Electrical synapses between Bergmann glial cells and Purkinje neurones in rat cerebellar slices

In the present study, we directly demonstrated electrical coupling between Bergmann glial cells (BG) and Purkinje neurones (PN) in acutely isolated cerebellar slices, prepared from 15 to 30 days old Sprague-Dawley rats. Electrical coupling between these two cells was identified by dual whole-cell voltage clamp, which allowed direct recording of junctional current. Whole-cell recordings from PN-PN, PN-BG and BG-BG pairs were made using Nomarski optics and infrared visualisation, which allowed precise morphological identification of cells. Junctional currents were recorded by applying hyper/and depolarising voltage sequences ranging from -120 to +40 mV (voltage step 10 mV) to one of the cells in the pair, while ion currents were measured from both cells. As has been shown before, junctional currents were frequently observed in BG-BG pairs: we found electrical coupling in 27 out of 34 pairs analysed. When the similar protocol was applied to the PN-BG pairs, junctional currents were found in 61 out of 87 pairs analysed. The electrical coupling was bi-directional as similar junctional currents were observed in PN when voltage step protocol was applied to BG. No electrical coupling was observed in PN-PN pairs (n = 21). To correlate the appearance of these currents with gap junctions we treated slices with octanol (200 microM) or halothane (500 microM)-known inhibitors of gap junction conductance. Both agents applied for 5 min resulted in a complete inhibition of junctional currents in PN-BG pair. The washout (15 min) led to a complete recovery of junctional currents after treatment with octanol; the action of halothane was irreversible. Finally, we found that filling the BG by Alexa Fluor 488 results in staining of adjacent PN (in 11 out of 23 pairs tested). We conclude therefore that cerebellar neurones and glial cells are directly connected via gap junctions.     

LM and EM immunolocalization of the gap junctional protein connexin 43 in rat brain

A site-specific antibody against the principal gap junctional protein in heart (connexin 43) was used to determine immunohistochemically the cellular localization of this protein in rat brain. Structures labelled with the antibody included gap junctional membranes between glial, ependymal, pial and arachnoid cells as well as cytoplasmic membranes and intracellular organelles in close proximity to junctions between these various cell types. No labelling was detected within cell bodies of oligodendrocytes and neurons and no labelled neuronal gap junctions were found. The results suggest that connexin 43 is one of the major gap junctional proteins utilized for junctional coupling between astrocytes and between cells lining the surfaces of the brain.     

Connexins and gap junctions of astrocytes and oligodendrocytes in the CNS

This review article summarizes early and recent literature on the structure, distribution and composition of gap junctions between astrocytes and oligodendrocytes, and the differential expression of glial connexins in adult and developing mammalian CNS. In addition to an overview of the topic, discussion is focused on the organization of homologous gap junctional interactions between astrocytes and between oligodendrocytes as well as on heterologous junctional coupling between astrocytes and oligodendrocytes. The homotypic and heterotypic nature of these gap junctions is related to the connexins known to be produced by glial cells in the intact brain and spinal cord. Emphasis is placed on the ultrastructural level of analysis required to attribute gap junction and connexin deployment to particular cell types and subcellular locations. Our aim is to provide a firm basis for consideration of anticipated rapid advances in understanding of structural relationships of gap junctions and connexins within the glial gap junctional syncytium. Conclusions to date suggest that the glial syncytium is more complex than previously appreciated and that glial pathways of junctional communication may not only be determined by the presence of gap junctions, but also by the connexin composition and conductance regulation of junctional channels.     

Panglial gap junctions between astrocytes and olfactory ensheathing cells mediate transmission of Ca2+ transients and neurovascular coupling

Astrocytes are arranged in highly organized gap junction-coupled networks, communicating via the propagation of Ca²⁺ waves. Astrocytes are gap junction-coupled not only to neighboring astrocytes, but also to oligodendrocytes, forming so-called panglial syncytia. It is not known, however, whether glial cells in panglial syncytia transmit information using Ca²⁺ signaling. We used confocal Ca²⁺ imaging to study intercellular communication between astrocytes and olfactory ensheathing glial cells (OECs) in in-toto preparations of the mouse olfactory bulb. Our results demonstrate that Ca²⁺ transients in juxtaglomerular astrocytes, evoked by local photolysis of “caged” ATP and “caged” tACPD, led to subsequent Ca²⁺ responses in OECs. This transmission of Ca²⁺ responses from astrocytes to OECs persisted in the presence of neuronal inhibition, but was absent when gap junctional coupling was suppressed with carbenoxolone. When Ca²⁺ transients were directly evoked in OECs by puff application of DHPG, they resulted in delayed Ca²⁺ responses in juxtaglomerular astrocytes, indicating that panglial transmission of Ca²⁺ signals occurred in a bidirectional manner. In addition, panglial transmission of Ca²⁺ signals from astrocytes to OECs resulted in vasoconstriction of OEC-associated blood vessels in the olfactory nerve layer. Our results demonstrate functional transmission of Ca²⁺ signals between different classes of glial cells within gap junction-coupled panglial networks and the resulting regulation of blood vessel diameter in the olfactory bulb.     

Evidence of involvement of neurone‐glia/neurone‐neurone communications via gap junctions in synchronised activity of KNDy neurones

Pulsatile secretion of gonadotrophin‐releasing hormone (GnRH)/luteinising hormone is indispensable for the onset of puberty and reproductive activities at adulthood in mammalian species. A cohort of neurones expressing three neuropeptides, namely kisspeptin, encoded by the Kiss1 gene, neurokinin B (NKB) and dynorphin A, localised in the hypothalamic arcuate nucleus (ARC), so‐called KNDy neurones, comprises a putative intrinsic source of the GnRH pulse generator. Synchronous activity among KNDy neurones is considered to be required for pulsatile GnRH secretion. It has been reported that gap junctions play a key role in synchronising electrical activity in the central nervous system. Thus, we hypothesised that gap junctions are involved in the synchronised activities of KNDy neurones, which is induced by NKB‐NK3R signalling. We determined the role of NKB‐NK3R signalling in Ca²⁺ oscillation (an indicator of neuronal activities) of KNDy neurones and its synchronisation mechanism among KNDy neurones. Senktide, a selective agonist for NK3R, increased the frequency of Ca²⁺ oscillations in cultured Kiss1‐GFP cells collected from the mediobasal hypothalamus of the foetal Kiss1‐green fluorescent protein (GFP) mice. The senktide‐induced Ca²⁺ oscillations were synchronised in the Kiss1‐GFP and neighbouring glial cells. Confocal microscopy analysis of these cells, which have shown synchronised Ca²⁺ oscillations, revealed close contacts between Kiss1‐GFP cells, as well as between Kiss1‐GFP cells and glial cells. Dye coupling experiments suggest cell‐to‐cell communication through gap junctions between Kiss1‐GFP cells and neighbouring glial cells. Connexin‐26 and ‐37 mRNA were found in isolated ARC Kiss1 cells taken from adult female Kiss1‐GFP transgenic mice. Furthermore, 18β‐glycyrrhetinic acids and mefloquine, which are gap junction inhibitors, attenuated senktide‐induced Ca²⁺ oscillations in Kiss1‐GFP cells. Taken together, these results suggest that NKB‐NK3R signalling enhances synchronised activities among neighbouring KNDy neurones, and that both neurone‐neurone and neurone‐glia communications via gap junctions possibly contribute to synchronised activities among KNDy neurones.     

TNFα Inhibits Schwann Cell Proliferation, Connexin46 Expression, and Gap Junctional Communication

Schwann cell responses to nerve injury are stimulated, in part, by inflammatory cytokines. This study compares changes in the phenotype of cultured Schwann cells after exposure to the cytokine tumor necrosis factor (TNF)-α or the mitogen neu differentiation factor (NDF)-β. TNFα inhibited proliferation in a dose-dependent manner without altering Schwann cell survival. TNFα also reduced both gap junctional conductance and Lucifer yellow dye coupling between Schwann cells. Moreover, both P₀and glial fibrillary acidic protein (GFAP) immunoreactivity were reduced. By contrast, NDFβ initially had little effect on cell division although it reduced junctional coupling within 8 h. However, by 48 h, NDFβ stimulated proliferation with a concomitant increase in coupling. Dividing Schwann cells (BrdU⁺) were preferentially dye coupled compared to nondividing cells, indicating an association between proliferation and coupling. Moreover, cultured Schwann cells expressed connexin46 mRNA and protein, and changes in the levels of the protein correlated with the degree of proliferation and coupling. The data thus provide evidence for cytokine-induced modulation of Schwann cell antigenic phenotype, proliferation, and gap junction properties. These observations suggest that enhanced gap junctional communication among Schwann cells after nerve injury could help to coordinate cellular responses to the injury, and that TNFα may be a signal which terminates proliferation as well as junctional communication.     

Glial ER and GAP junction mediated Ca2+ waves are crucial to maintain normal brain excitability

Astrocytes play key roles in regulating multiple aspects of neuronal function from invertebrates to humans and display Ca²⁺ fluctuations that are heterogeneously distributed throughout different cellular microdomains. Changes in Ca²⁺ dynamics represent a key mechanism for how astrocytes modulate neuronal activity. An unresolved issue is the origin and contribution of specific glial Ca²⁺ signaling components at distinct astrocytic domains to neuronal physiology and brain function. The Drosophila model system offers a simple nervous system that is highly amenable to cell-specific genetic manipulations to characterize the role of glial Ca²⁺ signaling. Here we identify a role for ER store-operated Ca²⁺ entry (SOCE) pathway in perineurial glia (PG), a glial population that contributes to the Drosophila blood–brain barrier. We show that PG cells display diverse Ca²⁺ activity that varies based on their locale within the brain. Ca²⁺ signaling in PG cells does not require extracellular Ca²⁺ and is blocked by inhibition of SOCE, Ryanodine receptors, or gap junctions. Disruption of these components triggers stimuli-induced seizure-like episodes. These findings indicate that Ca²⁺ release from internal stores and its propagation between neighboring glial cells via gap junctions are essential for maintaining normal nervous system function.     

Intercellular Ca2+ waves induce temporally and spatially distinct intracellular Ca2+ oscillations in glia

Mechanically induced intercellular Ca²⁺ waves propagated for approximately 300 μm in primary glial cultures. Following the wave propagation, 34% of the cells displayed Ca²⁺ oscillations in a zone 60–120 μm from the stimulated cell. The initiation, frequency, and duration of these Ca²⁺ oscillations were dependent on the cells' distance from the wave origin but were not dependent on the cell type nor on the magnitude of the Ca²⁺ wave. When an individual cell propagated two sequential intercellular Ca²⁺ waves originating from different sites, the characteristics of the Ca²⁺ oscillations initiated by each wave were determined by the distance of the cell from the origin of each wave. Each Ca²⁺ oscillation commonly occurred as an intracellular Ca²⁺ wave that was initiated from a specific site within the cell. The position of the initiation site and the direction of the intracellular Ca²⁺ wave were independent of the orientation of the initial intercellular Ca²⁺ wave. Because initiation and frequency of Ca²⁺ oscillations are dependent on the intracellular inositol trisphosphate concentration ([IP₃]_i), we propose that the zone of cells displaying Ca²⁺ oscillations is determined by an intercellular gradient of [IP₃]_i, established by the diffusion of IP₃ through gap junctions during the propagation of the intercellular Ca²⁺ wave. Exposure to acetylcholine, a muscarinic agonist that initiates IP₃ production, shifted the zone of oscillating cells about 45 μm farther away from the origin of the mechanically induced wave. These findings indicate that a glial syncytium can resolve information provided by a local Ca²⁺ wave into a distinct spatial and temporal pattern of Ca²⁺ oscillations. GLIA 28:97–113, 1999. © 1999 Wiley‐Liss, Inc.     

Electrical coupling between cultured glomus cells of the rat carotid body: observations with current and voltage clamping

Electrically coupled pairs of cultured rat glomus cells were used. In one group of experiments, both cells were current-clamped. Delivery of positive or negative pulses to Cell 1 elicited appreciable voltage noise in this cell and large action potentials (probably Ca²⁺ spikes) in about 10% of them. Both passive and active electrical events spread to Cell 2, presumably through the gap junctions between them. The coupling coefficient (K_c) was larger for the spikes than for non-regenerative voltage noise. In another group of experiments, Cell 1 was current-clamped and Cell 2 was voltage-clamped at Cell 1 E_M. Pulses of either polarity, delivered to Cell 1, produced current flow through the intercellular junction and allowed direct measurement of junctional currents (I_j) and total conductances (G_j). I_j had a mean value of about 12.5 pA and G_j of 391 pS. Unitary (presumably single channel) conductance (g_j) was about 78 pS.     

Gap junction protein connexin43 is heterogeneously expressed among glial cells in the adult rabbit retina.

The immunohistochemical distribution and ultrastructural immunolocalization of connexin43 (Cx43) in the neural retina of the rabbit was investigated. Cx43 immunolabeling appeared in the form of distinct puncta distributed on different kinds of glial cells and exclusively in the myelinated fiber region of the neural retina. Double-label immunohistochemistry showed that the most obvious Cx43 labeling occurred at processes of glial fibrillary acidic protein-positive astrocytes and on vimentin-positive Müller cells. Cx43-immunoreactive puncta were also evident on cell bodies and processes of 2'-3'-cyclic nucleotide phosphodiesterase-labeled oligodendrocytes. As shown by electron microscopy, immunoreactivity to Cx43 was restricted to gap junctions among the macroglial cell population. The homologous interastrocytic and Müller cell-to-Müller cell, as well as the heterologous astrocyte-to-Müller cell and astrocyte/Müller-to-oligodendrocyte gap junctions were symmetrically labeled. Our results indicate a specific expression of Cx43 at gap junctions between macroglial cells located in the myelinated streak. The extensive Cx43 immunolabeling suggests a substantial amount of gap junctional coupling that establishes a macroglial syncytium.     

Calcium Signalling in Mouse Bergmann Glial Cells Mediated by α1-adrenoreceptors and H1 Histamine - Receptors

The presence of adrenergic and histaminergic receptors in Bergmann glial cells from cerebellar slices from mice aged 20–25 days was determined using fura-2 Ca²⁺ microfluorimetry. To measure the cytoplasmic concentration of Ca²⁺ ([Ca²⁺]_i), either individual cells were loaded with the Ca²⁺-sensitive probe fura-2 using the whole-cell patch-clamp technique or slices were incubated with a membrane-permeable form of the dye (fura-2/AM) and the microfluorimetric system was focused on individual cells. The monoamines adrenalin and noradrenalin (0.1-10 μM) and histamine (10-100 μM) triggered a transient increase in [Ca²⁺]_i. The involvement of the α₁-adrenoreceptor was inferred from the observations that monoamine-triggered [Ca²⁺]_i responses were blocked by the selective α¹-adreno-antagonist prazosin and were mimicked by the α¹-adreno-agonist phenylephrine. The monoamine-induced [Ca²⁺]_i signals were not affected by β- and α₂-adrenoreceptor antagonists (propranolol and yohimbine), and were not mimicked by β- and α₂-adrenoreceptor agonists (isoproterenol and clonidine). Histamine-induced [Ca²⁺]_i responses demonstrated specific sensitivity to only H₁ histamine receptor modulators. [Ca²⁺]_i responses to monoamines and histamine did not require the presence of extracellular Ca²⁺ and they were blocked by preincubation of slices with thapsigargin (500 nM), indicating that the [Ca²⁺]_i increase is due to release from intracellular pools. No [Ca²⁺]_i responses were recorded after application of aspartate, bradykinin, dopamine, GABA, glycine, oxytocin, serotonin, somatostatin, substance P, taurine or vasopressin. We conclude that cerebellar Bergmann glial cells are endowed with α₁ -adrenoreceptors and H₁ histamine receptors which induce the generation of intracellular [Ca²⁺]_i signals via activation of Ca²⁺ release from inositol-l,4,5-trisphosphate-sensitive intracellular stores.     

Cx36, Cx43 and Cx45 in mouse and rat cerebellar cortex: species‐specific expression,compensation in Cx36 null mice and co‐localization in neurons vs. glia

Electrical synapses formed by connexin36 (Cx36)‐containing gap junctions between interneurons in the cerebellar cortex have been well characterized, including those formed between basket cells and between Golgi cells, and there is gene reporter‐based evidence for the expression of connexin45 (Cx45) in the cerebellar molecular layer. Here, we used immunofluorescence approaches to further investigate expression patterns of Cx36 and Cx45 in this layer and to examine localization relationships of these connexins with each other and with glial connexin43 (Cx43). In mice, strain differences were found, such that punctate labelling for Cx36 was differentially distributed in the molecular layer of C57BL/6 vs. CD1 mice. In mice with EGFP reporter representing Cx36 expression, Cx36‐puncta were localized to processes of stellate cells and other cerebellar interneurons. Punctate labelling of Cx45 was faint in the molecular layer of wild‐type mice and was increased in intensity in mice with Cx36 gene ablation. The vast majority of Cx36‐puncta co‐localized with Cx45‐puncta, which in turn was associated with the scaffolding protein zonula occludens‐1. In rats, Cx45‐puncta were also co‐localized with Cx36‐puncta and additionally occurred along Bergmann glial processes adjacent to Cx43‐puncta. The results indicate strain and species differences in Cx36 as well as Cx45 expression, possible compensatory processes after loss of Cx36 expression and localization of Cx45 to both neuronal and Bergmann glial gap junctions. Further, expression of both Cx43 and Cx45 in Bergmann glia of rat may contribute to the complex properties of junctional coupling between these cells and perhaps to their reported coupling with Purkinje cells.     

Inositol-trisphosphate-dependent intercellular calcium signaling in and between astrocytes and endothelial cells

Interactions between astrocytes and endothelial cells are believed to play an important role in the control of blood-brain barrier permeability and transport. Astrocytes and endothelial cells respond to a variety of stimuli with an increase of intracellular free calcium ([Ca²⁺]_i) that is propagated to adjacent cells as an intercellular Ca²⁺ wave. We hypothesized that intercellular Ca²⁺ signaling also occurs between astrocytes and endothelial cells, and we investigated this possibility in co-cultures of primary astrocytes and an endothelial cell line using caged messengers. Intercellular Ca²⁺ waves, induced by mechanical stimulation of a single cell, propagated from astrocytes to endothelial cells and vice versa. Intercellular Ca²⁺ waves could also be induced by flash photolysis of pressure-injected caged inositol trisphosphate (IP₃) and also by applying the flash to remote noninjected cells. Ca²⁺ waves induced by flash photolysis propagated from endothelial cells to astrocytes but not from astrocytes to endothelial cells even though caged IP₃ diffused between the two cell types. Flash photolysis of caged Ca²⁺ (NP-EGTA) resulted in an increase of [Ca²⁺]_i but did not initiate an intercellular Ca²⁺ wave. We conclude that an increase of IP₃ in a single cell is sufficient to initiate an intercellular Ca²⁺ wave that is propagated by the diffusion of IP₃ to neighboring cells and that can be communicated between astrocytes and endothelial cells in co-culture. By contrast, Ca²⁺ diffusion via gap junctions does not appear to be sufficient to propagate an intercellular Ca²⁺ wave. We suggest that intercellular Ca²⁺ waves may play a role in astrocyte-endothelial interactions at the blood-brain barrier. GLIA 24:398–407, 1998. © 1998 Wiley-Liss, Inc.     

Calcium signaling in cultured rat oligodendrocytes

The syntax of neuronal-glial or axonal-glial interaction is frequently communicated through transient changes in internal calcium (Ca_i). We examined mechanisms for Ca_i signaling and intercellular propagation of Ca_i responses in cultured oligodendrocytes (OLGs) derived form adult spinal cord (SC), postnatal day 21 (P21) SC, and P21 brain. We found that (1) cultured OLGs exhibited a heterogeneous responese to norepinephrine, carbachol, ATP, histamine, and glutamate; (2) receptor-mediated Ca_i increases were derived from both Ca²⁺ influx and intracellular Ca²⁺ release; (3) the percentage of responders to neuroligands varied as a function of cell origin; (4) cultured OLGs exhibited a thapsigargin-sensitive, but not a caffeine-sensitive, intracellular Ca²⁺ pool; and (5) gap junctional contacts between OLGs permitted limited intercellular propagation of mechanically stimulated Ca_i responses. Receptor-mediated Ca_i signaling appears to occur not only in cultured OLGs but also in acutely dissociated OLGs. The heterogeneity in Ca_i responses as a function of cell origin may reflect the existence of OLG subsets of differences in the maturation stage of OLGs. © 1995 Wiley-Liss, Inc.     

Effect of kainate on the membrane conductance of hilar glial precursor cells recorded in the perforated-patch configuration

The effects of kainate on membrane current and membrane conductance were investigated in presumed hilar glial precursor cells of juvenile rats. The perforated-patch configuration was used also to reveal possible second-messenger effects. Kainate evoked an inward current that was accompanied by a biphasic change in membrane conductance in 69% of the cells. An initial conductance increase with a time course similar to that of the inward current was followed by a second delayed conductance increase. This second conductance was absent in whole-cell-clamp recordings, suggesting that it was mediated by a second messenger effect. Analysis of the reversal potentials of the membrane current during both phases of the kainate-induced conductance change revealed that the first conductance increase reflected the activation of AMPA receptors. Several lines of evidence suggest that the delayed second conductance increase was due to the indirect activation of Ca²⁺-dependent K⁺ channels via Ca²⁺-influx through AMPA receptors. (1) the delayed second conductance increase was blocked by Ba²⁺ and the reversal of its underlying current was significantly shifted towards E_K+, suggesting that it is due to the activation of K⁺ channels. (2) The delayed second conductance increase disappeared in a Ca²⁺-free saline buffered with BAPTA, indicating that it depended on Ca²⁺-influx. (3) Co²⁺, Cd²⁺ and nimodipine failed to block the delayed second conductance increase excluding a major contribution of voltage-dependent Ca²⁺ channels. (4) The involvement of metabotropic glutamate receptors also appeared unlikely, because the kainate-induced delayed second conductance increase could not be blocked by a depletion of the intracellular Ca²⁺ stores with the Ca²⁺-ATPase inhibitor thapsigargin, and t-ACPD exerted no effect on membrane current and conductance. We conclude that kainate activates directly AMPA receptors in presumed hilar glial precursor cells. This results in a Ca²⁺ influx that could lead indirectly to the activation of Ca²⁺-dependent K⁺ channels. GLIA 23:35–44, 1998. © 1998 Wiley-Liss, Inc.     

Chloride-dependent transport of NH4+ into bee retinal glial cells

Mammalian astrocytes convert glutamate to glutamine and bee retinal glial cells convert pyruvate to alanine. To maintain such amination reactions these glial cells may take up NH₄⁺/NH₃. We have studied the entry of NH₄⁺/NH₃ into bundles of glial cells isolated from bee retina by using the fluorescent dye BCECF to measure pH. Ammonium caused intracellular pH to decrease by a saturable process: the rate of change of pH was maximal for an ammonium concentration of about 5 mm . This acidifying response to ammonium was abolished by the loop diuretic bumetanide (100 μm ) and by removal of extracellular Cl^–. These results strongly suggest that ammonium enters the cell by cotransport of NH₄⁺ with Cl^–. Removal of extracellular Na⁺ did not abolish the NH₄⁺-induced acidification. The NH₄⁺-induced pH change was unaffected when nearly all K⁺ conductance was blocked with 5 mm Ba²⁺ showing that NH₄⁺ did not enter through Ba²⁺-sensitive ion channels. Application of 2 mm NH₄⁺ led to a large increase in total intracellular proton concentration estimated to exceed 13.5 mEq/L. As the cell membrane appeared to be permeable to NH₃, we suggest that when NH₄⁺ entered the cells, NH₃ left, so that protons were shuttled into the cell. This shuttle, which was strongly dependent on internal and external pH, was quantitatively modelled. In retinal slices, 2 mm NH₄⁺ alkalinized the extracellular space: this alkalinization was reduced in the absence of bath Cl^–. We conclude that NH₄⁺ enters the glial cells in bee retina on a cotransporter with functional similarities to the NH₄⁺(K⁺)-Cl^– cotransporter described in kidney cells.     

Receptor-mediated calcium signalling in glial cells from mouse corpus callosum slices

We addressed the question of whether glial cells in intact white matter tracts express neurotransmitter receptors and we used Ca⁺⁺ signalling as a probe to detect the receptor activation. Corpus callosum slices from postnatal mice were bulk-loaded with the Ca⁺⁺- sensitive fluorescent dye fluo-3, and confocal microscopy was used to measure Ca⁺⁺ transients in response to neuroligands. Glial cell bodies were intensely dye-loaded and could be discriminated from the diffuse fluorescence of axons. Subpopulations of glial cells from slices obtained at postnatal days 3 to 7 responded with Ca⁺⁺ signals to ATP, glutamate, histamine, GABA, norepinephrine, serotonin, angiotensin II, bradykinin, and substance P. These subpopulations showed a distinct overlap; cells which were responsive to substance P always showed Ca⁺⁺ signalling in response to histamine, ATP, GABA, and high K⁺ (membrane depolarization). GABA-responsive cells almost always showed a [Ca⁺⁺], increase after membrane depolarization. In brain slices from postnatal day 11 to 18 animals, the Ca⁺⁺ responses were evident for glutamate, ATP, and norepinephrine, while GABA, substance P, serotonin, histamine, or angiotensin II rarely elicited a response. This study demonstrates that white matter glial cells in slices exhibit a large repertoire of neurotransmitter responses linked to Ca⁺⁺ signalling and that these receptor systems are differentially distributed on sub-populations of glial cells. © 1996 Wiley-Liss, Inc.     

Ca2+ influx into leech glial cells and neurones caused by pharmacologically distinct glutamate receptors

The effect of glutamatergic agonists on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) of neuropile glial cells and Retzius neurones in intact segmental ganglia of the medicinal leech Hirudo medicinalis was investigated by using iontophoretically injected fura-2. In physiological Ringer solution the [Ca²⁺]_i levels of both cell types were almost the ssame (glial cells: 58 ± 30 nM, n = 51; Retzius neurones: 61 ± 27 nM, n = 64). In both cell types glutamate, kainate, and quisqualate induced an increase in [Ca²⁺]_i which was inhibited by 6,7-dinitroquinoxaline-2,3-dione (DNQX). This increase was caused by a Ca²⁺ influx from the extracellular space because the response was greatly diminished upon removal of extracellular Ca²⁺. The glutamate receptors of neuropile glial cells and Retzius neurones differed with respect to the relative effectiveness of the agonists used, as well as with regard to the inhibitory strenght of DNQX. In Retzius neurones the agonist-induced [Ca²⁺]_i increase was abolished after replacing extracellular Na⁺ by organic cations or by mM amounts of Ni²⁺, whereas in glial cells the [Ca²⁺]_i increase was largely preserved under both conditions. It is concluded that in Retzius neurones the Ca²⁺ influx is predominantly mediated by voltage-dependent Ca²⁺ channels, whereas in neuropile glial cells the major influx occurs via the ion channels that are associated with the glutamate receptors.     

Gap junctions in the chicken pineal gland

The chicken pineal gland, which contains a heterogeneous cell population, sustains a circadian rhythm of activity. Synchronization of cellular activity of heterogeneous cells might be facilitated by gap junctional intercellular channels which are permeable to ions and second messengers. To test this possibility, we looked for morphologically identifiable gap junctions between the different pineal cells, used antibodies and cDNA probes to screen for the presence of connexins, and tested for functional intercellular coupling. By transmission electron microscopy and immunocytochemistry, gap junctions and connexins were observed between pinealocyte cell bodies, stromal cells, astrocytes, and astrocyte and pinealocyte processes. Two gap junctional proteins, connexin43 and connexin45, were detected by immunocytochemistry, immunoblotting and RNA blot analysis. Functional intercellular coupling was observed in the gland by transfer of low molecular weight dyes. Dye transferred between homologous and heterologous cells. These data suggest that homologous and heterologous gap junctions may provide a mechanism for coordination of the cellular responses of the elements of the biological clock which are induced by lighting cues to produce the circadian rhythm of pineal activity.