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

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.

Neurobeachin controls the asymmetric subcellular distribution of electrical synapse proteins.

The subcellular positioning of synapses and their specialized molecular compositions form the fundamental basis of neural circuits. Like chemical synapses, electrical synapses are constructed from an assortment of adhesion, scaffolding, and regulatory molecules, yet little is known about how these molecules localize to specific neuronal compartments. Here, we investigate the relationship between the autism- and epilepsy-associated gene Neurobeachin, the neuronal gap junction channel-forming Connexins, and the electrical synapse scaffold ZO1. Using the zebrafish Mauthner circuit, we find Neurobeachin localizes to the electrical synapse independently of ZO1 and Connexins. By contrast, we show Neurobeachin is required postsynaptically for the robust localization of ZO1 and Connexins. We demonstrate that Neurobeachin binds ZO1 but not Connexins. Finally, we find Neurobeachin is required to restrict electrical postsynaptic proteins to dendrites, but not electrical presynaptic proteins to axons. Together, the results reveal an expanded understanding of electrical synapse molecular complexity and the hierarchical interactions required to build neuronal gap junctions. Further, these findings provide novel insight into the mechanisms by which neurons compartmentalize the localization of electrical synapse proteins and provide a cell biological mechanism for the subcellular specificity of electrical synapse formation and function.

Recording Synaptic Transmission from Auditory Mixed Synapses on the Mauthner Cells of Developing Zebrafish.

The Mauthner cells are a pair of large reticulospinal neurons that organize sensory-evoked tail flip responses in fishes. An identifiable group of auditory "mixed" (electrical and chemical) synaptic contacts known as "Large Myelinated Club endings" on these cells have provided a valuable model for the study of synaptic transmission in the vertebrate brain. While most of studies were performed in adult fish, we describe here methods that make possible recording synaptic transmission from these contacts in developing zebrafish, a genetically tractable vertebrate species which is uniquely amenable for combining synaptic physiology with live imaging and behavioral analysis.

Trichloroacetic Acid Fixation and Antibody Staining of Zebrafish Larvae.

Larval zebrafish have been established as an excellent model for examining vertebrate biology, with many researchers using the system for neuroscience. Controlling a fast escape response of the fish, the Mauthner cells and their associated network are an attractive model, given their experimental accessibility and fast development, driving ethologically relevant behavior in the first five days of development. Here, we describe methods for immunostaining electrical and chemical synapse proteins at 3-7 days post fertilization (dpf) in zebrafish using tricholoracetic acid fixation. The methods presented are ideally suited to easily visualize neural circuits and synapses within the fish.

On the location of electrical synapses.

Location is of critical functional relevance for synapses, including electrical synapses, which are a form of neuronal communication mediated by cell-cell channels. In this issue of Developmental Cell, Palumbos et al. identify a mechanism that supports the localization and function of electrical synapses with subcellular specificity.

A forward genetic screen identifies Dolk as a regulator of startle magnitude through the potassium channel subunit Kv1.1.

The acoustic startle response is an evolutionarily conserved avoidance behavior. Disruptions in startle behavior, particularly startle magnitude, are a hallmark of several human neurological disorders. While the neural circuitry underlying startle behavior has been studied extensively, the repertoire of genes and genetic pathways that regulate this locomotor behavior has not been explored using an unbiased genetic approach. To identify such genes, we took advantage of the stereotypic startle behavior in zebrafish larvae and performed a forward genetic screen coupled with whole genome analysis. We uncovered mutations in eight genes critical for startle behavior, including two genes encoding proteins associated with human neurological disorders, Dolichol kinase (Dolk), a broadly expressed regulator of the glycoprotein biosynthesis pathway, and the potassium Shaker-like channel subunit Kv1.1. We demonstrate that Kv1.1 and Dolk play critical roles in the spinal cord to regulate movement magnitude during the startle response and spontaneous swim movements. Moreover, we show that Kv1.1 protein is mislocalized in dolk mutants, suggesting they act in a common genetic pathway. Combined, our results identify a diverse set of eight genes, all associated with human disorders, that regulate zebrafish startle behavior and reveal a previously unappreciated role for Dolk and Kv1.1 in regulating movement magnitude via a common genetic pathway.

Electrical synaptic transmission requires a postsynaptic scaffolding protein.

Electrical synaptic transmission relies on neuronal gap junctions containing channels constructed by Connexins. While at chemical synapses neurotransmitter-gated ion channels are critically supported by scaffolding proteins, it is unknown if channels at electrical synapses require similar scaffold support. Here, we investigated the functional relationship between neuronal Connexins and Zonula Occludens 1 (ZO1), an intracellular scaffolding protein localized to electrical synapses. Using model electrical synapses in zebrafish Mauthner cells, we demonstrated that ZO1 is required for robust synaptic Connexin localization, but Connexins are dispensable for ZO1 localization. Disrupting this hierarchical ZO1/Connexin relationship abolishes electrical transmission and disrupts Mauthner cell-initiated escape responses. We found that ZO1 is asymmetrically localized exclusively postsynaptically at neuronal contacts where it functions to assemble intercellular channels. Thus, forming functional neuronal gap junctions requires a postsynaptic scaffolding protein. The critical function of a scaffolding molecule reveals an unanticipated complexity of molecular and functional organization at electrical synapses.

Neurons 'talk' with each another at junctions called synapses, which can either be chemical or electrical. Communication across a chemical synapse involves a 'sending' neuron releasing chemicals that diffuse between the cells and subsequently bind to specialized receptors on the receiving neuron. These complex junctions involve a large number of well-studied molecular actors. Electrical synapses, on the other hand, are believed to be simpler. There, neurons are physically connected via channels formed of 'connexin' proteins, which allow electrically charged ions to flow between the cells. However, it is likely that other proteins help to create these structures. In particular, recent evidence shows that without a structurally supporting 'scaffolding' protein called ZO1, electrical synapses cannot form in the brain of a tiny freshwater fish known as zebrafish. As their name implies, scaffolding proteins help cells organize their internal structure, for example by anchoring other molecules to the cell membrane. By studying electrical synapses in zebrafish, Lasseigne, Echeverry, Ijaz, Michel et al. now show that these structures are more complex than previously assumed. In particular, the experiments reveal that ZO1 proteins are only present on one side of electrical synapses; despite their deceptively symmetrical anatomical organization, these junctions can be asymmetric, like their chemical cousins. The results also show that ZO1 must be present for connexins to gather at electrical synapses, whereas the converse is not true. This suggests that when a new electrical synapse forms, ZO1 moves into position first: it then recruits or stabilizes connexins to form the channels connecting the two cells. In many animals with a spine, electrical synapses account for about 20% of all neural junctions. Understanding how these structures form and work could help to find new treatments for disorders linked to impaired electrical synapses, such as epilepsy.

Regulatory Roles of Metabotropic Glutamate Receptors on Synaptic Communication Mediated by Gap Junctions.

Variations of synaptic strength are thought to underlie forms of learning and can functionally reshape neural circuits. Metabotropic glutamate receptors play key roles in regulating the strength of chemical synapses. However, information within neural circuits is also conveyed via a second modality of transmission: gap junction-mediated synapses. We review here evidence indicating that metabotropic glutamate receptors also play important roles in the regulation of synaptic communication mediated by neuronal gap junctions, also known as 'electrical synapses'. Activity-driven interactions between metabotropic glutamate receptors and neuronal gap junctions can lead to long-term changes in the strength of electrical synapses. Further, the regulatory action of metabotropic glutamate receptors on neuronal gap junctions is not restricted to adulthood but is also of critical relevance during brain development and contributes to the pathological mechanisms that follow brain injury.

Science, art, society and Klimt's University of Vienna paintings.

At the turn of the 19th century the Austrian artist Gustav Klimt was commissioned to decorate the ceiling of the Great Hall of the University of Vienna. However the three paintings he produced - Philosophy, Medicine and Jurisprudence - were rejected by the university and later destroyed by retreating German troops during World War II. The story of these paintings, and another called Goldfish, illuminates common ground between art and science, and highlights ongoing tensions in the relationships between art, science and society.

Neuroscience: The Hidden Diversity of Electrical Synapses.

The complete description of the expression of gap junction proteins in the nervous system of the worm reveals a great complexity of their distribution amongst different neuronal classes, opening an unprecedented opportunity to expose the functional diversity of electrical synapses.

Beyond plasticity: the dynamic impact of electrical synapses on neural circuits.

Electrical synapses are found in vertebrate and invertebrate nervous systems. The cellular basis of these synapses is the gap junction, a group of intercellular channels that mediate direct communication between adjacent neurons. Similar to chemical synapses, electrical connections are modifiable and their variations in strength provide a mechanism for reconfiguring neural circuits. In addition, electrical synapses dynamically regulate neural circuits through properties without equivalence in chemical transmission. Because of their continuous nature and bidirectionality, electrical synapses allow electrical currents underlying changes in membrane potential to leak to 'coupled' partners, dampening neuronal excitability and altering their integrative properties. Remarkably, this effect can be transiently alleviated when comparable changes in membrane potential simultaneously occur in each of the coupled neurons, a phenomenon that is dynamically dictated by the timing of arriving signals such as synaptic potentials. By way of this mechanism, electrical synapses influence synaptic integration and action potential generation, imparting an additional layer of dynamic complexity to neural circuits.

On the occurrence and enigmatic functions of mixed (chemical plus electrical) synapses in the mammalian CNS.

Electrical synapses with diverse configurations and functions occur at a variety of interneuronal appositions, thereby significantly expanding the physiological complexity of neuronal circuitry over that provided solely by chemical synapses. Gap junctions between apposed dendritic and somatic plasma membranes form "purely electrical" synapses that allow for electrical communication between coupled neurons. In addition, gap junctions at axon terminals synapsing on dendrites and somata allow for "mixed" (dual chemical+electrical) synaptic transmission. "Dual transmission" was first documented in the autonomic nervous system of birds, followed by its detection in the central nervous systems of fish, amphibia, and reptiles. Subsequently, mixed synapses have been detected in several locations in the mammalian CNS, where their properties and functional roles remain undetermined. Here, we review available evidence for the presence, complex structural composition, and emerging functional properties of mixed synapses in the mammalian CNS.

Two Forms of Electrical Transmission Between Neurons.

Electrical signaling is a cardinal feature of the nervous system and endows it with the capability of quickly reacting to changes in the environment. Although synaptic communication between nerve cells is perceived to be mainly chemically mediated, electrical synaptic interactions also occur. Two different strategies are responsible for electrical communication between neurons. One is the consequence of low resistance intercellular pathways, called "gap junctions", for the spread of electrical currents between the interior of two cells. The second occurs in the absence of cell-to-cell contacts and is a consequence of the extracellular electrical fields generated by the electrical activity of neurons. Here, we place present notions about electrical transmission in a historical perspective and contrast the contributions of the two different forms of electrical communication to brain function.

A Cyfip2-Dependent Excitatory Interneuron Pathway Establishes the Innate Startle Threshold.

Sensory experiences dynamically modify whether animals respond to a given stimulus, but it is unclear how innate behavioral thresholds are established. Here, we identify molecular and circuit-level mechanisms underlying the innate threshold of the zebrafish startle response. From a forward genetic screen, we isolated five mutant lines with reduced innate startle thresholds. Using whole-genome sequencing, we identify the causative mutation for one line to be in the fragile X mental retardation protein (FMRP)-interacting protein cyfip2. We show that cyfip2 acts independently of FMRP and that reactivation of cyfip2 restores the baseline threshold after phenotype onset. Finally, we show that cyfip2 regulates the innate startle threshold by reducing neural activity in a small group of excitatory hindbrain interneurons. Thus, we identify a selective set of genes critical to establishing an innate behavioral threshold and uncover a circuit-level role for cyfip2 in this process.

A Forward Genetic Screen in Zebrafish Identifies the G-Protein-Coupled Receptor CaSR as a Modulator of Sensorimotor Decision Making.

Animals continuously integrate sensory information and select contextually appropriate responses. Here, we show that zebrafish larvae select a behavioral response to acoustic stimuli from a pre-existing choice repertoire in a context-dependent manner. We demonstrate that this sensorimotor choice is modulated by stimulus quality and history, as well as by neuromodulatory systems-all hallmarks of more complex decision making. Moreover, from a genetic screen coupled with whole-genome sequencing, we identified eight mutants with deficits in this sensorimotor choice, including mutants of the vertebrate-specific G-protein-coupled extracellular calcium-sensing receptor (CaSR), whose function in the nervous system is not well understood. We demonstrate that CaSR promotes sensorimotor decision making acutely through Gαi/o and Gαq/11 signaling, modulated by clathrin-mediated endocytosis. Combined, our results identify the first set of genes critical for behavioral choice modulation in a vertebrate and reveal an unexpected critical role for CaSR in sensorimotor decision making.

Electrical synapses in mammalian CNS: Past eras, present focus and future directions.

Gap junctions provide the basis for electrical synapses between neurons. Early studies in well-defined circuits in lower vertebrates laid the foundation for understanding various properties conferred by electrical synaptic transmission. Knowledge surrounding electrical synapses in mammalian systems unfolded first with evidence indicating the presence of gap junctions between neurons in various brain regions, but with little appreciation of their functional roles. Beginning at about the turn of this century, new approaches were applied to scrutinize electrical synapses, revealing the prevalence of neuronal gap junctions, the connexin protein composition of many of those junctions, and the myriad diverse neural systems in which they occur in the mammalian CNS. Subsequent progress indicated that electrical synapses constitute key elements in synaptic circuitry, govern the collective activity of ensembles of electrically coupled neurons, and in part orchestrate the synchronized neuronal network activity and rhythmic oscillations that underlie fundamental integrative processes. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Electrical transmission: Two structures, same functions?

Electrical synapses are finding increasing representation and importance in our understanding of signaling in the nervous system. In contrast to chemical synapses, at which molecules are evolutionary conserved, vertebrate and invertebrate electrical synapses represent molecularly different structures that share a common communicating strategy that allows them to serve very similar functions. A better understanding of differences and commonalities regarding the structure, function and regulation of vertebrate and invertebrate electrical synapses will lead to a better understanding of the properties and functional diversity of this modality of synaptic communication. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 517-521, 2017.

The electrical synapse: Molecular complexities at the gap and beyond.

Gap junctions underlie electrical synaptic transmission between neurons. Generally perceived as simple intercellular channels, "electrical synapses" have demonstrated to be more functionally sophisticated and structurally complex than initially anticipated. Electrical synapses represent an assembly of multiple molecules, consisting of channels, adhesion complexes, scaffolds, regulatory machinery, and trafficking proteins, all required for their proper function and plasticity. Additionally, while electrical synapses are often viewed as strictly symmetric structures, emerging evidence has shown that some components forming electrical synapses can be differentially distributed at each side of the junction. We propose that the molecular complexity and asymmetric distribution of proteins at the electrical synapse provides rich potential for functional diversity. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 562-574, 2017.

The Variable Strength of Electrical Synapses.

In contrast to chemical synapses, less is known regarding the determinants of the strength of electrical synapses. New evidence from Szoboszlay et al. (2016) shows that the number of gap junctions is the dominant factor underlying the strength of electrical coupling between cerebellar interneurons.