Single-Synapse Analysis of a Diverse Synapse Population:
Proteomic Imaging Methods and Markers
Kristina D. Micheva, Brad Busse, Nicholas C. Weiler, Nancy O’Rourke, and Stephen J
Smith
Department of Molecular and Cellular Physiology, Stanford University, Stanford, California 94305
Summary
A lack of methods for measuring the protein compositions of individual synapses in situ has so far
hindered the exploration and exploitation of synapse molecular diversity. Here we describe the use
of array tomography, a new high-resolution proteomic imaging method, to determine the
composition of glutamate and GABA synapses in somatosensory cortex of Line-H-YFP Thy-1
transgenic mice. We find that virtually all synapses are recognized by antibodies to the presynaptic
phosphoprotein synapsin I, while antibodies to 16 other synaptic proteins discriminate amongst 4
subtypes of glutamatergic synapses and GABAergic synapses. Cell-specific YFP expression in the
YFP-H mouse line allows synapses to be assigned to specific presynaptic and postsynaptic
partners and reveals that a subpopulation of spines on layer 5 pyramidal cells receives both
VGluT1-subtype glutamatergic and GABAergic synaptic inputs. These results establish a means
for the high-throughput acquisition of proteomic data from individual cortical synapses in situ.
Introduction
Rapidly accumulating physiological and genetic evidence establishes that the molecular
diversity of synapses extends far beyond that envisioned by traditional classification
schemes based solely on neurotransmitter identity. For instance, it is now clear that within
each neurotransmitter category (e.g., glutamatergic, GABAergic, cholinergic) there is
substantial diversity in the expression of many intrinsic synaptic proteins, including
neurotransmitter transporters and receptors (Gupta et al., 2000; Hausser et al., 2000; Staple
et al., 2000; Cherubini and Conti, 2001; Craig and Boudin, 2001; Cull-Candy et al., 2001;
Grabowski and Black, 2001; Huang and Bergles, 2004; Mody and Pearce, 2004; Grant,
2006). Until synapse molecular diversity is properly fathomed, it is likely to be a
troublesome source of variability in physiological and neurodevelopmental experimentation.
Conversely, a systematic understanding of synapse diversity (i.e. the synaptome) is likely to
provide valuable new perspectives on the organization of synaptic circuitry (i.e. the
connectome), its development, plasticity and disorders. It is easy to envision, for instance,
that a potential catalog of molecular synapse types (Grant, 2007) would help explorations of
the synaptic basis of specific memory or disease processes to focus more fruitfully on
specific synapse subpopulations.
Correspondence: kmicheva@stanford.edu (K.D.M.); sjsmith@stanford.edu (S.J S.).
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Author Manuscript
Neuron. Author manuscript; available in PMC 2011 November 18.
Published in final edited form as:
Neuron. 2010 November 18; 68(4): 639–653. doi:10.1016/j.neuron.2010.09.024.
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To place a possible molecular catalog of synapse types on a firm footing, two broad
experimental challenges remain. First, it is essential that synapse populations be explored at
the single-synapse level. Until recently, the only way to reliably resolve and characterize
individual synapses was by way of electron microscopy (EM). While traditionally a time-
consuming and very volume-limited method, recent advances in EM (Denk and Horstmann,
2004; Harris et al., 2006; Knott et al., 2008; Anderson et al., 2009; Kasthuri and Lichtman,
2010) have greatly improved its throughput, even offering the possibility of detailed
neuronal circuit reconstruction. Nonetheless, EM still provides only very limited proteomic
discrimination (although Anderson et al., 2009, describe a very powerful new approach to
integrating small-molecule discrimination with EM). Secondly, synapse diversity must be
explored in situ, in ways that retain full fidelity to the intact tissue setting and allow for the
acquisition of as much information as possible about circuit context and cellular
morphology.
Array tomography (AT) is a high-resolution proteomic imaging method (Micheva and
Smith, 2007; Micheva et al., 2010) that exploits a combination of light and EM approaches
to resolve fine details at the level of synapses across large fields of view spanning entire
circuits. Of prime significance to the present application, AT allows the
immunofluorescence resolution of single synapses within cortical neuropil, where such
resolution is highly problematic for other optical methods. Additionally, AT can acquire
many more dimensions of immunofluorescence information about single synapses than
previous methods (up to 17 in the present work, as compared to the standard
immunofluorescence limit of three or four). AT also benefits from greatly improved
quantitative reliability, since both staining and imaging are completely independent of depth
within a tissue sample. Finally, AT delivers very high experimental throughput: our present
automated methods acquire image data at a rate of approximately one million synaptic
protein puncta per hour. Such throughput will help advance the analysis of synaptic diversity
from the anecdote to the realm of solid bioinformatics. AT thus seems uniquely suited to
meet the challenges of exploring the molecular diversity of cortical synapses.
Here, we describe array tomographic immunofluorescence methods for the single-synapse
analysis of mouse cortex, focusing on the discrimination and analysis of glutamatergic and
GABAergic synapses. Toward a goal of identifying every single cortical synapse as
unambiguously as possible, we evaluated antibody markers to presynaptic proteins likely to
be common to all synapses, such as synaptophysin, bassoon, and synapsin. We find that
antibodies to the presynaptic phosphoprotein synapsin I (De Camilli et al., 1983; Hilfiker et
al., 1999) are particularly robust and useful, labeling the vast majority of cortical synapses
with a minimum of labeling at non-synaptic loci. For increased confidence in synapse
identification, we also develop here a basis for conjoint use of multiple synaptic markers.
We argue that antibodies to the glutamatergic synaptic proteins VGluT1, VGluT2, PSD-95,
GluR2, NMDAR1 and the GABAergic synaptic proteins GAD, VGAT and gephyrin can be
used both to distinguish reliably between glutamatergic and GABAergic synapses and begin
the work of searching for finer synapse molecular subtypes within these broad categories.
Results
A note about color use
We have adopted a colorblind-friendly scheme in as many figures as possible. In figures
with only two immunofluorescence channels (Figures 3, 6, 7, 8C,D) we use magenta and
green as additive colors, such that regions of overlap display as white. When three or more
channels need to be displayed (Figures 1, 2, 4 and 8A,B), we represent each channel with a
non-transparent color plane. In this case, colors are non-additive, for example, white in such
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figures is not the result of magenta and green overlap but rather a distinct color representing
a distinct immunofluorescence channel.
AT resolves individual puncta of multiple synaptic proteins in mouse cortex
Figure 1 offers a panoramic view of a volume of somatosensory cortex from a YFP-H Thy-1
transgenic mouse (Feng et al., 2000) representative of the specimens used in the present
work. The volume image rendered here was acquired by automated AT imaging of a mosaic
of 52 fields per section over 60 serial sections (200 nm each) in three fluorescence spectral
channels, comprising a total of 9,360 individual image tiles. The tiles were stitched and
aligned in three-dimensions and rendered as described in Methods. This volume is 12 μm
thick, 0.5 mm wide and extends a distance of 1.4 mm from the pial surface of the cortex
through all cortical layers past the subcortical white matter and into a portion of the
underlying striatum. The three fluorescence channels represented are YFP fluorescence
(green), anti-tubulin (blue) and anti-synapsin I (magenta) immunofluorescence. The vast
information content of the volume presented in Figure 1 is better appreciated from dynamic
volume renderings as in Supplemental Movie S1.
YFP fluorescence in the cortex of line H mouse represents a soluble YFP marker
transgenically expressed in a large subset of layer 5 pyramidal neurons. This mouse line was
used because the YFP-expressing neurons provide a useful anatomical framework, with their
apical dendrites extending all the way to layer 1 and their axons forming conspicuous
bundles in the white matter. However, YFP fluorescence is not necessary for the subsequent
single synapse analysis, which can be performed also in a wild type mouse. In addition to
the YFP labeled neurons, the apical dendrites of pyramidal cells not expressing YFP are
evident from tubulin immunostaining of their core microtubule bundles (Figure 1D). Finally,
the presence of aggregates of synapsin I protein in the neuropil is apparent from the magenta
puncta, which can be individually discerned (Figure 1C–H). The spatial distribution of
synapsin immunoreactivity is consistent with that expected of neocortical synapses. For
example, cell bodies appear as circular spaces largely devoid of synapsin puncta. The few
puncta observed within such voids are actually situated in front or behind the cell bodies, as
the depth of this volume is larger than the diameters of most cortical cell bodies. There is no
staining in blood vessels and very few puncta in the white matter (Figure 1H). All of the
data that follow were collected from arrays similar to that represented in Figure 1, but image
acquisition was, for expediency’s sake, carried out in single fields of view in layers 4 and 5,
corresponding to the areas in Figure 1E and F, respectively. Thinner, 70 nm sections were
used to increase z-resolution and the sampling of each synapse.
While the distribution of synapsin puncta resembles that of cortical synapses, it is not clear
whether all synapsin puncta represent synapses and whether all synapses are
immunoreactive for synapsin. Synapsin I is highly concentrated in presynaptic boutons (De
Camilli et al., 1983) and has been used extensively as a general synaptic marker, but a one-
to-one relationship between synapsin puncta and synapses has not yet been demonstrated.
Therefore, it cannot be assumed that synapsin immunofluorescence data alone are sufficient
for synapse identification. Also, there are other proteins, for example synaptophysin and
bassoon, which are highly concentrated at presynaptic boutons and could be useful as
general markers for synapses. To evaluate candidate cortical synapse markers, we developed
a panel of antibodies that label a variety of pre- and postsynaptic proteins (Table 1, see
Supplemental Experimental Procedures for antibody characterization). Because of the
proteomic capability of AT to immunostain sections multiple times with different sets of
antibodies, we were able to measure numerous pre- and postsynaptic markers at every
putative synaptic locus. An example of multiple antibody labeling is shown in Figure 2,
which represents a volume rendering from layer 4 of the somatosensory cortex of a YFP-H
mouse immunostained with 10 different antibodies against synaptic proteins (synapsin,
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bassoon, VGluT1, VGluT2, PSD95, GluR2, NMDAR1, GAD, VGAT, gephyrin) and one
against tubulin. The sequence of antibody application is presented in Table S1 (dataset
KDM-SYN-090416). In addition, two other fluorescent labels (YFP and DAPI) were
imaged, making a total of 13 fluorescent channels collected from each section.
Synaptic protein distributions imaged by AT correlate as expected from synapse structure
Some of the antibody markers in Table 1 are expected to be present at all synapses in
cortical neuropil, while others are specific for particular synapse subtypes. For example, as
universal presynaptic proteins, synapsin and synaptophysin puncta overlap (Figure 3A). The
majority of synapsin puncta also overlap with VGluT1, known to be present in most cortical
glutamatergic synapses. In addition, synapsin puncta are closely apposed with PSD95 puncta
as would be anticipated from imaging pre- and postsynaptic proteins at single synapses.
GAD puncta, expected to label inhibitory GABAergic synapses, overlap with a small subset
of synapsin puncta, and synapsin levels are generally lower in these synapses.
To quantify globally the extent of spatial correlation amongst various synaptic marker
candidates, we designed a correlation matrix test based on the van Steensel method (van
Steensel et al., 1996; see Experimental Procedures for full description). The basic idea is to
test for the effect of very small relative displacements between pairs of marker images on a
measurement of image overlap. Because of the abundance of many synaptic markers,
overlapping spatial distributions might occur by chance. If the association between two
channels is real, however, then any shift of one channel relative to the other will decrease
the observed degree of colocalization. On the other hand, if two channels tend to be
mutually exclusive, then a shift will increase the degree of colocalization. Finally, if the
association between two channels is occurring by chance, then a shift will not substantially
affect the degree of colocalization. Using a 20 × 20 × 6.3 μm
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volume of neuropil from
dataset KDM-SYN-091207 (Table S1), we computed a cross-correlation score for pairs of
channels over a range of lateral offset distances. From the 17 antibodies used in this dataset,
we focused on the general presynaptic markers synapsin, synaptophysin and bassoon, as
well as several specific markers for glutamatergic (VGluT1, VGluT2, PSD95 and GluR2)
and GABAergic synapses (GAD and VGAT).
The cross-correlation score is represented in Figure 3B as a grid of false colored squares
with centers corresponding to the score at 0 offset and each pixel shift equal to 0.1 μm
offset. To visualize the data, different channel pairs are also shown as immunofluorescent
images from a small area of a single section of the same dataset. As can be seen in the
correlation matrix, both synapsin and synaptophysin, and to a lesser extent bassoon,
colocalize with all other synaptic markers, including those of smaller subsets of synapses
that contain VGluT2 or GAD. All synaptic markers are anticorrelated with tubulin, which
labels microtubules within dendrites and cell bodies. VGluT1 and VGluT2, found in cortical
glutamatergic synapses, do not colocalize with the GABAergic markers. PSD95 and GluR2,
both present at the postsynaptic side of glutamatergic synapses, correlate strongly with each
other and more weakly with the presynaptic glutamatergic markers. GAD and VGAT,
presynaptic markers for GABAergic synapses, show strong correlation. An interesting
distinction can be made between the presynaptic markers with respect to their colocalization
with postsynaptic markers. Presynaptic markers that are associated with synaptic vesicles
(e.g. synapsin, synaptophysin, VGluTs) show high colocalization among themselves, while
their colocalization with postsynaptic markers such as PSD95 and GluR2 is weaker. On the
other hand, the presynaptic marker bassoon, which labels the presynaptic active zone, shows
similar colocalization with both pre- and post-synaptic markers. This is due to the fact that
the synaptic vesicle cluster is situated far enough from the postsynaptic density to be
resolved by AT. On the other hand, the presynaptic active zone is only one synaptic cleft
(around 20 nm) away from the postsynaptic density which is below the resolution
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capabilities of AT. For example, in single section images in Figure 3B, synapsin puncta are
seen next to PSD95 and GluR2 puncta, while bassoon overlaps with these postsynaptic
markers.
AT immunofluorescence of synapsin is highly reliable as synapse marker
A single marker protein detectable at all synapses and only at synapses would be very useful
for many purposes, but thus far there has been no conclusive demonstration of any such
marker. While numerous markers, e.g., intrinsic proteins of synaptic vesicles, might be
localized at every chemical synapse, the usefulness of any such antibody marker would be
diminished if it were found at non-synaptic loci as well. From the colocalization matrix of
Figure 3B, it is evident that both synapsin and synaptophysin colocalize strongly with all
other synaptic markers and thus might be useful as general markers for synapses. Further
examination of the immunofluorescence images revealed, however, that synaptophysin
immunoreactivity is also fairly often detectable at obviously extrasynaptic sites, e.g., in cell
body and dendritic cytoplasm and nuclei (Figure 3A). Synaptophysin puncta moreover tend
to be smaller and less continuous than synapsin puncta. For these reasons, the synapsin I
antibody appeared to be the stronger candidate as a reliable synaptic marker and was
subjected to further evaluation.
Synapsin is detectable at virtually all dendritic spines—Almost all dendritic spines
in adult cortex receive synapses and therefore a general synaptic marker should be present at
these sites. To determine the distribution of synapsin puncta at spines, we reconstructed the
apical dendrites of YFP-positive layer 5 pyramidal cells extending through layer 4 in tissue
that was immunostained for both pre- and post-synaptic proteins (Figure 4)..
Immunofluorescence reveals PSD95 puncta within spine heads that are closely associated
with both synapsin and bassoon puncta. Two dendritic segments from dataset KDM-
SYN-091207 were used to quantify the number of spines contacted by synapsin puncta.
Only synaptic marker immunofluorescence within 0.5 μm of the YFP dendrites was
considered for this analysis. One of the dendritic segments was 45 μm in length, 2 μm in
width and had 131 spines (2.9 spines/μm). From the 116 spines included in their entirety
within the imaged volume, 114 had at least one synapsin punctum associated with them. The
other dendritic segment was 93 μm long, 1.7 μm wide and had 117 spines (1.3 spines/μm),
from which 110 were completely included in the volume. All of these spines were associated
with a synapsin punctum. Thus, more than 99% of the dendritic spines on layer 5 pyramidal
neurons were in the immediate vicinity of a synapsin punctum. Moreover, other pre- and
postsynaptic proteins colocalized with the synapsin puncta at these dendritic spines (100%
of synapsin puncta with pre- and 98% with postsynaptic markers).
EM analysis supports the identification of synapses with synapsin
immunoractivity—The use of the synapsin antibody as a general synaptic marker was
also assessed at the EM level. Postembedding immunoEM using synapsin and a secondary
antibody conjugated to colloidal gold (15 nm) labeled presynaptic boutons, as identified by
the presence of synaptic vesicles and adjacent postsynaptic densities (Figure 5C). The
relatively low density of immunogold labeling is likely due to the addition of 0.1%
glutaraldehyde and 0.1% OsO4 during tissue preparation, which is necessary for
ultrastructural preservation but significantly impairs immunogenicity. Indeed, synapsin
immunofluorescence on sections from tissue treated this way is much weaker than on our
conventional sections used for the rest of this study (Figure S2).
We then compared synapsin immunofluorescence with the corresponding ultrastructure to
assess what proportion of synapses identified at the EM level are fluorescently labeled.
Because the tissue preparation for EM significantly reduces synapsin immunolabeling this
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analysis will result in an underestimate of the presence of synapsin at synapses. Serial
sections from tissue prepared for EM observation and mounted on coverslips were first
immunofluorescently labeled with the synapsin antibody and imaged with the fluorescent
microscope. The sections were then poststained with uranyl acetate and lead citrate and
viewed in the SEM using the backscattered electron detector. The fluorescent and SEM
images were aligned using the DAPI signal and the nuclei as viewed in the SEM. The bright
DAPI-stained puncta in the nuclei correspond to the electron dense heterochromatin masses
(Figure 5A,B). A comparison of the ultrastructurally identified synapses with synapsin
immunofluorescence revealed that 91% of synapses (279 out of 305) were synapsin positive
on at least one section. The intensity of synapsin immunofluorescence did not correlate with
the size of the synapse as seen in the SEM. For example, some big presynaptic boutons
(asterisk on Figure 5B) were very weakly labeled. Thus, despite the reduced
immunoreactivity with conjugate immunofluorescence – SEM imaging, 91% of synapses
were synapsin-positive, which is consistent with synapsin being a reliable marker for
immunofluorescent imaging of cortical synapses.
Multiple synaptic proteins can be visualized volumetrically as a ‘synaptogram’ mosaic
The large number of immunofluorescence stains used with AT presents a challenge for
visualization. The traditional color-coding cannot be used for so many channels, and volume
reconstruction along any single axis can obscure weak labels or show false colocalization of
markers. Therefore, we devised a representation for multi-channel volumetric image data,
called a ‘synaptogram’ (Figure 6), that is useful for single-synapse analysis. All possible
synaptic loci are identified with synapsin immunostaining and represented by single channel
serial sections in a matrix where each section occupies a column and each channel a row.
Sections represent a 1×1μm
2
area centered on the centroid of the synapsin punctum. With
synaptograms many antibody labels can be visualized simultaneously and spatial
relationships among labeled structures can be examined with precision and relative ease. For
example, the synaptograms in Figure 6 use 18 different fluorescent signals: the general
synaptic markers synapsin, synaptophysin and bassoon (2 different antibodies), 8
glutamatergic markers, 4 GABAergic markers and 2 structural markers (Dataset KDM-
SYN-091207, Table S1). The glutamatergic synapse on the left has a distinct synaptogram
appearance compared to the GABAergic synapse on the right. Both synapses contain the
general synaptic markers synapsin, synaptophysin and bassoon. The glutamatergic synapse
contains presynaptic VGluT1 as well as a number of postsynaptic scaffold and receptor
markers (PSD95, MAGUK, GluR2, NMDA receptor subunits). The GABAergic synapse is
distinguished by the presence of presynaptic GAD and VGAT as well as the postsynaptic
scaffold protein gephyrin and GABAA receptor subunit. Both of the synapses are adjacent
to a YFP-positive process and it appears that the glutamatergic synapse is making a contact
with this process (postsynaptic markers overlap with the YFP signal), while the GABAergic
synapse is not (postsynaptic markers away from the YFP process).
The synaptogram makes it easy to check for the continuity of a given marker punctum from
one serial section to the next, and the 3D colocalization of multiple markers that would be
expected at a true synapse. The synaptogram can also be useful in identifying fluorescence
signals that are clearly not synaptic structures, such as staining artifacts, and excluding them
from the analysis. For example, the presence of a synaptic vesicle immunofluorescence
signal in just one isolated section is unlikely to originate from an actual synapse, because
synaptic vesicle clusters almost always have a minimum extent greater than the 70 nm
thickness.
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AT imaging discriminates multiple glutamatergic and GABAergic synapse subtypes
To begin characterizing the diversity of cortical synapses we focused on a panel of 10
antibodies. Synapsin and bassoon were used as general synaptic markers. VGluT1, VGluT2,
PSD95, GluR2 and NMDAR1 were used as markers for glutamatergic synapses. The
vesicular glutamate transporters VGluT1 and VGluT2 were included because their
expression reportedly varies depending on the intracortical or subcortical origins of the
synapses. In particular it is believed that VGluT2 is predominantly expressed in
thalamocortical synapses (Fremeau et al., 2001; De Gois et al., 2005; Graziano et al., 2008).
GAD, VGAT and gephyrin were used as markers for GABAergic synapses. This
combination of antibodies allowed the identification of two general types of synapses:
glutamatergic and GABAergic, with the glutamatergic synapses further subdivided into
subtypes containing VGluT1, both VGluT1 and 2, VGluT2 and other (lacking VGluT1 and
2) (Figure 7A).
Synapses formed by axons of layer 5 pyramidal neurons belonged to the glutamatergic
VGluT1 subtype (Figure 7D), as evidenced by examination of YFP-positive presynaptic
boutons. From 96 YFP synapses in layer 4 and 110 YFP synapses in layer 5, all had
associated VGluT1, not VGluT2, immunofluorescence.
To further evaluate the reliability of our approach to single-synapse analysis, we calculated
the proportion of synapses falling into each synaptic subtype from two experiments
performed on tissue sections from the same region of the same animal, but with a different
order of antibody application (Table S1, datasets KDM-SYN-090416 and KDM-
SYN-091207). A cluster of glutamatergic markers was considered to be a synapse only if
both pre- and postsynaptic markers were present. The requirement for the presence of pre-
and postsynaptic markers was not applied to the GABAergic synapses, because it is not yet
known whether the postsynaptic scaffold gephyrin is present at all synapses of this type
(Fritschy et al., 2008). Instead, we adopted the less stringent criterion of colocalization of
several presynaptic markers belonging to different compartments (e.g. cytoplasmic,
presynaptic active zone and vesicular). Thus, in addition to the presence of a ubiquitous
synaptic marker (e.g. synapsin or bassoon) and the cytoplasmic GAD, we required the
presence of the vesicular marker VGAT, which has been shown to specifically localize to
presynaptic boutons (Chaudhry et al., 1998). The results obtained from these 2 experiments
were very similar (Table 2). Approximately 84% synapses were glutamatergic and 16%
GABAergic. There were almost twice as many VGluT2 containing synapses (VGluT1 + 2
subtype and VGluT2 only subtype) in layer 4 compared to layer 5 (21.4% vs 12.9%). On
average, only 4% of synapsin puncta were not associated with other synaptic proteins and
therefore most likely do not represent synapses. The proportion of glutamatergic and
GABAergic synapses, as well as the preference of VGlut2 synapses for layer 4 are
consistent with previous studies (e.g. Micheva and Beaulieu, 1995;Fremeau et al.,
2001;Graziano et al., 2008). These results, reproducible between 2 separate experiments,
confirm the reliability of single-synapse analysis with AT. It should be noted that only one
animal was analyzed quantitatively, and the results are aimed at evaluating the technique,
not providing reference proportions of synapse types in the mouse somatosensory cortex.
This will, undoubtedly, require a larger number of animals and is beyond the scope of the
present study.
AMPA and NMDA receptors distributions vary at different glutamatergic
synapses—There is great variability in the expression levels, subunit composition and
localization of AMPA and NMDA receptors at synapses, which significantly affects their
functional properties (e.g. Craig and Boudin, 2001). To further characterize cortical
synapses based on the type of postsynaptic receptors present (Figure 7C), 110 synapses were
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randomly selected in each of layers 4 and 5 using synapsin immunostaining. Inhibitory
synapses were excluded from the analysis. The distribution of glutamatergic receptors was
very similar in both cortical layers. The great majority of excitatory synapses contained both
AMPA and NMDA receptors, as identified with antibodies against GluR2 and NMDAR1
respectively (85.4% in layer 4 and 84.5% in layer 5). In 11.8% of synapses in both layers
only AMPA receptors could be detected and in 2.7% of synapses in layer 4 and 3.6% in
layer 5 – only NMDA receptors. GluR2 and, to a lesser extent, NMDA labels often extended
through more sections than PSD95, or were observed in sections adjacent to PSD labeling.
Synapsin is present at all glutamatergic and GABAergic synapses, but in varying amounts
In addition to enabling the study of synaptic diversity, the establishment of markers for
synapse subtypes also allowed us to revisit the question of whether synapsin is expressed in
all cortical synapses. From the conjugate immunofluorescence – SEM analysis it was
observed that 91% of ultrastructurally identified synapses were labeled for synapsin, but, as
mentioned before, those were suboptimal conditions for immunostaining. To better
understand what proportion of synapses are labeled with synapsin in our tissue prepared for
immunofluorescence, synapses were identified using different combinations of pre- and
postsynaptic markers, excluding synapsin. Glutamatergic VGluT1 synapses were identified
by the combination of VGluT1 - PSD-95 antibodies, VGluT2 synapses with VGluT2 -
PSD-95 antibodies and GABAergic synapses with VGAT and gephyrin antibodies. One
hundred synapses from each group were chosen randomly and the synapsin
immunofluorescence associated with them was measured on all sections through the
synapse. Synapsin immunofluorescence was above background levels in all analyzed
synapses (Figure 7B). VGluT1 synapses contained the highest average synapsin levels (151
± 7 arbitrary units) compared to VGluT2 (111± 7 a.u.) and VGAT synapses (81 ± 4 a.u.). A
significant proportion of VGluT2 (17%) and VGAT synapses (12%) contained low levels of
synapsin immunofluorescence (below 40 a.u.) compared to only 2% of the VGluT1
synapses.
These data further confirm that synapsin I can be used as a general synaptic marker because
it appears to be present in all mouse glutamatergic and GABAergic cortical synapses.
However, synapsin content as detected with immunofluorescence varies depending on
synapse type with some VGluT2 and VGAT synapses exhibiting low levels of synapsin.
Thus, applying a simple intensity threshold of synapsin immunofluorescence should be
avoided because this can lead to underestimation of the synapse subtypes exhibiting low
synapsin levels.
Double innervated spines on layer 5 pyramidal neurons are contacted by a VGluT1 and a
GABAergic synapse
Double innervated dendritic spines are an intriguing synaptic arrangement involved in
cortical plasticity and are found in a variety of species and cortical areas. Until now they
have been observed only by EM (Jones and Powell, 1969; Micheva and Beaulieu, 1995;
Knott et al., 2002; Jasinska et al., 2010). Very little is known about the identity of either the
input or the target in this arrangement (Dehay et al., 1991; Kubota et al., 2007) especially in
the mouse somatosensory cortex. AT can resolve neighboring synapses, as can be seen on
Figure 5B, where adjacent synapses imaged in SEM are represented by separate
immunofluorescent synapsin puncta. We therefore used AT to further characterize dually
innervated spines. Examples of dendritic spines receiving 2 synaptic inputs, one
glutamatergic and one GABAergic, are presented in Figure 8. These spines emerge from the
apical dendrites of YFP-positive layer 5 pyramidal neurons. From 2 datasets (KDM-
SYN-090416 and KDM-SYN-091207), 22 unequivocal double innervated spines were
identified, i.e. both pre- and postsynaptic markers were present for the two inputs. Of those
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spines, 82 % received a glutamatergic VGluT1 input and a GABAergic input. The remaining
18% received a glutamatergic VGluT2-containing (VGluT2 only, 9%, and VGluT1 and 2,
9%) as well as a GABAergic input. The great majority of the glutamatergic synapses
contained both AMPA and NMDA receptors (91%), one spine was seen with NMDA
receptors only, and one spine had neither AMPA nor NMDA receptor markers present.
Thus, layer 5 pyramidal neurons have apical dendrites that contain spines innervated by both
excitatory and inhibitory synapses, and the glutamatergic input to these spines is
predominantly VGluT1-positive.
Discussion
Our results demonstrate that AT can be used for the efficient detection of individual
synapses within brain tissue. Because of its proteomic capabilities and high resolution, AT
allows a detailed characterization of individual synapses by their pre- and postsynaptic
molecular composition, targets and spatial relationship to adjacent synapses. In addition, this
technique can acquire very large volume images with high throughput which, together with
a future development of advanced computational methods for data analysis, can provide for
the first time the means for large-scale exploration of synaptic diversity.
In the present study, we identify a synapsin I antibody as a reliable marker for cortical
synapses. Synapsin I associates exclusively with small synaptic vesicles and is concentrated
in presynaptic boutons (De Camilli et al., 1983). The great majority of synapses are thought
to contain synapsin and this protein has been used extensively as a general synaptic marker.
However, the list of possible exceptions to this rule has grown recently and now includes
ribbon synapses in the retina (Mandell et al., 1990), reticulogeniculate synapses (Kielland et
al., 2006) and some GABAergic and VGluT2 containing synapses in the cerebral cortex
(Bragina et al., 2007). AT, because of its increased sensitivity of immunofluorescence
detection, ability to analyze multiple antibody stains and use immunofluorescence in
conjunction with EM, allowed us to examine in detail the relationship between synapsin
puncta and synapses in the mouse cerebral cortex.
For the synapsin antibody to be relied upon as a universal synapse marker, the following
conditions had to be confirmed: 1) Synapsin labels the great majority of synapses; 2)
Synapsin background labeling is minimal and can be differentiated from the labeling of
synapses; and 3) Synapsin imaging has the resolution for discerning adjacent synapses. This
study presents multiple lines of evidence that a rabbit polyclonal synapsin I antibody labels
the vast majority of synapses in the mouse somatosensory cortex. For example, conjugate
SEM of sections stained and imaged for synapsin immunofluorescence revealed that 91% of
ultrastructurally identified synapses are immunofluorescently labeled on at least one section.
Because the preparation of the tissue for EM sharply decreases antigenicity, this is a
conservative estimate of the number of synapsin positive synapses. In tissue prepared for
immunofluorescence, more than 99% of dendritic spines have an adjacent synapsin
punctum. Also, all glutamatergic and GABAergic synapses identified by different
combinations of pre- and postsynaptic markers contain synapsin label, albeit some at low
levels. The background synapsin immunofluorescence is very low and only occasional
puncta can be seen in cell body or large dendrite cytoplasm, or nuclei. Very few synapsin
puncta (around 4%) exist alone, away from other synaptic markers. Synapsin
immunofluorescence colocalizes with other presynaptic markers such as bassoon, VGluT1,
VGluT2, VGAT and GAD and is also found immediately adjacent to postsynaptic markers
such as PSD95, GluR2, NMDAR1 and gephyrin. These relationships were confirmed both at
the synapse population level using a normalized cross-correlation analysis and for individual
synapses using synaptograms. Finally, synapsin labeling with AT allows for the resolution
of juxtaposed synapses as can be seen from conjugate EM. At the light level, when multiple
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immunofluorescent labels are used, adjacent synaptic puncta can be observed that colocalize
with different sets of pre- and postsynaptic antibodies and thus clearly belong to 2 different
synapses.
The reliability of synapsin as a universal synaptic marker can be strengthened with the
concomitant use of multiple strategically chosen synaptic markers. This not only helps the
unequivocal identification of synapses, but also allows the positive identification of
glutamatergic and GABAergic synapses within cortical neuropil and reveals the existence of
several synaptic subtypes within those broad categories. Based on the presence of vesicular
glutamate transporters, the glutamatergic synapses could be divided into those containing
only VGluT1, only VGluT2, both VGluT1 and 2, and neither VGlut1 or 2. Synapsin
immunofluorescence was detected in all synapses, but varied in intensity depending on the
synapse type. It was highest in VGluT1 containing synapses, followed by VGluT2 synapses
and GABAergic synapses. Previous studies (e.g. Bragina et al., 2007) have noted this
heterogeneity of synapsin content in cortical synapses of rats. Using confocal microscopy in
Vibratome sections, synapsin was observed in ~90% of VGluT1 puncta and only 30–50% of
VGluT2 and VGAT puncta. This discrepancy with our results is probably due to the
inability to detect small synapsin puncta using confocal microscopy. Previously, we
observed fewer small synapsin puncta with confocal microscopy of Vibratome sections
compared to AT on LR White sections prepared from the same animal (Micheva and Smith,
2007). The varying synapsin content in different synapse types is probably related to the
different functional properties of the synapses. For example, release probability is low at
VGluT1 synapses and high at VGluT2 (Fremeau et al., 2001) and many VGAT synapses
(e.g. synapses made by parvalbumin-containing fast-spiking interneurons). Interestingly,
there is evidence that synaptophysin is also expressed at a lower level in GABAergic
synapses (Grønborg et al., 2010).
The proportions of different synaptic types was found to be very similar in layers 4 and 5,
with the exception of VGluT2 containing synapses which, as observed in previous studies
(Fremeau et al., 2001), were more prominently represented in layer 4. In addition, the
existence of a sizable population of glutamatergic synapses that contain both VGluT1 and 2
was detected. There were several-fold more synapses containing both VGluT1 and 2 (15%
in layer 4 and 10% in layer 5) than purely VGluT2 synapses (6% in layer 4 and 2.5% in
layer 5). It was previously thought that the expression of VGluT1 and 2 in synapses in the
adult animal is mostly complementary (Fremeau et al., 2001), but later studies have revealed
the existence of both VGluTs in the same cortical synapses (Nakamura et al., 2007),
particularly, the thalamocortical terminals in layer 4 (Graziano et al., 2008). VGluT1,
VGluT1 and 2, and VGluT2 containing synapses appear to have distinct intracortical and
subcortical origins, but the exact details of their identities are still being explored (Fremeau
et al., 2001; De Gois et al., 2005; Graziano et al., 2008). Interestingly, the expression of the
two vesicular glutamate transporters can be regulated by activity in opposite directions (De
Gois et al., 2005). Thus, determining the VGluT1 and 2 content of synapses may provide
information about their synaptic activity as well. Including more molecular markers in the
single-synapse analysis is expected to reveal additional synaptic categories and contribute
further to our understanding of synaptic diversity.
AT also allowed us to observe double innervated spines at the light level and to begin
characterizing their input. The YFP fluorescence expressed in pyramidal neurons in the
YFP-H mouse line conveniently outlines dendritic spines, but similar analysis can also be
performed in wild type mice on neurons labeled by intracellular microinjections of
fluorescent tracers or by in utero electroporation. Between 5 and 30% of cortical dendritic
spines in a variety of species are thought to receive two synaptic inputs, one excitatory and
one inhibitory (Jones and Powell, 1969; Le Vay and Gilbert, 1976; Micheva and Beaulieu,
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1995). This intriguing synaptic arrangement is involved in cortical plasticity and is an
example of a specific synapse subtype, namely inhibitory synapses on spines in layer 4, that
changes in response to modifications in sensory experience and/or learning (Micheva and
Beaulieu, 1995; Knott et al., 2002; Jasinska et al., 2010). Sensory deprivation induced by
whisker removal results in a selective reduction of inhibitory synapses on spines, while
increased sensory stimulation or classical conditioning involving the whiskers results in a
selective increase in inhibitory synapses on spines. No direct evidence existed about the
identity of either the dendritic spines or their inputs in mouse somatosensory cortex. A
recent study in the frontal cortex of young rats suggests that double-innervated spines are
preferentially targeted by VGluT2-containing thalamocortical afferents, while the inhibitory
input is from all subtypes of cortical interneurons (Kubota et al., 2007). Using AT, we were
able to observe the double innervated spines of layer 5 pyramidal neurons in mouse
somatosensory cortex at the light level and confirm that they receive one glutamatergic and
one GABAergic synapse. We also show for the first time that the glutamatergic input is
predominantly VGluT1-containing and that both AMPA and NMDA receptors are present at
the postsynaptic site. The addition of synaptic markers such as neuropeptide markers could
provide information about the identity of the inhibitory input.
Conclusion
Here we demonstrate the usefulness of AT for the proteomic examination of individual
synapses in natural brain tissue with full preservation of neuroanatomical and circuit context
information. As efficient automated analysis strategies are developed to complement the
inherently high throughput of array tomographic image acquisition, this tool should open
new doors to the large-scale bioinformatic exploration of the molecular diversity and
architecture of synapses. One likely consequence of such exploration could be the
development of new schemes for the differentiation and cataloging of molecular synapse
types. By isolating specific subsets of synapses, a synapse catalog could help enormously in
pinpointing the specific synapse changes involved in particular neurological disorders
(Luscher and Isaac, 2009) or forms of neural plasticity (Micheva and Beaulieu, 1995; Knott
et al., 2002; Hofer et al., 2009; Xu et al., 2009; Yang et al., 2009). AT’s unique abilities to
extract simultaneously rich proteomic and fine-scale structural information also suggests
that the method may substantially advance ongoing efforts to integrate the structural and
molecular views of neuronal microcircuit function.
Experimental Procedures
Tissue preparation
All procedures related to the care and treatment of animals were approved by the
Administrative Panel on Laboratory Animal Care at Stanford University. Four adult mice:
three C57BL/6J and one YFP-H (Feng et al., 2000), were used for this study. The animals
were anesthetized by halothane inhalation and their brains quickly removed and placed in
4% formaldehyde and 2.5% sucrose in phosphate-buffered saline (PBS) at room
temperature. Each cerebral hemisphere was sliced coronally into 3 pieces and fixed and
embedded using rapid microwave irradiation (PELCO 3451 laboratory microwave system
with ColdSpot; Ted Pella, Redding CA) as described in Micheva et al, 2010. To preserve
YFP fluorescence in the YFP-H mouse, the tissue was dehydrated only up to 70% ethanol.
For EM, the tissue was processed as above except that the fixative also contained 0.1 %
glutaraldehyde and a postfixation step was added with osmium tetroxide (0.1%) and
potassium ferricyanide (1.5%) with rapid microwave irradiation, 3 cycles of 1 min on −1
min off −1 min on at 100W, followed by 30 min at room temperature.
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LRWhite sections
Ribbons of serial ultrathin (70 nm) sections were cut with an ultramicrotome (EM UC6,
Leica Microsystems, Wetzlar, Germany) as described in (Micheva et al, 2010). The ribbons
were mounted on subbed coverslips (coated with 0.5% gelatin and 0.05% chromium
potassium sulphate) and placed on a hot plate (~ 60°C) for 30 min. For SEM imaging the
subbed coverslips were also carbon coated using a Denton Bench Top Turbo Carbon
Evaporator (Denton Vacuum, Moorestown, NJ). Subbed and carbon coated coverslips were
also prepared for mounting ribbons of sections to be used for multiple immunostaining
rounds (>6). For transmission electron microscope (TEM) the sections were collected on
formvar coated nickel grids.
Immunofluorescence staining and antibodies
Staining was performed as described in (Micheva et al.,, 2010). The coverslips with sections
were mounted using SlowFade Gold antifade with DAPI (Invitrogen, Carlsbad CA). To
elute the applied antibodies, the mounting medium was washed away with dH
2
O and a
solution of 0.2M NaOH and 0.02% SDS in distilled water was applied for 20 min. After an
extensive wash with Tris buffer and distilled water, the coverslips were dried and placed on
a hot plate (60°C) for 30 min.
The primary antibodies and their dilutions are listed in Table 1. Only well characterized
commercial antibodies were used and they were evaluated specifically for AT as described
in Supplemental Experimental Procedures. For immunofluorescence, Alexa Fluor 488, 594
and 647 secondary antibodies of the appropriate species, highly pre-adsorbed (Invitrogen,
Carlsbad CA) were used at a dilution 1:150. The sequence of antibody application in the
multi-round staining is presented in Supplemental Table 1.
ImmunoEM staining
The staining protocol was similar to the immunofluorescence staining with the addition of 2
steps in the beginning: treatment for 1 min with saturated sodium metaperiodate solution in
dH
2
O to remove osmium and 5 min with 1% sodium borohydride in Tris buffer to reduce
free aldehydes resulting from the presence of glutaraldehyde in the fixative. A 15 nm gold
labeled goat anti-rabbit IgG secondary antibody (SPI Supplies, West Chester, PA) was used
at 1:25 for 1h. After washing off the secondary antibody, the sections were treated with 1%
glutaraldehyde for 1 min to fix the antibodies in place and the sections were poststained with
5% uranyl acetate for 30 min and lead citrate for 1 min.
Fluorescence microscopy and image processing
Sections were imaged on a Zeiss Axio Imager.Z1 Upright Fluorescence Microscope with
motorized stage and Axiocam HR Digital Camera as described in Micheva et al., 2010.
Briefly, a tiled image of the entire ribbon of sections on a coverslip was obtained using a
10x objective and the MosaiX feature of the software. The region of interest was then
identified on each section with custom-made software and imaged at a higher magnification
with a Zeiss 63x/1.4 NA Plan Apochromat objective, using the image-based automatic focus
capability of the software. The resulting stack of images was exported to ImageJ, aligned
using the MultiStackReg plugin and imported back into the Axiovision software to generate
a volume rendering. When a ribbon was stained and imaged multiple times, the
MultiStackReg plugin was used to align the stacks generated from each successive imaging
session with the first session stacks based on the DAPI channel, then a second ‘within stack’
alignment was applied to all the stacks.
To reconstruct large volumes of tissue (Figure 1), we first used Zeiss Axiovision software to
stitch together the individual high-magnification image tiles and produce a single ‘mosaic’
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image of each antibody stain for each serial section in the ribbon. We created a z-stack of
mosaic images for each fluorescence channel, and then grossly aligned the stacks using the
MultiStackReg plugin. Finally, to remove non-linear physical warping introduced into the
ribbons by the sectioning process, we used a second ImageJ plugin, autobUnwarpJ
(available at http://www.stanford.edu/~nweiler), which adapts an algorithm for elastic image
registration using vector-spline regularization (Arganda-Carreras et al., 2006).
For the figures, images representing single sections were upscaled using bicubic
interpolation. No other image processing was used except for adjustment of brightness/
contrast in some of the channels (NMDA receptor subunits and gephyrin). Synapsin
immunofluorescence was not adjusted. For volume renderings, meant only for visual
appreciation, more extensive image processing was used to adequately illustrate the spatial
distribution and relationship between different markers. No quantifications or substantive
comparisons were based on these images.
Colocalization analysis
To examine the spatial relationships between synaptic markers, we developed a
colocalization detection function similar to the van Steensel method (van Steensel at al.,
1996). Using a 20 × 20 × 6.3 μm
3
volume of neuropil, for each pair of channels we
computed the three-dimensional normalized cross-correlogram (Lewis, 1995) for a range of
lateral offsets approximating the size of a synapse, using Eaton’s extension of the MATLAB
function normxcorr2 (http://www.cs.ubc.ca/~deaton/tut/normxcorr3.html). Pairs of labeled
channels with nonrandom associations (either positive or negative) should demonstrate a
nonzero correlation effect which asymptotically approaches 0 at offset ranges exceeding
their scale of interaction.
Transmission and Scanning electron microscopy
The immunostained TEM grids were imaged using a JEOL TEM1230 equipped with a
Gatan 967 slow-scan, cooled CCD camera. For SEM following fluorescence imaging, the
immunostained arrays were washed with dH
2
0 to remove the mounting medium and
poststained with 5% uranyl acetate in H
2
0 for 30 min and lead citrate for 1 min. The arrays
were imaged on a Zeiss Sigma scanning electron microscope equipped with field emission
gun using the backscattered electron detector at 10kV. SEM images were aligned with the
corresponding immunofluorescent images in ImageJ using the TurboReg plugin (Thevenaz
et al., 1998). The nuclei as viewed with DAPI fluorescence and the SEM defined the
identical regions in the two imaging modes.
Highlights
• Resolution of individual synapses in situ by high-throughput array tomography
• Measurement of multiple (e.g., 17) molecular markers at individual synapses
• Discrimination of defined subtypes of glutamate and GABA synapses
• Detection of both VGluT1-type glutamate and GABA synapses on single spines
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
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Acknowledgments
This work was supported by grants from the National Institutes of Health (NS063210), Gatsby Charitable Trust,
Howard Hughes Medical Institute, by funds from the Stanford’s BioX Program, Stanford’s Departments of
Neurosurgery and Neurology and Neurological Science, and by a gift from Dr. Lubert Stryer. We thank Nafisa
Ghori for her expert technical help and JoAnn Buchanan and Gordon Wang for help and advice. We thank Profs.
Liqun Luo, Miriam Goodman and Thomas Clandinin (Stanford University) and Bradley Hyman (Harvard Medical
School) for their very helpful comments on the manuscript.
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Figure 1.
Array tomographic synapsin I immunofluorescence in the cerebral cortex of an adult YFP-H
mouse is punctate and consistent with synapse identity. (A) A volume rendering of 60 serial
sections (200 nm each) through the entire cortical depth, including portions of the striatum.
While all subsequent experiments and analysis were performed on thinner, 70 nm sections,
the thicker sections in this case have allowed us to collect a larger volume and to better
visualize the extensive dendrites of pyramidal neurons. Synapsin (magenta), tubulin (blue),
and YFP (green). Scale bar, 50 μm. (B) A close up of layer 5 pyramidal neurons labeled
with YFP. (C–H) Zoomed-in view of layers 1 (C), 2/3 (D), 4 (E), 5a (F), 6a (G) and white
matter and striatum (H). Scale bar for B–H, 10 μm. See also Movie S1 for a more revealing
rendering of the same image volume and Figure S1 for comparison of different synapsin
antibodies.
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Figure 2.
Proteomic immunofluorescence AT of mouse somatosensory cortex yields staining patterns
consistent with synaptic protein distributions. Volume rendering from 20 sections, 70 nm
each, from an array stained with 11 antibodies (Table S1, dataset KDM-SYN-09041). (A)
Tubulin (blue), synapsin (magenta), YFP (green) and DAPI (grey) fluorescence. (B–D) The
boxed area in (A). DAPI (grey) and YFP (green). (B) Distribution of all presynaptic boutons
as labeled with synapsin (magenta). (C) Distribution of VGluT1 (red), VGluT2 (yellow) and
GAD (cyan) presynaptic boutons. (D) Postsynaptic labels: GluR2 (blue), NMDAR1 (white)
and gephyrin (orange) next to synapsin (magenta). Scale bar 10 μm. See also Table S1 for
sequence of antibody application.
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Figure 3.
Multiple synaptic proteins are colocalized in a fashion consistent with synaptic identity and
glutamatergic and GABAergic synapse subtype. (A) Volume rendering of 20 sections (70
nm) from the mouse somatosensory cortex immunostained for synapsin (magenta) and
synaptophysin, VGluT1, PSD95 or GAD (green). Colocalization of the magenta and green
channels is displayed as white. DAPI, blue. These volume renderings are from an array
stained with 17 antibodies (Table S1, dataset KDM-SYN-091207). Scale bar, 5 μm. (B)
Colocalization matrix of nine synaptic markers and tubulin (left) and corresponding pair-
wise representation of the channels on a small area (4 × 4 μm) of a single section (right). For
each pair of channels we computed a cross-correlation score over a range of lateral offset
distances for images in the two channels. The cross-correlation score is represented as a grid
of false colored squares with their center representing the score at 0 offset and each pixel
equal to 0.1 μm offset. (C) For a subset of channel comparisons, the cross-correlation score
is plotted as a function of the lateral offset. Each trace is obtained by averaging 16 equally-
spaced radii. Left, With no lateral shift the normalized cross-correlation is equal to the
Pearson correlation coefficient, and at shifts beyond the rough size of a synapse the
correlation drops to ~ 0 for all channels. Right, The same is normalized such that each curve
is 1.0 in the no-shift case. Pre-presynaptic and post-postsynaptic channel comparisons drop
off sharply, while pre-postsynaptic do not.
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Figure 4.
Dendritic spines in mouse cerebral cortex are contacted by synapsin puncta and colocalize
with other pre- and postsynaptic markers. Volume rendering of 45 sections from dataset
NAOR-081118 (Table S1). To better visualize the synaptic markers associated with
dendritic spines, only immunofluorescence within 0.5 μm of the YFP dendrite was
displayed. (A) In the left panel, a 20 μm long segment from a spiny dendrite of a layer 5
pyramidal cell (green) is shown as it traverses layer 4. In each subsequent panel the labeling
of a synaptic protein is added. PSD95 (blue), bassoon (yellow), and synapsin (magenta). The
postsynaptic protein PSD95 is found within spine heads and closely apposed to the
presynaptic proteins bassoon and synapsin (arrow). Scale bar, 2 μm. (B) The opposite side
of the spine marked with an arrowhead in (A) at higher magnification. Scale bar, 0.5 μm.
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Figure 5.
Ultrastructurally identified synapses are labeled with the synapsin antibody. (A, B)
Conjugate synapsin immunofluorescence and SEM of the adult mouse cerebral cortex.
Synapsin (magenta) and DAPI (blue) signal as obtained with the fluorescence microscope
are overlaid on the SEM image from the same section. (B) Four serial sections through the
boxed region in (A). Boxed region is section #2 in the series. The majority of presynaptic
boutons are consistently labeled from section to section (arrows), but some are labeled only
on few sections with a weak signal (asterisk). Scale bar, 0.5 μm. (C) A TEM image of
postembedding gold immuno-EM for synapsin. The 15 nm gold particles label presynaptic
terminals as identified by the presence of synaptic vesicles and postsynaptic density. Scale
bar, 0.5 μm. See also Figure S2 for effect of tissue processing on synapsin immunostaining.
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Figure 6.
Synaptograms are useful for viewing proteomic information from serially sectioned single
synapses. A glutamatergic (left) and a GABAergic synapse (right) are shown. Each square
represents an area of 1×1 μm from a single 70 nm section. Each section through the synapsin
punctum occupies a column and each antibody label – a row. See also Table S1 for sequence
of antibody application.
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Figure 7.
Proteomic imaging with AT reveals the diversity of cortical synapses. (A) Examples of
synaptograms representing the main synapse subtypes observed in mouse somatosensory
cortex with the current antibody panel. (B) Synapsin content of different synaptic subtypes.
For each subtype, 100 synapses were randomly selected using the VGluT1-PSD95, VGluT2-
PSD95 and VGAT-gephyrin channels and synapsin immunofluorescence was measured on
each section through the synapse. Top panel, Histograms of synapsin immunofluorescence
in the three synapse subtypes. Lower panel, Scatterplot of synapsin intensity versus the
respective vesicular transporter immunofluorescence for each synapse. (C) Examples of
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glutamatergic synapses with different postsynaptic receptor combinations. (D) Example of a
synapse made by the axon of a YFP-positive layer 5 pyramidal neuron.
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Figure 8.
Double innervated spines receive both a glutamatergic VGluT1 and GABAergic synapse.
(A, B) Volume rendering of dendritic spines from YFP-positive pyramidal cell dendrites
(green), each receiving 2 synaptic inputs on the head. The glutamatergic synapses are
represented by postsynaptic PSD95 label (blue) and presynaptic synapsin (magenta). The
GABAergic synapses are represented by postsynaptic gephyrin (orange) and presynaptic
GAD (cyan). The labels are added consecutively from left to right. Additional synapses not
contacting the spines are also observed within the reconstructed volume. (C, D), Single
sections through double innervated spines labeled with multiple antibodies. For each spine
the two adjacent sections where most of the markers were present was chosen. Each panel
shows the spine (green) and one synaptic marker (magenta). Direct overlap of the two labels
is seen as white. The punctuated line separates adjacent sections. C′ and D′ show a volume
rendering of the spines in C and D with the plane of the single sections represented in gray.
Scale bar, 1 μm.
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Micheva et al. Page 27
Table 2
Proportion of synapses from different synaptic subtypes.
Synaptic subtype Layer 4 Layer5
Exp. 1 Exp. 2 Exp. 1 Exp. 2
VGluT1 60.1% (149) 57.5% (100) 66.7% (156) 67.3% (113)
VGluT1+2 15.3% (38) 15.5% (27) 9.0% (21) 11.9% (20)
VGluT2 5.6% (14) 6.3% (11) 2.6% (6) 2.4% (4)
Other glutamatergic 3.6% (9) 4.6 (8) 5.6% (13) 2.4% (4)
GABAergic 15.3% (38) 16.1 (28) 16.2% (38) 16.1% 27)
All synapses 100% (248) 100% (174) 100% (234) 100% (168)
All synapsin puncta 262 183 242 172
Not synapse 5.3% (14) 4.9% (9) 3.3% (8) 2.3% (4)
Neuron. Author manuscript; available in PMC 2011 November 18.