke et al., 2012) and
002) were from Alomone Labs. The polyclonal Ab against
maturation (Duan et al., 2007; Shinoda et al., 2007; Niwa
the C-terminus of mouse DISC1 was previously described
et al., 2010). Even though DISC1 interacts with several signaling
(Kuroda et al., 2011). The polyclonal Ab against human DISC1
proteins known to regulate synaptic functions, relatively little
(ab59017) and monoclonal Ab against bassoon were from
is known about functions of DISC1 at the synapse. In
Abcam (ab82958).
particular, the impact of DISC1 on neurotransmitter release
remains largely unexplored, despite the fact that aberrant
dopamine and glutamate neurotransmission is a probable cause Mouse Lines, Primary Neuron Cultures and
of schizophrenia and other mood disorders (Howes et al., Transfection Protocols
2015). DISC1 (∆2–3) mice (C57BL/6JJmsSlc) have been described
Given the preferential expression of DISC1 in the before Kuroda et al. (2011). Heterozygous DISC1wt/∆2–3
hippocampus and the involvement of this brain structure mice were crossed to each other to obtain DISC1wt/wt and
in cognition and psychiatric disorders (Chubb et al., 2008), we DISC1∆2–3/∆2–3 lines from which hippocampal neurons were
set out to determine the impact of DISC1 on synaptic vesicle prepared from E17/E18 embryos, according to published
(SV) cycling in hippocampal neurons. We used two independent procedures using papain (Worthington) digestion (Huettner
genetic strategies to alter DISC1 expression—RNAi and a and Baughman, 1986). Primary rat hippocampal neurons
DISC1 KO mouse—and imaged SV cycling and Ca2+ dynamics were prepared from E18 embryos according to Kaech and

Frontiers in Synaptic Neuroscience | www.frontiersin.org June 2016 | Volume 8 | Article 15 | 8

Tang et al. DISC1 Enhances Synaptic Vesicle Cycling

Banker (2006) and as previously described in Garcia-Alvarez (Sigma-Aldrich). Cells were then incubated with 5% goat
et al. (2015). For live-cell imaging and immunocytochemistry serum to block non-specific binding sites and stained with the
experiments, neurons were grown on poly-L-Lysine coated glass primary and Alexa Fluor-conjugated secondary antibodies (Life
coverslips, on top of a glial feeder according to the Banker Technologies). Coverslips were then mounted on glass slides
Protocol (Kaech and Banker, 2006). For biochemical analysis, and imaged with an inverted laser scanning confocal microscope
cells were seeded on poly-L-Lysine coated 6-well culture dishes. (LSM710, Zeiss) with a Plan-Apochromat 63× (NA = 1.40)
Unless otherwise stated, neurons were cultured for 14–16 days. objective.
The vGpH and SyGC3 constructs were electroporated in
freshly-dissociated neurons using the Nucleofector kit (Amaxa
Biosystems, Lonza). Lentiviral particles expressing shRNAs
Image Analysis
For automated analysis of vGpH responses, we wrote a
were added on DIV2, at a MOI (multiplicity of infection) of
Matlab script that segments responsive boutons based on
1–3. All animal procedures were approved by the SingHealth
the difference between peak vGpH intensity during the
Institutional Animal Care and Use Committee (IACUC) of
first stimulation and baseline intensity prior to stimulation
Singapore.
(∆F = F peak − F baseline ). An intensity threshold for ∆F was
selected to resolve individual boutons and exclude those with
Live-Cell Confocal Imaging and ∆F below 5%. The same threshold was used for all conditions
in one independent experiment (i.e., one neuron preparation
Immunofluorescence with control vs. DISC1-depleted conditions). This threshold
Time-lapse confocal imaging was performed on an inverted
value was minimally adjusted (less than 10% change) across
Eclipse TE2000-E microscope (Nikon, USA), mounted with
all independent experiments described in this article. Binarized
a spinning-disk confocal scan head (CSU-10; Yokogawa,
boutons were then slightly dilated (Figure 1B) to ensure that
Japan), and equipped with a temperature controlled (36.5◦ C)
vGpH fluorescence was captured in its totality even after minor
stage and an autofocusing system (PFS; Nikon). Images were
lateral movement or change in shape. Time series with minor
acquired with an Orca-Flash 4.0 CCD camera (Hamamatsu
x–y drifts (originating from the stage) were re-aligned using a
Photonics, Japan) controlled by MetaMorph 7.8.6 (Molecular
script previously described (Thevathasan et al., 2013). Segmented
Devices, CA, USA) at 0.5 Hz or 20 Hz. Samples were
boutons were then size gated, with gating parameters kept
imaged using a 60× (NA 1.4) objective in Tyrode’s buffer
constant across all experiments. vGpH fluorescence intensity
(150 mM NaCl, 2.5 mM KCl, 1 mM CaCl2 , 1 mM MgCl2 ,
was then extracted in each segmented bouton across the time
6 mM glucose, 25 mM HEPES, pH 7.4) supplemented with
series and divided by the signal after NH4 Cl to normalize
25 µM 6-cyano-7-nitroquinoxaline-2,3-dione/CNQX (Tocris
for vGpH expression (Figure 1C). This segmentation strategy
Bioscience) and 50 µM D,L-2-amino-5-phosphonovaleric
ensures that the same boutons are analyzed during the two
acid/AP5 (Tocris Bioscience). Coverslips were mounted in an
consecutive AP trains. vGpH traces with significant baseline
RC-21BRFS chamber (Warner Instruments, USA) equipped
drifts between the first and second stimulation or after Baf
with platinum wire electrodes. Field stimulation was induced
application were excluded. A similar segmentation approach
by a square pulse stimulator (Grass Technologies, USA)
was used to analyze SyGC3 Ca2+ signals. SV exocytic rates
and monitored by an oscilloscope (TDS210, Tektronix,
were obtained by measuring the slope of the vGpH rise
USA). Trains of action potentials (APs) were generated
during the second stimulation (in the presence of Baf). The
by applying 20 V pulses (1 ms duration) at 10 or 20 Hz.
first six time points (during stimulation) were used for linear
Our typical stimulation paradigm for vGpH measurements
regression analysis. To measure endocytic rates, the vGpH
involved two consecutive trains of 300 APs at 10 Hz,
trace during the first stimulation was subtracted from that
separated by ∼5 min to allow synapses to recover. vGpH
obtained during the second stimulation. The resulting trace
responses between the first and second stimulation were
is a measure of endocytosis (Figure 1D). Endocytic rates
highly reproducible (data not shown). For measuring SV
were measured by linear fitting of six time points chosen
exocytic rates, the second stimulation was preceded (30 s
after stimulation onset when endocytosis kicks in. Presynaptic
earlier) by the addition of Bafilomycin A1 (Baf; AG scientific,
localization, abundance and density of Cav2.1 or Cav2.2 were
USA) 0.5 µM. To normalize for total expression of vGpH
analyzed with a modified version of a Matlab script described
in each individual bouton, 50 mM NH4 Cl was added at
previously (Poon et al., 2014). All Matlab scripts are available
the end of the time series. For SyGC3 measurements, Ca2+
upon request.
signals were normalized by adding 10 µM or 50 µM (for
Figure 4G) ionomycin (Sigma-Aldrich) at the end of the
stimulation protocol. ω-agatoxin TK125 nM (Tocris Bioscience) Immunoblotting
and ω-conotoxin GVIA 125 nM (Alomone Labs) were Cultured primary neurons or HEK293T cells were washed
used for experiment involving inhibition of P/Q-type Ca2+ with ice-cold PBS and lysed with RIPA buffer (10 mM Tris-
channels and N-type Ca2+ channels activity, respectively. HCl pH = 7.2, 150 mM NaCl, 1% TritonX-100, 0.1% SDS,
For immunofluorescence studies, neurons grown on glass 5 mM EDTA, 0.25% Na-deoxycholate) supplemented with
coverslips were fixed in 4% paraformaldehyde with 4% Complete protease inhibitor and PhosphoStop phosphatase
sucrose in PBS and permeabilized with 100 ng/ml Digitonin inhibitor (Roche). For analysis of hippocampal tissue, the

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Tang et al. DISC1 Enhances Synaptic Vesicle Cycling

FIGURE 1 | Extracting synaptic vesicle (SV) exo- and endocytic rates by population imaging of vGpH. (A) Snapshots of rat hippocampal neurons (DIV 15)
expressing vGpH and stimulated with two consecutive trains of action potentials (APs; 300 APs, 10 Hz) before (first two images) and after (last two images) addition
of Bafilomycin (Baf). Time is in sec. scale bar: 10 µm. (B) Automated segmentation of active boutons based on vGpH responses after the first stimulation.
(C) Individual (gray) and average vGpH (black) traces derived from segmented boutons. Shaded errorbar indicates 95% confidence interval. (D) Measurement of exo-
and endocytic rates based on vGpH responses before and after Baf (see text).

hippocampi from P10 mice were harvested and homogenized from −50 to 40 mV and fitted with a modified Boltzmann
in RIPA buffer using a Dounce tissue homogenizer. Lysates equation:
were cleared by centrifugation and boiled in Laemmli  V1/2act −V 
sample buffer. Equal amount of total proteins were loaded. I = Gmax (Erev − V)/ 1 + e kact
Samples were then analyzed by SDS-PAGE, transferred onto
nitrocellulose membranes, probed with appropriate primary and
HRP-labeled secondary antibodies and revealed by enhanced where, I = current density (in pA/pF), Gmax = maximum
chemiluminescence. conductance (in nS/pF), Erev = reversal potential, V = measured
potential, V1/2act = midpoint voltage for current activation, and
kact = the slope factor.
Whole-Cell Patch Clamp Recordings of We used a tail protocol to measure current density; cells were
Cav2.1 and Cav2.2 Currents depolarized using 10 mV voltage steps, from −60 to 60 mV.
Cav2.1 or Cav2.2, the auxiliary subunits (GFP-β2a and α2 δ1 ) Following depolarization, tail currents were evoked with a 10 ms
and DISC1 were transiently transfected in HEK 293 cells using pulse at −50 mV. The data were fitted with single Boltzmann
the calcium phosphate method (Huang et al., 2012). Whole-cell equation:
patch-clamp recordings were performed within 36–72 h after  V1/2inact −V 
transfection. The external solution contained (in mM) 10 HEPES,
I = I min + (Imax − I min )/ 1 + e kinact
140 TEA-MeSO3 and 5 BaCl2 (pH 7.4, 300–310 mOsm). The
glass pipette solution was backfilled with pipette solution (in
mM) 10 HEPES, 5 CsCl, 138 Cs-MeSO3 , 0.5 EGTA, 1 MgCl2 , 2 where, Imax and I min = maximal and minimal current
mg/ml MgATP (pH 7.3, 290–300 mOsm). HEK 293 cells were respectively, V1/2inact = the half-maximal voltage for current
held at −90 mV using the Axopatch 700B amplifier (Axon inactivation, kinact = slope of inactivation curve.
Instruments). The series resistance for all recordings was less
than 5 MΩ; 70–80% compensation on serial resistance and Statistics
cell membrane capacitance were applied. A P/4 protocol was The experimental design of this study implies that data
used to subtract leakage current. All recordings were obtained collected at individual synaptic terminals are clustered according
with an Axon Digidata 1440A data acquisition system, sampled to neuron preparations and imaged fields. Galbraith et al.
at 5–50 kHz and low pass-filtered at 1 kHz or 6 kHz. The (2010) demonstrated that such clustering can adversely affect
I-V curves were obtained from 10 mV voltage-steps ranging statistical inference when not accounted for. In order to

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Tang et al. DISC1 Enhances Synaptic Vesicle Cycling

probe for clustering effects (i.e., intra-field correlations), we to a stimulation paradigm that consists of two consecutive
tested whether bouton responses significantly vary from field trains of APs (Fernandez-Alfonso and Ryan, 2004; Burrone
to field. For this, we conducted Wald tests of the null et al., 2006). The amplitude of the first vGpH transient is
hypothesis of no difference in mean outcome across fields governed by the relative rates of SV exo- and endocytosis
within each condition, which revealed (p-value < 0.0001) during stimulation (Figure 1C). To separate the contributions
strong intra-field correlation. To account for these correlations, of exo- and endocytosis, we blocked SV re-acidification with
we performed our statistical analysis in the framework of the vacuolar H+ ATPase inhibitor Bafilomycin (Baf) during
linear mixed models (Laird and Ware, 1982) with normally- the second stimulation, which allows selective measurement
distributed random field effects and preparation fixed effects. of SV exocytosis (Figure 1C). To account for cell-to-cell
In experiments involving one genetically-modified condition variation in vGpH expression, we normalized each trace
and one control group, we tested the null hypothesis of based on the vGpH signal measured after NH4 Cl addition
no difference between the mean outcome of the groups via (Figure 1C). The endocytic component of SV cycling was then
2-sample t-tests. In experiments involving two genetically- computed by subtracting the first vGpH transient from the
modified conditions and one control group, we tested the vGpH response after Baf treatment (Figure 1D). Synaptic
null hypothesis of no difference between the mean outcome traces with baseline drifts between the first and second
of each group and the control group jointly using Wald stimulation or after Baf addition were excluded from the
tests. All tests were performed at the 5% level of statistical analysis (see ‘‘Materials and Methods’’ Section). Exocytosis
significance and carried out using the statistical software Stata largely dominates at the onset of stimulation, while endocytosis
version 13.2. Because our statistical approach (linear mixed kicks in towards the end of the AP train. Exo- and endocytic
model) is not a standard practice when analyzing synaptic rates were finally obtained by linear fitting of the exo- and
properties, we compared the p-values obtained with our method endo traces (Figure 1D). Exocytic rates are about three
with those measured by the more common field averaging times higher than endocytic rates under this stimulation
approach, where the information of a field is collapsed to protocol. To evaluate the variability of bouton responses
a single independent observation by taking the mean of across fields and neuron preparations, we compiled exocytic
bouton responses. Both methods are valid and gave comparable rates from six independent experiments (Supplementary
results (Supplementary Table T1), although the field averaging Figure S1). Statistical analysis of these responses revealed
approach is less statistically efficient as it diminishes the substantial field to field variation (see ‘‘Materials and Methods’’
information that can be obtained from the data by reducing Section), implying that the information collected from each
individual measurements in a field to one observation (Galbraith bouton does not constitute an independent measurement.
et al., 2010). To compare synaptic responses in different fields and
conditions we used a statistical approach that accounts for
correlations within fields and variations in neuron preparations
RESULTS (see ‘‘Materials and Methods’’ Section and Supplementary
Table T1).
DISC1 Loss-of-Function Slows Down SV We then silenced the DISC1 gene using a published shRNA
Cycling sequence (shRNA-E), targeting exon 2 (Duan et al., 2007) and
We opted for an imaging approach based on the synaptic a new shRNA sequence (shRNA-A) targeting exon 9. These
tracer vGpH to explore the impact of DISC1 on the SV cycle. two shRNAs target regions of the DISC1 gene that are fully
vGpH consists of the pH-sensitive GFP variant pHluorin conserved in rodents and are thus expected to silence rat
(Miesenbock et al., 1998) fused to the SV-resident glutamate and mouse DISC1 with the same efficiency. To probe the
transporter vGlut1. pHluorin, which faces the acidic lumen effect of these shRNAs on DISC1 protein levels, we used an
of SVs, undergoes a ∼20-fold increase in fluorescence antibody raised against the C-terminus of mouse DISC1, which
intensity when exposed to the neutral pH of the extracellular has previously been validated in a DISC1 KO mouse (Kuroda
milieu after membrane fusion. (Sankaranarayanan et al., et al., 2011). In our hands, this antibody poorly reacts with rat
2000). Following glutamate discharge and vGpH re-uptake, DISC1 (not shown). In mouse hippocampal neurons, however,
SVs are rapidly re-acidified and vGpH fluorescence is it detects one major protein at ∼100 kDa corresponding to the
quenched. This property has made vGpH a valuable tool predicted full length DISC1 (Kuroda et al., 2011) and which is
to monitor both exo- and endocytosis of SVs at single substantially reduced in both shRNA-E and -A transduced cells
synapses. (Figure 2A).
Due to the inherent variability in the properties of individual Next, we probed the impact of these shRNAs on the
synaptic boutons (Ariel et al., 2013) we measured SV cycling SV cycle in rat hippocampal neurons. We initially chose
in large synapse populations. For this, we developed an image to work with rat, rather than mouse neurons, because we
analysis algorithm that identifies all responding boutons in can harvest substantially more cells from a rat embryonic
a given field (Figures 1A,B), and imaged 16–18 fields from litter. vGpH signals in shRNA-E and -A expressing neurons
at six independent rat hippocampal neuron cultures, yielding display a slower rise and lower amplitude during the first
close to a thousand synaptic boutons for each condition. We and second stimulation, relative to control cells expressing a
employed this algorithm to monitor SV cycling in response scramble shRNA (Figure 2B). Analysis of exocytosis shows

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Tang et al. DISC1 Enhances Synaptic Vesicle Cycling

FIGURE 2 | DISC1 silencing by RNAi slows down SV exocytosis. (A) Immunoblot analysis of DISC1 in mouse hippocampal neurons (DIV 8) transduced with
scramble, DISC1-A and DISC1-E shRNAs. (B) Average vGpH traces derived from neurons expressing scr (1204 boutons, 18 fields), -E (960 boutons, 18 fields) and
-A (924 boutons, 16 fields) shRNAs from six independent experiments. (C,D) Analysis of SV exocytosis after Baf treatment. (C) Exocytic profiles of neurons
expressing the indicated shRNAs. (D) Boxplot of exocytic rates. (E,F) Analysis of SV endocytosis. (E) Endocytic profiles. (F) Boxplot of endocytic rates. (G) Exocytic
profile of cells expressing the indicated shRNAs in response to a stimulation (1200 APs, 10 Hz) that depletes the total releasable pool.

substantial synapse-to-synapse variation in all conditions, but display SV cycling defects that are remarkably similar to
reveals a marked decrease in exocytic rates (Figures 2C,D) and these observed with RNAi. The rates and amplitudes of
amplitudes (Figure 2C) in both shRNA-E and -A expressing exocytic responses were substantially reduced relative to wt
neurons. DISC1 silencing also resulted in slightly slower cells (Figures 3B–D), while endocytic rates were marginally,
SV endocytosis, although this effect did not reach statistical but not significantly diminished (Figures 3E,F). Together, these
significance (Figures 2E,F). results show that both genetic ablation and RNAi knockdown
Reduced amplitude of the exocytic response prompted us of DISC1 selectively disrupts SV exocytosis at glutamatergic
to test whether DISC1 also regulates the size of the total synapses with no detectable impact on the total releasable
releasable pool. For this, we applied a stimulus strong enough pool.
(1200 APs at 10 Hz) to maximally deplete releasable SVs
from presynaptic terminals (Ariel and Ryan, 2010). Under this
stimulation regime, vGpH responses reached the same plateau DISC1 Stimulates Ca2+ Influx and
in DISC1-silenced and control neurons, albeit with different Regulates Cav2.2-Dependent SV Release
kinetics, arguing against an effect on the total releasable pool but Because SV exocytosis is initiated by Ca2+ influx through
confirming the stimulatory function of DISC1 in SV exocytosis VGCCs, we measured evoked Ca2+ transients at presynaptic
(Figure 2G). terminals using the SV-targeted Ca2+ sensor SyGC3 (Li
To rule out the possibility of RNAi off-target effects—shRNA- et al., 2011). Ca2+ signals evoked by 300 APs (10 Hz)
E has recently been suggested to inhibit neuron migration or 200 APs (20 Hz) show a rapid rise and partial decay
in the developing cortex independently of DISC1 (Tsuboi during the stimulus (Figures 4A,B,D,E), as observed
et al., 2015)—we examined SV cycling in a DISC1 KO by others (Li et al., 2011). These Ca2+ transients were
mouse that lacks exons 2 and 3 of the DISC1 gene (Kuroda significantly reduced in shRNA-E and -A expressing
et al., 2011). Homozygous DISC1∆2–3/∆2–3 mice show no neurons, under the same stimulation conditions used
detectable levels of the major isoform of DISC1 (Figure 3A). for vGpH measurements (Figures 4A,C), or in response
Hippocampal neurons derived from DISC1∆2–3/∆2–3 mice to a higher frequency stimulus (Figure 4B). Similarly,

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Tang et al. DISC1 Enhances Synaptic Vesicle Cycling

FIGURE 3 | Attenuated SV release in hippocampal neurons from DISC1∆2–3/∆2–3 mice. (A) Immunoblot analysis of DISC1 in hippocampal lysates prepared
from P10 DISC1∆2–3/∆2–3 , DISC1wt/∆ 2–3 and DISC1wt/wt mice, confirming the ablation of full-length DISC1 (∼100 kD). (B) Average vGpH traces in DISC1wt/wt
(575 boutons, 7 fields) and DISC1∆2–3/∆2–3 (682 boutons, 9 fields) neurons in response to two consecutive trains of APs and obtained from two independent
experiments. (C,D) Analysis of SV exocytosis from vGpH responses after Baf. Average kinetics (C) and rates (D) of vGpH exocytic responses. (E,F) Analysis of SV
endocytosis. Average kinetics (E) and rates (F) of vGpH endocytic responses.

DISC1∆2–3/∆2–3 neurons show lowered Ca2+ transients channel-specific toxins to determine the contribution of
compared to wt cells (Figures 4D–F). We were concerned each subtype to SV release in hippocampal neurons. ω-
that, under such prolonged stimulations, Ca2+ concentration Conotoxin GVIA (an N-type blocker) reduced SV exocytic
in terminals might reach levels that partially saturate SyGC3. rates by 73 ± 5% (Figures 5A,B), while ω-Agatoxin TK
Ca2+ signals were therefore also examined in response to (a P/Q-type blocker) resulted in a 42 ± 3% decrease in
shorter stimulations (20 APs, 20 Hz) under conditions SV exocytic rates (Figures 5C,D), in a good agreement
well below SyGC3 saturation (Tian et al., 2009; Akerboom with a recent study (Ariel et al., 2013). Note that in these
et al., 2012). A clear decrease in the amplitude of Ca2+ experiments, exocytic rates were approximated by measuring
transients was also observed in DISC1-silenced neurons the initial slope of the vGpH response in the absence of Baf
under this stimulation regime (Figure 4G). Together, these (see Figure 1D). Importantly, blockade of Cav2.2 almost
Ca2+ imaging data hint at a Ca2+ influx defect in DISC1- completely occluded the effect of DISC1 silencing on SV
inactivated cells, although abnormal clearance of Ca2+ from the exocytosis (Figures 5A,B,E). Blockade of Cav2.1, on the
presynaptic terminal could also be involved (see ‘‘Discussion’’ other hand, slightly increased the inhibitory impact of
Section). DISC1 knockdown on SV release rates (Figures 5C–E).
To determine whether reduced Ca2+ influx is the primary We conclude from these results that DISC1 selectively
cause of SV cycling defects, we attempted to rescue SV exocytosis stimulates Cav2.2-dependent SV release at hippocampal
in DISC1-silenced neurons by elevating extracellular Ca2+ synapses.
concentration. Shifting extracellular Ca2+ concentration from 2 We then asked whether DISC1 regulates Cav2.2 activity
to 4 mM increases AP-induced Ca2+ entry (Ariel and Ryan, 2010) by controlling its presynaptic localization and/or abundance.
and restored the vGpH response (Figure 4H), in line with a role Co-localization studies of endogenous Cav2.2 with the active
of DISC1 in facilitating Ca2+ influx. zone marker bassoon revealed extensive presence of Cav2.2 in
At central synapses, the P/Q-type (Cav2.1) and N-type synaptic boutons, both in control and DISC1-silenced neurons
(Cav2.2) Ca2+ channels are the main sources of Ca2+ for (Supplementary Figures S2A–C). We found no significant
initiation of SV exocytosis (Catterall et al., 2013). We used difference between these two groups in the fraction of boutons

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Tang et al. DISC1 Enhances Synaptic Vesicle Cycling

FIGURE 4 | DISC1 loss-of-function reduces evoked Ca2+ transients at nerve terminals. SyGC3 imaging in rat hippocampal neurons (DIV14–16) in response
two different trains of APs. (A) Average Ca2+ transients in neurons expressing scr (1052 boutons, 15 fields, 5 experiments (exps)), DISC1-E (1836 boutons, 9 fields,
3 exps) and -A (754 boutons, 11 fields, 3 exps) shRNAs, in response to 300 APs, 10 Hz. The DISC1-E and DISC-A groups are significantly different than the scr
group (p = 0.0057). (B) Average Ca2+ transients in neurons expressing scr (861 boutons, 9 fields, 5 exps), DISC1-E (2118 boutons, 7 fields, 3 exps) and -A (1549
boutons, 9 fields, 3 exps) shRNAs, in response to 200 APs, 20 Hz. (C) Cumulative probability of SyGC3 peak intensity from individual boutons corresponding to
(A). (D) Average Ca2+ transients in DISC1 wt/wt (1090 boutons, 11 fields, 3 exps) and DISC1∆2–3/∆2–3 (727 boutons, 9 fields, 3 exps) neurons, in response to 300
APs, 10 Hz. The DISC1 wt/wt and DISC1∆2–3/∆2–3 groups are statistically different (p = 0.0182). (E) Average Ca2+ transients in DISC1 wt/wt (1365 boutons, 9 fields, 3
exps) and DISC1∆2–3/∆2–3 (1233 boutons, 8 fields, 3 exps) neurons, in response to 200 APs, 20 Hz. (F) Cumulative probability of SyGC3 peak intensity from
individual boutons corresponding to (D). (G) Average Ca2+ transients in neurons expressing scr (290 boutons, 6 fields, 2 exps), and DISC1-E (390 boutons, 6 fields,
2 exps) shRNAs, in response to 20 APs, 20 Hz. The scr and DISC1-E groups are statistically different (p = 0.0036). (H) Average vGpH traces in scr (180 boutons, 5
fields, 2 exps) and DISC1-E (488 boutons, 6 fields, 2 exps) shRNA-expressing neurons during two consecutive trains of APs (300 AP, 10 Hz) in the presence of 2 or
4 mM extracellular Ca2+ .

containing Cav2.2, or in the intensity of Cav2.2 staining in We finally examined the effect of DISC1 on Cav2 currents
presynaptic terminals (Supplementary Figures S2B,C). Nor by whole cell patch-clamp recordings. Co-transfection of
did we find evidence for altered presynaptic localization and recombinant DISC1 (Figure 6A) in HEK293T cells together
intensity of Cav2.1 (Supplementary Figures S2D–F or reduced with Cav2.2 and its auxiliary subunits (see ‘‘Materials and
density of presynaptic terminals (Supplementary Figure S2G) Methods’’ Section) resulted in a 27% increase in Cav2.2
in DISC1 knockdown neurons. Thus, synaptic targeting and peak current density (Figures 6B,C). Likewise, tail current
abundance of Cav2 channels does not seem to be regulated by density of Cav2.2 was substantially elevated in DISC1-
DISC1. overexpressing cells (Figures 6D,E). Current density can be

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Tang et al. DISC1 Enhances Synaptic Vesicle Cycling

FIGURE 5 | DISC1 regulates Cav2.2-dependent SV exocytosis. (A) Average vGpH traces in scr (101 boutons, 6 fields, 2 exps) and DISC1-E (87 boutons,
5 fields, 2 exps) shRNA-expressing neurons during consecutive trains of APs (300 AP, 10 Hz) in the absence or presence of the Cav2.2 blocker ω-Conotoxin GVIA
(125 nM). (B) Boxplot of SV exocytic rates before and after ω-Conotoxin GVIA application. Exocytic rates were measured by linear fitting of the first six time points of
the vGpH response. (C) Average vGpH traces in scr (1294 boutons, 9 fields, 3 exps) and DISC1-E (1282 boutons, 9 fields, 3 exps) shRNA-expressing neurons during
consecutive trains of APs (300 AP, 10 Hz) in the absence or presence of the Cav2.1 blocker ω-Agatoxin TK (125 nM). (D) Boxplot of SV exocytic rates before and
after ω-Agatoxin TK application. (E) Table showing the percentage of inhibition of exocytosis rate by DISC1 knockdown before and after Cav2.2- or Cav2.1 blockade.

affected by a change in channel gating or by the number that restricts the activity of DISC1 to Cav2.2-dependent SV
of channels at the cell surface. To test the first possibility, exocytosis.
we examined the voltage-dependent properties of Cav2.2
current activation and saw no difference between control DISCUSSION
and DISC1-overexpressing cells (Figure 6F). This suggests
that DISC1 promotes surface delivery and/or stabilize We used here two independent gene-targeting approaches
surface expression of Cav2.2. Recording of Cav2.1 currents together with large-scale imaging of presynaptic function to
revealed a similar, voltage-independent, potentiation effect determine the impact of the mental disease gene DISC1
of DISC1 (Figures 6G–K). Thus, DISC1 equally augments on the glutamate release machinery. Our results show that
both Cav2.2 and Cav2.1 currents in this heterologous DISC1 accelerates SV exocytosis and thus enhances presynaptic
system, presumably by increasing surface expression of performance. This boosting effect is mediated by N-type Ca2+
these Ca2+ channels. These results also imply the presence channels, establishing the first link between DISC1 and VGCC
of an additional layer of regulation in hippocampal neurons activity.

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Tang et al. DISC1 Enhances Synaptic Vesicle Cycling

FIGURE 6 | DISC1 enhances Cav2.2 and Cav2.1 currents. (A) Western blot showing expression of ectopic (human) DISC1 in HEK293 cells. (B,C) Cav2.2
Current-voltage (I-V) curves for hDISC1-expressing and control cells. (B) Stimulation protocol and individual current responses shown at three different voltages
(−30, 0 and 30 mV) (C). Average I-V plots for hDISC1 (peak = 54.2 ± 4.5 pA/pF, 13 cells) and control (peak = 39.2 ± 4.9 pA/pF, 12 cells), p = 0.033. (D–F) Cav2.2
activation curves in response to the tail protocol. (D) Illustration of the tail protocol and individual tail currents measured at −50 mV after three different voltage steps
(−20, 0 and 40 mV). (E) Average current density based on tail currents for DISC1 (164.3 ± 7.3 pA/pF, 12 cells) and control (110.5 ± 6.3 pA/pF, 12 cells), ∗ p < 0.001.
(F) Normalized activation curve from tail currents showing no significant difference between DISC1 (V50 : −6.98 ± 3.43 mV, 12 cells) and control (V50 : 0.86 ± 3.62
mV, 12 cells), p = 0.13. (G,H) Cav2.1 Current-voltage (I-V) curves for hDISC1-expressing and control cells. (G) Stimulation protocol and individual current responses
shown at three different voltages (−30, 0 and 30 mV). (H) Average I-V plots for hDISC1 (peak = 79.9 ± 8.3 pA/pF, 15 cells) and control (peak = 56.3 ± 7.4 pA/pF,
13 cells), p = 0.046. (I–K) Cav2.1 activation curves in response to the tail protocol. (I) Illustration of the tail protocol and individual tail currents measured at −50 mV
after three different voltage steps (−20, 0 and 40 mV). (J) Average current density based on tail currents for DISC1 (156.3 ± 5.7 pA/pF, 25 cells) and control
(123.2 ± 7.4 pA/pF, 17 cells), ∗ p = 0.003. (K) Normalized activation curve from tail currents showing no significant difference between DISC1 (V50 : −5.39 ± 0.49
mV, 24 cells) and control (V50 : −4.41 ± 0.93 mV, 17 cells), p = 0.31.

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Tang et al. DISC1 Enhances Synaptic Vesicle Cycling

DISC1 inactivation in hippocampal neurons results in The efficacy of neurotransmitter release determines not only
decreased Ca2+ transients at hippocampal terminals in response the strength of synaptic excitation, but also dictates various forms
to AP firing, consistent with a deficit in Ca2+ entry. Because of short-term plasticity (Abbott and Regehr, 2004), suggesting
these Ca2+ signals are shaped by both Ca2+ entry and broad functions of DISC1 in synaptic computation and neural
clearance processes, we cannot exclude a role of DISC1 in circuit performance. Although we have not directly measured
regulating Ca2+ extrusion or buffering. A stimulatory effect release probability (Pr ), the impact of DISC1 on Ca2+ entry
of DISC1 on Ca2+ entry is further supported, however, by and SV cycling suggests a positive influence on Pr (see Maher
its positive impact on Cav2.2 and Cav2.1 currents, the two and Loturco, 2012). Because synaptic facilitation and depression
main VGCCs underlying the initiation of neurotransmission depend largely on the initial Pr (high Pr favors depression while
at hippocampal synapses. How DISC1 enhances Cav2 currents low Pr favors facilitation), our results predict altered short-
is unclear. The lack of an effect on gating suggests that term plasticity in DISC1-deficient neural circuits. Of interest,
DISC1 promotes surface expression of these Ca2+ channels, abnormal short-term plasticity has been associated with deficits
although we cannot exclude a role of DISC1 in regulating in moment-to-moment information processing and working
single-channel conductance or open probability. Our immuno- memory, two hallmarks of schizophrenia (Crabtree and Gogos,
localization studies revealed no detectable impact of DISC1 2014). Reduced efficacy in glutamate release may also be relevant
on presynaptic abundance of Cav2 channels. This does not for forms of long-term potentiation (LTP) that have clear
rule out, however, the possibility that DISC1 regulates the presynaptic components, such as LTP at the perforant path-
number of functional Ca2+ channels at the surface of the granule cell synapse in the dentate gyrus (Errington et al.,
terminal. 1987). Notably, this form of LTP is impaired in DISC1∆2–3/∆2–3
Although overexpression of DISC1 enhances both Cav2.1 mice—a stronger tetanic stimulus is required for the expression
and Cav2.2 currents in a heterologous system, our findings of LTP (Kuroda et al., 2011). Our findings suggest that this
point to a selective influence of DISC1 on Cav2.2-dependent LTP deficit could be due to a failure of the presynaptic
SV exocytosis. How is this selectivity achieved? It is possible terminal to undergo activity-dependent increase in release
that both Cav2.1 and Cav2.2 compete for DISC1 regulation at probability.
presynaptic terminals. The dominant contribution of Cav2.2 to Several genes encoding VGCC subunits, including
SV release probably results (at least in part) from increased CACNA1C, CACNB2 and CACNA1I have repeatedly been
levels of Cav2.2 (relative to Cav2.1) at hippocampal terminals associated with schizophrenia and other psychiatric disorders
(Ariel et al., 2013). Thus, preferential regulation of Cav2.2 by (Ferreira et al., 2008; Cross-Disorder Group of the Psychiatric
endogenous levels of DISC1 combined with greater abundance Genomics, 2013; Hamshere et al., 2013; Ripke et al., 2013;
of Cav2.2 may explain why DISC1-dependent SV exocytosis Schizophrenia Working Group of the Psychiatric Genomics,
appears to be exclusively mediated by Cav2.2. Specificity could 2014). Although CACNA1A (Cav2.1) and CACNA1B (Cav2.2)
also arise, however, from functions of DISC1 downstream are not typically associated with risk loci, RIM1 (also called
of Ca2+ entry. For example, DISC1 could be involved in RIMS1)—a presynaptic scaffold that regulates density of
positioning Cav2.2 in close proximity to the SV release P/Q- and N-type Ca2+ channels and SV docking at the
machinery at the active zone, a process that dictates speed and active zone (Han et al., 2011)—was recently identified as a
fidelity of neurotransmission. While the role of Ca2+ entry candidate gene for schizophrenia in the largest GWAS study
in SV exocytosis is undisputed, its impact on SV endocytosis conducted to date (Schizophrenia Working Group of the
remains somewhat controversial. Recent evidence suggests, Psychiatric Genomics, 2014). Together these findings point
however, that Ca2+ couples rates of SV exo- and endocytosis to neurotransmitter release as a central process targeted in
and optimizes endocytic rates during AP bursts (Armbruster schizophrenia.
et al., 2013). We observed reduced endocytic rates in both In conclusion, our results shed light on a novel
DISC1-silenced and DISC1∆2–3/∆2–3 neurons. Although this mechanism by which a major susceptibility gene for mental
effect was small and did not reach statistical significance, illness enhances the efficacy of glutamate release, and
our results suggest that the influence of DISC1 on Cav2.2 provide further support for a central role of glutamate
impacts both exo- and endocytosis of SVs at hippocampal neurotransmission in schizophrenia and other major mood
terminals. disorders.
Our findings extend on two recent studies implicating
DISC1 in presynaptic function. In glutamatergic neurons AUTHOR CONTRIBUTIONS
differentiated from human iPS cells and derived from members
of a family with a frameshift mutation in DISC1, deficits WT and JVT performed and analyzed all imaging and
in SV release were observed after KCl-induced depolarization biochemical experiments. QL performed and analyzed whole-cell
(Wen et al., 2014). In a separate report, RNAi knockdown patch-clamp recordings. MB carried out the statistical analysis.
of DISC1 in layer 2/3 neocortical neurons increases paired- KBL made the original observation implicating DISC1 in SV
pulse facilitation and appears to reduce probability of glutamate cycling. KKu and KKa made the DISC1 ∆2–3 mouse and
release (Maher and Loturco, 2012). Collectively, these results the DISC1 C-terminus antibody. WT and MF wrote Matlab
clearly identify DISC1 as a positive modulator of glutamate scripts. TWS and MF supervised the project. MF wrote the
release. article.

Frontiers in Synaptic Neuroscience | www.frontiersin.org June 2016 | Volume 8 | Article 15 | 17

Tang et al. DISC1 Enhances Synaptic Vesicle Cycling

ACKNOWLEDGMENTS FIGURE S2 | Presynaptic localization and abundance of Cav2 channels

are unaffected by DISC1 silencing. (A,D) Immunolocalization of
We thank Bo Lu for critical reading of the manuscript. We are endogenous Cav2.2 (A) or Cav2.1 (D) and bassoon in rat hippocampal
neurons (DIV 14–15) expressing scr and DISC1-E shRNAs. Arrowheads
in debt to Durgadevi Alagappan and Tan Li-Ting for excellent indicate boutons positive for Cav2.2 and bassoon (A) or Cav2.1 and bassoon
technical assistance. This work was supported by a grant to (D). Scale bar 10 µm. (B) Percentage of Cav2.2-positive boutons in scr (5
MF from the Ministry of Education Academic Research Fund cells) and DISC1-E (5 cells) shRNA-expressing neurons. (C) Cav2.2
(MOE2013-T2-1-053). fluorescence intensity measured at individual presynaptic boutons (based on
bassoon staining) in (B). (E) Percentage of Cav2.1-positive boutons in scr (8
cells) and DISC1-E (8 cells) shRNA-expressing neurons. (F) Cav2.1
SUPPLEMENTARY MATERIAL fluorescence intensity measured at individual presynaptic boutons (based on
bassoon staining) in (E). (G) Quantification of the density of presynaptic
terminals (based on bassoon staining) in neurons expressing scr (24 cells),
The Supplementary Material for this article can be found
DISC1–E (21 cells) and -A (17 cells) shRNAs from four independent
online at: http://journal.frontiersin.org/article/10.3389/fnsyn. experiments.
2016.00015/abstract
TABLE T1 | Statistical analysis of synaptic responses. Column 1:
FIGURE S1 | Variability of SV exocytic responses. Scatter plot showing datasets. Column 2: p values derived from the field averaging approach, with
SV exocytic rates in individual boutons across six (color-coded) independent each field weighted according to its number of boutons (see “Materials and
preparations. Each preparation comprises three fields colored light to dark. Methods” Section). Column 3: p values derived from the linear mixed model,
These rates were measured in hippocampal neurons (DIV14–16) expressing where intra-field correlations and variations in neuron preparations are taken
scr shRNA and correspond to the data shown in Figure 2. into account (see “Materials and Methods” Section).

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nn.3984
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(2006). Distinct endocytic pathways control the rate and extent of synaptic and Fivaz. This is an open-access article distributed under the terms of the Creative
vesicle protein recycling. Neuron 51, 71–84. doi: 10.1016/j.neuron.2006. Commons Attribution License (CC BY). The use, distribution and reproduction in
05.027 other forums is permitted, provided the original author(s) or licensor are credited
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515, 414–418. doi: 10.1038/nature13716 comply with these terms.

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 ORIGINAL RESEARCH
published: 26 July 2016
doi: 10.3389/fnsyn.2016.00021

Visualizing Presynaptic Calcium

Dynamics and Vesicle Fusion with a
Single Genetically Encoded Reporter
at Individual Synapses
Rachel E. Jackson and Juan Burrone *
Centre for Developmental Neurobiology, King’s College London, London, UK

Synaptic transmission depends on the influx of calcium into the presynaptic

compartment, which drives neurotransmitter release. Genetically encoded reporters are
widely used tools to understand these processes, particularly pHluorin-based reporters
that report vesicle exocytosis and endocytosis through pH dependent changes in
fluorescence, and genetically encoded calcium indicators (GECIs) that exhibit changes
in fluorescence upon binding to calcium. The recent expansion of the color palette
of available indicators has made it possible to image multiple probes simultaneously
within a cell. We have constructed a single molecule reporter capable of concurrent
imaging of both presynaptic calcium influx and exocytosis, by fusion of sypHy, the vesicle
associated protein synaptophysin containing a GFP-based pHluorin sensor, with the
red-shifted GECI R-GECO1. Due to the fixed stoichiometry of the two probes, the ratio of
the two responses can also be measured, providing an all optical correlate of the calcium
dependence of release. Here, we have characterized stimulus-evoked sypHy-RGECO
Edited by: responses of hippocampal synapses in vitro, exploring the effects of different stimulus
Marc Fivaz, strengths and frequencies as well as variations in external calcium concentrations. By
Duke-NUS Graduate Medical School,
Singapore combining live sypHy-RGECO imaging with post hoc fixation and immunofluorescence,
Reviewed by: we have also investigated correlations between structural and functional properties of
Kirill Volynski, synapses.
University College London, UK
Michael Alan Cousin, Keywords: presynaptic, calcium, neurotransmitter release, pHluorin, RGECO, vesicle
University of Edinburgh, UK
*Correspondence:
Juan Burrone INTRODUCTION
[email protected]
Synaptic transmission depends on the influx of calcium into the presynaptic compartment, driving
Received: 14 May 2016 the release of vesicles containing neurotransmitter, which will bind to receptors on the postsynaptic
Accepted: 13 July 2016 cell. Electrophysiological studies have been fundamental in understanding the relationship between
Published: 26 July 2016 presynaptic calcium and neurotransmitter release (Dodge and Rahamimoff, 1967) but are limited
Citation: in spatial resolution and cannot provide information about individual presynaptic terminals, unless
Jackson RE and Burrone J (2016) employed at large synapses that can be accessed directly (Heidelberger and Matthews, 1992;
Visualizing Presynaptic Calcium
Schneggenburger and Neher, 2000; Vyleta and Jonas, 2014). For studying individual synapses,
Dynamics and Vesicle Fusion with
a Single Genetically Encoded
optical strategies using either chemical indicators or genetically encoded reporters have therefore
Reporter at Individual Synapses. become the method of choice. Genetically encoded reporters offer the advantage that neural activity
Front. Synaptic Neurosci. 8:21. can be monitored in defined populations of cells, both in vitro and in vivo, and the probe can be
doi: 10.3389/fnsyn.2016.00021 localized to specific subcellular compartments (reviewed in Broussard et al., 2014).

Frontiers in Synaptic Neuroscience | www.frontiersin.org July 2016 | Volume 8 | Article 21 | 21

Jackson and Burrone Imaging Calcium and Neurotransmitter Release

The largest family of single color genetically encoded calcium overall release probability of the synapse. Whilst this elegant
indicators (GECIs) is the GCaMP family, based on a circularly study was highly informative, the techniques used are limited
permuted GFP linked to calmodulin (CaM) and it’s binding to relatively small numbers of cells and cannot be transferred
peptide M13 (Nakai et al., 2001). The original sensor has to an in vivo situation. With genetically encoded reporters,
undergone multiple rounds of both random and structure-guided visualization of both calcium influx and neurotransmitter release
mutagenesis to improve stability, brightness, signal to noise ratio has been achieved by co-expression of a red-shifted reporter of
and kinetics (Tian et al., 2009; Akerboom et al., 2012; Chen vesicle exocytosis, either VGLUT-mOr2 or sypHTomato, with a
et al., 2013) and several variants have been effectively targeted to green calcium indicator, such as members of the GCaMP family
the neuronal presynaptic terminal by fusion with synaptophysin (Li et al., 2011; Li and Tsien, 2012). However, in this situation
(Dreosti et al., 2009; Li et al., 2011; Nikolaou et al., 2012). To the two probes were expressed separately, resulting in possible
enable multicolor imaging in single cells, red-shifted GECIs have differences in the level of expression of each reporter and in
been developed, including RCaMP (Akerboom et al., 2013) and their subcellular location. Crucially, it also precludes the use of a
R-GECO1 (Zhao Y. et al., 2011), for which a presynaptically ratiometric approach to measuring sypHy responses (Rose et al.,
targeted version (SyRGECO) has also been characterized (Walker 2013), complicating the direct comparison of responses across
et al., 2013). Much like the GCaMPs, red GECIs are constantly cells.
evolving, with new variants recently produced in both RCaMP We have generated a new genetically encoded probe that
and R-GECO families (Inoue et al., 2015; Dana et al., 2016). brings together the measure of calcium and of exocytosis, in
The most widely used reporters of synaptic vesicle exocytosis addition to a ratiometric measure of sypHy signals. To do this
and endocytosis consist of a synaptic vesicle protein fused to we combined sypHy, a well characterized pHluorin sensor of
pHluorin, a pH-sensitive form of GFP (Miesenbock et al., 1998). synaptic vesicle exocytosis (Granseth et al., 2006), with the red-
Crucially in these constructs, pHluorin is localized to the acidic shifted calcium indicator R-GECO1 (Zhao Y. et al., 2011) in a
lumen of synaptic vesicles, so that its fluorescence is quenched single molecule (sypHy-RGECO) to enable concurrent imaging
at rest, but increases by up to 20-fold upon fusion with the of calcium influx and neurotransmitter release. The fixed 1:1 ratio
plasma membrane and exposure to the more basic pH of the of the two reporter molecules allows not only the normalization
extracellular medium (Sankaranarayanan et al., 2000). Several of sypHy signals to baseline or maximum RGECO fluorescence,
variants exist including fusions to the intraluminal domains of but also the use of the ratio of the two responses as a measure
VAMP2, known as synaptopHluorin, (Miesenbock et al., 1998), of the calcium dependence of release at individual synapses.
VGLUT1 (Voglmaier et al., 2006), and synaptophysin, known We systematically varied the number and frequency of action
as sypHy (Granseth et al., 2006). Recently, efforts have been potentials, as well as the external calcium concentrations, and
made to improve the normalization of presynaptic pHluorin measured sypHy and RGECO signals in individual synapses. We
responses, which are complicated by the presence of a fraction of found that while the amplitude of both responses changed, the
the protein at the cell surface, particularly for synaptopHluorin distribution of the ratios remained constant. Interestingly, the
and sypHy. At rest, surface stranded protein dominates the calcium dependence of release was found to vary considerably
baseline fluorescence due to its increased brightness compared between synapses, even within the same cell, suggesting the
to quenched protein localized to synaptic vesicles and is sensitivity of neurotransmitter release to calcium is synapse-
highly variable between boutons, preventing the use of baseline specific. We also combined live sypHy-RGECO imaging with
fluorescence as a normalizing factor for exocytic responses. By immunofluorescence for structural markers and observed that
fusing tdimer2 to the C-terminus of sypHy Rose et al. (2013) the amplitudes of calcium influx and exocytosis at individual
generated ratio-sypHy, in which the red fluorescence provides an synapses were positively correlated with the levels of the active
invariant signal proportional to the expression of the probe rather zone protein RIM but were less well correlated with levels of the
than the surface fraction. The ratio of green to red fluorescence synaptic vesicle calcium sensor synaptotagmin-1.
is fixed and allows normalization of sypHy responses to the
red baseline, simplifying comparisons across cells with different
expression levels and at different depths within a tissue. Finally, MATERIALS AND METHODS
red-shifted reporters of vesicle fusion have also been generated,
including VGLUT-2XmOr2 (Li et al., 2011), synaptobrevin 2- Generation of syn::sypHy-RGECO
mOrange (Ramirez et al., 2012), and sypHTomato (Li and Tsien, Plasmid
2012), again offering the possibility of combined imaging with SypHy was amplified by PCR using primers containing HindIII
other spectrally distinct reporters. and NotI restriction sites from CMV::sypHy A4 (a gift from
Despite the development of these tools, relatively few studies Leon Lagnado, Addgene plasmid # 24478). The resulting PCR
have been carried out in which both presynaptic calcium influx fragment was subcloned in to a modified version of the
and vesicle exocytosis have been imaged in the same small, en- pEGFP-N2 plasmid (Clontech Laboratories, USA), in which the
passant presynaptic terminals. Ermolyuk et al. (2012) used FM CMV promoter had been replaced with the human synapsin I
dyes to monitor vesicle exocytosis in conjunction with a calcium promoter. To amplify RGECO from SyRGECO (Walker et al.,
indicator introduced via a patch pipette, to examine the major 2013), a forward primer containing an XmaI site followed by a
parameters of presynaptic function including the size of the RRP, 5 amino acid linker (GSGGT) was used in conjunction with a
the probability of single vesicle fusion, calcium influx and the reverse primer containing a NotI site. When subcloned into these

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Jackson and Burrone Imaging Calcium and Neurotransmitter Release

sites in the syn::sypHy-EGFP-N2 plasmid the EGFP was replaced were imaged in HBS pH5.5 and HBS pH7.4 ±50 mM NH4 Cl
to generate syn::sypHy-RGECO. and the surface fraction calculated as (GpH7.4 –GpH5.5 )/(Gmax –
GpH5.5 ). Synapses in which the surface fraction was calculated to
Hippocampal Neuronal Culture and be >100% were not included (<1% of synapses).
Transfection Analysis of Live Images
Dissociated hippocampal neurons were prepared from Sprague– Images were analyzed using custom written Matlab codes
Dawley rats at embryonic day 18. Dissected hippocampi were (Mathworks, USA). Regions of interest (ROIs, 6 × 6 pixels)
treated with 5 mg/ml trypsin (Worthington, UK) for 5 min at 37 were selected for each puncta of sypHy fluorescence. Mean
and triturated with fire-polished Pasteur pipettes. Neurons were background-subtracted fluorescence intensity values were
plated on 18mm glass coverslips (Hecht Assistent, Germany) or calculated for each ROI in both sypHy (G) and RGECO (R)
35 mm Grid500 µ-dishes (Ibidi, Germany) coated with poly-D- channels. Traces were smoothed by averaging over a sliding
lysine (50 µg/ml) and laminin (20 µg/ml, both Sigma–Aldrich, window of 4 frames. Baseline fluorescence (G0 and R0 ) was
UK). Cultures media were maintained in neurobasal media measured as the average of ten frames prior to the stimulus.
supplemented with 2% B27, 2% fetal bovine serum, 1% glutamax 1G and 1R values were calculated by the change in signal
(all ThemoFisher Scientific, UK) and 1 % penicillin/streptomycin intensity from the baseline, with the peak responses defined as
(Sigma), at 37◦ C in a humidified incubator with 5% CO2 . After the maximum 1G and 1R within 40 frames of the stimulus.
3 days in vitro (DIV) the media was exchanged for serum-free Puncta in which the 1R response to the first 10 AP 20 Hz
media. At 7DIV neurons were transfected with sypHy-RGECO stimulus was greater than three times the standard deviation
using Effectene (QIAGEN, UK). After transfection neurons of the baseline were considered responding synapses and were
were maintained in serum-free media without antibiotics. Only analyzed in further images or stimulation conditions. 1G and
schedule 1 procedures performed by a competent individual 1R values for each synapse were averaged across trials and any
were used in these studies, which are exempt under the Animals synapse in which the mean peak response did not reach 3 SDs of
(Scientific Procedures) Act 1986. the mean baseline in either channel were also discarded. Before
pooling, mean 1G and 1R responses were normalized to either
Imaging of sypHy-RGECO R0 or Rmax , defined as the average of 10 images in 200 µM
Neurons were imaged at 17–21DIV in HEPES buffered saline ionomycin.
(HBS; 139 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 10 mM
D-Glucose, 2 mM CaCl2 , 1.3 mM MgCl2 ; pH 7.3, 290 mOsmol) Immunofluorescence
with 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f ]quinoxaline- After fixation cells were permeabilized with 0.2% Triton X-100 in
7-sulfonamide (NBQX), 0.025 mM amino-5-phosphonovaleric PBS for 5 min, washed three times in PBS and placed in blocking
acid (APV) and 6-imino-3-(4-methoxyphenyl)-1(6H)-pyrida- solution (3% BSA in PBS + 0.05% NaN3 ) for 60 min at room
zinebutanoic acid hydrobromide (Gabazine) (all Tocris, UK). temperature (RT), or for longer at 4◦ C. Cells were incubated in
Coverslips were placed in a field stimulation chamber containing primary antibodies diluted in blocking solution for 60 min at RT
platinum electrodes (RC49-MFS, Warner Instruments, USA). then washed five times in PBS. Secondary antibodies in blocking
For Ibidi dishes a stimulation insert (RC-37WS, Warner solution were applied for 60 min at RT and cells were washed five
Instruments) was placed inside the dish. Neurons were imaged times in PBS. Cells were mounted using Ibidi mounting medium.
on an inverted Olympus IX71 microscope, equipped with Cells were stained with chicken anti-GFP (1:1000, Abcam,
a 60x/1.42 NA oil objective. Imaging was performed with a UK) to amplify the sypHy signal, and either rabbit anti-RIM1/2
dual band-pass filter set optimized for EGFP and mCherry (1:500, Synaptic Systems, Germany) or mouse anti Synaptogmin-
(Chroma, cat number 59022) and with LED excitation light 1 (1:100, Synaptic Systems). Appropriate secondary antibodies
sources of 470 and 585 nm (CoolLED, UK) for pHluorin and tagged with Alexa 488 or 647 (ThermoFisher Scientific) were used
RGECO fluorophores, respectively. Time-lapse images were at 1:1000.
acquired at approximately 9.4 Hz using an Evolve 512 EMCCD
camera (Photometrics, USA) controlled by Slidebook software Fixed Cell Imaging and Analysis
(Intelligent Imaging Innovations, USA). Each frame consisted of The regions previously imaged live were located using the grid
a 20 ms exposure of the 470 and 585 nm LEDs sequentially, at coordinates and imaged on an inverted Nikon Eclipse Ti spectral
50 and 70% power, respectively, with 2 × 2 binning. Stimulation confocal microscope equipped with a 60x/1.40 NA oil objective
consisted of 1 ms 80 V pulses, which approximate single APs using NIS Elements software. Excitation wavelengths were 405,
(Zhao C. et al., 2011), delivered by an SD9 Stimulator (Grass 488, and 636 nm with bandpass emission filters set to 425–
Technologies, USA), controlled by Slidebook. At the end of 475 nm, 500–550 nm, and 662–737 nm. Microscope settings were
the imaging session 200 µM ionomycin was applied (Cayman kept the same for all images of the same antibodies. Z-stacks
Chemicals, USA) in either standard HBS, or HBS in which were generated from 0.15 µm optical sections, and maximum
50 mM of NaCl was replaced with 50 mM NH4Cl, to maximize projections were produced in Image-J. To align the live and fixed
sypHy-RGECO fluorescence. Neurons in Ibidi dishes were fixed images landmarks were selected in the GFP channel of both
in 4% PFA + 1% sucrose after imaging and were not treated with images, followed by affine transformation of the fixed image.
ionomycin. For calculations of surface fluorescence, synapses ROIs from the live image were scaled and overlaid on the

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Jackson and Burrone Imaging Calcium and Neurotransmitter Release

registered GFP image for manual confirmation of the position. (Supplementary Figure S1B). A similar correlation was observed
The GFP image was then thresholded using the ‘Moments’ when ionomycin treatment was combined with application of
algorithm in ImageJ to produce a mask, which was applied to HBS containing 50mM NH4 Cl to alkalinize all compartments
the other channels of the fixed image to avoid the inclusion of and maximize sypHy fluorescence (Supplementary Figure S1C).
neighboring puncta in the ROI. Intensity values for each ROI This contrasts with the less tight correlations observed between
were then calculated from the masked images. the baseline fluorescence values of the two channels, G0 and
R0 (Supplementary Figure S1D), and between G0 and Gmax
Statistical Analysis (Supplementary Figure S1E), which are likely affected by
Mean responses are displayed ±SEM unless otherwise stated, heterogeneity in the baseline fluorescence of sypHy. To directly
and n = the total number of synapses. As data are not normally measure the surface fraction of sypHy-RGECO, we compared the
distributed, non-parametric statistical tests have been used. difference in sypHy fluorescence in external solutions at pH5.5
and pH7.4 to the sypHy fluorescence maximized by application
of 50mM NH4 Cl. The surface fraction varied between synapses
RESULTS (Supplementary Figure S1F, mean = 30.8%, median = 24.8%) and
was slightly higher than that reported for ratio-sypHy (median
To generate a single molecule reporter of both presynaptic f surf = 20%, Rose et al., 2013). This may be due to the fact that
calcium influx and synaptic vesicle exocytosis, the R-GECO1 as basal RGECO fluorescence is low, it was necessary to identify
fragment was amplified from SyRGECO (Walker et al., 2013) transfected cells using basal sypHy fluorescence, potentially
and subcloned downstream of sypHy (Granseth et al., 2006) with biasing toward brighter cells with higher surface fractions.
a short linker sequence. The whole sequence was placed under SypHy-RGECO responses to a 10 AP stimulus from multiple
control of the human synapsin I promoter to drive expression cells were normalized and pooled, and a positive non-linear
in neurons. The resultant syn::sypHy-R-GECO1 plasmid will correlation between 1G/Rmax and 1R/Rmax was observed
hereafter be referred to as sypHy-RGECO (Figure 1A). With this (Figure 1E), as expected from the non-linear relationship
probe, presynaptic calcium influx is reported by an increase in between calcium and exocytosis (Dodge and Rahamimoff, 1967)
red fluorescence and exocytosis of synaptic vesicles by an increase and similar to that seen in other imaging studies (Zhao C.
in green fluorescence (Figure 1A). In dissociated hippocampal et al., 2011; Ariel et al., 2012; Ermolyuk et al., 2012). The spread
neurons transfected with sypHy-RGECO, punctate fluorescence of the responses suggested that individual synapses differed
in presynaptic boutons was observed in both green and red in the amount of neurotransmitter released for a given influx
channels (Figure 1B). A field stimulation of 10 action potentials of calcium, which can be evaluated by the ratio 1G/1R. We
(APs) at 20 Hz was applied and individual boutons responded observed a range of ratio values, the majority of which fell within
with increases in fluorescence in both channels (Figure 1C), a normal distribution, although a small number of synapses
the amplitudes of which (1G and 1R) were highly correlated displayed very high release in comparison to calcium influx, an
(Figure 1D). order of magnitude higher than the lowest ratios (Figure 1F).
In order to pool responses from multiple cells or synapses, Thus, there is heterogeneity in the calcium dependence of release
baseline fluorescence is often used to normalize reporter between synapses.
responses. However, for pHluorins this value can be dominated When using ratiometric imaging, differential bleaching of
by protein localized to the plasma membrane, which is the fluorophores could pose a potential problem. In boutons
approximately 20-fold brighter than the quenched vesicular imaged without stimulation, sypHy fluorescence decayed by
protein (Sankaranarayanan et al., 2000), and is highly variable only 2% during the imaging period. This decay could be fit
between synapses. One method that has been used to overcome with a double exponential, with time constants of 1.64 s and
this problem involved fusion of the red fluorescent protein 3172 s (Supplementary Figure S2A). Frames within the first
tdimer2 to the C-terminus of sypHy, generating a reporter time constant were therefore disregarded and frames from 1 s
termed ratio-sypHy (Rose et al., 2013). The fixed stoichiometry before the stimulus were used to calculate baseline fluorescence.
of the two fluorophores in this probe allowed normalization of The slow phase of bleaching, which accounted for 98.5% of the
the sypHy signals to the tdimer2 fluorescence. Using the same decay, had a negligible effect on either baseline fluorescence or
reasoning, RGECO can be used not only as a calcium indicator, maximum 1G amplitude and was not corrected for, although
but to normalize both 1G and 1R responses. As baseline this would need to be considered if imaging over a long period
RGECO fluorescence (R0 ) is relatively dim, we applied ionomycin or using this probe to measure vesicular endocytosis rates.
in 2mM extracellular calcium to maximize the RGECO signal RGECO did not exhibit bleaching, instead showing an initial
within the bouton (Rmax ). Both values (R0 and Rmax ) were highly rapid increase in fluorescence that plateaued, the reasons for
correlated (Supplementary Figure S1A) and were subsequently which will be discussed in more detail below (Supplementary
used as a normalizing factor for sypHy and RGECO responses. Figure S2B). The rapid initial changes in fluorescence suggested
After ionomycin treatment, sypHy fluorescence also gradually that sequential images may be affected by differential bleaching.
increased to a plateau presumably due to vesicle release caused To test this, cells were stimulated with 10 AP stimuli five times,
by the large influx of calcium. The maximum fluorescence in with a 1 min interval between images. The average 1G and 1R
green and red channels was also highly correlated, reflecting responses both decreased over the trials, with a slightly greater
the fixed ratio of the two fluorophores at all synapses decrease in the calcium response (Supplementary Figure S2C).

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Jackson and Burrone Imaging Calcium and Neurotransmitter Release

FIGURE 1 | Generation and characterization of sypHy-RGECO. (A) Schematic of the sypHy-RGECO construct and its function. Presynaptic calcium influx is
reported by an increase in RGECO fluorescence, situated on the cytoplasmic tail of synaptophysin, whilst exocytosis is reported by an increase in pHluorin
fluorescence situated in the vesicle lumen. (B) Representative images of a dissociated hippocampal cell expressing sypHy-RGECO. Scale bar 20 µm. Inset shows
merge of the two channels for the boxed region. (C) Changes in sypHy (i, 1G) and RGECO (ii, 1R) fluorescence in response to a field stimulus of 10 APs at 20 Hz.
(n = 65 synapses from 1 coverslip, traces representing individual synapse responses shown in green/red, mean response of all synapses shown in black, stimulation
period shown by black bar). (D) 1G and 1R responses are correlated (Spearman’s rank correlation r s = 0.766, p < 0.001). (E) Cells were stimulated five times with
a 10 AP 20 Hz stimulus. A mean response across trials was calculated and normalized to the maximum RGECO fluorescence in 200 µM ionomycin for each
synapse. 1G/Rmax and 1R/Rmax responses from multiple cells show a non-linear correlation (r s = 0.727, p < 0.0001, n = 526 synapses, from 9 coverslips and 5
independent cultures). The black line shows a fit to a power function of the form axb +c, where b = 2.18. (F) Distribution of values for the ratio 1G/1R, a measure of
the calcium dependence of release (for the same synapses as in E).

The reasons for these decreases are not clear, and may be ratio of the two responses remained relatively stable across
different for the two responses. RGECO did not exhibit bleaching trials, increasing by only 9% between the first and fifth repeat
across trials, whilst the baseline fluorescence of sypHy decreased (Supplementary Figure S2C). To avoid any inherent bias due to
by approximately 20% (Supplementary Figure S2C). It has the effect of repeated trials we interspersed different stimulation
previously been suggested that GECIs targeted to the presynaptic conditions, such as number or frequency of AP stimuli.
compartment can exhibit rundown in responses (Walker et al., It has previously been reported that R-GECO1 displays
2013), whereas the responses of synaptic pHluorins are similar photoactivation when illuminated with 488 nm light (Akerboom
across repeated trials (Burrone et al., 2006). However, the et al., 2013), which may explain the increase in red fluorescence

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Jackson and Burrone Imaging Calcium and Neurotransmitter Release

observed in unstimulated boutons (Supplementary Figure S2B). 1994; Gasparini et al., 2001). The contribution of different
To examine if this phenomenon affected sypHy-RGECO calcium channel subtypes to the presynaptic calcium signal varies not
responses, single channel images, illuminated with 585 nm only between synapses, but also with stimulation frequency
light only, were compared to our usual imaging conditions, (Ricoy and Frerking, 2014). We stimulated neurons expressing
alternating 470 and 585 nm light. 1R responses were larger in sypHy-RGECO with 10 APs at 5, 20, and 83 Hz, to examine
images taken with 585 nm light only (Supplementary Figure whether stimulation frequency affected the relationship between
S2C, upper trace), although when scaled for comparison, the 1G and 1R responses. Pooled 1G and 1R responses across
kinetics of the response were the same in both imaging all frequencies showed the expected non-linear relationship
conditions (Supplementary Figure S2C, lower trace). The (Figure 3D). Both calcium influx and vesicle exocytosis were
response amplitudes and baseline fluorescence in single channel significantly reduced at 83 Hz stimulation, which could be
and two channel images were highly correlated and, importantly, due to the failure of the cell to fire action potentials at
scaled linearly (Supplementary Figures S2D,E). These results high frequency, or to the inactivation of calcium channels
suggested that in our imaging conditions, fast alterations between (Figures 3D,E). However, the distributions of 1G/1R ratios were
blue and green excitation light reduces the size of the RGECO indistinguishable between stimulation frequencies (Figure 3F),
responses in a multiplicative manner, without the introduction of suggesting frequency did not alter the relationship between
non-linear artifacts. calcium influx and vesicle release.
To characterize the sypHy-RGECO response to different Synaptic boutons are heterogeneous in both structural and
stimulus strengths, stimuli ranging from 1 AP to 20 AP at functional parameters. Studies of the relationship between the
20 Hz were delivered by field stimulation. Both responses two show that bouton volume is a poor predictor of functional
increased linearly over the range of stimulus strengths tested properties such as presynaptic calcium influx and the synaptic
(Figures 2A,B), as has been previously reported for SyRGECO probability of release (Ermolyuk et al., 2012; Holderith et al.,
(Walker et al., 2013). Peak 1G and 1R responses to different 2012), whereas active zone size strongly correlates with function
numbers of action potentials also showed a strong linear (Holderith et al., 2012). Additionally, levels of the active zone
correlation (Figure 2C). The mean ratio between vesicle proteins RIM1/2 are highly correlated with active zone size
exocytosis and calcium influx did not differ between 5, 10, and (Holderith et al., 2012). To examine the relationship between
20 AP stimuli (Figure 2D). The mean ratio was significantly sypHy-RGECO responses and active zone size, neurons were
increased at lower stimulus strengths, although many synapses first imaged live, whilst stimulating with 10 APs at 20 Hz,
that responded at higher stimulus strengths fell below the and subsequently fixed and stained for RIM1/2. Live and fixed
detection threshold in the 1 AP and 2 AP stimuli, potentially images were aligned to allow the comparison of functional and
biasing toward synapses with higher release probabilities in structural data from individual identified synapses (Figure 4A
these groups. At higher numbers of APs, the longer stimulation and Supplementary Figure S4). We observed significant positive
times required would begin to overlap with the endocytosis correlations between RIM levels and both sypHy and RGECO
and reacidification of synaptic vesicles, making the amplitude responses (Figure 4Bi,ii). Whilst significant, these correlations
of sypHy responses harder to interpret. However, to check for are weaker than those found in ultrastructural studies, most
potential saturation of the probe we also delivered 40 and likely due to limitations in the alignment process and the lower
100 AP stimuli at 20 Hz. The calcium response indicated by resolution of light microscopy. Interestingly, RIM levels also
RGECO was saturated at 40 APs and displayed no further correlated significantly, albeit weakly, with the ratio 1G/1R
increase in amplitude to a 100 AP stimulus, whilst sypHy (Figure 4Biii), possibly reflecting the role of RIM, along with
responses continued to increase with longer stimulation trains RIM binding protein (RBP), in localizing calcium channels to
(Supplementary Figure S3). the active zone (Kaeser et al., 2011), vesicle docking (Han et al.,
We next tested the effect of changing the external calcium 2011) and coupling of calcium channels to release (Kaeser et al.,
concentration, recording sypHy-RGECO responses in both 2012).
0.5 mM and 2 mM calcium for the same set of synapses. As Similar correlations were also carried out with staining for
expected, both the calcium influx and level of vesicle release the putative calcium sensor for synchronous neurotransmitter
were significantly reduced in 0.5 mM calcium (Figure 3A), release, synaptotagmin-1 (Syt1) (Geppert et al., 1994; Fernandez-
although the positive correlation between the two responses was Chacon et al., 2001). We found that the levels of Syt1
maintained (Figure 3B). For synapses which responded above were positively correlated with the magnitude of the RGECO
threshold in both conditions, the distribution of 1G/1R ratios response, and showed a trend toward a positive correlation
was not significantly different between the two concentrations with neurotransmitter release that did not reach significance
(Figure 3C), although the ratios measured for 0.5 mM calcium (Figures 4Ci,ii). However, the ratio of release to calcium
showed a skewed distribution toward higher values. This influx at each synapse was not related to the level of Syt1
discrepancy is likely due to the saturation of the vesicular sensor (Figure 4Ciii). As the neurons were stimulated with a train of APs
of exocytosis for higher calcium concentrations, as reported at high frequency (20 Hz), both synchronous and asynchronous
previously (Ermolyuk et al., 2012). forms of release are expected to operate (Hagler and Goda,
Calcium influx at the presynaptic terminal is predominantly 2001), and thus the levels of other calcium sensors may be
mediated by CaV 2.1 and 2.2 channels, with some contribution more important in determining the total release that occurs in
from CaV 2.3 (Takahashi and Momiyama, 1993; Wheeler et al., response to this type of stimulus. Furthermore, approximately

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Jackson and Burrone Imaging Calcium and Neurotransmitter Release

FIGURE 2 | SypHy-RGECO responses to different stimulus strengths. A range of AP stimuli were delivered by field stimulation at 20 Hz. Each stimulus was
repeated twice, varying the order in which different numbers of APs were given, and a mean of the two responses was taken. (A) Mean sypHy and RGECO
responses to different numbers of APs, the black bar indicates the start of stimulation. (B) Peak 1G/Rmax (green) and 1R/Rmax responses (red) increase linearly over
the range of AP stimuli tested (colored lines indicate linear regression fit: sypHy, green, r 2 = 0.986, RGECO, red, r 2 = 0.961). (C) Correlation between peak 1G/Rmax
and 1R/Rmax responses over the range of APs tested (black line indicates linear fit, r 2 = 0.989). (D) Ratio 1G/1R for each of the stimulus strengths tested. Only the
ratios for 1 and 2 APs show a significant difference from the other stimuli (one way ANOVA,∗∗ p < 0.01,∗∗∗ p < 0.001). All plots show mean ±SEM, n = 171
synapses from 4 coverslips and two independent cultures.

20% of total Syt1 is found on the surface of the bouton, 2011), fused to the C-terminus of sypHy, a reporter of
not on vesicles (Fernandez-Alfonso et al., 2006), thus the vesicle exocytosis and endocytosis (Granseth et al., 2006).
level of Syt1 present at the bouton may not accurately reflect Combining the two reporters in one molecule offers several
the size of the vesicle pool. Together, our data is in broad advantages. First, it provides two independent readouts of
agreement with the general principle that synapses that have presynaptic function, without the need for co-expression of
larger active zones also show higher levels of calcium influx multiple probes. Second, as the probes are not separated
and neurotransmitter release. Although the overall levels of Syt1 spatially and a fixed ratio of the two fluorophores is present
at the synapse also broadly correlated with these functional at all synapses, it provides a way of normalizing sypHy
measures, they were a worse predictor of synapse function signals across synapses and cells, in a manner similar to
than RIM. that employed for ratio-sypHy (Rose et al., 2013). This is
particularly important for sypHy signals, where resting levels
of fluorescence vary quite dramatically from bouton to bouton
DISCUSSION and do not correlate directly with the amount probe at the
synapse, but rather to its surface levels. Finally, the fixed
We have generated a single molecule reporter, sypHy-RGECO, stoichiometry also allows a direct comparison of the two
for concurrent imaging of presynaptic calcium influx and fluorescence responses, thereby providing a direct measure of
synaptic vesicle recycling. This hybrid molecule consisted the calcium dependence of release within single presynaptic
of a red calcium indicator, R-GECO1 (Zhao Y. et al., boutons.

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Jackson and Burrone Imaging Calcium and Neurotransmitter Release

FIGURE 3 | Effects of altering stimulation conditions. (A–C) Dissociated hippocampal neurons in external solution (HBS) containing 0.5 mM calcium were
stimulated with a train of 10 APs at 20 Hz repeated five times 1 min apart. The solution was replaced with HBS containing 2 mM calcium and the stimulus set
repeated. Responses across trials were averaged for each synapse. (A) Fluorescence changes in sypHy (upper traces) and RGECO (lower traces) in 0.5 and 2 mM.
Traces show mean ± SEM for n = 131 synapses from 3 coverslips from 3 independent cultures. (B) Correlation between peak 1G/Rmax and 1R/Rmax responses
for these synapses (Spearman’s rank correlation r s = 0.873, p < 0.001, the black line indicates a fit to a power function of the form axb +c, where b = 1.30.)
(C) Distribution of 1G/1R ratios for synapses which responded above threshold at both calcium concentrations, ratios shown are averaged from the last two
0.5 mM trials (gray) and the first two 2 mM trials (black) (n = 47 synapses). The distribution is not significantly different between concentrations (Kolmogorov–Smirnov
test, p = 0.093). (D–F) Hippocampal neurons were stimulated with 10 APs at 5, 20, and 83 Hz. Each set of stimuli was repeated three times, varying the order in
which the frequencies were delivered and the mean of the trials was calculated. (D) Correlation between peak 1G/Rmax and 1R/Rmax values at 5 Hz (blue), 20 Hz
(red), and 83 Hz (green). Mean responses to each frequency are shown in the larger, brighter points (n = 134 synapses from 3 coverslips and three independent
cultures, the black line indicates a fit to a power function of the form axb +c, where b = 1.91). (E) Distribution of 1G/Rmax (left) and 1R/Rmax (right) responses for
these synapses show a decrease in the responses at 83 Hz compared to 20 Hz (Kolmogorov–Smirnov test, 1G/Rmax p = 0.0013, 1R/Rmax p < 0.001) but no
change in the response at 5 Hz (Kolmogorov–Smirnov test 1G/Rmax p = 0.537, 1R/Rmax p = 0.240). (F) The distribution of 1G/1R ratios does not change
between stimulus frequencies (Kolmogorov–Smirnov test 5 Hz p = 0.703, 83 Hz p = 0.282).

We observed a strong non-linear correlation between calcium both calcium and exocytosis signals. A possible source of this
influx and vesicle exocytosis across synapses that was maintained variation may be the repertoire of presynaptic calcium channel
in response to different stimulation conditions, in agreement with subtypes, which can vary between synapses (Reid et al., 1997;
many previous studies (Ariel et al., 2012; Ermolyuk et al., 2012). Nimmervoll et al., 2013). In support of this, individual boutons
Using the ratio of the sypHy response to the RGECO response displayed differential sensitivity to pharmacological blockade of
(1G/1R), we were also able to measure the calcium-dependence CaV 2.1 and 2.2 channels in both calcium influx measured by
of release at individual presynaptic boutons, a feature that has GCaMP3 and exocytosis measured by sypHTomato, when these
been difficult to study in these small CNS synapses. Interestingly, reporters were co-expressed in the same cell (Li and Tsien, 2012).
we observed heterogeneity in this value, suggesting that calcium Similarly, in another study in which calcium and vesicle dynamics
triggers vesicle fusion with different efficiencies at different were imaged in separate cells, pharmacological inhibition of
synapses. It is not yet clear what underlies this variation, as the CaV 2.1 or 2.2 led to variable blockade of neurotransmitter release
distribution of 1G/1R ratios did not change significantly under between individual synapses, although this did not alter the
different stimulation conditions, such as stimulation frequency relationship between calcium and exocytosis measured across
and extracellular calcium, despite changes in the amplitude of the synapse population (Ariel et al., 2012). However, neither

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Jackson and Burrone Imaging Calcium and Neurotransmitter Release

FIGURE 4 | Combining functional imaging with sypHy-RGECO and immunofluorescence. Neurons were stimulated five times with 10 APs at 20 Hz and the
responses averaged across trials. Cells were then fixed and stained for presynaptic markers and imaged using confocal microscopy. Live and fixed images were
registered and fluorescence intensities for individual synapses were measured. (A) Example of a live image of the green channel of sypHy-RGECO (left) and fixed
image (middle) of GFP and synaptotagmin-1 (Syt1) for the same cell. Right, magnified view of individual synapses. (B) Positive correlations between levels of RIM
and sypHy responses (i, green, p < 0.001), RGECO responses (ii, red, p < 0.001), and the 1G/1R ratio (iii, gray, p = 0.039), (n = 85 synapses from 3 dishes from
independent cultures). (C) Levels of Syt1 are not significantly correlated with sypHy responses (i, p = 0.059), or the 1G/1R ratio (iii, p = 0.323), but are correlated
with the calcium response (ii, p = 0.005) (n = 62 synapses from three dishes from independent cultures).

of these studies directly examined the relationship between An alternative possibility is that individual boutons differ
channel subtypes and the calcium dependence of release in in the levels or components of the molecular machinery that
individual boutons, which could be addressed using sypHy- links calcium influx to vesicle release. Of the markers that we
RGECO. examined using post hoc immunofluorescence, the level of the

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Jackson and Burrone Imaging Calcium and Neurotransmitter Release

calcium sensor synaptotagmin-1 was not related to the 1G/1R (Akerboom et al., 2013). When combined in the single molecule
ratio, although a weak correlation was observed with the level sypHy-RGECO, both reporters displayed a linear increase in
of RIM, an active zone protein with roles in the localization of the amplitude of the response across the range of 1–20 AP
calcium channels and vesicle docking (Han et al., 2011). Whilst stimuli delivered at 20 Hz, but at higher stimulus strengths the
absolute RIM levels are only weakly predictive of the ratio, it RGECO response became saturated. Whilst sypHy responses
is possible that more nuanced features such as the size and continued to increase to both 40 and 100 AP stimuli, the
shape of the active zone relative to the bouton volume, and amplitude of these responses will be affected by the decrease
positioning of elements such as calcium channels within it may in fluorescence caused by endocytosis and reacidifcation of
affect the calcium dependence of release at individual synapses. vesicles. The dynamic ranges of the two reporters are therefore
Combining functional imaging with structural imaging by well matched and suited to reporting activity up to 20 action
immunofluorescence, super-resolution, or electron microscopy potentials.
will enable investigation of these possibilities. Such methods One potential disadvantage of combining R-GECO1 with
could also be used to examine if differences in the calcium green sensors of vesicle exocytosis is its photoswitching behavior
dependence of release exist in different cell types, or to study in response to 488 nm light (Akerboom et al., 2013). Indeed,
the relationship between presynaptic function and postsynaptic we found that blue light exposure in our imaging conditions
properties at individual connections. did cause an offset in the basal RGECO fluorescence, as
Finally, it would be interesting to examine if the calcium well as a reduction in the calcium response. However, these
dependence of release is fixed for an individual synapse or is decreases scaled linearly with the original R-GECO1 response,
subject to regulation. Calcium influx and vesicle exocytosis have and both the kinetics of the calcium transient and the range
been measured individually after chronic changes in network of responses across synapses were preserved. SypHy-RGECO
activity, and the changes observed indicate that the relationship is therefore a useful addition to the expanding toolbox of
between the two is not altered at the population level following genetically encoded reporters, and will enable a detailed study of
homeostatic plasticity (Zhao C. et al., 2011). However, this is yet the relationship between calcium influx and vesicle dynamics at
to be studied simultaneously in single synapses. Furthermore, it individual synapses. Whilst we have shown proof of principle in
is unknown if short-term plasticity mechanisms involve changes hippocampal synapses in vitro, this reporter could also be used to
in the efficiency with which calcium triggers vesicle fusion. Tools study these processes in more intact systems, such as brain slices
such as sypHy-RGECO will enable optical interrogation of these and in vivo.
questions.
Several spectrally distinct genetically encoded reporters of
calcium and vesicle recycling have been developed, offering the AUTHOR CONTRIBUTIONS
opportunity to combine probes with different characteristics.
We selected to combine sypHy and R-GECO1 for several JB and RJ designed experiments, RJ performed experiments,
reasons; synaptophysin has previously been shown to tolerate analyzed data, and drafted the manuscript, JB and RJ edited and
addition of both pHluorin in the intravesicular loop with a approved the manuscript.
second fluorophore at the C-terminus (Rose et al., 2013), and
both sypHy and SyRGECO have previously been characterized
individually (Granseth et al., 2006; Walker et al., 2013). SypHy FUNDING
has a superior signal-to-noise ratio than synaptopHluorin and
whilst VGLUT-pHluorin offers further improvement in this This work was funded by an ERC consolidator grant, a Wellcome
aspect (Voglmaier et al., 2006), there is a risk that VGLUT1 Trust Investigator award and a Lister Institute Prize to JB.
overexpression may cause an increase in quantal content (Wojcik
et al., 2004; Wilson et al., 2005), and therefore alter the strength
of synaptic connections. Additionally, although VGLUT based ACKNOWLEDGMENTS
probes have been used in inhibitory neurons, it may impart
GABAergic boutons with unwanted properties resulting from We would like to thank Mideia Kotsogianni for the preparation
the overexpression of a transporter not usually found in these of dissociated hippocampal cultures, Robert E. Campbell for the
neurons. R-GECO1 is a sensitive calcium indicator with a R-GECO1 plasmid (Addgene plasmid 32444) and Leon Lagnado
wide dynamic range that exhibits a larger fluorescence change for the sypHy plasmid (Addgene plasmid 24478).
to field stimuli than the RCaMP1 sensors (Akerboom et al.,
2013; Walker et al., 2013). RCaMP2 (Inoue et al., 2015) offers
greater sensitivity than R-GECO1 but saturates at approximately SUPPLEMENTARY MATERIAL
8 APS and was therefore less suitable for combination with
sypHy, for which longer stimulus trains are typically used. The Supplementary Material for this article can be found
The 1F/F responses of R-GECO1 are also linear over a online at: http://journal.frontiersin.org/article/10.3389/fnsyn.
greater range of calcium concentrations than RCaMP1 sensors 2016.00021

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Jackson and Burrone Imaging Calcium and Neurotransmitter Release

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Wojcik, S. M., Rhee, J. S., Herzog, E., Sigler, A., Jahn, R., Takamori, S., et al. (2004). Conflict of Interest Statement: The authors declare that the research was
An essential role for vesicular glutamate transporter 1 (VGLUT1) in postnatal conducted in the absence of any commercial or financial relationships that could
development and control of quantal size. Proc. Natl. Acad. Sci. U.S.A. 101, be construed as a potential conflict of interest.
7158–7163. doi: 10.1073/pnas.0401764101
Zhao, C., Dreosti, E., and Lagnado, L. (2011). Homeostatic synaptic plasticity Copyright © 2016 Jackson and Burrone. This is an open-access article distributed
through changes in presynaptic calcium influx. J. Neurosci. 31, 7492–7496. doi: under the terms of the Creative Commons Attribution License (CC BY). The use,
10.1523/JNEUROSCI.6636-10.2011 distribution or reproduction in other forums is permitted, provided the original
Zhao, Y., Araki, S., Wu, J., Teramoto, T., Chang, Y. F., Nakano, M., et al. (2011). author(s) or licensor are credited and that the original publication in this journal
An expanded palette of genetically encoded Ca2+ indicators. Science 333, is cited, in accordance with accepted academic practice. No use, distribution or
1888–1891. doi: 10.1126/science.1208592 reproduction is permitted which does not comply with these terms.

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 METHODS
published: 19 August 2016
doi: 10.3389/fnsyn.2016.00024

Flash-and-Freeze: Coordinating
Optogenetic Stimulation with Rapid
Freezing to Visualize Membrane
Dynamics at Synapses with
Millisecond Resolution
Shigeki Watanabe 1, 2*
1
Department of Cell Biology, Johns Hopkins University, Baltimore, MD, USA, 2 The Solomon H. Snyder Department of
Neuroscience, Johns Hopkins University, Baltimore, MD, USA

Electron microscopy depicts subcellular structures at synapses exquisitely but only

captures static images. To visualize membrane dynamics, we have developed a
novel technique, called flash-and-freeze, which induces neuronal activity with a flash
of light and captures the membrane dynamics by rapid freezing. For characterizing
membrane movements during synaptic transmission, a light-sensitive cation channel,
channelrhodopsin, is heterologously expressed in mouse hippocampal neurons or
in Caenorhabditis elegans motor neurons. A brief pulse of blue light activates
channelrhodopsin and induces an action potential, leading to synaptic transmission.
Following the light stimulation, neurons are frozen at different time intervals ranging from
10 ms to 20 s. Electron micrographs are then acquired from each time point to visualize
the morphological changes. Using this approach, we have characterized a novel form of
Edited by:
George Augustine, endocytosis, ultrafast endocytosis, which rapidly removes excess membrane added to
Nanyang Technologicial University, the surface during neurotransmission. The flash-and-freeze approach can be adapted
Singapore
to study other cellular phenomena that can be induced by light-sensitive genetic or
Reviewed by:
pharmacological tools.
Tom Reese,
National Institutes of Health, USA Keywords: flash-and-freeze, synaptic transmission, synaptic cell biology, synaptic vesicle exocytosis, synaptic
Ruud Toonen, vesicles, optogenetics, high-pressure freezing, electron microscopy
Vrije Universiteit Amsterdam,
Netherlands
*Correspondence: INTRODUCTION
Shigeki Watanabe
[email protected] Imaging Neurons
To process information at synapses, membrane trafficking takes place on a millisecond time
Received: 25 May 2016 scale. When a nerve impulse arrives at synaptic terminals, synaptic vesicles fuse with the plasma
Accepted: 03 August 2016 membrane within 1 ms (Heuser and Reese, 1981). Subsequently, vesicle membrane and proteins
Published: 19 August 2016 are recovered from the plasma membrane to reconstitute new vesicles (Dittman and Ryan, 2009;
Citation: Saheki and De Camilli, 2012). One major challenge in studying membrane dynamics at synapses is
Watanabe S (2016) Flash-and-Freeze: visualizing these rapid events at the spatial resolution necessary to resolve individual vesicles and
Coordinating Optogenetic Stimulation
temporal resolution relevant to synaptic functions. Several methods have been developed over the
with Rapid Freezing to Visualize
Membrane Dynamics at Synapses
years including electrophysiological, optical, and electron microscopy methods.
with Millisecond Resolution. Capacitance measurements of synaptic terminals allow monitoring of membrane flux at
Front. Synaptic Neurosci. 8:24. the plasma membrane with sub-millisecond temporal resolution (Hamill et al., 1981; Neher
doi: 10.3389/fnsyn.2016.00024 and Marty, 1982). The capacitance across a membrane is proportional to the membrane area.

Frontiers in Synaptic Neuroscience | www.frontiersin.org August 2016 | Volume 8 | Article 24 | 33

Watanabe Flash-and-Freeze

Direct patch-clamping of synaptic boutons shows an increase synaptic functions, but it has two major drawbacks. First, the
in the capacitance during exocytosis and a decrease during specimen must be physically attached to the electrical wire, and
endocytosis (von Gersdorff and Matthews, 1994; Sun et al., thus experiments can only be performed ex vivo. Second, only the
2002; Delvendahl et al., 2016). This method has a single vesicle surface of specimens (∼5 µm) can be frozen without ice crystal
sensitivity. However, it is blind to the locations of the membrane damage. These two factors limit its utility as a versatile tool for
insertion and removal. Furthermore, membrane trafficking studying membrane trafficking at synapses. To overcome these
within the cytosol cannot be visualized by this technique. limitations, we have developed a novel technique, flash-and-
Optical imaging methods aim to label functional pools of freeze, that combines optogenetics with high-pressure freezing
vesicles in the terminals and visualize their fates. The spatial (Watanabe et al., 2013a,b, 2014a). With this technique, non-
resolution of optical microscopy is limited to ∼200 nm by invasive light stimulation is used to induce synaptic transmission.
the diffraction of light. Because synaptic vesicles are small Specimens as thick as 200 µm can be properly frozen without ice
(∼40 nm in diameter) and hundreds of these vesicles are crystal formation. Therefore, intact animals like Caenorhabditis
clustered in a confined space, optical microscopy cannot resolve elegans (C. elegans hereafter) or an entire population of neurons
individual vesicles in the terminal. Therefore, several tricks in a dish can be studied. Here, I will describe the methods in detail
have been developed to visualize specific subsets of vesicles. and discuss its potential applications in synaptic cell biology.
For example, fluid phase markers like dextran, ferritin, and
quantum dots can be applied exogenously to label endocytosed High-Pressure Freezing
vesicles and visualize their dynamics in the terminal (Zhang An electron microscope operates under high vacuum to avoid
et al., 2007b; Park et al., 2012). Similarly, lipophilic dyes the scattering of electrons by gaseous molecules in the air. Thus,
like FM dyes and mCling can be internalized via endocytosis to observe a specimen in a transmission electron microscope,
and mark the newly reconstituted vesicles (Betz et al., 1992; it must be fixed and dehydrated. In addition, electron beam
Revelo et al., 2014). Finally, pH-sensitive fluorescent molecules must penetrate through the specimen, requiring extremely flat
such as pHluorin (Miesenböck et al., 1998) can be used to specimens with a thickness of ∼30–70 nm. For this reason,
monitor synaptic vesicle cycle. Fluorescence from pHlourin is the specimen is embedded in plastic and sectioned ultrathin.
quenched in the lumen of synaptic vesicles due to the low The sample preparation for electron microscopy often leads
pH. When exposed to the extracellular space by exocytosis, to the generation of artifacts. Fixation using aldehyde-based
the protein becomes fluorescent. The pHluorin molecules are chemicals cross-links proteins and aggregates them. Worse yet,
quenched again once they are internalized and vesicles are fully this reaction can induce fusion of synaptic vesicles (Smith and
re-acidified. These techniques allow visualization of particular Reese, 1980). Furthermore, dehydration leads to the shrinkage
vesicles (Sankaranarayanan and Ryan, 2000; Balaji and Ryan, of the membrane-bound structures and the overall changes in
2007; Armbruster et al., 2013). However, like in the capacitance the morphological architecture of the cells. Therefore, a better
measurement, it is difficult to visualize intracellular trafficking approach must be used to study membrane trafficking events at
events, limiting the interpretation of the results. synapses.
Electron microscopy, on the other hand, depicts all the One approach to avoid these artifacts is to immobilize cells
membrane-bound structures within the terminal, but only physically by rapid freezing. The freezing process, however,
captures a static image. To overcome this limitation, neurons leads to formation of ice crystals that can damage the cellular
are typically stimulated and fixed at defined time points after architecture by directly penetrating through the membrane.
the stimulation (Ceccarelli et al., 1972; Heuser and Reese, 1973; Alternatively, the solutes segregated from ice crystals can burst
Ferguson et al., 2007; Clayton et al., 2008; Matthews and Sterling, membrane due to the local changes in the osmotic pressure. To
2008; Hoopmann et al., 2010; Schikorski, 2014; Wu et al., prevent water molecules from forming ice crystals, a freezing
2014). However, the temporal resolution is limited to seconds to rate of at least 10,000 K/s must be achieved. At this rate, water
minutes for two reasons. First, to ensure the capture of endocytic molecules cannot rearrange to form ice crystals and are frozen
events, neurons are often stimulated with prolonged intense in an unordered state. The cooling rate by liquid nitrogen can
stimulation that lasts seconds to minutes. Second, diffusion exceed 16,000 K/s. Unfortunately, the heat conductance of water
and reaction of aldehyde-based chemicals are slow, requiring is poor, reducing the rate to 1000 K/s within 10 µm from the
an additional time. To visualize membrane dynamics on a point of the contact. Under high pressure (2100 bar), however,
millisecond time scale, Heuser and Reese developed the “freeze a freezing rate of 100 K/s is sufficient to freeze water in an
slammer,” which freezes tissues near instantaneously following unordered state due to the supercooling effect (Moor, 1987;
electrical stimulation (Heuser et al., 1979; Heuser and Reese, Dubochet, 2007). Thus, high-pressure freezing allows freezing of
1981). Using this device, they were able to observe vesicles fusing tissues up to 200 µm thickness or intact animals like C. elegans,
with the plasma membrane 5 ms after an electrical pulse (Heuser (∼70 µm thick) without ice crystal damage.
and Reese, 1981; Heuser et al., 1979). Although individual images
are static, membrane dynamics can be visualized in electron Optogenetics
micrographs by inducing a particular activity and freezing To visualize membrane dynamics during neurotransmission,
neurons at multiple defined time points. neurons must be stimulated and frozen at defined time points
The freeze slamming approach allows visualization of after stimulation. In the freeze slammer, specimens are physically
membrane dynamics at the temporal resolution relevant to attached to the stimulating wire, limiting the specimen choice.

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Watanabe Flash-and-Freeze

Furthermore, the configuration of the high-pressure freezer feeder layer, cortices are treated with 0.05% Trypsin-EDTA for
makes it difficult to apply electrical field stimulation. To 20 min at 37◦ C. The dissociated astrocytes are then cultured in
overcome these limitations, we have coupled optogenetics with DMEM containing 10% FBS and 0.1% penicillin-streptomycin
high-pressure freezing. Channelrhodopsin is a light-sensitive for 2 weeks in a T-75 flask. Then astrocytes are transferred
cation channel isolated from Chlamydomonas reinhardtii (Nagel to the 12-well plate containing sapphire disks (5 × 104 / well)
et al., 2003). A flash of blue light opens the channel, allowing and grown for a week. Astrocyte mitosis is arrested a few
cations to flow into the cell, thereby depolarizing it. When the hours before neuron plating using fluorodeoxyuridine (80 mm).
channel is heterologously expressed in neurons, a short pulse of Media are exchanged with Neurobasal, a media containing 2%
light triggers an action potential, leading to synaptic transmission B27, 1% glutamax, and 0.1% penicillin-streptomycin, prior to
(Boyden et al., 2005; Nagel et al., 2005). Therefore, non-invasive neuron plating. Isolated hippocampi are treated with papain
stimulation can be applied to a population of neurons in a dish or (20 U/ ml) for 1 h and plated (5 × 104 /well) on top of the
intact animals. feeder layer. Neurons are allowed to grow for 2–3 weeks.
To couple optogenetic stimulation with high-pressure On DIV1-3, neurons are infected with lentivirus containing
freezing (“flash-and-freeze”), we have developed a device that Channelrhodopsin expressed from the neuron specific synapsin
interfaces with the computer of high-pressure freezer as well promoter.
as with an LED (Watanabe et al., 2013a,b, 2014a). This device
allows application of light pulses at defined time points before Caenorhabditis elegans
the specimen is frozen (see Section Materials and Methods). Transgenic animals expressing ChIEF, a variant of
Using this approach, we can visualize the membrane trafficking channelrhodopsin-2, are grown on an agar plate (50 mm)
events at synapses with a millisecond temporal resolution. seeded with 250 µl of E. coli OP50. Larval stage 4 (L4) transgenic
animals are transferred to agar plates seeded with bacteria
MATERIALS AND METHODS and 4 µl trans-retinal solution (100 mM) a day prior to the
experiment. These animals are kept in the dark before the
All experiments are performed according to the guidelines for experiments.
the animal use by the National Institute of Health. The animal
protocol is approved by the Animal Care and Use Committee Freezing
at Johns Hopkins University, School of Medicine. The graphical We have used three different high-pressure freezers: Leica EM
representations of the workflow is shown in Figure 1. The step- PACT2, HPM100, and ICE. C. elegans has been tested in the EM
by-step protocol can be found in the Supplementary information. PACT2, whereas neuronal cultures are frozen with the HPM100
or ICE. For the EM PACT2, a membrane carrier with a 100 µm-
Cell Cultures deep well is used to mount transgenic worms (Figure 2A). First,
For flash-and-freeze experiments, specimens must be mounted the well of the membrane carrier is filled with M9 worm buffer
on a substrate that is translucent like a glass coverslip but can containing 20% bovine serum albumin (BSA) as cryo-protectant.
withstand the extreme pressure application. Sapphire disks are Approximately 10–15 young adult animals are transferred into
ideal substrates, meeting these two criteria. In addition, thermal the solution. The membrane carrier is then mounted into the
conductivity of sapphire is extremely high, particularly at low modified specimen pod.
temperature, making it the perfect substrate. For culturing cells To mount cultured neuronal cells, the following procedures
on sapphire disks, the following procedures are performed. are carried out (Figure 2B). First, a sapphire disk with neurons
First, we sputter carbon on one side of 6 mm sapphire disks so is transferred into pre-warmed (37◦ C) recording solution
that we can distinguish the surface that the cells are cultured containing 140 mM NaCl, 2.4 mM KCl, 10 mM HEPES (pH
on. Then, we scratch out a letter “4” on the carbon-coated 7.5), 10 mM glucose, 4 mM CaCl2 , 1 mM MgCl2 , 3 µM
surface with a diamond scribe. Alternatively, a finder grid can NBQX, and 30 µM bicuculline. The addition of NBQX and
be placed on a sapphire disk as a mask. The sapphire disks bicuculline blocks the recurrent network activity following
are baked at 120◦ C overnight. After a brief dip in ethanol, channelrhodopsin stimulation. The sapphire disk is mounted
two sapphire disks are placed in each well of a 12-well plate on the countersink of a 6-mm middle plate. A spacer ring
and air-dried in a biosafety cabinet. Poly-D-Lysine solution (100 µm thickness) is placed on the sapphire disk so that
(acetic acid 3 parts, rat tail collagen 1 part, and poly-D-lysine cells are not crushed when the assembly is capped with
1 part) is applied directly on each sapphire disk. After 5 min, another sapphire disk. To cap the assembly, a sapphire disk
the excess solution is removed, and the plates are dried under is first dipped in the recording solution so that one side
the laminar flow. Prior to use, the 12-well plates containing of the disk carries a drop of solution. This sapphire disk
sapphire disks are sterilized by exposure to UV light for is then placed on top of the spacer ring gently so that air
20 min. bubbles are not trapped in between the disks. Two more
For hippocampal cultures, we first culture astrocytes as a spacer rings (200 µm each) are then placed to fill up the
feeder layer and then plate the neurons on top of the astrocytes. remaining space. The excess solution should be removed
Mouse brains are dissected out from newborn C57/BL6J mice by gently tapping with a small piece of filter paper. The
immediately after decapitation. Cortices and hippocampi are middle plate assembly is then sandwiched between two
isolated from each brain, in cold HBSS solution. For the astrocyte transparent plastic half cylinders. Note that light is shone

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Watanabe Flash-and-Freeze

FIGURE 1 | Schematic drawings showing the experimental procedures. Specimens are mounted in the appropriate carrier and frozen immediately following
light stimulation. The freeze-substition is carried out in an automated freeze-substitution unit. Once the freeze-substitution is complete, specimens are embedded in
resin and cured for 48 h. The region of interest (the dotted line) is located using a stereoscope. The blue line indicates the direction of sectioning. The ultrathin sections
are mounted on a grid and imaged on a transmission electron microscope.

from the bottom side of the specimen assembly in the Programming Light Stimulation
HPM100 while it is from the top in the ICE. The video The modifications we have made for the EM PACT2 are described
demonstrating sample loading can be found on the Leica in detail in previous publications (Watanabe et al., 2013a,b,
website (http://www.leica-microsystems.com/science-lab/video- 2014a). For controlling the light stimulation and freezing, we
tutorials-filling-and-assembling-of-different-carriers-for-high- constructed an Arudino Uno based device that sends out 5V
pressure-freezing/). TTL signals to the LED and the high-pressure freezer at the

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Watanabe Flash-and-Freeze

FIGURE 2 | Schematic drawings showing sample loading in the EM PACT2 (A) and in the HPM 100 or the EM ICE (B). For the EM PACT2, the membrane
carrier must be sealed with a sapphire anvil before the experiments. Typically, the liquid is slightly overfilled so that no air bubbles are trapped when sealing with the
sapphire anvil. For the HPM 100 and the EM ICE, liquid should be carried with the top sapphire disk to seal the assembly with no bubbles. For the EM PACT2, the
pressure is applied from the bottom of the membrane carrier while liquid nitrogen is applied from the side of the specimen. For the HPM 100 and the EM ICE,
pressurized liquid nitrogen is applied to the specimen from both sides.

desired time point. This device is driven by custom firmware applied to the specimen, the specimen is frozen to −20◦ C in 8 ms,
to produce any pattern of light pulses (i.e., single stimulus, 10 according to standard thermodynamic equations. To account for
Hz stimulation) and send out the “start” signal that triggers the variability in timing, we calculate the actual time interval post-
the freezing process (this device can be purchased from Marine hoc based on the pressure peak recorded by the accelerometer.
Reef International). Once the signal is sent, the freezer applies We have found the actual time interval to typically be within
hydraulic fluid to the specimen and pressurize the specimen. 33.6 ± 4.6 ms of the intended time.
When the pressure reaches 2000 bar, the liquid nitrogen is To introduce light stimulation capability in the HPM100,
immediately applied to the specimen. The initiation of this a similar Arudino Uno based device was constructed. The
freezing process needs to be timed so that the desired time operation of the HPM100 is fundamentally different from that
interval between the light stimulation and freezing is achieved. of the EM PACT2 in that pressurized liquid nitrogen is applied
To monitor the precise timing when the pressure is applied to to the specimen instead of a hydraulic fluid. After sending the
the specimen, we installed an accelerometer on the sample holder “start” signal, it takes 370 ms to compress the liquid nitrogen.
(also known as a “specimen bayonet”). When the pressure is The pressurized liquid nitrogen reaches the specimen in precisely
applied to the specimen, the sample holder jolts, producing a 72 ms. The specimen is frozen to −20◦ C in 8 ms from the time the
distinctive peak in the accelerometer recording. We found that it pressurized liquid nitrogen hits the specimen surface. Therefore,
requires about 170 ms for the EM PACT2 to apply the pressure to a total of 450 ms delay is expected. To monitor the precise timing,
the specimen after sending the ‘start’ signal. After the pressure is light stimulation device record the internal signal of the machine

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Watanabe Flash-and-Freeze

that opens the liquid nitrogen valve in each shot. This signal using a glass knife. We collect ribbons of 250 sections from each
initiates almost invariably at 370 ms after the start signal. The animal. For mouse hippocampal neurons, we first remove the
actual timing of the freeze is adjusted post-hoc based on the valve sapphire disks from the sample by submerging them in the liquid
opening signal. nitrogen for a few seconds. About 40 sections are cut from the
For EM ICE, Leica microsystems has integrated the light exposed surface (Figure 1, bottom) and collected. These sections
stimulation control into the freezer. The machine is capable of are stained with 2.5% uranyl acetate in 70% methanol for 4 min
freezing specimen at the desired timing after the light stimulation prior to imaging.
with 1–2 ms variability.
Imaging
Freeze-Substitution A transmission electron microscope is operated at 80–120 keV.
Following high-pressure freezing, vitrified water from the Images are acquired with a digital camera. Roughly 200–300
specimen must be substituted with organic solvent to avoid images are collected from each time point.
crystallization of vitrified water as the specimen warms up to
room temperature for further processing. The freeze-substitution Image Analysis
is carried out in cryotubes containing fixatives (1% osmium The morphological analysis is performed in ImageJ using a
tetroxide, 1% glutaraldehyde, 1% milliQ water in anhydrous custom-written macro. This macro records X- and Y-coordinates
acetone) using an automated freeze-substitution unit (Leica of hand-traced membrane structures such as the plasma
AFS2). The cryotubes containing fixatives are stored under liquid membrane, active zone, membrane invaginations, synaptic
nitrogen to avoid the cross-reaction between osmium tetroxide vesicles, dense-core vesicles, large vesicles, and endosomes.
and glutaraldehyde. The sapphire disks containing the cells The positional information is first exported as text files
typically remain associated with the middle plate. To release the and then imported into MATLAB (MathWorks). We have
sapphire disks, the middle plate is quickly transferred to a cup written scripts in MATLAB to analyze the coordinates and
containing acetone, which is precooled to −90◦ C in AFS. Once calculate several statistics: distribution of vesicles from the
the middle plate reaches −90◦ C in the acetone, the sapphire plasma membrane, distribution of vesicles from the active zone,
disk often dissociates from the middle plate spontaneously. If diameter of vesicles, and number of vesicles. A normality
not, it can be released by a gentle tap. The sapphire disk is then test is performed on these data, and P-values are calculated
transferred into the cryotube containing the fixatives at −90◦ C. using student’s T-test for normally distributed data and Mann–
For C. elegans, the frozen specimens can be transferred under Whitney U-test for skewed data. The confidence level is set at
liquid nitrogen into the cryotubes containing fixatives. The 95%. For multiple comparisons, the Bonferroni correction is
tubes should be transferred quickly into the AFS. The following applied.
program is used for the freeze-substitution: −90◦ C for 5–30 h,
5◦ C/h to −20◦ C, 12 h at −20◦ C, 10◦ C/h to 20◦ C. The cryotubes RESULTS
are agitated at least twice a day during the substitution process.
Accuracy of Timing
Plastic Embedding To test the accuracy of the timing for light stimulation, we applied
Once the freeze-substitution is complete, specimens are a single stimulus and froze tissues at desired time points. We
embedded in Epon/Araldite resin. The resin is as composed of calculated the actual time at which the specimens were frozen
4.4 g Araldite 502, 6.2 g Eponate 12, 12.2 g dodecenyl succinic by monitoring the time when the pressure was applied to the
anhydrite (DDSA), and 0.8 ml benzyldimethylamine (BDMA). specimens. The pressure application can be monitored using an
First, the fixative solution is carefully removed from the cryotube, accelerometer for EM PACT 2 (Watanabe et al., 2013b, 2014a).
and specimens are washed with acetone a few times. Specimens For HPM 100, we recorded the signal that triggers opening
are then treated with 0.1% uranyl acetate in acetone for 1 h of the valve that allows pressurized liquid nitrogen to pass
to enhance the membrane contrast. Following several acetone through because specimens are frozen exactly 80 ms after the
washes, infiltration is carried out in the same cryotubes on an valve opening (see Section Materials and Methods; Watanabe
orbital shaker: 30% for 2–5 h, 70% for 3–6 h, and 90% overnight et al., 2013a). On the EM ICE, we can measure the interval
at room temperature. The next day, specimens are transferred between the end of the desired stimulation program and the
into the caps of BEEM capsules containing 100% Epon/Araldite time at which the sensor, which is located close to the specimen,
resin. The resin is replaced three times (2 h each). The specimens reaches 0◦ C. The sensor is placed before the specimen, and it
are cured in an oven (60◦ C) for 48 h. requires additional 3-6 ms for the specimen to experience the
same magnitude of cooling.
Sectioning We recorded the values from each machine on at least three
For observation in a transmission electron microscope, different experimental days. Out of 52 shots taken on the EM
specimens must be sliced thin so that electrons can penetrate PACT2 (4 experiments, 13 shots/experiment), the desired time
through the tissue and generate an image. Ultrathin sections point was achieved in only 5 shots (Figure 3). The actual timing
(40 nm) of specimens are cut using a diamond knife and collected was variable, ranging from −102 to 110 ms. Because of this
on pioloform-coated single slot grids. For C. elegans, we orient variability, the actual timing is always calculated post-hoc for each
the animal so that it is perpendicular to the sectioning surface shot on the EM PACT2. On the HPM100, nearly two thirds of
(Figure 1, bottom). We trim the animal to the reflex of the gonad the shots (57/90) achieved the desired time point (3 experiments,

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Watanabe Flash-and-Freeze

30 shots/experiment). The rest were either 5 ms before or after Exocytosis

the desired time point, indicating that the variation is small To further test the accuracy of the timing, we stimulated
in the HPM 100. However, the signal for the valve opening is C. elegans neuromuscular junctions for 20 ms and froze them
measured at 5 ms time interval (200 Hz), and thus each recording at the end of the pulse using the EM PACT2. Based on
has an uncertainty of ±2.5 ms. The EM ICE, on the other hand, the electrophysiological recordings, channelrhodopsin induced
records the temperature and pressure sensors’ signals at 2000 Hz. neurotransmission is close to the peak at this time point in the
The data from the EM ICE indicated that 56 out of 72 shots C. elegans motor neurons (Watanabe et al., 2013b). From a total
(3 experiments, 24 shots/experiment) landed between 1.5 and of 13 shots taken, only 1 shot achieved this time point. To further
2.5 ms after the desired time point. The maximum delay was 4 ms, test the accuracy, we also imaged the samples that were frozen
and this maximum delay occurred on the very first shot on each 10 ms before the onset of the light pulse and 30 ms after the end
experimental day. The rest of the shots were within 1.5–3.5 ms of the light pulse, as calculated post-hoc. We were able to capture
after the desired time point (average = 2.2 ± 0.08 ms). Given exocytic omega figures at neuromuscular junctions frozen 20 ms
that the additional 3 ms is required to freeze specimens at 5 µm after the light onset (Figure 4A). However, these structures were
deep from the surface, a total of 4.5–6.5 ms is expected. These not observed in the samples frozen 10 ms before the light onset or
results indicate that millisecond temporal control can be achieved 30 ms after the end of the light pulse. These results indicate that
in HPM 100 and EM ICE. the EM PACT2, although unreliable, can be used to capture fast
membrane dynamics at synaptic terminals.
To test if the exocytic omega figures can be captured with
the HPM 100 and the EM ICE, we froze mouse hippocampal
neurons expressing channelrhodopsin. We applied a single 10 ms
light pulse to the specimen and froze it 5 ms after the end of
the light pulse. In the neurons frozen on the HPM 100, we
found at least one omega figure in 19% of synapses at this
time point (Figure 4B; 193 profiles; Watanabe et al., 2013a).
Similarly, omega figures were captured in 18% of synapses by
EM ICE (Figure 4C; 105 profiles). These results indicate that
the flash-and-freeze approach can reliably capture the membrane
dynamics at synapses with milliseconds temporal resolution.

FIGURE 3 | A bar graph showing the time difference between the DISCUSSION
desired time point and the actual time point at which specimens are
frozen. The variation is largest in the EM PACT2, with the average delay of Comparisons among Three Instruments
33.6 ± 4.6 ms (N = 4 experiments; n = 52 shots). The average delay of the To visualize membrane dynamics at synapses, we developed
HPM100 is 1.9 ± 0.3 ms (N = 3 experiments; n = 90 shots) while that of the
EM ICE is 2.2 ± 0.1 (N = 3 experiments; n = 72 shots).
a technique, “flash-and-freeze,” which couples optogenetic
stimulation of neurons with high-pressure freezing. Here, we

FIGURE 4 | Representative micrographs showing exocytic omega figures from the flash-and-freeze experiments. (A) C. elegans neuromuscular junctions,
stimulated for 20 ms, and frozen at the end of the light pulse. Mouse hippocampal neurons, stimulated for 10 ms, frozen 5 ms after the end of the pulse using HPM
100 (B), and EM ICE (C). These structures are rarely observed in non-stimulated control or in specimens frozen at later time points after the stimulation.

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Watanabe Flash-and-Freeze

compared three instruments that allow such experiments: EM that take place during these manipulations can be preserved
PACT2, HPM100, and EM ICE. There are advantages and through the flash-and-freeze approach and visualized with a
disadvantages for each instrument. The major advantage of the millisecond temporal resolution. Furthermore, there are neurons
EM PACT 2 is in its potential application - higher intensity like photoreceptors that are naturally sensitive to light. The
of ultraviolet light can be applied to specimens because light flash-and-freeze approach can be readily adapted to address the
only has to penetrate through a clear sapphire anvil. Most caged mechanism underlying tonic release of neurotransmitters from
compounds are uncaged with ultraviolet light. In both HPM100 these terminals and how exocytosis is mediated by synaptic
and EM ICE, light must travel through an optical fiber and ribbons, electron dense proteinaceous protrusion in the center of
penetrate through a plastic sample holder that absorbs ultraviolet the active zone (LoGiudice and Matthews, 2009).
light. Thus, experiments using caged compound are more feasible
in the EM PACT2. However, the EM PACT2 required many Potential Difficulties
trials before the desired time point was reached. Besides the Membrane dynamics are inferred by observing the membrane
unreliability, the major disadvantage of the EM PACT2 is the morphology at defined time points, but individual electron
size of the specimen cup. A specimen cup has a dimension of 1.6 micrographs still capture static images of cells. The flash-and-
mm diameter × 100 or 200 µm depth. Cells must be cultured on freeze technique cannot follow the membrane dynamics in a
sapphire disks with a diameter of 1.4 mm. These small sapphire single cell. Therefore, to reconstruct the membrane dynamics,
disks float in culture media, making it difficult to culture cells. hundreds of images must be acquired and analyzed from each
On the other hand, sapphire disks as large as 6 mm can be time point. For membrane trafficking at synapses, an even
loaded into the HPM100 or the EM ICE. Both the HPM100 and higher number may be required due to the heterogeneity in the
the EM ICE are essentially comparable in terms of the freezing release probability (Rosenmund and Stevens, 1996)—not every
quality and the temporal precision of light stimulation. However, action potential leads to fusion of synaptic vesicles. We have
HPM100 is more flexible in terms of its potential applications, estimated that about 20–30% of synapses are activated with a
as the light stimulation device and programs are customizable. single light pulse in mouse hippocampal neurons (Watanabe
With the expansion in the repertoire of optogenetic tools or light- et al., 2013a, 2014b). To produce data with statistical significance,
sensitive compounds, cellular activity can be controlled using we have analyzed over 10,000 images. These analyses were
different wavelengths of light, and it is possible to develop a performed blind to genotypes and time points, and thus the
multi-color system for the HPM100. The major disadvantages average frequency of morphological features at a particular
of HPM 100 are that it requires an external device and that time point can be determined from these images. To expedite
the actual freezing point must be calculated based on when analysis and increase the statistical power, automation is in an
the liquid nitrogen valve opens each time. On the contrary, immediate need. However, with the recent advances in methods
EM ICE does not require a custom device or an external light for automated image acquisition and analysis (Potter et al., 1999;
source—everything is integrated. Furthermore, it achieves the Denk and Horstmann, 2004; Knott et al., 2008; Hayworth et al.,
most precise control of the light stimulation and the fastest 2014), achieving such a goal is likely within reach.
freezing rate, requiring only 3 ms from the initial temperature
drop to −20◦ C. Despite these differences among instruments, AUTHOR CONTRIBUTIONS
exocytosis of synaptic vesicles, which occurs on a millisecond
time scale, were captured in all three instruments, suggesting SW performed all the experiments, analyzed the data, and wrote
that any of these machines are compatible with flash-and-freeze the manuscript.
experiments. Therefore, the choice should be made depending on
the potential applications. ACKNOWLEDGMENTS
Potential Applications I would like to thank Erik Jorgensen, Christian Rosenmund, and
The flash-and-freeze approach can be applied to study many all the members in their laboratories for the help in performing
cell biological problems. We have been using channelrhodopsin the original experiments. The light stimulation device for Leica
to stimulate neuronal activity and capture membrane dynamics EM PACT 2 and HPM 100 is designed and developed by M.
at synapses. Neuronal activity or cellular functions can be Wayne Davis. I would like to thank the Grass Foundation and
manipulated using light-sensitive molecules such as bacterial the Marine Biological Laboratory at Woods Hole for space,
opsins (Han and Boyden, 2007; Zhang et al., 2007a; Berthold equipment and funding for performing these experiments. I
et al., 2008; Chow et al., 2010), caged compounds (Walker et al., would like to thank Sumana Raychaudhuri and Edward Hujber
1986; Tsien et al., 1987; Milburn et al., 1989; Adams and Tsien, for a critical reading of the manuscript. The research is supported
1993; Wieboldt et al., 1994), light-sensitive proteins (Levskaya by Johns Hopkins University startup fund.
et al., 2009; Wu et al., 2009; Yazawa et al., 2009; Idevall-Hagren
et al., 2012; Zhou et al., 2012; Guntas et al., 2015), and photo- SUPPLEMENTARY MATERIAL
isomerizable molecule (Banghart et al., 2004; Kramer et al., 2009).
These tools can be used to inhibit or activate neuronal activity, The Supplementary Material for this article can be found
protein function, lipid composition, signaling cascades, and ion online at: http://journal.frontiersin.org/article/10.3389/fnsyn.
composition in vesicular structures. The morphological changes 2016.00024

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Watanabe Flash-and-Freeze

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 PERSPECTIVE
published: 19 August 2016
doi: 10.3389/fnsyn.2016.00023

The Postsynaptic Density: There Is

More than Meets the Eye
Ayse Dosemeci 1 *, Richard J. Weinberg 2 , Thomas S. Reese 1 and Jung-Hwa Tao-Cheng 3
1
Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke (NINDS), National Institutes
of Health (NIH), Bethesda, MD, USA, 2 Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill,
Chapel Hill, NC, USA, 3 Electron Microscopy Facility, National Institute of Neurological Disorders and Stroke (NINDS),
National Institutes of Health (NIH), Bethesda, MD, USA

The postsynaptic density (PSD), apparent in electron micrographs as a dense lamina just
beneath the postsynaptic membrane, includes a deeper layer, the “pallium”, containing
a scaffold of Shank and Homer proteins. Though poorly defined in traditionally prepared
thin-section electron micrographs, the pallium becomes denser and more conspicuous
during intense synaptic activity, due to the reversible addition of CaMKII and other
proteins. In this Perspective article, we review the significance of CaMKII-mediated
recruitment of proteins to the pallium with respect to both the trafficking of receptors
and the remodeling of spine shape that follow synaptic stimulation. We suggest that
the level and duration of CaMKII translocation and activation in the pallium will shape
activity-induced changes in the spine.
Keywords: postsynaptic density, PSD, pallium, electron microscopy, EM, Shank, Homer, CaMKII

Excitatory glutamatergic synapses examined with the electron microscope typically display a
pronounced postsynaptic density (PSD), which appears in conventional electron micrographs as
an approximately 30 nm thick electron-dense structure applied to the postsynaptic membrane
(Palay, 1956). The large majority of excitatory synapses in the vertebrate CNS release glutamate
as neurotransmitter. Ionotropic glutamate receptors concentrate at the PSD, where specialized
Edited by: molecules anchor them and regulate their trafficking; modulation of their expression and
George Augustine, trafficking plays a key role in synaptic plasticity. Decades of research show that much of the
Nanyang Technologicial University
story of this modulation resides in the PSD.
(NTU), Singapore
Using special stains, Valtschanoff and Weinberg (2001) uncovered a distinct layer ∼50 nm thick
Reviewed by: immediately adjacent and deep to the PSD. Though previously reported, this ‘‘subsynaptic web’’
Eric Hanse,
University of Gothenburg, Sweden
(DeRobertis, 1964) had been largely ignored for decades, because it is difficult to distinguish from
Anne McKinney, the rest of the spine cytoplasm after standard histological procedures. Extending this work, Petralia
McGill University, Canada et al. (2005) reported high levels of immunoEM label for both Shank and Homer in the same
*Correspondence: region, which was referred to as the ‘‘subjacent’’ area.
Ayse Dosemeci The subsynaptic web becomes more prominent after short bouts of stimulation, reflecting
[email protected] the reversible incorporation of additional proteins. Because this part of the PSD comprises
different proteins and displays a more labile organization than the ‘‘core’’ layer lining the synaptic
Received: 17 May 2016
Accepted: 25 July 2016
membrane, it merits a distinct name; we propose to designate this separate structural and functional
Published: 19 August 2016 layer of the PSD as its pallium or mantle (Figure 1). Here we present a perspective on PSD structure
Citation:
that highlights potential functions carried out in the pallium.
Dosemeci A, Weinberg RJ, Reese TS
and Tao-Cheng J-H (2016) The
Postsynaptic Density: There Is More
CORE LAYER OF THE PSD
than Meets the Eye.
Front. Synaptic Neurosci. 8:23. Though highly variable, PSDs in the rodent forebrain contain on average 300–400 copies of PSD-95
doi: 10.3389/fnsyn.2016.00023 and related membrane-associated guanylate kinase (MAGUK) molecules (Chen et al., 2005;

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Dosemeci et al. The Postsynaptic Density

BOX 1 | Major scaffold proteins in the PSD.

Names used in the present article are listed along with synonyms.

MAGUKs (membrane-associated guanylate kinases).

The most abundant MAGUK at the mammalian PSD is PSD-95 (DLG4,
SAP90); others include SAP97 (DLG1), PSD-93 (DLG2, Chapsyn-110), and
SAP102 (DLG3).

GKAP (guanylate kinase-associated protein) also called SAPAP (SAP90/PSD-

95-associated protein), or disks large-associated protein.

Shank (SH3 and multiple ankyrin repeat domains protein) also called ProSAP
(Proline-rich synapse-associated protein), Synamon and CortBP (Cortactin
binding protein).

Homer also called Vesl, Cupidin, and PSD-Zip45.

2015). Thus, the dense matrix of PSD-95/MAGUK filaments

capped by GKAPs provides a stable platform for the subjacent
pallium.

PALLIAL LAYER OF THE PSD

FIGURE 1 | Excitatory synapse. Excitatory glutamatergic synapses typically Early biochemical analyses indicated that the PSD complex
display a prominent electron-dense zone juxtaposed to the postsynaptic contains several types of scaffolding molecules in addition
membrane, traditionally known as the postsynaptic density (PSD). Extending to MAGUKs and GKAPs (Box 1). Two-hybrid and
deeper from the PSD core is a specialized region we call the PSD pallium.
co-immunoprecipitation experiments led to the identification
Scale bar: 0.1 µm.
of the Shank/ProSAP family of proteins, which bind to GKAPs
(Boeckers et al., 1999a,b; Naisbitt et al., 1999). Similar two-
Sugiyama et al., 2005). PSD-95 is localized in the core layer hybrid studies identified the Homer family of proteins as binding
of the PSD. Comparing immunogold labeling with antibodies partners for Shanks and the two types of proteins were shown
against its N- and C-termini shows that PSD-95 is oriented with to co-immunoprecipitate from brain extracts (Tu et al., 1999).
its N-terminus near the plane of the postsynaptic membrane Both Shank and Homer family proteins are highly enriched
and its C-terminus deep in the spine (Chen et al., 2008). in PSD fractions (Xiao et al., 1998; Naisbitt et al., 1999) and
Electron microscopic tomography reveals vertical filaments co-purify as large complexes, with PSD-95, GKAP and other
extending from the postsynaptic membrane in numbers PSD constituents (Collins et al., 2006; Dosemeci et al., 2007).
and dimensions expected for PSD-95 (Chen et al., 2008), While biochemical studies identify Shank and Homer as part
and these filaments are depleted by acute knockdown of of the PSD complex, immunoEM studies show that label for
PSD-95 (Chen et al., 2011). Thus, PSD-95-containing filaments these proteins concentrates outside the electron-dense material
oriented vertically to the postsynaptic membrane represent a conventionally defined as the PSD (Figure 2). Although the
major structural component of the core layer (Chen et al., reported distributions of label for these proteins differ somewhat
2008). This close apposition to the membrane puts PSD- between groups, immunoEM studies typically find that much of
95 in a position to bind neurotransmitter receptors. Indeed, the postsynaptic immunogold label for both Shank and Homer
there is substantial evidence indicating the association of lie in a layer immediately below the PSD core (Tu et al., 1999;
PSD-95 with both NMDA and AMPA types of glutamate Valtschanoff and Weinberg, 2001; Petralia et al., 2005; Rostaing
receptors. et al., 2006; Tao-Cheng et al., 2010, 2014a).
EM tomography also reveals filaments of different lengths, These data imply that a network containing Shanks and
oriented parallel to the postsynaptic membrane, that appear to Homer extends the PSD scaffold well beyond the classically
contact the deep (C-terminal) ends of PSD-95 filaments (Chen recognized electron-dense material. This second layer, which
et al., 2008). The dimensions of one frequently-encountered we term the pallium, is likely pegged to the PSD core through
type of horizontal filament corresponds to that expected for GKAPs, which can simultaneously associate with PSD-95 and
guanylate kinase-associated protein (GKAP), consistent with Shanks. In vitro studies report that Shanks can associate with each
biochemical work demonstrating that GKAP can bind PSD- other through their SAM domains to form sheet-like structures
95 near its C-terminal (Kim et al., 1997). GKAP is in the (Baron et al., 2006) and Homers can polymerize into tetramers
PSD at a ratio of approximately one GKAP for two PSD- that can cross-link Shanks (Hayashi et al., 2009). Furthermore,
95s (Lowenthal et al., 2015) so a layer of GKAP cross-linked purified C-terminal Shank and Homer form a mesh-like matrix
with PSD-95 could provide an interface between the core layer when mixed together (Hayashi et al., 2009). Thus, Shank and
and the pallium of the PSD. Like PSD-95 (Yang et al., 2011), Homer proteins in the pallium are likely to form an extended
GKAP stays put during brief stimulation (Tao-Cheng et al., scaffold. The side of this scaffold facing the cleft is continuous

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Dosemeci et al. The Postsynaptic Density

FIGURE 2 | Label for two PSD scaffold proteins, Shank and Homer, lies mostly outside the PSD core. (A) Electron micrographs of asymmetric synapses in
cultured hippocampal neurons immunogold labeled with antibodies for three major PSD scaffold proteins. Silver-enhanced gold label appears as black particles of
heterogeneous size (EM in middle panel from Tao-Cheng et al., 2015). The PSD core with PSD-95 label and the PSD pallium with Shank and Homer labels are
marked by brackets on the left and middle panels respectively. Scale bar: 0.1 µm. (B) Frequency histograms depicting the distribution of label at the postsynaptic
compartment in a typical experiment. While label for PSD-95 is within the electron-dense material, label for Shank and Homer is concentrated in a deeper region we
designate as the pallium of the PSD.

with the PSD core, whereas the cytoplasmic side, characterized Role of CaMKII as a Kinase
by an extremely rough surface (Petersen et al., 2003) merges CaMKII, a Ca2+ -regulated serine-threonine protein kinase, is
imperceptibly into the central zone of the spine head. present at very high concentrations in neurons, with a relative
abundance reaching ∼1–2% of total protein in the cerebral cortex
and hippocampus (Erondu and Kennedy, 1985). Within neurons,
ACTIVITY-INDUCED CHANGES AT THE CaMKII is especially prominent in the PSD pallium (Petralia
PALLIUM et al., 2005; Ding et al., 2013). Activation of CaMKII plays
a pivotal role in certain types of synaptic modification, most
The Pallium Becomes More Electron- notably NMDA-dependent LTP (reviews: Lisman et al., 2012;
Dense as CaMKII Accumulates Shonesy et al., 2014).
Thin-section EM of cultured hippocampal neurons reveals The CaMKII holoenzyme is a dodecamer made of individually
increased electron density at the pallium under excitatory active subunits, with α- and β-isoforms prevalent in brain.
conditions (Figure 3: top panels), a phenomenon we have Subunits within a single holoenzyme can phosphorylate
described as PSD ‘‘thickening’’ (Dosemeci et al., 2001). one another, a process called autophosphorylation. Upon
The increase in electron density likely reflects the addition autophosphorylation in the presence of Ca2+ , CaMKII becomes
of proteins, and immunoEM shows significant increase in autonomous, allowing enzymatic activity to persist beyond
CaMKII immunolabel within the PSD complex under excitatory the duration of Ca2+ elevation (reviews: Hell, 2014; Shonesy
conditions (Figure 3: bottom panels, Dosemeci et al., 2001). et al., 2014). Autonomous CaMKII can remain at the PSD
Similar morphological changes at the PSD, accompanied by the pallium after the cessation of excitatory stimuli (Dosemeci et al.,
accumulation of CaMKII and other proteins, also occur under 2002; Otmakhov et al., 2004). Continued co-localization of
the excitotoxic conditions promoted by ischemia (Suzuki et al., autonomous CaMKII with its substrates at the pallium would
1994; Hu et al., 1998; Martone et al., 1999). The duration of the counteract dephosphorylation by phosphatases and thus help
morphological and compositional changes at the PSD appears maintain phosphorylation of CaMKII substrates.
to vary according to the type of stimulation; importantly, more The accumulation and activation of CaMKII at the pallium
sustained modification is observed following an LTP-inducing triggers further changes at the PSD. Some of these have been
protocol (Otmakhov et al., 2004). studied in depth, such as the incorporation of AMPA receptors

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Dosemeci et al. The Postsynaptic Density

the spine cytoplasm serve as actin bundling elements (Okamoto

et al., 2007) and dissociation of CaMKII from F-actin upon
autophosphorylation destabilizes the actin cytoskeleton (Kim
et al., 2015). Thus the translocation of CaMKII from the actin
network to the PSD pallium can trigger synchronized changes at
the PSD and actin cytoskeleton.

SynGAP Levels Decrease in the PSD Core

and Increase in the Pallium
SynGAP is an enzymatic regulator of Ras, a small GTPase.
However, as one of the most abundant proteins at the PSD (at
levels exceeding even those of PSD-95, its binding partner at
the PSD core), SynGAP is likely to also play a non-enzymatic
role. ImmunoEM shows that SynGAP label is concentrated at the
PSD core (Yang et al., 2011, 2013). After strong depolarization,
labeling for SynGAP significantly decreases at the PSD core while
increasing at the pallium (Yang et al., 2011, 2013). Under the
same conditions, label for PSD-95 does not change its localization
at the PSD (Yang et al., 2011), implying dissociation of the two
molecules. Further studies indicate that the release of SynGAP
FIGURE 3 | The PSD pallium becomes electron-dense as CaMKII and from PSD-95 and its exit from the PSD core require CaMKII-
other proteins are recruited under excitatory conditions. Electron mediated phosphorylation (Yang et al., 2013; Araki et al., 2015).
micrographs of asymmetric synapses in cultured hippocampal neurons under
NMDA- or high K+ -induced changes in the distribution
basal conditions (left) and after depolarization in medium containing high K+
(right). Upon intense synaptic activity the pallium of the PSD becomes of SynGAP are reversed within 30 min after cessation of the
electron-dense due to the accumulation of proteins, including CaMKII (bottom excitatory conditions. In contrast, a stimulation protocol (glycine
panels). Scale bar: 0.1 µm. in the absence of Mg2+ ) that leads to sustained increases in
synaptic efficacy (chem-LTP) also leads to a sustained exclusion
into the postsynaptic membrane (review: Lisman et al., 2012). of SynGAP from the spine (Araki et al., 2015) indicating
ImmunoEM studies also reveal activity-induced increases in the a correlation between LTP and the removal of SynGAP. It
levels of a number of proteins at the pallium, including SynGAP, appears likely that a wide range of excitatory stimuli promotes
AIDA-1 and Shanks, in parallel with CaMKII. Importantly, the translocation of SynGAP out of the PSD core, but that only
redistribution of all these PSD components is blocked by the under LTP-promoting conditions is the molecule removed on
application of a specific CaMKII inhibitor (Yang et al., 2013; Tao- a long-term basis. The extent and maintenance of SynGAP
Cheng et al., 2014b; Dosemeci et al., 2016), indicating that the phosphorylation at the pallium may determine its subsequent
activation of CaMKII is required for the redistribution of these movement. In this regard, it is interesting that SynGAP is most
PSD components. efficiently phosphorylated by the autonomous form of CaMKII
in the absence of Ca2+ (Dosemeci and Jaffe, 2010).
What might be the functional implications of activity-induced
Role of CaMKII as a Dynamic Structural Element
redistribution of SynGAP at the PSD? Both SynGAP and
In addition to its function as a protein kinase, accumulating
members of the transmembrane AMPA receptor regulatory
evidence suggests that CaMKII in the pallium may act as
protein family (TARPs) can bind to the same PDZ domains
a scaffold. Bingol et al. (2010) show that activity-induced
on PSD-95 (Kim et al., 1998; Schnell et al., 2002; Dakoji et al.,
translocation of autophosphorylated α-CaMKII, but not
2003) and thus may compete for association with PSD-95.
its kinase activity, is responsible for the translocation
Considering its high abundance in the PSD, SynGAP is likely to
of proteasomes into synapses. Similarly, activity-induced
block TARP binding to PDZ domains on PSD-95 under basal
CaMKII translocation is responsible for parallel accumulation
conditions. Thus, the removal of SynGAP during activity may
of the deubiquitinase CYLD, specific for K63-linked
be a prerequisite for the anchoring of AMPA receptors to the
polyubiquitins, to the PSD (Thein et al., 2014). Since K63-
PSD, explaining the well-documented role of SynGAP as an
linked polyubiquitination inhibits interaction of proteins
inhibitor of AMPA receptor insertion (Rumbaugh et al., 2006).
with proteasomes (Nathan et al., 2013) CYLD activity is
This function may account for the remarkable abundance of
expected to facilitate protein binding to proteasomes. Thus,
SynGAP at the PSD.
CaMKII accumulated at the pallium appears to constitute
a structural platform that brings together components that
may work synergistically to promote local degradation of AIDA-1 Levels Decrease in the PSD Core
proteins. and Increase in the Pallium
Activity-induced translocation of CaMKII also may mediate Quantitative mass spectrometric analysis of PSD fractions
modifications in spine morphology. CaMKII holoenzymes at reveals that Amyloid-β protein precursor Intracellular Domain-

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Dosemeci et al. The Postsynaptic Density

Associated 1 (AIDA-1, also known as EB-1 and ANKS1B) is a TABLE 1 | Redistribution of postsynaptic density (PSD) components
major component of the PSD complex, with a PSD-95/AIDA- during activity.
1/GKAP stoichiometry of 2:1:1 (Lowenthal et al., 2015). Like
CaMKII SynGAP AIDA-1 Shank CYLD
SynGAP, AIDA-1 binds directly to PSD-95 at the same domains
that bind TARPs (Jordan et al., 2007) and therefore could also Core decrease decrease
interfere with the anchoring of AMPA receptors. By immunoEM, Pallium increase increase increase increase increase
the AIDA-1 label is mostly located within the PSD core at rest Changes observed in immunogold label density for selected proteins following
(Jacob et al., 2010; Dosemeci et al., 2015). Under excitatory treatment of cultured hippocampal neurons with media containing NMDA or
conditions AIDA-1 label at the PSD core is significantly high K+ .
reduced with a concomitant increase at the pallium (Dosemeci
et al., 2015). The reversible CaMKII-mediated redistribution increases the density of mushroom-shaped spines; importantly,
of AIDA-1 at the PSD under excitatory conditions (Dosemeci its association with Shank is necessary for Abp1-mediated
et al., 2016) parallels that of SynGAP. We speculate that different regulation of spine morphology (Haeckel et al., 2008).
regulatory mechanisms may trigger dissociation of SynGAP and Another protein that associates with both Shanks and
AIDA-1 from PSD-95. actin is cortactin, although immunoEM shows that cortactin
concentrates mainly at the central region of the spine (Racz
and Weinberg, 2004). In contrast to Abp1, cortactin exits the
Shank Levels Increase in the Pallium spines during activity (Hering and Sheng, 2003). While Abp1
Shanks, a protein scaffold family concentrated in the PSD, are overexpression increases mushroom spine density (Haeckel et al.,
encoded by three genes, Shank1, Shank2 and Shank3, each giving 2008), cortactin overexpression causes elongation of spines
rise to multiple splice variants. Shank mutations have been linked (Hering and Sheng, 2003).
to autism and other neurodevelopmental/neuropsychiatric The above considerations lead us to suggest a model for
disorders (reviews: Grabrucker et al., 2011; Sala et al., 2015). activity-induced modification of spine morphology: under basal
All Shank isoforms have a similar molecular organization, with conditions, cytosolic Shanks within the spine are pegged to
specialized domains that can associate with GKAPs, Homers, the actin cytoskeleton through cortactin and held outside of
and other Shanks. Analysis of immunoEM data suggests that the pallium. During activity, CaMKII-mediated phosphorylation
Shanks at the PSD are organized into a proximal pool, close releases Shanks from cortactin. The Shank thus released
enough to the interface between the core and pallium to associate accumulates at the pallium, while cortactin exits the spine.
with GKAP, and a distal (deeper) pool that may be stabilized Shank accumulated at the pallium could then recruit Abp1
through association with Homers and/or with other Shanks. to promote remodeling of the actin cytoskeleton around the
Under excitatory conditions, Shanks accumulate preferentially in pallium.
the distal pool (Tao-Cheng et al., 2015).
Shanks promote maturation of dendritic spines and the ACTIN AND THE PSD
enlargement of spine heads (Sala et al., 2001), although the
precise mechanism remains unclear. Considering that changes There are several reports that filaments of F-actin, the primary
in spine shape and size involve reorganization of the actin cytoskeletal component of the spine, contact the PSD (Gulley and
cytoskeleton, it is likely that Shanks regulate spine morphology Reese, 1981; Landis and Reese, 1983; Fifková, 1985), especially
through interaction with actin. Indeed, Shanks associate with at its periphery (Burette et al., 2012), though these contacts are
three actin-regulating proteins, Insulin Receptor Substrate likely highly variable, considering the dynamic nature of F-actin.
Protein 53 (IRSp53), Abp1 and cortactin. While the molecular basis of the attachment of actin filaments
The actin binding protein IRSp53 (also called BAIAP2; see to the PSD remains uncertain, a number of PSD molecules are
review by Kang et al., 2016) is a major PSD component, with plausible candidates. As discussed above, two Shank binding
a molar ratio of IRSp53 to PSD-95 of 1:4 (Lowenthal et al., proteins, IRSp53 and Abp1, may provide a link between the PSD
2015). IRSp53 is located between layers containing Shank and and the actin cytoskeleton.
PSD-95, relatively close to the postsynaptic membrane (Burette Yet another point of interaction of actin with the PSD may
et al., 2014), where it may function as a linker between the actin be through the ubiquitous actin binding protein α-actinin. In
cytoskeleton and the plasma membrane (Scita et al., 2008; Burette vitro assays show that α-actinin can form a ternary complex
et al., 2014). Indeed, IRSp53 contains an actin-binding BAR-like with the PSD proteins, densin and CaMKII (Walikonis et al.,
domain that can induce changes in membrane curvature (Zhao 2001). Loss of α-actinin-2 in rat hippocampal neurons creates an
et al., 2011) and thus may be involved in activity-induced changes increased density of immature, filopodia-like protrusions that fail
in the curvature of the synapse (Burette et al., 2014). to mature into a mushroom-shaped spine during development
Abp1, which can associate simultaneously with actin and (Hodges et al., 2014).
Shanks, may link the actin cytoskeleton to the PSD (Qualmann
et al., 2004). Abp1 preferentially interacts with dynamic rather CONCLUSION
than static F-actin structures (Kessels et al., 2000) and more
Abp1 becomes incorporated into Shank-positive synapses during The pallium is an extension of the electron-dense PSD
activity (Qualmann et al., 2004). Overexpression of Abp1 core, demarcated by strong immunolabeling for two PSD

Frontiers in Synaptic Neuroscience | www.frontiersin.org August 2016 | Volume 8 | Article 23 | 47

Dosemeci et al. The Postsynaptic Density

scaffold proteins, Shank and Homer. This specialized zone, of the actin cytoskeleton (Kim et al., 2015). Subsequent
sandwiched between the electron-dense PSD core and the accumulation of CaMKII at the pallium docks elements that
actin ‘‘spinoskeleton’’, is highly dynamic, exhibiting striking regulate protein turnover (Bingol et al., 2010; Thein et al.,
changes during synaptic activity. Under conditions of strong 2014).
excitation, the pallium becomes electron-dense, with the addition In summary, activation of CaMKII and its translocation to
of CaMKII and several other proteins (Table 1). Activation the pallium under excitatory conditions trigger a chain of events
and/or translocation of CaMKII is necessary for the recruitment poised to elicit profound effects on the organization of the PSD
of other components to the pallium. complex and of the actin cytoskeleton that could synchronize
The pallium can be viewed as a hub where several receptor trafficking with changes in spine morphology. We
proteins converge during activity. Accumulating evidence on speculate that the degree and duration of CaMKII accumulation
the movements of individual proteins suggests a mechanism for and activity at the pallium, promoted by different types of
concerted insertion of receptors to the PSD and re-organization excitatory stimuli, may determine the type and level of synaptic
of the actin spinoskeleton, both mediated by CaMKII. Upon modification.
synaptic stimulation, translocation and activation of CaMKII
cause SynGAP and AIDA-1 to move out of the PSD AUTHOR CONTRIBUTIONS
core and accumulate at the pallium (Yang et al., 2013;
Dosemeci et al., 2015, 2016). We propose that the movement All authors contributed to the writing of the manuscript.
of SynGAP (and perhaps also of AIDA-1) vacates ‘‘slots’’
on PSD-95, providing a window of opportunity for the ACKNOWLEDGMENTS
insertion of AMPA receptors. Simultaneous CaMKII-mediated
accumulation of Shanks at the pallium (Tao-Cheng et al., We would like to thank Dr. John Lisman for stimulating
2014b), on the other hand, may enable actin reorganization discussions and for a critical reading of the manuscript. This
around the PSD. CaMKII also acts as a dynamic structural work was supported by the Intramural Research Program of
element whose activity-induced translocation changes the the National Institute of Neurological Disorders and Stroke
molecular organization within the spine. Dissociation of (NINDS)/National Institutes of Health (NIH) and NINDS/NIH
CaMKII from F-actin causes destabilization and reorganization NS 039444 grant to RJW.

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 REVIEW
published: 05 August 2016
doi: 10.3389/fnsyn.2016.00022

Optogenetic Monitoring of Synaptic

Activity with Genetically Encoded
Voltage Indicators
Ryuichi Nakajima 1† , Arong Jung 1,2† , Bong-June Yoon 2 and Bradley J. Baker 1,3 *
1
Center for Functional Connectomics, Korea Institute of Science and Technology, Seongbuk-gu, Seoul, South Korea,
2
College of Life Sciences and Biotechnology, Korea University, Seongbuk-gu, Seoul, South Korea, 3 Department of
Neuroscience, Korea University of Science and Technology, Daejeon, South Korea

The age of genetically encoded voltage indicators (GEVIs) has matured to the point that
changes in membrane potential can now be observed optically in vivo. Improving the
signal size and speed of these voltage sensors has been the primary driving forces during
this maturation process. As a result, there is a wide range of probes using different
voltage detecting mechanisms and fluorescent reporters. As the use of these probes
transitions from optically reporting membrane potential in single, cultured cells to imaging
populations of cells in slice and/or in vivo, a new challenge emerges—optically resolving
the different types of neuronal activity. While improvements in speed and signal size are
still needed, optimizing the voltage range and the subcellular expression (i.e., soma only)
of the probe are becoming more important. In this review, we will examine the ability of
recently developed probes to report synaptic activity in slice and in vivo. The voltage-
sensing fluorescent protein (VSFP) family of voltage sensors, ArcLight, ASAP-1, and the
Edited by:
George Augustine, rhodopsin family of probes are all good at reporting changes in membrane potential, but
Nanyang Technological University, all have difficulty distinguishing subthreshold depolarizations from action potentials and
Singapore
detecting neuronal inhibition when imaging populations of cells. Finally, we will offer a few
Reviewed by:
Jason D. Shepherd, possible ways to improve the optical resolution of the various types of neuronal activities.
University of Utah, USA
Christian Wilms, Keywords: genetically-encoded voltage indicators, synaptic activity, optogenetics, brain slices, in vivo
Scientifica Ltd., UK

*Correspondence:
Bradley J. Baker
INTRODUCTION
[email protected]
†
As developers of genetically-encoded voltage indicators (GEVIs) we are often asked for our best
These authors have contributed
probe. Until recently, a good GEVI would have been any that gave a voltage-dependent, optical
equally to this work.
signal in mammalian cells (Dimitrov et al., 2007; Lundby et al., 2008, 2010; Perron et al., 2009a,b).
Received: 06 May 2016 Now the experimenter has several probes to choose from that differ in their voltage-dependencies,
Accepted: 25 July 2016 speed, signal size, and brightness (Akemann et al., 2012; Jin et al., 2012; Kralj et al., 2012; Han et al.,
Published: 05 August 2016 2013; St-Pierre et al., 2014; Zou et al., 2014; Gong et al., 2015; Piao et al., 2015; Abdelfattah et al.,
Citation: 2016). The combinations of these varying characteristics result in strengths and weaknesses of
Nakajima R, Jung A, Yoon B-J and every GEVI available. There is no perfect probe that can optically resolve action potentials, synaptic
Baker BJ (2016) Optogenetic activity, and neuronal inhibition in vivo. Some GEVIs will give large, voltage-dependent optical
Monitoring of Synaptic Activity with
signals but are very dim limiting their usefulness in vivo. Others will give large optical signals but
Genetically Encoded
Voltage Indicators. are very slow reducing their ability to resolve fast firing action potentials. So now, when asked
Front. Synaptic Neurosci. 8:22. which is the best probe, the answer is simply another question. What do you want to measure? To
doi: 10.3389/fnsyn.2016.00022 fit with the theme of this edition, we will assume that the answer to that question is synaptic activity.

Frontiers in Synaptic Neuroscience | www.frontiersin.org August 2016 | Volume 8 | Article 22 | 51

Nakajima et al. Optogenetic Monitoring of Brain Activity

Several reviews have been published comparing the signal

size, speed, and brightness of the GEVIs currently available
at the time of publication (Wachowiak and Knöpfel, 2009;
Akemann et al., 2012, 2015; Knöpfel, 2012; Mutoh et al., 2012;
Perron et al., 2012; Mutoh and Knöpfel, 2013; Emiliani et al.,
2015; Knöpfel et al., 2015; St-Pierre et al., 2015; Storace et al.,
2015b, 2016; Antic et al., 2016). In this review, we will shift
the focus to one of the lesser considered characteristics of
a GEVI, the voltage-sensitivity of the probe. Of course, the
other characteristics, especially signal size and brightness, are
still important, but the range and steepness of the voltage
sensitivity of the optical response have extremely important
consequences on which type of neuronal activity a GEVI
reports well. For instance, a GEVI with a voltage range from
−20 mV to +30 mV would be perfect for monitoring action
potentials but not ideal for observing synaptic potentials.
Even the shape of the slope of the optical response over the
voltage range of the probe will affect its performance. The
consequences of the slope and voltage range should also be
considered when choosing probes for monitoring neuronal
activity.

A BRIEF DESCRIPTION OF CURRENTLY

AVAILABLE GEVIs
There now exist several GEVIs with multiple mechanisms
of converting membrane potential changes into an optical
signal. These GEVIs fall into two main classes. One class
utilizes bacterial rhodopsin to detect alterations in voltage,
while the other class relies on a voltage-sensing domain
(VSD) from voltage-sensing proteins. Another viable alternative
for optical, neuronal recordings is hybrid voltage sensor
(hVOS) which consists of a genetically encoded component,
a farnesylated fluorescent protein (FP), and a quenching
compound, dipicrylamine (DPA; Chanda et al., 2005; Wang
et al., 2010, 2012; Ghitani et al., 2015). The requirement for the
treatment with an exogenous chemical limits hVOS use in vivo
but still has value for imaging voltage in slice preparations. FIGURE 1 | Various types of genetically-encoded voltage indicators
The molecular schematics of representative probes from (GEVIs) and their voltage sensitivities. The voltage sensitivities of different
GEVIs are compared. Typical voltage ranges of inhibitory postsynaptic
these classes and their corresponding voltage ranges are shown potential (IPSP), excitatory postsynaptic potential (EPSP) and action potential
in Figure 1. The voltage range of a generic mammalian are indicated as blue, yellow and red, respectively. The vertical scale bar with
neuron is color coded to represent different neuronal minus ∆F/F indicates that the fluorescence dims upon depolarization of the
activities. The inhibitory postsynaptic potential (IPSP) plasma membrane. The voltage-sensitivity curves were as reported in: Arch
voltage range is shown in blue. The excitatory postsynaptic (D95N; modified with permision from Kralj et al., 2012, Figures 3B, 5);
Ace2N-mNeon (modified with permission from Gong et al., 2015, Figure 1D);
potential (EPSP) voltage range is shown in yellow. Voltages ASAP-1 (modified with permission from St-Pierre et al., 2014, Figure 1D);
corresponding to action potentials are color coded red. As ArcLight (modified with permission from Jin et al., 2012, Figure 1C); Butterfly
can be seen from Figure 1, the slopes of these optical voltage 1.2 (modified with permission from Akemann et al., 2012, Figure 2C); hybrid
responses are significantly different. This is an important voltage sensor (hVOS; modified with permission from Chanda et al., 2005,
Figure 1D).
consideration when measuring synaptic potentials. For instance,
Butterfly 1.2 has nearly reached its maximal fluorescent
change at −40 mV which would imply that differentiating thing for imaging membrane potential. First developed in Adam
subthreshold potentials from action potentials will be very Cohen’s lab, the intrinsic fluorescence of rhodopsin as a Schiff
difficult. base being protonated or deprotonated in response to voltage
was used to image changes in membrane potential (Maclaurin
Class I—The Rhodopsin-Based Probes et al., 2013). This probe, Arch, was extremely fast having a
Channel rhodopsin has revolutionized neuroscience. The tau under 1 ms. The fast optical response is due in part
rhodopsin-based voltage sensors are promising to do the same to the fact that the chromophore resides in the voltage field

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Nakajima et al. Optogenetic Monitoring of Brain Activity

enabling a nearly instantaneous response. The signal size was the probe activity (Flytzanis et al., 2014; St-Pierre et al.,
also large giving roughly a 70% ∆F/F optical signal per 100 mV 2014).
membrane depolarization (Kralj et al., 2012; Figure 1, Arch The weak fluorescence of Arch limits its use to single
(D95N)). cell in culture studies or to C. elegans (Kralj et al., 2012;
Arch excelled in speed and signal size but suffered from some Flytzanis et al., 2014) for two main reasons. The first is that
serious weaknesses. The first weakness was that the original the intrinsic fluorescence of higher order neuro-systems will
version had an associated, light-induced current. The D95N mask the fluorescence of Arch-type probes. The second is that
mutation drastically reduced this current but also resulted in a ∆F in addition to ∆F/F is an important characteristic of the
slower probe (Kralj et al., 2012). The second weakness was that GEVI when it comes to the signal to noise ratio. An example
it does not traffic well to the plasma membrane. Even with the of this is shown in Figure 2. The HEK cell in Figure 2 is
addition of endoplasmic reticulum and Golgi network release expressing a GEVI from which the ∆F and the ∆F/F traces
motifs, every image of a rhodopsin probe in the literature exhibits from three different light levels are shown (Lee et al., 2016).
high intracellular fluorescence (Kralj et al., 2012; Flytzanis et al., As can be seen from this comparison, a high ∆F/F value
2014; Gong et al., 2014, 2015; Hochbaum et al., 2014; Hou can be achieved by a large change in fluorescence or a small
et al., 2014). The third and most devastating weakness was change in fluorescence when the probe is dim. Notice the
that Arch is very dim. The best versions of Arch and related increased noise in trace 3, a telltale sign of poor expression/dim
probes are still at least 5× dimmer than the green fluorescent fluorescence.
protein (GFP) requiring exceptionally strong illumination, at An ingenious solution to compensate for the poor
least 700× the light intensity required for ASAP-1 to visualize fluorescence of the rhodopsin voltage probes was developed

FIGURE 2 | Varying light levels affect ∆F and ∆F/F values. (A) An HEK 293 cell expressing a single fluorescent protein (FP) based GEVI, Bongwoori, is shown in
resting light intensity (RLI), (B) the fluorescence traces of averaged ∆F (Fx -F0 ) values from three different regions with varying intensity, (C) the fluorescence traces
showing averaged ∆F/F values from the same regions in (B; modified with permission from Lee et al., 2016, Figure 6).

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Nakajima et al. Optogenetic Monitoring of Brain Activity

simultaneously by the Adam Cohen and Mark Schnitzer the brighter signal (Wilt et al., 2013). It is also difficult to only
laboratories. By fusing an FP to the rhodopsin protein, förster excite the donor chromophore and not the acceptor as well.
resonance energy transfer (FRET) enabled the rhodopsin These factors combined with the relatively low signal size of
chromophore to affect the fluorescence of the fused FP. This FRET-based probes prohibit any reliable absolute measurement
design reduced the excitation light intensity needed to visualize of membrane potential.
the GEVI while maintaining the speed of the optical response The second design involves a circularly-permuted fluorescent
since the voltage-sensing chromophore was still in the voltage protein (cpFP) attached to the VSD. Initial designs fused the
field. These probes could also cover different wavelengths since cpFP downstream of the VSD so that the chromophore was in
many different FPs could be fused to rhodopsin and give a the cytoplasm (Gautam et al., 2009; Barnett et al., 2012). Electrik
signal (Gong et al., 2014; Zou et al., 2014). While this made the PK gave very small signals less than 1% ∆F/F per 100 mV
rhodopsin probes better, the optical signal sometimes could only depolarization but were very fast having a tau under 2 ms. A
indirectly report neuronal activity by determining the frequency substantial increase in signal size was achieved when the cpFP
of the noise in the optical recording (See Supplementary Figure was placed between the S3 transmembrane segment and the S4
5 in Gong et al., 2014). Now, an exciting new version using the transmembrane segment of the VSD putting the chromophore
FP, mNeonGreen, has recently been reported (Gong et al., 2015). outside of the cell (St-Pierre et al., 2014). This probe, ASAP-1,
mNeonGreen is a very bright FP (Shaner et al., 2013) enabling is one of the better GEVIs giving a fast and robust optical signal
Ace2N-mNeon to resolve action potentials in vivo in both flies (tau = 1–2 ms and about 20% ∆F/F per 100 mV depolarization
and mice. in HEK cells). ASAP-1 has a very broad voltage range which
is virtually linear over much of the physiologically relevant
potentials of neurons.
Class II—VSD Containing GEVIs The third design of GEVIs that utilize a VSD simply fuses
The second class of GEVIs is also the oldest. The original GEVI, the FP at the carboxy-terminus which puts the chromophore in
Flash (Siegel and Isacoff, 1997), was the result of inserting the cytoplasm. During a systematic test of different FPs fused at
GFP downstream of the pore domain of the voltage-gated different linker lengths from the VSD done in collaboration by
potassium channel, Shaker. Like the rhodopsin-based probes, the Vincent Pieribone’s lab and Larry Cohen’s lab, a point mutation
first generation of VSD-based probes had significant drawbacks on the outside of the FP, Super Ecliptic pHlorin (Miesenbock
making them useless in mammalian cells (Baker et al., 2007). et al., 1998; Ng et al., 2002) converted an alanine to an aspartic
The main problem was that the GEVIs did not traffic to the acid improving the optical signal 15 fold from 1% ∆F/F to 15%
plasma membrane. In 2007, one of the biggest advancements in per 100 mV depolarization of the plasma membrane (Jin et al.,
GEVI development was achieved by the Knöpfel laboratory when 2012). This negative charge on the outside of the β-can seems to
they fused FPs to the VSD of the voltage-sensing phosphatase affect the fluorescence of a neighboring chromophore when S4
gene from Ciona intestinalis (Murata et al., 2005). This probe, moves since mutations that favor the monomeric form of the FP
voltage-sensing fluorescent protein (VSFP) 2.1 trafficked well reduce the voltage-dependent optical signal substantially (Kang
to the plasma membrane which resulted in the first voltage- and Baker, 2016). Further development of ArcLight has gotten
dependent optical signals from cultured neurons (Dimitrov et al., signals as high as 40% ∆F/F per 100 mV depolarization step (Han
2007). et al., 2013). While ArcLight has the drawback of being slow, its
Another issue with VSD-based GEVIs is that the brightness and signal size make it one of the better probes for
chromophore resides outside of the voltage field so the optical imaging in vivo and in slice. In 2015, two publications improving
signal relies on the conformational change of the VSD. These the speed of this sensor were published. One dramatically
probes are therefore generally slower than the rhodopsin-based improved the off rate called Arclightening but reduced the signal
probes, but a recently developed red-shifted GEVI is extremely size to under 10% ∆F/F per 100 mV depolarization (Treger
fast having taus under 1 ms (Abdelfattah et al., 2016). et al., 2015). The other, Bongwoori, improved the speed of
There are three different designs for GEVIs that utilize a the sensor and shifted the voltage response to more positive
VSD. The first design uses a FRET pair flanking the VSD. An potentials which improved the resolution of action potentials but
example is Butterfly 1.2 (Akemann et al., 2012). This probe decreased the signal size for synaptic potentials (Piao et al., 2015).
is somewhat slow and gives a very small optical signal, less The reduced optical signal response for sub-threshold potentials
than 3% ∆F/F per 100 mV depolarization. A butterfly style gives Bongwoori a better ‘‘contrast’’ for optically resolving action
probe that gives a faster and larger optical signal was developed potentials.
last year called Nabi (Sung et al., 2015). An advantage of A final design for researchers to consider when choosing a
FRET-based probes is that the ratiometric imaging can remove GEVI is the genetically encoded, hVOS (Chanda et al., 2005;
movement artifacts due to respiration and blood flow in vivo. Wang et al., 2010, 2012; Ghitani et al., 2015). First developed
Theoretically, a ratiometric measurement could also be used to in the Bezanilla lab, hVOS consists of an FP anchored to the
determine the absolute value of the membrane potential since plasma membrane with the addition of a small charged molecule,
the ratio is concentration independent. In practice, however, the DPA, that binds to the plasma membrane effectively acting as
relative fluorescence of the two chromophores differ substantially a fluorescent quencher. Since DPA is a lipophilic anion, the
resulting in a potential increase in the noise for the analysis of quenching agent will move from the outer surface of the plasma
the optical signal. Often the experimenter should only analyze membrane to the inner surface upon membrane depolarizations

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Nakajima et al. Optogenetic Monitoring of Brain Activity

generating a voltage-responsive fluorescent signal. Like the other

sensors, hVOS also has drawbacks which are primarily due to
the fact that an exogenous chemical must be administered to
the sample to be imaged. This is not a trivial process since
too much DPA will significantly increase the capacitance of the
plasma membrane and alter the neuronal activity of the cell.
However, once the appropriate conditions are determined, hVOS
gives optical signals for subthreshold potentials as well as action
potentials in slice from populations of cells (Wang et al., 2012)
or individual cells when expression of the FP is sparser (Ghitani
et al., 2015).

SYNAPTIC ACTIVITY MONITORING IN

SLICES WITH GEVIs
Brain slices are invaluable for studying in detail the cellular,
molecular, and circuitry activity of neuronal functions (Ting
et al., 2014). GEVIs can expand this information since every pixel
potentially becomes an electrode. There are not many examples FIGURE 3 | Comparison of voltage indicators for synaptic imaging in
of synaptic potential recordings from GEVIs in slice. Most brain slices. (A) Fluorescence and ratiometric signals of voltage-sensing
fluorescent protein (VSFP) Butterfly 1.2 in cortical brain slices (modified with
examples are proof-of-principle type of recordings in the original
persmission from Akemann et al., 2012, Figures 3I,J). Left: wide-field
publication of a new sensor to demonstrate its potential. The fluorescence image with indicated position of the stimulation electrode (blue
VSFP family of GEVIs are the most published recordings in brain arrow). Scale bar, 150 µm. Middle and right: a single-pulse synaptic stimulus
slice (Akemann et al., 2013; Scott et al., 2014; Carandini et al., (middle: 100 µA; right: 200 µA) induced a depolarizing response as indicated
2015; Empson et al., 2015; Mutoh et al., 2015). Here, we compare by a transient decrease in mCitrine (yellow) and increase in mKate2 (red)
emission from the pixels indicated by a red circle. The ratio of the two
optical synaptic recordings in brain slices from VSFP Butterfly
emission spectra is in black. (B) Stimulus-evoked fluorescence changes
1.2 and hVOS. (∆F/F) and their dipicrylamine (DPA) dependence (modified with permission
from Wang et al., 2012, Figure 3). A slice expressing hVOS 1.5 from mouse
hippocampus. All recordings were from the striatum-radiatum (sr) of the CA1
FRET Signals of Butterfly in Cortical Brain region. Left: a slice from hVOS 1.5 line 602 with images and fluorescence
Slices traces superimposed. Traces are from the three numbered locations before
Figure 3A shows the population imaging in coronal cortical and after addition of DPA. The stimulation site is indicated by the asterisk. All
traces of hVOS signals are averages of 10 trials.
slices prepared from a mouse brain electroporated in utero with
VSFP-Butterfly 1.2 (Akemann et al., 2012). To explore voltage
imaging from populations of cells, cortical slices were imaged could be seen throughout the field of view (Wang et al.,
at low magnification while delivering a single electrical stimulus 2012).
(Figure 3A, left panel). The amplitude of the evoked optical VSFP-Butterfly 1.2, and hVOS can all generate an optical
signal ranged from 1 to 1.5% ∆R/R0 (Figure 3A). Disinhibition signal corresponding to synaptic responses in acute brain slices.
with 25 mM gabazine increased the signal to 11% ∆R/R0 hVOS has the larger ∆F/F. VSFP-Butterfly 1.2 does not require
(Akemann et al., 2012). Since VSFP-Butterfly 1.2 is a FRET additional drug application to detect voltage changes in the
probe, the ratio of the fluorescent change can be reported, but neuron. Other sensors can also give optical signals in slice but
in slice the advantage of a ratiometric recording is of lesser value those recordings have focused on action potentials in individual
since movement artifacts due to respiration and blood flow do cells and are not shown here. The brightness, signal size, and
not exist. Despite this advantage, the voltage-dependent change voltage range of ASAP-1 make it a potentially useful sensor
in fluorescence is quite small, less than 0.5% ∆F/F which requires for imaging synaptic potentials in slice. While there are no
multiple trials to improve the signal to noise ratio. reports in the literature of ArcLight being used to analyze
neuronal activity in brain slices, the brightness, signal size and
hVOS Signal in the Hippocampal Slice voltage-sensitivity are also ideal for optically recording synaptic
Figure 3B shows the hVOS signal in a hippocampal slice. potentials.
The electrical stimulation evoked clear fluorescence changes
only when 4 µM DPA was present. This concentration of SYNAPTIC ACTIVITY MONITORING
DPA provided excellent signal up to 2 h with minimum IN VIVO WITH GEVIs
pharmacological action (Wang et al., 2010). The hVOS probe
fluorescence decreases with membrane depolarization because While slice recordings are extremely valuable for deciphering
DPA moves to the inner surface of the cell membrane where neuronal circuitry, the ultimate goal of voltage imaging is
hVOS probes are anchored; the arrival of DPA quenches to detect neuronal activity in a behaving animal. This is an
the probe fluorescence. Responses of approximately 1–3% ambitious endeavor with very few examples, but some GEVIs

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Nakajima et al. Optogenetic Monitoring of Brain Activity

FIGURE 4 | Continued
images of ArcLight demonstrate membrane localization (arrow). The FP,
mCherry, is localized to the nucleus to facilitate identification of transduced
neurons. (d–f) Low magnification of the olfactory bulb—onl, olfactory nerve
layer; gl, glomerular layer; epl, external plexiform layer; mcl, mitral cell layer. (g)
Wide-field resting fluorescence intensity. (h) Glomerular patterns of activation
after odor stimulation. (i) Odor-evoked optical signals from the region of
interest marked with a red circle in (g,h). (j) Six unfiltered single trials aligned to
the first sniff of odorant.

are now capable of giving a robust signal that allows in vivo

imaging.
Proof of principle for in vivo voltage imaging was established
by the Knöpfel lab using the VSFP family of probes (Akemann
et al., 2012, 2013). Figure 4A shows single trial responses
in the barrel cortex during whisker stimulation. Clearly, a
stimulus evoked voltage signal could be detected in single trials
even though the signal size is very small. Asterisks denote
potential spontaneous voltage transients. However, unlike the
stimulus evoked optical response, these potential transients
exhibit different start times and kinetics. Having a low signal
to noise ratio undermines the confidence in reliably detecting
neuronal activity trial to trial (Carandini et al., 2015). Another
drawback with VSFP Butterfly 1.2 is that the V1/2 is roughly
−70 mV with maximal fluorescent change occurring at −40
mV (Figure 1) making it virtually impossible to distinguish
synaptic activity from action potentials based solely on signal
size.
ArcLight has also been tested in vivo in mice and flies (Cao
et al., 2013; Storace et al., 2015a). The V1/2 for ArcLight is
around −30 mV making it ideal to detect neuronal activity
in flies whose action potentials range from a resting potential
of −40 mV to a final excitation of −10 mV. As a side note
this is why our probe, Bongwoori, should not be used for
imaging neuronal activity in flies since the V1/2 has been
shifted to around 0 mV (Piao et al., 2015). Figure 4B shows
a recording from a mouse expressing ArcLight in the olfactory
bulb. As can be seen, respiration causes an optical artifact, but
since ArcLight gives a large signal, identifying regions of the
olfactory bulb responding to an odor is still possible. Again,
though, it is not possible to resolve synaptic activity from action
potentials.
The rhodopsin-based GEVIs have also been shown to elicit
an optical signal in vivo. An example is shown in Figure 5A
FIGURE 4 | Comparison of voltage indicators for synaptic imaging in from the Gradinaru lab (Flytzanis et al., 2014). With the dim
vivo. (A) Butterfly 1.2 signal in the somatosensory cortex during whisker fluorescence of the GEVI, Archer, C. elegans is one of the
stimulation (modified with permission from Akemann et al., 2012, Figures few multicellular organisms one would be able to record from.
6B,D,E). (a,b) Fluorescence image of mCitrine (a) and mKate2 (b) from a
After odorant stimulation, there is a slight variation in the ∆F
mouse expressing VSFP-Butterfly 1.2. White rectangle indicates area imaged
in (c). Scale bar, 2 mm. (c,d) Ratio images obtained at times before and after compared to control. While there appears to be a slight signal,
brief deflection of the D1 whisker performed at time 0. (c) 10 ms before the low signal to noise ratio again undermines one’s confidence
(d) 90 ms after the deflection. (e) Single-sweep (1, 2, and 3) mCitrine (yellow) in being able to reliably detect neuronal activity from trial to
and mKate2 (red) optical signals sampled from the region of interest indicated trial.
by white circle in (c), together with the 10-trial average (bottom). Asterisks
mark signals corresponding to potential spontaneously occurring voltage
The last example of in vivo recordings is the best example
transients. (f) Ratio (∆R/R0 ) signals corresponding to the traces in (e). of resolving action potentials. Ace2N-4AA-mNeon has been
(B) Odor-evoked signals of ArcLight in olfactory bulb (modified with permission imaged in flies and mice (Gong et al., 2015). This probe is
from Storace et al., 2015a, Figures 1, 2). (a–c) High magnification confocal extremely fast showing the best fit of optical data to voltage yet
(Continued)
(Figure 5B). The red arrow shows an optical response to a 5 mV

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Nakajima et al. Optogenetic Monitoring of Brain Activity

FIGURE 6 | Consequence of internal fluorescence when imaging

population of cells. (A) Schematic of a neuron under high magnification with
plasma membrane and internal fluorescence in green. The cell is projected
onto multiple pixels enabling the experimenter to choose pixels with optical
activity. In this example the red, highlighted pixels labeled 1–4 have a large
∆F/F while pixels highlighted in blue, labeled 5–8, exhibit low or no change in
fluorescence upon depolarization of the plasma membrane. (B) Same cell
under low magnification now only projects onto a few pixels. The pixel
highlighted in black, labeled 9, is a summation of pixels 1–8 in (A). The
FIGURE 5 | Comparison of rhodopsin-based voltage indicators for experimenter is no longer able to avoid the non-responsive, internal
synaptic imaging in vivo. (A) Archer1 expressed in worms. (a) C. elegans fluorescence.
expressing Archer1 shows fluorescence (λ = 655 nm; I = 880 mWmm−2 ,
100 ms exposure). Scale bar, 20 mm. (b) Top: experimental conditions:
worms are stimulated with odorant (Isoamyl alcohol, IAA) for 5 min, flow is
switched to buffer (S Basal) for 30 s, and then odorant flow is restored. The the experimenter wants to image any neuronal activity from a
control conditions are performed on the same worm. Bottom traces: imaging population of cells in brain slice, the recommendations would be
of Archer1 fluorescence (250 Hz) (modified with permission from Flytzanis hVOS, ArcLight, and ASAP-1. All have a broad voltage range,
et al., 2014, Figures 5B,C). (B) Imaging single action potentials and traffic to the membrane well and give relatively large signals.
subthreshold membrane voltage by Ace2N-4AA-mNeon (modified with
permission from Gong et al., 2015, Figures 2A, 3D). (a) Optical resolution of
ArcLight and ASAP-1 will have some difficulty in separating
action potentials of cultured hippocampal neurons under current clamp synaptic activity from action potentials due to their voltage
exhibiting best fit of optical data to electrical recording to date. Arrow denotes sensitivities, but this could theoretically be overcome by co-
5 mV depolarization. (b) Optical traces from a cortical V1→LM neuron in an expression of a red calcium sensor to verify action potential
awake mouse, showing visually evoked responses to drifting gratings.
activity if the neuron tested has an action potential-induced
calcium transient. If one wants to measure neuronal activity
of individual cells in slice, then one should also consider
depolarization. However, comparing the optical signal at the
Ace2N-4AA-mNeon.
spike to the subthreshold potential, one can see that the optical
Imaging single cells vs. a population of cells will also affect the
response is skewed towards action potential activity. This gives a
choice of GEVI to be used. When imaging single cells, probes
fantastic response when imaging the visual cortex in response to
with broad voltage ranges will enable the optical detection of
visual stimuli. Action potentials are easily discernible. The ability
inhibition, synaptic potentials, and action potentials. However,
to optically report synaptic activity is less clear but still promising.
these same probes when imaging large populations of cells
are potentially less informative since the depolarization of a
CONCLUSION subgroup of neurons could swamp the small, hyperpolarizing
signals from inhibited neurons.
GEVIs come in many flavors. As demonstrated, the signal size, Inefficient trafficking or high intracellular expression will
speed, and voltage sensitivity affect the neuronal activity a affect the voltage imaging of a population of cells more so than
GEVI can resolve. Many probes will give an optical signal in when imaging individual cells. The reason for this is that the
slice and in vivo but some signals will be more informative. If spatial representation of the cell under high magnification onto

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Nakajima et al. Optogenetic Monitoring of Brain Activity

the pixels of the camera has changed. Under high magnification, is to use only the most abundant codons for rapid translation
a researcher can choose only pixels that correspond to regions of of the protein. This has been shown to be effective for
the cell that exhibited a fluorescent response. When imaging a cytoplasmic proteins. However, for membrane proteins slowing
population of cells, a pixel will be less likely to capture only the the translation to allow proper folding and insertion into the
responsive fluorescence. This situation is depicted in Figure 6. translocon may also be important (Norholm et al., 2012; Yu et al.,
When imaging a single cell, it is much easier to avoid the internal, 2015). Finally, limiting the expression of the GEVI to subcellular
non-responsive fluorescence and maximize the signal to noise components (i.e., the soma, dendrites, etc.) could also focus the
ratio. optical signal to the desired region of the neuron again improving
While the GEVIs currently available have shown significant the signal to noise ratio.
improvement in their ability to optically detect neuronal activity,
there is still much room for improvement. Refining the voltage- AUTHOR CONTRIBUTIONS
sensitivity will enable maximizing the optical signal. For instance,
a probe that only responded to hyperpolarization of the RN and AJ wrote the manuscript and contributed figures. B-JY
plasma membrane would make identifying the inhibited parts and BJB helped to write the manuscript.
of a neuronal circuit much easier. Improving the membrane
expression of the GEVI will decrease the nonresponsive ACKNOWLEDGMENTS
fluorescence in a population of cells, thereby improving the
signal to noise ratio. Most efforts to improve trafficking involve This work was funded by the Korea Institute of Science and
the addition of endoplasmic reticulum and Golgi release motifs. Technology (KIST) Institutional Program Multiscale Functional
Codon optimization is another approach which for membrane Connectomics, 2E26190 and KIST Institutional Program
proteins may be a misnomer. The idea of codon optimization 2E26170.

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proteins for monitoring membrane potential. Front. Mol. Neurosci. 2:5. doi: 10. Conflict of Interest Statement: The authors declare that the research was
3389/neuro.02.005.2009 conducted in the absence of any commercial or financial relationships that could
Perron, A., Mutoh, H., Launey, T., and Knöpfel, T. (2009b). Red-shifted voltage- be construed as a potential conflict of interest.
sensitive fluorescent proteins. Chem. Biol. 16, 1268–1277. doi: 10.1016/j.
chembiol.2009.11.014 Copyright © 2016 Nakajima, Jung, Yoon and Baker. This is an open-access article
Piao, H. H., Rajakumar, D., Kang, B. E., Kim, E. H., and Baker, B. J. (2015). distributed under the terms of the Creative Commons Attribution License (CC BY).
Combinatorial mutagenesis of the voltage-sensing domain enables the optical The use, distribution and reproduction in other forums is permitted, provided the
resolution of action potentials firing at 60 Hz by a genetically encoded original author(s) or licensor are credited and that the original publication in this
fluorescent sensor of membrane potential. J. Neurosci. 35, 372–385. doi: 10. journal is cited, in accordance with accepted academic practice. No use, distribution
1523/jneurosci.3008-14.2015 or reproduction is permitted which does not comply with these terms.

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 ORIGINAL RESEARCH
published: 22 July 2016
doi: 10.3389/fnsyn.2016.00019

Control of Transmembrane Protein

Diffusion within the Postsynaptic
Density Assessed by Simultaneous
Single-Molecule Tracking and
Localization Microscopy
Tuo P. Li and Thomas A. Blanpied*

Department of Physiology and Program in Neuroscience, University of Maryland School of Medicine, Baltimore, MD, USA

Postsynaptic transmembrane proteins are critical elements of synapses, mediating

trans-cellular contact, sensitivity to neurotransmitters and other signaling molecules,
and flux of Ca and other ions. Positioning and mobility of each member of this large
class of proteins is critical to their individual function at the synapse. One critical
example is that the position of glutamate receptors within the postsynaptic density
(PSD) strongly modulates their function by aligning or misaligning them with sites of
presynaptic vesicle fusion. In addition, the regulated ability of receptors to move in or out
of the synapse is critical for activity-dependent plasticity. However, factors that control
receptor mobility within the boundaries of the synapse are not well understood. Notably,
PSD scaffold molecules accumulate in domains much smaller than the synapse. Within
these nanodomains, the density of proteins is considerably higher than that of the
synapse as a whole, so high that steric hindrance is expected to reduce receptor
Edited by: mobility substantially. However, while numerical modeling has demonstrated several
George Augustine, features of how the varying protein density across the face of a single PSD may
Nanyang Technological
University, Singapore modulate receptor motion, there is little experimental information about the extent of
Reviewed by: this influence. To address this critical aspect of synaptic organizational dynamics, we
Michele H. Jacob, performed single-molecule tracking of transmembrane proteins using universal point
Tufts University, USA
Jason D. Shepherd, accumulation-for-imaging-in-nanoscale-topography (uPAINT) over PSDs whose internal
University of Utah, USA structure was simultaneously resolved using photoactivated localization microscopy
*Correspondence: (PALM). The results provide important experimental confirmation that PSD scaffold
Thomas A. Blanpied
[email protected]
protein density strongly influences the mobility of transmembrane proteins. A protein
with a cytosolic domain that does not bind PSD-95 was still slowed in regions of high
Received: 30 April 2016
Accepted: 05 July 2016
PSD-95 density, suggesting that crowding by scaffold molecules and perhaps other
Published: 22 July 2016 proteins is sufficient to stabilize receptors even in the absence of binding. Because
Citation: numerous proteins thought to be involved in establishing PSD structure are linked to
Li TP and Blanpied TA (2016) Control
of Transmembrane Protein Diffusion
disorders including autism and depression, this motivates further exploration of how
within the Postsynaptic Density PSD nanostructure is created. The combined application PALM and uPAINT should
Assessed by Simultaneous
Single-Molecule Tracking and
be invaluable for distinguishing the interactions of mobile proteins with their nano-
Localization Microscopy. environment both in synapses and other cellular compartments.
Front. Synaptic Neurosci. 8:19.
doi: 10.3389/fnsyn.2016.00019 Keywords: uPAINT, PALM, synapse, nanodomain, glutamate receptor, macromolecular crowding

Frontiers in Synaptic Neuroscience | www.frontiersin.org July 2016 | Volume 8 | Article 19 | 60

Li and Blanpied PALM-PAINT of Diffusion in the Postsynaptic Density

INTRODUCTION is also concentrated (Fukata et al., 2013; MacGillavry et al.,

2013). In previous work, we modeled diffusion within PSDs
Transmembrane proteins such as receptors diffuse on the cell where the heterogeneous distribution of PSD-95 was measured,
surface to reach their sites of action. In doing so, they must and found that the clustered nature of this scaffold could
make their way through complex environments typified by strongly limit the ability of transmembrane proteins to enter
varying densities of obstacles and potential binding partners. The (or escape) the crowded regions of the PSD (Li et al., 2016).
average behavior of proteins moving through such environments Thus, nanoscale regional variation in protein composition
has been well characterized (Frick et al., 2007). However, within a single PSD may have strong impact on synaptic
on small spatial scales or within small compartments, the transmission by controlling the subsynaptic distribution of
local organization of potential interactors will dominate the receptors.
influence on receptor motion paths (Kusumi et al., 2014). For A major impediment to progress on this issue is the
instance, locally high concentrations of steric obstacles create a technical challenge of simultaneously measuring the nanoscale
phenomenon called macromolecular crowding (Ryan et al., 1988) distribution of the protein environment while simultaneously
that can slow mobility and result in anomalous diffusion (Saxton, measuring protein motion through it. To address this, we
1994; Santamaria et al., 2010). Thus, high-resolution information developed a combined single-molecule imaging approach
about the distribution of even non-binding obstacles is necessary that uses single-particle tracking photoactivated localization
to understand motion trajectories of transmembrane proteins on microscopy (sptPALM; Manley et al., 2008) to map the positions
small scales. of PSD-95 molecules within the synapse (MacGillavry et al.,
Perhaps the most complex compartment of the plasma 2013), while simultaneously tracking the motion of proteins
membrane in neurons is the postsynaptic density (PSD). The in the plasma membrane by universal point-accumulation-for-
PSD of glutamatergic synapses concentrates numerous receptor imaging-in-nanoscale-topography (uPAINT; Giannone et al.,
types aligned to the presynaptic active zone. Despite the small size 2010). Using this strategy, we could directly investigate the
of the average PSD (∼0.08 µm2 × 50 nm; Harris and Weinberg, influence of both obstacle density and protein binding on
2012), roughly 500 species of proteins can be found in this motion through the PSD. The results provide direct experimental
compartment (Husi et al., 2000; Sheng and Hoogenraad, 2007). confirmation that macromolecular crowding within the PSD
Because of the high local density of transmembrane proteins, can strongly limit the motion of even small transmembrane
receptor-binding proteins such as PSD-95, and juxtamembrane proteins, likely helping to establish the distribution and dynamic
cytosolic molecules, protein motion within the membrane at exchange characteristics of glutamate receptors and other
the synapse is likely extremely obstructed (Santamaria et al., molecules.
2010). This complicated environment is critical to understand,
because protein organization in the PSD directly regulates
MATERIALS AND METHODS
synaptic transmission in many ways. The number of glutamate
receptors present in the PSD sets an upper limit on the strength
of the synapse (Huganir and Nicoll, 2013), and receptors
Neuron Culture and Transfection
Dissociated hippocampal neuron cultures were prepared from
exchange continuously by diffusion between the PSD and the
E18 rat embryos as described previously (Frost et al., 2010).
perisynaptic plasma membrane (Opazo and Choquet, 2011;
All procedures conformed to the guidelines established by
Choquet and Triller, 2013). Further, alterations to the PSD are
Animal Welfare Act, Public Health Service, and the United
a critical component of activity-driven plasticity mechanisms
States Department of Agriculture, and were approved by
regulating receptor number (Inoue and Okabe, 2003; Bosch
the Institutional Animal Care and Use Committee at the
et al., 2014). Thus, understanding mechanisms within the
University of Maryland, Baltimore. Prior to plating the cells on
PSD that control motion of glutamate receptors is critical
coverslips, the coverslips were first cleaned as reported previously
for determining how receptor number is modulated during
(MacGillavry et al., 2013), subsequently coated with lateral-drift
plasticity.
tracking, yellow-green fluorescent 100 nm beads (F8803; Thermo
Even beyond the clear importance of the number of receptors,
Fischer Scientific) diluted 1:25,000 in 100% ethanol (dried within
however, their distribution within the synapse in the plane
25 min in the hood), and then coated overnight with poly-
of the membrane is a vital regulator of synaptic strength
L-lysine (Sigma). Cells were transfected at DIV10–13 using
(MacGillavry et al., 2011). This is because when glutamatergic
Lipofectamine 2000 (Thermo Fischer Scientific) and imaged
vesicles fuse with the presynaptic plasma membrane, the
72–96 h later (unless stated otherwise). Individual coverslips
result is a highly concentrated but narrow spike of released
were transfected with 0.5–0.75 µg of cDNA for each expression
neurotransmitter. The rapid dissipation of this spike by
construct.
glutamate diffusion means that receptors laterally displaced
from the site of fusion even by less than 100 nm often
fail to activate (Xie et al., 1997; Raghavachari and Lisman, Expression Constructs
2004; Santucci and Raghavachari, 2008; Freche et al., 2011). Plasmid cDNAs were obtained as follows (with original sources):
Amplifying this effect, receptors in the PSD are concentrated the binding and nonbinding probes, Super ecliptic phluorin
in ∼80 nm subdomains (MacGillavry et al., 2013; Nair et al., (SEP)-transmembrane (TM)-Bind and SEP-TM-Nonbind
2013) where the principle receptor-binding scaffold PSD-95 (Li et al., 2016), the PSD-95-mEos2 replacement plasmid

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Li and Blanpied PALM-PAINT of Diffusion in the Postsynaptic Density

shrPSD-95-mEos2 (MacGillavry et al., 2013). We have Single-Molecule Localization, Tracking

previously measured that this construct produces a mild Analysis, and PSD Nanostructure Analysis
1.4× overexpression. Interpretations of the results from All data analysis was performed offline using custom routines
the PALM-PAINT assay require control experiments to in MATLAB (The MathWorks). The algorithms for determining
ensure that the expressed protein is organized similarly molecule location and criteria for filtering molecules to be
to its endogenous counterpart. To this end, we have also considered for further analysis were applied as previously
compared the PSD-95 nanostructure of untransfected cells and described (MacGillavry et al., 2013). In addition to filtering
those expressing the replacement construct PSD-95-mEos2 by localization precision, elliptical form, and brightness, we
(MacGillavry et al., 2013; Tang et al., in press). These were also utilized a Voronoi-based segmentation program SR-Tesseler
not different in terms of PSD area, number of subsynaptic (Levet et al., 2015) to filter out spurious localizations outside
nanoclusters, and the size of those nanoclusters. Thus, we of putative neuronal border. Criteria for defining a track were
believe the mEos2 tag does not affect PSD nanostructure, described by Li et al. (2016). Instantaneous effective diffusion
at least in the absence of severe PSD-95 overexpression. coefficients (Deff ) at individual track time points were calculated
Furthermore, mEos2 is fused at the c-terminal end of the for tracks that persisted at least eight frames (the duration
PSD-95, not altering the positions of the various protein- in which the mean mean squared displacement (MSD) was
protein interaction motifs. Thus, we believe tagging itself linear; a more detailed description can be found in Lu et al.,
does not affect the interaction of PSD-95 with other 2014). For comparing diffusion inside and outside of PSDs,
proteins. tracks that entered or exited the PSD were divided into two
portions, a synaptic and an extrasynaptic subtrack, the Deff of
Two-Color Single-Molecule Imaging which were calculated by averaging the instantaneous Deff
PALM-PAINT, a combination of PALM (Betzig et al., 2006; for the tracked localizations therein. For the rare tracks that
Hess et al., 2006) and uPAINT (Giannone et al., 2010), was entered and exited PSDs multiple times, synaptic Deff was
performed through a Photometrics DV2 on an Olympus IX81 determined by averaging the instantaneous Deff of all the
ZDC2 inverted microscope that was described by MacGillavry tracked locations inside the PSD border; vice versa to calculate
et al. (2013). Cells expressing the indicated constructs were the extrasynaptic Deff . To calculate the Deff of subtracks in
imaged in a previously described extracellular buffer (Li et al., other cases (e.g., within a particular range of PSD-95 regional
2016). SEP-containing probes were labeled with ATTO647N- density, or below the detection limit of Deff ), we calculated
conjugated anti-green fluorescent protein (GFP) nanobodies the average of instantaneous Deff of localizations meeting the
(GFPBooster-647N, Chromotek), bath applied to a final criteria. The lower detection limit of Deff was determined
concentration of 0.5–2 nM once the first stretch of synapses was conservatively by calculating the Deff of a theoretical immobile
identified for each coverslip. Cells remained at 25◦ C for no more particle displaced as much as the average trajectory error ∼30 nm
than 30 min per imaging session. (Savin and Doyle, 2005; Lu et al., 2014) per time frame, which
We imaged the red and far-red bands by interleaving amounted to 0.003 µm2 /s; this value is indicated by a gray
excitations of 561 and 640 nm. Imaging was conducted at vertical dotted or dotted-dashed line in cumulative frequency
29 Hz (14.5 Hz per color), with 10-ms duration excitation per graphs of Deff .
frame for 5000–20,000 frames per color. The two emissions The PALM PSD border was determined by taking the convex
were overlaid based on calibration images of TetraSpeck beads hull of the PSD-95 molecular positions. To determine the
(100 nm; Thermo Fischer Scientific) deposited on an acellular Gaussian-blurred border of each PSD, we first constructed a
coverslip as described by MacGillavry et al. (2013). Yellow-green 2-dimensional molecular density map of 25 × 25 nm subpixels
beads (100 nm) ethanol-diluted and dried onto the coverslips from PSD-95 molecules for each PSD. We then convolved it
prior to plating the cells (F8803; Thermo Fischer Scientific) were with a constant-amplitude Gaussian image profile (σ = 125 nm,
excited and captured once every 1000 frames to monitor lateral image width = 900 nm) that is similar to an ideal microscope
drift. To correct lateral drift, we localized fiducials post hoc from point-spread function with a full-width at half max (FWHM) of
images of the yellow-green beads. We screened for spurious ∼250 nm. To determine the FWHM border of the blurred PSD,
localizations by the duration of fluorescence and mobility. we thresholded the convolved image at half-maximum intensity.
Namely, the bead ought to be present on the first frame, persist To determine the 95%-border, we thresholded it at 5% of the
for as long as each imaging session, and displace <100 nm maximum intensity.
(1 pixel) per 1000 frames. Such a filtering process provided
a list of localizations that correspond to fiducials. From this Determination of Regional PSD-95 Density
list we calculated the sample lateral drift as the weighted To quantify the regional density of PSD-95, we measured the
average of the displacements of all fiduciary localizations between number of molecules surrounding each position of a tracked
each set of 1000 frames. For weights, we used the inverse probe molecule. PSD-95 localizations appearing in consecutive
of the estimated localization uncertainty (Thompson et al., frames separated by no more than 200 nm were considered
2002) of each fiducial. The single linear correction in drift one molecule, and its position was taken from the first frame it
we applied to each subset of 1000 frames was the average appeared. We counted the number of PSD-95 molecules within
correction obtained from the estimates of 2–10 fiducials in the a 30 nm radius (the average trajectory error) of each position
field of view. in the track of a probe molecule. Because we wished primarily

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Li and Blanpied PALM-PAINT of Diffusion in the Postsynaptic Density

to relate Deff to regional density, we used the same tracked

locations to calculate each measure. That is, for the regional
density of PSD-95 surrounding the probe at each position, we
calculated the forward running average of regional densities
for eight frames, the same number of frames used to calculate
Deff . The absolute density of molecules in all calculations was
adjusted by the average expected number of blinks (one) of
mEos2 in our experimental conditions (Annibale et al., 2011;
MacGillavry et al., 2013). The final density when averaged
across synapses is likely somewhat higher than endogenous
PSD-95 densities due to the mild (∼1.4-fold) overexpression
obtained with our shRNA knockdown/replacement approach.
However, we did not attempt to measure the potentially quite
variable ratio of endogenous and tagged PSD-95 at each synapse
analyzed, and did not take this into account for calculations.
Data of immunostaining done in cells not intended for PALM-
PAINT showed that endogenous PSD-95 molecules resistant
to the knockdown did not form dense clusters outside those
formed by the expressed PSD-95 molecules, indicating that
though the endogenous PSD-95 might be present in the
synapse, they likely raise the absolute number of PSD-95
molecules without changing the spatial variation in regional
density. FIGURE 1 | Photoactivated localization microscopy (PALM)-point
accumulation-for-imaging-in-nanoscale-topography (PAINT),
single-molecule tracking during PALM imaging. (A) (Left) Super ecliptic
Statistics phluorin (SEP)-TM-Bind molecules tracked for at least eight frames
Where means are presented, the accompanying errors are super-imposed on molecules of shrPSD-95, accumulated from 5000–20,000
the standard error of the mean; additionally, these data were frames. (Right) A typical example of tracked probes superimposed on
positions of shrPSD-95 molecules. The first and last localized positions are
normally distributed according to the Shapiro-Wilk normality
indicated as filled and open circles, respectively. (B) Mean squared
test. Where box-and-whisker plots are presented, the middle displacement (MSD) over time of connected sub-segments of tracks
bar represents the median, the upper and lower limit of (subtracks) that are lasted at least 15 frames (n = 156 synaptic and 2907
the boxes denote the interquartile range, and the whiskers extrasynaptic tracks/113 PSDs/13 fields/11 cells/3 cultures). (C) Cumulative
extend to 5% and 95% of the distribution; additionally, frequency distributions of Deff for subtracks that are at least eight frames long
(n = 654 synaptic and 3349 extrasynaptic tracks/113/13/11/3).
these data were not normally distributed according to the ∗∗∗∗
p < 0.0001.
Shapiro-Wilk normality test. Different sets of statistical tests
were used for normally and non-normally distributed data.
Pairwise statistical tests were performed using unpaired t-test targeting PSD-95 along with an RNAi-resistant, mEos2-tagged
with Welch’s correction for normally distributed data; they PSD-95 (MacGillavry et al., 2013). To track motion of
were performed using Mann-Whitney U test for non-normally SEP-TM-Bind at the cell surface, we used uPAINT and applied
distributed data. Where two-way analysis of variance (ANOVA) anti-GFP nanobodies carrying Atto647N in the chamber to a
was used, a Bonferroni correction was used for post hoc final concentration of 0.5–2 nM. Concurrently, we localized
pairwise comparisons. Kolmogorov-Smirnov tests were applied the positions of PSD-95-mEos2 using conventional sptPALM
for cumulative frequency distributions. In all cases, means methods (Figure 1A).
(or medians) were considered significantly different if the test The SEP-TM-Bind probe exhibited clearly different mobility
reported p < 0.05. Most statistical tests and all graphing were inside and outside of the PSD (Figure 1A right), as expected
done using Prism (GraphPad Software). Two-way ANOVA was based on the behavior of AMPA-type glutamate receptors (Bats
done in MATLAB (The MathWorks). et al., 2007; Hoze et al., 2012), intercellular adhesion molecules
such as neuroligin and leucine rich repeat transmembrane
RESULTS neuronal 2 (LRRTM2; Chamma et al., 2016), and NMDA-type
glutamate receptors (Dupuis et al., 2014). We compared the
To perform single-molecule tracking during superresolution diffusion patterns of molecules inside and outside of the PSD,
imaging of the PSD, we co-transfected 13–17 DIV hippocampal when the PSD border was defined by the convex-hull border
neurons with two cDNA constructs. The first encoded a of PSD-95 positions. Probes outside of the PSD displayed
single-pass TM protein composed of an extracellular SEP, near-free diffusion as evidenced by an almost linear plot
the TM domain, and the intracellular carboxy terminus of MSD (Figure 1B). However, probes within the PSD showed
stargazin, which enables this protein to bind PSD-95. Thus, a much slower mobility and appeared highly confined in
we refer to it as SEP-TM-Bind (Li et al., 2016). This was their motion, as evidenced by saturation of the relationship
co-transfected with shrPSD-95-mEos2, which expresses shRNA between MSD and time. This curve approached a plateau

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Li and Blanpied PALM-PAINT of Diffusion in the Postsynaptic Density

of 104 ± 35 nm2 , suggesting confinement within <60 nm

diameter regions of the PSD (Ehlers et al., 2007). The effective
diffusion coefficients (Deff ) of the synaptic subtracks were ∼2
orders of magnitude slower than those of the extrasynaptic
subtracks (Figure 1C). This differential is similar to that
seen for AMPARs (Bats et al., 2007; Hoze et al., 2012),
suggesting that AMPAR motion within synapses likely is
regulated by mechanisms that also impact many other types of
molecules.
The improved resolution of the PSD border obtained by
imaging the positions of individual PSD-95 molecules as opposed
to using widefield or confocal microscopy should improve
discrimination of which molecules are within the synapse. The
average PSD area (0.085 ± 0.006 µm2 , n = 263 PSDs/21
neurons) was within the ranges as previously detected by PALM
(MacGillavry et al., 2013; Nair et al., 2013) and by electron
microscopy (Harris and Stevens, 1989; Schikorski and Stevens,
1997; Shinohara et al., 2008). To test whether diffraction-limited
PSD borders could perform as well as PALM of PSDs at
segregating synaptic and extrasynaptic probes, we simulated
diffraction by blurring the PSD-95 molecular density maps
with a Gaussian point-spread function. Taking the FWHM
of this intensity distribution as the border of the diffraction-
blurred PSDs, as was done by Li et al. (2016), performed
nearly as well as the PALM’ed PSD border in segregating
synaptic and extrasynaptic probe movements. Taking the 95%
border of the blurred PSDs diminished the difference between
synaptic and extrasynaptic Deff (Figure 2). Thus, how the PSD
border is defined in diffraction-limited approaches can influence
the accuracy of segregating diffusing molecules in different
sub-compartments of the cell.

Synaptic TM Protein Diffusion is not

Influenced by PSD Size or Whole-Synapse
PSD-95 Density FIGURE 2 | Better discrimination of postsynaptic density (PSD) border
Interestingly, the Deff distribution within different synapses reveals strong reduction of mobility within synapses. (A) Typical tracks
varied widely, and individual molecules exhibited Deff spanning of the binding probe (magenta) and positions of shrPSD-95 (black circles)
super-imposed. Convex hull border of PSD-95 positions (solid line), full-width
more than five orders of magnitude. This difference did not half max (FWHM) border of Gaussian-blurred shrPSD-95 positions (dashed
stem from neuron-to-neuron variability, as the median synaptic line), and full-width 5% max (95%) border (dotted lines). (B) Cumulative
Deff of the binding probes measured in different neurons frequency distribution of synaptic (black lines) and extrasynaptic (gray lines)
differed by only up to 2-fold. We reasoned that this broad tracks segregated using the synaptic borders of PSDs determined using
PALM’ed shrPSD-95 (solid lines), FWHM of Gaussian-blurred shrPSD-95
range of Deff may arise because the diffusion environment
positions (dashed lines), or full-width 5% max (95% border) of the
within the PSD might vary based on synaptic size or geometry, Gaussian-blurred shrPSD-95 positions (dotted lines) (n = 654 synaptic/3349
or because the density of binding sites could influence how extrasynaptic tracks for PALM’ed PSDs, 658/3345 FWHM, 1197/3125 95%
likely a probe is able to be bound at any given time. To border; 113 PSDs, 11 cells, 3 cultures).
test this, we first examined the relationship between the area
of the PSD and the median Deff of probes found within it. of the binding probes, we examined the relationship between
Based on linear regression analysis, we found no statistically the density of PSD-95-mEos2 localizations and the synaptic
significant correlation (Figure 3A). However, our previous study median Deff of probes in each PSD. The absolute density of
found that the fluorescence recovery of these probes after localizations in all calculations was adjusted by the average
photobleaching spines was negatively correlated with PSD area expected number of blinks (one) of mEos2 in our experimental
(Li et al., 2016). Combined with this finding, this suggests that conditions (Annibale et al., 2011; MacGillavry et al., 2013).
the size of the synapse correlates with the rate with which It should be noted that this measure of density does not
these probes enter and exit the spine, but does not influence incorporate the unknown fraction of total PSD-95 molecules
their diffusion within the synapse. To determine whether overall that were mapped, and also ignores the numerous other binding
PSD-95 density within the synapse can determine the diffusion partners of SEP-TM-Bind that may not correlate with the

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Li and Blanpied PALM-PAINT of Diffusion in the Postsynaptic Density

could obscure the effect that PSD-95 molecular density can

have on the diffusion of probes when measured at the level
of the entire synapse. Consistent with this notion, computer
modeling has demonstrated that measured arrangements of
PSD-95 molecules can prevent a larger fraction of TM proteins
from escaping the synapse than homogeneously distributed
PSD-95 molecules, without changing the overall density of
PSD-95 (Li et al., 2016).
To test whether the density of PSD-95 immediately
surrounding the probe can influence its diffusion within
the synapse, we defined a subsynaptic metric termed ‘‘regional
PSD-95 density’’ to be the number of PSD-95 molecules
FIGURE 3 | PSD size and density do not correlate with intrasynaptic surrounding a tracked probe position. We measured the regional
mobility. (A) PSD area and median Deff within each of the PSDs; linear density using a fixed radius based on the average positional error
regression test (n = 113 PSDs/11 cells/3 cultures). (B) PSD-95 density and of the tracked molecules (30 nm, see ‘‘Materials and Methods’’
median Deff within each of the PSDs; linear regression test (n = same as in A).
Section). However, the results of the following analyses depended
only very weakly on the radius over the range of 15–80 nm (data
measured density of PSD-95-mEos2 as well as the likelihood not shown). We subdivided tracks into subsegments (subtracks)
of slight overexpression compared to endogenous protein level based on the regional PSD-95 density at each of their positions,
(∼1.4×, see MacGillavry et al., 2013). Nevertheless, we found no and plotted the Deff for subtracks based on their regional density.
statistically significant correlation (Figure 3B), suggesting that, This analysis revealed that the higher the regional PSD-95
given these caveats, the overall density of PSD-95 within the PSD density, the slower the diffusion coefficients of the subtracks in
does not influence the median diffusion of binding TM proteins that area of the PSD (Figures 4A,B). In fact, the median probe
in the synapse. Deff within the synapse was strongly correlated with the regional
density of PSD-95 (Figure 4C).
The Control of TM Protein Diffusion
by Binding and Steric Hindrance Within The Control of TM Protein Diffusion
the PSD by Steric Hindrance Alone Within the PSD
Though the overall measured density of PSD-95 did not correlate At a first glance, this result may not be surprising, as it supports
with the diffusion coefficient of SEP-TM-Bind, it would be the idea that the more scaffold binding partners there are in
surprising if this key scaffolding protein did not affect the the synapse, the more likely the probe will be bound and
mobility of its binding partners at all. We thus considered that thus immobilized before diffusing further. However, this effect
the distribution of PSD-95 molecules is highly heterogeneous is more difficult to interpret if we consider that PSD-95 not
within single synapses (Fukata et al., 2013; MacGillavry et al., only binds this probe, but can serve as a steric obstacle. In
2013; Nair et al., 2013; Broadhead et al., 2016) and can display fact, PSD-95 is a hub for binding many other proteins that
multiple regions of high density within the synapse. Namely, can serve as additional obstacles which could, without binding
two synapses of the same PSD-95 density can have very the probe, hinder its diffusion. To isolate the effect of steric
different arrangement of PSD-95 molecules, an organization that hindrance from the combined effect of steric hindrance and

FIGURE 4 | Nanoscale regional density of PSD-95 within the synapse correlates with probe diffusion coefficient. (A) Typical example of tracks and
PSD-95 positions. Tracks pseudo-colored purple, PSD-95 positions pseudo-colored by regional density. (B) Cumulative frequency distributions of probe Deff binned
in increasing regional densites of PSD-95 surrounding the subtracks (n = 448 subtracks in 0–5 PSD-95, 281 in 5–10, 159 in 10–15, 106 in 15–20, 59 in 20–25, 22 in
25–30, 24 in 30+). (C) PSD-95 regional density and the median Deff of the binding probe; relationship tested by linear regression.

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Li and Blanpied PALM-PAINT of Diffusion in the Postsynaptic Density

FIGURE 5 | A non-binding transmembrane protein enters and slows within the synapse, but not as much as if it can bind PSD-95. (A) Typical examples
of tracked probes (purple) superimposed on shrPSD-95 positions (pseudo-colored on a scale of regional density same as Figure 4A; binding probe SEP-TM-Bind
(left), nonbinding probe SEP-TM-Nonbind (right). (B) Cumulative frequency distributions of the binding probe and nonbinding probe Deff within PSDs (n = 654
tracks/113 PSDs/11 cells/3 cultures for SEP-TM-Bind, 519/91/10/3 SEP-TM-Nonbind). (C) Cumulative frequency distributions of the binding probe and nonbinding
probe Deff outside of PSDs (n = 3349 tracks for SEP-TM-Bind, 4470 SEP-TM-Nonbind). (D) (Left) PSD area and median Deff within each of the PSDs; linear
regression test (n = 91 PSDs/10 cells/3 cultures of SEP-TM-Nonbind, same as in Figure 3 for SEP-TM-Bind). (Right) Overall synaptic PSD-95 density and median
Deff within each of the PSDs; linear regression test (n = as in left panel).

probe-scaffold binding, we performed PALM-PAINT on a probe binding probe (Figure 5B). On the other hand, the extrasynaptic
variant that cannot bind to PSD-95 (SEP-TM-Nonbind from diffusion of the nonbinding probe was not different from that of
Li et al., 2016). Interestingly, SEP-TM-Nonbind still entered the binding probe (Figure 5C). It is interesting to note that using
synapses and diffused within them, but did not enrich within sptPALM (i.e., by photoactivating and tracking an mEos3 fusion
the PSD nearly as greatly as SEP-TM-Bind (Figure 5A and see protein rather than using an anti-GFP PAINT approach as here)
also Li et al., 2016). Notably, the diffusion of this nonbinding there was a very similar mobility differential between the binding
probe within the synapse was dramatically faster than that of the and nonbinding probes despite a substantial absolute difference

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Li and Blanpied PALM-PAINT of Diffusion in the Postsynaptic Density

in Deff arising from the poorer localization precision of the fusion that despite the lack of a PSD-95-binding motif, the probe still
protein compared to the organic dye (Li et al., 2016). Thus, diffused more slowly within higher density regions of the PSD
the mobility difference between the two probes is quite robust. (Figure 6A). The effect appeared to saturate at low Deff since the
Intriguingly, the shoulder-like shape of the cumulative Deff mobility of these slowly moving molecules is below our detection
distribution suggests that probes undergo multiple influences limit (0.003 µm2 /s).
on their diffusion within the synapse. However, neither PSD If steric obstruction influences probe mobility, it may
area nor whole-synapse PSD-95 density correlated with probe influence the overall pattern of probe position within the synapse.
diffusion within the PSD (Figure 5D). We thus compared the fraction of subtracks found in different
By labeling SEP-tagged TM proteins with nanobodies, we add regional PSD-95 densities (Figure 6B). Interestingly, though the
minimal but appreciable bulk to the extracellular domain of the binding and the nonbinding probes were distributed similarly
diffusing entity. SEP is fused to the extracellular domain of the through most density values, the nonbinding probes appeared
TM probes, adding approximately 3–5 nm of bulk; nanobody to be preferentially excluded from the highly dense subregions
labeling of the GFP adds an additional 3–5 nm of bulk. While (i.e., >25 regional molecules) of the synapse. Because of the
additional extracellular bulk has previously been shown to slow small number of subtracks in these bins, however, this difference
receptor diffusion (Groc et al., 2007), we expect the effect to be was not significant. Note also that the distribution of tracked
identical for both the binding and the nonbinding probes and molecules may not completely faithfully represent the total
thus not change our conclusions about the effect of postsynaptic steady-state distribution of probe molecules, since molecules
steric hindrance. immobilized in the synapse for long periods are less likely to be
We next considered whether the regional density of PSD-95 recognized by a nanobody and be tracked by uPAINT.
immediately surrounding the nonbinding probe can sterically It appeared that regional densities of PSD-95 higher than
control the probe diffusion. To test this, we first subdivided the 10 had minimal effect on diffusion of the nonbinding probe
tracks into subtracks and binned them into increasing regional in the Deff range below our detection limit (Figure 6C),
densities of PSD-95 molecules, as in Figure 4B. This revealed whereas the regional density of PSD-95 linearly correlated with

FIGURE 6 | Subsynaptic regional density of PSD-95 influences the mobility of a probe that does not bind PSD-95. (A) Cumulative frequency distributions
of the nonbinding probe Deff binned in increasing regional densites of PSD-95 surrounding the subtracks (n = 480 subtracks in 0–5 PSD-95, 204 in 5–10, 148 in
10–15, 81 in 15–20, 32 in 20–25, 12 in 25–30, 13 in 30+). (B) Fraction of subtracks in different regional densities of PSD-95. (C) PSD-95 regional density and the
median Deff of the nonbinding probe. (D) Fraction of tracks with subsegments that were slowed below the detection limit per PSD (n = 113 PSDs for SEP-TM-Bind,
91 SEP-TM-Nonbind; ∗ p = 0.0123 Mann-Whitney U test). (E) (Left) Cartoon highlighting the PSD-95 molecules surrounding subtrack durations that diffused below
the detection limit. The open and closed purple circles indicate the beginning and end of a track, the circles pseudo-colored by regional density were within 30 nm of
sub-detection limit subtracks. We calculated the median regional density of PSD-95 of all subtrack durations that diffused below the detection limit with every PSD.
(Right) Median regional density of PSD-95 surrounding subtracks that were below the detection limit (n same as in D; ∗ p = 0.0325 K-S test). (F) Median regional
density of PSD-95 surrounding subtracks that above the detection limit (n same as in D; ns, Not significant, p = 0.158 K-S test).

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Li and Blanpied PALM-PAINT of Diffusion in the Postsynaptic Density

the diffusion of the binding probe (Figures 4B,C). Thus we

wondered whether the two probes required different degrees
of steric hindrance in order to be stabilized. To test this,
we first noted that during multiple visits to a synapse, or
even within a single visit, probe molecules could display
both slow and fast periods of motion. Considering only the
subtracks within synapses, SEP-TM-Bind had a higher fraction
than SEP-TM-Nonbind of these subtracks for which Deff was
below the detection limit (Figure 6D). This indicates that the
binding probes were more often slowed down while within
the synapse than were the non-binding probes. We could then
further exploit the ability afforded by PALM with uPAINT
to examine the very local environment of the molecules as
they moved within the synapse. Specifically, we noted that
if steric hindrance slows mobility of the both the binding
and non-binding probes, but binding is only able to slow
SEP-TM-Bind, then SEP-TM-Bind would be expected to show
a greater tendency to slow its mobility in relatively less
dense PSD subregions. That is, even sparse binding partners FIGURE 7 | Estimating how dense PSD-95 is when protein mobility is
could potentially capture and immobilize SEP-TM-Bind whereas slowed sterically. Deff of SEP-TM-Nonbind and of SEP-TM-Bind within
higher concentrations of molecules would be required to synaptic subregions of different PSD-95 densities (n of SEP-TM-Nonbind
sterically obstruct SEP-TM-Nonbind. Consistent with this, when same as in Figure 6B; that of SEP-TM-Bind same as in Figure 4B; two-way
ANOVA, effect of binding F 1,6 = 15.27, p < 0.001; effect of regional PSD-95
we analyzed the sub-detection-limit portion of the nonbinding
density F 1,6 = 13.53, p < 0.0001; post hoc Bonferroni multiple comparisons
probe subtracks, we found that these were preferentially in locales tests between probes in increasing regional densities ∗ p < 0.05, ns, Not
of higher regional density of PSD-95 compared to those of the significant).
binding probes (Figure 6E). Interestingly, the fraction of the
subtracks above the detection limit did not show any difference the diffusion of the TM probe. This influence was apparent
in regional PSD-95 density (Figure 6F), with even a trend to the even in the absence of probe-scaffold binding, indicating
opposite relationship. steric hindrance by macromolecular crowding can complement
Altogether, these results suggest that crowding by scaffold protein-protein binding interactions in organizing TM proteins
molecules and perhaps other proteins is sufficient to stabilize TM within the synapse.
proteins in the absence of binding. How dense does the molecular
environment have to be in order to slow the TM probes
sterically as much as the combined influence of steric hindrance The Roles of Receptor-Scaffold Binding
and probe-scaffold binding? To estimate an answer to this and Macromolecular Crowding in
question, we compared the diffusion coefficients of the binding Subsynaptic Organization
and nonbinding probes within increasing regional densities of The mobility of AMPARs in the synapse is increased when
PSD-95 (Figure 7). As expected based on Figures 4, 6, the the binding of their accessory subunit Stargazin to PSD-95 is
synaptic Deff of both probes decreased gradually with increasing disrupted (Bats et al., 2007; Sainlos et al., 2011), providing strong
PSD-95 regional density. However, the Deff of SEP-TM-Nonbind evidence that receptors are acutely stabilized by PSD-95 binding.
decreased precipitously over the range of 0–15; yet, it did However, even in these conditions some stabilization of receptors
not decrease further at higher densities. Furthermore, the Deff in the synapse occurs, and our results indicate specifically that
of SEP-TM-Nonbind plateaued at the Deff value displayed by even small probes carrying a cytosolic tail unable to bind PSD-95
SEP-TM-Bind at very low PSD-95 densities. Thus, by this is still slowed substantially in the synapse. What controls this
analysis, ∼15 PSD-95 molecules per region of 30 nm radius stabilized fraction even in the absence of binding has been a
(∼5000 molecules/µm2 ) is the threshold beyond which the steric mystery. Mechanisms such as additional binding interactions
hindrance is as strong as both steric and binding influences have been proposed, which is not unlikely considering that
combined. AMPARs have numerous auxiliary subunits (Tomita et al., 2003;
Cho et al., 2007; Soto et al., 2009; Kalashnikova et al., 2010;
DISCUSSION von Engelhardt et al., 2010; Erlenhardt et al., 2016) that can
bind to various scaffolding proteins. However, even a cytosolic
Using simultaneous single-molecule tracking and localization domain composed of just a GFP-type molecule is slowed within
microscopy enabled by uPAINT and PALM, we demonstrated the synapse (Li et al., 2016). Thus, we propose a more general
that the subsynaptic regional density of a scaffold protein PSD-95 mechanism that likely applies not only to glutamate receptors
can stabilize the surface membrane diffusion and positional but also to other TM proteins critically important for synaptic
organization of a single-pass transmembrane protein probe. function. In this model, receptor-scaffold binding is a ticket
The denser the regional density of PSD-95, the slower was to entry and exit; PSD morphing redistributes even bound

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Li and Blanpied PALM-PAINT of Diffusion in the Postsynaptic Density

receptors within the synapse; and macromolecular crowding in with sites of neurotransmitter release, as indicated by their
combination with binding stabilizes the receptors at subsynaptic transsynaptic alignment with RIM1/2 molecules which in
domains highly packed with other proteins important for turn correlate with presynaptic vesicle fusion locations (Tang
synaptic transmission. et al., in press). Thus, we speculate that the enhanced
Interestingly, though the TM binding probe is much smaller crowding within these high-density subdomains will slow
than AMPARs, its distribution of Deff within the synapse and help limit the escape of receptors from points in the
appeared as slow as, or even slower than, AMPAR diffusion synapse where they are most likely to be activated during
measured in other studies using dye-conjugated antibodies or neurotransmission.
quantum-dot-conjugated nanobodies (Nair et al., 2013; Cai et al., Crowding within high-density subdomains is likely not
2014). It is possible that these various labeling approaches due only to postsynaptic scaffolding proteins. Indeed,
preferentially sample receptors that exit the synapse and diffuse transmembrane adhesion molecules, which associate with
more freely in the perisynaptic space, which may tend to one another across the synaptic cleft, will enhance crowding
obscure real differences in the relative numbers of low-Deff further in the extracellular, transmembrane, and intracellular
molecules that occupy the synapse for long periods. On the domains. The distribution of these adhesion molecules may
other hand, taken at face value, the similarity is consistent regulate the alignment between neurotransmitter receptors and
with our previous report using sptPALM that a substantial release sites, though this is not known. The TM probes employed
and nearly identical fraction of mEos-tagged AMPARs and here share commonalities with some of the intercellular adhesion
binding probes diffused with Deff < 0.02 µm2 /s (Li et al., molecules in size (e.g., single-pass TM proteins) or the ability
2016), further supporting the notion that much of the synapse to bind PSD-95, yet synaptic adhesion proteins display quite
is so crowded it stabilizes and organizes both large and small divergent patterns of expression within the synapse. SynCAM
TM proteins. is distributed in clusters surrounding the border of PSD-95
Previously, we demonstrated using partial synapse molecules (Perez de Arce et al., 2015), as are some members
Fluorescence Recovery after Photobleaching (FRAP), of the cadherin/catenin system (Uchida et al., 1996). On the
smtPALM alone, and uPAINT alone that the protein bulk other hand, neuroligin1 and LRRTM2 are inside the synapse and
of the TM probe decreased its diffusion within the synapse. distributed in clusters reminiscent of PSD-95 (Chamma et al.,
However, previous approaches were limited in ability to 2016).
assess how the complex structure within the PSD could Surprisingly, LRRTM2, though smaller in both extracellular
influence the mobility and organization of TM proteins in and intracellular length than neuroligin1, in fact diffuses slower
a living synapse. Results from PALM-PAINT indicate that a than neuroligin1 (Chamma et al., 2016). Moreover, LRRTM2
particular degree of postsynaptic crowding, which we estimate is more compactly distributed and in the synaptic center than
as 5000 molecules/µm2 , can be sufficient to stabilize TM the larger neuroligin1, suggesting that LRRTM2 is more likely
protein diffusion in the absence of binding. This density associated with dense regions of PSD-95 which are often found
translates to an average ∼14 nm inter-PSD-95 distance, very near the center of the PSD (MacGillavry et al., 2013; Tang
similar to the mean nearest-neighbor distance of ∼13 nm et al., in press). This paradoxical result confirms that the
between the ‘‘vertical filaments’’ corresponding to PSD-95 arrangements of TM proteins cannot be predicted by their
as measured in EM tomography (Chen et al., 2008). Note bulk alone. Interestingly, however, AMPARs associate with
that we deduce this as an average spacing, but could not LRRTM2 through their extracellular domains (de Wit et al.,
directly measure it around individual moving probes. The 2009), potentially regulating the mobility of each protein by
similarity between these values suggests that rather subtle the addition of further bulk and protein-protein interactions.
variations in scaffold density across the lateral extent of the Further experiments are needed to tease out how the various
synapse could change TM protein mobility substantially. This interactions on diverse synaptic TM protein species dictate one
high density packing is similar to what has been measured another’s spatial arrangement.
for AMPA receptors (e.g., 2000–4000/µm2 , see Levet et al., The diffusion of TM proteins appeared complicated outside
2015). Indeed, receptor-scaffold binding may facilitate the of the synapse particularly within few hundred nanometers from
assembly of this tight packing. Though the fractional time the border of the PSD. In some cases, the nonbinding probe
synaptic AMPARs spend bound to PSD-95 is not known, moves over a large area in the extrasynaptic regions, but in other
macromolecular crowding is likely to augment maintenance cases, they can appear confined or immobilized as the binding
of this architecture once assembled, because receptors in probes. Proteins other than scaffolding proteins can certainly
crowded areas that dissociate from scaffolds will face a longer affect the mobility of these probes outside the synapse. Some
escape time from the region and thus are more likely to factors could affect both probes roughly equally: regions with
rebind PSD-95. high density of endocytic adaptor molecules (Blanpied et al.,
If domains of high PSD-95 density tend to accumulate 2002; Petralia et al., 2003; Racz et al., 2004), zones of dense
not only probes such as used here but also receptors, cortical cytoskeleton (He et al., 2016), sites of plasma membrane-
then their impact on synaptic transmission will depend on ER apposition (Spacek and Harris, 1997). In addition, puncta
where they are with respect to sites of neurotransmitter adherens or clusters of adhesion molecules (Perez de Arce et al.,
release (MacGillavry et al., 2011). Recently, we have found 2015) may slow transit of all TM proteins. Less frequently,
that nanoclusters of PSD-95 frequently align transsynaptically undetected regions could exist that would selectively affect the

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Li and Blanpied PALM-PAINT of Diffusion in the Postsynaptic Density

binding probe. For example, small and low-density regions below alignment of the uPAINT and PALM data was subject to residual
the PSD detection criteria (<10 molecules and <30 nm in errors that may have diminished a larger underlying effect. The
diameter) could be situated within nanometers of the detected two color channels faced an alignment error of ∼6 nm, which
PSD border. These could come from small segments of multi- would somewhat blur our measurement of regional PSD-95
segmented PSDs (Spacek and Hartmann, 1983; Stewart et al., density around individual tracked locations. In addition, the
2005), but these types of PSDs are rare. In addition, though uPAINT data is subject to error stemming from the finite
the density of PSD-95 outside the PSD border is quite low precision of individual localizations. The Atto647N we used
(Zhang and Diamond, 2009; Perez de Arce et al., 2015), its for tracking is a relatively bright organic dye and helps to
extrasynaptic mobility has not been measured and there may maximize this precision and thus minimize error in the estimate
be enough PSD-95 in the perisynaptic region to bind and of Deff . However, brighter, longer-lasting fluorophores could
immobilize the binding probes. We speculate that the overall be advantageous. Nanobody-labeled small quantum dots (Wang
extrasynaptic diffusion of both probes are not different because et al., 2014) have been used to track AMPARs in and around
few extrasynaptic regions selectively affect the binding probe but synapses, and have the additional advantage of being so bright
not the nonbinding probe. as to facilitate tracking in 3D (Cai et al., 2014). However, 3D
mapping of the PSD would require high localization numbers
and longer imaging durations (Legant et al., 2016; and see below),
Role of Crowding in Synaptic Plasticity
and the z resolution normally obtainable without 4pi detection is
The many types and time scales of ongoing and triggered
usually worse than 100 nm for fluorescent proteins, making this
PSD plasticity that have been documented (Okabe et al., 1999;
difficult to implement.
Blanpied et al., 2008; MacGillavry et al., 2013; Bosch et al.,
In our application of PALM-PAINT, there was only limited
2014) suggest that PSD reorganization during plasticity will
temporal relationship between individual tracks (generally
affect accumulation not just of TM proteins like AMPARs
lasting <1 s and the PALM map (aggregated over the imaging
but additional molecules contributing to crowding as well. For
session of generally 4–6 min)). Though lateral drift was corrected
instance, PSD-95 content in spines has been shown to decrease
during this time (to an error we estimated as <10 nm), ongoing
transiently after an LTP induction protocol in hippocampal slice
morphing and internal reorganization of the PSD (Kerr and
cultures (Steiner et al., 2008). This may directly lead to loss of
Blanpied, 2012; MacGillavry et al., 2013) presumably degraded
AMPARs as their binding partners are lost. However, the loss
many details of the PSD-95 distribution in our final images.
of PSD-95 may decrease crowding, which could prompt a net
The reduced precision in capturing the true regional density
loss of even TM proteins with minimal direct binding to PSD-95
of PSD-95 molecules would diminish the difference we saw
(e.g., desensitized AMPARs uncoupled from stargazin Constals
between probes in different regional densities, and also reduce
et al., 2015). This further loss of molecular crowders could then
the difference between binding and nonbinding probe. Further,
further facilitate receptor exit. However, whether the transient
probes in a similar subsynaptic space but tracked early vs. late
loss of spine PSD-95 reflects a disruption of high-density areas
in the mapping might have not truly experienced the same
of the synapse is unknown.
degree of steric hindrance. However, the differences we observed
On the other hand, speculating further, if the transient
were robust even in the face of these errors. Ideally, to capture
decrease in synaptic PSD-95 during LTP induction reflects
the true effect size, one would need to monitor lateral drift
primarily loss from the PSD edge, and thus is not correlated
continuously (Bon et al., 2015) and achieve more rapid mapping
with significant de-crowding at high-density regions, then
(Huang et al., 2013). However, in structures with low protein
the continued presence of additional nonbinding proteins at
copy number, a large fraction of the proteins must be mapped
these high-density regions could in fact obstruct the entry of
to achieve statistical reliability (MacGillavry et al., 2013; Legant
AMPARs to these regions and limit the changes in AMPARs
et al., 2016), which may preclude time-lapse imaging except if the
level, at least over certain kinetic phases. Thus, it would be
protein exchange rate is high compared to the photobleaching
tempting to speculate in this case that the nonbinding proteins
rate induced by imaging.
could affect LTP induction kinetics but not maintenance, as
We hope the combined approach of PALM-PAINT will
morphing dynamics and internal mixing of the PSD would
help answer many key questions regarding synapse architecture
eventually enable synapses to reach their new steady state
and plasticity. One key issue is what mechanisms assemble
capacity of AMPARs on a time scale of minutes. Resolving these
the particular organization of PSD-95, a pattern that appears
many possibilities in the future will require close examination
to dictate receptor number and position (Opazo et al., 2012).
of the kinetics of protein redistribution and exchange during
One possibility is that the more deeply positioned multi-domain
plasticity.
proteins in the PSD, such as the Shank and GKAP families
(Valtschanoff and Weinberg, 2001; Dani et al., 2010), may
Advantages and Disadvantages establish a platform of loose spacing with which the more
of PALM-PAINT superficial proteins such as PSD-95 may interact (Chen et al.,
PALM of the PSD border improves discrimination of those 2008). Interestingly, in this case, a close interaction of the
molecules definitively within the synapse proper. However, deeper PSD with cytoskeleton (Frost et al., 2010; MacGillavry
we suspect that the effect of crowding may have been et al., 2016) may thus provide a link between activity-dependent
underestimated in our analysis because spatial and temporal plasticity of spine and PSD structure. Alternatively, cleft-resident

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Li and Blanpied PALM-PAINT of Diffusion in the Postsynaptic Density

adhesion molecules have distinct organizational patterns (Perez FUNDING

de Arce et al., 2015; Chamma et al., 2016), that may guide
intracellular protein organization in both the presynaptic and This work was supported by the National Institutes of
postsynaptic cells. Dissecting these possibilities, which require Health Grant F30MH102891 to TPL and R01MH080046 and
nanoscale resolution of position and mobility of multiple R01MH096376 to TAB and the Kahlert Foundation to TAB.
proteins, may be aided by future PALM-PAINT applications.
ACKNOWLEDGMENTS
AUTHOR CONTRIBUTIONS
We thank Sridhar Raghavachari and members of the Thompson,
TPL and TAB designed research; TPL performed research and Jurado, and Blanpied laboratories helpful discussion, and
data analysis; TPL and TAB wrote the article. Minerva Contreras for help with cultures.

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1126/science.1184178
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 REVIEW
published: 28 July 2016
doi: 10.3389/fnsyn.2016.00020

The Emergence of NMDA Receptor

Metabotropic Function: Insights
from Imaging
Kim Dore 1 , Jonathan Aow 2 and Roberto Malinow 1 *
1
Center for Neural Circuits and Behavior, Department of Neuroscience and Section for Neurobiology, Division of Biology,
University of California at San Diego, San Diego, CA, USA, 2 Genome Institute of Singapore, Singapore, Singapore

The NMDA receptor (R) participates in many important physiological and pathological
processes. For example, its activation is required for both long-term potentiation (LTP)
and long-term depression (LTD) of synaptic transmission, cellular models of learning and
memory. Furthermore, it may play a role in the actions of amyloid-beta on synapses
as well as in the signaling leading to cell death following stroke. Until recently, these
processes were thought to be mediated by ion-flux through the receptor. Using a
combination of imaging and electrophysiological approaches, ion-flux independent
functions of the NMDAR were recently examined. In this review, we will discuss the
role of metabotropic NMDAR function in LTD and synaptic dysfunction.
Keywords: ion-flux independent, FRET-FLIM, long-term depression (LTD), NMDAR interactions with CaMKII,
amyloid-beta induced depression, PP1, excitotoxicity

INTRODUCTION
Transmembrane receptors have traditionally been divided into two classes: ionotropic and
Edited by: metabotropic. Ionotropic glutamate receptors (iGluRs) form channels that allow the passage of
George Augustine, ions into the cell to drive signaling, while metabotropic glutamate receptors (mGluRs) generate
Nanyang Technological University, downstream effects without ion-flux. The boundary between these two classes is not completely
Singapore distinct, as there has been evidence that several iGluRs are capable of producing effects in the
Reviewed by: absence of ion-flux. For example, the N-terminal domain of GluA2, a subunit of the AMPA
Kei Cho, receptor (AMPAR), is sufficient to promote spine formation in hippocampal neurons (Passafaro
University of Bristol, UK
et al., 2003). Another iGluR, the kainate receptor, can modulate GABA transmission without
William N. Green,
University of Chicago, USA ion-flux (Rodríguez-Moreno and Lerma, 1998).
The NMDA receptor (NMDAR), a member of the iGluR family, is ubiquitously expressed
*Correspondence:
Roberto Malinow
and plays numerous roles in the brain (Traynelis et al., 2010). Given its ability to conduct
[email protected] calcium ions (Ca2+ ) well, it has been assumed that downstream signaling triggered by NMDARs
was mediated by Ca2+ influx and increased cytoplasmic Ca2+ . However, to allow Ca2+ entry
Received: 25 April 2016 through the receptor, several conditions have to be fulfilled: (1) glutamate must bind to
Accepted: 06 July 2016 GluN2 subunits; (2) glycine, the co-agonist must bind to GluN1 subunits; and (3) neurons
Published: 28 July 2016 must be sufficiently depolarized to eliminate the voltage-dependent magnesium ion (Mg2+ )
Citation: block of the channel. During high-frequency stimulation (HFS) these three conditions are
Dore K, Aow J and Malinow R (2016) met resulting in long-term potentiation (LTP; Bliss and Collingridge, 1993). For long-term
The Emergence of NMDA Receptor
depression (LTD), however, the role of the NMDAR is not as clear. A long-standing model
Metabotropic Function: Insights
from Imaging.
has proposed that while LTP requires a large increase in cytoplasmic Ca2+ , a moderate rise
Front. Synaptic Neurosci. 8:20. in cytoplasmic Ca2+ would produce LTD (Lisman, 1989; Malenka, 1994). However, several
doi: 10.3389/fnsyn.2016.00020 recent studies indicate that NMDARs can induce LTD without ion-flow through the receptor

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Dore et al. NMDA Receptor Metabotropic Function

(Nabavi et al., 2013; Dore et al., 2015; Stein et al., 2015; Carter and during chemical LTD induction. This ligand-driven decrease
Jahr, 2016). Other publications have shown that excitotoxicity in FRET required NMDAR conformational movement but
as well as amyloid-beta-induced synaptic depression depend not PP1 activity (Aow et al., 2015). It is possible that
on NMDAR activity but are likewise independent of ion-flow the transient movement of PP1 relative to the NMDAR
(Kessels et al., 2013; Tamburri et al., 2013; Birnbaum et al., 2015; cytoplasmic domain exposes the catalytic active site of PP1
Weilinger et al., 2016). In this review, we will discuss how these to a target unavailable under basal conditions. One potential
studies probed NMDAR metabotropic activity with an emphasis target is calcium-calmodulin dependent protein kinase II
on the imaging techniques used. (CaMKII; Strack et al., 1997), which is recruited to the
NMDAR complex during LTP stimuli (Otmakhov et al.,
NMDAR-DEPENDENT LTD CAN BE 2004) and whose activity is required for both LTP (Malenka
INDUCED INDEPENDENTLY OF ION-FLUX et al., 1989; Malinow et al., 1989; Silva et al., 1992) and
LTD (Coultrap et al., 2014). By monitoring FRET between
Interestingly, evidence for ion-flux independent LTD can fluorescently-tagged GluN1 and CaMKII, a delayed decrease
be observed in data from older literature. Over 20 years in the NMDAR-CaMKII interaction was observed during ion-
ago, data were published indicating that MK-801, which flux independent LTD (Aow et al., 2015). This effect depended
blocks NMDAR channels, blocked LTP but failed to block on PP1 activity and was not seen with a CaMKII mutant
LTD (Mayford et al., 1995). A similar effect was obtained that cannot be dephosphorylated at Thr-286 (CaMKII-Thr-
by a different group (Scanziani et al., 1996). Surprisingly, 286-Asp), suggesting that dephosphorylation of Thr-286 is
these observations were not discussed in either study. The necessary to modify the NMDAR-CaMKII interaction (Aow
ion-flux dependence of LTD was recently examined more et al., 2015). Co-immunoprecipitation experiments additionally
closely (Nabavi et al., 2013). Low-frequency stimulation revealed that the amount of total CaMKII bound to the NMDAR
(LFS) produced LTD in the presence of either MK-801 or was unaffected by ion-flow independent LTD, whereas levels
7-chloro-kynurenate (7CK, a competitive GluN1 antagonist; of phosphorylated Thr-286 were reduced both during and
see Figure 1A) but not APV (a competitive GluN2 antagonist); after LTD induction (Aow et al., 2015). These results are
all three antagonists effectively blocked ion-flux through the consistent with a model for ion-flux independent LTD in which
NMDAR. Moreover, LTD was observed in experiments in which glutamate binding to the NMDAR induces a conformational
intracellular Ca2+ was clamped to basal levels, suggesting that change in the NMDAR intracellular domain that facilitates
a rise in intracellular Ca2+ is not required for LTD. It was PP1 access to and dephosphorylation of CaMKII at Thr-286,
thus proposed that glutamate binding to the NMDAR could thereby repositioning the CaMKII holoenzyme within the
induce a conformational change in the cytoplasmic domain NMDAR complex. The relocated CaMKII could in turn
of the NMDAR that triggers downstream signaling resulting potentially act on a novel site of the GluA1 subunit (Ser-567)
in LTD. that undergoes phosphorylation during LTD (Coultrap et al.,
To test if ligand binding could drive movement of the 2014). Consistent with this model, CaMKII phosphorylation of
NMDAR intracellular domain, FRET-FLIM [Forster resonance GluA1-Ser-567 does not require Ca2+ or calmodulin (Coultrap
energy transfer measured by fluorescence lifetime imaging of the et al., 2014). Ultimately, this process could increase AMPAR
FRET donor, see Toolbox and (Wallrabe and Periasamy, 2005; endocytosis (Lüscher et al., 1999; Lin et al., 2000; Kim
Yasuda, 2006)] was employed (Dore et al., 2015). Recombinant et al., 2001; Shi et al., 2001) and lead to depressed synaptic
GluN1 subunits of the NMDAR were tagged with GFP or transmission.
mCherry at their carboxyl(c)-terminus and co-expressed in Ion-flow independent NMDAR activation of downstream
neurons. As the magnitude of FRET is very sensitive to signaling pathways has also been linked to shrinkage of
the distance and orientation of the interacting fluorophores, dendritic spines (Stein et al., 2015). Stein et al. used 2-
nanometer-scale changes in distance can be reliably detected. photon laser scanning microscopy (TPLSM) to monitor
Bath application or uncaging of glutamate in the presence structural changes in the dendritic spines of GFP-
of MK-801 or 7CK, but not APV, produced a transient expressing hippocampal neurons. Low-frequency uncaging
change in FRET consistent with conformational movement of glutamate produced a ∼20% decrease in spine size
of the NMDAR cytoplasmic domain (Figure 1B). Infusing that was independent of ion-flow through the NMDAR
neurons with a GluN1 c-terminus antibody through a patch (Figure 1C). While high-frequency glutamate uncaging
pipette blocked the ligand-driven FRET change as well as LTD, produced an increase in spine volume, the same stimulus
suggesting that this conformational change is required for LTD in the presence of either 7CK or MK-801 led to spine
induction. shrinkage. This result is consistent with the finding that
Downstream signaling events were also examined using HFS (delivered electrically) in the presence of MK-801
FRET-FLIM (Aow et al., 2015). Protein-phosphatase 1 (PP1) produces LTD instead of LTP (Nabavi et al., 2013). Spine
is one of the first molecules whose activity was shown shrinkage was also abolished when p38 MAPK activity
to be required for LTD (Mulkey et al., 1993) and it was blocked (Stein et al., 2015), which is again consistent
co-immunoprecipitates with the NMDAR complex (Husi with the observation that levels of phosphorylated p38
et al., 2000). FRET between GluN1-GFP and PP1-mCherry, (which is the active form) were increased during ion-flow
observed in baseline conditions, was transiently reduced independent LTD (Nabavi et al., 2013). In the future, it will

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Dore et al. NMDA Receptor Metabotropic Function

be important to elucidate how the initial movement in the antagonists were typically acutely washed in and then out
NMDAR cytoplasmic domain subsequently affects signaling of the preparation during LTD induction (Volianskis et al.,
molecules, such as cofilin, calcineurin and p38, implicated 2015), instead of being present throughout the experimental
in LTD. duration. Furthermore, control pathways, which monitor
An ion-flux-independent mechanism for NMDAR- transmission onto the same neurons but do not receive the
dependent LTD has been challenged by some recent studies conditioning stimulus, were generally not included (Babiec
(Babiec et al., 2014; Volianskis et al., 2015; Sanderson et al., et al., 2014; Volianskis et al., 2015; Sanderson et al., 2016). These
2016) but confirmed by others (Kim et al., 2015; Stein differences in methodology are significant and can make an
et al., 2015; Carter and Jahr, 2016). It is notable that the impact in the outcome and interpretation of results (Nabavi
experimental conditions used in these recent studies that failed et al., 2014). Therefore, it will be important to compare carefully
to detect ion-flux-independent LTD were not identical to the experimental conditions employed by different studies.
those supporting this form of LTD. For instance, NMDAR Nevertheless, it remains possible that two different, independent

TOOLBOX | FRET measurements using fluorescence lifetime imaging microscopy.

FRET is a non-radiative energy transfer mechanism between two fluorescent molecules. There are two main requirements for successful FRET. First the fluorescence
emission of the FRET donor must overlap with the FRET acceptor absorption spectrum; and second, these fluorescent molecules must be no more than ∼10 nm
apart from each other (Lakowicz, 2006). This spatial requirement of FRET is very sensitive; it can thus be used as a “molecular ruler” to assess protein structure
(Gustiananda et al., 2004) or to monitor subtle conformational changes (Dore et al., 2015).

FRET can be measured by acquiring a series of images in different combinations of excitation and emission channels or by photobleaching of the FRET acceptor.
However, these methods are prone to errors and are generally not well suited for measurements in living cells expressing fluorescent proteins (Selvin, 2000;
Yasuda, 2006; Piston and Kremers, 2007). Fluorescence lifetime is defined as the average time a molecule stays in its excited state before emitting a photon (A).
Because lifetime is an intrinsic property of fluorophores, it is independent of experimental conditions such as concentration, excitation intensity and photobleaching
(Lakowicz, 2006; Yasuda, 2006). Importantly, by adding an additional route for the donor fluorophore to return to ground state, the degree of FRET makes the
fluorescence lifetime of the donor proportionally shorter (A,B). To measure fluorescence lifetimes, the most common approach is time correlated single photon
counting (TCSPC; Becker et al., 2004) which calculates the time delay between the detection of fluorescence photons and laser excitation pulses (B). When TCSPC is
combined with laser scanning microscopy, it is possible to obtain fluorescence lifetimes, and hence detect changes in donor-acceptor distances, at every pixel of an
image (B,C).

FRET-FLIM. (A) Jablonski diagram of FRET donor and acceptor energy levels. After excitation by a 1-photon (blue arrow) or 2-photon (brown arrows) laser,
the FRET donor can return to ground state by emitting a photon (green arrow) or by transferring its energy to a nearby acceptor (dashed green arrows). (B) The
fluorescence lifetime of the FRET donor, which becomes shorter with increased proximity of the FRET acceptor, is calculated with the fluorescence decay curve.
(C) Time-correlated single photon counting (TCSPC) records fluorescence decay curves for each pixel of an image. By fitting these curves, the fluorescence lifetime
of the FRET donor can be assessed. The FLIM image is then color coded according to the FRET donor lifetime at each pixel.

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Dore et al. NMDA Receptor Metabotropic Function

FIGURE 1 | Ion-flux independent long-term depression (LTD). (A) APV, but not 7-chloro-kynurenate (7CK), blocks LTD. LTD (1 Hz, black bar) was delivered to
the test pathway (black symbols), while the control pathway (red symbols) received no stimulus; the slice was incubated in APV (top) or 7CK (bottom) throughout the
experiment. Note that there was no change to the control or test pathway in APV, as expected, whereas the test pathway was significantly depressed after LTD
induction in 7CK; representative of N = 15, APV, p > 0.05 LTD; N = 15, 7CK, p < 0.01 LTD. Insets: superimposed traces obtained before and after LTD in the test
(black line) and control (red line) pathways. Scale bars 0.3 mV, 5 ms. Modified from Nabavi et al. (2013). (B) Chemical LTD induction (+NMDA) resulted in spines that
exhibit less Förster Resonance Energy Transfer (FRET; and higher GFP fluorescence lifetimes; ps = picoseconds) between the GFP- and mCherry-tagged GluN1
subunits (see Toolbox ), indicating movement of the NMDAR cytoplasmic domain. Modified from Dore et al. (2015). (C) A low frequency glutamate uncaging
protocol (LFU), similar to the 1 Hz electrical stimulation in (A), induced spine shrinkage both in vehicle or 7CK-incubated GFP-expressing neurons, indicating that
ion-flux through the NMDAR is not required for spine shrinkage. Modified from Stein et al. (2015).

forms of NMDAR-dependent LTD exist: one that requires of glycine was sufficient to prime NMDARs for subsequent
ion-flow through NMDARs and one that does not. Different use-dependent endocytosis, again leading to a decline in
experimental conditions could selectively recruit either of these NMDA currents (Nong et al., 2003). Moreover, synaptic
two forms. replacement of GluN2B- with GluN2A-containing NMDARs, an
important developmentally controlled process (Hestrin, 1992;
TRAFFICKING OF NMDAR IS REGULATED Monyer et al., 1994; Sheng et al., 1994; Stocca and Vicini,
BY SYNAPTIC ACTIVITY BUT NOT 1998; Tovar and Westbrook, 1999), required ligand binding
ION-FLUX without ion flux (Barria and Malinow, 2002). Interestingly,
the replacement of synaptic GluN2B-containing NMDARs by
In addition to its more recently described role in LTD, a newly synthetized GluN2B-containing NMDARs did not require
few older studies have indicated that ligand binding to the ligand binding. It is important to note that these effects
NMDAR, in the absence of ion-flux, could control NMDAR on NMDAR trafficking required both agonist and co-agonist
trafficking (Vissel et al., 2001; Barria and Malinow, 2002; binding to NMDARs as they were blocked by antagonists
Nong et al., 2003). The Westbrook lab showed that even to the glutamate binding site on GluN2 subunits or to the
with its pore blocked, ligand binding to the NMDAR drove glycine binding site on GluN1 subunits (Vissel et al., 2001;
tyrosine dephosphorylation of GluN2A subunits, resulting Barria and Malinow, 2002; Nong et al., 2003); in contrast, LTD
in NMDAR endocytosis and decreased NMDA currents. only requires ligand binding to GluN2 subunits (Nabavi et al.,
Another group separately observed that an initial application 2013).

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Dore et al. NMDA Receptor Metabotropic Function

FIGURE 2 | Metabotropic NMDAR activity can induce synaptic dysfunction. (A) APP-CT100 overexpression depresses synaptic transmission without ion-flux.
Model figure for a dual whole-cell recording of an infected and a neighboring uninfected CA1 neuron (top left) and an example trace of evoked AMPA receptor
(AMPAR) currents of such a recording (top right). Bottom, results from paired-recordings performed in ACSF containing no drug (N = 41), Ro 25-6981 (N = 37) or
MK-801 (N = 25); ∗∗ indicates p < 0.001. Modified from Kessels et al. (2013). (B) Spine density is reduced in a transgenic mouse model of Alzheimer’s disease (AD),
APV blocks the effect but not memantine. Scale bar, 5 µm. Modified from Birnbaum et al. (2015). (C) Sustained NMDA application causes excito-toxic insults to CA1
neurons in the form of blebbing of dendrites (left panels). This effect is blocked by APV and CGP-78608 (middle panels) but not MK-801 (right panels). Scale bar,
25 µm. Modified from Weilinger et al. (2016).

METABOTROPIC NMDAR ACTIVITY CAN shown using TPLSM or confocal microscopy that endogenously
INDUCE SYNAPTIC DYSFUNCTION expressed or exogenously applied amyloid-beta reduces spine
density in GFP-expressing neurons (Hsieh et al., 2006; Shrestha
Recent results have suggested a role for metabotropic NMDAR et al., 2006; Calabrese et al., 2007; Shankar et al., 2007; Wei
activity in amyloid-beta mediated synaptic dysfunction, which et al., 2010; Zempel et al., 2010), which may explain the
may contribute to hippocampal deficits in Alzheimer’s disease electrophysiologically observed decreased frequency of miniature
(AD) and precede neurological symptoms by a decade or excitatory postsynaptic currents (Kamenetz et al., 2003; Hsieh
more (Terry et al., 1991; Reiman et al., 1996). A number et al., 2006). Therefore, amyloid-beta induces synaptic insults
of studies using electrophysiology and imaging have reported that can be directly observed through imaging.
that amyloid-beta impairs LTP, depresses synaptic transmission A mechanism proposed to account for synaptic impairment
and induces synapse loss in various hippocampal preparations by amyloid-beta is enhanced ionotropic glutamate receptor
(Chapman et al., 1999; Larson et al., 1999; Walsh et al., 2002; endocytosis. Indeed, there is evidence that inhibiting endocytic
Wang et al., 2002; Kamenetz et al., 2003; Snyder et al., 2005; signaling pathways or overexpressing mutant endocytic-resistant
Hsieh et al., 2006; Shankar et al., 2007; Wei et al., 2010; receptors can ameliorate the reduction in AMPAR and/or
Birnbaum et al., 2015). The effect of intracellularly delivered NMDAR currents (Snyder et al., 2005; Hsieh et al., 2006; Knafo
amyloid-beta is not clear, as one report indicated synaptic et al., 2016). Notably, Kessels et al. (2013) reported that despite
depression (Ripoli et al., 2014) while another indicated synaptic block of ion flux, not all NMDAR antagonists prevented amyloid-
potentiation (Whitcomb et al., 2015). In many of these studies beta-induced depression of AMPAR-mediated transmission.
the electrophysiological results could be corroborated using The GluN2 antagonists (R)-CPP, Ro25-6981, and ifenprodil
imaging. For instance, Wei et al. (2010) used TPLSM to show afforded a complete block; whereas the GluN1 antagonist
that GFP-filled dendritic spines close to axons or dendrites 7CK and the NMDAR pore blocker MK-801 had no effect
overexpressing amyloid-beta displayed a smaller increase in (Figure 2A; Kessels et al., 2013). Thus, the block of depression
spine volume following a chemically-induced LTP protocol correlated with actions on different NMDAR subunits rather
as compared to more distant spines, suggesting that secreted than block of ion-flux. In another model, amyloid-beta
amyloid-beta impaired LTP. Hsieh et al. (2006) used TPLSM of oligomers exogenously applied to organotypic hippocampal
AMPARs tagged with the pH-sensitive GFP-variant SEP (Super- slices acutely depressed AMPAR-mediated transmission in
Ecliptic-Phluorin) to measure surface AMPARs and found that a manner that was dependent on synaptic stimulation and
amyloid-beta reduced surface GluA1 and GluA2. Likewise, NMDAR activation but not NMDAR ion-flux (Tamburri
immunostaining and imaging primary cultures treated with et al., 2013). Both studies therefore suggest that amyloid-
amyloid-beta revealed a reduction in surface NMDARs (Snyder beta activates a metabotropic NMDAR signaling pathway that
et al., 2005) and AMPARs (Almeida et al., 2005; Alfonso et al., depresses synaptic transmission. The evidence that this pathway
2014). The decrease in synaptic AMPAR and NMDAR currents could then be involved in eventual spine loss comes from
correlates, therefore, with a decrease in surface receptors as three other studies using imaging techniques. Two studies
determined with optical techniques. Finally, several groups have (Shankar et al., 2007, 2008) showed using TPLSM that (R)-CPP

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Dore et al. NMDA Receptor Metabotropic Function

FIGURE 3 | Model of NMDAR metabotropic functions. In baseline conditions, the NMDAR interacts with both protein-phosphatase 1 (PP1) and
calcium-calmodulin dependent protein kinase II (CaMKII) which is phosphorylated at Thr286. Upon ligand binding, movement in the NMDAR cytoplasmic domain
permits PP1 catalytic access to phospho-CaMKII-T286, this movement also inducing activation and phosphorylation of p38 MAPK. These signaling molecules (along
with others) will eventually lead to AMPAR removal. In the context of LTD, these events will produce spine shrinkage whereas in the case of sustained amyloid-beta
presence, spine elimination will occur.

prevented amyloid-beta-induced spine loss in GFP-expressing receptor is responsible for inducing cell death (Choi, 1995;
organotypic slice neurons. Birnbaum et al. (2015) subsequently Tu et al., 2010). Interestingly, recent findings suggest that a
demonstrated that the competitive GluN2 antagonist APV metabotropic NMDAR ‘‘signalsome’’—involving the NMDAR,
also blocked spine loss in transgenic AD mice (as well as the pannexin-1 channel (Panx1) and src kinase—is capable
in hippocampal slices incubated in amyloid-beta oligomers), of inducing cellular dysfunction in response to excessive
whilst MK-801, memantine, another NMDAR pore blocker, NMDAR stimulation (Weilinger et al., 2016). TPLSM was
and buffering postsynaptic calcium ions with BAPTA had used to image fluorescently labeled CA1 neurons in acute
no effect (Figure 2B). That study (Birnbaum et al., 2015) rat hippocampal slices treated with a high dose of NMDA.
also showed an amyloid-beta-induced reduction in PSD-95 This protocol induced blebbing in the dendrites of CA1
and synaptophysin levels that was blocked by APV but not neurons as well as mitochondrial dysfunction, an effect that
by MK-801 or memantine. Moreover, they demonstrated was blocked by co-application of the GluN1 antagonist CGP-
that p38 MAPK phosphorylation was increased by amyloid- 78608 and APV, but not by MK-801, indicating that ligand
beta in a NMDAR ion-flux independent manner and that binding to the NMDAR was capable of damaging neuronal
spine loss depended on p38 MAPK activity (Birnbaum et al., morphology independently of ion flux through the receptor
2015), supporting a link, previously examined (Hsieh et al., (Figure 2C). Interestingly, Panx1 channels appear to be
2006), between LTD and amyloid-beta-induced depression. located almost exclusively at the PSD (Zoidl et al., 2007),
Taken together, these imaging results are in agreement with which suggests that this metabotropic NMDAR ‘‘signalsome’’
electrophysiological experiments and support the hypothesis is synaptic. Additional experiments demonstrated that ligand
that amyloid-beta toxicity depresses synaptic transmission binding, but not NMDAR ion flux, was necessary for
via metabotropic NMDAR signaling that results in eventual downstream activation of Panx1 (Thompson et al., 2006, 2008;
spine loss. Weilinger et al., 2012). Metabotropic NMDAR activity did
The role of NMDARs in mediating excitotoxicity has been not change the degree of interaction between GluN1 and
extensively described (reviewed in Choi, 1992), and it has Panx1, but it did increase Src kinase binding to GluN1,
been widely suggested that excessive Ca2+ influx through the Src activation, as well as Src-dependent phosphorylation

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Dore et al. NMDA Receptor Metabotropic Function

of Panx1. Peptides that either disrupted the GluN1-Src mechanism is engaged during LTD. Ion-flux independent LTD
interaction (Src48 ) or interfered with Panx1 phosphorylation appears to be mediated by a movement in the NMDAR
(Tat-Panx308 ) were neuroprotective in vitro, and injection of cytoplasmic domain that affects its interactions with at least
Tat-Panx308 reduced brain lesion volume in an in vivo model two signaling proteins, PP1 and CaMKII. Subsequent signaling,
of stroke. Indeed, as ischemia in the brain is believed to drive with increased p38 MAPK phosphorylation likely, leads to
subsequent excitotoxicity, these results suggest that targeting AMPAR removal, shrinkage of dendritic spines and depressed
Src or Panx1 in a clinical setting could be therapeutically synaptic transmission (Figure 3). Interestingly, it seems that
effective. if the stimulus recruiting metabotropic NMDAR function is
sustained, as in the contexts of amyloid-beta overproduction
CONCLUDING REMARKS or excitotoxic conditions following ischemia, metabotropic
NMDAR activity can also lead to synaptic and neuronal
A number of studies have provided evidence that physiological dysfunction.
and pathological processes can be triggered by ligand binding
to the NMDAR, without requiring flow of ions through AUTHOR CONTRIBUTIONS
its pore. It will be important to determine conditions that
control whether an ionotropic or metabotropic NMDAR KD, JA and RM wrote the manuscript. KD designed figures.

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 REVIEW
published: 23 August 2016
doi: 10.3389/fnsyn.2016.00028

Correlative Light Electron

Microscopy: Connecting Synaptic
Structure and Function
Isabell Begemann 1,2 and Milos Galic 1,2*
1
DFG Cluster of Excellence ‘Cells in Motion’, (EXC 1003), University of Muenster, Muenster, Germany, 2 Institute of Medical
Physics and Biophysics, University Hospital Münster, University of Muenster, Muenster, Germany

Many core paradigms of contemporary neuroscience are based on information obtained

by electron or light microscopy. Intriguingly, these two imaging techniques are often
viewed as complementary, yet separate entities. Recent technological advancements
in microscopy techniques, labeling tools, and fixation or preparation procedures
have fueled the development of a series of hybrid approaches that allow correlating
functional fluorescence microscopy data and ultrastructural information from electron
micrographs from a singular biological event. As correlative light electron microscopy
(CLEM) approaches become increasingly accessible, long-standing neurobiological
questions regarding structure-function relation are being revisited. In this review, we
will survey what developments in electron and light microscopy have spurred the
advent of correlative approaches, highlight the most relevant CLEM techniques that
are currently available, and discuss its potential and limitations with respect to neuronal
and synapse-specific applications.
Keywords: correlative light electron microscopy, CLEM, synapse, neuron, fluorescence microscopy, electron
microscopy, TEM, SEM
Edited by:
Marc Fivaz,
Duke NUS Graduate Medical School,
Singapore INTRODUCTION
Reviewed by:
Jeff Lichtman,
Neurons transmit the majority of trans-cellular signals via synaptic contacts. To correctly and
Harvard University, USA reliably respond to various stimuli (e.g., different input frequencies), each synapse hosts an
Christian Wilms, elaborate machinery to regulate signal transmission in a context-dependent manner. On the
Scientifica Ltd., UK molecular level, synaptic transmission in its most simple form relies on fusion of ∼40 nm large
*Correspondence: vesicles at the presynaptic site, diffusion of released neuro-transmitters across the 30 nm wide
Milos Galic synaptic cleft, and subsequent ligand-specific activation of receptors at the postsynaptic site.
[email protected] Considering the small size of this signaling unit, it is not surprising that major advancements in
understanding synaptic function have been closely correlated with progress in imaging technology.
Received: 23 March 2016 For instance, the notion that communication across synaptic clefts relies on chemical signals
Accepted: 12 August 2016 would not have been possible without detailed information from electron micrographs on synapse
Published: 23 August 2016
ultrastructure (Derobertis and Bennett, 1955; Pappas and Bennett, 1966) and vesicle dynamics
Citation: (Heuser and Reese, 1973). Likewise, our understanding about the function of individual synaptic
Begemann I and Galic M (2016)
proteins has substantially advanced with the introduction of ion and protein markers (Shimomura
Correlative Light Electron Microscopy:
Connecting Synaptic Structure
et al., 1962; Tsien, 1981; Heim et al., 1994) for fluorescence light microscopy. Intriguingly, and
and Function. despite the fact that today’s neuroscience is building up on information obtained with electron and
Front. Synaptic Neurosci. 8:28. light microscopy, strategies to combine these two approaches only recently have started gaining
doi: 10.3389/fnsyn.2016.00028 traction.

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Begemann and Galic CLEM in Synapses

The emergence of such dual approaches, termed correlative heavy elements (i.e., high atomic number) in the uncoated sample
light electron microscopy (CLEM), from a sparsely known can also be used to render images comparable to transmission
branch of imaging to center stage can be linked to a series electron micrographs of ultrathin sections, an approach that is
of landmark papers from the nineties (Deerinck et al., 1994; of particular relevance for embedded sections on an electron-
Svitkina et al., 1995). Since then, fueled by advances in imaging opaque surface or samples that are investigated ‘en block’
and labeling techniques, publications using CLEM approaches (Briggman et al., 2011).
have steadily been rising. In this review, we will revisit what A single cross-section, or the surface of the sample, is lacking
developments have incited the advent of correlative imaging information on the three-dimensional organization within the
approaches, and highlight the most relevant dual microscopy biological specimen. Here, cryo-fracture, where frozen samples
techniques that are currently available. We begin by giving an are broken to expose cell structures along the fracture line
overview of electron and light-based imaging methods that can be (Castejon and Caraballo, 1980; Castejon, 1996), or unroofing,
used for CLEM, followed by a section describing how individual where intracellular structures are uncovered by brief bursts of
approaches can be successfully combined to create correlative ultrasound (Lang, 2003), can be used together with deep-etching
approaches. Next, we will reflect on technical concerns that need (Heuser and Salpeter, 1979) to gain insights into subcellular
to be taken care of when designing dual imaging experiments, organization. While useful, these strategies lack the capability for
before closing with a discussion on current limitations and future systematic three-dimensional sample reconstruction that can be
challenges associated with CLEM. achieved with serial sectioning, where successive ultrathin slices
are imaged. Early attempts of serial sectioning reach back to
the late 60s (De Rosier and Klug, 1968), and have since then
ELECTRON AND LIGHT-BASED continuously been used to investigate neuronal networks (White
TECHNIQUES USED FOR CLEM et al., 1986) and synaptic connection (Mishchenko et al., 2010).
In modern serial section TEM (ssTEM), sections of 60 ± 20 nm
CLEM describes a continuously growing number of procedures are cut with an ultra-microtome and placed manually on a metal
that allow merging electron and light-based images from the support grid (Harris et al., 2006). More recently, automated tape-
same object. Thus, at least in theory, each existing electron and collecting ultra-microtome SEM (ATUM-SEM) has emerged as a
light microscopy technique where ultrastructure remains intact powerful alternative (Hayworth et al., 2006; Kasthuri et al., 2007,
could be paired to generate a CLEM image. To illustrate this 2015; Terasaki et al., 2013; Morgan et al., 2016). Here, sections
combinatorial potential, and to expound existing limitations, we of 30 nm are cut by an ultra-microtome and collected from the
begin this section by discussing electron and light microscopy water bath using a conveyor-belt like support tape. As the support
techniques suitable for correlative approaches, before proceeding tape is electron-opaque, a finely focused SEM beam is applied
to examples where CLEM was successful applied to study to raster the surface of the sample and backscattered electrons
neuronal and synaptic function. are used to reconstruct the image. Thus, compared to ssTEM,
ATUM-based strategies not only allow thinner sectioning and
Electron Microscopy–Visualizing the rapid imaging of larger areas but also substantially reduce errors
Ultrastructure associated with manual sample handling (Hayworth et al., 2014).
Electron micrographs in CLEM rely to a large extent on Alternatively, an embedded tissue block can also be sectioned
transmission electron microscopy (TEM) and scanning electron directly within the SEM vacuum chamber, either using a diamond
microscopy (SEM). TEM, which enables visualization of 50– knife (serial block-face SEM, SBEM; Leighton, 1981; Denk and
100 nm thick cross-sections of samples with a resolution of Horstmann, 2004) or by milling with a focused ion-beam (FIB-
down to a few Angstrom (Pierce and Buseck, 1974; Ruska, SEM; Watkins et al., 1986; Knott et al., 2008). In the latter, a
1987; Erni et al., 2009), was critically involved in the detailed scanning electron microscope is used to image the surface of
characterization of synaptic structures (Gray, 1959; Pappas and the embedded sample, while a high current focused ion beam
Bennett, 1966; Fifkova and Delay, 1982; Landis and Reese, 1983; continuously slices off sections perpendicular to the SEM axis,
Watanabe et al., 2013, 2014), and has helped to advance our thus allowing 3D reconstruction of the sample. Compared to
understanding on age and disease-dependent changes in synaptic diamond-based sectioning, FIB-SEM is thus not only faster but
properties (DeKosky and Scheff, 1990; Navlakha et al., 2013). also permits sectioning samples with a step size in the single nm
Contrary to TEM, where electron shadows are used to range (Knott et al., 2008; Merchan-Perez et al., 2009; Lehmann
create the image, SEM-based strategies utilizes the interaction et al., 2014).
of electrons with molecules in the sample to recreate the Another strategy for detailed three-dimensional
image. One commonly applied SEM strategy, where secondary reconstructions relies on electron tomography (ET). In this
electrons are used to determine the surface topography of objects approach, a tilt series (usually ranging from −60◦ to +60◦ )
with a precision of ∼1 nm (Vernon-Parry, 2000), was central of two-dimensional images is generated to reconstruct the
for investigating surface features such as the morphology of three-dimensional shape of an object within the single slice
neuromuscular junctions (Desaki and Uehara, 1981) as well as (Crowther et al., 1970; Hoppe et al., 1974; Penczek, 2010; Ercius
structural reorganization during exploratory dendritic filopodia et al., 2015). ET was successfully used to resolve ultrastructural
formation in hippocampal neurons (Galic et al., 2014). In features of the presynapse (Perkins et al., 2015) and synaptic
addition, back-scattered electrons created by interaction with vesicle populations in saccular hair cells (Lenzi et al., 1999). Yet,

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Begemann and Galic CLEM in Synapses

while ET is suitable for atomic-resolution, radiation damage, among others subcellular protein localization (Chalfie et al., 1994;
and limitations in interpretability for thicker sections need to be Heim et al., 1994; Kilgore et al., 2013), protein activity (Adams
considered when preparing the sample (Tocheva et al., 2010). et al., 1991), ion dynamics (Tsien, 1980, 1981; Tsien et al., 1982;
Finally, we would like to note that further electron-based Minta and Tsien, 1989; Nakai et al., 2001), pH (Tanasugarn
imaging strategies exist (e.g., STEM (Crewe et al., 1970; Engel, et al., 1984; Miesenbock et al., 1998), membrane potential or
1978), tSEM (Kuwajima et al., 2013)). While not commonly used voltage (Davila et al., 1973; Siegel and Isacoff, 1997; Zochowski
in CLEM studies, it is plausible to envision that some of these et al., 2000), or lipid species (Stauffer et al., 1998). Intriguingly,
additional types of electron microscopy may be beneficial for light is also suitable to actively manipulate the biological sample
a particular question. For readers interested in learning more in a precise spatio-temporal manner. One commonly used
on electron microscopy techniques, we recommend reading one approach relies on light-activation of caged substrates. Relevant
of the many excellent reviews available on this exciting topic for neurobiology, strategies for uncaging calcium (Ellisdavies and
(Briggman and Bock, 2012; Knott and Genoud, 2013; Miranda Kaplan, 1994), IP3 (Wang and Augustine, 1995), and various
et al., 2015; Perkins et al., 2015). neurotransmitters (Ellis-Davies, 2011) have been realized. More
recently, light has also been used to regulate protein function
(Cao et al., 2008; Tischer and Weiner, 2014). This approach,
Light Microscopy–Monitoring and coined optogenetics, has provided among others tools for
Manipulating Cell Function controlling neuronal and synaptic activity with unprecedented
While the electron microscopy techniques described above are spatio-temporal precision (Boyden et al., 2005; Fenno et al.,
well suited to investigate ultrastructural features with high 2011; Rost et al., 2015). Finally, it is worth mentioning that even
axial resolution, they lack spatio-temporal information available physical cell parameters, such as shape or membrane tension, can
with light microscopy. To increase penetration depth and be altered by light in living samples, using for instance optical
reduce background compared to whole field illumination (Epi), tweezers (Ashkin, 1970; Zhang and Liu, 2008). In summary,
CLEM studies frequently rely on confocal microscopy, where when combined with electron microscopy, these light-based
a pinhole is used to reduce out-of-focus light (Minsky, 1988), approaches allow precisely altering a specific parameter while
light sheet microscopy, where a light beam perpendicular to monitoring the cellular responses followed by analysis of the
the objective illuminates only the focal plane (Engelbrecht and corresponding ultrastructural features.
Stelzer, 2006; Keller et al., 2008), or two-photon microscopy,
where simultaneous absorption of two photons is used for
spatially controlled illumination (Denk et al., 1990). Resolution Correlative Light and Electron
in light and electron microscopy is based on numerical aperture Microscopy in Neuroscience
and wavelength. However, unlike in electron microscopy, where Although the potential of combining ultrastructural information
resolution is limited by the spot size (SEM) or the grain of the with functional studies was early noticed, first attempts to
detector (TEM), the limiting factors in light microscopy is the combine these microscopy techniques were limited to depicting
wavelength (Rayleigh, 1879; Abbe, 1883). To reach beyond the separately prepared and imaged biological samples (Porter et al.,
diffraction limit, several approaches have been introduced over 1945). Experiments where fluorescence and EM images of
the last decade. Among the most prominent super-resolution subcellular structures from the same cell were aligned started
microscopy techniques used in CLEM studies are stimulated appearing in the 1970s (Hollander, 1970; Nakai and Iwashita,
emission depletion microscopy (STED), where a depletion laser 1976), and have since then been applied to investigate a variety
limits the width of the emitting light source (Hell and Wichmann, of neurobiological questions.
1994; Klar et al., 2001), and stochastic techniques, such as To fully understand neuronal or synaptic function, the cellular
fluorescent photo-activation localization microscopy (PALM; context needs to be considered. It is thus not surprising, that large
Betzig and Chichester, 1993; Betzig et al., 2006) or stochastic efforts have been put into elucidating ultrastructural information
optical reconstruction microscopy (STORM; Rust et al., 2006), within the intact tissue. In situ CLEM, where data from life-
where light emitted from sequentially activated fluorophores is cell fluorescence imaging is combined with EM micrographs,
fitted to determine its precise localization. As before, we would has advanced our understanding of neuronal circuits (Bock
like to note that additional light-based approaches [e.g., total et al., 2011; Briggman et al., 2011; Lee et al., 2016), and
internal reflection fluorescence microscopy (Axelrod, 1981) or has also shed light on subcellular behaviors such as axosome
structured illumination microscopy (Neil et al., 1997)] can be shedding (Figure 1A) (Bishop et al., 2004). An alternative
used in CLEM approaches. Readers interested to learn more strategy, frequently applied to gain information of individual
about fluorescence microscopy techniques, we refer to one of the neurons in situ, relies on array tomography (AT), where plastic-
many excellent reviews focused on these topics (Lichtman and embedded tissue samples are sliced with an ultra-microtome,
Conchello, 2005; Combs, 2010; Huang et al., 2010). bonded array-wise onto a glass coverslip, stained, and finally
Fluorescence light microscopy not only allows to visualize cells imaged by fluorescence and electron microscopy (Figure 1B)
within neuronal circuits (Livet et al., 2007), but a continuously (Micheva and Smith, 2007; Rah et al., 2013; Collman et al.,
growing number of molecular probes and genetically encoded 2015). By combining super-resolution techniques and serial
markers also provide tools to study a variety of neuronal and sectioning, it was even possible to detect presynaptic dense
synaptic properties. Parameters that can be analyzed include projection proteins at a lateral resolution of 35–65 nm (Watanabe

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Begemann and Galic CLEM in Synapses

et al., 2011). While serial sectioning-based CLEM approaches penetration of the antibody. In the post-embedding method,
allow studying biological samples with high axial and lateral the antigen–antibody reaction is performed after (i.e., post)
resolution, sample preparation, and 3D image alignment can be plastic embedding. As labeling takes place on thin tissue slices,
challenging (Micheva and Smith, 2007; Collman et al., 2015). antigens are more easily accessible. However, OsO4 , which is
Here, SBEM and FIB-SEM based approaches, where en-block often used as fixative and stain for membrane structures, can
EM imaging and fluorescence approaches are merged to obtain quench the fluorescence signal, and epoxy-based resins used
CLEM images, have helped to streamline in situ studies focused due to its good preservation and ultra-sectioning properties
on the complexity of the nervous system (Briggman et al., 2011; partially inhibit photo-switching of fluorophores (Kim et al.,
Blazquez-Llorca et al., 2015), or to reconstruct whole cortical 2015). Here, a number of modifications, such as using acrylic
neurons (Maco et al., 2013) and single synapses (Bosch et al., resins as embedding material or replacing OsO4 with uranyl
2015). acetate, tannic acid or p-phenylenediamine, have helped to
Orthogonal to in situ approaches described above, CLEM enhance compatibility and immunoreactivity (Phend et al.,
can also be applied to neurons isolated from brain tissues and 1995; Osamura et al., 2000; Akagi et al., 2006; Kim et al.,
cultured in vitro (Banker and Cowan, 1977). As such, cultured 2015). Complementing these slice-based assays, cells can also be
neurons are compatible with each CLEM technique described prepared for analysis of surface features. As before, samples are
above (Figure 1C) (Al Jord et al., 2014; Paez-Segala et al., 2015; fixed. However, rather than embedding the sample, a platinum
Fares et al., 2016). Although these cells lack the physiological replica of the surface is generated (Heuser et al., 1976), or the
context usually encountered by neurons within a tissue, cultured sample is subjected to critical point drying and surface staining,
neurons may still yield advantages compared to in situ samples prior to image acquisition via SEM. Finally, one last concern
depending on the biological question. One such example, and is that chemical fixation is not instantaneous (Szczesny et al.,
complementing the section-based CLEM assays described so 1996). To circumvent this issue, quick-freezing, where samples
far, is the analysis of processes at the cellular surface, such as are ‘slammed’ on a super-cold block of metal sprayed with
curvature-dependent protein recruitment to deforming plasma liquid helium (Heuser et al., 1976) or high-pressure freezing,
membranes (Figure 1D) (Galic et al., 2014). where samples are frozen in milli seconds while pressure
is increased to avoid water crystallization (Hunziker et al.,
1984; Dahl and Staehelin, 1989), have improved preservation
TECHNICAL CONSIDERATIONS quality.

In this section, we will focus on technical possibilities and Picking the Right Markers for CLEM
limitations that need to be taken into consideration when Unfortunately, not all markers are equally well suited for
designing a CLEM experiment. We start by discussing challenges individual CLEM approaches. To account for these limitations,
arising from sample preparation. Next, we will survey what a series of labeling techniques has emerged. Considering its
markers are suitable for which technique, and discuss strategies widespread availability, the most commonly used markers for
for alignment of corresponding fluorescence images and electron CLEM are genetically encoded fluorescence tags, such as GFP,
micrographs with respect to their potential and drawbacks. that can be detected in electron micrographs via immuno-
gold labeling (Figure 2A) (Kukulski et al., 2011; Galic et al.,
Sample Preparation for CLEM Images 2012). In order to distinguish several proteins in the same
The quality of correlative image alignment critically relies on sample, immuno-gold particles of variable sizes directed against
the ability to maintain the native organization of the cell different fluorescence tags can be used. However, since sample
during fixation and subsequent sample preparation. Thus, CLEM fixation may interfere with genetically encoded fluorescence
techniques not only need to be optimized for signal strength, but markers (Muller-Reichert and Verkade, 2014) and gold particles
also for shape preservation. Formaldehyde, glutaraldehyde, and may quench the fluorophore in parallel fluorescence/electron
osmium tetroxide (OsO4 ), have been the standard fixatives for imaging (Kandela and Albrecht, 2007), additional markers are
decades, including for brain tissues that are due to their softness desirable. One such alternative relies on quantum dots (QDs;
easily damaged during fixation and subsequent processing. Figure 2B), which were shown to have a 10 times higher labeling
Classical fixation protocols include whole animal perfusion via efficiency than immuno-gold (Giepmans et al., 2005; Kuipers
the vascular system for larger specimens (e.g., whole brain) et al., 2015). These semiconductor nanocrystals consist of a
as well as immersion of tissue slice and cultured cells. Once cadmium selenide core surrounded by a zinc sulfide shell coated
the biological sample is fixed, the actual preparation for the with affinity ligands (e.g., antibodies) for targeting the desired
respective EM technique is rendered. For slice-based assays, biomolecules (Alivisatos, 1996). Intriguingly, as the fluorescence
two different approaches exist: the pre-embedding method and wavelength of these photo-stable nanocrystals depends on their
the post-embedding method. For both methods, correlative core size, it is possible to label samples with fluorescently
approaches have been reported (Watanabe et al., 2011; Kopek different QDs, and then to distinguish individual QDs in electron
et al., 2012). In the pre-embedding method, the antigen–antibody micrographs by size (Giepmans et al., 2005; Kuipers et al., 2015).
reaction is performed before (i.e., pre) plastic embedding and A minor disadvantage of QDs, however, lies in the difficulty to
subsequent ultrathin sectioning. While better for preserving precisely measure sizes of small QDs due to its low contrast
ultra-structures, this approach is often hindered by poor in EM (Brown and Verkade, 2010). While widely used, both

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Begemann and Galic CLEM in Synapses

FIGURE 1 | Examples of in situ and in vitro correlative light electron microscopy (CLEM) approaches. (A) In situ CLEM of confocal and transmission
electron microscopy (TEM) images showing axonal retreat from neuromuscular junctions. Top left image shows confocal image depicting axonal bulb (arrow) present
25 um from the neuromuscular junction site (in red). Middle left image illustrating surface rendering of the bulb indicated above. Top right image shows TEM, shows
that the bulb is sheathed by Schwann cells. TEM images at the bottom depict neurofilament disorganization in axonal bulb. (B) In situ CLEM of confocal and
scanning electron microscopy (SEM) images of 70 nm section from the mouse cerebral cortex. From left to right: immunostaining of ultrathin sections for tubulin,
GABA, SNAP-25, β-actin, and SEM image. Below, the boxed region is shown at a higher magnification. (C) In vitro CLEM of confocal and TEM images of
hippocampal neurons. Ultrastructure of hα-Syn inclusions in cultured neurons from SNCA+/− mice shown in TEM (top), confocal image (middle), and as merged
CLEM image (bottom). For illustration, the nucleus is rendered in blue, the cytosol in purple, and inclusions in yellow. To the right, high-resolution images of inclusions
(red boxes 1–3), with arrowheads indicating filamentous structures, are shown. (D) In vitro CLEM of confocal and SEM images of cultured hippocampal neurons.
Alignment of SEM and fluorescence signal for actin in cultured hippocampal neurons (top) and magnified sections of actin-rich convoluted nodes that form along
dendritic arbors (bottom) are shown. Scale bars: (A) 25 µm at the top left, 1 µm at the top right, 0.25 µm at the bottom; (B) 10 µm at the top and 2 µm at the
bottom; (C) 5 µm; (D) 2 µm. Pictures reprinted with permission from: (A) (Bishop et al., 2004); (B) (Micheva and Smith, 2007); (C) (Fares et al., 2016); (D) (Galic
et al., 2014).

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Begemann and Galic CLEM in Synapses

FIGURE 2 | Examples of markers commonly used for CLEM. (A) To the left, cartoon depicting immune-gold labeling of genetically encoded fluorescent protein.
Note difference between the relative position of signal in fluorescence (yellow) and electron microscope (blue) images caused by antibodies. To the right, an example
showing fluorescence and electron micrographs of HIV particle labeled with MA-EGFP on MDCK cells expressing RFP-tagged Histone 2B. (B) To the left, cartoon
depicting protein and signal from QD in fluorescence (yellow) and electron microscope (blue) images. Note difference between protein epitope recognized by
antibody and QD signal position. To the right, example depicting RFL6 fibroblasts fixed and stained with primary antibodies followed by secondary antibodies linked
to QDs. QDs identify Cx43 at gap junctions and trafficking intermediates (green) and α-tubulin in microtubules (red). (C) To the left, cartoon depicting genetically
encoded MiniSOG, as well as the relative position and signal shape for fluorescence (yellow) and electron microscope (blue) images. To the right, an example
showing fluorescence and electron micrographs of HeLa cells expressing miniSOG labeled α-actinin. (D) To the left, cartoon depicting HaloTag labeling of protein as
well as position and signal shape for fluorescence (yellow) and electron microscope (blue) images. To the right, an example showing fluorescence and electron
micrographs of Hela cell transfected with Palmitoyl-HaloTag-meGFP. (E) Dendrites of medium-size spiny neurons in the rat neostriatum labeled with
membrane-targeted GFP and immunolabeled with Cy5 against vesicular glutamate transporter2 (VGluT2; top). After detection by fluorescence microscopy, GFP and
VGluT2 immunoreactivities were further developed for focused ion-beam SEM (FIB-SEM) via immunogold/silver enhancement and immunoperoxidase/DAB
methods, respectively (bottom). Scale bars: (A) 5 µm to the left and 100 nm to the right; (B) 5 µm; (C) 2 µm to the left and 1 µm to the right; (D) 1 µm to the left
and 500 nm to the right; (E) 3 µm. Pictures reprinted with permission from: (A) (Kukulski et al., 2011); (B) (Giepmans et al., 2005); (C) (Shu et al., 2011); (D) (Liss
et al., 2015); (E) (Sonomura et al., 2013).

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Begemann and Galic CLEM in Synapses

methods are hampered by a localization error due to spatial post-embedding strategies. In both approaches, the region of
separation between the protein epitope and the electro-dense interest in the embedded sample is identified using a previously
particle that is visualized in light and electron micrographs determined set of reference points. Here both, biological
(Kandela et al., 2007; van Donselaar et al., 2007). To bypass (e.g., blood vessels and dirt) and artificial (e.g., silica beads
this imprecision, Aptamers directed against the fluorescence and gold markers) landmarks have proven to be useful for
tag (Shui et al., 2012) linked to gold (Javier et al., 2008; navigation. Once identified, individual EM sections are made
Chang et al., 2013), or nano-bodies directed against fluorescent and, if desired, 3D reconstruction of individual slices can be
proteins that can be conjugated to gold nanoparticles (Van de prepared by manual or automatic alignment using fiduciary
Broek et al., 2011; Ries et al., 2012) may provide promising landmarks present in adjacent sections (Bishop et al., 2004;
alternatives. Bock et al., 2011; Lee et al., 2016). Finally, electron micrographs
Orthogonal to these approaches, several strategies have (2D or 3D) are merged with the corresponding fluorescence
emerged that take advantage of singlet oxygen generators. images either manually or automatically using large features
Genetically encoded singlet oxygen generators are of particular visible in both images (e.g., blood vessels and silica beads) for
relevance for samples that are investigated ‘en block’ (i.e., coarse alignment. For subsequent fine-adjustment, subcellular
SBEM and FIB-SEM) and thus preclude post-staining of structures (e.g., gold fiduciary markers, nucleus, and filopodia)
individual sections. One such example is the genetically can be used. To estimate how accurate the alignment is, cross-
encoded miniSOG (Figure 2C), which not only fluoresces correlation analysis between light and electron micrographs can
when illuminated by blue light, but also yields an osmophilic be performed.
reaction product by catalyzing the polymerization of 3,30 - In vitro CLEM approaches permit the generation of additional
Diaminobenzidine (DAB) into electron-dense polymers that are reference systems for image alignment on the plating substrate
detectable in electron micrographs (Shu et al., 2011). The same (i.e., glass and plastic). For example, a unique reference point
strategy is also applicable using the Halo-Tag system, where a can be created at the position where the fluorescence image
modified haloalkene dehalogenase (Los et al., 2008) is used to is acquired, using for instance laser etching (Colombelli et al.,
covalently bind to specific ligands such as tetramethylrhodamine 2008; Bishop et al., 2011; Urwyler et al., 2015) or a scratching
(TMR; Figure 2D). Like miniSOG, TMR fluoresces upon device such as diamond objective markers (Sochacki et al.,
illumination and is visible in EM due to DAB oxidation 2014). Upon processing, this reference point can then be used
(Liss et al., 2015). Other self-labeling systems are provided for navigation on the electron microscope and for subsequent
by SNAP/CLIP-tags, in which the enzyme domain is bound image alignment. Another popular reference system relies on
to the protein of interest and labeled with a cell–permeable pre-formed structured (Svitkina, 2009; Benedetti et al., 2014)
fluorescent ligand (Liss et al., 2015). Finally, cells can also and stochastic (Begemann et al., 2015) micro-patterns or fiducial
be transfected with APEX, a non-fluorescent peroxidase that landmarks (Kukulski et al., 2011) for re-finding the region of
withstands strong fixation to yield light-independently EM interest on the EM as well as subsequent image alignment.
contrast (Martell et al., 2012). However, considering that the Intriguingly, such reference points are not only suitable for
reaction product can diffuse, these approaches may yield best manual alignment, but also for software-assisted solutions
results when staining enclosed structures. Notably, multiple (Begemann et al., 2015), rendering these approaches an attractive
procedures can be combined within a sample for visualization entry point compared to more sophisticated CLEM strategies.
(Figure 2E). Finally, simultaneous fluorescence and electron imaging may
provide an alternative strategy. Here, a number of custom-made
(Liv et al., 2013; Nishiyama et al., 2014; Peddie et al., 2014; de
Strategies of Aligning Fluorescence Boer et al., 2015) and commercial (e.g., from Fei and Zeiss)
Image and Electron Micrograph in CLEM instruments have been presented. These instruments, which are
Correlation of fluorescence images and electron micrographs equally suitable for in situ and in vitro samples, integrate light-
can be challenging for a number of reasons. For one, the and electron imaging in one apparatus (iCLEM), thus ensuring
region of interest can cover less than 0.0004% of the total dual imaging of the region of interest without need of later
sample area (Begemann et al., 2015), thus offering only image alignment. However, as sample preparation for iCLEM
on a limited number of reference points for navigation. is restricted to techniques suitable for parallel LM and EM
And even after the region of interest acquired at the light (Agronskaia et al., 2008), and due to the limited availability
microscope is found again on the electron microscope, rotation, of such instruments, iCLEM has not yet reached the optional
magnification, and tilting angle still need to be adjusted for image users.
alignment. This is of particular relevance in pre-embedding
approaches, where, unlike to post-embedding strategies, the
angle of the respective acquisition planes may differ. To CLEM–POTENTIAL AND LIMITATIONS
tackle these obstacles, several strategies for navigation and
image alignment have emerged for in situ and in vitro In this review, we have discussed possible combinations for
approaches. correlating optical and electron microscopy approaches. We
For in situ CLEM, various fiduciary markers have been mentioned how improvements in imaging techniques, sample
described that are equally suitable for pre-embedding and preparation, markers, and image alignment have facilitated

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Begemann and Galic CLEM in Synapses

FIGURE 3 | Graphical summary of existing CLEM approaches. Electron microscopy (blue) and light-based techniques (yellow) that were successfully combined
in correlative approaches are depicted by white lines connecting the respective methods. References corresponding to individual publications using specific CLEM
approaches (numbered 1–50) can be found in the Supplementary Materials (Supplementary Table S1).

development of novel CLEM approaches, and showed examples continuous increase in sophistication and computational power
where these strategies were successfully used for structure- used for image analysis have captured the imagination of people.
function analysis in neurons and synapses. In order to visualize While it remains elusive whether these and other emerging
the combinatorial potential, we provide a chart summarizing techniques will merge or replace CLEM-based studies, they
published CLEM pairings (Figure 3). are exemplary for an ongoing push toward visualization and
While correlative approaches have revealed aspects of comprehensive analysis of biological function on the structural
neuronal and synaptic function that would not have been level.
possible without it, challenges still remain due to the limited Taken together, the possibilities of applying correlative
temporal and spatial resolution of CLEM. From this perspective, approaches to study neuronal and synapse function are growing
recent work on liquid cell electron microscopy (Ross, 2015), thanks to the continuous progress in techniques, tools and
where electron-permeable membranes are used to host aqueous protocols for combining these two types of microscopy.
solutions under atmospheric pressure conditions for TEM and Alas, while correlative approaches in neurobiology have
SEM imaging (Danilatos et al., 2011; Ominami et al., 2015), substantially increased in quality and availability, truly artifact-
cryo-ET to study the supramolecular architecture of cellular free preparation techniques for precise molecule-localization are
structures (Medalia et al., 2002; Beck et al., 2007), and the yet to be achieved.

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Begemann and Galic CLEM in Synapses

AUTHOR CONTRIBUTIONS ACKNOWLEDGMENT

IB and MG conceived the idea, prepared illustrations for The authors thank members of the Galic and Matis labs for
publication and wrote the manuscript. critical reading of the manuscript.

FUNDING SUPPLEMENTARY MATERIAL

The authors acknowledge funding from the Cluster of Excellence The Supplementary Material for this article can be found
‘Cells in Motion’ (DFG, EXC-1003) to IB (PP-2014-04) and MG online at: http://journal.frontiersin.org/article/10.3389/fnsyn.
(FF-2016-03). 2016.00028

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Svitkina, T. M., Verkhovsky, A. B., and Borisy, G. G. (1995). Improved procedures conducted in the absence of any commercial or financial relationships that could
for electron microscopic visualization of the cytoskeleton of cultured cells. be construed as a potential conflict of interest.
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Szczesny, P. J., Walther, P., and Muller, M. (1996). Light damage in rod outer Copyright © 2016 Begemann and Galic. This is an open-access article distributed
segments: the effects of fixation on ultrastructural alterations. Curr. Eye. Res. under the terms of the Creative Commons Attribution License (CC BY). The use,
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 REVIEW
published: 30 June 2016
doi: 10.3389/fnsyn.2016.00016

Advanced Fluorescence
Protein-Based Synapse-Detectors
Hojin Lee 1,2† , Won Chan Oh 1† , Jihye Seong 2,3 * and Jinhyun Kim 1,2 *
1
Center for Functional Connectomics, Korea Institute of Science and Technology, Seoul, South Korea, 2 Neuroscience
Program, Korea University of Science and Technology, Daejeon, South Korea, 3 Center for Diagnosis Treatment Care of
Dementia, Korea Institute of Science and Technology, Seoul, South Korea

The complex information-processing capabilities of the central nervous system emerge

from intricate patterns of synaptic input-output relationships among various neuronal
circuit components. Understanding these capabilities thus requires a precise description
of the individual synapses that comprise neural networks. Recent advances in
fluorescent protein engineering, along with developments in light-favoring tissue clearing
and optical imaging techniques, have rendered light microscopy (LM) a potent candidate
for large-scale analyses of synapses, their properties, and their connectivity. Optically
imaging newly engineered fluorescent proteins (FPs) tagged to synaptic proteins or
microstructures enables the efficient, fine-resolution illumination of synaptic anatomy
and function in large neural circuits. Here we review the latest progress in fluorescent
protein-based molecular tools for imaging individual synapses and synaptic connectivity.
We also identify associated technologies in gene delivery, tissue processing, and
computational image analysis that will play a crucial role in bridging the gap between
synapse- and system-level neuroscience.
Keywords: fluorescent protein sensors, synaptic connectivity, synapses, gene delivery, mapping and localization,
light microscopy
Edited by:
George Augustine,
National Technical University,
INTRODUCTION
Singapore
The synapse is the primary site for neurons to make functional contacts for exchanging information.
Reviewed by: The term ‘‘synapse’’, meaning conjunction in Greek (synapsis = together + to fasten), was coined
Lucas Pozzo-Miller,
The University of Alabama at
in 1897 by the eminent physiologist Charles Scott Sherrington (Nobel Laureate 1932). But the idea
Birmingham, USA that synapses play critical roles as dynamically polarized, communication contacts was proposed
Maurizio Giustetto,
University of Torino, Italy Abbreviations: 30 UTR, 30 untranslated region; AMPAR, α-amino-hydroxy-5-methyl-4-isoxazolepropionic acid
*Correspondence: receptor; CA3, Cornu Ammonis 3; CALI, chromophore-assisted light inactivation; CaMKII, calcium/calmodulin-
Jihye Seong dependent kinase II; CFP, Cyan fluorescent protein; CD4, cluster of differentiation 4; ChR, Channelrhodopsin; CRISPR,
[email protected] clustered regularly-interspaced short palindromic repeats; ENABLED, endogenous labeling via exon duplication;
Jinhyun Kim EM, electron microscopy; FMN, flavin mononucleotide; FP, fluorescent protein; fSPIM, fluorescent selective plane
[email protected] illumination microscopy; GFP, green fluorescent protein; HA, hemagglutinin; HSV-1, Herpes simplex virus-1; iDISCO,
†
immunolabeling-enabled three-dimensional imaging of solvent-cleared organs; ID-PRIME, Interaction-Dependent
Co-first author.
Probe Incorporation Mediated by Enzymes; InSynC, Inhibition of Synaptic Release with CALI; KI, knock-in; LAP,
Received: 09 May 2016 lipoic acid acceptor peptide; LM, light microscopy; LOV2, light, oxygen, and voltage 2; LpIA, lipoic acid ligase; mGluR,
Accepted: 13 June 2016 metabotropicglutamatereceptors;mGRASP,MammalianGFPReconstitutionAcrossSynapticPartners;miniSOG,mini
Published: 30 June 2016 small singlet oxygen generator; NCBI, National Center for Biotechnology Information; NS3, Nonstructural protein 3;
Citation: OFP, Orange fluorescent protein; PDZ, PSD-95, Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1
Lee H, Oh WC, Seong J and Kim J protein (zo-1); PSD-95, postsynaptic density protein-95; rAAV, recombinant adeno-associated virus; SM protein,
(2016) Advanced Fluorescence Sec1/Munc18-like protein; SNARE, SNAP (Soluble N-ethylmaleimide-sensitive factor attachment protein) Receptor;
Protein-Based Synapse-Detectors. syb, synaptobrevin; spGFP, split-GFP; sypHTomato, Synaptophysin-fused pHTomato; TimeSTAMP, Time-Specific
Front. Synaptic Neurosci. 8:16. Tagging for the Age Measurement of Proteins; VAMP2, vesicle-associated membrane protein 2; VenusNT, Venus
doi: 10.3389/fnsyn.2016.00016 N-terminal; VenusCT, Venus C-terminal; VGluT, vesicular glutamate transporter; YFP, Yellow fluorescent protein.

Frontiers in Synaptic Neuroscience | www.frontiersin.org June 2016 | Volume 8 | Article 16 | 95

Lee et al. Advanced Fluorescence Protein-Based Synapse-Detectors

by Santiago Ramon y Cajal (Nobel Laureate 1906). Since then, kinase II (CaMKII; Shen et al., 2000), α-amino-hydroxy-5-
the synapse as a structural and functional communication unit methyl-4-isoxazolepropionic acid receptor (AMPAR; Zamanillo
has been in the spotlight of neuroscientific inquiry (Cowan et al., et al., 1999) and so forth, that had been tagged with FPs.
2001). In fact, many studies have demonstrated that synaptic Recently, sophisticated molecular engineering has allowed even
events, such as changes in molecular composition, structure, more precise and detailed visualization of synaptic structure,
efficacy, and potentiation, play important roles in brain functions composition, and physiology (Chen et al., 2014; Fortin et al.,
including memory formation, perception, and other complex 2014; Figure 1, Table 1).
behaviors (Tsien et al., 1996; Markram et al., 1997; Malinow
and Malenka, 2002; Russo et al., 2010; Caroni et al., 2014). An
important focus has been to visualize the synapse and to measure pH-Sensitive FPs for Visualizing Vesicle
its activity. In 1954, DeRobertis and Palay first observed synapses Release: pHluorin, pHTomato, and pHuji
independently by electron microscopy (EM) and George Gray Although previous, straightforward, FP-based detection of
suggested there may be different types of synapses, i.e., excitatory synaptic distribution revealed many important details of synaptic
and inhibitory (De Robertis and Bennett, 1955; Palay and Palade, physiology, synaptic vesicle release/recycling and neural activity-
1955; Palay, 1956; Gray, 1959). EM provides enough resolution driven changes in membrane-bound synaptic proteins are
for nanometer-scale imaging of the synaptic ultrastructure, difficult to be detected by regular FP-tagging. Special sensors
something that cannot be achieved by light microscopy (LM) of vesicle secretion and neurotransmission have been developed
because of its diffraction limit. However, despite recent advances by linking vesicle membrane proteins with pH-sensitive mutants
that have reduced the time needed for image acquisition and of GFP called pHlourin (Miesenböck et al., 1998). The
reconstruction, EM remains inherently time-consuming, labor- fluorescence intensity of pHluorin largely depends on the
intensive, and volume-limited for large neural circuits. pH of its biochemical environment: in acidic environments
With the recent engineering of fluorescent proteins (FPs) with pH below 6.5, pHluorin is mostly nonfluorescent in
and new developments in light-favoring tissue clearing and 480 nm of light illumination, but becomes highly fluorescent
advanced optical methods, LM is rising as a potent alternative in neutral environments with pH around 7.4. This special
tool for investigating individual synapses in the context of feature of pHluorin was achieved by several mutations on
neural networks (Gray, 1959; Keller et al., 2008; Kim et al., residues important for the proton-relay of tyrosine 66 in the
2012; Tomer et al., 2012; Chung et al., 2013; Richardson chromophore (S147D, N149Q, T161I, S202F, and Q204T). These
and Lichtman, 2015). Imaging the synapse with LM by using amino acid substitutions, which set the pKa of pHluorin to
newly engineered FPs tagged to synaptic proteins or targeted around 7.0, can facilitate the pH-dependent switching of the
to synaptic structures enables fine-resolution illumination of electrostatic environment of the chromophore, allowing the pH-
synaptic anatomy and function in large neural circuits, possibly dependent changes in fluorescent intensity (Sankaranarayanan
in real-time. In this review, we describe recent FP-based and Ryan, 2000). When pHluorin is fused to presynaptic
molecular tools for imaging individual synapses and synaptic vesicle proteins such as VAMP2 (Miesenböck et al., 1998),
connectivity in the contexts of single- and dual component synaptophysin (Zhu et al., 2009), and vesicular glutamate
synaptic detection. We also identify crucial technologies: gene transporter (vGluT; Voglmaier et al., 2006), the release and
delivery of molecular synapse detector; tissue clearing for recycle of synaptic vesicles can be monitored as the pH inside
whole-brain imaging; and computational analysis, whose parallel the synaptic vesicles (∼5.5) transitions to the pH of the
development has potential to bridge synaptic sensor engineering extracellular environment (∼7.4). Thus, by tracking changes
and systems neuroscience. We end by proposing a scheme in pHluorin fluorescence intensity, one can detect real-time
of technological integration for synaptic neuroscience at the presynaptic exocytosis in living, active neurons. Similarly,
systems level. postsynaptic endo-/exo-cytosis and related receptor dynamics
can be visualized by pHluorin-fused mGluRs, for instance
SINGLE COMPONENT SYNAPTIC (Pelkey et al., 2007).
DETECTION More recently, red pH-sensitive FPs such as pHTomato (Li
and Tsien, 2012) and pHuji (Shen et al., 2014) have been
The discovery of green fluorescent protein (GFP) and its developed, allowing for the simultaneous monitoring of multiple
derivatives revolutionized the visualization of biological synaptic activities when combined with the green GFP-based
phenomena, including the individual synapse and its functions. sensors (e.g., GCaMP). pHTomato (pKa ∼ 7.8) was derived from
GFP and other FPs are relatively inert and small (27 kDa) and mStrawberry by introducing six mutations (F41T, F83L, S182K,
can be used to tag synaptic proteins while minimally interfering I194K, V195T, and G196D). Synaptophysin-fused pHTomato
with their normal functions. In fact, the distribution, trafficking, (sypHTomato) has been shown to be suitable for simultaneously
and physiological changes of synaptic proteins caused by neural monitoring the fusion of synaptic vesicles and, when paired
activity became evident in the last two decades, largely through with GCaMP3, postsynaptic Ca2+ changes in living neurons.
observations of synaptic proteins, such as synaptophysin (Li In addition, multiple synaptic events have been successfully
and Murthy, 2001) vesicle-associated membrane protein 2 measured by using sophisticated combinations of these pH-
(VAMP2; Ahmari et al., 2000), postsynaptic density protein-95 sensitive FP-fused synaptic proteins and spectrally distinct
(PSD-95; Nelson et al., 2013) calcium/calmodulin-dependent variants of optogentic stimulators (ChR2-T2A-vGluT-pHlourin

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Lee et al. Advanced Fluorescence Protein-Based Synapse-Detectors

FIGURE 1 | Scheme of single component synapse detectors. (A) Graphical depiction of the conventional green fluorescent protein (GFP) tagging scheme and
summary of the expressed carrier-sensor construct. GFP (green circle) and other fluorescent proteins (FPs) are directly tagged to synaptic proteins in the presynaptic
terminal or postsynaptic spines. Major targets for tagging include presynaptic vesicle proteins (e.g., vesicle-associated membrane protein 2 (VAMP2) and
synaptophysin), postsynaptic receptors (e.g., α-amino-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, AMPAR) and postsynaptic density protein-95 (PSD-95).
(B) pH-sensitive FP mutants are fused to synaptic vesicle membrane proteins, such as VAMP2, to visualize vesicle secretion/recycling and neurotransmission. A
pH-sensitive GFP variant, pHluorin does not fluoresce (gray circle) when inside the acidic chemical environment of the synaptic vesicle, but becomes highly
fluorescent (green circle) when the vesicle is released and exposed to the neutral extracellular environment. (C) Inhibition of Synaptic Release with CALI (InSynC):
attached to target SNARE proteins, molecular actuators such as mini small singlet oxygen generator (miniSOG; light blue filled-in circle) selectively inactivate specific
synaptic proteins that regulate vesicle release and other synaptic events. When illuminated with blue light, miniSOG stimulates generation of reactive oxygen species
(small, dark blue filled-in circles), which then oxidizes susceptible amino acid residues in target vesicle proteins and deactivates protein functions. (D) TimeSTAMP
effectively tracks spatiotemporally controlled protein synthesis and trafficking in living neurons. In the presence of a membrane-permeable protease inhibitor, NS3
protease (gray oval) activity is inhibited, and Venus C-terminus (Venus CT) and Venus N-terminus (Venus NT) reconstitute as fluorescent Venus (yellow circle).
Reconstituted Venus accumulates in the postsynaptic spine, the trafficking destination of the fused PSD-95. When the protease inhibitor is present, however, NS3
protease cleaves the protease target sites (gray circles flanking NS3), preventing Venus reconstitution.

and VChR1-T2A-synpHTomato). However, the pH-sensitivity Inhibition of Synaptic Release with CALI
of pHTomato is relatively low (3-fold change in pH 5.5–7.5). (InSynC)
Thus, a new red pH-sensitive FP, named pHuji, has been more Beyond merely visualizing the distributions and endo-/exocytosis
recently derived from mApple (Shaner et al., 2008) by including of synaptic proteins by tagging them with FPs and their pH-
a K163Y mutation, resulting in high pH sensitivity (20-fold sensitive variants, genetically encoded chromophore-assisted
change in pH 5.5–7.5). The use of different colored pH-sensitive light inactivation (CALI) has been developed to selectively
FPs together with a Ca2+ indicator and/or a spectrally distinct inactivate specific synaptic protein functions that regulate
optogenetic modulator offers a new promising readout system synaptic events such as synaptic release (Lin et al., 2013). CALI
for complex, coordinated synaptic events. is based on light-induced generation of reactive oxygen and the

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Lee et al. Advanced Fluorescence Protein-Based Synapse-Detectors

consequent inactivation of nearby attached synaptic proteins. chosen because it is small (19 kDa) and monomeric, specific
Its original agents were synthetic chromophores for example to its substrate, and not cytotoxic, and most of all, NS3
malachite green (Jay, 1988), fluorescein (Beck et al., 2002), FlAsH shows high selectivity and efficiency of its cell-permeable
(Marek and Davis, 2002), ReAsH (Tour et al., 2003), and eosin inhibitor.
(Takemoto et al., 2011) and FPs such as KillerRed (Bulina et al., Although the first version of TimeSTAMP has worked
2006). To precisely inhibit synaptic release with improved target successfully in primary hippocampal neurons to track PSD-
specificity and inactivation efficiency compared to these CALI 95 accumulation during synaptic growth, and in Drosophila
agents, inhibition of synaptic release with CALI (InSynC) has for whole-brain mapping of newly synthesized CaMKII,
been recently developed using a newly engineered flavoprotein it required post hoc immunostaining against epitope tags,
called mini small singlet oxygen generator (miniSOG), fused limiting the benefits of pulse-chase labeling through time.
with the SNARE proteins VAMP2 and synaptophysin (Lin Thus, TimeSTAMP2 has been introduced, replacing HA-
et al., 2013). miniSOG was originally derived from the light, tag with split-fluorescence proteins (e.g., Yellow fluorescent
oxygen, and voltage 2 (LOV2) domain of phototropin, a protein, YFP; Orange fluorescent protein, OFP). In fluorescent
blue light photoreceptor (Shu et al., 2011). Under blue light TimeSTAMP2, for instance, Venus yellow fluorescent protein
illumination, this photoreceptor binds to and excites flavin is separated by an NS3 domain flanked by NS4A/B target
mononucleotide (FMN), which then functions as an oxygen sites into VenusNT (1-158 aa) and VenusCT (159-238 aa)
generator in cells. miniSOG contains the single amino acid for drug-dependent fluorescence. In the absence of BILN-
substitution of FMN-binding residue Cys426 to Gly on the LOV2 2061, VenusCT is cleaved and degraded by the active NS
domain, allowing for more efficient energy transfer to FMN, domain resulting in no yellow fluorescence, while VenusNT
and contains further mutations for enhanced brightness. When and CT are reconstituted as fluorescent forms after drug
illuminated by blue light, synaptic proteins can be selectively application. This allows optical pulse labeling of synaptic proteins
inactivated by miniSOG-mediated oxidization of susceptible such as PSD-95 and Neuroligin with a drug-defined temporal
residues such as tryptophan, tyrosine, histidine, cysteine, and resolution. Additionally, photo-oxidizing TimeSTAMP using
methionine. InSynC by miniSOG can selectively inhibit vesicle miniSOG inserted into TS:YFP can be used to visualize new
release at individual synapses in vitro and in vivo thanks proteins at an EM-based ultrastructural level (Butko et al.,
to the high efficiency of its light-induced oxygen generation, 2012).
independence of exogenous cofactors, and small size (106
residues, 14 kDa; Lin et al., 2013). However, further engineering DUAL COMPONENT SYNAPTIC
is required for investigating the functional dynamics of synaptic
DETECTION
circuits with physiologically relevant temporal resolutions, as
inactivation by InSynC persists relatively long (∼1 h) after light Thus far, we have described methods for detecting
stimulation. synapses by labeling single components, either pre- or
post-synaptic. Although these single-component tools
allow for detecting, measuring, and manipulating synaptic
Time-Specific Tagging for the Age structures and activities, they do not address the critical
Measurement of Proteins (TimeSTAMP) fact that synapses are bilateral microstructures involving
Another new strategy beyond merely visualizing synaptic both presynaptic terminal and postsynaptic density. For
proteins by tagging with FPs is time-specific tagging for reliable synapse detection, therefore, several recent studies
the age measurement of proteins (TimeSTAMP; Lin et al., introduced new methods for labeling synaptic interactions
2008). As spatiotemporally controlled protein synthesis between pre- and post-synaptic components, such as
and trafficking are critical for synaptogenesis, synaptic using split-FPs or enzymes to label neurexin-neuroligin
connectivity, and long-lasting changes in synapses, TimeSTAMP interactions. Here we review dual-component methods using
is beneficial for tracking important, newly synthesized synaptic handshaking-like transmembrane molecular interaction
proteins such as PSD-95 and CaMKII in living neurons. across the synaptic cleft, with particular attention to GFP
TimeSTAMP is a drug-controllable, time-specific tagging Reconstitution Across Synaptic Partners (GRASP; Figure 2,
strategy based on the hepatitis C virus NS3 protease and Table 1).
its cell-permeable inhibitor BILN-2061. PSD-95-GFP, for
example, was fused to NS3 protease flanked by NS4A/B
target sites and the C-terminal HA tag (PSD-95-GFP-TS- Mammalian GFP Reconstitution Across
HA). In the absence of the specific inhibitor BILN-2061, Synaptic Partners (mGRASP)
NS3 protease cleaves the NS4A/B target sites allowing the To detect particular synaptic connections with LM, the
C-terminal HA-tag to be cleaved from PSD-95-GFP and Mammalian GFP Reconstitution Across Synaptic Partners
degraded; but drug application will allow the HA-tag to (mGRASP) technique takes advantage of the complementarity
be accumulated. This allows distinguishing between newly of two non-fluorescent split-GFP fragments, each of which is
and previously synthesized PSD-95 by measuring HA/GFP tethered specifically to the pre- and postsynaptic membrane,
signals at a time defined by the drug application. For respectively (Kim et al., 2012). When two neurons, each
the TimeSTAMP strategy, the NS3 protease domain was expressing one of the fragments, are closely opposed across a

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Lee et al. Advanced Fluorescence Protein-Based Synapse-Detectors

FIGURE 2 | Scheme of dual component synapse detectors. (A) GFP reconstitution-dependent molecular tools detect pre- and postsynaptic membrane
apposition through the reconstitution of spGFP fragments each attached to the extracellular ends of membrane protein carriers. Synapse-targeted GFP
Reconstitution Across Synaptic Partners (GRASP) utilizes a presynaptic component of spGFP11 (spG11, circular wedge) linked to a CD4-neurexin-1β C-terminus
(NxCT) fusion through flexible peptide linkers (gray zigzag), and a postsynaptic component of spGFP1-10 (spG1-10) and truncated neuroligin-1 (tNg) connected with
a linker. SpG11 and spG1-10 unite at synapses, not random membrane contacts. In activity-dependent X-RASP the presynaptic split-XFP1-10 (spX1-10) is fused to
SNARE proteins such as synaptobrevin (syb), and unites with CD4-bound spG11 when presynaptic activity triggers vesicle release. (B) Neurexin (Nx)-neuroligin (Ng)
interaction-dependent molecular tools detect individual synapses through fluorescent protein (FP)-based (SynView) and enzymatic reaction-based
(Interaction-Dependent Probe Incorporation Mediated by Enzymes, ID-PRIME) direct visualization of Nx-Ng interaction. In SynView, spG11 is inserted between the
esterase and proximal extracellular domain of Ng, and spG1-10 in the middle of the LNS domain of Nx. GFP reconstitutes when Nx and Ng unites. ID-PRIME uses
the identical set of synaptic protein carriers, but tandem LAP tags (3xLAP) and lipoic acid ligase A (LplA) are attached to C-terminus of Ng esterase domain and Nx
LNS domain, respectively. Nx-Ng interaction initiates LplA-mediated lipoic acid tagging to 3xLAP, and signals useful for imaging originate from antibody-fluorophore
conjugates.

synaptic cleft, the split fragments are reconstituted as fluorescent approximately 20 nm-wide synaptic cleft without gross changes
GFP in that location. This dual component synapse detection in endogenous synaptic organization and physiology. Based
bypasses the Abbe’s diffraction limit and allows relatively on published sequences from NCBI, we generated synapse-
rapid and accurate synapse mapping with LM. This method specific chimeric carriers. Both the pre- and postsynaptic
takes advantage of the fast folding kinetics and stability after mGRASP components share a common six-subcomponent
maturation of superfolder GFP (Pédelacq et al., 2006), split framework: (1) a signal peptide; (2) a split-GFP fragment;
into two fragments, namely spGFP1-10 (first 214 residues (3) an extracellular domain; (4) a transmembrane domain;
comprising ten β barrels) and spGFP11 (16 residues, 11th β- (5) an intracellular domain; and (6) a fluorescent protein for
barrel strand). The GRASP technique was initially implemented neurite and soma visualization. The presynaptic mGRASP
in C. elegans (Feinberg et al., 2008; Park et al., 2011) and component consists of the signal peptide of nematode β-
later in Drosophila (Gordon and Scott, 2009; Fan et al., 2013; integrin (PAT-3, residues 1–29), GFP β-strand 11 (spGFP11,
Gorostiza et al., 2014). In the original GRASP constructs, 16 residues), two flexible GGGGS linkers, the extracellular
membrane carriers (human CD4) for split-GFP fragments were and transmembrane domains of human CD4-2 (residues
not synapse-specific—fluorescence could arise wherever any 25–242). Rat neurexin-1β (residues 414–468) containing
membranes expressing fragment pairs were closely apposed. the PDZ-binding motif constitutes the intracellular domain
This became a critical issue for mammalian synapse detection for maintenance of correct localization at the presynaptic
because the mammalian nervous system contains much more site. mCerulean is fused to the intracellular end of the
compactly intermingled neurites than the invertebrate nervous presynaptic mGRASP to visualize axonal expression. The
system. postsynaptic mGRASP component is based primarily on
In a previous study, we engineered and successfully applied mouse neuroligin-1, which interacts with presynaptic adhesion
mGRASP for mapping fine-scale synaptic connectivity in proteins including β-neurexins, and mediates the formation
the mouse brain (Kim et al., 2012). The primary features of and maintenance of synapses between neurons. To prevent
mGRASP include targeting specific synapses, and matching the nonspecific synaptogenesis through interactions with its

Frontiers in Synaptic Neuroscience | www.frontiersin.org June 2016 | Volume 8 | Article 16 | 99

 Lee et al.

TABLE 1 | Summarized methods of single and dual component synapse detection.

Single component Dual component

Synaptic InSynC TimeSTAMP GRASP mGRASP Syb:GRASP XRASP SynView ID-PRIME

release-detecting FPs

Detection/working pH-sensitive FPs: pHluorin, miniSOG- Reconstitution of Reconstitution of Reconstitution of Reconstitution of Reconstitution of Enzymatic
module pHTomato generated reactive VenusNT and spGFP1-10 and spGFP11 spGFP1-10 and spXFP1-10 spGFP1-10 interaction of LpIA
oxygen VenusCT spGFP11 and spXFP11 and spGFP11 and LAP

Synaptic targeting Synaptophysin, VAMP2, Synaptophysin, PSD-95, CaMKII, Human CD4, PTP-3A, Rat neurexin1-β, Synaptobrevin, Rat neurexin-1β, Human

Frontiers in Synaptic Neuroscience | www.frontiersin.org

module PSD-95, CaMKII, vGLUT1, VAMP2 Neuroligin nlg-1 mouse neuroligin-1 human CD4 rat neuroligin-1, 2 neurexin-3β, mouse
mGluRs neurexin-1β, rat
neuroligin-1

Model Systems
In vitro HEK, primary cultured Primary cultured HEK, primary N/A N/A HEK HEK, COS-7, primary HEK, primary
neurons, organotypic slices neurons, cultured neurons cultured neurons cultured neurons
organotypic slices
In vivo N/A N/A N/A C.elegans, Drosophila Mouse Drosophila N/A N/A

Remarks Tracking of presynaptic Optically inhibiting Optical pulse-chase in vivo synaptic detection in in vivo synaptic Activity-dependent Synapse labeling by Enzyme-based
vesicle release, synaptic release labeling of synaptic invertebrate circuits detection in vertebrate labeling of multiple direct interaction of visualization of
postsynaptic receptor with good spatial proteins with high circuits synaptic inputs neurexin-neuroligin direct interaction of
trafficking resolution spatiotemporal neurexin-neuroligin
resolution
Lacks in vivo application Lacks Awaits mammalian Limited to apply Limited to detect Limited to apply Lacks in vivo Requires separate
with quantitative analysis physiologically in vivo application mammalian system functional synaptic mammalian system, verification and introduction of
relevant temporal connections limited temporal application exogenous ligase
resolutions due to resolutions and antibody-
slow recovery fluorophore
conjugates

References Miesenböck et al. (1998), Lin et al. (2013) Lin et al. (2008) Feinberg et al. (2008), Feng et al. (2012), Karuppudurai et al. Tsetsenis et al. Liu et al. (2013)
Sankaranarayanan and and Butko et al. Gordon and Scott (2009), Kim et al. (2012) (2014), (2014)
Ryan (2000), (2012) Gong et al. (2010), and Druckmann et al. Frank et al. (2015),
Kopec et al. (2006), Park et al. (2011), (2014) Macpherson et al. (2015)
Voglmaier et al. (2006) Yuan et al. (2011), and Li et al. (2016)
and Li and Tsien (2012) Fan et al. (2013),
Pech et al. (2013)
and Gorostiza et al. (2014)

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Lee et al. Advanced Fluorescence Protein-Based Synapse-Detectors

endogenous partner, neurexin, the extracellular esterase 2-photon imaging together with Ca2+ indicators and for
domain of neuroligin is deleted in the main skeleton of the fine-scale synapse labeling from multiple inputs within the
postsynaptic mGRASP. Similar to presynaptic mGRASP, single neuron, because it has proved difficult to separate
post-mGRASP is composed a signal peptide from the CFP/GFP/YFP signals in the complex mammalian nerve
esterase-truncated neuroligin-1 (residues 1–49), GFP β- system.
strand 1-10 (spGFP1-10, 648 residues), the extracellular,
transmembrane, and intracellular regions of neuroligin (71, Engineering Carriers
19, and 127 residues, respectively), followed by the self- for Activity-Dependent GRASP
cleavable 2A peptide-fused dTomato for visualizing postsynaptic To understand functional organizations underlying complex
neuronal morphology. This optimized mGRASP enabled the brain functions that go beyond structural connectivity patterns,
comprehensive synaptic connectivity mapping of hippocampal it will be essential to identify active synaptic connectivity at
CA3-CA1 and identified spatially structured patterns of synaptic defined times and conditions. For the activity-dependent GRASP
connectivity (Druckmann et al., 2014). It is important to note system, synaptobrevin (syb), a key constituent of synaptic
that brain-wide synapse detection for comprehensive fine-scale vesicle membrane, was used as a synaptic carrier instead of
mapping becomes achievable not only by advanced molecular constantly membrane-targeted carriers in the previous GRASP
engineering to label the synapse such as mGRASP, but also by systems. The straightforward fusion of syb and spGFP1-10
appropriately engineered computational analysis (Feng et al., (syb:spGFP1-10) with the original CD4:spGFP11 can together
2012, 2015). form activity-dependent GRASP, called the syb:GRASP system.
Current GRASP technology has proved to be a tool suitable Given activity-dependent interactions of syb with SNARE-SM
for rapid and accurate mapping of synaptic connectivity in protein complex triggering synaptic vesicle release, syb:GRASP
nematode (Feinberg et al., 2008), fruit fly (Gordon and Scott, showed preferential labeling of active synapses as a boost of
2009), and mouse (Kim et al., 2012; Druckmann et al., 2014). Yet, GRASP fluorescence signals in well-studied thermosensory and
further improvements to GRASP such as multi-colored FPs for olfactory circuits in the fruit fly (Macpherson et al., 2015). This
analyzing convergent synaptic inputs, various carriers for neural new strategy for mapping active synaptic connectivity might
activities, and tailored computational analyses will provide a clear expedite the mapping of functional connectivity at the synapse
overview of complex synaptic connectivity and its operation. We level in a way previously achieved only by difficult combinations
next discuss recent efforts in these directions. of Ca2+ imaging and exhaustive EM reconstruction (Bock
et al., 2011; Briggman et al., 2011). Yet, these new techniques
Engineering FPs for Multi-Color GRASP raise basic concerns about possible side effects caused by
A neuron oftentimes receives multiple inputs from different the overexpression of key synaptic vesicle proteins, and need
presynaptic neurons of distinct cell types and/or various further optimization before they can be applied to mammalian
brain areas. For comprehensive mapping of complex synaptic networks.
circuits, multiple synaptic detection is beneficial, as described
earlier in the section describing pH-sensitive FPs. The
current version of GRASP restricts convergent synaptic FP- and Enzyme-Based Visualization
mapping because it relies on only a single pair of spGFP of Neuroligin-Neurexin Interaction
fragments such that spectral overlap of its signals hinders An alternative way to detect synapses has been suggested
simultaneous imaging with previously well-established GFP- by imaging neurexin-neuroligin interactions. Presynaptic
based tools such as GCaMP. Therefore, the multi-color neurexin and postsynaptic neurolign are transmembrane
GRASP (XRASP) components have been developed recently adhesion proteins and their interaction at the synaptic
(Macpherson et al., 2015; Li et al., 2016). Given that Cyan cleft has been believed to be a key process for synaptic
fluorescent protein (CFP), GFP, and YFP have identical formation, maintenance, and connectivity (Li and Sheng,
structures except for several residues in the chromophore 2003; Graf et al., 2004; Chen et al., 2010; Krueger et al.,
located in beta barrels of 1-10, C-RASP and Y-RASP were 2012). Therefore, it is thought that identifying sites of
generated by color-shifting mutations in spGFP1-10 (Y66W, neurexin-neuroligin interactions could provide selective
S72A, F145A∗ , N146I, and H148D for C-RASP, ∗ additional visualization of synapses. Similar to the GRASP approaches,
mutation in Li et al., 2016, T65G, V68L, S72A and T203Y this method, based on neuroligin-neurexin interactions using
for Y-RASP) paired with the unaltered spGFP11 (Li et al., splitGFP, is called SynView. It is composed of spGFP1-
2016). Multi-color GRASP (XRASP) was tested in vivo in 10-inserted neurexin-1β between residues N275-D276 or
several circuits, including the Kenyon cells of the mushroom A200-G201 for the presynaptic component, and the split-
body, and projection neurons of the thermosensory and GFP11-inserted neuroligin-1 in the C-terminal end of the
olfactory systems in the fruit fly. Reconstructed CFP and YFP extracellular esterase (between Q641-Y642) for the postsynaptic
signals showed minimal spectral overlap with the GRASP component (Tsetsenis et al., 2014). Another method for
emission and excitation spectra. Extended choices of multi- visualizing neuroligin-neurexin interactions is based on
color GRASP (XRASP) will allow simultaneous imaging of mutated bacterial lipoic acid ligase (LpIA) and lipoic acid
multiple, convergent connectivity and functional activity. acceptor peptide (LAP) tags (Uttamapinant et al., 2010, 2012),
However, more red-shifted XRASP is required for in vivo and is called Interaction-Dependent Probe Incorporation

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Lee et al. Advanced Fluorescence Protein-Based Synapse-Detectors

Mediated by Enzymes (ID-PRIME; Liu et al., 2013). Using of Cre recombinase. When this PSD-95-ENABLED mouse
the same principle of SynView, ID-PRIME was designed to line is crossed with a dopaminergic cell-type specific DAT-
detect trans-synaptic contacts of neurexin and neuroligin Cre line, for instance, PSD-95mVenus is expressed specifically
enzymatically using lipoic acid. These two methods successfully in dopaminergic neurons. PSD-95-ENABLED was shown to
imaged the direct interactions of neurexin-neuroligin synaptic functionally replace wild-type PSD-95 and to sparsely label
adhesive molecules. However, these methods seem restricted a particular cell-type, allowing insights into the detailed
to particular investigations of the dynamics of neurexin- distribution and dynamics of synapses. In applying this
neuroligin, rather general synaptic mapping. In addition, strategy to a wide variety of synaptic proteins, one concern
it is known that overexpressing these synaptic adhesion with using the ENABLED strategy is that it is limited to
molecules causes substantial structural and physiological synaptic proteins that are compatible with C-terminal FP-
perturbations of normal synaptic compartments and cleft tagging.
structures. Thanks to incredibly fast developments and improvements in
genetic manipulation technologies such as clustered regularly-
BRIDGING TOOLS BETWEEN interspaced short palindromic repeats (CRISPR) and effector
MOLECULAR SYNAPSE DETECTORS nucleases Cas system (Cong et al., 2013; Wang et al., 2013;
AND BRAIN-WIDE SYNAPSE MAPPING Fujii et al., 2014; Aida et al., 2015), the generation of synapse
detector KI mouse lines is becoming time- and cost-efficient, and
Thus far, we have described methods for detecting synapses is dramatically facilitating synapse mapping.
focusing on molecular engineering. To exert these genetically
encoded synapse detectors to brain-wide synaptic mapping Viral System-Based Gene Delivery
at the system level, there need to be critically partnered Spatially targeted gene delivery into the mature brain can
technologies such as gene delivery, advanced imaging, and digital be achieved through stereotactic microinjection of viral
representation platform that are appropriate for neural system vectors expressing FP-tagged proteins. Diverse virus families
(Figure 3). have been recruited into this effort: Retroviridae (e.g.,
lentivirus), Parvoviridae (e.g., rAAV), Adenoviridae (e.g.,
Gene Delivery System for Synapse canine adenovirus), and Alphaviridae (e.g., sindbis virus),
including some that cross the synapse, such as Rhabdoviridae
Detectors in the Mouse Brain (e.g., rabies virus) and Herpesviridae (e.g., HSV-1 and
Improving the targeting specificity for types of cells and
pseudorabies virus; Nassi et al., 2015). Because of their low
scaling up the scope of synaptic sensor delivery are among
cytotoxicity and stable expression, lentiviruses and rAAVs
the crucial initial steps needed to expand single-synapse
are the most widely used viral vectors in neuroanatomical
level analysis to systems level neuroscience. Gene delivery
tracing studies and human clinical trials testing gene therapy.
technologies, particularly for the nervous system, have
In fact, spatially restricted injection of Cre-(in)dependent
developed at a breathtaking pace over the past decades,
rAAV vectors have been used for mGRASP-assisted synaptic
establishing methodologies that can be classified into
mapping.
two main categories: germ line manipulation and viral
In parallel, ongoing efforts include searching for and
injection.
engineering new types of virus to allow transduction efficiency/
specificity and retrograde infection. Canine adenovirus has
Genetic Manipulation-Based Gene Delivery drawn attention because of its strong retrograde transport
To avoid side effects that can sometimes be caused by the capability and relatively large payload size (∼30 kb); further,
overexpression of FP-tagged synaptic proteins, and to mimic several successful applications of Canine adenovirus in the mouse
expression patterns of endogenous proteins, gene knock- brain suggest a powerful complement to the lentivirus and rAAV
in (KI) technology has been used to substitute wild-type (Bru et al., 2010; Ekstrand et al., 2014; Junyent and Kremer,
genes with FP-tagged copies. For synaptic detectors based 2015; Schwarz et al., 2015). Additionally, systemic delivery of
on universal synaptic proteins such as PSD-95, however, viral vectors in animal models has proved to be effective and
this standard KI method leads to expression of FP-tagged safe for various serotypes of AAV (Foust et al., 2009; Bevan
synapse detectors globally, throughout the brain, which makes et al., 2011; Gray et al., 2011; Yang et al., 2014; Deverman et al.,
it difficult to acquire high resolution images of a particular 2016).
cell type. Therefore, a recent study introduced a conditional
KI strategy called endogenous labeling via exon duplication
(ENABLED; Fortin et al., 2014). In the ENABLED strategy, Advanced Brain-Wide Imaging and Digital
a knocked-in gene cassette is composed of the floxed last Representation of Synaptic Detectors
exon and 3’UTR of PSD-95 followed by its mVenus-tagged Once FP-based synaptic detectors are introduced into the
duplicated last exon and 3’UTR. The FP-tagged duplicated brain as described above, appropriate brain-wide imaging and
gene will be selectively expressed only in Cre-expressing digital representation technologies are necessary to map synaptic
neurons because its translation is designed to be blocked by connectivity. Happily, there has been remarkable progress
translation stop signals in the endogenous copy in the absence in tissue clearing methodology such as BABB (Dodt et al.,

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Lee et al. Advanced Fluorescence Protein-Based Synapse-Detectors

FIGURE 3 | Convergence of technologies for synaptic neuroscience at the systems level. (A) Synapse-detector genes can be delivered to target neural
components through germline manipulation-based and viral system-based methods. Exogenous genetic materials can be effectively introduced to the germline with
high expression specificity via Cre-mediated conditional knock-in (KI) strategies and the homology directed repair (HDR) pathway of the clustered
regularly-interspaced short palindromic repeats (CRISPR)/Cas system. In a Cre-mediated conditional KI strategy called endogenous labeling via exon duplication
(ENABLED), a KI cassette including duplicates of exons 19 and 20 and the 3’UTR of PSD-95 is generated. The first duplicate is flanked by head-to-tail oriented loxP
sites (black arrows), while the second duplicate has monovalent Venus (mV) inserted between exon 20 and the 3’UTR. The two duplicate sequences, along with
exon 18, are flanked by a set of homology arms (h arm), which mediates KI cassette insertion through homologous recombination. Cre-lox recombination excises the
first duplicate containing translation stop signals and polyadenylation sequences, and activates mV expression only in Cre-expressing neurons. Transient expression
of sensor genes using viral vectors benefits from efforts to engineer expression through Cre-dependent expression cassettes e.g., Cre-dependent Mammalian
GRASP (mGRASP) constructs and newly engineered serotypes that have more selective tropism and transduction efficiency (e.g., AAV-PHP.B). Local tissue or
systemic injection of such viral systems can lead to flexible, versatile gene delivery in mature organisms. (B) Combination of light-favoring brain clearing, whole-brain
imaging, and computational techniques for three-dimensional synapse mapping enables single-synapse level analysis of synaptic profiles across the whole brain.
Further improvements in lipid extraction, refractive index matching, advanced light-sheet microscopy, and large-scale data processing and 3D reference space
generation will accelerate systems neuroscience at the synaptic scale.

2007), CUBIC (Susaki et al., 2014) 3DISCO (Ertürk et al., sectioning. These new clearing techniques, together with
2012), immunolabeling-enabled three-dimensional imaging of advanced optical methods such as fluorescent selective plane
solvent-cleared organs (iDISCO; Renier et al., 2014), SeeDB illumination microscopy (fSPIM; Huisken et al., 2004), will
(Ke et al., 2013) and CLARITY (Chung et al., 2013; Tomer allow high-throughput whole brain imaging. Also, once images
et al., 2014) that are needed to prepare the intact brain are acquired, digital representation programs are necessary
for imaging while avoiding distortions caused by physical to reliably extract synaptic wiring information from the

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Lee et al. Advanced Fluorescence Protein-Based Synapse-Detectors

images, and to bring data from different sections and animals brain will provide firm grounds for further anatomical and
into register with one another (Johnson et al., 2010; Oh functional studies and such mapping is essential for analyzing
et al., 2014). Improvements in this software will be greatly large-scale information processing phenomena. We believe that
beneficial. rapid, scalable synaptic cartography with the triad of single-
In our view, new clearing methods, optics, and tailored synapse resolution, brain-wide scope, and cell-type-specific
computational analysis platforms together with advanced connectivity requires a synergistic combination of advanced
synaptic detectors such as mGRASP are very promising technologies, and that FP-based neurosensors are the key to this
developments for the complete mapping of mammalian synaptic grand integrative project.
connectivity.
AUTHOR CONTRIBUTIONS
CONCLUSION AND PERSPECTIVE
HL, WCO, JS, and JK wrote this manuscript.
Here we reviewed new FP-based synapse detection techniques,
which are useful for rapidly imaging individual synapses and ACKNOWLEDGMENTS
synaptic connectivity in the whole brain with LM. Recent
technical developments have allowed a focus of neuroscience to HL, WCO, JK, and JS are supported by the Korea Institute of
move from individual synapses to ensemble interactions among Science and Technology (KIST) Institutional Program (2E26190
neurons of various cell types through multiple synaptic pathways. and 2N41660, respectively) and the Samsung Science and
Comprehensively mapping individual synapses in the whole Technology Foundation.

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 MINI REVIEW
published: 16 September 2016
doi: 10.3389/fnsyn.2016.00029

Organelle-Specific Sensors for

Monitoring Ca2+ Dynamics in
Neurons
Seok-Kyu Kwon, Yusuke Hirabayashi and Franck Polleux*

Department of Neuroscience, Mortimer B. Zuckerman Mind Brain Behavior Institute, Kavli Institute for Brain Science,
Columbia University Medical Center, New York, NY, USA

Calcium (Ca2+ ) plays innumerable critical functions in neurons ranging from regulation
of neurotransmitter release and synaptic plasticity to activity-dependent transcription.
Therefore, more than any other cell types, neurons are critically dependent on spatially
and temporally controlled Ca2+ dynamics. This is achieved through an exquisite
level of compartmentalization of Ca2+ storage and release from various organelles.
The function of these organelles in the regulation of Ca2+ dynamics has been
studied for decades using electrophysiological and optical methods combined with
pharmacological and genetic alterations. Mitochondria and the endoplasmic reticulum
(ER) are among the organelles playing the most critical roles in Ca2+ dynamics in
neurons. At presynaptic boutons, Ca2+ triggers neurotransmitter release and synaptic
plasticity, and postsynaptically, Ca2+ mobilization mediates long-term synaptic plasticity.
To explore Ca2+ dynamics in live cells and intact animals, various synthetic and
genetically encoded fluorescent Ca2+ sensors were developed, and recently, many
groups actively increased the sensitivity and diversity of genetically encoded Ca2+
Edited by: indicators (GECIs). Following conjugation with various signal peptides, these improved
Marc Fivaz, GECIs can be targeted to specific subcellular compartments, allowing monitoring
Duke NUS Graduate Medical School,
Singapore of organelle-specific Ca2+ dynamics. Here, we review recent findings unraveling
Reviewed by: novel roles for mitochondria- and ER-dependent Ca2+ dynamics in neurons and
Lucas Pozzo-Miller, at synapses.
University of Alabama at Birmingham,
USA Keywords: synapse, mitochondria, endoplasmic reticulum, circuit function, calcium dynamics
William N. Green,
University of Chicago, USA

*Correspondence:
INTRODUCTION
Franck Polleux
[email protected] Calcium (Ca2+ ) ions govern prevalent physiological processes in various cell types (Rizzuto
and Pozzan, 2006; Clapham, 2007). This is especially prominent in excitable cells like neurons
Received: 13 June 2016 where Ca2+ influx through the plasma membrane and release of Ca2+ from internal stores
Accepted: 30 August 2016 transduce the effects of changes in membrane polarization and therefore mediate faithful
Published: 16 September 2016 transfer or storage of information over various timescales (milliseconds to minutes/hours).
Citation: Therefore, regulation of intracellular Ca2+ homeostasis is central to the proper function of
Kwon S-K, Hirabayashi Y and neuronal circuits. The maintenance of baseline levels of intracellular Ca2+ levels is regulated
Polleux F (2016) Organelle-Specific
in part through exchangers and pumps such as the plasma membrane Ca2+ -ATPase (PMCA
Sensors for Monitoring Ca2+
Dynamics in Neurons.
pump), the Na+ /Ca2+ exchanger (NCX), and the Na+ /Ca2+ -K+ exchanger (NCKX) which
Front. Synaptic Neurosci. 8:29. extrude Ca2+ through the plasma membrane into the extracellular space. In addition to
doi: 10.3389/fnsyn.2016.00029 these mechanisms, intracellular organelles, such as mitochondria and endoplasmic reticulum

Frontiers in Synaptic Neuroscience | www.frontiersin.org September 2016 | Volume 8 | Article 29 | 107

Kwon et al. Organelle-Specific Calcium Dynamics in Neurons

(ER), are able to regulate cytoplasmic Ca2+ ([Ca2+ ]c ) [Ca2+ ]c . This has been characterized in various species,
through mitochondrial calcium uniporter (MCU) and smooth neuronal cell types and circuits (Figure 1A). At the Drosophila
endoplasmic reticulum Ca2+ -ATPase (SERCA), respectively. neuromuscular junction (NMJ), the GTPase dMiro mutant lacks
In neurons, mitochondria and ER play important presynaptic mitochondria through impaired axonal transport
physiological roles via [Ca2+ ]c regulation, thereby diverse (Guo et al., 2005; Wang and Schwarz, 2009). During prolonged
synaptic functions including basal synaptic transmission, stimulation, these mutants lacking presynaptic mitochondria
presynaptic short-term plasticity, and long-term plasticity can displayed subtle, but significantly increased presynaptic Ca2+
be regulated by these organelles (Verkhratsky, 2005; Bardo et al., accumulation and display decrease forms of sustained synaptic
2006; Mattson et al., 2008; Vos et al., 2010). In addition, impaired transmission or synaptic ‘‘fatigue’’ (Guo et al., 2005). Drosophila
Ca2+ homeostasis in the nervous system has been proposed Drp1 mutants also deplete presynaptic mitochondria at NMJ
to play an important function in the physio-pathological and exhibit elevated presynaptic Ca2+ levels in resting and
mechanisms underlying Alzheimer’s disease, Parkinson’s evoked states. However, spontaneous release (mini Excitatory
disease, and spinocerebellar ataxia (Verkhratsky, 2005; Mattson junctional potential, mEJP) was not altered, but the evoked
et al., 2008; Schon and Przedborski, 2011). synaptic transmission was impaired during high frequency
To monitor Ca2+ dynamics, various fluorescent Ca2+ dyes stimulation, and this defect was partially rescued by ATP
and genetically encoded Ca2+ indicators (GECIs) were developed (Verstreken et al., 2005) suggesting that mitochondria plays a role
and applied both in vitro and in vivo. Also, GECIs tagged with in synaptic transmission through their ability to generate ATP
target peptide sequences have allowed imaging of Ca2+ dynamics through oxidative phosphorylation. Although mitochondrial
in specific organelles (Rizzuto et al., 1992; Palmer et al., 2004; Ca2+ uptake has limited effects on Drosophila NMJ neurons, in
Palmer and Tsien, 2006). mammalian NMJ terminals, acute inhibition of mitochondrial
Previously published reviews have already summarized the Ca2+ uptake causes rapid depression of the endplate potential
usefulness and limitations of various Ca2+ sensors and GECIs (EPP) and increased asynchronous release (David and Barrett,
applied to neuronal and non-neuronal cells (Palmer and Tsien, 2003). Furthermore, in synapses of the mammalian central
2006; Knopfel, 2012; Tian et al., 2012; Rose et al., 2014). In this nervous system (CNS), mitochondria-dependent Ca2+ uptake
review, we only describe recently uncovered insights about Ca2+ accelerates the recovery from synaptic depression in the calyx of
dynamics and its regulation by mitochondria and ER, and we Held (Billups and Forsythe, 2002). Other studies in mammalian
discuss how these organelle-specific Ca2+ sensors have been used hippocampal neurons claimed that impaired mitochondrial
for the exploration of the role of these subcellular compartments anchoring at presynaptic sites increases presynaptic Ca2+ during
in the regulation of Ca2+ homeostasis and synaptic function in repetitive stimulation and produces short-term facilitation (STF),
neurons. and insulin-like growth factor-1 receptor (IGF-1R) signaling
regulates resting mitochondrial Ca2+ level and spontaneous
UNVEILED SYNAPTIC FUNCTIONS OF transmission (Kang et al., 2008; Gazit et al., 2016). Although
MITOCHONDRIA-DEPENDENT Ca2+ most pharmacological studies employed uncoupling agents as
HOMEOSTASIS mitochondrial Ca2+ influx blocker, which may affect ATP
production, these reports support presynaptic control via
Mitochondrial Ca2+ uptake has been studied since the 1950s mitochondrial Ca2+ import (Ly and Verstreken, 2006). A recent
from studies of rat heart muscle and kidney (Slater and study demonstrates that presynaptic boutons associated with
Cleland, 1953; Deluca and Engstrom, 1961). In the nervous mitochondria display lower levels of [Ca2+ ]c accumulation than
system, mitochondria were described at presynaptic terminals presynaptic boutons not associated with mitochondria (Kwon
and dendrites of various neuronal subtypes using the light and et al., 2016). Furthermore, acute inhibition of mitochondria
electron microscope (EM) several decades ago (Bartelmez and calcium import increased [Ca2+ ]c accumulation at presynaptic
Hoerr, 1933; Palay, 1956; Gray, 1963; Shepherd and Harris, 1998; boutons occupied by mitochondria. In the same study, we
Rowland et al., 2000). In axons, mitochondria are short and demonstrate that this mitochondria-dependent regulation of
sparsely distributed, and interestingly, several studies showed [Ca2+ ]c plays an important role in regulating presynaptic release
that half of presynaptic boutons are occupied by mitochondria properties including spontaneous release, asynchronous release
(Shepherd and Harris, 1998; Kang et al., 2008). In contrast, and short-term synaptic plasticity.
dendritic mitochondria have tubular shapes and they are rarely In addition to regulation of [Ca2+ ]c clearance, Ca2+ release
observed in postsynaptic spines in the excitatory neurons (Sheng from mitochondria plays important roles at presynaptic
and Hoogenraad, 2007; Kasthuri et al., 2015). sites (Figure 1A). Following the sustained high frequency
At presynaptic boutons and terminals, synaptic vesicle stimulation, an enhancement of synaptic transmission
(SV) fusion with the plasma membrane occurs following lasting tens of seconds to minutes is observed and which
increase of [Ca2+ ]c following opening of voltage-sensitive Ca2+ is called post-tetanic potentiation (PTP; Zucker, 1989).
channels (VSCC) followed by Ca2+ binding to sensors like Mitochondrial Ca2+ release is suggested as one of the underlying
synaptotagmins (Schneggenburger and Neher, 2005; Neher and mechanisms for this prolonged enhancement of synaptic
Sakaba, 2008; Jahn and Fasshauer, 2012; Südhof, 2012). The transmission. Pharmacological inhibition of mitochondrial
ability of mitochondria to import Ca2+ into the mitochondrial Ca2+ uptake and release at the crayfish NMJ impaired PTP
matrix ([Ca2+ ]m ) plays a role in regulating presynaptic (Tang and Zucker, 1997; Zhong et al., 2001). Furthermore,

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Kwon et al. Organelle-Specific Calcium Dynamics in Neurons

FIGURE 1 | Synaptic functions regulated by endoplasmic reticulum (ER) and mitochondria-dependent Ca2+ homeostasis. (A) Schematic diagram
depicting the presynaptic functions regulated by ER- and mitochondria-dependent Ca2+ dynamics. Ca2+ release from ER can modulate spontaneous
neurotransmitter release, short-term facilitation (STF) and long-term depression (LTD). Ca2+ re-uptake by the ER controls the spontaneous release and STF.
Presynaptic mitochondria also play important roles in regulating spontaneous neurotransmitter release, STF and post-tetanic potentiation (PTP) through their ability to
regulate Ca2+ clearance. (B) A simplified schematic diagram depicting the postsynaptic functions regulated by ER- and mitochondria-dependent Ca2+ dynamics.
Ca2+ release from ER via IP3 -induced Ca2+ release (IICR) and Ca2+ -induced Ca2+ release (CICR) controls long-term potentiation (LTP) and LTD. In fact, depending
on neuronal and synaptic subtypes, IP3 R and RyR show differential distribution and distinct synaptic functions. Dendritic mitochondrial Ca2+ influx can regulate ATP
synthesis, Ca2+ homeostasis and dendritic development. In non-neuronal cell types, direct Ca2+ exchange between ER and mitochondria have been described, but
their role in neurons has not yet been documented. IP3 R, IP3 receptor; RyR, ryanodine receptor; SERCA, smooth endoplasmic reticulum Ca2+ -ATPase; VGCC,
voltage-gated Ca2+ channel; PMCA, plasma membrane Ca2+ -ATPase; NCX: the Na+ /Ca2+ exchanger; mPTP, mitochondrial permeability transition pore; MCU,
mitochondrial calcium uniporter; VDAC, voltage-dependent anion channel; mGluR, metabotropic glutamate receptor; GluN, NMDA receptor.

similar phenotypes were observed at mouse NMJ and contain mitochondria (Chicurel and Harris, 1992). However, a
hippocampal mossy fiber synapses with blocking the physiological role of these postsynaptic mitochondria is largely
mitochondrial NCX, which mediates mitochondrial Ca2+ unknown. In general, mitochondria are distributed primarily in
release (García-Chacón et al., 2006; Lee et al., 2007). dendrite shaft and therefore localized microns away from the
In contrast to presynaptic boutons and terminals, the postsynaptic density, but might still be able to buffer [Ca2+ ]c
postsynaptic function of mitochondrial Ca2+ regulation is less mobilized through Ca2+ -channels and glutamate receptors
well-documented. In mouse hippocampal pyramidal neurons (Thayer and Miller, 1990; White and Reynolds, 1995;
(Li et al., 2004), a minority (<5%) of dendritic spines contains Wang and Thayer, 2002). This mitochondrial calcium import
mitochondria. Also, large branched spines in hippocampal CA3 can stimulate tricarboxylic acid (TCA) cycle and might

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Kwon et al. Organelle-Specific Calcium Dynamics in Neurons

increase ATP production (Kann and Kovács, 2007) and Ca2+ (Gazit et al., 2016; Kwon et al., 2016; Marland et al.,
may also regulate other ATP-dependent Ca2+ pumps like 2016). Both sensors displayed action potential (AP)-dependent
PMCA and SERCA. While it is still unclear whether or not mitochondrial Ca2+ import. In addition, red fluorescent GECIs
mitochondria play significant roles in regulating postsynaptic by replacing cpEGFP with cpmApple or cpmRuby (mtRCaMP1e
[Ca2+ ]c under physiological conditions of neurotransmission, and LAR-GECO1.2) revealed mitochondrial Ca2+ import
they might play a role in pathophysiological contexts. For simultaneously with cytosolic Ca2+ (Akerboom et al., 2013; Wu
example, neurons lacking LRRK2, a protein associated with et al., 2014).
Parkinson’s disease, show impaired dendritic Ca2+ homeostasis However, these fluorescent proteins have some limitations,
through mitochondrial defects and thought to cause defective for example, they are affected by pH and mitochondrial matrix
mitochondrial depolarization and reduction in dendritic pH (pHm ) can be changed by Ca2+ influx (Abad et al., 2004;
complexity (Figure 1B; Cherra et al., 2013). Poburko et al., 2011; Chouhan et al., 2012; Marland et al.,
Overall, mitochondria-dependent Ca2+ clearance and release 2016). In addition to this point, [Ca2+ ]m can span broad ranges
in neurons plays important physiological and developmental (0.05–300 µM) depending on cell types and stimulation protocol
roles pre- and post-synaptically but their functional importance (Arnaudeau et al., 2001; Palmer and Tsien, 2006). Thus, Kd
seems to depend on the neuronal subtypes and the structure/size value for Ca2+ of mitochondrial GECI should be considered for
of the pre- and postsynaptic compartments. experimental purposes because high affinity (low Kd ) sensors can
be easily saturated by high [Ca2+ ]m and low affinity (high Kd )
MITOCHONDRIAL Ca2+ -IMAGING IN sensors may not be sensitive enough to detect small [Ca2+ ]m
NEURONS AND AT SYNAPSES changes. Several studies reported low affinity mitochondrial
Ca2+ probes for avoiding saturation (Arnaudeau et al., 2001;
To investigate organelle-specific Ca2+ dynamics, various Ca2+ Suzuki et al., 2014).
sensors are developed (Table 1). One of the first method In conclusion, these mitochondria-targeted GECIs allow
developed to monitor mitochondrial Ca2+ dynamics was imaging of mitochondria Ca2+ dynamics in neurons and have
established using rhod-2, a cationic chemical Ca2+ -binding revealed interesting, synapse-specific properties of mitochondria
fluorophore preferentially accumulating in the mitochondrial in the regulation of [Ca2+ ]c and neurotransmitter release
matrix presumably because of the highly negative membrane properties.
potential across the mitochondrial inner membrane (Minta
et al., 1989). Then, in the calyx of Held, rhod-2 and rhod-
FF (low affinity version) were used to visualize presynaptic REGULATION OF SYNAPTIC Ca2+
mitochondrial Ca2+ transient (Billups and Forsythe, 2002). DYNAMICS BY THE ENDOPLASMIC
However, these dyes cannot be precisely targeted to these RETICULUM
organelles. Therefore, GECIs have recently become the
preferred method to image Ca2+ in specific organelles including Neurons are among the most polarized cell types in our body
mitochondria. For mitochondrial matrix localization, the and consists of a soma, relatively short dendrites and long
targeting presequence of subunit VIII of human cytochrome c axons. ER is found throughout the entire length of neuronal
oxidase (COXVIII) was tagged to GECIs (Rizzuto et al., 1992). processes, and usually rough ER is prominent in the cell
Mitochondria-targeted aequorin (mt-AEQ), a luminescent body and proximal dendrites, whereas smooth ER is dominant
Ca2+ indicator, was first employed to monitor the neuronal in distal dendrites, spines and axons (Spacek and Harris,
mitochondrial Ca2+ , and this probe showed NMDA-induced 1997; Verkhratsky, 2005). ER imports and sequesters large
mitochondrial Ca2+ increase in hippocampal neurons (Baron amount of Ca2+ ([Ca2+ ]er ∼500 µM) through SERCA and
et al., 2003). However, this probe needs a chemical reaction store-operated Ca2+ entry (SOCE) mechanism (Verkhratsky,
characterized by a modest turnover rate and has very limited 2005; Bardo et al., 2006). Ca2+ release from ER is mediated
dynamic range (Palmer and Tsien, 2006). Other GECIs have by two major mechanisms, called Ca2+ -induced Ca2+ release
been developed and tested in various neuronal subtypes (CICR) and IP3 -induced Ca2+ release (IICR; Verkhratsky, 2005;
with the same targeting sequence. Mitochondrial-targeted Bardo et al., 2006). CICR is caused by the cytosolic Ca2+
ratiometric pericam (2mtRP) consists of circularly permutated increase through N-Methyl-D-Aspartate receptors (NMDAR,
Enhanced yellow fluorescent protein (cpEFYP) conjugated GluN receptors) and voltage-gated Ca2+ channels (VGCCs),
with Ca2+ -responsive calmodulin (CaM) and its binding whereas IICR is triggered by IP3 , which is generated via activation
peptide (Nagai et al., 2001; Robert et al., 2001). This probe of phospholipase C (PLC) depending on metabotropic glutamate
has a bimodal excitation spectrum and the relative emission receptors (mGluRs) or other receptors like receptor tyrosine
intensity is dependent on Ca2+ -binding. In hippocampal kinases (Figure 1).
neurons, the use of 2mtRP described mitochondrial Ca2+ Ryanodine receptors (RyRs) are involved in CICR, and they
uptake and also determined cytosolic Ca2+ rise upon synaptic have three major subtypes; RyR1, RyR2, and RyR3. All of these
activation via dual imaging with cytosolic Ca2+ dye (fura-red isoforms are detected in the brain, and show region-specific
AM; Young et al., 2008). Other CaM conjugated cpEGFPs expression (Sharp et al., 1993; Furuichi et al., 1994; Giannini
called GCaMPs (mito-GCaMP2, 2mtGCaMP6m, and mito- et al., 1995; Verkhratsky, 2005; Bardo et al., 2006; Baker et al.,
GCaMP5G) were used to monitor axonal mitochondrial 2013). Similar to RyRs, IP3 receptor (IP3 R), which mediate IICR,

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Kwon et al. Organelle-Specific Calcium Dynamics in Neurons

TABLE 1 | Organelle-specific Ca2+ sensors in neurobiology.

Organelle Sensors Neuron type Kd for Excitation Emission Dynamic Reference

Ca2+ (µM) used (nm) filter (nm) Range
(Fmax /Fmin ,
Rmax /Rmin )

Mitochondria Dye The calyx of Held 0.57, 19 575 590 3.4 Billups and Forsythe (2002)
Rhod-2, Rhod-FF

GECI
mito-aequorin Hippocampal 1–2 Luminescence Baron et al. (2003)
(Hp) neuron
2mtRP (ratioPericam) Hp neuron 1.7 Ratiometric, 535/20 10 Young et al. (2008)
405/485
mito-GCaMP2 Hp neuron 0.195 488 507 5 Marland et al. (2016)
2mtGCaMP6m Hp neuron 0.167 488 510 38 Patron et al. (2014),
Gazit et al. (2016)
mtRCaMP1e Cortical neuron 1.6 572 592.5 6.5 Akerboom et al. (2013)
LAR-GECO1.2 DRG and Hp 12 561 589 Wu et al. (2014)
neurons
ER Dye
Mag-Fura-2 Sensory neuron 53 Ratiometric, 510 25 Solovyova et al. (2002)
340/380
GECI
D1ER Hp neuron 0.8, 60 FRET, 450 475/40, 1.6 Zhang et al. (2010)
535/25
erGAP1 DRG neuron, Hp 12 Luminescence, 510 3∼4 Rodriguez-Garcia et al. (2014)
slice 403/470
G-CEPIA1er Cerebellar Purkinje 672 488 511 4.7 ± 0.3 Suzuki et al. (2014)
cell
GCaMPer (10.19) Cortical neurons 400 490 540/50 14 Henderson et al. (2015)

consist of three isoforms, IP3 R1, IP3 R2, and IP3 R3, but IP3 R1 et al., 2000; Nishiyama et al., 2000; Raymond and Redman, 2002).
is the dominant form in the brain (Sharp et al., 1993, 1999; Hippocampal mossy fiber pathway (MF, dentate gyrus to CA3)
Verkhratsky, 2005; Bardo et al., 2006; Baker et al., 2013). shows IICR- and CICR-dependent LTP and LTD, however, there
Long-term synaptic plasticity is regulated by Ca2+ - are conflicting results regarding the underlying mechanisms
dependent signaling mechanisms such as Ca2+ /calmodulin- (Figure 1B; Yeckel et al., 1999; Itoh et al., 2001; Kapur et al., 2001;
dependent kinase II (CaMKII), calcineurin (a Ca2+ -dependent Mellor and Nicoll, 2001; Lauri et al., 2003; Lei et al., 2003).
phosphatase), protein phosphatase 1 (PP1) and protein kinase C Presynaptic ER-dependent Ca2+ release is also detected and
(PKC; Malenka and Nicoll, 1999; Yang et al., 1999; Lüscher and contributes to changes in neurotransmitter release properties
Malenka, 2012). Therefore, Ca2+ release from intracellular stores and short-term synaptic plasticity at various inhibitory and
like the ER regulates long-term synaptic plasticity in specific excitatory synapses including basket cell to Purkinje cell
circuits. synapses, hippocampal MF pathway, SC-CA1 and CA3-CA3
In cerebellar Purkinje cell dendrites, mGluR-IP3 -dependent pyramidal neuron synapses (Figure 1A; Llano et al., 2000;
Ca2+ increase is observed during parallel fiber (PF) stimulation Emptage et al., 2001; Liang et al., 2002; Galante and Marty, 2003;
and this mediates long-term depression (LTD) of PF-Purkinje Lauri et al., 2003; Sharma and Vijayaraghavan, 2003; Unni et al.,
cell pathway (Finch and Augustine, 1998; Takechi et al., 1998; 2004; Mathew and Hablitz, 2008).
Miyata et al., 2000; Wang et al., 2000). At synapses made In addition to Ca2+ efflux, Ca2+ uptake by ER via SERCA
by hippocampal Schaffer collateral (SC) onto CA1 pyramidal pump affects STF at SC-CA1 presynapses and NMJ (Figure 1A;
neurons, both long-term potentiation (LTP) and LTD are linked Castonguay and Robitaille, 2001; Scullin and Partridge, 2010;
to IP3 -dependent signaling (Oliet et al., 1997; Nishiyama et al., Scullin et al., 2010). Stromal interaction molecules (STIMs)
2000; Raymond and Redman, 2002; Nagase et al., 2003). In and Orai1, which allow SOCE, are localized to neuronal
addition, CICR is also observed in CA1 pyramidal neuronal compartment including dendritic spines, and impaired SOCE
spines, and LTD is abolished in RyR3-deficient mice and alters α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
following application of RyR inhibitor (ryanodine) although receptor (AMPAR) trafficking, neuronal Ca2+ signaling and LTP
the connection between CICR and LTP is controversial in SC- in CA1 pyramidal neuron and cerebellar Purkinje neurons (Baba
CA1 pathway (Reyes and Stanton, 1996; Emptage et al., 1999; et al., 2003; Hartmann et al., 2014; Korkotian et al., 2014; Garcia-
Futatsugi et al., 1999; Sandler and Barbara, 1999; Kovalchuk Alvarez et al., 2015; Segal and Korkotian, 2015).

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Kwon et al. Organelle-Specific Calcium Dynamics in Neurons

IMAGING NEURONAL ER Ca2+ DYNAMICS is only beginning to be explored. Genetically-encoded Ca2+

sensors targeted to intracellular organelle and/or to specific
As mentioned above, ER contains high levels of Ca2+ , therefore, synapses as well as functional indicators (like pHluorin-tagged
in order to monitor Ca2+ dynamics in the ER lumen, low affinity synaptophysin or GluRs) will lead to the identification of
sensors were employed. Mag-Fura-2, a low affinity membrane- synapse- and circuit-specific roles of mitochondria and ER Ca2+
permeable dye (K d = 53 µM), has been the first applied for in neurons.
neuronal ER Ca2+ measurement, and after loading this dye in MCU has been recently shown to be associated with multiple
the cytoplasm and organelles, cytosolic dye was removed by regulatory proteins, which seems to modify or gate its gating
perfusion with dye-free pipette solution (Solovyova et al., 2002). properties and can prevent or enhance mitochondrial Ca2+
This allowed for the first time the visualization of caffeine- uptake upon changes in cytosolic Ca2+ dynamics (Perocchi
induced Ca2+ release and reuptake in the ER of dorsal root et al., 2010; Mallilankaraman et al., 2012; Csordás et al., 2013;
ganglia (DRG) neurons (Table 1). Plovanich et al., 2013; Raffaello et al., 2013; Sancak et al., 2013;
However, this method can non-specifically label internal De Stefani et al., 2015). In addition, MCU activity can be
compartments, therefore, genetically targeted sensors have been differentially controlled in different tissues (Fieni et al., 2012).
developed more recently for the visualization of ER-derived Ca2+ Therefore, future investigations should probe the function of this
dynamics (Table 1). The signal sequence of calreticulin, a Ca2+ - MCU-regulatory complex in neurons and test if MCU and/or
binding protein in ER, and an ER retention sequence, KDEL, MCU-associated proteins can act as neuronal subtype-specific
lead GECIs into the ER lumen, and to optimize for measuring and/or synapse-specific functional modifiers.
a massive amount of [Ca2+ ]er , various mutations were applied In non-neuronal cells, ER and mitochondria establish
to the CaM domain or EF-hand motif of existing GECIs for focal connections which play a key role in Ca2+ transfer
reducing their Ca2+ affinity. A fluorescence resonance energy from ER to mitochondria which has been characterized
transfer (FRET)-based Ca2+ sensor, D1ER, showed altered via intra- and inter-organelle Ca2+ imaging (Rizzuto et al.,
ER Ca2+ leak function in hippocampal neurons of presenilin 1993, 2012; Csordás et al., 2010; Kornmann, 2013). This
double knockout and Alzheimer’s disease model mice (Zhang transfer modulates ATP production in mitochondria and
et al., 2010). Also, bioluminescence-based sensor, GFP-Aequorin may also affect lipid exchange between these two organelles
protein (GAP), was modified and targeted to ER of DRG and (Voelker, 1990; Cárdenas et al., 2010; Fujimoto and Hayashi,
hippocampal neurons, and showed 3- to 4-fold larger ratio 2011). At present, in neurons, the role of Ca2+ translocation
change than D1ER (Rodriguez-Garcia et al., 2014). In addition, between ER and mitochondria is largely unknown. Although
recently established GCaMP variants for ER Ca2+ detection, immuno-EM images in vivo and Ca2+ imaging with dyes
calcium-measuring organelle-entrapped protein indicator one in respiratory motor neurons suggested ER-mitochondria
in the ER (CEPIA1er) and GCaMPer (10.19), characterized Ca2+ crosstalk, future work will need to establish the
ER Ca2+ uptake and release in cortical neurons and cerebellar context in which ER-mitochondria interface regulates
Purkinje cells (Suzuki et al., 2014; Henderson et al., 2015). Ca2+ dynamics and synaptic function (Takei et al., 1992;
Interestingly, cerebellar Purkinje cells displayed differential ER Shoshan-Barmatz et al., 2004; Mironov and Symonchuk,
Ca2+ dynamics in postsynaptic compartment depending on 2006).
the nature of synaptic inputs (Okubo et al., 2015). These
lumenal ER Ca2+ indicators also revealed interesting dynamics
AUTHOR CONTRIBUTIONS
in dendritic spines, which suggest that [Ca2+ ]er and therefore
[Ca2+ ]c can undergo synapse-specific regulation (Suzuki et al., All three authors co-wrote the manuscript.
2014; Henderson et al., 2015).
FUNDING
FUTURE PERSPECTIVES
This work was partially funded by support from the NIH-
Recent studies characterized the roles of presynaptic NINDS (R01NS067557, FP), Japan society for the promotion of
mitochondria and circuit-specific ER Ca2+ mobility in dendrites science fellowship for research abroad and The Uehara Memorial
directly via live imaging (Okubo et al., 2015; Kwon et al., 2016), Foundation (YH), and a grant from the Human Frontier Science
however, organelle-specific Ca2+ dynamics at local synapses Program long-term fellowship (S-KK).

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489–505. or reproduction is permitted which does not comply with these terms.

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 REVIEW
published: 29 June 2016
doi: 10.3389/fnsyn.2016.00018

Understanding Synaptogenesis and

Functional Connectome in C. elegans
by Imaging Technology
Jung-Hwa Hong 1, 2 and Mikyoung Park 1, 3*
1
Center for Functional Connectomics, Korea Institute of Science and Technology, Seoul, South Korea, 2 Department of Life
Sciences, Korea University, Seoul, South Korea, 3 Department of Neuroscience, Korea University of Science and Technology,
Daejeon, South Korea

Formation of functional synapses is a fundamental process for establishing neural circuits

and ultimately for expressing complex behavior. Extensive research has interrogated
how such functional synapses are formed and how synapse formation contributes to
the generation of neural circuitry and behavior. The nervous system of Caenorhabditis
elegans, due to its relatively simple structure, the transparent body, and tractable genetic
system, has been adapted as an excellent model to investigate synapses and the
functional connectome. Advances in imaging technology together with the improvement
of genetically encoded molecular tools enabled us to visualize synapses and neural
circuits of the animal model, which provide insights into our understanding of molecules
and their signaling pathways that mediate synapse formation and neuronal network
modulation. Here, we review synaptogenesis in active zones and the mapping of local
connectome in C. elegans nervous system whose understandings have been extended
Edited by: by the advances in imaging technology along with the genetic molecular tools.
George Augustine,
Nanyang Technological University, Keywords: presynaptic assembly, synaptic specificity, synaptogenesis, functional connectome, neural circuits,
Singapore imaging, C. elegans

Reviewed by:
Fabrice Ango,
University of Montpellier, France INTRODUCTION
Antonio Malgaroli,
Università Vita-Salute San Raffaele, One of the fundamental goals of neuroscience is to understand the generation of functional nervous
Italy system that underlies neural basis of behavior and cognition. Extensive research has attempted
*Correspondence: to interrogate the molecular and cellular mechanisms of synapse formation and functional
Mikyoung Park neural circuit development. Ever since it was proposed by Sydney Brenner in the mid 1960’s
[email protected]; (Brenner, 1974), the nematodes Caenorhabditis elegans (C. elegans) has been considered as an
[email protected] ideal model organism to study synaptic development and neural circuitry. The organism has
relatively simple nervous system, having 302 neurons and its neurochemistry and genetics are
Received: 25 April 2016 similar to those of mammals. Moreover, the complete structure and connectivity of C. elegans
Accepted: 17 June 2016 nervous system have been deciphered through genetic screens and reconstruction of electron
Published: 29 June 2016
micrographs (EM) of serial sections, which led to discovery of novel molecules important for
Citation: development and maintenance of functional synaptic connectivity (White et al., 1986). C. elegans
Hong J-H and Park M (2016)
with its transparent body was the first animal in which the green fluorescent protein (GFP)
Understanding Synaptogenesis and
Functional Connectome in C. elegans
was expressed (Chalfie et al., 1994). Combined with its stable expression of fluorescently tagged
by Imaging Technology. proteins (Mello et al., 1991; Frokjaer-Jensen et al., 2008), studies with C. elegans have made major
Front. Synaptic Neurosci. 8:18. contributions to our knowledge on neural development, axonal migration, and synapse formation.
doi: 10.3389/fnsyn.2016.00018 Recently, selective plane illumination microscopy (SPIM) techniques such as tiling light-sheet

Frontiers in Synaptic Neuroscience | www.frontiersin.org June 2016 | Volume 8 | Article 18 | 116

Hong and Park Imaging C. elegans Synaptogenesis and Connectome

SPIM (TLS-SPIM) (Fu et al., 2016) and inverted SPIM (iSPIM) Many studies using C. elegans have investigated the role of
(Wu et al., 2011) have been developed and utilized to achieve various proteins localized at active zone in synapse formation
high spatiotemporal resolution 3-dimensional live imaging of (Yeh et al., 2005; Watanabe et al., 2011). Classical EM
C. elegans embyos with no detectable phototoxicity, which could analysis has provided initial assessment of C. elegans synaptic
enable studies on synaptogenesis and axon guidance during components but its requirement for ultrathin sectioning of
embryogenesis in C. elegans. Another recent work adopting samples approximately 50 nm thickness (White et al., 1986)
complementation-activated light microscopy (CALM) in which limits the resolution and impairs detailed visualization of fine
proteins are conjugated with non-fluorescent split-fluorescent structures. The multifunctional synaptic scaffolding protein
proteins, which become to be fluorescent when complemented SYD-2/liprin-α is one of the key proteins identified to regulate
with synthetic peptides enabled single-molecule imaging with a synaptic development in C. elegans and Drosophila (Zhen
precision of 30 nm within synapses in live worms (Zhan et al., and Jin, 1999). The loss-of-function analysis on SYD-2/liprin-
2014). α and uncoordinated-10 (UNC-10)/Rab3-interacting molecule
Rapid developments of advanced imaging technologies have (RIM), which is another dense-projection components (Weimer
expanded our understanding of the molecular and cellular basis et al., 2006) revealed reduced vesicle recruitment at active zone
of synaptogenesis with great depth, taking a huge step closer (Stigloher et al., 2011; Kittelmann et al., 2013), and smaller dense-
to revealing functional neural connectome. Here, we discuss projection due to loss of SYD-2/liprin-α function (Kittelmann
on the synaptogenesis in presynaptic active zones revealed by et al., 2013) unlike the finding showing an expanded dense-
both conventional and advanced imaging set-ups and review projection (Zhen and Jin, 1999). One suggested explanation
recent work utilizing advanced imaging technology to unravel for variability in syd-2 mutant synaptic ultrastructure is due to
the functional connectome of neural circuits. Rather than the differences in fixation procedure (Kittelmann et al., 2013).
dealing with the mechanistic aspects of synapse formation Nevertheless, it is certain that advanced and optimized imaging
and neural circuits development, this review will mainly technique led to identification of regulatory proteins to retain
focus on how synaptic ultrastructure, synaptic formation, and synaptic vesicle at active zone.
functional neural connectome have been sophisticated by the A method which comprises of correlative fluorescence
advanced imaging technology. For more in-depth reviews on the electron microscopy was developed and optimized to observe
mechanism of synaptogenesis, synaptic specificity, and neural the nanoscopic localization of SYD-2/liprin-α in C. elegans
circuits development, see Campbell et al. (2015), Cherra and Jin active zone (Watanabe et al., 2011). The technique employed
(2015), Jin (2015), Zhen and Samuel (2015), Yogev and Shen both stimulated emission depletion (STED) microscopy and
(2014), Chia et al. (2013), and Park and Shen (2012). photoactivated localization microscopy (PALM) on ultrathin
sections for protein localization at super-resolution nanoscale
level and subsequently correlate the protein localization with
IMAGING SYNAPSE ASSEMBLY ultrastructures by electron microscope. The localization of
SYD-2/liprin-α to the C. elegans presynaptic dense-projection
Chemical synapses are specialized intercellular junctions with observed by this technique (Watanabe et al., 2011) was consistent
two apposed compartments, the pre-synaptic terminal and with the earlier finding from the immunoelectron micrograph
the postsynaptic target, and the synaptic cleft which is about (Yeh et al., 2005) but the result was more advanced to provide
20 nm gap between the pre- and postsynapses (Cowan and the precise localization of the proteins in small and dense
Kandel, 2001). Proper organization of pre- and postsynaptic structures likely within the synapse at the level of nanoscale
components with precise regulation underlies formation of super-resolution.
functional synapses. For the past decades, tremendous details In addition, studies using advanced EM tomography of
regarding the morphology and assembly of C. elegans synaptic 250 nm thick sections combined with high-pressure freezing
structure have been revealed with development of genetic tools (HPF) and freeze substitution (Stigloher et al., 2011; Kittelmann
and imaging technology. This section focuses on presynaptic et al., 2013) have resolved the highly complex structure of dense-
assembly and synaptic specificity revealed by genetically encoded projections at cholinergic neuromuscular junctions (NMJs) of
molecular tools and imaging technologies. C. elegans, revealing composition of building units forming bay-
like structures in which synaptic vesicles are docked to the
Presynaptic Active Zone Imaging active zone membrane. Furthermore, serial reconstruction of
The presynaptic compartment in C. elegans exhibits an overall HPF EM sections and EM tomography enabled the construction
structural organization similar to that in vertebrates, with of a high-resolution 3D model of presynaptic ultrastructure,
synaptic vesicles clustered in and around the electron-dense overcoming resolution limitation raised by the conventional EM
membrane structure called active zone known to serve as a and revealing a physical link between dense-projections and
major site of neurotransmitter release. Ultrastructural analysis synaptic vesicles within C. elegans presynaptic active zone.
have shown that, despite the variations among the appearances,
synapses of various organisms commonly display synaptic vesicle Presynaptic Assembly Imaging
docking and fusion at active zone that can be identified by darkly Cell type-specific tagging of synaptic proteins with fluorescent
stained membrane structures (Zhai and Bellen, 2004; Ackermann reporter has been a key reagent to study synaptogenesis and its
et al., 2015). regulation in C. elegans (Nonet, 1999; Shen and Bargmann, 2003;

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Hong and Park Imaging C. elegans Synaptogenesis and Connectome

Sieburth et al., 2005; Yeh et al., 2005). Hierarchical assembly to ectopic localizations of presynaptic components, including
of presynaptic active zone was observed in C. elegans HSNL RAB-3, SYD-1, SYD-2/liprin-α, GIT, and ELKS-1/ERC/CAST
synapses by fluorescently labeling the multiple active zone in the HSNL regions where the secondary vulval epithelial cells
proteins and expressing them in the various mutant animals made contacts (Figure 1A). This supported the idea that along
(Patel et al., 2006). Fluorescent protein fused with a synaptic with SYG-2/Nephrin as an upstream signal of SYG-1/Neph1,
vesicle-associated protein RAB-3 visualized synaptic vesicle SYG-1/Neph1 defines the presynaptic localization and is
clusters and confirmed the presynaptic localization of various sufficient to recruit presynaptic components, including the two
active zone components, including SYD-1, SYD-2/liprin-α, key scaffold molecules SYD-1 and SYD-2/liprin-α (Zhen and Jin,
ELKS-1/ERC/CAZ-associated structural protein (CAST), GIT, 1999) to the regions defined by its localization (Patel et al., 2006;
and SAD-1 kinase in the HSNL synapses (Patel et al., 2006). Figure 1B).
Altering the location of SYG-1/Neph1 by ectopically expressing In syd-1 and syd-2 mutants, the presynaptic components,
SYG-2/Nephrin in the secondary vulval epithelial cells, led including RAB-3, ELKS-1/ERC/CAST, GIT, SAD-1,

FIGURE 1 | Synaptic specificity regulated by non-neuronal factors. (A) Synaptic connectivity of neurons and muscles associated in the egg-laying circuit of C.
elegans. HSNL forms synapses with vulval muscle 2 (vm2) and ventral cord (VC) motor neurons, VC4 and VC5 specifically to the regions immediately adjacent to the
primary epithelial cells (1◦ ) which secretes SYG-2/Nephrin. Mutations in SYG-1/Neph1 or SYG-2/Nephrin disrupt synaptic specificity of HSNL and cause ectopic
synapse formation with select body wall muscle (BWM). Ectopic positioning of SYG-2/Nephrin to the secondary epithelial cells (2◦ ) recruited SYG-1/Neph1 to HSNL
near the secondary epithelial cells, which was shown to be sufficient to form synapses ectopically at the sites where SYG-1/Neph1 is recruited (yellow circles). (B)
Pathway for HSNL synapse assembly. SYG-2/Nephrin ensures proper localization of SYG-1/Neph1 which defines presynaptic localization of the active zone proteins.
ELKS-1/ERC/CAST could function redundantly with SYD-1 or other unidentified presynaptic proteins that positively regulate synapse assembly (gray lines). In the
presence of SYD-1, the SYD-2/liprin-α and ELKS-1/ERC/CAST interaction was enhanced (red arrow). In the presence of RSY-1, SYD-1, and ELKS-1/ERC/CAST
interaction is weakened (solid green), suggesting RSY-1 as a negative regulator in the HSNL presynaptic assembly process likely by weakening the SYD-2/liprin-α and
ELKS-1/ERC/CAST interaction (dotted green) indirectly through the RSY-1 and SYD-1 interaction. Plain lines indicate biochemical interactions. (C) Synaptic
connectivity of AIY and RIA interneurons regulated by ventral cephalic sheath cells (CEPshV) at C. elegans nerve ring. Synapses between AIY and RIA are formed en
passent as they are ensheathed in zone 2 by CEPshV, which secretes UNC-6/Netrin that regulates UNC-40/DCC activity in AIY. Abnormal distend positioning of
CEPshV toward zone 1 causes ectopic localizations of both presynapses (red circles) and UNC-40/DCC (purple triangles) in zone 1 of AIY. (D) Pathways for AIY and
RIA connectivity. CEPshV secretes UNC-6/Netrin, which regulates both positioning of presynapses in AIY and axon guidance of postsynaptic RIA through
UNC-40/DCC activity to the location specified by CEPshV.

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Hong and Park Imaging C. elegans Synaptogenesis and Connectome

UNC-57/endophilin, and SNN-1/synapsin-1 were failed to Inaki et al., 2007) or from a guidepost cell (Christopherson
be assembled, identifying those presynaptic components et al., 2005; Colon-Ramos et al., 2007). In C. elegans, synaptic
as downstream molecules of SYD-1 and SYD-2/liprin-α in the contacts are usually formed en passant, in which synapses are
active zone assembly process (Patel et al., 2006). Gain-of-function formed along the adjacent processes but not its terminus (White
mutation (Dai et al., 2006) or overexpression of SYD-2/liprin-α et al., 1986). Synaptic specificity studies in C. elegans have been
(Patel et al., 2006) in syd-1 mutants completely restored the accelerated upon the development of the expression tool of
synaptic accumulation of SNB-1/synaptobrevin, whereas the fluorescently tagged proteins in specific cell types driven by cell
SYD-1 overexpression in syd-2 mutants was not sufficient to type-specific promoters, which enabled researchers to specifically
induce the rescue effect (Patel et al., 2006), illustrating the SYD-1 label pre-, postsynapses, and neighboring guidepost cells (Nonet,
and SYD-2/liprin-α mediated presynaptic assembly with the 1999; Shen and Bargmann, 2003; Grunwald et al., 2004; Francis
SYD-1 as an upstream of SYD-2/liprin-α (Figure 1B). Although et al., 2005; Sieburth et al., 2005; Yeh et al., 2005; Hoerndli et al.,
the loss of ELKS-1 function by itself did not induce apparent 2013).
defects in synapse assembly in C. elegans HSNL synapses (Dai A specific synaptic connectivity between amphid interneuron
et al., 2006; Patel et al., 2006), synapse formation in the syd-2 Y (AIY) and ring interneuron A (RIA) in C. elegans nerve ring,
gain-of-function and syd-1 double mutants exhibited a high considered as brain of the animal, was fluorescently visualized
dependency on ELKS-1 expression (Dai et al., 2006), suggesting by expressing presynaptic RAB-3 in AIY and postsynaptic
that ELKS-1 functions redundantly with SYD-1 or other glutamate receptors GLR-1 in RIA (Colon-Ramos et al., 2007;
presynaptic proteins that positively regulate synapse assembly Shao et al., 2013) (Figure 1C). The localization of synaptic
(Figure 1B). connectivity between AIY and RIA has shown to be restricted
Regulator of synaptogenesis-1 (RSY-1) was cloned as a in the zone 2 of AIY axon (Figure 1C) and such specificity
negative regulator of synapse formation for its deletion mutants is achieved by activation of both UNC-6/Netrin, a well-known
to lead to extra synapse formation and exhibit increased axon guidance molecule that is exclusively expressed by glia-
accumulation of SNB-1/synaptobrevin at presynaptic sites in like ventral cephalic sheath cells (CEPshV) (Wadsworth et al.,
the HSNL (Patel and Shen, 2009). A single-cell in situ 1996) and the netrin receptor UNC-40/Deleted in Colorectal
protein-protein interaction assay revealed enhanced interaction Cancer (DCC) (Colon-Ramos et al., 2007), supporting the idea
between SYD-2/liprin-α and ELKS-1/ERC/CAST in presence that secreted molecules from glia govern synaptic specificity.
of SYD-1 while direct interaction between SYD-1 and ELKS- Confocal microscopy revealed the projection of the CEPshV
1/ERC/CAST is weakened in presence of RSY-1, suggesting processes with respect to the region of innervation between
RSY-1 as a negative regulator of C. elegans HSNL synapse AIY and RIA (Figure 1C). Loss-of-function in either UNC-
assembly likely by weakening the SYD-2/liprin-α and ELKS- 34/enabled, a regulator of the actin cytoskeleton (Colon-Ramos
1/ERC/CAST interaction indirectly through its interaction with et al., 2007) or circuit maintenance abnormal protein (CIMA-
SYD-1 (Figure 1B). Together, presynaptic differentiation at 1), a regulator of synaptic maintenance in C. elegans (Shao
C. elegans HSNL synapses was initiated by SYG-1/Neph1, et al., 2013), caused morphological alterations in CEPshV which
a synaptic specificity molecule that defines the location of migrated toward further posteriorly to ensheath AIY axon in
presynaptic sites along the HSNL axon, leading to activate zone 1 (Figure 1C). Morphological alterations in CEPshV led
the presynaptic assembly process by recruiting the two key to ectopic localization of both UNC-40/DCC and presynaptic
scaffolding proteins SYD-1 and SYD-2/liprin-α. SYD-2/liprin- components in zone 1 (Figure 1C) due to the existence of UNC-
α-centered assembly of presynaptic components was achieved 6/Netrin secreted from CEPshV in zone 1 (Colon-Ramos et al.,
through the inter-communications among positive (SYD- 2007). The process of RIA in unc-34 mutants also abnormally
1 and ELKS-1/ERC/CAST) and negative (RSY-1) regulators migrated toward zone 1 where the ectopic synapses were formed
(Figure 1B). (Figure 1C). Together, UNC-40/DCC plays two independent
roles in each neuron, which are positioning of presynapses in
AIY and axon guidance of postsynaptic RIA to the location
IMAGING SYNAPTIC SPECIFICITY specified by CEPshV (Figure 1D). These findings further support
the model of non-neuronal contribution to the regulation of
During the event of synapse formation, a precise apposition precise localization of synaptogenesis.
between the presynaptic release sites and postsynaptic receptors Earlier than the AIY-RIA synaptic specificity study, the
must be accomplished to ensure a rapid neurotransmitter C. elegans egg-laying circuit, which is predominantly innervated
release and reliable synaptic response. Neurons can select by the two hermaphrodite-specific motor neurons (HSNs), HSNL
subpopulations of neurons they form synapses onto and can also and HSNR, and the two ventral cord (VC) motor neurons, VC4
select the defined specific subcellular sites to establish synapses and VC5 has been reported to be regulated by non-neuronal
(Akins and Biederer, 2006; White, 2007; Margeta and Shen, factor. HSNL and HSNR synapse onto vulval muscle cells and
2010). Such synaptic specificity is achieved by trans-synaptic onto the VC4 and VC5 neurons, while VC4 and VC5 neurons also
adhesion between pre- and postsynaptic neurons (Yamagata synapse onto the vulval muscle cells. Despite the direct contact
et al., 2002; Graf et al., 2004; Choe et al., 2006), adhesion between HSN and VC processes, synapses formed between these
between the presynaptic neuron and a guidepost cell (Shen cells are only restricted to the regions adjacent to the vulva
and Bargmann, 2003; Shen et al., 2004), molecules secreted (White et al., 1986) (Figure 1A). The specific positioning of
from pre- or postsynaptic neurons (Umemori et al., 2004; synapses and the recognition between HSNL and its target

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Hong and Park Imaging C. elegans Synaptogenesis and Connectome

were determined by adjacent vulva epithelial guidepost cells that light-sensitive proteins such as channelrhodopsins (Nagel et al.,
express SYG-2/Nephrin. SYG-2/Nephrin interacts with SYG- 2003, 2005), halorhodopsins (Han and Boyden, 2007; Zhang
1/Neph1 expressed in the HSNL, to recruit SYG-1/Neph1 to the et al., 2007; Husson et al., 2012b), and archaerhodopsins (Ihara
site along the HSNL axon where presynaptic sites are developed et al., 1999) as optogenetic actuators to either activate or inhibit
(Shen and Bargmann, 2003; Shen et al., 2004) (Figure 1A). neuronal activity via light and genetically encoded sensors such
More recently, introduction of the GFP reconstitution as GCaMP calcium indicator (Tian et al., 2009) and Clomeleon
across synaptic partners (GRASP) developed in C. elegans chloride indicator (Kuner and Augustine, 2000; Berglund et al.,
has overcome the challenges addressed by labor-intensive 2006) as optogenetic sensors to monitor responses to the synaptic
conventional EM analysis and increased the spatial resolution inputs. This section will discuss various experimental imaging
to visualize the pre- and postsynaptic contacts. GRASP is based approaches to interrogate the neural connection using the C.
on functional complementation between two non-fluorescent elegans nervous system.
split-GFP fragments separately expressed in the pre- and The initial optogenetics was applied to manipulate the
postsynaptic neurons, which label synapses between two cells behavior of C. elegans (Nagel et al., 2005). Expression of
of close proximity in living animals (Feinberg et al., 2008). Channelrhodopsin-2 (ChR2), a blue light-gated depolarizing
Using GRASP, specific visualization of synaptic contacts between cation channel used to activate neural activity in C. elegans
AIY and RIA was observed with high spatial resolution (Shao body muscles caused blue light-evoked contractions (Nagel et al.,
et al., 2013). In addition, GRASP revealed restricted synaptic 2003, 2005), whereas expression of NpHR, a yellow light-gated
localization between AIY and CEPshV (Shao et al., 2013), which hyperpolarizing chloride ion pump applied to inhibit neural
is consistent with the published EM data (White et al., 1986). activity in C. elegans muscle cells caused an extension of the
Formation of ectopic synapses between AIY and CEPshV due worm’s body length and locomotion defects by whole-field
to morphological alteration in CEPshV was confirmed as well illumination of yellow-green light (Zhang et al., 2007; Husson
(Shao et al., 2013) (Figure 1C). GRASP application has also et al., 2012b). Aside from NpHR, a yellow-green light-sensitive
confirmed the SYG-1/Neph1 and SYG-2/Nephrin as synaptic archaerhodopsin-3 (Arch) (Ihara et al., 1999) and a green-
specificity regulators of HSN synapses with vulval muscles and blue light-sensitive Mac (Waschuk et al., 2005) have also been
VC neurons. Analyzing GRASP fluorescence in wild-type and expressed in C. elegans and induced a stronger optical silencing
syg-1 or syg-2 mutants recapitulated the synaptic connectivity effect than NpHR likely due to efficient protein trafficking to
of HSN neurons (Feinberg et al., 2008) (Figure 1A). Besides the plasma membrane (Chow et al., 2010; Husson et al., 2012b).
the C. elegans nervous system, the GRASP has also been widely The simultaneous use of Arch and Mac enabled inhibition
adapted by other model systems, such as Drosophila (Gordon and of two different neuronal subpopulations, depending on the
Scott, 2009; Gong et al., 2010) mouse (Kim et al., 2012; Yamagata illuminating lights used.
and Sanes, 2012) and the cultured hippocampal neuronal system Light-sensitive probes expressed in C. elegans in vivo are
(Tsetsenis et al., 2014). Lately, newly modified GRASP strategies, mostly under the control of a promoter sequence. However,
involving activity-dependent synaptic GRASP and multi-color promoter-driven single cell expression of optogenetic protein
fluorescence reconstitution across synapses (X-RASP) have been is challenging to achieve due to the lack of single cell-specific
validated in Drosophila, allowing preferential labeling of active promoter and instead proteins are diversely expressed, eliciting
synapses and multi-color labeling of active synapses in one robust behavioral responses upon whole-field illumination
animal (Macpherson et al., 2015; Li et al., 2016). Continuous (Husson et al., 2013). Although it may be useful for inspecting
development of GRASP shows the potential to expand the utility a novel optogenetic protein, optical manipulation of individual
of GRASP to identify and map synaptic connectivity of neural neurons needs to be accomplished in order to obtain insights
circuits in the living animal with high resolution. into individual contribution by single neurons in functional
connectivity. To this aim, new methods have been adapted
in C. elegans to drive selective optical manipulation, either
IMAGING FUNCTIONAL NEURAL by genetically modulated single cell-specific expression of
CIRCUITS optogenetic protein (Ezcurra et al., 2011; Schmitt et al., 2012; Cho
and Sternberg, 2014; Guo et al., 2015) or selective illumination of
An underlying goal of neuroscience is to understand the neural target neurons with a high spatial and temporal resolution (Guo
connectome that are responsible for synaptic function and et al., 2009; Leifer et al., 2011; Stirman et al., 2011; Husson et al.,
neuronal basis of behavior. Anatomical structural connectome 2012a,b; Kocabas et al., 2012; Cohen et al., 2014; Luo et al., 2014;
of the whole nervous system of C. elegans, which has been fully Shipley et al., 2014; Trojanowski et al., 2014) in order to dissect
mapped by EM of serial sections (White et al., 1986), has served functional connections within the neural circuits (Table 1).
as a useful resource for researchers to study circuit function, Mainly adapted approach to specifically deliver light-sensitive
thus making the C. elegans nervous system as an excellent opsins to individual neurons of C. elegans restricts the opsin
model to investigate functional connectome of neural circuits. expression by genetic application using Cre or FLP recombinases
For the past decade, optogenetics has been widely adapted (Ezcurra et al., 2011; Schmitt et al., 2012; Cho and Sternberg,
to manipulate neural circuits and examine the corresponding 2014; Guo et al., 2015) (Figure 2). The recombinase-dependent
changes in synaptic function and behavior (Fang-Yen et al., gene expression is driven by a set of two promoters, a first
2012; Husson et al., 2013). Optogenetics uses genetically encoded promoter driving the expression of opsin conjugated with a

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Hong and Park Imaging C. elegans Synaptogenesis and Connectome

TABLE 1 | Cell-specific optogenetic applications in C. elegans

Single-cell stimulation approach Circuit Cells manipulated References

Cre or FLP Avoidance ASH, AVA Ezcurra et al., 2011

recombinase application
Locomotion ASH, AVA, PVC Schmitt et al., 2012

Avoidance circuit ASH, AVA, Cho and Sternberg, 2014

during sleep behavior RIM, RIG

Nociception and avoidance ASH, ASI*, ADF* Guo et al., 2015

Selective illumination Locomotion DB, VB Leifer et al., 2011

Avoidance ASH, RIM Guo et al., 2009

ALM, AVM, PLM Stirman et al., 2011

Leifer et al., 2011
Shipley et al., 2014

Nociception AQR, FLP, PVD Husson et al., 2012a

ASH, ALM, AVM Husson et al., 2012b

PVD Cohen et al., 2014

Chemotaxis AIB, AIY, AIZ, RME, SMB Kocabas et al., 2012

ASER Luo et al., 2014

Feeding MC, M1, M2, M4 Trojanowski et al., 2014

*Optogenetic manipulation driven by neuronal type-specific promoters rather than Cre/FLP recombinase application.

fluorophore along with or without a bicistronic fluorescent ASH to the interneurons AVA and AVD and the connections
reporter and a second promoter driving the expression of Cre between the interneurons RIM and AVA have been monitored
or FLP recombinase. In the first promoter-containing construct, (Guo et al., 2009; Table 1).
a transcription termination sequence flanked by recombinase Improvement in microscopic analysis and optogenetic
recognition sequences, loxP or FRT that are recognized by Cre illumination system allowed manipulation of neural activity in a
or FLP recombinase is enclosed in front of opsin. The Cre or freely behaving C. elegans with a high spatiotemporal resolution,
FLP recombinase-mediated recombination of loxP or FRT sites providing an in-depth analysis on functional neural circuits
excised the stop sequence and allows conditional expression underlying behavior at a single-cell level. A modified three-
of opsin only in the target cell where both promoters are panel liquid crystal display (3-LCD) projector for simultaneous
active (Husson et al., 2013) (Figure 2). Using Cre and FLP multicolor illumination and a motorized X-Y stage for keeping
system, ChR2 were specifically expressed in PVC interneurons the unrestrained worm centered in the camera’s field of view
which evoked forward locomotion and in AVA interneuron and with a standard inverted epifluorescence microscope were
ASH sensory neurons which evoked backward-movement upon systemized (Stirman et al., 2011; Husson et al., 2012b) and
photostimulation (Ezcurra et al., 2011; Schmitt et al., 2012) the Colbert system was equipped to control locomotion and
(Table 1). Further effort to isolate exclusive expression of the behavior in real time (Leifer et al., 2011; Luo et al., 2014;
light-sensitive proteins in a single cell (Ezcurra et al., 2011) would Shipley et al., 2014). Spatial regulation of optical illumination
need to define the role of individual single neurons in functional is controlled either by estimating the coordinates of targeted
neural circuits. cells using the machine-vision algorithms (Leifer et al., 2011;
Instead of using genetically generated system and whole-field Trojanowski et al., 2014) or by calculating the anterior-posterior
illumination, spatiotemporally patterned illumination of neurons (A-P) axis (Stirman et al., 2011). Both systems have been
expressing light-sensitive optogenetic proteins in immobilized instrumental in defining neural coding of several behaviors
C. elegans was used by for the first time in vivo using a digital in C. elegans linked to the motor circuit, avoidance circuit,
micromirror device (DMD) whose individual mirrors can be nociceptive circuit, chemotaxis circuit, and feeding circuits of
controlled independently to precisely determine the location freely moving worms (Table 1). Using AIY expressing ChR2 and
and size of the regions to be illuminated while simultaneously targeted illumination by the DMD technology, it was shown that
recording the calcium levels using a genetically encoded calcium optogenetic manipulation of AIY activity alone was sufficient
sensor, GCaMP to analyze the functional connections among to evoke chemotactic behavior in freely moving C. elegans, and
neurons. Combining the optogenetic actuator ChR2 and the was suggested that AIY is plausible to act as a control node
sensor GCaMP with the patterned illumination via a DMD for coordinating other taxis behaviors as well (Kocabas et al.,
technology, the functional connections from the sensory neuron 2012). Another report using the Colbert system equipped with

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Hong and Park Imaging C. elegans Synaptogenesis and Connectome

FIGURE 2 | Restricted expression of light-sensitive opsin mediated by Cre or FLP recombinases. Promoter 1-containing construct is designed to drive
expression of opsin with a fluorescent reporter. Promoter 2 drives expression of Cre or FLP recombinase. Conditional expression of opsin is mediated by the Cre or
FLP recombinases by removing a transcription termination sequence flanked by loxP or FRT only in target cell where the both promoters are active.

the DMD investigated an experience-dependent salt chemotaxis As genetically encoded fluorescent proteins have been rapidly
circuit. Optogenetic manipulation of neuronal activity of the developed for the past decades since the GFP was introduced
ASER sensory neuron expressing ChR2 was shown to be in the field, it is also expected that the number of optogenetic
connected to positive and negative chemotaxis in response to salt tools will rapidly increase to likely provide optogenetic proteins
concentrations, indicating that ASER sensory neuron encodes with different spectral properties (Zhang et al., 2008; Gradinaru
the perception of salt concentration and the memory of the et al., 2010) and ionic specificities (Han and Boyden, 2007;
chemotactic set point in a chemotaxis circuit of C. elegans (Luo Zhang et al., 2007) and help expand the understanding of
et al., 2014). In addition, optogenetic manipulations of specific synaptic function and neural circuits. During such processes,
pharyngeal neurons MC, M2, M4, and I1 in freely behaving it is confidently predicted that C. elegans will provide a
worms by adopting ChR2 for optical stimulation and Mac for systematic in vivo platform to test the optogenetic tools newly
optical silencing along with the DMD for targeted illumination developed and to ultimately apply to the synaptic function and
revealed a pharyngeal pumping/feeding circuit and identified the functional connectome studies. Together with the improvement
regulation of feeding rate by nicotinic and muscarinic receptors of fluorescent and optogenetic tools, continuous development in
through the pharyngeal neuronal network (Trojanowski et al., C. elegans imaging technology will promise a breakthrough in
2014). Furthermore, multispectral illumination (Stirman et al., deciphering functional neural connectome.
2011) enables simultaneous application of optical stimulation In addition to the monitoring and controlling of existing
and inhibition to an individual animal. Emerging studies have neuronal circuits via optogenetic applications and advanced
successfully facilitated multimodal optogenetic manipulation on microscopy systems as described in this review, it is very
C. elegans to independently excite different neurons in a single plausible to develop the ways to actively manipulate neural
worm (Erbguth et al., 2012; Husson et al., 2012b; Schild and circuits for instance by inserting new connections or removing
Glauser, 2015). existing connections, resulting in the reprogramming of neural
circuits. Indeed, a recent study on artificial modifications
PERSPECTIVES of neural circuits was reported in C. elegans by expressing
transgenically targeted heterologous connexin to insert
C. elegans is currently the best organism to study synapses and a new electrical synapse between normally unconnected
neuronal circuits because the connectivity of its 302 neurons neurons in intact animals, which resulted in altered salt
has been well-defined by serial reconstruction of EM (White taste and olfactory chemotaxis behavior (Rabinowitch
et al., 1986), the body is transparent, and it is a genetically et al., 2014). Conversely, laser ablation method can be
tractable animal model. C. elegans was one of the first organisms used to remove existing connection (Sulston and White,
that GFP was expressed to label protein (Chalfie et al., 1994), 1980; Bargmann et al., 1993; McIntire et al., 1993; Fang-
GRASP was utilized to visualize specific synaptic contacts Yen et al., 2012; Rabinowitch et al., 2013). Such artificial
(Feinberg et al., 2008), optogenetics was applied to manipulate modification of neural circuits not only help understand
behavior of live animals (Nagel et al., 2005), and more recently, fundamental functions of neuronal connectivity underlying
sonogenetics using low-pressure ultrasound was challenged to complex behavior but could also be applied to disease
activate specific ultrasonically sensitized neurons and modify brain circuits with the purpose of therapeutics at the circuit
locomotory behavior (Ibsen et al., 2015). level.

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Hong and Park Imaging C. elegans Synaptogenesis and Connectome

AUTHOR CONTRIBUTIONS ACKNOWLEDGMENTS

All authors listed, have made substantial, direct and intellectual The work in M. Park laboratory was supported by the KIST
contribution to the work, and approved it for publication. Institutional Programs (Project No. 2E26190 and 2E26170).

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Yogev, S., and Shen, K. (2014). Cellular and molecular mechanisms of synaptic Conflict of Interest Statement: The authors declare that the research was
specificity. Annu. Rev. Cell Dev. Biol. 30, 417–437. doi: 10.1146/annurev- conducted in the absence of any commercial or financial relationships that could
cellbio-100913-012953 be construed as a potential conflict of interest.
Zhai, R. G., and Bellen, H. J. (2004). The architecture of the active zone
in the presynaptic nerve terminal. Physiology (Bethesda) 19, 262–270. doi: Copyright © 2016 Hong and Park. This is an open-access article distributed under the
10.1152/physiol.00014.2004 terms of the Creative Commons Attribution License (CC BY). The use, distribution or
Zhan, H., Stanciauskas, R., Stigloher, C., Dizon, K. K., Jospin, M., Bessereau, J. reproduction in other forums is permitted, provided the original author(s) or licensor
L., et al. (2014). In vivo single-molecule imaging identifies altered dynamics of are credited and that the original publication in this journal is cited, in accordance
calcium channels in dystrophin-mutant C. elegans. Nat. Commun. 5, 4974. doi: with accepted academic practice. No use, distribution or reproduction is permitted
10.1038/ncomms5974 which does not comply with these terms.

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