Tanycytes are specialized glial cells lining the lateral walls and the floor of the third ventricle behind the optic chiasm. In addition to functioning as barrier cells, they also have an important role in the regulation of neuroendocrine axes and energy homeostasis. To determine whether tanycytes communicate with each other via Connexin 43 (Cx43) gap junctions, individual tanycytes were loaded with Lucifer yellow (LY) through a patch pipette. In all cases, LY filled a larger group of tanycytes as well as blood vessels adjacent to tanycyte processes. The Cx43-blocker, carbenoxolone, inhibited spreading of LY. The greatest density of Cx43-immunoreactive spots was observed in the cell membrane of α-tanycyte cell bodies. Cx43-immunoreactivity was also present in the membrane of β-tanycyte cell bodies, but in lower density. Processes of both types of tanycytes also contained Cx43-immunoreactivity. At the ultrastructural level, Cx43-immunoreactivity was present in the cell membrane of all types of tanycytes including their ventricular surface, but gap junctions were more frequent among α-tanycytes. Cx43-immunoreactivity was also observed in the cell membrane between contacting tanycyte endfeet processes, and between tanycyte endfeet process and axon varicosities in the external zone of the median eminence and capillaries in the arcuate nucleus and median eminence. These results suggest that gap junctions are present not only among tanycytes, but also between tanycytes and the axons of hypophysiotropic neurons. Cx43 hemichannels may also facilitate the transport between tanycytes and extracellular fluids, including the cerebrospinal fluid, extracellular space of the median eminence and bloodstream.

Following brain injury, and during the process of neurodegeneration, a reactive astrocytic proliferation occurs. This is accompanied by an increase in the synthesis of neuropeptides, cytokines, growth factors and glial fibrillary acidic protein (GFAP), a cell-specific marker for reactive astrocytes. Astrocytes are extensively coupled by gap junctions of the Cx43 connexin subtype. Several studies have shown that in severe trauma, coupling between astrocytes may add to the spread of the damaged area. In this study we ask whether the astrocytosis which is a feature of other neurodegenerative diseases also occurs in mesial temporal lobe epilepsy (MTLE) and whether it is accompanied by an increase in astrocytic communication through an upregulation of Cx43 gap junction channel proteins. In order to examine the astrocytic response and the expression pattern of Cx43 protein, double immunohistochemical labeling studies were undertaken using antibodies against GFAP and Cx43 applied to human hippocampal tissue resected from patients with MTLE, and to normal human control hippocampal tissue. Immunofluorescent labeling of astrocytes and Cx43 was examined using confocal laser scanning microscopy. The images obtained were quantitatively analysed and reconstructed using three-dimensional volume rendering. The results of this study have established that not only is astrocytosis greater in MTLE-affected tissues than previously suggested, but it is accompanied by a highly significant increase in astrocytic Cx43 protein levels. We hypothesize that this surprisingly large upregulation in Cx43 may exacerbate generalized seizures in the progression of MTLE.

Electrical synaptic transmission via gap junctions has become an accepted feature of neuronal communication in the mammalian brain, and occurs often between dendrites of interneurons in major brain structures, including the hippocampus. Electrical and dye-coupling has also been reported to occur between pyramidal cells in the hippocampus, but ultrastructurally-identified gap junctions between these cells have so far eluded detection. Gap junctions can be formed by nerve terminals, where they contribute the electrical component of mixed chemical/electrical synaptic transmission, but mixed synapses have only rarely been described in mammalian CNS. Here, we used immunofluorescence localization of the major gap junction forming protein connexin36 to examine its possible association with hippocampal pyramidal cells. In addition to labeling associated with gap junctions between dendrites of parvalbumin-positive interneurons, a high density of fine, punctate immunolabeling for Cx36, non-overlapping with parvalbumin, was found in subregions of the stratum lucidum in the ventral hippocampus of rat brain. A high percentage of Cx36-positive puncta in the stratum lucidum was localized to mossy fiber terminals, as indicated by co-localization of Cx36-puncta with the mossy terminal marker vesicular glutamate transporter-1, as well as with other proteins that are highly concentrated in, and diagnostic markers of, these terminals. These results suggest that mossy fiber terminals abundantly form mixed chemical/electrical synapses with pyramidal cells, where they may serve as intermediaries for the reported electrical and dye-coupling between ensembles of these principal cells. This article is part of a Special Issue entitled Electrical Synapses.

Retinal amacrine cells of the same class in cyprinid fish are homotypically connected by gap junctions. The permeability of their gap junctions examined by the diffusion of Neurobiotin into neighboring amacrine cells under application of dopamine or cyclic nucleotides to elucidate whether electrical synapses between the cells are regulated by internal messengers. Neurobiotin injected intracellularly into amacrine cells in isolated retinas of goldfish, and passage currents through the electrical synapses investigated by dual whole-patch clamp recordings under similar application of their ligands. Control conditions led us to observe large passage currents between connected cells and adequate transjunctional conductance between the cells (2.02±0.82nS). Experimental results show that high level of intracellular cyclic AMP within examined cells block transfer of Neurobiotin and suppress electrical synapses between the neighboring cells. Transjunctional conductance between examined cells reduced to 0.23nS. However, dopamine, 8-bromo-cyclic AMP or high elevation of intracellular cyclic GMP leaves gap junction channels of the cells permeable to Neurobiotin as in the control level. Under application of dopamine (1.25±0.06nS), 8-bromo-cyclic AMP (1.79±0.51nS) or intracellular cyclic GMP (0.98±0.23nS), the transjunctional conductance also remains as in the control level. These results demonstrate that channel opening of gap junctions between cyprinid retinal amacrine cells is regulated by high level of intracellular cyclic AMP.

In the neocortex, gap junctions are expressed at very early developmental stages, and they are involved in many processes such as neurogenesis, neuronal migration and synapse formation. Connexin43 (Cx43), a gap junction protein, has been found to be abundantly expressed in radial glial cells, excitatory neurons and astrocytes. Although accumulating evidence suggests that Cx43-mediated gap-junctional coupling between astrocytes plays an important role in the central nervous system, the function of Cx43 in early excitatory neurons remains elusive. To investigate the impact of Cx43 deficiency in excitatory neurons at early postnatal stages, we conditionally knocked out Cx43 in excitatory neurons under the Emx1 promoter by tamoxifen induction. We found that deletion of Cx43 around birth did not impair the laminar distribution of excitatory neurons in the neocortex. Moreover, mice with Cx43 deletion during the early postnatal stages had normal anxiety-like behaviors, depression-related behaviors, learning and memory-associated behaviors at adolescent stages. However, Cx43 conditional knockout mice exhibited impaired motor-learning behavior. These results suggested that Cx43 expression in excitatory neurons at early postnatal stages contributes to short-term motor learning capacity.

Organotypic slice cultures obtained from immature brain tissue represent a well-established model system for neuroscience research. Current culture methods, however, do not allow long-term culture of mature brain slices. Slice cultures from mature animals would provide an in vitro experimental environment suitable for investigation of neuropathologies, which in human, predominate in aged individuals. We hypothesized that damage, incurred by slicing of the brain, is propagated through intercellular connexin43 (Cx43) gap junction channels and that this damage is not easily repaired in mature central nervous system (CNS) tissue that lacks the pluripotency of immature tissue. We investigated the role of Cx43 gap junctions in long-term survival of mature brain tissue using antisense oligodeoxynucleotide (AsODN) technology. The application of Cx43 AsODN immediately after slicing of the mature brain led to a significant but transient knockdown of Cx43 protein. This treatment was associated with the long-term survival of hippocampal neurons with normal morphology within whole brain slices taken from 14 and 40-day-old adult rats.

Arachidonic acid (AA) induced a concentration- and time-dependent reduction in gap junction-mediated dye coupling between cultured astrocytes. The effect was greatly diminished by inhibition of cyclooxygenases and lipoxygenases. The action of a low concentration of AA (5 microM) was also prevented by Ca2+-free extracellular solution or a high concentration of melatonin, a potent free radical scavenger, but not by Nomega-nitro-l-arginine, a nitric oxide (NO) synthase inhibitor. Thus, this effect may depend on Ca2+ influx and oxygen free radicals but not on NO generation. Cellular uncoupling induced by a high (100 microM), but not a low, AA concentration was rapidly reversed by washing with albumin containing solution. After reversal from 5 min but not 2.5 min inhibition with a high AA concentration dye coupling between astrocytes became refractory to a low concentration of AA, suggesting desensitization of the response elicited by a low concentration of the fatty acid. Dye uncoupling occurred without changes in levels and state of phosphorylation (immunoblotting and 32P-incorporation) of connexin43, the main astrocyte gap junctional protein. However, maximal cell uncoupling induced by a low (Slow action) but not by a high (Fast action) AA concentration was paralleled by a reduction in connexin43 (immunofluorescence) at cell-to-cell contacts. It is proposed that the AA-induced dye uncoupling is mediated by byproducts that induce rapid channel closure or slow removal of connexin43 gap junctions.

Electrically coupled neurons communicate through channel assemblies called gap junctions, which mediate the transfer of current from one cell to another. Electrical synapses ensure spike synchronization and reliable transmission, which influences bursting patterns and firing frequency. The present study concerns an electrically coupled two-neuron network in the gastropod mollusc, Lymnaea stagnalis. The neurons, designated Visceral Dorsal 1 (VD1) and Right Parietal Dorsal 2 (RPD2), are peptidergic, innervate aspects of the cardio-respiratory system, and show strong coupling, such that they fire synchronously. Using dual sharp-electrode current-clamp recording and morphological staining in isolated brain preparations, the hypothesis that the electrical synapse is necessary for accurate network output was tested. We found that both cells make extensive projections within and out of the brain, including across the visceral-parietal connective, which links VD1 and RPD2. Cutting this connective uncoupled the neurons and disrupted the firing rate and pattern of RPD2 more than VD1, consistent with VD1 being the master and RPD2 the follower. The electrical synapse was inhibited by select gap junction blockers, with niflumic acid and 5-nitro-2-(3-phenylpropylamino) benzoic acid decreasing the VD1→RPD2 and RPD2→VD1 coupling coefficients, whereas carbenoxolone, α-glycyrrhetinic acid, meclofenamic acid, and quinine were ineffective. There was little-to-no impact on VD1↔RPD2 firing synchrony or frequency when coupling was reduced pharmacologically. However, in the presence of gap junction blockers, suppressing the activity of VD1 by prolonged hyperpolarization revealed a distinct, low-frequency firing pattern in RPD2. This suggests that strong electrical coupling is key to maintaining a synchronous output and proper firing rate.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) play important roles in non-image forming photoreception and participate in the regulation of the circadian rhythm and the pupillary light reflex. The aim of the present work was to characterize the light response of ipRGCs at two developmental stages of the embryonic chick. The electrophysiological study was based on comparative multielectrode array recordings from acute retinal slices. To ensure that light was the only source of excitation, intercellular activity modulation by gap junctions and chemical synapses was inhibited by carbenoxolone and bafilomycin A1, respectively. Action potentials evoked by blue light were detected as early as day 13 of embryonic development, which is notably earlier than the completion of the maturation process of functional rods and cones. Three different response types were distinguished by their response latency and sensitivity to different illumination intensities. At this point it is not clear whether these types just represent different maturation stages or have different morphologies and functions with respect to the non-image forming visual system and circadian entrainment.

The neural substrate of brain stimulation reward (BSR) has eluded identification since its discovery more than a half-century ago. Notwithstanding the difficulties in identifying the neuronal integrator of BSR, the mesocorticolimbic dopamine (DA) system originating in the ventral tegmental area (VTA) of the midbrain has been implicated. We have previously demonstrated that the firing rate of a subpopulation of gamma-aminobutyric acid (GABA) neurons in the VTA increases in anticipation of BSR. We show here that GABA neurons in the VTA, midbrain, hypothalamus, and thalamus of rats express connexin-36 (Cx36) gap junctions (GJs) and couple electrically upon DA application or by stimulation of the internal capsule (IC), which also supports self-stimulation. The threshold for responding for IC self-stimulation was the threshold for electrical coupling between GABA neurons, the degree of responding for IC self-stimulation was proportional to the magnitude of electrical coupling between GABA neurons, and GJ blockers increased the threshold for IC self-stimulation without affecting performance. Thus, a network of electrically coupled GABA neurons in the ventral brain may form the elusive neural integrator of BSR.

The effects of different adrenoceptor agonists were investigated on mechanically induced Ca2+ waves in astroglial cells in astroglial-neuronal mixed cultures from rat hippocampus. In the initial part of the study some properties of the waves were characterized. The results show that the initiation of the Ca2+ waves was not critically dependent on extracellular Ca2+ but both the calcium signal and the propagation area of the calcium wave were significantly reduced when the experiments were performed in Ca2+-free buffer. In addition, using the phospholipase C (PLC) inhibitor U-73122 (1 microM) and the gap junction uncoupler octanol (1 mM), the results showed that the Ca2+ wave propagation required PLC activation and functional gap junctions. Further, the data also showed that the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA 150 nM) reduced the spreading of the waves. The adrenoceptor agonists isoproterenol (iso; beta), phenylephrine (phe; alpha1) and clonidine (clon; alpha2) were evaluated for their short-term (<30 s) effects on the wave propagation. The propagation area was persistently decreased 1, 3 and 5 min after removal of phe. No effects were observed after incubation with iso or clon. Furthermore, using U-73122 or PMA together with phe, shortly incubated, the experiments showed that PLC was a central regulator in the initial phase of the initiation procedure of wave propagation. However, under these conditions PKC was shown not to be involved. Instead it appeared that PKC exerted its inhibitory action on the Ca2+ waves in a latter phase, after prolonged phe exposure. Taken together, the results show that the propagation of Ca2+ waves between astroglial cells in primary cultures can be inhibited/regulated in two principally different ways which involve a pronounced time component. The results also further point out the adrenergic signaling system as an important mediator of dynamic neuron-astroglial information exchange.