The components of an electrical synapse as revealed by expansion microscopy of a single synaptic contact

Abstract

Most nervous systems combine both transmitter-mediated and direct cell-cell communication, known as 'chemical' and 'electrical' synapses, respectively. Chemical synapses can be identified by their multiple structural components. Electrical synapses are, on the other hand, generally defined by the presence of a 'gap junction' (a cluster of intercellular channels) between two neuronal processes. However, while gap junctions provide the communicating mechanism, it is unknown whether electrical transmission requires the contribution of additional cellular structures. We investigated this question at identifiable single synaptic contacts on the zebrafish Mauthner cells, at which gap junctions coexist with specializations for neurotransmitter release and where the contact defines the anatomical limits of a synapse. Expansion microscopy of these contacts revealed a detailed map of the incidence and spatial distribution of proteins pertaining to various synaptic structures. Multiple gap junctions of variable size were identified by the presence of their molecular components. Remarkably, most of the synaptic contact's surface was occupied by interleaving gap junctions and components of adherens junctions, suggesting a close functional association between these two structures. In contrast, glutamate receptors were confined to small peripheral portions of the contact, indicating that most of the synaptic area works as an electrical synapse. Thus, our results revealed the overarching organization of an electrical synapse that operates with not one, but multiple gap junctions, in close association with structural and signaling molecules known to be components of AJs. The relationship between these intercellular structures will aid in establishing the boundaries of electrical synapses found throughout animal connectomes and provide insight into the structural organization and functional diversity of electrical synapses.

Keywords: N-cadherin; adherens junction; connexin; gap junction; glutamate; ß-catenin.

Figure 1.. Expansion microscopy of Club ending contact areas in larval zebrafish.

(A) The cartoon illustrates the spatial distribution of auditory afferents that terminate as single Club endings (CEs), each containing both gap junctions (GJs, green) and specializations for chemical transmission (vesicles), on the distal portion of the lateral dendrite of the Mauthner (M-) cell. (B) Confocal image with anti-GFP (purple) and anti-Cx35/36 (green) showing a long stretch of the lateral dendrite (projection of 34 confocal Z-sections, totaling 13.5 µm), revealing the contact areas of several CEs. (C-D) Contact areas of individual CEs labeled with anti-Cx35/36 (C, green; projection of 12 sections, totaling 4.7 µm) and anti-ZO1 (D, red; projection of 4 sections, totaling 1.6 µm). (E) Expansion microscopy (ProExM) with anti-Cx35.5 increases the size of CE synaptic contact areas, enabling the visualization of intrasynaptic components (projection of 19 sections, totaling 10.5µm).

Figure 2.. Electrical and chemical transmitting areas are mutually exclusive.

(A,B) Expanded synaptic contact areas labeled with anti-Cx35.5 (green) and anti-GluR2 (magenta). (C) ‘En face’ view of an expanded synaptic contact area showing that GluR2 labeling is restricted to the periphery of the contact, whereas Cx35.5 labeling is distributed throughout the whole contact area. (D) Graph shows the lack of colocalization between Cx35.5 and GluR2 fluorescence at individual CE contacts, determined by the Mander’s Colocalization Coefficient: GluR2/Cx35.5 0.078 ± 0.05 (x axis); Cx35.5/GluR2 0.098 ± 0.06 (y axis), n= 14 (mean ± SEM). (E) Distribution of labeling. Quantification of labeling for Cx35.5 and GluR2 at the periphery and the center of the CE contact area (Student’s t-test <0.05, n=4 on face views, p=<0.0001). Error bars denote ± SEM. GluR2 fluorescence is higher in the periphery, while Cx35.5 fluorescence is significantly higher at the center of the contact area. (F) Graphical description of the center vs. periphery distribution of Cx35.5 and GluR2 for the data described in panel E.

Figure 3.. Labeling for gap junction proteins reveals the presence of multiple puncta at expanded CE synaptic contact areas.

(A) CE synaptic contact areas labeled with anti-Cx34.1 and anti-Cx35.5. (B) Contact areas labeled with anti-Cx35.5 and anti-ZO1. Same experiment as Fig. 1E. (C) Labeling with anti-Cx34.1 and anti-Cx35.5. (D) Magnification of the boxed region in panel B showing a side view of an expanded synaptic contact area labeled for Cx35.5 and ZO1. (E) Graph showing colocalization of Cx35.5 and Cx34.1 fluorescence at individual CEs determined by the Mander’s Coefficient: Cx34.1/Cx35.5 0.80 ± 0.08 (x axis); Cx35.5/Cx34.1 0.84 ± 0.06 (y axis), n=14. (F) Colocalization of Cx35.5 and ZO1 fluorescence at individual CEs. Mander’s Coefficient: ZO1/Cx35.5 0.71 ± 0.04 (x axis); Cx35.5/ZO1 0.82 ± 0.07 (y axis), n=14.

Figure 4.. Expansion microscopy reveals the molecular components of gap junction plaques at CE synaptic contact areas.

(A) Schematic representation of the molecular organization of GJs between CEs (pre-synaptic) and the M-cell (post-synaptic). The presynaptic and postsynaptic hemichannels are formed by Cx35.5 and Cx34.1, respectively. The scaffolding protein ZO1 is postsynaptic and interacts with Cx34.1. (B) Cartoon of a CE terminal illustrating the concavity of its contact area with the M-cell. The concavity determines differences in the relative position of presynaptic (green) vs. postsynaptic (red) labeling at different points throughout the contact area. Puncta located in the periphery of the contact are ideally aligned to determine colocalization of fluorescence at individual puncta (line scan, inset). (C-E) Line scan of puncta at expanded contact areas showing colocalization of presynaptic Cx35.5 and postsynaptic Cx34.1 (C), and presynaptic Cx35.5 and ZO1 (D-E). The example in C is part of the experiment illustrated in Fig. 3A. The magenta lines indicate the position of the line scan in each case. The fluorescence intensity profiles for each fluorophore are illustrated on the right side of each panel. As a control, secondary antibodies were swapped in E. (F) Bar graph illustrates the distance between the peaks of fluorescence intensity profiles for Cx35.5-Cx34.1 labeling (either 647Atto or 546Alexa-Cx35.5 vs. either 647Atto or 546Alexa -Cx34.1: 0.03 ± 0.001 µm, n=38) and Cx35.5-ZO1 labeling (546Alexa-ZO1 vs. 647Atto-35.5: 0.21 ± 0.002 µm, n=39). Secondary antibodies were swapped as control (647Atto-ZO1 vs. 546Alexa-Cx35.5: 0.20 ± 0.001 µm, n=30). Bars represent ± SEM. Anova analysis with Tukey’s multiple comparison test correction p<0.001.

Figure 5.. Expansion reveals the presence of multiple, variably sized, gap junctions.

(A-B) ‘En face’ views of expanded CE contact areas labeled with anti-Cx35.5 showing multiple puncta with high variability of their size. (C) Magnification of the CE contact area at the top of panel B (light gray). The area enclosed by the orange box illustrates the wide variability in puncta size, labeled 1 to 10 (with the goal of highlighting puncta size variation, the image delimited by the orange box was cropped from and placed on the same region of the lighter image). Panels B and C are the same experiment as Figs. 1E and 3B–C, demonstrating the ability of expansion microscopy for providing multiple layers of information in the same experiment. (D) Frequency histograms illustrating the number and size distribution of puncta labeled for Cx35.5 within individual CEs. Histogram shows the distribution of puncta size at three CEs obtained in the same cell (dendrite), illustrating overlap in different shades of blue. Three different examples are illustrated from left to right. Each histogram shows similar variability in number and size for all nine terminals. (E) Frequency histogram of number and size distribution of Cx35.5 puncta for the nine reconstructed contacts in panel D.

Figure 6.. Gap junctions at CEs are associated with adherens junctions.

(A) Electron micrograph of a CE obtained in a 6 dpf zebrafish showing a GJ (g, highlighted in green) surrounded by AJs (p, encircled in red). From Kimmel et al., 1981 (with permission) (49). (B) GJ and AJ proteins do not colocalize. Left: Cx35.5 and N-Cadherin labeling show a low index of colocalization. Mander’s coefficient: N-Cadherin/Cx35.5 0.35 ± 0.003 (x axis); Cx35.5/N-Cadherin 0.32 ± 0.003 (y axis), n=28. Right: Cx35.5 and β-catenin labeling also show low colocalization. Mander’s coefficient: Cx35.5/β-catenin 0.43 ± 0.004 (x axis); β-catenin/Cx35.5 0.36 ± 0.008 (y axis), n=25. (C) Expansion microscopy of a CE contact area labeled for N-cadherin (red) and Cx35.5 (green). (D) Fluorescence intensity profiles for N-cadherin and Cx35.5 obtained with a line scan (magenta line in C) are mutually exclusive. The low degree of colocalization observed in panel B is likely due to fluorophore amplification and the close spatial association between GJs and AJs, as shown in panel A. (E) Image shows an expanded CE contact area labeled for β-Catenin (red) and Cx35.5 (green). (F) Line scan (magenta line in E) shows that labeling for β-Catenin and Cx35.5 are also mutually exclusive. (G,H) ‘En face’ view of the expanded contact area double-labeled for N-Cadherin and Cx35.5, and β-Catenin and Cx35.5, respectively, showing the close association of GJs and AJs throughout the synaptic contact area. Insets: the boxed areas in the ‘En face’ images highlight the mutually exclusive labeling.

Figure 7.. The electrical synapse at the CE combines multiple gap junctions with adherens junctions.

(A) Double labeling with anti-Cx35.5 and anti-ZO1 show similar proportion of fluorescence at CEs (ZO1 43.3% ± 1.49; Cx35.5 38.25% ± 1.49, n=8). (B) Double labeling for N-Cadherin and Cx35.5 (left), and for β-Catenin and Cx35 (right) also show similar proportionality (N-Cad 41.88% ± 0.82, n=13; β-Catenin 39.84% ± 0.49; Cx35.5 38.25% ± 0.59, n=11). (C) Double labeling for Cx35.5 and GluR2 shows lack of proportionality, with Cx35.5 fluorescence occupying the majority of the CE contact area (GluR2 18.9% ± 0.79; Cx35.5 35.91% ± 1.25; n=11). (D) Tree plot illustrating the area occupancy (fluorescence per contact area) for AJ (N-Cad=29.1%), GJ (Cx35=30.8%), and glutamatergic (GluR2=18.9%) components at individual CEs. The unlabeled area represents 21.3% of the contact’s surface. (E) The cartoon summarizes the synaptic components identified at a single CE contact. While chemical synapses are restricted to a small and peripheral area of the contact (presynaptic vesicles and release sites are represented in grey, postsynaptic receptor areas in magenta), most of its contact surface is occupied by multiple gap junctions (GJs; green) of variable size, which are interleaved and closely associated to adherens junctions (AJs; red).