Neurons communicate with each other through specialized structures known as synapses. At chemical synapses, the cells do not physically interact as they rely instead on molecules called neurotransmitters to pass along signals. At electrical synapses, however, neurons are directly connected via gap junctions, which are clusters of intercellular channels that allow ions and other small compounds to move from one cell to another.

Both electrical and chemical synapses play critical roles in neural circuits, and both exhibit some amount of plasticity – they weaken or strengthen depending on how often they are used, an important feature for the brain to adapt to the needs of the environment. Yet the structure and molecular organization of electrical synapses have remained poorly understood compared to their chemical counterparts.

In response, Cárdenas-García, Ijaz and Pereda took advantage of a new approach known as expansion microscopy to examine the electrical synapse that connects neurons bringing sound information to a pair of unusually large neurons in the brain of most bony fish. With this method, a biological sample is prepared in such a way that its size increases, but the relative position of its components is preserved. This allows scientists to better observe structures that would otherwise be too difficult to capture using traditional microscopy techniques.

Experiments in larval zebrafish revealed that contrary to previous assumptions, the electrical synapse was formed of not one but multiple gap junctions of various sizes closely associated with a range of structural and signaling molecules typically found in adherens junctions (a type of structure that physically links cells together). The team suggests that these molecular actors could work to ensure that the multiple gap junctions act in concert at the synapse. Overall, these findings offer a new perspective on how electrical synapses are organized and regulated, which refines our understanding of how the nervous system functions both in health and in disease.

Synapses are specialized cell–cell contacts where two neurons can share relevant functional information. The exchange of information can occur directly through cell–cell channels, known as ‘electrical synapses’, or indirectly, via the release of a chemical messenger, known as ‘chemical synapses’ (Pereda, 2014). While the molecular complexity of chemical synapses with structurally distinct pre- and postsynaptic components has long been recognized (Wichmann and Kuner, 2022; Wilhelm et al., 2014), less is known regarding the molecular and structural complexity of electrical synapses. Electrical synapses are a modality of neuronal communication mediated by structures known as ‘gap junctions’ (GJs) (Goodenough and Paul, 2009). These structures contain intercellular channels formed by the apposition of two hemichannels, each provided by one of the connected cells, and which cluster together into GJ ‘plaques’ (Goodenough and Paul, 2009). Hemichannels are formed by proteins called ‘connexins’ (Goodenough and Paul, 2009; Söhl and Willecke, 2004) in vertebrates and ‘innexins’ (Phelan et al., 1998; Phelan, 2005) in invertebrates that, though unrelated in sequence, share a similar membrane topology that allows them to assemble into intercellular channels. While GJs are ubiquitous and present in virtually every tissue of the organism providing metabolic coupling (Goodenough and Paul, 2009), they additionally serve as a pathway of low resistance for the spread of electrical currents between neurons (and cells of the heart), the main form of signaling in the brain, which is fast enough to operate within the time frame required for decision-making by neural circuits (Bennett, 1997; Alcamí and Pereda, 2019). Electrical synapses are generally perceived as structurally simpler than chemical synapses and exclusively involving the function of intercellular channels. However, recent data indicates that the function of these channels is under the control of their supporting molecular scaffold (Flores et al., 2008; Lasseigne et al., 2021; Martin et al., 2020; Martin et al., 2023), suggesting that neuronal GJs are complex molecular structures whose function requires the contribution of multiple molecular components (Miller and Pereda, 2017). Such molecular complexity is likely to underlie plastic changes in the strength of electrical synapses (Pereda et al., 2013; O’Brien and Bloomfield, 2018), which are capable of dynamically reconfiguring neural circuits (O’Brien and Bloomfield, 2018; Bloomfield and Völgyi, 2009).

Thus far, investigations of the properties of vertebrate electrical transmission have solely considered the functional properties of the channel-forming proteins, the connexins, the molecules that regulate them, and their interactions at the GJ plaque. However, could a single neuronal GJ per se be considered an electrical synapse? Alternatively, does electrical transmission rely on additional structural components? The identification of the structural components of a chemical synapse is facilitated by the presynaptic bouton, which anatomically defines its limit (Sotelo, 2020). In contrast, neuronal GJs are typically found connecting cell somata or other neuronal processes (Alcamí and Pereda, 2019; Sotelo, 2020; Sotelo and Llinás, 1972), such as dendrites and axons, making it more difficult to define the exact anatomical boundaries that constitute an electrical synapse. Neuronal GJs can also occur at synaptic boutons, generally coexisting with specializations for chemical transmission (Sotelo, 2020; Martin and Pilar, 1963). This is the case for auditory afferents terminating as single ‘large myelinated club endings’ (Bartelmez, 1915; Bartelmez and Hoerr, 1933) or ‘club endings’ (CEs) on the lateral dendrite of the teleost Mauthner cells (a pair of large reticulospinal neurons involved in tail-flip escape responses in fish) (Pereda and Faber, 2011; Faber and Pereda, 2011), each containing GJs and specializations for chemical transmission (Robertson et al., 1963). Because of their experimental accessibly and functional properties, these terminals are considered a valuable model to study vertebrate electrical transmission as they more easily allow for the correlation between structure and function of synaptic features (Pereda et al., 2013; Robertson et al., 1963; Furshpan, 1964; Flores et al., 2012). Since the bouton marks the anatomical limits of a synapse, these contacts offer the opportunity to examine the anatomical structures that together make an electrical synapse.

Here, we used expansion microscopy (Tillberg et al., 2016) to expose the presence and spatial arrangement of synaptic components in CEs of larval zebrafish. CEs from larval zebrafish share comparable morphological and functional properties with those of adult fish (Lasseigne et al., 2021; Yao et al., 2014; Miller et al., 2017; Echeverry et al., 2022), and due to their genetic accessibility, allow for the opportunity to investigate the functional link between the structures enabling electrical transmission and its regulation. Expansion revealed the presence of multiple well-defined puncta distributed throughout the contact area, which, consistent with the notion that they represent GJ plaques, each exhibited labeling for the fish homologs of the widespread mammalian connexin 36 (Cx36), Cx35, and Cx34, as well as for the GJ scaffolding protein zonula occludens (ZO1) (Flores et al., 2008; Lasseigne et al., 2021; Miller et al., 2017; Rash et al., 2013). Strikingly, expansion following staining with N-cadherin and ß-catenin antibodies, protein components of ‘adherens junctions’ (AJs), showed that these proteins are also distributed all throughout the contact area, but in a fashion that is mutually exclusive with connexin. This suggests that the subsynaptic topography of electrical synapses is carefully and intimately coordinated. Finally, double labeling with Cx and glutamate receptor antibodies showed that, while GJs are distributed throughout the entire contact area, a much smaller number of glutamatergic sites are restricted to the periphery occupying a small fraction (~19%) of the contact’s surface. Thus, our data suggest that synaptic communication at electrical synapses results from not one but the coordinated action of multiple GJs of variable size, which may require the functional contribution of additional structures, such as AJs.

A group of auditory afferents, each of which terminate as a single synaptic contact, known as a club ending (CE), on the distal portion of the lateral dendrite of the Mauthner (M-) cell (Bartelmez, 1915; Bartelmez and Hoerr, 1933; Figure 1A). Because of their unusual large size and experimental accessibility, CEs represent a valued model for the correlation of synaptic structure and function. Ultrastructural analysis of CEs in adult goldfish (Robertson et al., 1963; Tuttle et al., 1986; Kohno and Noguchi, 1986) and larval zebrafish (Yao et al., 2014) revealed the presence of GJs coexisting with specializations for neurotransmitter release. Consistent with these synaptic specializations, stimulation of CEs evokes a synaptic response that combines electrical and chemical transmission (Furshpan, 1964; Yao et al., 2014; Echeverry et al., 2022; Lin and Faber, 1988). A wealth of evidence indicates that GJs at these terminals consist of heterotypic intercellular channels created by the apposition of a presynaptic hemichannel formed by connexin Cx35.5 and a postsynaptic hemichannel formed by Cx34.1, two of the Cx35 (Cx35.1 and Cx35.5) and Cx34 (Cx34.1 and Cx34.7) orthologs (Miller et al., 2017; Rash et al., 2013). These junctions also contain the scaffolding protein ZO1 (Flores et al., 2008; Lasseigne et al., 2021), which regulates channel function. The synaptic contact areas of CEs (Figure 1A) can be visualized and unambiguously identified by immunolabeling for these GJ proteins, which are revealed as large fluorescent oval areas at the distal portion of the lateral dendrite of the M-cell (Figure 1B–D).

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Immunolabeling is commonly used to define the biochemical composition of a synapse by allowing detection of the presence of specific proteins. We combined this approach with a protein-retention expansion microscopy protocol (proExM; Tillberg et al., 2016) to explore not only the presence but the relative distribution of the structures formed by various synaptic proteins throughout the contact areas of CEs in 5 days post fertilization (dpf) zebrafish. Tissue expansion with anti-Cx35.5 revealed the presence of multiple Cx35.5-positive puncta at these contacts forming a concave oval area (Figure 1E, Figure 1—video 1), reminiscent of that observed at goldfish CEs, known to correspond to GJ plaques (Flores et al., 2008; Pereda et al., 2003). The main diameter of the oval synaptic contact area defined by labeling of Cx35.5, either using anti-Cx35/36 (which recognizes both Cx35.5 and Cx35.1) or a specific anti-Cx35.5, or of ZO1 with anti-ZO1, increased about fourfold (3.9× expansion factor), from 2.11 ± 0.037 µm in non-expansion experiments (n = 38 CEs from 26 fish) to 8.25 ± 0.225 µm in expansion experiments (n = 40 CEs from 13 fish) (mean ± SEM). Moreover, expansion led to an about 13-fold increase in the area of the contact (13.4×), from 2.63 ± 0.055 µm² (n = 38 CEs from 26 fish) to 35.13 ± 1.266 µm² (n = 40 CEs from 13 fish). A concern when using proExM is whether this procedure leads to the distortion of normal anatomical features. Therefore, to determine the degree of isotropy of the expansion, we measured the ratio between the short and long diameters of the CE oval areas (S/L ratio) in non- and post-expanded samples. The short and long diameters in non-expanded tissue averaged 1.62 ± 0.026 µm and 2.11 ± 0.037 µm (n = 38 CEs from 26 fish), respectively, and 5.16 ± 0.142 µm and 8.25 ± 0.225 µm (n = 40 CEs from 13 fish), respectively, in expanded terminals. Analysis showed that the average S/L ratio decreased in the expanded tissue, with the decrease reaching statistical significance (non-expanded, 0.77 ± 0.018 [n = 38 CEs from 26 fish]; expanded, 0.64 ± 0.019 [n = 40 CEs from 13 fish]; p<0.01). However, the decrease in the average S/L ratio was less than 18%. Moreover, the distribution of S/L ratios from expanded tissue was nearly identical to the distribution from non-expanded tissue, and both were well described by normal distributions (Figure 1—figure supplement 1). This result indicates that the expansion procedure had no selective effects on CEs, but rather affected all uniformly. The difference between expanded and non-expanded S/L ratios might also result from an underestimation of the small diameter due to a minor tilting along the long diameter in ‘en face’ views of expanded CEs, which are more difficult to obtain because of their larger size. (In contrast to adult animals, ‘en face’ views are harder to obtain in larval zebrafish because the diameter of the CE contact in larvae is comparable to the diameter of the lateral dendrite of the M-cell [Yao et al., 2014], which, together with the presence of a smaller number of these afferents in larvae, makes ‘en face’ views less likely to be found and measured.) Together, with our synaptic alignment findings (see below), these results show that the expansion procedure had minimal effects on synaptic structure. Thus, expansion of these single synapses resulted in a more than tenfold increase of the synaptic contact area, allowing for a more detailed visualization of the relative distribution of its synaptic components.

CEs combine electrical with chemical transmission. To investigate the area occupied by each form of transmission, we labeled for Cx35.5 as a marker of GJs and glutamate receptor 2 (GluR2) as a marker of glutamatergic transmission (Figure 2A–C). As expected, colocalization analysis (Manders’ coefficient analysis) revealed that labeling for Cx35.5 and GluR2 was mutually exclusive (Figure 2D). Yet, while Cx35.5 labeling covered the majority of the area, labeling for GluR2 was substantially lower and limited to the contact’s peripheral margin (Figure 2A–C). To quantify this differential distribution, we defined two regions of interest (ROIs) at ‘en face’ views of the CE contact: a central, oval ROI representing ¾ of the area (‘center’) and an annular ROI representing the peripheral, remaining ¼ of the area (‘periphery’). Estimates of the relative intensity of Cx35.5 vs. GluR2 in each ROI showed that, while GluR2 is constrained to the periphery, Cx35.5 is homogeneously distributed through the contact area (Figure 2E and F; see figure legend and ‘Materials and methods’ section for statistical analysis). This distribution is consistent with previous EM reconstruction of CEs in adult goldfish (Tuttle et al., 1986), at which specializations for transmitter release were found to be restricted to the periphery of the contact. Thus, while GJs occupy most of the surface, chemical transmission occupies a smaller and peripheral portion, suggesting that most of the contact operates as an electrical synapse.

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Rather than diffusely distributed, labeling for Cx35.5 in expansion samples was characterized by well-defined puncta distributed throughout the contact area, suggesting that they each might represent an individual GJ plaque. Consistent with this interpretation, double labeling for Cx35.5 and Cx34.1 (Figure 3A and C) showed a high degree of colocalization at CE contact areas (Figure 3E). A high degree of colocalization (Figure 3B and D, Figure 3—video 1) was also found between labeling for Cx35.5 and ZO1 (Figure 3F; see figure legend for statistical analysis). Moreover, labeling for Cx35.5 and Cx34.1 colocalized at single puncta, as indicated by line scan of individual puncta. Given the characteristic concavity of the CE contact area, the relationship between labeled pre- and postsynaptic GJ proteins (Figure 4A) was easier to establish and accurately measure at the periphery of the labeled areas (Figure 4B). Line scan of samples labeled for Cx35.5 and Cx34.1 indicated the presence of labeling for these proteins at single punctum (Figure 4C). Strikingly, while the peak of maximum intensity for the pre-and postsynaptic connexins was aligned (Figure 4C), the peak of maximum intensity for Cx35.5 always preceded that of ZO1 (Figure 4D). This is consistent with the fact that, while Cx35.5 is presynaptically localized, ZO1b, one of the two zebrafish orthologs of ZO1, was reported to be postsynaptic. The observed distance between the peaks of fluorescence was not due to the differences in the wavelength of the fluorophores due to uncompensated chromatic aberration as it remained when secondary antibodies were swapped (Figure 4D and E). These differences are quantified in the graph of Figure 4F (see figure legend for statistical analysis). The finding confirms previous conclusions reached with biochemical and chimera analysis indicating that ZO1b is only located at postsynaptic hemiplaques (Lasseigne et al., 2021). Although a distance between Cx35.5 and Cx34.1 labeling peaks was observed in some samples (~0.15 µm), it was still too small to be consistently detected with this method due to the spatial amplification produced by the fluorophores. By increasing the distance between pre- and postsynaptic sites, expansion increases resolution, making it possible to explore differences in the composition of GJ hemiplaques. Thus, altogether, our findings indicate that each puncta correlates to a GJ.

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Puncta labeled for Cx35.5 (Figure 5A and B), which we established each represent a GJ, showed a high degree of variability in number and size (see inset of Figure 5C). We then quantified the number of puncta and their individual area in ‘en face’ views of CEs, at which this analysis was possible. (Panels B and C in Figure 5 are the same experiment as Figures 1E and 3B and C, illustrating the ability of expansion microscopy for providing multiple layers of information within the same experiment.) Figure 5D illustrates the variability in puncta area observed between neighboring CEs within the same M-cell lateral dendrite in three different fish. Histograms show a similar variability in number and size at all terminals. Overall, the number of GJs per CE averaged 36.73 ± 1.287 (n = 11 CEs from four fish), and their area ranged from 0.06 to 1.99 µm². The number of GJs might have been slightly underestimated because fluorophore spatial amplification could have caused a big punctum to form from two closely spaced small GJs. This limitation could result in two nearby GJs appearing to merge. After correcting for the expansion factor (13×), the areas of the GJs were estimated to be between 4 and 148 nm². Assuming that connexons in GJ plaques are organized in a crystalline fashion with a density of 12,000 connexons/µm² (Kamasawa et al., 2006), we estimated that GJs at CEs contain 49–1775 connexons, and the total junctional area represents an average of 12,425 ± 493.76 connexons per CE. Thus, electrical transmission at zebrafish CEs is mediated by multiple GJs containing a variable number of channels.

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Our labeling with Cx35.5 and GluR2 showed that most of the contact area of a CE operates as an electrical synapse. We then asked what other associated anatomical structures might be contributing to electrical transmission. Previous electron microscopy analysis exposed the presence of AJs in close proximity to neuronal GJs, including those at CEs in goldfish and larval zebrafish (Figure 6A). AJs are known to initiate and mediate the maturation and maintenance of cell–cell contacts, including GJs, at which a wealth of evidence suggests a close functional interaction (Shaw et al., 2007; Defourny and Thiry, 2021; Thomas et al., 2021). Therefore, we decided to investigate the incidence, association, and spatial distribution between these two structures at CEs. Recent immunohistochemical analysis revealed the association between components of AJs and Cx36 in various mammalian structures (Nagy and Lynn, 2018). Expansion for Cx35.5, transmembrane protein N-cadherin, and the intracellular protein ß-catenin, the latter two being major structural components of AJs, showed that labeling for AJs components seems equally distributed through the synaptic contact (Figure 6B–D). In contrast to Cx35.5, which is characterized by well-defined puncta reminiscent of GJs, labeling for N-cadherin and ß-catenin was diffuse, less structured, and did not colocalize with Cx35.5 (Figure 6B; see figure legend for statistical analysis). Rather, the labeling for N-cadherin (Figure 6C) and ß-catenin (Figure 6E) was interleaved with Cx35.5 puncta, as illustrated by line scan analysis in the margins of CE contacts (Figure 6D and F). Furthermore, in ‘en face’ views, labeling for N-cadherin (Figure 6G) and ß-catenin (Figure 6H) was broadly distributed throughout the surface of the contact, appearing to engulf Cx35.5-labeled puncta. While labeling for N-cadherin and ß-catenin was interleaved with Cx35.5 labeling, we observed some degree of colocalization (Figure 6B) likely due to fluorophore amplification of the close spatial association between GJs and AJs (see Figure 6A).

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The spatial distribution of the labeling for components of AJs suggests that their function is associated with electrical transmission, rather than chemical transmission. To establish this association, we measured the relative proportion of labeling fluorescence of various synaptic components at CE contact areas. As expected, labeling for Cx35.5 and the GJ scaffold ZO1 was equally proportional (Figure 7A). Similar proportionality was found for fluorescence of N-cadherin and Cx35.5, and ß-catenin and Cx35.5 (Figure 7B). In contrast, fluorescence of GluR2 represented a smaller fraction than that of Cx35.5 labeling (Figure 7C; see figure legend for statistical analysis). Finally, we normalized the fluorescence to the area of the CE contact to calculate the area occupancy for these three synaptic structures (Figure 7D). Less than 19% was occupied by GluR2, while around 73% of the contact was occupied by similar amounts of Cx35.5 and AJs. About 8% of the contact area was not labeled for these antibodies, either corresponding to the surface membrane between the structures recognized by labeling or to the presence of additional synaptic components. Thus, while chemical synapses are restricted to a small and peripheral area of the contact, most of the contact surface is occupied by multiple GJs of variable size which are interleaved and closely associated to AJs (Figure 7E).

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“One of the most difficult problems in the correlation of structural and functional concepts is that of accurately defining the synapse” (Robertson et al., 1963).

Because they support both electrical and chemical synaptic transmission, CEs on the M-cells enabled the correlation of their synaptic properties with structural specializations for each of these modalities of communication (Robertson et al., 1963). In addition to specializations observed at purely chemically transmitting contacts, electron microscopy analysis by Robertson, 1963 revealed areas of close membrane apposition that at ‘en face’ views were round-shaped and exhibited a characteristic reticular pattern. These ‘synaptic discs’ provided early evidence for the cellular structures that we now know as ‘gap junctions’ (GJs): clusters of intercellular channels that provide the mechanism of communication for electrical transmission. However, in his seminal paper (Robertson et al., 1963), Robertson warned about the limitations of correlating structural and functional notions to define the components of an electrical synapse. In contrast to chemical synapses, it is challenging to define exactly what anatomically constitutes an electrical synapse as neuronal GJs are often found connecting cell somata or other neuronal processes, such as dendrites and axons (Nagy et al., 2018). Because the presynaptic bouton anatomically marks the limit of a synapse, CEs provided the ideal opportunity to investigate the components of an electrical synapse. By applying expansion microscopy to these single terminals, we found evidence suggesting that, in contrast to the general perception, electrical synapses might have a more complex structural organization. Our data indicates that the CE electrical synapse operates with not one but multiple (~35) GJs that are in close association with structural and signaling molecules known to be components of AJs (Figure 7E). This extended notion of the overarching organization of an electrical synapse might contribute to a better understanding of their functional diversity and structural configurations.

All data generated and analyzed for Figures 2D-F; 3E-F; 4C-F; 5D-E; 6B,D, F; 7A-D; and Figure 1—figure supplement 1B is available as source data Excel files on G-Node (https://doi.org/10.12751/g-node.8ljsii).

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Author details

1. Sandra P Cárdenas-García

   Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Methodology, Writing - original draft, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7001-4446

2. Sundas Ijaz

   Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, United States

   Contribution

   Conceptualization, Formal analysis, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0005-8199-7598

3. Alberto E Pereda

   Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   alberto.pereda@einsteinmed.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8283-8768

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