Gap junctions

Electrical coupling through gap junctions constitutes a mode of signal transmission between neurons (electrical synaptic transmission). Originally discovered in invertebrates and in lower vertebrates, electrical synapses have recently been reported in immature and adult mammalian nervous systems. This has renewed the interest in understanding the role of electrical synapses in neural circuit function and signal processing. The present review focuses on the role of gap junctions in shaping the dynamics of neural networks by forming electrical synapses between neurons. Electrical synapses have been shown to be important elements in coincidence detection mechanisms and they can produce complex input-output functions when arranged in combination with chemical synapses. We postulate that these synapses may also be important in redefining neuronal compartments, associating anatomically distinct cellular structures into functional units. The original view of electrical synapses as static connecting elements in neural circuits has been revised and a considerable amount of evidence suggests that electrical synapses substantially affect the dynamics of neural circuits.     

Horizontal cells of turtle retina: a neurotransmitter control of electrical junctions?

Recent experiments suggest that GABA and dopamine can modulate the permeability of the electrical junctions between horizontal cells of the turtle retina. The possible significance and functional advantages of a chemical control of the electrical junctions are briefly discussed. A similar mechanism could combine in a network of interacting elements the low energy cost and low intrinsic noise typical of the electrical synapses with the great functional plasticity typical of the chemical transmission.     

The differential regulation of formation of chemical and electrical connections in Helisoma

Novel chemical and electrical connections form between neurons not normally connected in the buccal ganglia of the snail Helisoma. We examined the cellular and environmental conditions required for the formation of each type of connection. Previous work in situ showed that novel electrical connections could form in response to axotomy. We have now found that axotomy can evoke the formation of novel unidirectional chemical connections between neurons B5 and B4 in addition to a novel electrical connection. The novel chemical connections display all of the normal properties of chemical synapses in Helisoma ganglia. These connections, however, are transient in nature and break 4 days following axotomy. Previous work has shown that conjoint outgrowth is required for the formation of electrical connections. In cell culture we have investigated whether conjoint outgrowth is also required for chemical synaptogenesis. Using neurons B5 and B19 we have found that when neuron pairs make contact in cell culture, under conditions of synchronous neurite extension, both electrical and chemical synapses form. However, if one neuron has ceased extension prior to contact by a growing neuron, electrical synapses never form (Hadley et al., 1983, 1985) but chemical synapses do form. Furthermore, the addition of serotonin (10(-6) M) to culture medium to inhibit neurite extension of B19, but not that of B5, selectively prevents the formation of electrical connections while permitting the formation of chemical synapses. Thus, the timing of contact in relation to the state of neurite extension can specify the type of connection a given neuron can form.     

Ultrastructural analysis of chemical synapses and gap junctions between Drosophila brain neurons in culture

Dissociated cultures from many species have been important tools for exploring factors that regulate structure and function of central neuronal synapses. We have previously shown that cells harvested from brains of late stage Drosophila pupae can regenerate their processes in vitro. Electrophysiological recordings demonstrate the formation of functional synaptic connections as early as 3 days in vitro (DIV), but no information about synapse structure is available. Here, we report that antibodies against pre-synaptic proteins Synapsin and Bruchpilot result in punctate staining of regenerating neurites. Puncta density increases as neuritic plexuses develop over the first 4 DIV. Electron microscopy reveals that closely apposed neurites can form chemical synapses with both pre- and postsynaptic specializations characteristic of many inter-neuronal synapses in the adult brain. Chemical synapses in culture are restricted to neuritic processes and some neurite pairs form reciprocal synapses. GABAergic synapses have a significantly higher percentage of clear core versus granular vesicles than non-GABA synapses. Gap junction profiles, some adjacent to chemical synapses, suggest that neurons in culture can form purely electrical as well as mixed synapses, as they do in the brain. However, unlike adult brain, gap junctions in culture form between neuronal somata as well as neurites, suggesting soma ensheathing glia, largely absent in culture, regulate gap junction location in vivo. Thus pupal brain cultures, which support formation of interneuronal synapses with structural features similar to synapses in adult brain, are a useful model system for identifying intrinsic and extrinsic regulators of central synapse structure as well as function.     

Electrotonic synapses in the mammalian spinal cord

By means of light and electron microscopy methods structural peculiarities of motor nuclei have been studied in the rat spinal cord (17 animals) on the 1st-3d and on the 10th-18th days of postnatal ontogenesis. Synaptic junctions of the gap type are revealed; they are considered as electrotonic synapses. Dendro-somatic and dendrodendritic synaptic junctions of the gap type are found. Together with the electrotonic synapses, morphologically mixed synapses of axo-somatic and axo-axonal types are disclosed; they contain, besides organells, specific for chemical synapses, close opposition areas of pre- and postsynaptic membranes of the gap junction type. Morphologically mixed synapses occur in neuropil of the motor nuclei of the spinal cord in young rats of all age groups studied. Homologous synapses are detected in the motor nuclei of the white mouse spinal cord. Synaptic junctions of the gap type in the mammalian spinal cord could be a substrate of electrical interaction between its motor neurons.     

Electrical Transmission between Mammalian Neurons is Supported by a Small Fraction of Gap Junction Channels

Electrical synapses formed by gap junctions between neurons create networks of electrically coupled neurons in the mammalian brain, where these networks have been found to play important functional roles. In most cases, interneuronal gap junctions occur at remote dendro-dendritic contacts, making difficult accurate characterization of their physiological properties and correlation of these properties with their anatomical and morphological features of the gap junctions. In the mesencephalic trigeminal (MesV) nucleus where neurons are readily accessible for paired electrophysiological recordings in brain stem slices, our recent data indicate that electrical transmission between MesV neurons is mediated by connexin36 (Cx36)-containing gap junctions located at somato-somatic contacts. We here review evidence indicating that electrical transmission between these neurons is supported by a very small fraction of the gap junction channels present at cell-cell contacts. Acquisition of this evidence was enabled by the unprecedented experimental access of electrical synapses between MesV neurons, which allowed estimation of the average number of open channels mediating electrical coupling in relation to the average number of gap junction channels present at these contacts. Our results indicate that only a small proportion of channels (~0.1?%) appear to be conductive. On the basis of similarities with other preparations, we postulate that this phenomenon might constitute a general property of vertebrate electrical synapses, reflecting essential aspects of gap junction function and maintenance.     

Short-range functional interaction between connexin35 and neighboring chemical synapses

Auditory afferents terminating as mixed, electrical, and chemical, synapses on the goldfish Mauthner cells constitute an ideal experimental model to study the properties of gap junctions in the nervous system as well as to explore possible functional interactions with the other major form of interneuronal communication--chemically mediated synapses. By combining confocal microscopy and freeze-fracture replica immunogold labeling (FRIL), we found that gap junctions at these synapses contain connexin35 (Cx35), the fish ortholog of the neuron-specific human and mouse connexin36 (Cx36). Conductance of gap junction channels at these endings is known to be dynamically modulated by the activity of their co-localized chemically mediated glutamatergic synapses. By using simultaneous pre- and postsynaptic recordings at these single terminals, we demonstrate that such functional interaction takes place in the same ending, within a few micrometers. Accordingly, we also found evidence by confocal and FRIL double-immunogold labeling that the NR1 subunit of the NMDA glutamate receptor, proposed to be a key regulatory element, is present at postsynaptic densities closely associated with gap junction plaques containing Cx35. Given the widespread distribution of Cx35- and Cx36-mediated electrical synapses and glutamatergic synapses, our data suggest that the local functional interactions observed at these identifiable junctions may also apply to other electrical synapses, including those in mammalian brain.     

Expression and functions of neuronal gap junctions

Gap junctions are channel-forming structures in contacting plasma membranes that allow direct metabolic and electrical communication between almost all cell types in the mammalian brain. At least 20 connexin genes and 3 pannexin genes probably code for gap junction proteins in mice and humans. Gap junctions between murine neurons (also known as electrical synapses) can be composed of connexin 36, connexin 45 or connexin 57 proteins, depending on the type of neuron. Furthermore, pannexin 1 and 2 are likely to form electrical synapses. Here, we discuss the roles of connexin and pannexin genes in the formation of neuronal gap junctions, and evaluate recent functional analyses of electrical synapses that became possible through the characterization of mouse mutants that show targeted defects in connexin genes.     

Short-Range Functional Interaction Between Connexin35 and Neighboring Chemical Synapses

Auditory afferents terminating as mixed, electrical, and chemical, synapses on the goldfish Mauthner cells constitute an ideal experimental model to study the properties of gap junctions in the nervous system as well as to explore possible functional interactions with the other major form of interneuronal communication—chemically mediated synapses. By combining confocal microscopy and freeze-fracture replica immunogold labeling (FRIL), we found that gap junctions at these synapses contain connexin35 (Cx35), the fish ortholog of the neuron-specific human and mouse connexin36 (Cx36). Conductance of gap junction channels at these endings is known to be dynamically modulated by the activity of their co-localized chemically mediated glutamatergic synapses. By using simultaneous pre- and postsynaptic recordings at these single terminals, we demonstrate that such functional interaction takes place in the same ending, within a few micrometers. Accordingly, we also found evidence by confocal and FRIL double-immunogold labeling that the NR1 subunit of the NMDA glutamate receptor, proposed to be a key regulatory element, is present at postsynaptic densities closely associated with gap junction plaques containing Cx35. Given the widespread distribution of Cx35- and Cx36-mediated electrical synapses and glutamatergic synapses, our data suggest that the local functional interactions observed at these identifiable junctions may also apply to other electrical synapses, including those in mammalian brain.     

Gap junctions in excitable cells

Gap junction channels are an integral part of the conduction or propagation of an action potential from cell to cell. Gap junctions have rather unique gating and permeability properties which permit the movement of molecules from cell to cell. These molecules may not be directly linked to action potentials but can alter nonjunctional processes within cells, which in turn can affect conduction velocity. The data described in this review reveal that, for the majority of excitable cells, there are two limiting factors, with respect to gap junctions, that affect the conduction/propagation of action potentials. These are (1) the total number of channels and (2) the selective permeability of the channels. Interestingly, voltage dependence and the time course of voltage inactivation (kinetics) are not rate limiting steps under normal physiological conditions for any of the connexins studied so far. Only specialized rectifying electrical synapses utilize strong voltage dependence and rapid kinetics to permit or deny the continued propagation of an action potential.     

Electrical synapses between GABA-releasing interneurons

Although gap junctions were first demonstrated in the mammalian brain about 30 years ago, the distribution and role of electrical synapses have remained elusive. A series of recent reports has demonstrated that inhibitory interneurons in the cerebral cortex, thalamus, striatum and cerebellum are extensively interconnected by electrical synapses. Investigators have used paired recordings to reveal directly the presence of electrical synapses among identified cell types. These studies indicate that electrical coupling is a fundamental feature of local inhibitory circuits and suggest that electrical synapses define functionally diverse networks of GABA-releasing interneurons. Here, we discuss these results, their possible functional significance and the insights into neuronal circuit organization that have emerged from them.     

Ultrastructure, histological distribution, and freeze-fracture immunocytochemistry of gap junctions in rat brain and spinal cord

The historical development of concepts of gap junctions as sites for electrical, ionic, and metabolic coupling is reviewed, from the initial discovery of gap junctions linking heart cells, to the current concepts that gap junctions represent 'electrotonic synapses' between neurons. The ultrastructure and immunocytochemistry of gap junctions in heart, brain, and spinal cord of adult rats is examined using conventional thin sections, negative staining, grid-mapped freeze-fracture replicas, and immunogold-labeled freeze-fracture replicas. We review evidence for neuronal gap junctions at 'mixed' (combined electrical and chemical) synapses throughout adult rat spinal cord. We also show immunogold labeling of connexin43 in astrocyte and ependymocyte gap junctions and of connexin32 in oligodendrocyte gap junctions. Ultrastructural and freeze-fracture immunocytochemical methods have provided for definitive determination of the number, size, histological distribution, and connexin composition of gap junctions between neurons in all regions of the central nervous systems of vertebrate species.     

Ephaptic interactions within a chemical synapse: hemichannel-mediated ephaptic inhibition in the retina

The two best-known types of cell-cell communication are chemical synapses and electrical synapses, which are formed by gap junctions. A third, less well known, form of communication is ephaptic transmission, in which electric fields generated by a specific neuron alter the excitability of neighboring neurons as a result of their anatomical and electrical proximity. Ephaptic communication can be present in a variety of forms, each with their specific features and functional implications. One of these is ephaptic modulation within a chemical synapse. This type of communication has recently been proposed for the cone-horizontal cell synapse in the vertebrate retina. Evidence indicates that the extracellular potential in the synaptic terminal of photoreceptors is modulated by current flowing through connexin hemichannels at the tips of the horizontal cell dendrites, mediating negative feedback from horizontal cells to cones. This example can be added to the growing list of cases of ephaptic communication in the central nervous system.     

Synergism and antagonism of neurons caused by an electrical synapse

To investigate the role of electrical junctions in the nervous system, a model system consisting of two nearly identical neurons electrotonically coupled is studied. We assume that each neuron discharges a train of impulses or bursts either spontaneously or under constant stimulus via chemical synapses. It is known that not only an electric current but also chemical substances whose molecular weight is about 1000 can pass through the junction of an electrical synapse (gap junction). So, our model system is regarded as a set of non-linear oscillators coupled by diffusion, and it may be described by a system of ordinary differential equations. Neurons are excited constantly when they are stimulated by an electric current above the threshold level. Therefore, we expect Hopf bifurcation to occur at the critical magnitude of a stimulating electric current in the system of differential equations which describes the dynamics of a single neuron. Studying our model system according to the theory of Hopf bifurcation, we found regions of diffusion constants of the electrical junction which give two kinds of periodic solutions. One is the solution where two neurons oscillate in phase synchrony. The other is where two neurons oscillate 180° out of phase. In the case where one neuron is described by the BVP model, the following was found by computer simulation. When the initial difference between the phase of two neurons is small, the two neurons come to oscillate synchronously. If the initial difference is large, however, the two come to be excited alternately. The physiological implications of these results are discussed.Department of Behaviorology, Faculty of Human Sciences     

Clathrin-mediated endocytosis at synapses

Neurons are communication specialists that convert electrical into chemical signals at specialized cell-cell junctions termed synapses. Arrival of an action potential triggers calcium-regulated exocytosis of neurotransmitter (NT) from small synaptic vesicles (SVs), which then diffuses across the synaptic cleft and binds to postsynaptic receptors to elicit specific changes within the postsynaptic cell. Endocytosis of pre- and postsynaptic membrane proteins including SV components and postsynaptic NT receptors is essential for the proper functioning of the synapse. During the past several years, we have witnessed enormous progress in our understanding of the mechanics of clathrin-mediated endocytosis (CME) and its role in regulating exo-endocytic vesicle cycling at synapses. Here we summarize the molecular machinery used for recognition of synaptic membrane protein cargo and its clathrin-dependent internalization, and describe the inventory of tools that can be used to monitor vesicle cycling at synapses or to inhibit CME in a stage-specific manner.     

Inter- and intramyotomal gap junctions in the axolotl embryo.

In early tailbud embryos of the axolotl (Ambystoma mexicanum), cells of the anterior myotomes begin to elongate and align along the longitudinal axis of the animal. Soon thereafter, gap junctions appear between the differentiating myotubes. These junctions occur between adjacent cells within a myotome (intramyotomal) and between the cells of adjacent myotomes which are separated from one another by narrow connective tissue septa (intermyotomal). The latter are found at the ends of the elongating cells where muscle-tendon insertion will occur and nerve-muscle synapses will form. The gap junctions are transient: They appear with the onset of myofibrillar formation at the time that nerve fibers enter the intermyotomal septa. The junctions last until the cells have differentiated into mature striated muscle cells and neuromuscular synapses are fully developed.These gap junctions may provide a means for the direct intercellular spread of electrical excitation between the differentiating muscle cells and so account for the observed myogenic contraction of myotomes. We also suggest that these junctions may form a means for cellular communication and interaction during the development of the axial musculature.     

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.     

Gap Junctions and Epileptic Seizures – Two Sides of the Same Coin?

Electrical synapses (gap junctions) play a pivotal role in the synchronization of neuronal ensembles which also makes them likely agonists of pathological brain activity. Although large body of experimental data and theoretical considerations indicate that coupling neurons by electrical synapses promotes synchronous activity (and thus is potentially epileptogenic), some recent evidence questions the hypothesis of gap junctions being among purely epileptogenic factors. In particular, an expression of inter-neuronal gap junctions is often found to be higher after the experimentally induced seizures than before. Here we used a computational modeling approach to address the role of neuronal gap junctions in shaping the stability of a network to perturbations that are often associated with the onset of epileptic seizures. We show that under some circumstances, the addition of gap junctions can increase the dynamical stability of a network and thus suppress the collective electrical activity associated with seizures. This implies that the experimentally observed post-seizure additions of gap junctions could serve to prevent further escalations, suggesting furthermore that they are a consequence of an adaptive response of the neuronal network to the pathological activity. However, if the seizures are strong and persistent, our model predicts the existence of a critical tipping point after which additional gap junctions no longer suppress but strongly facilitate the escalation of epileptic seizures. Our results thus reveal a complex role of electrical coupling in relation to epileptiform events. Which dynamic scenario (seizure suppression or seizure escalation) is ultimately adopted by the network depends critically on the strength and duration of seizures, in turn emphasizing the importance of temporal and causal aspects when linking gap junctions with epilepsy.     

Synapse formation and function: Insights from identified leech neurons in culture

Identified leech neurons in culture are providing novel insights to the signals underlying synapse formation and function. Identified neurons from the central nervous system of the leech can be removed individually and plated in culture, where they retain their characteristic physiological properties, grow neurites, and form specific synapses that are directly accessible by a variety of approaches. Synapses between cultured neurons can be chemical or electrical (either rectifying or not) or may not form, depending on the neuronal identities. Furthermore, the characteristics of these synapses depend on the regions of the cells that come into contact. The formation and physiology of synapses between the Retzius cell and its partners have been well characterized. Retzius cells form purely chemical, inhibitory synapses with pressuresensitive (P) cells where serotonin (5-HT) is the transmitter. Retzius cells synthesize 5-HT, which is stored in vesicles that recycle after 5-HT is secreted on stimulation. The release of 5-HT is quantal, calcium-dependent, and shows activity-dependent facilitation and depression. Anterograde and retrograde signals during synapse formation modify calcium currents, responses to 5-HT, and neurite outgrowth. The nature of these synaptogenic signals is being elucidated. For example, contact specifically with Retzius cells induces a localized selection of transmitter responses in postsynaptic P cells. This effect is signaled by tyrosine phosphorylation prior to synapse formation. © 1995 John Wiley & Sons, Inc.     

Neural Organization of the Median Ocellus of the Dragonfly : II. Synaptic structure

Two types of presumed synaptic contacts have been recognized by electron microscopy in the synaptic plexus of the median ocellus of the dragonfly. The first type is characterized by an electron-opaque, button-like organelle in the presynaptic cytoplasm, surrounded by a cluster of synaptic vesicles. Two postsynaptic elements are associated with these junctions, which we have termed button synapses. The second synaptic type is characterized by a dense cluster of synaptic vesicles adjacent to the presumed presynaptic membrane. One postsynaptic element is observed at these junctions. The overwhelming majority of synapses seen in the plexus are button synapses. They are found most commonly in the receptor cell axons where they synaptically contact ocellar nerve dendrites and adjacent receptor cell axons. Button synapses are also seen in the ocellar nerve dendrites where they appear to make synapses back onto receptor axon terminals as well as onto adjacent ocellar nerve dendrites. Reciprocal and serial synaptic arrangements between receptor cell axon terminals, and between receptor cell axon terminals and ocellar nerve dendrites are occasionally seen. It is suggested that the lateral and feedback synapses in the median ocellus of the dragonfly play a role in enhancing transients in the postsynaptic responses.