Gap Junction Connections Research Articles

内リンパ水腫とＮａ｀＋´‐Ｋ｀＋´‐２Ｃｌ｀−´共輸送体 モルモットか牛の免疫組織化学的検討

Na+-K+-2Cl- cotransporter 1 (NKCC1) and c-Jun N-terminal kinase (JNK) were immunohistochemically studied in guinea pig cochlea following the induction of endolymphatic hydrops by blocking the endolymphatic duct. Postoperative survivals were set to 1 day (n=3), 3 days (n=4), 1 week (n=3), 2 weeks (n=4), 1 month (n=4) and 3 months (n=4). The most striking finding was an increase in the immunoreactivity for NKCC1 at type 1 fibrocytes of the spiral ligament throughout the survival times. This suggests that the affected cells shrink as the result of surgical intervention and upregulate NKCC1 to restore the volume of the cells. The shrinkage of type 1 fibrocytes should reduce gap junctional connectivity among cells of the ligament, which could reduce K+ supply into the stria vascularis and disturb ionic homeostasis in the endolymph. On the other hand, JNK, which has been reported to activate NKCC1 by phosphorylation, showed a consistent decrease in immunoreactivity in the types 1 and 2 fibrocytes in hydropic ears, indicating that JNK does not play an important role in the activation of NKCC1 in the cochlea.The findings in the present study suggested that endolymphatic homeostasis depends on cell integrity within the spiral ligament and that NKCC1 in type 1 fibrocytes is crucial in maintaining cochlear fluid homeostasis.

Endogenous dopaminergic regulation of horizontal cell coupling in the mammalian retina.

Horizontal cells in an isolated wholemount preparation of the mouse retina were injected with Lucifer yellow and neurobiotin to characterize both the pattern of gap junctional connectivity and its regulation by dopamine. The injected horizontal cells had a uniform morphology of a round cell body, a compact dendritic tree, and an axon, which could sometimes be traced to an expansive terminal system. The dendro-dendritic gap junctions between neighboring cells mediated both weak Lucifer yellow dye coupling and strong neurobiotin tracer coupling. The extent of the tracer coupling was decreased by either exogenous dopamine (100 microM) or cyclic adenosine monophosphate (cAMP) analogs and was significantly increased by the D1 antagonist SCH 23390 (10 microM). These results provide the first evidence in the mammalian retina that the gap junctions between horizontal cells are endogenously regulated by dopamine, which acts through D1 receptors to increase the intracellular cAMP. It has been proposed that the gap junctional coupling between horizontal cells is mediated by connexin 32 (Cx32), but the pattern and dopaminergic regulation of horizontal cell coupling were unaffected in Cx32-knockout mice, ruling out the possible involvement of Cx32. Every tracer-coupled horizontal cell showed calbindin immunoreactivity, and vice versa, providing strong evidence that the horizontal cells in the mouse retina comprise a single cell type. Like the axonless horizontal cells in other mammalian retinas, the axon-bearing horizontal cells in the mouse retina are coupled by gap junctions that are permeable to Lucifer yellow and dopamine sensitive, suggesting that the mouse horizontal cells have hybrid properties to compensate for the absence of axonless horizontal cells.

Connexin 26 Expression and Extensive Gap Junctional Coupling in Cultures of GT1-7 Cells Secreting Gonadotropin-Releasing Hormone

Gap junctions (GJs) are transmembrane channels that permit rapid intercellular transit of various small molecules including ions, second messengers and metabolites. GJs promote communication and coordinated activity between coupled neurons, and may help facilitate the synchronous release and pulsatile secretion of neurohormones. A previous study using GnRH-secreting GT1-7 cells reported that connexin 26 was the major GJ subunit present, and that about 20% of the cultured cells engaged in GJ coupling as assayed by fluorescence recovery after photobleaching of 5,6-carboxyfluorescein diacetate (MW 460 D). To reassess GJ connectivity with a more permeant probe, we grew GT1-7 cells to 70% confluency on Matrigel-coated glass coverslips and microinjected Neurobiotin^TM (MW 322 D) into single cells. Dye was allowed to diffuse for 30 min before cultures were fixed, and subsequently immunostained for Neurobiotin with 3,3′-diaminobenzidine HCl and examined by light microscopy. Dye coupling between 2 or more GT1-7 cells was observed after 75% of all microinjections. Connectivity involved the somata and neurites of an average of 6.6 ± 2.0 adjoining cells, but in one instance was seen in a group of 32 GT1-7 neighbors. Western blotting and immunofluorescence staining confirmed that connexin 26 was the predominant GJ subunit expressed by GT1-7 cultures. Our results using Neurobiotin suggest these GJ channels may be smaller than anticipated. In addition, functional GJ connectivity between subconfluent GT1-7 cells is more extensive than previously reported, occurring with higher frequency and coupling significantly greater numbers of cultured cells. Since cAMP, IP3, and Ca²⁺ are able to pass through GJs and can elicit secretion of GnRH by GT1 cell cultures, GJs may play an important role in the coordination and synchronization of GnRH release.

Distribution of the glycine transporter glyt-1 in mammalian and nonmammalian retinae.

We have examined the distribution of the glycine transporter glyt-1 in retinae of macaques, cats, rabbits, rats, and chickens. In all species, all glycine-containing amacrine cells expressed immunoreactivity for glyt-1, though the intensity of immunoreactivity for glyt-1 did not appear to directly correlate with the intensity of immunoreactivity for glycine in individual cells. A small subpopulation of glycine-immunoreactive displaced amacrine cells or ganglion cells also expressed glyt-1 in retinae from macaques, cats, chickens, and rats but not in retinae from rabbits. In addition, in all species examined, some displaced amacrine cells also contained glycine but did not express glyt-1. In monkeys, cats, and rats, populations of cells which we interpret as being glycine-containing interplexiform cells expressed glyt-1: these cells lacked a content of glutamate, suggesting they are not bipolar cells. The glycine-containing bipolar cells did not express glyt-1, suggesting that these cells probably acquired their content of glycine by other means such as via gap junctional connections with glycine-containing amacrine cells.

In vitro long-term potentiation of electrotonic responses of goldfish Mauthner cells is accompanied by ultrastructural changes at afferent mixed synapses

The potentiated afferent mixed synapses of the Mauthner cells of fry and adult goldfish in stumps of the medulla oblongata incubated long-term in vitro were studied by electrophysiological and electron microscopic methods. It was shown that brief high-frequency stimulation of posterior branches of the eighth nerve induced a long-term potentiation of electrotonic transmission at large and small mixed club endings. It was about 135% upon subthreshold stimulation and about 200% upon suprathreshold stimulation. The ultrastructural analysis of ultrathin sections of potentiated mixed synaptic endings revealed an increase in the dimensions of desmosome-like contacts which was proportional to the degree of potentiation, about 135% or 200%, depending on the type of stimulation. The dimensions of gap junctions remained unchanged. The dimensions of active zones at potentiated synapses were reduced two-fold as compared with their unpotentiated counterparts, irrespective of the type of stimulation. Considering that desmosome-like contacts consist predominantly of F-actin, a molecule which possesses electroconductivity, it can be assumed that this cytoskeletal protein is involved in the process of potentiation. The increase in the synapse electrical conductivity can be mediated either directly, by shunting the synaptic junction with polymer actin filaments in the region of desmosome-like contacts, or indirectly, via the interaction of actin with gap junction connections situated nearby.

Electrical coupling underlies high-frequency oscillations in the hippocampus in vitro.

Coherent oscillations, in which ensembles of neurons fire in a repeated and synchronous manner, are thought to be important in higher brain functions. In the hippocampus, these discharges are categorized according to their frequency as theta (4-10Hz), gamma (20-80 Hz) and high-frequency (approximately 200 Hz) discharges, and they occur in relation to different behavioural states. The synaptic bases of theta and gamma rhythms have been extensively studied but the cellular bases for high-frequency oscillations are not understood. Here we report that high-frequency network oscillations are present in rat brain slices in vitro, occurring as a brief series of repetitive population spikes at 150-200 Hz in all hippocampal principal cell layers. Moreover, this synchronous activity is not mediated through the more commonly studied modes of chemical synaptic transmission, but is in fact a result of direct electrotonic coupling of neurons, most likely through gap-junctional connections. Thus high-frequency oscillations synchronize the activity of electrically coupled subsets of principal neurons within the well-documented synaptic network of the hippocampus.

Myocardial architecture and ventricular arrhythmogenesis.

Conduction of the cardiac impulse is dependent on both the active membrane properties of cardiac cells (generating the action potential) and the passive properties determined by architectural features of the myocardium. The influence of tissue architecture on conduction is determined principally by the size, shape, and packing of individual myocytes and by the quantity, three-dimensional distribution, and physiological behavior of the specialized intercellular junctions responsible for impulse propagation from cell to cell, the gap junctions.1 It has long been recognized that abnormalities in conduction of the cardiac impulse are an important cause of arrhythmias2 in that they alter the relationships between conduction velocity, path length, and recovery of excitability (the determinants of reentrant excitation). However, it was not until more recently that myocardial architecture was considered important in determining patterns of activation and conduction velocity3 4 and was therefore thought to have a central role in arrhythmogenesis,5 and some of the evidence for this forms the basis of this review. ### Myocardial Syncytium: Anisotropy of Structure and Function The early observation that conduction properties in myocardium were different in different directions,6 with more rapid conduction in the direction parallel to the myocardial fiber axis than in the transverse direction (the definition of the anisotropic conduction characteristic of heart muscle [Figure 1⇓]), is attributable principally to the lower resistivity of myocardium in the longitudinal than the transverse direction. Gap-junctional channels create continuity between the cytoplasmic compartments of abutting myocytes but act as resistive discontinuities to the cytoplasmic current flow between the intracellular compartments of the cells (the “intracellular” conduction pathway). Longitudinal resistivity is lower than transverse because this intracellular pathway encounters fewer cell boundaries per unit distance in the longitudinal than the transverse direction. In normal ventricular myocardium, conduction in the direction parallel to the long axis of the myocardial fiber bundles …

Mechanisms of electrical coupling between pyramidal cells.

Direct electrical coupling between neurons can be the result of both electrotonic current transfer through gap junctions and extracellular fields. Intracellular recordings from CA1 pyramidal neurons of rat hippocampal slices showed two different types of small-amplitude coupling potentials: short-duration (5 ms) biphasic spikelets, which resembled differentiated action potentials and long-duration (>20 ms) monophasic potentials. A three-dimensional morphological model of a pyramidal cell was employed to determine the extracellular field produced by a neuron and its effect on a nearby neuron resulting from both gap junctional and electric field coupling. Computations were performed with a novel formulation of the boundary element method that employs triangular elements to discretize the soma and cylindrical elements to discretize the dendrites. An analytic formula was derived to aid in computations involving cylindrical elements. Simulation results were compared with biological recordings of intracellular potentials and spikelets. Field effects produced waveforms resembling spikelets although of smaller magnitude than those recorded in vitro. Gap junctional electrotonic connections produced waveforms resembling small-amplitude excitatory postsynaptic potentials. Intracellular electrode measurements were found inadequate for ascertaining membrane events because of externally applied electric fields. The transmembrane voltage induced by the electric field was highly spatially dependent in polarity and wave shape, as well as being an order of magnitude larger than activity measured at the electrode. Membrane voltages because of electrotonic current injection across gap junctions were essentially constant over the cell and were accurately depicted by the electrode. The effects of several parameters were investigated: 1) decreasing the ratio of intra to extracellular conductivity reduced the field effects; 2) the tree structure had a major impact on the intracellular potential; 3) placing the gap junction in the dendrites introduced a time delay in the gap junctional mediated electrotonic potential, as well as deceasing the potential recorded by the somatic electrode; and 4) field effects decayed to one-half of their maximum strength at a cell separation of approximately 20 micron. Results indicate that the in vitro measured spikelets are unlikely to be mediated by gap junctions and that a spikelet produced by the electric field of a single source cell has the same waveshape as the measured spikelet but with a much smaller amplitude. It is hypothesized that spikelets are a manifestation of the simultaneous electric field effects from several local cells whose action potential firing is synchronized.

Computer modeling of heterogeneous beta-cell populations.

β-cells differ significantly with respect to their electrical and secretory properties (Pipeleers, 1992), leading us to consider whether heterogeneity may have functional significance for the behavior of β-cell populations, such as the natural unit, the islet of Langerhans. On the other hand, β-cells in islets behave essentially synchronously, at least with regard to electrical activity (Eddlestone et al., 1984) and cytosolic Ca2+ levels (Santos et al., 1991). Several questions arise: How does the islet synchronize? Are the reported size and density of gap junctional connections adequate to produce synchrony? What is the role of cell heterogeneity? Is it important for building the graded response to glucose or merely an obstacle that must be overcome in order to achieve coordinated behavior? Does glucose increase insulin secretion by recruiting more cells (Pipeleers, 1992) into activity or by increasing the output of each cell (Beigelman et al., 1977)? How is the oscillatory activity of the islet generated in the absence of a pacemaker?

Altered patterns of cardiac intercellular junction distribution in hypertrophic cardiomyopathy.

To examine the distribution pattern of intercellular junctions (the mechanically coupling desmosomes and the electrically coupling gap junctions) in hypertrophic cardiomyopathy (HCM) hearts showing myofibre disarray. Samples from six necropsied hearts were studied, representing the interventricular septum and the free walls of the left and right ventricles. Immunohistochemical labelling of desmoplakin was used as a marker for desmosomes, and of connexin43 as a marker for gap junctions, in single and double stainings. The slides were examined by confocal laser scanning microscopy. Marked disorganisation of intercalated discs was observed in areas featuring myofibre disarray. Besides overall derangement, localised abnormalities in desmosome organisation were evident, which included: (1) the formation of abnormally enlarged megadiscs; (2) the presence of intersecting disc structures; and (3) aberrant side to side desmosomal connections. Gap junctional abnormalities included: (1) random distribution of gap junctions over the surface of myocytes, rather than localisation to intercalated discs; (2) abundant side to side gap junction connections between adjacent myocytes; and (3) formation of abnormally shaped gap junctions. Circles of myocytes continuously interconnected by gap junctions were also observed. Regions of the diseased hearts lacking myofibre disarray, and control hearts of normal patients and patients with other cardiac diseases, did not show these alterations. The disorganisation of the intercellular junctions associated with myofibre disarray in HCM may play an important role in the pathophysiological manifestations of the disease. The remodelling of gap junction distribution may underlie the formation of an arrhythmogenic substrate, thereby contributing to the generation and maintenance of cardiac arrhythmias associated with HCM.

Passover eliminates gap junctional communication between neurons of the giant fiber system in Drosophila. off.

The Passover-related gene family plays significant roles in cellular connectivity. Mutations in three family members from Drosophila and from Caenorhabditis elegans alter a few specific electrical synapses. The passage of cobalt between Drosophila neurons was used to assay the presence of gap junctional connections. The giant fiber in the wild type has specific gap junctional connections in the brain and in the thorax. In flies mutant for Passover, cobalt cannot pass into or out of the giant fiber in either the anterograde or the retrograde directions. A large number of other gap junctional connections remain unaffected. This demonstrates that the Passover gene is necessary for gap-junctional communication between the neurons of the Drosophila giant fiber system.

Passover eliminates gap junctional communication between neurons of the giant fiber system in Drosophila

The Passover-related gene family plays significant roles in cellular connectivity. Mutations in three family members from Drosophila and from Caenorhabditis elegans alter a few specific electrical synapses. The passage of cobalt between Drosophila neurons was used to assay the presence of gap junctional connections. The giant fiber in the wild type has specific gap junctional connections in the brain and in the thorax. In flies mutant for Passover, cobalt cannot pass into or out of the giant fiber in either the anterograde or the retrograde directions. A large number of other gap junctional connections remain unaffected. This demonstrates that the Passover gene is necessary for gap-junctional communication between the neurons of the Drosophila giant fiber system. © 1996 John Wiley & Sons, Inc.

Dye-coupling in the root epidermis of Arabidopsis is progressively reduced during development

ABSTRACT Epidermal cells of the Arabidopsis thaliana root are derived from a ring of 16 or so initials (stem cells) that are located just below the quiescent centre of four central cells. Derivatives of these initials divide transversely in the meristem-atic region of the root to produce files of cells that subsequently elongate and differentiate. There are two kinds of cell file in the epidermis, hair cells and non-hair cells, that are organised in an ordered pattern and become distinguishable within a few divisions of the initials. It is likely that local intercellular communication plays an important role in the early establishment of this pattern, enabling cells to register their position and fate. We have therefore examined the extent to which cells in the root apex are sym-plastically coupled through plasmodesmata. Using a variety of dye loading methods we show that the undifferentiated cells are dye-coupled i.e. cells in the meristem and the elongation zone are symplastically connected, but that cells gradually become symplastically isolated as the cells differentiate. By the time that visible hair outgrowth is observed the cells of the epidermis are symplastically isolated. This resembles the pattern of gap-junction connectivity observed in many animal systems where cells are coupled at the stage that cell patterns become established, but become progressively more isolated as they differentiate.

Astrocytes exhibit regional specificity in gap-junction coupling.

Astrocytes are coupled to each other via gap-junctions both in vivo and in vitro. Gap-junction coupling is essential to a number of astrocyte functions including the spatial buffering of extracellular K+ and the propagation of Ca2+ waves. Using fluorescence recovery after photo-bleach, we quantitatively assayed and compared the coupling of astrocytes cultured from six different central nervous system (CNS) regions in the rat: spinal cord, cortex, hypothalamus, hippocampus, optic nerve, and cerebellum. The degree of fluorescence recovery (% recovery) and time constant of recovery (tau) served as quantitative indicators of coupling strength. Gap-junction coupling differed markedly between CNS regions. Coupling was weakest in astrocytes derived from spinal cord (43% recovery, tau approximately 400 s) and strongest in astrocytes from optic nerve (91% recovery, tau approximately 226 s) and cerebellum (95% recovery, tau approximately 100 s). As indicated by the degree of recovery, coupling strength among CNS regions could be ranked as follows: spinal cord < cortex < hypothalamus < hippocampus = optic nerve = cerebellum. Gap-junction coupling also differed between CNS regions with respect to its sensitivity to inhibition by the uncoupling agent octanol. Kd values for 50% inhibition by octanol ranged from 188 microM in spinal cord astrocytes to 654 microM in hippocampal astrocytes. Sensitivity of gap-junctions to octanol could be ranked as follows: spinal cord = cortex = hypothalamus > cerebellum > optic nerve > hippocampus. The observed differences in coupling indicate differences in the number of gap-junction connections in astrocytes cultured from the six CNS regions. These differences may reflect the adaptation of astrocytes to varying functional requirements in different CNS regions.

Gap junctional connections between hair cells, supporting cells and nerves in a vestibular organ

The pattern of gap-junctional connections between cells in the vestibular neuroepithelium of the posterior semicircular duct of the alligator lizard are described based upon the study of freeze fracture replicas and ultrathin sections with a transmission electron microscope. Both type I and type II hair cells are coupled to adjacent supporting cells by a series of small macular gap junctions located in a ring around the hair cell at the level of the apical circumferential belt of actin filaments. Adjacent supporting cells are extensively interconnected by gap junctions. A few cases of gap junctions between afferent dendrites and supporting cells, and between afferent dendrites and calyceal nerve endings were seen. These morphological observations together with data from other studies in the literature suggest a possible role for supporting cells in altering the micromechanical properties of the hair cell receptor organs during stimulation.

Lateral gap junction connections between retinal amacrine cells summating sustained responses.

Vertebrate retinal amacrine cells produce transient or sustained responses. Sustained depolarizing amacrine cells in the dace retina were identified by their intracellularly recorded responses to light flashes. The response amplitude produced a notable spatial summation which exceeded that of individual dendritic arbors. When sustained type amacrine cells were intracellularly injected with Lucifer Yellow and biocytin, there was extensive transfer of biocytin, but not Lucifer Yellow, to surrounding cells with similar cellular morphology. Ultrastructural analysis of the interconnections by electron microscopy revealed the presence of gap junctions at the contact area, which did not include conventional synapses. Present results demonstrate that sustained response amacrine cells make direct electrical connections between the cells of the same type and electrical coupling may contribute to extension of their receptive fields.

Rapid communication between neurons and astrocytes in primary cortical cultures

The identification of neurotransmitter receptors and voltage-sensitive ion channels on astrocytes (reviewed by Barres, 1991) has renewed interest in how these cells respond to neuronal activity. To investigate the physiology of neuron astrocyte signaling, we have employed primary cortical cultures that contain both neuronal and glial cells. As the neurons in these cultures exhibit synchronous spontaneous synaptic activity, we have used both calcium imaging and whole-cell recording techniques to identify physiological activity in astrocytes related to neuronal activity. Whole-cell voltage-clamp records from astrocytes revealed rapid inward currents that coincide with bursts of electrical activity in neighboring neurons. Calcium imaging studies demonstrate that these currents in astrocytes are not always associated with slowly propagating calcium waves. Inclusion of the dye Lucifer yellow within patch pipettes confirmed that astrocytes are extensively coupled to each other but not to adjacent neurons, indicating that the currents observed are not due to gap junction connections between these cell types. These currents do not reflect widespread diffusion of glutamate or potassium released during neuronal activity since a population of small, round, multipolar presumed glial cells that are not dye coupled to adjacent cells did not display electrical currents coincident with neuronal firing, even though they respond to locally applied glutamate and potassium. These findings indicate that, in addition to the relatively slow signaling conveyed by calcium waves, astrocytes also display rapid electrical responses to neuronal activity.

Gap junctional coupling between the JGA and the glomerular tuft.

The juxtaglomerular apparatus (JGA) in the rabbit kidney was examined by transmission electron microscopy and by freeze fracturing. It was found, that the Goormaghtigh cells of the JGA are extensively coupled with the mesangial cells within the glomerular tuft by gap junctions. A broad band of gap junctions starting within the Goormaghtigh cells, traversing the transitional area at the root of the glomerular tuft and continuing along the mesangial cells has been revealed by freeze fracturing. No gap junctional connections to the macula densa cells have been found. In accordance with data from literature it may be stated that all smooth muscle derived cell groups at the vascular pole of the glomerulus (smooth muscle cells of the vas afferens and efferens, granular cells, Goormaghtigh cells, mesangial cells) are extensively coupled by gap junctions with each other. It is supposed that this cell system may act as a synchronized functional unit.

The morphology of the Limulus visual system. VI. Connectivity in the ocellus.

An ocellus of the horseshoe crab, Limulus polyphemus, has been serially sectioned for light and electron microscopy, its sensory cells have been indexed, and the interconnections of a third of these traced. The ocellus contains 155 retinula cells and 26 arhabdomeric cells, which are secondary sensory neurons. Of these, 55 retinula cells constitute 7 quasi-ommatidial assemblages, each innervated by at least one and a total of 9 arhabdomeric cells. When known electrotonic coupling patterns are compared with gap-junctional connections, retinula cells sensitive to visible or ultraviolet light can be tentatively identified. Retinula cell axons contribute collaterals to a synaptic plexus, in which the arhabdomeric cells apparently do not participate.