Dye-coupling visualizes networks of large-field motion-sensitive neurons in the fly

In the fly, visually guided course control is accomplished by a set of 60 large-field motion-sensitive neurons in each brain hemisphere. These neurons have been shown to receive retinotopic motion information from local motion detectors on their dendrites. In addition, recent experiments revealed extensive coupling between the large-field neurons through electrical synapses. These two processes together give rise to their broad and elaborate receptive fields significantly surpassing the extent of their dendritic fields. Here, we demonstrate that the electrical connections between different large-field neurons can be visualized using Neurobiotin dye injection into a single one of them. When combined with a fluorescent dye which does not cross electrical synapses, the injected cell can be identified unambiguously. The Neurobiotin staining corroborates the electrical coupling postulated amongst the cells of the vertical system (VS-cells) and between cells of the horizontal system (HS-cells and CH-cells). In addition, connections between some cells are revealed that have so far not been considered as electrically coupled.     

Variability and frequent failure of lucifer yellow to pass between two electrically coupled neurons in Lymnaea stagnalis

The electrically coupled giant neurosecretory neurons VD1 and RPD2 of Lymnaea stagnalis were found to have coupling coefficients ranging from ca. 0.1-0.6. When the fluoroescent dye Lucifer Yellow was injected intracellularly into one of the neurons, in most preparations no dye was observed to pass through into the coupled cell body or the process leading to it. There was no apparent correlation between the amount of dye coupling and the length of time allowed for diffusion of the dye in the cells. In eight preparations, the electrical coupling coefficient was measured before dye was injected. There was no correlation between dye coupling and the electrical coupling coefficient.     

Functional gap junctions in the early sea urchin embryo are localized to the vegetal pole.

Using the whole-cell voltage-clamp technique we have studied electrical coupling and dye coupling between pairs of blastomeres in 16- to 128-cell-stage sea urchin embryos. Electrical coupling was established between macromeres and micromeres at the 16-cell stage with a junctional conductance (G(j)) of 26 nS that decreased to 12 nS before the next cleavage division. G(j) between descendants of macromeres and micromeres was 12 nS falling to 8 nS in the latter half of the cell cycle. Intercellular current intensity was independent of transjunctional voltage, nondirectional, and sensitive to 1-octanol and therefore appears to be gated through gap junction channels. There was no significant coupling between other pairs of blastomeres. Lucifer yellow did not spread between these electrically coupled cell pairs and in fact significant dye coupling between nonsister cells was observed only at the 128-cell stage. Since 1-octanol inhibited electrical communication between blastomeres at the 16- to 64-cell stage and also induced defects in formation of the archenteron, it is possible that gap junctions play a role in embryonic induction.     

Characteristics and plasticity of electrical synaptic transmission

Electrical synapses are an omnipresent feature of nervous systems, from the simple nerve nets of cnidarians to complex brains of mammals. Formed by gap junction channels between neurons, electrical synapses allow direct transmission of voltage signals between coupled cells. The relative simplicity of this arrangement belies the sophistication of these synapses. Coupling via electrical synapses can be regulated by a variety of mechanisms on times scales ranging from milliseconds to days, and active properties of the coupled neurons can impart emergent properties such as signal amplification, phase shifts and frequency-selective transmission. This article reviews the biophysical characteristics of electrical synapses and some of the core mechanisms that control their plasticity in the vertebrate central nervous system.     

Differential modulation of chemical and electrical components of mixed synapses in the lobster stomatogastric ganglion

1. Two pairs of neurons in the pyloric network of the spiny lobster, Panulirus interruptus, communicate through mixed graded chemical and rectifying electrical synapses. The anterior burster (AB) chemically inhibits and is electrically coupled to the ventricular dilator (VD); the lateral pyloric (LP) and pyloric (PY) neurons show reciprocal chemical inhibition and electrical coupling. We examined the effects of dopamine (DA), serotonin (5HT) and octopamine (Oct) on these mixed synapses to determine the plasticity possible with opposing modes of synaptic interaction.
2. Dopamine increased net inhibition at all three pyloric mixed synapses by both reducing electrical coupling and increasing chemical inhibition. This reversed the sign of the net synaptic interaction when electrotonic coupling dominated some mixed synapses, and activated silent chemical components of other mixed synapses.
3. Serofonin weakly enhanced LP → PY net inhibition, by reducing electrical coupling without altering chemical inhibition. Serotonin reduced AB→ VD electrical coupling, but variability in its effect on the chemical component made the net effect non-significant.
4. Octopamine enhanced LP→ PY and PY→ LP net inhibition by enhancing the chemical inhibitory component without altering electrical coupling.
5. Differential modulation of chemical and electrical components of mixed synapses markedly changes the net synaptic interactions. This contributes to the flexible outputs that modulators evoke from anatomically defined neural networks.

     

Bursting synchronization dynamics of pancreatic β-cells with electrical and chemical coupling

Based on bifurcation analysis, the synchronization behaviors of two identical pancreatic β-cells connected by electrical and chemical coupling are investigated, respectively. Various firing patterns are produced in coupled cells when a single cell exhibits tonic spiking or square-wave bursting individually, irrespectively of what the cells are connected by electrical or chemical coupling. On the one hand, cells can burst synchronously for both weak electrical and chemical coupling when an isolated cell exhibits tonic spiking itself. In particular, for electrically coupled cells, under the variation of the coupling strength there exist complex transition processes of synchronous firing patterns such as “fold/limit cycle” type of bursting, then anti-phase continuous spiking, followed by the “fold/torus” type of bursting, and finally in-phase tonic spiking. On the other hand, it is shown that when the individual cell exhibits square-wave bursting, suitable coupling strength can make the electrically coupled system generate “fold/Hopf” bursting via “fold/fold” hysteresis loop; whereas, the chemically coupled cells generate “fold/subHopf” bursting. Especially, chemically coupled bursters can exhibit inverse period-adding bursting sequence. Fast–slow dynamics analysis is applied to explore the generation mechanism of these bursting oscillations. The above analysis of bursting types and the transition may provide us with better insight into understanding the role of coupling in the dynamic behaviors of pancreatic β-cells.     

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.     

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

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.     

Intercellular communication inAzolla roots: II. Electrical coupling

Summary There is a predictable and well defined variation in numbers of plasmodesmata in roots ofAzolla. As the apical cell of the root ages, it lays down walls with progressively fewer plasmodesmata, thereby gradually cutting itself off from the rest of the root (Gunning 1978). Electrical coupling was examined between the apical cell and an adjacent merophyte in roots of various lengths. The apical cell becomes increasingly electrically isolated from the rest of the root as it ages. Electrical coupling is strongly correlated with the number of the plasmodesmata between the coupled cells. The resistance of a plasmodesma, as estimated from equivalent electrical circuits, was 150–600 times more resistive than a value based on theoretical considerations. No evidence was found for a change in the physiology of plasmodesmata as the root ages. Coupling experiments, both on root hairs and at the apex, gave some suggestion that plasmodesmata may be less resistive towards the apical cell than away from it.     

Embryonic electrical connections appear to prefigure a behavioral circuit in the leech CNS

During development, many embryos show electrical coupling among neurons that is spatially and temporally regulated. For example, in vertebrate embryos extensive dye coupling is seen during the period of circuit formation, suggesting that electrical connections could prefigure circuits, but it has been difficult to identify which neuronal types are coupled. We have used the leech Hirudo medicinalis to follow the development of electrical connections within the circuit that produces local bending. This circuit consists of three layers of neurons: four mechanosensory neurons (P cells), 17 identified interneurons, and approximately 24 excitatory and inhibitory motor neurons. These neurons can be identified in embryos, and we followed the spatial and temporal dynamics as specific connections developed. Injecting Neurobiotin into identified cells of the circuit revealed that electrical connections were established within this circuit in a precise manner from the beginning. Connections first appeared between motor neurons; mechanosensory neurons and interneurons started to connect at least a day later. This timing correlates with the development of behaviors, so the pattern of emerging connectivity could explain the appearance first of spontaneous behaviors (driven by a electrically coupled motor network) and then of evoked behaviors (when sensory neurons and interneurons are added to the circuit).     

Estimating conductances of dual-recorded neurons within a network of coupled cells

Simultaneous pre- and postsynaptic cell recordings are used to calculate gap junction conductance based on an equivalent electrical circuit of an electrically coupled pair of cells. This calculation is imprecise when recording from a cell pair that is coupled to neighboring cells providing indirect conductance paths between the recorded cells. Despite this imprecision, junctional conductance has been calculated for coupled cell networks during the past 40 years since a more accurate method was lacking. The present study simulated a three-dimensional network of electrically coupled heterogeneous neurons and used mathematical modeling to reduce the complexity to the simplest equations that could more accurately estimate the electrical properties of dual-recorded cells in the network. Analyses of the simulations showed that knowledge of the number of unrecorded cells directly linked to the recorded cells and of the voltage responses of these recorded cells were largely sufficient to accurately predict the direct junctional resistance linking the recorded cells as well as the input resistance of the recorded cells that would exist in the absence of junctional coupling. All model parameters could be obtained from real dual-intracellular penetrations which allow electrophysiological recordings and intracellular staining.     

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.     

Developmental uncoupling between blastoderm and yolk cell in the embryo of the teleost Fundulus

During cleavage and blastula stages of embryos of the teleost Fundulus heteroclitus all of the cells are both electotonically coupled and dye coupled to one another, as determined by microelectrode impalements and spread of Lucifer Yellow. At about the time that gastrulation begins we observed a specific loss of junctional coupling between the yolk cell and cells of the blastoderm. Passage of Lucifer Yellow between the yolk cell and blastoderm was reduced at stage 12 (late blastula), and not detected at stage 13 and thereafter, although cells of the blastoderm remain dye coupled to one another through gastrula stages. Also, junctional electrical coupling between the yolk cell and blastoderm became substantially reduced at stage 13 and thereafter. The loss of coupling at this specific cell apposition and time and the large size of the yolk cell may prove useful in analyzing the underlying cellular mechanisms.     

A quantum of neurotransmitter causes minis in multiple postsynaptic cells at the Caenorhabditis elegans neuromuscular junction

The polyadic synapse, where a single presynaptic active zone associates with two or more postsynaptic cells, exists in both mammals and invertebrates. An important but unresolved question is whether synaptic transmission occurs between the presynaptic site and its various postsynaptic partners. Using the dual whole-cell voltage clamp technique, we analyzed miniature postsynaptic currents (mPSCs or minis) at the C. elegans neuromuscular junction (NMJ), which is a polyadic synapse. We found that neighboring muscle cells at the same position along the body axis had high frequencies of concurrent mPSCs, which could not be explained by pure chance. Although body-wall muscle cells are electrically coupled, the high frequency of concurrent mPSCs was not due to electrical coupling because there was no correlation between the frequency of concurrent mPSCs and the degree of electrical coupling; the rise time of concurrent mPSCs was identical to that of nonconcurrent mPSCs but distinct from that of junctional currents (I(j)); and a mutant defective in electrical coupling showed normal frequency of concurrent mPSCs. Our analyses suggest that a single quantum of neurotransmitter may cause mPSCs in multiple postsynaptic cells at polyadic synapses, and that high-fidelity synaptic transmission occurs between the presynaptic site and its various postsynaptic partners. Thus, polyadic synapses could be a distinct mechanism for synaptic divergence and for synchronizing activities of postsynaptic cells.     

The Role of Cellular Coupling in the Spontaneous Generation of Electrical Activity in Uterine Tissue

The spontaneous emergence of contraction-inducing electrical activity in the uterus at the beginning of labor remains poorly understood, partly due to the seemingly contradictory observation that isolated uterine cells are not spontaneously active. It is known, however, that the expression of gap junctions increases dramatically in the approach to parturition, by more than one order of magnitude, which results in a significant increase in inter-cellular electrical coupling. In this paper, we build upon previous studies of the activity of electrically excitable smooth muscle cells (myocytes) and investigate the mechanism through which the coupling of these cells to electrically passive cells results in the generation of spontaneous activity in the uterus. Using a recently developed, realistic model of uterine muscle cell dynamics, we investigate a system consisting of a myocyte coupled to passive cells. We then extend our analysis to a simple two-dimensional lattice model of the tissue, with each myocyte being coupled to its neighbors, as well as to a random number of passive cells. We observe that different dynamical regimes can be observed over a range of gap junction conductances: at low coupling strength, corresponding to values measured long before delivery, the activity is confined to cell clusters, while the activity for high coupling, compatible with values measured shortly before delivery, may spread across the entire tissue. Additionally, we find that the system supports the spontaneous generation of spiral wave activity. Our results are both qualitatively and quantitatively consistent with observations from in vitro experiments. In particular, we demonstrate that the increase in inter-cellular electrical coupling observed experimentally strongly facilitates the appearance of spontaneous action potentials that may eventually lead to parturition.     

Rhythmic coupling among cells in the suprachiasmatic nucleus

In mammals, the part of the nervous system responsible for most circadian behavior can be localized to a pair of structures in the hypothalamus known as the suprachiasmatic nucleus (SCN). Previous studies suggest that the basic mechanism responsible for the generation of these rhythms is intrinsic to individual cells. There is also evidence that the cells within the SCN are coupled to one another and that this coupling is important for the normal functioning of the circadian system. One mechanism that mediates coordinated electrical activity is direct electrical connections between cells formed by gap junctions. In the present study, we used a brain slice preparation to show that developing SCN cells are dye coupled. Dye coupling was observed in both the ventrolateral and dorsomedial subdivisions of the SCN and was blocked by application of a gap junction inhibitor, halothane. Dye coupling in the SCN appears to be regulated by activity-dependent mechanisms as both tetrodotoxin and the GABA(A) agonist muscimol inhibited the extent of coupling. Furthermore, acute hyperpolarization of the membrane potential of the original biocytin-filled neuron decreased the extent of coupling. SCN cells were extensively dye coupled during the day when the cells exhibit synchronous neural activity but were minimally dye coupled during the night when the cells are electrically silent. Immunocytochemical analysis provides evidence that a gap-junction-forming protein, connexin32, is expressed in the SCN of postnatal animals. Together the results are consistent with a model in which gap junctions provide a means to couple SCN neurons on a circadian basis.     

Electrical coupling and uncoupling of exocrine acinar cells

The electrical communication network in the mouse pancreatic acinar tissue has been investigated using simultaneous intracellular recording with two separate microelectrodes and direct microscopical control of the localizations of the microelectrode tips. All cells within one acinus were electrically coupled, and the coupling coefficient (the electrotonic potential change in a cell neighboring to the cell into which current is injected [V2] divided by the electrotonic potential change in the cell of current injection [V1]) between two cells near each other (less than 50 micron) was always close to 1. Cells farther apart (50-100 micron) were, in some cases, coupled; in other cases, there was no coupling at all. Coupling coefficients varied between 0 and 1. There was rarely electrical coupling over distances of more than 110 micron. Using microiontophoretic acetylcholine (ACh) application, it was possible to evoke almost complete electrical uncoupling of two previously coupled pancreatic or lacrimal acinar cells from different acini or within one acinus. The effects were fully and quickly reversible. While the ACh-evoked uncoupling in the pancreas was associated with membrane depolarization, ACh caused hyperpolarization in the lacrimal acinar cells. The uncoupling was associated with a very marked reduction in electrical time constant, indicating a reduction in input capacitance (effective surface cell membrane area). The concentrations of stimulants needed to evoke reduction in pancreatic cell-to-cell coupling were 1 micron for ACh, 0.14 nM for caerulein, and 3 nM for bombesin. These concentrations are smaller than those required to evoke maximal enzyme secretion.     

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.     

Transcellular ionic currents studied by intracellular potential recordings in Neurospora crassa hyphae. Transfer of energy from proximal to apical cells

Membrane potentials, input resistances, and electric coupling in the apical parts of N. crassa growing hyphae were recorded with the aid of intracellular microelectrodes. It was revealed that the apical cells were always depolarized by 10 to 30 mV as compared to the adjacent proximal cells. The septal pore maintained an electrical resistance of 4 to 6 M omega. The calculated values of the endogenous electrical current passing through the septal pore varied between 0.5 and 1 nA. Electrical isolation of the apical cells resulted in their depolarization from 120-150 mV to 40-60 mV, characteristics of the membrane potential value of N. crassa adult hyphae with completely blocked electrogenic pumps. A simultaneous increase in the input resistance value from 15-20 M omega to 40-80 M omega was observed. The above data can be explained assuming that H+-ATPase activity was greatly lowered in the apical cells. Thus in the intact hyphae with electrically coupled cells energy is transferred from the proximal hyphal compartments to the apical ones.