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.     

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

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.     

'Non-synaptic' mechanisms in seizures and epileptogenesis

The role of 'non-synaptic' mechanisms (i.e. those mechanisms that are independent of active chemical synpases) in the synchronization of neuronal activity during seizures and their possible contribution to chronic epileptogenesis are summarized. These 'non-synaptic' mechanisms include electrotonic coupling through gap junctions, electrical field effects (i.e. ephaptic transmission), and ionic interactions (e.g. increases in the extracellular concentration of K(+)). Several lines of evidence indicate that granule cells and pyramidal cells of the hippocampus, and probably other cortical neurons, can generate synchronized electrical activity after active chemical synaptic transmission has been blocked. This synchronized activity is sensitive to alterations in the size of the extracellular space, thus suggesting that electrical field effects and ionic mechanisms contribute to this synchronized activity. Recent studies also indicate that 'non-synaptic' synchronization is quite prominent early in development. Electrophysiological data from hippocampal and neocortical slices have led to a re-interpretation of the fast prepotentials (i.e. partial spikes) recorded in cortical pyramidal cells, suggesting that they may not be due to dendritic spike generation. Improvement in freeze-fracture ultrastructural techniques have led to a re-assessment of previous data on gap junctions in the nervous system and opened new approaches to the quantitative analysis and characterization of gap junctions on glia and neurons. Finally, new methods of dye/tracer coupling have the potential to provide a more rigorous basis for evaluating gap junctions and electrotonic communication between neurons in the mammalian central nervous system. Therefore, recent data continue to suggest that gap junctions and electrotonic coupling play an important role in neural integration, although additional studies using new techniques will be needed to address some of the controversial issues that have arisen over the last several decades.     

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.     

A STUDY OF THE INNERVATION OF THE TAENIA COLI

An electrophysiological and anatomical study of the guinea pig taenia coli is reported. Changing the membrane potential of single cells cannot modulate the rate of firing action potentials but does reveal electrical coupling between the cells during propagation. The amplitude of the junction potentials which occur during transmission from inhibitory nerves is unaffected in many cells during alteration of the membrane potential, indicating electrical coupling during transmission. The taenia coli is shown to consist of smooth muscle bundles which anastomose. There are tight junctions between the cells in the bundles, and these probably provide the pathway for the electrical coupling. The smooth muscle cells towards the serosal surface of the taenia coli are shown electrophysiologically to have an extensive intramural inhibitory innervation, but a sparse sympathetic inhibitory and cholinergic excitatory innervation. These results are in accordance with the distribution of these nerves as determined histochemically. As single axons are only rarely observed in the taenia coli, it is suggested that the only muscle cells which undergo permeability changes during transmission are those adjacent to varicosities in the nerve bundles. The remaining muscle cells then undergo potential changes during transmission because of electrical coupling through the tight junctions.     

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.     

Electrical coupling and neuronal synchronization in the Mammalian brain

Certain neurons in the mammalian brain have long been known to be joined by gap junctions, which are the most common type of electrical synapse. More recently, cloning of neuron-specific connexins, increased capability of visualizing cells within brain tissue, labeling of cell types by transgenic methods, and generation of connexin knockouts have spurred a rapid increase in our knowledge of the role of gap junctions in neural activity. This article reviews the many subtleties of transmission mediated by gap junctions and the mechanisms whereby these junctions contribute to synchronous firing.     

Gap junctions as electrical synapses

Gap junctions are the morphological substrate of one class of electrical synapse. The history of the debate on electrical vs. chemical transmission is instructive. One lesson is that Occam’s razor sometimes cuts too deep; the nervous system does its operations in a number of different ways and a unitarian approach can lead one astray. Electrical synapses can do many things that chemical synapses can do, and do them just as slowly. More intriguing are the modulatory actions that chemical synapses can have on electrical synapses. Voltage dependence provides an important window on structure function relations of the connexins, even where the dependence may have no physiological role. The new molecular approaches will greatly advance our knowledge of where gap junctions occur and permit experimental manipulation with high specificity.     

Connexin36 mediates spike synchrony in olfactory bulb glomeruli

Neuronal synchrony is important to network behavior in many brain regions. In the olfactory bulb, principal neurons (mitral cells) project apical dendrites to a common glomerulus where they receive a common input. Synchronized activity within a glomerulus depends on chemical transmission but mitral cells are also electrically coupled. We examined the role of connexin-mediated gap junctions in mitral cell coordinated activity. Electrical coupling as well as correlated spiking between mitral cells projecting to the same glomerulus was entirely absent in connexin36 (Cx36) knockout mice. Ultrastructural analysis of glomeruli confirmed that mitral-mitral cell gap junctions on distal apical dendrites contain Cx36. Coupled AMPA responses between mitral cell pairs were absent in the knockout, demonstrating that electrical coupling, not transmitter spillover, is responsible for synchronization. Our results indicate that Cx36-mediated gap junctions between mitral cells orchestrate rapid coordinated signaling via a novel form of electrochemical transmission.     

Mechanisms of elimination, remodeling, and competition at frog neuromuscular junctions

Mechanisms governing synapse elimination, synaptic remodeling, and polyneuronal innervation were examined in anatomical and electrophysiological studies of frog neuromuscular junctions. There was a substantial level of polyneuronal innervation in adult junctions and this varied seasonally. Nerve terminal retraction and synapse elimination occurred during normal growth and following reinnervation. Synapse elimination was not inevitable, however. Repeated in vivo observations of some identified junctions showed that polyneuronal innervation could persist for over a year, while at other junctions it arose de novo by terminal sprouting. We concluded that polyneuronal innervation in adult muscles was governed by an equilibrium between processes of retraction and elimination on one hand, and sprouting and synaptogenesis on the other. Other observations revealed that structural remodeling was a common feature of adult junctions. Most often, remodeling involved the simultaneous growth and retraction of different parts of the same junction. The net result was usually junctional growth that, in small frogs, appeared to provide a good match between synaptic size and the electrical demands of transmission. In larger animals, pre- and postsynaptic sizes were not as well matched, providing morphological evidence for a growth-associated decline in synaptic efficacy. Finally, electrophysiology was used to describe some of the functional correlates and consequences of competitive interactions between the terminals of different axons. These results are explained by a hypothetical mechanism that involves trophic support provided by the muscle to the motoneuron, the overall level of nerve-muscle activity, and the synchrony of pre- and postsynaptic activity.     

Revisiting the Stimulus-Secretion Coupling in the Adrenal Medulla: Role of Gap Junction-Mediated Intercellular Communication

The current view of stimulation-secretion coupling in adrenal neuroendocrine chromaffin cells holds that catecholamines are released upon transsynaptic sympathetic stimulation mediated by acetylcholine released from the splanchnic nerve terminals. However, this traditional vertical scheme would merit to be revisited in the light of recent data. Although electrical discharges invading the splanchnic nerve endings are the major physiological stimulus to trigger catecholamine release in vivo, growing evidence indicates that intercellular chromaffin cell communication mediated by gap junctions represents an additional route by which biological signals (electrical activity, changes in intracellular Ca²⁺ concentration,…) propagate between adjacent cells and trigger subsequent catecholamine exocytosis. Accordingly, it has been proposed that gap junctional communication efficiently helps synapses to lead chromaffin cell function and, in particular, hormone secretion. The experimental clues supporting this hypothesis are presented and discussed with regards to both interaction with the excitatory cholinergic synaptic transmission and physiopathology of the adrenal medulla.     

Close apposition of muscle cells in the longitudinal bands of the body wall of a holothurian,Isostichopus badionotus

Summary Electron microscopy reveals that sarcolemmata of adjacent muscle cells form pentalaminar junctions by fusion of apposed trilaminar double leaflet membranes. These junctions appear to be candidates for low resistance pathways between muscle fibers. The muscles depolarize slowly when bathed in solutions containing elevated concentrations of KCl, and the sucrose gap method can then be used to measure the potential difference between polarized and depolarized regions. Thus the junctions which we have observed may provide the structural basis for electrical transmission through the sucrose gap.Contribution No. 916 from the Bermuda Biological Station for Research, Inc. Supported by N.S.F. Grant POM 7613459 to the Bermuda Biological Station     

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent^1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions^3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling^3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation^7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading^9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas^{7, 8,13}, and can be used to study coupling between other retinal neurons, as described here.     

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.     

Electrical synapses: a dynamic signaling system that shapes the activity of neuronal networks

Gap junctions consist of intercellular channels dedicated to providing a direct pathway for ionic and biochemical communication between contacting cells. After an initial burst of publications describing electrical coupling in the brain, gap junctions progressively became less fashionable among neurobiologists, as the consensus was that this form of synaptic transmission would play a minimal role in shaping neuronal activity in higher vertebrates. Several new findings over the last decade (e.g. the implication of connexins in genetic diseases of the nervous system, in processing sensory information and in synchronizing the activity of neuronal networks) have brought gap junctions back into the spotlight. The appearance of gap junctional coupling in the nervous system is developmentally regulated, restricted to distinct cell types and persists after the establishment of chemical synapses, thus suggesting that this form of cell-cell signaling may be functionally interrelated with, rather than alternative to chemical transmission. This review focuses on gap junctions between neurons and summarizes the available data, derived from molecular, biological, electrophysiological, and genetic approaches, that are contributing to a new appreciation of their role in brain function.     

Pyramid power: Principal cells of the hippocampus unite!

Electrical transmission in the mammalian brain is now well established. A new study by Thomson and colleagues elegantly demonstrates coupling between CA1 hippocampal pyramidal cells, which is far more common than previously supposed. Although the history of coupling is extensive, doubt, predjudice, and technical issues long kept it from wide acceptance. Here “spikelets” or “fast prepotentials” are found when two cells are coupled and in this situation result from electrical transmission of impulses from one coupled cell to the other. Interesting questions remain as to whether connexin or pannexin gap junctions serve as the molecular substrate of transmission, and the role of electrical transmission in hippocampal physiology is uncertain. Increased coupling could well contribute to the known tendency of the hippocampus to exhibit seizure activity.     

Coordination of neuronal activity by gap junctions in the developing neocortex

During embryonic development, gap junctions link cells into functional communication compartments characterized by a common development fate. Increasing evidence for gap junctions between immature neurons suggest that similar mechanisms may also be at work in the developing vertebrate brain, where gap junction-coupled neuronal assemblies often precede synaptically-linked functional networks. Recent experiments in the developing mammalian neocortex demonstrated the presence of gap-junction mediated second messenger waves, similar to those in non-neuronal cells. The primary function of neuronal gap junctions, therefore, might be to coordinate biochemical activity, rather than to act as purely electrical synapses. Thus, gap junctions may serve to amplify neuronal activity produced by weak synaptic stimulation.     

On-line analysis of gap junctions reveals more efficient electrical than dye coupling between islet cells

Pancreatic beta-cells constitute a well-communicating multicellular network that permits a coordinated and synchronized signal transmission within the islet of Langerhans that is necessary for proper insulin release. Gap junctions are the molecular keys that mediate functional cellular connections, which are responsible for electrical and metabolic coupling in the majority of cell types. Although the role of gap junctions in beta-cell electrical coupling is well documented, metabolic communication is still a matter of discussion. Here, we have addressed this issue by use of a fluorescence recovery after photobleaching (FRAP) approach. This technique has been validated as a reliable and noninvasive approach to monitor functional gap junctions in real time. We show that control pancreatic islet cells did not exchange a gap junction-permeant molecule in either clustered cells or intact islets of Langerhans under conditions that allowed cell-to-cell exchange of current-carrying ions. Conversely, we have detected that the same probe was extensively transferred between islet cells of transgenic mice expressing connexin 32 (Cx32) that have enhanced junctional coupling properties. The results indicate that the electrical coupling of native islet cells is more extensive than dye communication. Dye-coupling domains in islet cells appear more restricted than previously inferred with other methods.     

Gap junctions between dendrites and somata of neurons in the primate sensori-motor cortex.

Gap junctions have been found infrequently between two dendrites or a dendrite and a cell soma in the deep layers of both the motor and somatic sensory cortices of the primate. At these junctions the outer leaflets of the plasma membranes of both profiles are intimately apposed with a gap of 2 nm between them which shows a structure of hexagonal subunits in tangential sections. These gap junctions occur mainly between the dendrites or dendrites and somata of large stellate cells but are also associated in some examples with a dendro-dendritic synapse and thus occur between large stellate dendrites and presynaptic dendrites; a desmosome may also occur in association with a gap junction and dendro-dendritic synapse. Gap junctions have been identified as sites of electrical transmission between cells in a number of sites and it is therefore suggested that some neurons in the sensori-motor cortex are electrotonically couples.