Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers

Abstract

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 among 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.

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.

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.

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.

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.

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.

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.

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 glutamatergic synapses with different postsynaptic receptor combinations. (D) Example of a synapse made by the axon of a YFP-positive layer 5 pyramidal neuron.

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.