Some murine thymic lymphocytes can form gap junctions

Analysis of thymic lymphocytes isolated from weanling mice has revealed a minority population able to form permeable, intercellular (gap) junctions. This population is largest in mice aged between 3 and 6 weeks, much smaller in fetal and new-born mice and undetectable in mice aged 12 weeks or more. Fractionation of the thymocytes on Percoll gradients or with peanut agglutinin (PNA) shows the cells able to form junctions are enriched in lower density fractions and agglutinated by PNA, suggesting they are among the most immature. Fractionation by complement mediated cytotoxicity (CMC) and by fluorescence activated cell sorting (FACS) using monoclonal antibodies to specific cell surface determinants shows the junction forming cells are Lyt-1+/Lyt-2- and that the phenotype is associated with both high and low Thy-1 and H-2K epitope densities.     

Neuronal gap junctions between intraglomerular mitral/tufted cell dendrites in the mouse main olfactory bulb

In the mouse main olfactory bulb (MOB) gap junction-forming processes in glomeruli were analyzed by means of the serial electron microscopical reconstruction. Gap junctions were encountered between diverse types of dendritic processes and thus confirming our previous study on gap junctions in the rat MOB. Importantly, among more than 30 gap junctions examined in serial sections, we encountered 3 gap junctions made between mitral/tufted cell dendrites in the glomerulus. Then we must consider both direct coupling between mitral/tufted cells via gap junctions and indirect coupling between mitral/tufted cells via intervening interneuronal processes as suggested previously.     

Ultrastructural study of gap junctions between dendrites of parvalbumin-containing GABAergic neurons in various neocortical areas of the adult rat

Parvalbumin (PV)-containing GABAergic neurons in the hippocampus form dual networks linked by both dendrodendritic gap junctions and mutual inhibitory synapses. Recent physiological studies have demonstrated similar functional connectivity among cortical GABAergic neurons, but the corresponding structures have not been fully analyzed at the electron microscopic level. In this study we examined detailed ultrastructural features of gap junctions between PV neurons in the mature neocortex. Light microscopic observations and confocal laser scanning microscopy revealed frequent dendrodendritic contacts between PV neurons. Electron microscopic analysis provided direct morphological evidence for the existence of gap junctions between 22 pairs of PV-immunoreactive dendrites in the visual, auditory, and somatosensory cortices. Their ultrastructural features that were characteristic of immunolabeled profiles were consistent with the general structure of gap junctions. In one case a gap junction coexisted with a dendrodendritic chemical synapse, making a mixed synapse. Importantly, we also encountered a gap junction between PV positive and negative, presumptive non-principal cell-derived, dendrites. Quantitative analysis was made in 16 pairs of PV positive dendrites forming gap junctions in the infragranular layers of the somatosensory cortex. Diameters of these dendrites ranged from 0.3 to 2.7 microm, suggesting diverse locations of gap junctions along the proximal-distal axis of dendritic trees, but the majority (81%) were less than 1 microm. The mean size of gap junctions along apposing membranes was 0.22+/-0.09 microm. By using this size, the theoretical value of a junctional conductance was estimated to be 2.1-5.3 nS. Dendrites of PV neurons in the infragranular layers of the somatosensory cortex were reconstructed light microscopically and the sites of contacts with other PV neurons were mapped. Although these contacts do not necessarily imply gap junctional coupling, their number (5.3+/-2.3 per cell, n=11) suggested the degree of connectivity of less than 10 coupling from single PV neurons with others. Sholl analysis revealed that only 38% of their dendrites occurred within 200 microm from the soma. The present study demonstrated detailed ultrastructural features of gap junctions between mature cortical PV neurons. These features will facilitate not only identification of gap junctions in variously labeled neurons but also analysis of their functional aspects by enabling theoretical estimate of their junctional conductances.     

Diversity and molecular anatomy of gap junctions

In animal tissues, most cells are connected via intercellular cytoplasmic channels called gap junctions. Various electron microscopy techniques have made a crucial contribution to our understanding of the function and structure of gap junction channels. Tracer studies and freeze-fracture replica observations indicate that the connexon, the unit gap junction channel, is a pair of hemichannels apposed in the narrow intercellular gap between neighboring cell membranes. Recent advances in cellular biology have shown that connexon hemichannels are composed of hexamers of connexin proteins. Purification of the gap junction membrane and cDNA cloning analysis indicate the diversity of the connexin protein family, which contains more than 18 members, and their tissue- and cell type-specific distributions. Defects in some connexin genes may cause various hereditary diseases, such as X-linked Charcot-Marie-Tooth disease (Cx32), nonsyndromic autosomal deafness (Cx26), and cataract (Cx50). Analysis of gene knockout mice indicates that certain types of connexin play important roles in differentiation and development at crucial times in specific tissues and cell types.     

Clustering of cell bodies, bundling of dendrites, and gap junctions: morphological substrate for electrical coupling in the prepacemaker nucleus

The possible morphological basis for electrical coupling between neurons of the prepacemaker nucleus was studied in weakly electric gymnotiform fish at the ultrastructural level. Three structural characteristics were found: Extremely dense clustering of cell bodies; 'bundling' of dendrites; and gap junctions between neurons. Electrical coupling may take place through gap junctions and the spatial arrangement of elements in the prepacemaker nucleus, which could enable ephaptic interactions. Such mechanisms may also be used for averaging the responses of individual neurons in the whole assembly in order to render more predictable behavioral reactions.     

Short duty cycle destabilizes a half-center oscillator, but gap junctions can restabilize the anti-phase pattern

Mutually inhibitory pacemaker neurons with duty cycle close to 50% operate as a half-center oscillator (anti-phase coordination, i.e., 180 degrees out of phase), even in the presence of weak to modest gap junctional coupling. For electrical coupling strength above a critical value synchronization occurs. But, as shown here with modeling studies, the effects of electrical coupling depend critically on a cell's duty cycle. Instead of oscillating either in-phase or anti-phase, model cells with short duty cycle express additional rhythmic patterns, and different transitions between them, depending on electrical coupling strength. For weak or no electrical coupling, cells do not oscillate in anti-phase but instead exhibit almost in-phase activity. Strengthening this weak coupling leads to stable anti-phase activity. With yet stronger electrical coupling stable inphase (synchrony) emerges but it coexists with the anti-phase pattern. Thus the network shows bistability for an intermediate range of coupling strength. For sufficiently strong electrical coupling synchrony is the network's only attracting rhythmic state. Our results, numerical and analytical (phase plane analysis), are based on a minimal but biophysically motivated pacemaker model for the slowly oscillating envelope of bursting neurons. However, illustrations for an Hodgkin-Huxley model suggest that some of our results for short duty cycle may extend to patterning of repetitive spikes. In particular, electrical coupling of intermediate strength may promote anti-phase activity and provide bistability of anti-phase and in-phase spiking.     

Tight and gap junctions in a vertebrate inner ear

The auditory organ of the alligator lizard has been investigated with the transmission electron microscope using methods which distinguish between tight and gap junctions. There is a continuous zone of tight junctions located near the endolymphatic surface of the organ forming a boundary between the endolymph in scala media and the interstitial spaces between the cells. No such tight junctions were observed between the perilymph of scala tympani and the interstitial fluid within the organ. Small gap junctions occur between hair cells and supporting cells and large gap junctions occur between adjacent supporting cells. The locations of the tight junctions suggest that the composition of the intercellular fluid in the receptor organ is probably more like perilymph than like endolymph. The presence of gap junctions between hair cells and supporting cells provides a possible morphological basis for the occurrence of intracellular responses to sound in supporting cells, and for electric coupling of receptor cells.     

Neuro-epitheliomuscular cell and neuro-neuronal gap junctions inHydra

Summary Gap junctions have been described ultrastructurally between neurons and epitheliomuscular cells and between neurons and their processes in the hypostome peduncle and basal disc ofHydra. All gap junctions examined inHydra exhibit two apposed plasma membranes having a 2–4 nm gap continuous with the extracellular space. The gap junctions are variable in length from 0.1–1.6 m and appear linear or V-shaped in section. Neuronal gap junctions inHydra occur infrequently as compared to chemical synapses. Electron microscopy of serial sections has demonstrated the presence of adjacent electrical and chemical synapses (neuromuscular junctions) formed by the same neuron. In addition multiple gap junctions were present between two neurons. This is the first ultrastructural demonstration of electrical synapses in the nervous system ofHydra. Such synapses occur in neurons previously characterized as sensory-motor-interneurons on the basis of their chemical synapses; these neurons appear to represent a type of stem cell characterized by having both electrical and chemical synapses.     

Oxidative stress, lens gap junctions, and cataracts

The eye lens is constantly subjected to oxidative stress from radiation and other sources. The lens has several mechanisms to protect its components from oxidative stress and to maintain its redox state, including enzymatic pathways and high concentrations of ascorbate and reduced glutathione. With aging, accumulation of oxidized lens components and decreased efficiency of repair mechanisms can contribute to the development of lens opacities or cataracts. Maintenance of transparency and homeostasis of the avascular lens depend on an extensive network of gap junctions. Communication through gap junction channels allows intercellular passage of molecules (up to 1 kDa) including antioxidants. Lens gap junctions and their constituent proteins, connexins (Cx43, Cx46, and Cx50), are also subject to the effects of oxidative stress. These observations suggest that oxidative stress-induced damage to connexins (and consequent altered intercellular communication) may contribute to cataract formation.     

Optical analysis of depolarization waves in the embryonic brain: a dual network of gap junctions and chemical synapses

Correlated neuronal activity plays a fundamental role in the development of the CNS. Using a multiple-site optical recording technique with a voltage-sensitive dye, we previously described a novel type of depolarization wave that was evoked by cranial or spinal nerve stimulation and spread widely over the whole brain region in the chick embryo. We have now investigated developmental expression and neuronal network mechanisms of this depolarization wave by applying direct stimulation to the brain stem or upper cervical cord of E5-E11 embryos, which elicited wave activity similar to that evoked by nerve stimulation. Spatial distribution patterns of the depolarization wave changed dynamically with development, and this change appeared to be related to the regional differences in neuronal differentiation. The depolarization wave was completely eliminated by application of either gap junction blockers or an N-methyl-D-aspartate (NMDA)-receptor antagonist, indicating that functions of both gap junctions and NMDA receptors are indispensable for wave propagation. A possible interpretation of the results is that dual networks of gap junctions and chemical synaptic coupling mediate large-scale depolarization waves in the developing chick CNS.     

Symmetric interactions within a homogeneous starburst cell network can lead to robust asymmetries in dendrites of starburst amacrine cells

Starburst amacrine cells in the mammalian retina respond asymmetrically to movement along their dendrites; centrifugal movement elicits stronger responses in each dendrite than centripetal movement. It has been suggested that the asymmetrical response can be attributed to intrinsic properties of the processes themselves. But starburst cells are known to release and have receptors for both GABA and acetylcholine. We tested whether interactions within the starburst cell network can contribute to their directional response properties. In a computational model of interacting starburst amacrine cells, we simulated the response of individual dendrites to moving light stimuli. By setting the model parameters for "synaptic connection strength" (cs) to positive or negative values, overlapping starburst dendrites could either excite or inhibit each other. For some values of cs, we observed a very robust inward/outward asymmetry of the starburst dendrites consistent with the reported physiological findings. This is the case, for example, if a starburst cell receives inhibition from other starburst cells located in its surround. For other values of cs, individual dendrites can respond best either to inward movement or respond symmetrically. A properly wired network of starburst cells can therefore account for the experimentally observed asymmetry of their response to movement, independent of any internal biophysical or biochemical properties of starburst cell dendrites.     

Proximally targeted GABAergic synapses and gap junctions synchronize cortical interneurons

Networks of GABAergic interneurons are implicated in synchronizing cortical activity at gamma frequencies (30-70 Hz). Here we demonstrate that the combined electrical and GABAergic synaptic coupling of basket cells instantaneously entrained gamma-frequency postsynaptic firing in layers 2/3 of rat somatosensory cortex. This entrainment was mediated by rapid curtailment of gap junctional coupling potentials by GABAA receptor-mediated IPSPs. Electron microscopy revealed spatial proximity of gap junctions and GABAergic synapses on somata and dendrites. Electrical coupling alone entrained postsynaptic firing with a phase lag, whereas unitary GABAergic connections were ineffective in gamma-frequency phasing. These observations demonstrate precise spatiotemporal mechanisms underlying action potential timing in oscillating interneuronal networks.     

The bicellular and reflexive membrane junctions of renomedullary interstitial cells: Functional implications of reflexive gap junctions

Renomedullary interstitial cells are loosely organized within the interstitial space surrounding collecting ducts, limbs of Henle, and capillaries of the rat renal medulla. These cells possess long processes, which interact with each other and with cell bodies to form bicellular tight, intermediate, and gap junctions. In addition, both cell bodies and cell processess “reflexive” gap and intermediate junctions. Possible functions of renomedullary interstitial cell membrane junctions are discussed. Particular attention is given to a consideration of the functional significance of “reflexive” gap junctions.     

Connexins and gap junctions in the EDHF phenomenon and conducted vasomotor responses

It is becoming increasingly evident that electrical signaling via gap junctions plays a central role in the physiological control of vascular tone via two related mechanisms (1) the endothelium-derived hyperpolarizing factor (EDHF) phenomenon, in which radial transmission of hyperpolarization from the endothelium to subjacent smooth muscle promotes relaxation, and (2) responses that propagate longitudinally, in which electrical signaling within the intimal and medial layers of the arteriolar wall orchestrates mechanical behavior over biologically large distances. In the EDHF phenomenon, the transmitted endothelial hyperpolarization is initiated by the activation of Ca²⁺-activated K⁺ channels channels by InsP₃-induced Ca²⁺ release from the endoplasmic reticulum and/or store-operated Ca²⁺ entry triggered by the depletion of such stores. Pharmacological inhibitors of direct cell-cell coupling may thus attenuate EDHF-type smooth muscle hyperpolarizations and relaxations, confirming the participation of electrotonic signaling via myoendothelial and homocellular smooth muscle gap junctions. In contrast to isolated vessels, surprisingly little experimental evidence argues in favor of myoendothelial coupling acting as the EDHF mechanism in arterioles in vivo. However, it now seems established that the endothelium plays the leading role in the spatial propagation of arteriolar responses and that these involve poorly understood regenerative mechanisms. The present review will focus on the complex interactions between the diverse cellular signaling mechanisms that contribute to these phenomena.     

The role of myocardial gap junctions in electrical conduction and arrhythmogenesis.

Electrical activation of the heart requires cell-cell transfer of current via gap junctions, arrays of densely packed protein channels that permit intercellular passage of ions and small molecules. Because current transfer occurs only at gap junctions, the spatial distribution and biophysical properties of gap junction channels are important determinants of the conduction properties of cardiac muscle. Gap junction channels are composed of members of a multigene family of proteins called connexins. As a general rule, individual cells express multiple connexins, which creates the potential for considerable functional diversity in gap junction channels. Although gap junction channels are relatively nonselective in their permeability to ions and small molecules, cardiac myocytes actively adjust their level of coupling by multiple mechanisms including changes in connexin expression, regulation of connexin trafficking and turnover, and modulation of channel properties. In advanced stages of heart disease, connexin expression and intercellular coupling are diminished, and gap junction channels become redistributed. These changes have been strongly implicated in the pathogenesis of lethal ventricular arrhythmias. Ongoing studies in genetically engineered mice are revealing insights into the role of individual gap junction channel proteins in normal cardiac function and arrhythmogenesis.     

Intercellular calcium signaling between astrocytes and oligodendrocytes via gap junctions in culture

To understand further how oligodendrocytes regulate brain function, the mechanism of communication between oligodendrocytes and other cell types needs to be explored. An important mode of communication between various cell types in the nervous system involves gap junctions. Astroglial cells are extensively connected through gap junctions forming the glial syncytium. Although the presence of gap junctions between oligodendrocytes and astrocytes have been well documented, evidence for gap junction-mediated calcium transfer between these two glial populations is still missing. To measure functional coupling between astrocytes and oligodendrocytes and to test whether this coupling is mediated by gap junctions we used laser photostimulation and monitored Ca²⁺ propagation in cultures from transgenic animals in which oligodendrocytes express enhanced green fluorescent protein (eGFP). We show that waves of Ca²⁺ spread from astrocytes to oligodendrocytes and that these waves are blocked by the broad-spectrum gap junction blocker carbenoxolone, but not the neuron-specific gap junction blocker quinine. We also show that the spread of Ca²⁺ waves between astrocytes and oligodendrocytes is bi-directional. Thus, increase of Ca²⁺ concentration in astrocytes triggered by surrounding neuronal activity may feed back onto different neuronal populations through oligodendrocytes.     

Summation of excitatory and inhibitory synaptic inputs by motoneurons with highly active dendrites

We investigated summation of steady excitatory and inhibitory inputs in spinal motoneurons using an in vivo preparation, the decerebrate cat, in which neuromodulatory input from the brain stem facilitated a strong persistent inward current (PIC) in dendritic regions. This dendritic PIC amplified both excitatory and inhibitory synaptic currents two- to threefold, but within different voltage ranges. Amplification of excitatory synaptic current peaked at voltage-clamp holding potentials near spike threshold (about –55 to –50 mV), whereas amplification of inhibitory current peaked at significantly more depolarized levels (about –45 to –40 mV). Thus the linear sum of excitatory and inhibitory currents tended to vary from net excitatory to net inhibitory as holding potential was depolarized. The actual summed currents, however, diverged from the predicted linear currents. At the peak of excitation, summation averaged about 15% sublinear (actual sum was less positive than the linear sum). In contrast, at the peak of inhibition, summation averaged about 18% supralinear (actual more positive than linear). Moreover, these nonlinear effects were substantially larger in cells where the variation from peak excitation to peak inhibition for linear summation was larger. When descending neuromodulatory input was eliminated by acute spinalization, PIC amplification was not observed and summation tended to be either sublinear or approximately linear, depending on input source. Overall, in cells with strong PICs, nonlinear summation of excitation and inhibition does occur, but this nonlinearity results in a more consistent relationship between membrane potential and the summed excitatory and inhibitory current.     

Influence fields: a quantitative framework for representation and analysis of active dendrites

Neuronal dendrites express numerous voltage-gated ion channels (VGICs), typically with spatial gradients in their densities and properties. Dendritic VGICs, their gradients, and their plasticity endow neurons with information processing capabilities that are higher than those of neurons with passive dendrites. Despite this, frameworks that incorporate dendritic VGICs and their plasticity into neurophysiological and learning theory models have been far and few. Here, we develop a generalized quantitative framework to analyze the extent of influence of a spatially localized VGIC conductance on different physiological properties along the entire stretch of a neuron. Employing this framework, we show that the extent of influence of a VGIC conductance is largely independent of the conductance magnitude but is heavily dependent on the specific physiological property and background conductances. Morphologically, our analyses demonstrate that the influences of different VGIC conductances located on an oblique dendrite are confined within that oblique dendrite, thus providing further credence to the postulate that dendritic branches act as independent computational units. Furthermore, distinguishing between active and passive propagation of signals within a neuron, we demonstrate that the influence of a VGIC conductance is spatially confined only when propagation is active. Finally, we reconstruct functional gradients from VGIC conductance gradients using influence fields and demonstrate that the cumulative contribution of VGIC conductances in adjacent compartments plays a critical role in determining physiological properties at a given location. We suggest that our framework provides a quantitative basis for unraveling the roles of dendritic VGICs and their plasticity in neural coding, learning, and homeostasis.     

Induction of gap junctions and brain endothelium-like tight junctions in cultured bovine endothelial cells: local control of cell specialization

The final development of specializations by brain capillary endothelial cells, which characterize them as distinct from non-central nervous system (CNS) endothelium, is thought to be controlled by astrocyte-derived factors produced locally within the CNS. One specialization, the complex intercellular tight junction, which is unique to these cells and a major component of the blood-brain barrier, is controlled by an astrocyte-derived factor(s) and a "competent' extracellular matrix (Arthur et al., 1987). In order to test whether these factors can also trigger development of brain endothelium-like tight junctions in non-CNS microvessel endothelial cells, passaged bovine aorta and pulmonary artery endothelial cells were cultured in either 50% astrocyte-conditioned medium and 50% alpha-MEM, or in alpha-MEM alone (control). Only endothelial cells maintained in conditioned medium exhibited ultrastructural features indicative of synthesis and plasma membrane-insertion of junction components (Shivers et al., 1985). No assembled tight junctions were seen in these cells. Endothelial cells plated onto coverslips coated with ECM (Cedarlane Labs., Hornby, Ont.) and maintained in astrocyte-conditioned medium, displayed large, complex tight junctions and extraordinarily large gap junctions. Cells plated onto plastic or fibronectin-coated substrates possessed no tight or gap junctions. Results of this study show that CNS astrocytes produce a soluble factor(s) that promotes synthesis and insertion of tight junction components in non-CNS endothelial cells. Moreover, an intact, endothelial-derived extracellular matrix is required for assembly of tight junctions to complete development of this brain capillary-like specialization. This study confirms the notions that: a) the final fine-tuning of cell differentiation is under local control, and b) that endothelial cells in general do not express their final destination-specific differentiated features until those features are induced by local environment-produced conditions.