Connexins and information transfer through glia.
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Animals↗
Biomedical subjects
C Giaume
Publications and source records attributed to C Giaume.
Metabolic coupling and the role played by astrocytes in energy distribution and homeostasis.
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Animals↗
Anandamide and WIN 55212-2 inhibit cyclic AMP formation through G-protein-coupled receptors distinct from CB1 cannabinoid receptors in cultured astrocytes.
The effects of anandamide and the cannabinoid receptor agonists WIN 55212-2 and CP 55940 on the evoked formation of cyclic AMP were compared in cultured neurons and astrocytes from the cerebral cortex and striatum of mouse embryos. The three compounds inhibited the isoproterenol-induced accumulation of cyclic AMP in neuronal cells, and these responses were blocked by the selective CB1 receptor antagonist SR 141716A. The three agonists were more potent in cortical than striatal neurons. Interestingly, WIN 55212-2, CP 55940 and anandamide also inhibited the isoproterenol-evoked accumulation of cyclic AMP in astrocytes but, in contrast to WIN 55212-2 and CP 55940, anandamide was much more potent in striatal than cortical astrocytes. Inhibition was prevented by pertussis toxin pretreatment, but not blocked by SR 141716A. Therefore, G-protein-coupled receptors, distinct from CB1 receptors, are involved in these astrocytic responses. Moreover, specific binding sites for [3H]-SR 141716A were found in neurons but not astrocytes. Furthermore, using a polyclonal CB1 receptor antibody, staining was observed in striatal and cortical neurons, but not in striatal and cortical astrocytes. Taken together, these results suggest that glial cells possess G-protein-coupled receptors activated by cannabinoids distinct from the neuronal CB1 receptor, and that glial cells responses must be taken into account when assessing central effects of cannabinoids.
Adrenergic beta-Agonists↗
Distinct pharmacological properties of ET-1 and ET-3 on astroglial gap junctions and Ca(2+) signaling.
Astrocytes represent a major target for endothelins (ETs), a family of peptides that have potent and multiple effects on signal transduction pathways and can be released by several cell types in the brain. In the present study we have investigated the involvement of different ET receptor subtypes on intercellular dye diffusion, intracellular Ca(2+) homeostasis, and intercellular Ca(2+) signaling in cultured rat astrocytes from hippocampus and striatum. Depending on the ET concentration and the receptor involved, ET-1- and ET-3-induced intracellular Ca(2+) increases with different response patterns. Both ET-1 and ET-3 are powerful inhibitors of gap junctional permeability and intercellular Ca(2+) signaling. The nonselective ET receptor agonist sarafotoxin S6b and the ET(B) receptor-selective agonist IRL 1620 mimicked these inhibitions. The ET-3 effects were only marginally affected by an ET(A) receptor antagonist but completely blocked by an ET(B) receptor antagonist. However, the ET-1-induced inhibition of gap junctional dye transfer and intercellular Ca(2+) signaling was only marginally blocked by ET(A) or ET(B) receptor-selective antagonists but fully prevented when these antagonists were applied together. The ET-induced inhibition of gap junction permeability and intercellular Ca(2+) signaling indicates that important changes in the function of astroglial communication might occur when the level of ETs in the brain is increased.
Animals↗
Potassium currents and excitability in second-order auditory and vestibular neurons.
Potassium channels are involved in the control of neuronal excitability by fixing the membrane potential, shaping the action potential, and setting firing rates. Recently, attention has been focused on identifying the factors influencing excitability in second-order auditory and vestibular neurons. Located in the brainstem, second-order auditory and vestibular neurons are sites for convergence of inputs from first-order auditory or vestibular ganglionic cells with other sensory systems and also motor areas. Typically, second-order auditory neurons exhibit two distinct firing patterns in response to depolarization: tonic, with a repetitive firing of action potentials, and phasic, characterized by only one or a few action potentials. In contrast, all mature vestibular second-order neurons fire tonically on depolarization. Already, certain fundamental roles have emerged for potassium currents in these neurons. In mature auditory and vestibular neurons, I(K), the delayed rectifier, is required for the fast repolarization of action potentials. In tonically firing auditory neurons, I(A), the transient outward rectifier, defines the discharge pattern. I(DS), a delayed rectifier-like current distinguished by its low threshold of activation, is found in phasically firing auditory and some developing vestibular neurons where it limits firing to one or a few spikes, and also may contribute to forming short-duration excitatory postsynaptic potential (EPSPs). Also, I(DS) sets the threshold for action potential generation rather high, which may prevent spontaneous discharge in phasically firing cells. During development, there is a gradual acquisition and loss of some potassium conductances, suggesting developmental regulation. As there are similarities in membrane properties of second-order auditory and vestibular neurons, investigations on firing pattern and its underlying mechanisms in one system should help to uncover fundamental properties of the other.
Action Potentials↗
Gap junctional communication and pharmacological heterogeneity in astrocytes cultured from the rat striatum.
Indo-1 and fluo-3 imaging techniques were used to investigate the role of gap junctions in the changes in cytosolic calcium concentrations ([Ca2+]i) induced by several receptor agonists. Subpopulations of confluent cultured astrocytes from the rat striatum were superfused with submaximal concentrations of endothelin-1 (Et1) and the alpha 1-adrenergic and muscarinic receptor agonists, methoxamine and carbachol, respectively. 2. Combined binding and autoradiographic studies indicated that all striatal astrocytes possess binding sites for Et1. In contrast, alpha 1-adrenergic and muscarinic binding sites were found to be heterogeneously distributed. In agreement with these findings, Et1 induced fast calcium responses in all cells while only subsets of striatal astrocytes responded to the application of methoxamine or carbachol. 3. Halothane, heptanol and octanol, which are commonly used as gap junction inhibitors, drastically reduced the amplitude of Et1-induced calcium responses. In contrast, 18-alpha-glycyrrhetinic acid (alpha GA) used at a concentration known to block gap junction permeability in astrocytes had no significant effect on the amplitude of these calcium responses. 4. As demonstrated by quantitative and topological analysis, Et1 application similarly increased [Ca2+]i levels in all astrocytes in both the absence and presence of alpha GA. 5. In control conditions, subpopulations of cells responding to methoxamine or carbachol exhibited two main types of calcium responses which differed in their shape and kinetic characteristics. In the presence of alpha GA the number of cells responding to these receptor agonists was significantly reduced. Indeed, responses characterized by their long latency, slow rise time and weak amplitude disappeared in the presence of alpha GA while responses with short latency and fast rise time were preserved. 6. These results indicate that permeable gap junction channels tend to attenuate the pharmacological and functional heterogeneity of populations of astrocytes, while their inhibition restricts calcium responses in astrocytes expressing high densities of transmitter receptors coupled to phospholipase C.
Animals↗
The differential expression of low-threshold sustained potassium current contributes to the distinct firing patterns in embryonic central vestibular neurons.
The principal cells of the chick tangential nucleus are second-order sensory neurons that participate in the three-neuron vestibulo-ocular and vestibulocollic reflexes. In postnatal animals, second-order vestibular neurons fire repetitively on depolarization. Previous studies have shown that, although this is an important feature for normal reflex function, it is only acquired gradually during embryonic development. Whereas at 13 embryonic days (E13) the principal cells accommodate after firing a single spike, at E16 a few principal cells repetitively can fire multiple action potentials on depolarization. Finally, in the hatchling, the vast majority of principal cells is capable of nonaccommodating firing on depolarization. As a first step in understanding the mechanisms underlying developmental change in excitability of these second-order vestibular neurons, we analyzed the outward potassium currents and their role in accommodation, using brainstem slices at E16. The principal cells exhibited transient and sustained potassium currents, with both of these containing calcium-dependent components. Further, both high- and low-threshold sustained potassium currents have been distinguished. The low-threshold dendrotoxin-sensitive sustained potassium current (IDS) is associated with principal cells that accommodate and is not expressed in those that fire repetitively. Finally, blocking of IDS transforms accommodating cells into neurons capable of firing trains of action potentials on depolarization. These findings indicate that suppression of IDS during development is sufficient to transform accommodating principal cells into nonaccommodating firing neurons and suggests that developmental regulation of this current is necessary for the establishment of normal vestibular function.
Action Potentials↗
Intercellular calcium signaling and gap junctional communication in astrocytes.
Two main characteristics of astrocytes are their elaborated intracellular calcium signaling and their high degree of intercellular communication mediated by gap junctional channels. In these cells a number of studies have contributed to demonstrate that the combination of these two properties provides a basis for a long-range signaling system within the brain. Intercellular calcium signaling, also termed calcium waves, allows astrocytes to communicate with each other and to interact with adjacent neurons. Most of the intra- and inter-cellular events involved in the initiation and propagation phases of this process has now been identified. This sequence of events includes the permeability of gap junction channels, which at the time-scale for calcium waves propagation, are likely permeated rather than closed by Ca2+ and/or related signaling molecules like IP3. In addition, in some studies an external component have been reported to participate to the propagation process. Finally, the control of the spread of intercellular calcium signaling has been demonstrated to occur at several levels including phospholipase C, IP3 receptors, intracellular Ca2+ stores, and cytoplasmic Ca2+ buffering. Accordingly, normal and pathological situations that affect one or several of these steps can be predicted to influence on astrocytic calcium waves.
Animals↗
Altered gap junctional communication, intercellular signaling, and growth in cultured astrocytes deficient in connexin43.
Astrocytes are characterized by extensive intercellular communication mediated primarily by gap junction channels composed of connexin43. To examine this junctional protein in astrocytic functions, astrocytes were cultured from embryonic mice with a null mutation in the connexin43 gene (Reaume et al.: Science 267:1831-1834, 1995). Using anti-Cx43 antibodies, immunoblotting and immunostaining indicated that homozygous null astrocytes were devoid of Cx43. They are also deficient in intercellular dye transfer. Astrocytes cultured from heterozygous embryos express significantly lower Cx43 compared to wild type, and their dye coupling is reduced. Markers of glial differentiation, such as glial fibrillary acidic protein and S100, appeared similar in all genotypes. Measurement of intercellular calcium concentration following mechanical stimulation of confluent astrocytes revealed that the number of cells affected by a rise in intracellular calcium was reduced in homozygous cultures compared to wild type. In fact, the calcium response in homozygous astrocytes was similar to that observed in wild-type astrocytes in the presence of a gap junction blocker. The growth rate of astrocytes lacking Cx43 was reduced compared to wild-type astrocytes. These results suggest that gap junctional intercellular communication mediated by Cx43 is not critical for astrocyte differentiation but is likely involved in the regulation of intercellular calcium signaling and cell growth.
Animals↗
Ontogeny of electrophysiological properties and dendritic pattern in second-order chick vestibular neurons.
The pattern of development of several subpopulations of second-order vestibular neurons was investigated by using intracellular recordings from chicken brain slices to define the timing of morphological and electrophysiological changes occurring at 3 critical ages. Two embryonic stages, embryonic day 13 (E13) and E15-16, and also newborn chicks were selected according to previous anatomical findings showing the differentiation of primary vestibular afferents and their synapses within a distinctive brainstem vestibular nucleus, the tangential nucleus. The responses of these cells to depolarizing and hyperpolarizing current pulses and their postsynaptic responses to vestibular nerve stimulation were recorded, while simultaneously biocytin was injected for subsequent morphogenetic analysis. From this study, developmental schedules of membrane properties, synaptic responses, and dendritic differentiation were established for the 2 cell populations of the tangential nucleus and other neurons located in the surrounding vestibular nuclei. Compared with all other second-order vestibular neurons, the principal cells of the tangential nucleus exhibited a distinctive schedule. Mainly, this includes their pattern of firing on depolarization, the shape and duration of the vestibular-evoked excitatory postsynaptic potential, and the time of onset of dendritic outgrowth. In regard to these observations, E15-16 appears to be a turning point in principal cell ontogeny, whereas these features occur earlier in development for other second-order vestibular neurons. These findings, which indicate that the principal cells may have distinct membrane properties at specific ages, are discussed in light of their unique vestibular innervation and the possible consequences for vestibular signal processing.
Animals↗
Mechanism involved in initiation and propagation of receptor-induced intercellular calcium signaling in cultured rat astrocytes.
The mechanisms involved in the initiation and the propagation of intercellular calcium signaling (calcium waves) were studied in cultured rat astrocytes. The analysis of calcium waves, induced either by mechanical stimulation or by focal application of ionomycin, indicated that initiation was dependent on the presence of external calcium. In addition, pharmacological experiments indicate that intercellular propagation required PLC activation, integrity of IP3-sensitive internal calcium stores, and functional gap junctions. An extracellular action of ATP or glutamate and participation of voltage-dependent Ca2+ channels were tested by using enzymatic degradation, receptor antagonists, and channel blockers, respectively. Because neither the speed of propagation nor the extent of the calcium waves was affected by these treatments, these alternate mechanisms were excluded from playing a role in intercellular calcium signaling. Biochemical assays and focal applications of several agonists (methoxamine, carbachol, glutamate) of membrane receptors to neurotransmitters and peptides (endothelin 1) demonstrated that their ability to trigger regenerative calcium waves depended on phospholipase C activity and inositol phosphate production. Thus, in rat astrocytes, initiation and propagation of calcium waves involve a sequence of intra- and intercellular steps in which phospholipase C, inositol trisphosphate, internal calcium stores, and gap junction channels play a critical role. The identification of these different events allows us to determine several targets at which the level of long-range signaling in astrocytes may be controlled.
Animals↗
(R)-methanandamide inhibits receptor-induced calcium responses by depleting internal calcium stores in cultured astrocytes.
The effect of (R)-methandamide, a chiral analog of the endogenous cannabimimetic anandamide, was investigated on calcium signalling in cultured rat astrocytes loaded with Indo-1. Pretreatment of astrocytes with (R)-methandamide resulted in the inhibition of calcium responses induced by endothelin-1 or glutamate. Test of the filling level of internal calcium stores and biochemical assays of phospholipase C activity suggested that this inhibition resulted from the depletion of internal pools. This effect occurred in a reversible time- and dose-dependent manner, and was prevented by treating the cells with pertussis toxin (PTX) but was not reproduced by similar concentrations of arachidonic acid. Altogether, these observations demonstrate that this stable analog of anandamide controls calcium signalling in astrocytes through a PTX-sensitive mechanism which leads to the depletion of fast mobilizable internal stores.
Animals↗
Nicotine inhibits slowly inactivating K+ currents in rat cultured striatal neurons.
As shown on cultured striatal neurons recorded in whole-cell configuration, both acetylcholine (in the presence of atropine) and nicotine reduced voltage-dependent outward currents. Although, at early postnatal ages, outward currents in these cells are mainly carried by rapidly and slowly inactivating K+ channels, these inhibitions resulted from a selective and reversible effect on the slowly inactivating K+ conductance (IK+). This action was blocked by the nicotinic antagonist dihydro-beta-erythroïdine and reproduced by nicotinic agonists. When neurons were recorded under current-clamp conditions, nicotine increased reversibly their firing rate generated by step depolarizations. Therefore, in addition to its well documented muscarinic effects, acetylcholine also controls K+ currents in striatal neurons through mechanisms mediated by nicotinic receptors.
Acetylcholine↗
Lack of biocytin transfer at gap junctions in the chicken vestibular nuclei.
In vivo experiments were designed to test for functional gap junctions at 'mixed' synapses that were morphologically characterized between the large-diameter, primary vestibular fibers and second-order vestibular neurons in the chicken, Gallus gallus. In previous intracellular recordings and dye injections into these neurons from brain slice preparations of chick embryos (E15/16) and also newborn hatchlings (HI-2), no evidence was obtained for functional gap junctions. Therefore, biocytin, a low molecular weight tracer that permeates gap junction channels, was extracellularly applied to either the ampullary nerves or to the vestibular ganglion of 3-6 day old hatchlings and adult chickens (9 months). This procedure resulted in the uptake of the dye and heavy staining of both the thick and thin fibers composing the vestibular nerve and in loading of vestibular efferent neurons. However, no dye transfer was observed between the large-diameter, primary vestibular fibers and second-order vestibular neurons. This observation, which was performed using a relatively non-invasive approach on intact animals, suggests that the gap junctions at these mixed synapses are probably not functional under the conditions of these experiments.
Animals↗
Metabolic trafficking through astrocytic gap junctions.
Astrocytes are interposed between the pericapillary space and neuronal membranes. Consequently, they may represent an important intermediary element between the source of energetic substrates and the main site of energy-consuming elements, respectively, the blood circulation and the neurons. A typical feature of astrocytes is the connections they establish between each other by specialized membrane structures, defined as gap junctions. These intercellular junctions allow direct cell-to-cell exchanges of ions and small molecules, including several compounds involved in major metabolic pathways occurring in astrocytes. The permeability of astrocytes gap junction channels is controlled by several endogenous compounds released by astrocytes themselves or by other brain cell types, including neurons and endothelial cells. In primary cultures of astrocytes, the intercellular diffusion, the utilization and the uptake of glucose and derivates are modified when gap junctional permeability is inhibited by uncoupling agents. Altogether these observations indicate that intercellular pathways constituted by groups of coupled astrocytes could participate to the metabolism and the distribution of energetic substrates throughout the brain.
Animals↗
Dendritic growth and changes in electrophysiological properties during development of chick vestibular neurons.
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Animals↗
Endothelin-1 regulates glucose utilization in cultured astrocytes by controlling intercellular communication through gap junctions.
The role played by endothelin-1 and intercellular communication mediated by gap junctions in the regulation of glucose disposal by astrocytes has been studied in primary culture. Endothelin-1 increased glucose uptake by astrocytes as did one of its putative messenger arachidonic acid and the non-physiological gap junction uncoupler alpha-glycyrrhetinic acid (AGA). None of these agents increased glucose uptake by C6 glioma cells, a cell line in which gap junction proteins are poorly expressed. In confluent astrocytes, the inhibition of gap junction permeability caused by AGA doubled the activity of the pentose phosphate shunt with minimal changes in the activity of the pyruvate dehydrogenase-catalyzed reaction and that of the tricarboxylic acid cycle. By contrast, these effects were not observed in dissociated astrocytes in which intercellular communication is lacking. The scraped loading dye transfer technique was modified to follow the passage of glucose and its metabolites through astrocyte gap junctions. The diffusion of glucose, the phosphorylated derivative glucose-6-phosphate, the phosphorylisable but not metabolisable derivative ortho-methyl-glucose, and the anaerobic glycolytic product L-lactate was much higher in astrocytes than in C6 glioma cells and was inhibited by the inhibition of gap junction permeability caused by endothelin-1, arachidonic acid, octanol, or AGA. It is concluded that gap junction permeability may regulate brain metabolism by controlling the uptake, utilization, and intercellular distribution of glucose and its metabolites in astrocytes.
Animals↗
Control of gap-junctional communication in astrocytic networks.
Astrocytes, which constitute the most abundant cell type in mammalian brain, are extensively coupled to one another through gap junctions composed mainly of connexin43. In regions exhibiting high levels of connexin43 expression, tens of astrocytes are labeled following single-cell intracellular injection. Importantly, both the expression and the permeability of gap junctions are tightly regulated. Such long- and short-term regulations indicate that astrocytic networks might be subject to remodeling and to some plasticity. Since evidence for neuro-glial interaction exists, the degree of coupling between astrocytes could participate to set the tone of neuronal activity and to determine the sphere of influenced neurons. Research in this area is still at its early stages and significant progress requires a transition from the understanding of basic properties to the study of function.
Animals↗