Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by Connexin43

Abstract

Intercellular Ca^{2 +} wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca^{2 +} wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca^{2 +} waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca^{2 +} wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca^{2 +} waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca^{2 +} waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.     

The dual face of connexin-based astroglial Ca communication: A key player in brain physiology and a prime target in pathology

For decades, studies have been focusing on the neuronal abnormalities that accompany neurodegenerative disorders. Yet, glial cells are emerging as important players in numerous neurological diseases. Astrocytes, the main type of glia in the central nervous system , form extensive networks that physically and functionally connect neuronal synapses with cerebral blood vessels. Normal brain functioning strictly depends on highly specialized cellular cross-talk between these different partners to which Ca^2 +, as a signaling ion, largely contributes. Altered intracellular Ca^2 + levels are associated with neurodegenerative disorders and play a crucial role in the glial responses to injury. Intracellular Ca^2 + increases in single astrocytes can be propagated toward neighboring cells as intercellular Ca^2 + waves, thereby recruiting a larger group of cells. Intercellular Ca²⁺ wave propagation depends on two, parallel, connexin (Cx) channel-based mechanisms: i) the diffusion of inositol 1,4,5-trisphosphate through gap junction channels that directly connect the cytoplasm of neighboring cells, and ii) the release of paracrine messengers such as glutamate and ATP through hemichannels (‘half of a gap junction channel’). This review gives an overview of the current knowledge on Cx-mediated Ca^2 + communication among astrocytes as well as between astrocytes and other brain cell types in physiology and pathology, with a focus on the processes of neurodegeneration and reactive gliosis. Research on Cx-mediated astroglial Ca^2 + communication may ultimately shed light on the development of targeted therapies for neurodegenerative disorders in which astrocytes participate. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Regulation of gap junctional communication by astrocytic mitochondrial K(ATP) channels following neurotoxin administration in in vitro and in vivo models

It is known that neuronal ATP-sensitive potassium (K(ATP)) channels and astrocytic gap junctions (GJs) are involved in the mechanism underlying neurodisorders. The K(ATP) channels exist also in glial cells, and the objective of this study was to determine whether the astrocytic K(ATP) channels exert their effect on neurotoxin-induced neurodysfunction through regulating the astrocytic GJ function. The results showed that diazoxide, a selective mitochondrial K(ATP) (mitoK(ATP)) channel opener, enhanced the GJ coupling, but 5-hydroxydecanoate, a selective mitoK(ATP) channel blocker that significantly inhibits GJ coupling in vitro did not. Activation of astrocytic mitoK(ATP) channels alleviated kainic acid-induced dysfunction of GJ intercellular communication. Finally, activation of mitoK(ATP) channels improved the astrocytic GJ coupling in the hippocampus after seizures due to the colabeling of GJ subunit connexin 43 and connexin 45 with glial marker and was increased substantially by the administration of diazoxide. Western blot demonstrated that the mitoK(ATP) channels regulated the expression of connexin 43 (P2; active form) and connexin 45 in the epileptic hippocampus. These findings demonstrate that activation of astrocytic mitoK(ATP) channels improves the GJ function in astrocytes, indicating that the effect of the astrocytic mitoK(ATP) channels on neurotoxin-induced neurodysfunction might be, in part, through the regulation of the GJ-coupled spatial buffering in the hippocampus.     

ATP-Induced Oscillations of Cytosolic Ca2+ Activity in Cultured Astrocytes from Rat Brain are Modulated by Medium Osmolarity Indicating a Control of [Ca2+]i Oscillations by Cell Volume

Oscillations of cytosolic Ca²⁺ activity ([Ca²⁺]_i) induced by stimulation with ATP in rat astrocytes in primary cultures were analysed. Astrocytes, prepared from the brains of newborn rats, loaded with the fluorescent Ca²⁺ indicator fura-2/AM, were continuously stimulated with ATP (10 M). ATP caused a large initial [Ca²⁺ peak, followed by regular [Ca²⁺]_i oscillations (frequencies 1–5/min). Astrocytes were identified by glial fibrillary acidic protein staining of cells after [Ca²⁺]_i recording. The oscillations were reversibly blocked by the P₂ purinoceptor antagonist suramin (30 M). Influx of extracellular Ca²⁺ and mobilization of Ca²⁺ from intracellular stores both contributed to the oscillations. The effects of hypertonic and hypotonic superfusion medium on ATP-induced [Ca²⁺]_i oscillations were examined. Hypertonic medium (430 mOsm) reversibly suppressed the ATP-induced oscillations. Hypotonic medium (250 mOsm), in spite of having heterogeneous effects, most frequently induced a rise in [Ca²⁺]_i, or reversibly increased the frequency of the oscillations. Thus, a change in cell volume might be closely connected with [Ca²⁺]_i oscillations in astrocytes indicating that [Ca²⁺]_i oscillations in glial cells play an important role in regulatory volume regulation in the brain.     

Exocytosis of ATP From Astrocytes Modulates Phasic and Tonic Inhibition in the Neocortex

Communication between neuronal and glial cells is important for many brain functions. Astrocytes can modulate synaptic strength via Ca²⁺-stimulated release of various gliotransmitters, including glutamate and ATP. A physiological role of ATP release from astrocytes was suggested by its contribution to glial Ca²⁺-waves and purinergic modulation of neuronal activity and sleep homeostasis. The mechanisms underlying release of gliotransmitters remain uncertain, and exocytosis is the most intriguing and debated pathway. We investigated release of ATP from acutely dissociated cortical astrocytes using “sniff-cell” approach and demonstrated that release is vesicular in nature and can be triggered by elevation of intracellular Ca²⁺ via metabotropic and ionotropic receptors or direct UV-uncaging. The exocytosis of ATP from neocortical astrocytes occurred in the millisecond time scale contrasting with much slower nonvesicular release of gliotransmitters via Best1 and TREK-1 channels, reported recently in hippocampus. Furthermore, we discovered that elevation of cytosolic Ca²⁺ in cortical astrocytes triggered the release of ATP that directly activated quantal purinergic currents in the pyramidal neurons. The glia-driven burst of purinergic currents in neurons was followed by significant attenuation of both synaptic and tonic inhibition. The Ca²⁺-entry through the neuronal P2X purinoreceptors led to phosphorylation-dependent down-regulation of GABAA receptors. The negative purinergic modulation of postsynaptic GABA receptors was accompanied by small presynaptic enhancement of GABA release. Glia-driven purinergic modulation of inhibitory transmission was not observed in neurons when astrocytes expressed dn-SNARE to impair exocytosis. The astrocyte-driven purinergic currents and glia-driven modulation of GABA receptors were significantly reduced in the P2X4 KO mice. Our data provide a key evidence to support the physiological importance of exocytosis of ATP from astrocytes in the neocortex.     

Oxygen-glucose deprivation-induced enhancement of extracellular ATP-P2Y purinoceptors signaling for the propagation of astrocytic calcium waves

Waves of elevated intracellular free Ca²⁺ that propagate between neighboring astrocytes (Ca²⁺ waves) are important for the communication among astrocytes. We have previously revealed that focal photolysis of a caged calcium ionophore results in an increase in the concentration of intracellular Ca²⁺ in the target astrocytes, then the increase propagates to neighboring astrocytes through gap junctions. The extracellular ATP-purinoceptors signaling pathways are not primarily responsible for the propagation of the photolytic flash-induced Ca²⁺ waves. Here we examined whether and if so how the dynamics of Ca²⁺ waves changed after treatment with sublethal simulated ischemia; oxygen-glucose deprivation (OGD). OGD treatment increased the astrocytic expression of P2Y₁ and P2Y₂ receptors early during reperfusion, resulting in an increase in the propagating waves speed. In contrast, the expression of a gap junction protein was not changed significantly by the OGD suggesting that the extracellular ATP-P2Y receptors signaling pathways were preferentially enhanced after OGD. The present method to induce Ca²⁺ waves by focal photolysis of a caged calcium ionophore may provide a valuable tool with which to analyze glial Ca²⁺ waves under not only normal but also pathologic conditions.     

Extracellular Calcium Sensing by Glial Cells: Low Extracellular Calcium Induces Intracellular Calcium Release and Intercellular Signaling

Abstract: Glial cells in primary mixed cultures or purified astrocyte cultures from mouse cortex respond to reduced extracellular calcium concentration ([Ca²⁺]_e) with increases in intracellular calcium concentration ([Ca²⁺]_i) that include single-cell Ca²⁺ oscillations and propagated intercellular Ca²⁺ waves. The rate and pattern of propagation of low [Ca²⁺]_e-induced intercellular Ca²⁺ waves are altered by rapid perfusion of the extracellular medium, suggesting the involvement of an extracellular messenger in Ca²⁺ wave propagation. The low [Ca²⁺]_e-induced Ca²⁺ response is abolished by thapsigargin and by the phospholipase antagonist U73122. The low [Ca²⁺]_e-induced response is also blocked by replacement of extracellular Ca²⁺ with Ba²⁺, Zn²⁺, or Ni²⁺, and by 100 µM La³⁺. Glial cells in lowered [Ca²⁺]_e(0.1–0.5 mM) show an increased [Ca²⁺]_i response to bath application of ATP, whereas glial cells in increased [Ca²⁺]_e (10–15 mM) show a decreased [Ca²⁺]_i response to ATP. These results show that glial cells possess a mechanism for coupling between [Ca²⁺]_e and the release of Ca²⁺ from intracellular stores. This mechanism may be involved in glial responses to the extracellular environment and may be important in pathological conditions associated with low extracellular Ca²⁺ such as seizures or ischemia.     

Fluctuations in the Concentration of Extracellular ATP Synchronized with Intracellular Ca2+ Oscillatory Rhythm in Cultured Cardiac Myocytes

Isolated and cultured neonatal cardiac myocytes contract spontaneously and cyclically. The intracellular concentration of free Ca²⁺ also changes rhythmically in association with the rhythmic contraction of myocytes (Ca²⁺ oscillation). Both the contraction and Ca²⁺ oscillatory rhythms are synchronized among myocytes, and intercellular communication via gap junctions has been considered primarily responsible for the synchronization. However, a recent study has demonstrated that intercellular communication via extracellular ATP‐purinoceptor signaling is also involved in the intercellular synchronization of intracellular Ca²⁺ oscillation. In this study, we aim to elucidate whether the concentration of extracellular ATP changes cyclically and contributes to the intercellular synchronization of Ca²⁺ oscillation among myocytes. In almost all the cultured cardiac myocytes at four days in vitro (4 DIV), intracellular Ca²⁺ oscillations were synchronized with each other. The simultaneous measurement of the concentration of extracellular ATP and intracellular Ca²⁺ revealed the extracellular concentration of ATP actually oscillated concurrently with the intracellular Ca²⁺ oscillation. In addition, power spectrum and cross‐correlation analyses suggested that the treatment of cultured cardiac myocytes with suramin, a blocker of P2 purinoceptors, resulted in the asynchronization of Ca²⁺ oscillatory rhythms among cardiac myocytes. Treatment with suramin also resulted in a significant decrease in the amplitudes of the cyclic changes in both intracellular Ca²⁺ and extracellular ATP. Taken together, the present study demonstrated the possibility that the concentration of extracellular ATP changes cyclically in association with intracellular Ca²⁺, contributing to the intercellular synchronization of Ca²⁺ oscillation among cultured cardiac myocytes.     

Exocytosis in Astrocytes: Transmitter Release and Membrane Signal Regulation

Astrocytes, a type of glial cells in the brain, are eukaryotic cells, and a hallmark of these are subcellular organelles, such as secretory vesicles. In neurons vesicles play a key role in signaling. Upon a stimulus—an increase in cytosolic concentration of free Ca²⁺ ([Ca²⁺]_i)—the membrane of vesicle fuses with the presynaptic plasma membrane, allowing the exit of neurotransmitters into the extracellular space and their diffusion to the postsynaptic receptors. For decades it was thought that such vesicle-based mechanisms of gliotransmitter release were not present in astrocytes. However, in the last 30?years experimental evidence showed that astrocytes are endowed with mechanisms for vesicle- and non-vesicle-based gliotransmitter release mechanisms. The aim of this review is to focus on exocytosis, which may play a role in gliotransmission and also in other forms of cell-to-cell communication, such as the delivery of transporters, ion channels and antigen presenting molecules to the cell surface.     

Neurotransmitters and Integration in Neuronal-Astroglial Networks

Two major neural cell types, glia, astrocytes in particular, and neurones can release chemical transmitters that act as soluble signalling compounds for intercellular communication. Exocytosis, a process which depends on an increase in cytosolic Ca²⁺ levels, represents a common denominator for release of neurotransmitters, stored in secretory vesicles, from these neural cells. While neurones rely predominately on the immediate entry of Ca²⁺ from the extracellular space to the cytosol in this process, astrocytes support their cytosolic Ca²⁺ increases by appropriating this ion from the intracellular endoplasmic reticulum store and extracellular space. Additionally, astrocytes can release neurotransmitters using a variety of non-vesicular pathways which are mediated by an assortment of plasmalemmal channels and transporters. Once a neuronal and/or astrocytic neurotransmitter is released into the extracellular space, it can activate plasma membrane neurotransmitter receptors on neural cells, causing autocrine and/or paracrine signalling. Moreover, chemical transmission is essential not only for homocellular, but also for heterocellular bi-directional communication in the brain. Further detailed understanding of chemical transmission will aid our comprehension of the brain (dys)function in heath and disease.     

Simulation of Spontaneous Ca Oscillations in Astrocytes Mediated by Voltage-Gated Calcium Channels

The purpose of this computational study was to investigate the possible role of voltage-gated Ca²⁺ channels in spontaneous Ca²⁺ oscillations of astrocytes. By incorporating different types of voltage-gated Ca²⁺ channels and a previous model, this study reproduced typical Ca²⁺ oscillations in silico. Our model could mimic the oscillatory phenomenon under a wide range of experimental conditions, including resting membrane potential (−75 to −60 mV), extracellular Ca²⁺ concentration (0.1 to 1500 μM), temperature (20 to 37°C), and blocking specific Ca²⁺ channels. By varying the experimental conditions, the amplitude and duration of Ca²⁺ oscillations changed slightly (both <25%), while the frequency changed significantly (∼400%). This indicates that spontaneous Ca²⁺ oscillations in astrocytes might be an all-or-none process, which might be frequency-encoded in signaling. Moreover, the properties of Ca²⁺ oscillations were found to be related to the dynamics of Ca²⁺ influx, and not only to a constant influx. Therefore, calcium channels dynamics should be used in studying Ca²⁺ oscillations. This work provides a platform to explore the still unclear mechanism of spontaneous Ca²⁺ oscillations in astrocytes.     

Comparative analysis of optogenetic actuators in cultured astrocytes

Astrocytes modulate synaptic transmission via release of gliotransmitters such as ATP, glutamate, d-serine and l-lactate. One of the main problems when studying the role of astrocytes in vitro and in vivo is the lack of suitable tools for their selective activation. Optogenetic actuators can be used to manipulate astrocytic activity by expression of variants of channelrhodopsin-2 (ChR2) or other optogenetic actuators with the aim to initiate intracellular events such as intracellular Ca²⁺ ([Ca²⁺]_i) and/or cAMP increases. We have developed an array of adenoviral vectors (AVV) with ChR2-like actuators, including an enhanced ChR2 mutant (H134R), and a mutant with improved Ca²⁺ permeability (Ca²⁺ translocating channelrhodopsin, CatCh). We show here that [Ca²⁺]_i elevations evoked by ChR2(H134R) and CatCh in astrocytes are largely due to release of Ca²⁺ from the intracellular stores. The autocrine action of ATP which is released under these conditions and acts on the P2Y receptors also contributes to the [Ca²⁺]_i elevations. We also studied effects evoked using light-sensitive G-protein coupled receptors (opto-adrenoceptors). Activation of optoα1AR (Gq-coupled) and optoβ2AR (G_s-coupled) resulted in astrocytic [Ca²⁺]_i increases which were suppressed by blocking the corresponding intracellular signalling cascade (phospholipase C and adenylate cyclase, respectively). Interestingly, the bulk of [Ca²⁺]_i responses evoked using either optoAR was blocked by an ATP degrading enzyme, apyrase, or a P2Y1 receptor blocker, MRS 2179, indicating that they are to a large extent triggered by the autocrine action of ATP. We conclude that, whilst optimal tools for control of astrocytes are yet to be generated, the currently available optogenetic actuators successfully initiate biologically relevant signalling events in astrocytes.     

Voltage-gated calcium channel types in cultured C. elegans CEPsh glial cells

The four cephalic sensilla sheath (CEPsh) glial cells are important for development of the nervous system of Caenorhabditis elegans. Whether these invertebrate glia can generate intracellular Ca²⁺ increases, a hallmark of mammalian glial cell excitability, is not known. To address this issue, we developed a transgenic worm with the specific co-expression of genetically encoded red fluorescent protein and green Ca²⁺ sensor in CEPsh glial cells. This allowed us to identify CEPsh cells in culture and monitor their Ca²⁺ dynamics. We show that CEPsh glial cells, in response to depolarization, generate various intracellular Ca²⁺ increases mediated by voltage-gated Ca²⁺ channels (VGCCs). Using a pharmacological approach, we find that the L-type is the preponderant VGCC type mediating Ca²⁺ dynamics. Additionally, using a genetic approach we demonstrate that mutations in three known VGCC α₁-subunit genes, cca-1, egl-19 and unc-2, can affect Ca²⁺ dynamics of CEPsh glial cells. We suggest that VGCC-mediated Ca²⁺ dynamics in the CEPsh glial cells are complex and display heterogeneity. These findings will aid understanding of how CEPsh glial cells contribute to the operation of the C. elegans nervous system.     

Possible Involvement of Extracellular ATP-P2Y Purinoceptor Signaling in Ischemia-induced Tolerance of Astrocytes in Culture

Extracellular adenosine 5′-triphosphate (ATP) activates specific G protein-coupled purinoceptors (P2Y), and ATP-P2Y signaling pathways induces intracellular Ca²⁺ mobilization resulting in changes in the gene expression of a variety of proteins in astrocytes. This study investigated whether the exposure of cultured astrocytes to sublethal ischemia produced resistance to subsequent lethal ischemic stress, and if so, whether the extracellular ATP-P2Y signaling pathways were responsible for the tolerance. Ischemia-like insults, sublethal oxygen-glucose deprivation (sOGD), produced tolerance to subsequent lethal OGD stress in cultured astrocytes. Early during reperfusion after sOGD, the amount of extracellular ATP and the expression of both P2Y₁ and P2Y₂ receptors were increased, leading to enhanced activation of the extracellular ATP-P2Y signaling pathways. The occurrence of intracellular spontaneous Ca²⁺ oscillations was also increased. In addition, sOGD treatment enhanced the expression of the phosphorylated form of extracellular signal-regulated protein kinases 1 and 2 (p-ERK 1/2), and treatment with an inhibitor of ERK significantly attenuated the sOGD-induced ischemic tolerance of astrocytes.     

Propagation of Mechanically Induced Intercellular Calcium Waves via Gap Junctions and ATP Receptors in Rat Liver Epithelial Cells

Mechanical stimulation was used to initiate Ca²⁺waves in rat liver epithelial cells in order to ascertain the degree to which gap junctional intercellular communication (GJIC) is involved in communication of Ca²⁺to adjacent cells and to assess alternative Ca²⁺signaling pathways that may be present between these cells. In both WB-F344 cells, which show a high degree of GJIC, and WB-aB1 cells, which are GJIC deficient, mechanical stimulation of a single cell induced a Ca²⁺wave which propagated away from the point of stimulation, across cell borders, to neighboring cells directly or indirectly in contact with the stimulated cell. In addition, the Ca²⁺wave was transmitted to nearby isolated cells that exhibited no direct or indirect contact with the stimulated cell. Treatment of cells with 18β-glycyrrhetinic acid, a compound that has been shown to block GJIC, did not significantly affect propagation of the Ca²⁺wave. In contrast, treatment with suramin, a P₂-purinergic receptor inhibitor, significantly reduced both the rate and the extent of Ca²⁺wave propagation in WB-F344 cells and completely blocked its propagation in WB-aB1 cells. Cotreatment with suramin and glycyrrhetinic acid was found to completely block the mechanically induced Ca²⁺wave in both cell lines. These studies indicate that mechanically induced cell injury in rat liver epithelial cells initiates signaling through at least two pathways, involving intercellular communication via gap junctions and extracellular communication via ATP activation of purinergic receptors.     

Cross-Talk of the Receptors for Bradykinin, Serotonin, and ATP Shown by Single Cell Ca2+ Responses Indicating Different Modes of Ca2+ Activation in a Neuroblastoma × Glioma Hybrid Cell Line

Abstract: Modes of Ca²⁺ activation by bradykinin, serotonin, and ATP and the possible receptor cross-talk were investigated in mouse neuroblastoma × rat glioma hybrid cells (108CC15) by monitoring fura-2 fluorescence in single cells. A transient rise of cytosolic Ca²⁺ activity was induced by short pulses of the hormones. Brief exposure of cells to ionomycin, which depletes intracellular Ca²⁺ stores, reduced the size of subsequent responses to bradykinin or ATP, but not to serotonin. Superfusion of the cells with Ca²⁺-free medium abolished the Ca²⁺ response to serotonin, whereas the responses to bradykinin and to ATP were only slightly reduced. This indicates that ATP, like bradykinin, Induces the release of Ca²⁺ from intracellular stores. Serotonin, in contrast, activates Ca²⁺ entry from the extracellular space. To investigate whether ATP releases Ca²⁺ from the same stores as bradykinin, we examined the interaction of the hormones by applying them consecutively. When ATP was applied after bradykinin, the nucleotide did not evoke any response, irrespective of the presence or absence of extracellular Ca²⁺. The application of ATP before that of bradykinin reduced the size of a following bradykinin-induced Ca²⁺ response in Ca²⁺-free medium, but not in Ca²⁺-containing medium. This suggests that bradykinin may interact with the ATP-activated mechanism by cross-desensitization. Possibly, bradykinin receptors are coupled to additional Ca²⁺ stores not accessible to ATP that are refilled by extracellular Ca²⁺. Cyclic AMP and cyclic GMP apparently do not affect the Ca²⁺ responses to bradykinin and serotonin, as shown by the lack of influence of preincubation of the cells with forskolin or sodium nitroprusside.     

Mechanisms for L-channel-mediated increase in [Ca]i and its reduction by anti-bipolar drugs in cultured astrocytes combined with its mRNA expression in freshly isolated cells support the importance of astrocytic L-channels

The importance of Ca²⁺ signaling in astrocytes is undisputed but a potential role of Ca²⁺ influx via L-channels in the brain in vivo is disputed, although expression of these channels in cultured astrocytes is recognized. This study shows that an increase in free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in astrocytes in primary cultures in response to an increased extracellular K⁺ concentration (45 mM) is inhibited not only by nifedipine (confirming previous observations) but also to a very large extent by ryanodine, inhibiting ryanodine receptor-mediated release of Ca²⁺, known to occur in response to an elevation in [Ca²⁺]_i. This means that the actual influx of Ca²⁺ is modest, which may contribute to the difficulty in demonstrating L-channel-mediated Ca²⁺ currents in astrocytes in intact brain tissue. Chronic treatment with any of the 3 conventional anti-bipolar drugs lithium, carbamazepine or valproic acid similarly causes a pronounced inhibition of K⁺-mediated increase in [Ca²⁺]_i. This is shown to be due to an inhibition of capacitative Ca²⁺ influx, reflected by decreased mRNA and protein expression of the ‘transient receptor potential channel’ (TRPC1), a constituent of store-operated channels (SOCEs). Literature data are cited (i) showing that depolarization-mediated Ca²⁺ influx in response to an elevated extracellular K⁺ concentration is important for generation of Ca²⁺ oscillations and for the stimulatory effect of elevated K⁺ concentrations in intact, non-cultured brain tissue, and (ii) that Ca²⁺ channel activity is dependent upon availability of metabolic substrates, including glycogen. Finally, expression of mRNA for Ca_v1.3 is demonstrated in freshly separated astrocytes from normal brain.     

A Novel Subtype of Astrocytes Expressing TRPV4 (Transient Receptor Potential Vanilloid 4) Regulates Neuronal Excitability via Release of Gliotransmitters

Astrocytes play active roles in the regulation of synaptic transmission. Neuronal excitation can evoke Ca²⁺ transients in astrocytes, and these Ca²⁺ transients can modulate neuronal excitability. Although only a subset of astrocytes appears to communicate with neurons, the types of astrocytes that can regulate neuronal excitability are poorly characterized. We found that ∼30% of astrocytes in the brain express transient receptor potential vanilloid 4 (TRPV4), indicating that astrocytic subtypes can be classified on the basis of their expression patterns. When TRPV4⁺ astrocytes are activated by ligands such as arachidonic acid, the activation propagates to neighboring astrocytes through gap junctions and by ATP release from the TRPV4⁺ astrocytes. After activation, both TRPV4⁺ and TRPV4⁻ astrocytes release glutamate, which acts as an excitatory gliotransmitter to increase synaptic transmission through type 1 metabotropic glutamate receptor (mGluR). Our results indicate that TRPV4⁺ astrocytes constitute a novel subtype of the population and are solely responsible for initiating excitatory gliotransmitter release to enhance synaptic transmission. We propose that TRPV4⁺ astrocytes form a core of excitatory glial assembly in the brain and function to efficiently increase neuronal excitation in response to endogenous TRPV4 ligands.     

Astroglial excitability and gliotransmission: an appraisal of Ca2+ as a signalling route

Astroglial cells, due to their passive electrical properties, were long considered subservient to neurons and to merely provide the framework and metabolic support of the brain. Although astrocytes do play such structural and housekeeping roles in the brain, these glial cells also contribute to the brain''s computational power and behavioural output. These more active functions are endowed by the Ca²⁺-based excitability displayed by astrocytes. An increase in cytosolic Ca²⁺ levels in astrocytes can lead to the release of signalling molecules, a process termed gliotransmission, via the process of regulated exocytosis. Dynamic components of astrocytic exocytosis include the vesicular-plasma membrane secretory machinery, as well as the vesicular traffic, which is governed not only by general cytoskeletal elements but also by astrocyte-specific IFs (intermediate filaments). Gliotransmitters released into the ECS (extracellular space) can exert their actions on neighbouring neurons, to modulate synaptic transmission and plasticity, and to affect behaviour by modulating the sleep homoeostat. Besides these novel physiological roles, astrocytic Ca²⁺ dynamics, Ca²⁺-dependent gliotransmission and astrocyte–neuron signalling have been also implicated in brain disorders, such as epilepsy. The aim of this review is to highlight the newer findings concerning Ca²⁺ signalling in astrocytes and exocytotic gliotransmission. For this we report on Ca²⁺ sources and sinks that are necessary and sufficient for regulating the exocytotic release of gliotransmitters and discuss secretory machinery, secretory vesicles and vesicle mobility regulation. Finally, we consider the exocytotic gliotransmission in the modulation of synaptic transmission and plasticity, as well as the astrocytic contribution to sleep behaviour and epilepsy.     

Dopaminergic Neurotoxicants Cause Biphasic Inhibition of Purinergic Calcium Signaling in Astrocytes

Dopaminergic nuclei in the basal ganglia are highly sensitive to damage from oxidative stress, inflammation, and environmental neurotoxins. Disruption of adenosine triphosphate (ATP)-dependent calcium (Ca²⁺) transients in astrocytes may represent an important target of such stressors that contributes to neuronal injury by disrupting critical Ca²⁺-dependent trophic functions. We therefore postulated that plasma membrane cation channels might be a common site of inhibition by structurally distinct cationic neurotoxicants that could modulate ATP-induced Ca²⁺ signals in astrocytes. To test this, we examined the capacity of two dopaminergic neurotoxicants to alter ATP-dependent Ca²⁺ waves and transients in primary murine striatal astrocytes: MPP⁺, the active metabolite of 1-methyl 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and 6-hydroxydopamine (6-OHDA). Both compounds acutely decreased ATP-induced Ca²⁺ transients and waves in astrocytes and blocked OAG-induced Ca²⁺ influx at micromolar concentrations, suggesting the transient receptor potential channel, TRPC3, as an acute target. MPP⁺ inhibited 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced Ca²⁺ transients similarly to the TRPC3 antagonist, pyrazole-3, whereas 6-OHDA only partly suppressed OAG-induced transients. RNAi directed against TRPC3 inhibited the ATP-induced transient as well as entry of extracellular Ca²⁺, which was augmented by MPP⁺. Whole-cell patch clamp experiments in primary astrocytes and TRPC3-overexpressing cells demonstrated that acute application of MPP⁺ completely blocked OAG-induced TRPC3 currents, whereas 6-OHDA only partially inhibited OAG currents. These findings indicate that MPP⁺ and 6-OHDA inhibit ATP-induced Ca²⁺ signals in astrocytes in part by interfering with purinergic receptor mediated activation of TRPC3, suggesting a novel pathway in glia that could contribute to neurotoxic injury.