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.     

Mechanisms of intercellular calcium signaling in glial cells studied with dantrolene and thapsigargin

Mechanical stimulation of a single cell in a primary mixed glial cell culture induced a wave of increased intracellular calcium concentration ([Ca²⁺]_i) that was communicated to surrounding cells. Following propagation of the Ca²⁺ wave, many cells showed asynchronous oscillations in [Ca²⁺]_i. Dantrolene sodium (10 μM) inhibited the increase in [Ca²⁺]_i associated with this Ca²⁺ wave by 60-80%, and prevented subsequent Ca²⁺ oscillations. Despite the markedly decreased magnitude of the increase in [Ca²⁺]_i, the rate of propagation and the extent of communication of the Ca²⁺ wave were similar to those prior to the addition of dantrolene. Thapsigargin (10 nM to 1 μM) induced an initial increase in [Ca²⁺]_i ranging from 100 nM to 500 nM in all cells that was followed by a recovery of [Ca²⁺]_i to near resting levels in most cells. Transient exposure to thapsigargin for 2 min irreversibly blocked communication of a Ca²⁺ wave from the stimulated cell to adjacent cells. Glutamate (50 μM) induced an initial increase in [Ca²⁺]_i in most cells that was followed by sustained oscillations in [Ca²⁺]_i in some cells. Dantrolene (10 μM) inhibited this initial [Ca²⁺]_i increase caused by glutamate by 65-90% and abolished subsequent oscillations. Thapsigargin (10 nM to 1 μm) abolished the response to glutamate in over 99% of cells. These results suggest that while both dantrolene and thapsigargin inhibit intracellular Ca²⁺ release, only thapsigargin affects the mechanism that mediates intercellular communication of Ca²⁺ waves. These findings are consistent with the hypothesis that inositol trisphosphate (IP₃) mediates the propagation of Ca²⁺ waves whereas Ca²⁺ -induced Ca²⁺ release amplifies Ca²⁺ waves and generates subsequent Ca²⁺ oscillations.     

Plasma albumin induces calcium waves in rat cortical astrocytes

Changes in intracellular calcium were monitored in cultured cortical astrocytes stimulated with albumin. Albumin elicited intracellular calcium mobilisation from intracellular stores, inducing repetitive intracellular calcium oscillations. The oscillations were not blocked by ryanodine, a blocker of the Ca-induced Ca release mechanism, and the release occurred from the same store as is accessed by glutamate and bradykinin, both of which release calcium by an IP₃-dependent mechanism. Calcium signals induced by albumin appear therefore to occur via a pure IP₃-dependent mechanism. When albumin was applied to confluent monolayers of astrocytes, the oscillations in individual cells were initially unsynchronised, but after several minutes of application, the Ca²⁺ oscillations were observed to synchronise and spread through the astrocyte network as a wave. These intercellular calcium waves were inhibited by the gap junction blocker halothane. Using the fluorescence recovery after photobleaching (FRAP) technique, we demonstrate that the development of propagated waves with prolonged exposure to albumin does not result from an increase in cell coupling. The development of calcium waves on exposure to albumin may be important in the formation of glial scars in the CNS after breakdown of the blood-brain barrier. GLIA 19:343–351, 1997. © 1997 Wiley-Liss, Inc.     

Inositol 1,4,5-trisphosphate activates non-selective cation conductance via intracellular Ca2+ increase in isolated frog taste cells

The effect of intracellular Ca²⁺ increase was analysed in isolated frog taste cells under the whole-cell patch clamp. External application of a Ca²⁺-ionophore, ionomycin (3 μm ) induced the sustained inward current of ?200 ± 17 pA (mean ± SE, n = 23) at – 50 mV in taste cells. The ionomycin-induced response was observed in most of the cells exposed in the drug, but not when 10 mm BAPTA (1,2-bis (o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) was included in the pipette (eight cells). Steady-state I–V relationships of ionomycin-induced currents were almost linear and reversed at – 8 ± 1 mV (n = 23). The simultaneous removal of Na⁺ and Ca²⁺ from the external solution eliminated the response completely (three cells). Intracellular dialysis with 1 mm Ca²⁺ or 50 μm inositol 1,4,5-trisphosphate (IP₃) in K⁺-internal solution also induced an inward current in the taste cells. The Ca²⁺-induced and IP₃-induced responses were observed in 82% and 36% of the cells dialysed with the drugs, respectively. The Ca²⁺-induced and IP₃-induced currents were inhibited by external Cd²⁺ (1–2 mm ). The reversal potentials of the inward currents were – 15 ± 3 mV (n = 9) in Ca²⁺ dialysis and – 11 ± 3 mV (n = 13) in IP₃ dialysis. The half-maximal Ca²⁺ concentration in the pipette to induce the inward current was ≈ 170 μm . The results suggest that IP₃ can depolarize the taste cell with mediation by intracellular Ca²⁺.     

Capacitative Ca2+ influx in glial cells is inhibited by glycolytic inhibitors

In non-excitable cells, stimulation of phosphoinositide (PI) turnover and inhibition of the endoplasmic reticulum (ER) Ca²⁺-ATPase are methods commonly used to deplete calcium stores, resulting in a capacitative Ca²⁺ influx (i.e., Ca²⁺ release-activated Ca²⁺ influx). Since this Ca²⁺ influx in glial cells has not been thoroughly investigated, we have used C₆ glioma cells as a glial cell model to study this phenomenon. On adding cyclopiazonic acid (CPA) or thapsigargin (TG) (two ER Ca²⁺-ATPase inhibitors) in Ca²⁺-free medium, only a small transient increase in intracellular Ca²⁺ was seen. After depletion of the stored Ca²⁺, a marked Ca²⁺ influx, followed by a prolonged plateau, was seen on re-addition of extracellular Ca²⁺ ions (2 mM), i.e., capacitative Ca²⁺ influx. A similar effect was seen on adding ATP, known to deplete the inositol triphosphate (IP₃)-sensitive Ca²⁺ store in C₆ cells. After various degrees of store depletion, the amplitude of the capacitative Ca²⁺ influx was found to be highly dependent on the amount of Ca²⁺ remaining in the store. This Ca²⁺ influx was markedly inhibited by (1) La³⁺ and Ni²⁺, (2) SK&F 96365, econazole, and miconazole, and (3) membrane depolarization, clearly showing that this Ca²⁺ influx after store depletion in C₆ cells is a capacitative mechanism. Interestingly, the capacitative Ca²⁺ influx can be inhibited by a reduction in intracellular ATP (ATP_i) levels in glial cells. The role of ATP_i in the capacitative Ca²⁺ influx is discussed. GLIA 21:315–326, 1997. © 1997 Wiley-Liss, Inc.     

Apoptosis induced by Aβ25–35 peptide is Ca2+‐IP3 signaling‐dependent in murine astrocytes

Although the accumulation of the neurotoxic peptide β‐amyloid (Aβ) in the central nervous system is a hallmark of Alzheimer's disease, whether Aβ acts in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that cytosolic Ca²⁺ dysregulation, induced by a neurotoxic fragment (Aβ25–35), caused apoptosis in a concentration‐dependent manner, leading to cytoplasmic Ca²⁺ mobilization from extra‐ and intracellular sources, mainly from the endoplasmic reticulum (ER) via IP₃ receptor activation. This mechanism was related to Aβ‐mediated apoptosis by the intrinsic pathway because the expression of pro‐apoptotic Bax was accompanied by its translocation in cells transfected with GFP‐Bax. Aβ‐mediated apoptosis was reduced by BAPTA‐AM, a fast Ca²⁺ chelator, indicating that an increase in intracellular Ca²⁺ was involved in cell death. Interestingly, the Bax translocation was dependent on Ca²⁺ mobilization from IP₃ receptors because pre‐incubation with xestospongin C, a selective IP₃ receptor inhibitor, abolished this response. Taken together, these results provide evidence that Aβ dysregulation of Ca²⁺ homeostasis induces ER depletion of Ca²⁺ stores and leads to apoptosis; this mechanism plays a significant role in Aβ apoptotic cell death and might be a new target for neurodegeneration treatments.     

Astrocytic IP3Rs: Contribution to Ca2+ signalling and hippocampal LTP

Astrocytes regulate hippocampal synaptic plasticity by the Ca²⁺ dependent release of the N‐methyl d ‐aspartate receptor (NMDAR) co‐agonist d ‐serine. Previous evidence indicated that d ‐serine release would be regulated by the intracellular Ca²⁺ release channel IP₃ receptor (IP₃R), however, genetic deletion of IP₃R2, the putative astrocytic IP₃R subtype, had no impact on synaptic plasticity or transmission. Although IP₃R2 is widely believed to be the only functional IP₃R in astrocytes, three IP₃R subtypes (1, 2, and 3) have been identified in vertebrates. Therefore, to better understand gliotransmission, we investigated the functionality of IP₃R and the contribution of the three IP₃R subtypes to Ca²⁺ signalling. As a proxy for gliotransmission, we found that long‐term potentiation (LTP) was impaired by dialyzing astrocytes with the broad IP₃R blocker heparin, and rescued by exogenous d ‐serine, indicating that astrocytic IP₃Rs regulate d ‐serine release. To explore which IP₃R subtypes are functional in astrocytes, we used pharmacology and two‐photon Ca²⁺ imaging of hippocampal slices from transgenic mice (IP₃R2^?/? and IP₃R2^?/?;3^?/?). This approach revealed that underneath IP₃R2‐mediated global Ca²⁺ events are an overlooked class of IP₃R‐mediated local events, occurring in astroglial processes. Notably, multiple IP₃Rs were recruited by high frequency stimulation of the Schaffer collaterals, a classical LTP induction protocol. Together, these findings show the dependence of LTP and gliotransmission on Ca²⁺ release by astrocytic IP₃Rs. GLIA 2017;65:502–513     

The inhibitory neurotransmitter GABA evokes long‐lasting Ca2+ oscillations in cortical astrocytes

Studies over the last decade provided evidence that in a dynamic interaction with neurons glial cell astrocytes contribut to fundamental phenomena in the brain. Most of the knowledge on this derives, however, from studies monitoring the astrocyte Ca²⁺ response to glutamate. Whether astrocytes can similarly respond to other neurotransmitters, including the inhibitory neurotransmitter GABA, is relatively unexplored. By using confocal and two photon laser‐scanning microscopy the astrocyte response to GABA in the mouse somatosensory and temporal cortex was studied. In slices from developing (P15‐20) and adult (P30‐60) mice, it was found that in a subpopulation of astrocytes GABA evoked somatic Ca²⁺ oscillations. This response was mediated by GABA_B receptors and involved both G_i/o protein and inositol 1,4,5‐trisphosphate (IP₃) signalling pathways. In vivo experiments from young adult mice, revealed that also cortical astrocytes in the living brain exibit GABA_B receptor‐mediated Ca²⁺ elevations. At all astrocytic processes tested, local GABA or Baclofen brief applications induced long‐lasting Ca²⁺ oscillations, suggesting that all astrocytes have the potential to respond to GABA. Finally, in patch‐clamp recordings it was found that Ca²⁺ oscillations induced by Baclofen evoked astrocytic glutamate release and slow inward currents (SICs) in pyramidal cells from wild type but not IP₃R2^?/? mice, in which astrocytic GABA_B receptor‐mediated Ca²⁺ elevations are impaired. These data suggest that cortical astrocytes in the mouse brain can sense the activity of GABAergic interneurons and through their specific recruitment contribut to the distinct role played on the cortical network by the different subsets of GABAergic interneurons. GLIA 2016;64:363–373     

Characterization of ryanodine receptors in oligodendrocytes,type 2 astrocytes,and O-2A progenitors

In this study we have investigated the expression of ryanodine receptors (RyRs), and the ability of caffeine to evoke RyR-mediated elevation of intracellular Ca²⁺ levels ([Ca²⁺]_i) in glial cells of the oligodendrocyte/type 2 astrocyte lineage. Immunocytochemistry with specific antibodies identified ryanodine receptors in cultured oligodendrocytes, type 2 astrocytes, and O-2A progenitor cells, at high levels in the perinuclear region and in a variegated pattern along processes. Glia acutely isolated from rat brain and in aldehyde-fixed sections of cortex were similarly found to express RyRs. Caffeine (5–50 mM) caused an increase in [Ca²⁺]_i in most cultured type 2 astrocytes and in 50% of oligodendrocytes. Responses elicited by caffeine were inhibited by pretreatment with ryanodine (10 μM) or thapsigargin (1 μM), and the peak response was unaffected by removal of [Ca²⁺]_o. O-2A progenitor cells, in contrast, were largely unresponsive to caffeine treatment. Pretreatment with kainate (200 μM) to activate Ca²⁺ entry increased the magnitude of caffeine-evoked [Ca²⁺]_i elevations in type 2 astrocytes and oligodendrocytes, and caused caffeine to activate responses in a significant proportion of previously non-responding O-2A progenitors. In both type 2 astrocytes and oligodendrocytes, caffeine evoked Ca²⁺ changes which propagated as wavefronts from several initiation sites. These wave amplification sites were characterized by significantly higher local Ca²⁺ release kinetics. Our results indicate that several glial cell types express RyRs, and that their functionality differs within different cell types of the oligodendrocyte lineage. In addition, ionotropic glutamate receptor activation fills the caffeine-sensitive Ca²⁺ stores in these cells. J. Neurosci. Res. 52:468–482, 1998. © 1998 Wiley-Liss, Inc.     

Modulation of two functionally distinct Ca2+ stores in astrocytes: Role of the plasmalemmal Na/Ca exchanger

Mechanisms that regulate the amount of releasable Ca²⁺ in intracellular stores of cultured mouse astrocytes were investigated using digital imaging of fura-2 loaded cells. At rest, the cytoplasmic Ca²⁺ concentration, [Ca²⁺]_cyt, was about 110 nM. In the absence of extracellular Ca²⁺, cyclopiazonic acid (CPA), an inhibitor of the endoplasmic reticulum (ER) Ca²⁺-ATPase, induced a transient, four-fold increase in [Ca²⁺]_cyt due to the release of Ca²⁺ from inositol triphosphate (IP₃) sensitive stores. Caffeine (CAF), which releases Ca²⁺ from Ca²⁺-sensitive stores, induced a two-fold increase in [Ca²⁺]_cyt. The CPA- and CAF-sensitive stores could be released independently. Changes in the amplitudes of the Ca²⁺ transients were taken as a measure of changes in store content. Removal of extracellular Na⁺ or addition of ouabain, which inhibit Ca²⁺ extrusion and promote Ca²⁺ entry across the plasmalemma via the Na/Ca exchanger, caused minimal increases in resting [Ca²⁺]_cyt but greatly potentiated both CPA- and CAF-induced Ca²⁺ transients. The amount of Ca²⁺ releasable from the IP₃ (CPA) sensitive store was directly proportional to cytosolic Na⁺ concentration (i.e., inversely proportional to the transmembrane Na⁺ electrochemical gradient). Under these reduced Na⁺ gradient conditions, little, if any, Ca²⁺ destined for the ER stores enters the cells through voltage-dependent Ca²⁺ channels. These results demonstrate that mouse astrocytes contain two distinct ER Ca²⁺ stores, the larger, IP₃- (CPA-) sensitive, and the smaller, Ca²⁺- (CAF-) sensitive. The Ca²⁺ content of both ER stores can be regulated by the Na/Ca exchanger. Thus, the magnitude of cellular responses to signals that are mediated by Ca²⁺ release induced by the two second messengers, IP₃ and Ca²⁺, can be modulated by factors that affect the net transport of Ca²⁺ across the plasmalemma. © 1996 Wiley-Liss, Inc.     

Inositol 1,4,5-trisphosphate receptor type 2-independent Ca2+ release from the endoplasmic reticulum in astrocytes

Accumulating evidence indicates that astrocytes are actively involved in the physiological and pathophysiological functions of the brain. Intracellular Ca²⁺ signaling, especially Ca²⁺ release from the endoplasmic reticulum (ER), is considered to be crucial for the regulation of astrocytic functions. Mice with genetic deletion of inositol 1,4,5-trisphosphate receptor type 2 (IP₃R2) are reportedly devoid of astrocytic Ca²⁺ signaling, and thus widely used to explore the roles of Ca²⁺ signaling in astrocytic functions. While functional deficits in IP₃R2-knockout (KO) mice have been found in some reports, no functional deficit was observed in others. Thus, there remains a controversy regarding the functional significance of astrocytic Ca²⁺ signaling. To address this controversy, we re-evaluated the assumption that Ca²⁺ release from the ER is abolished in IP₃R2-KO astrocytes using a highly sensitive imaging technique. We expressed the ER luminal Ca²⁺ indicator G-CEPIA1er in cortical and hippocampal astrocytes to directly visualize spontaneous and stimulus-induced Ca²⁺ release from the ER. We found attenuated but significant Ca²⁺ release in response to application of norepinephrine to IP₃R2-KO astrocytes. This IP₃R2-independent Ca²⁺ release induced only minimal cytosolic Ca²⁺ transients but induced robust Ca²⁺ increases in mitochondria that are frequently in close contact with the ER. These results indicate that ER Ca²⁺ release is retained and is sufficient to increase the Ca²⁺ concentration in close proximity to the ER in IP₃R2-KO astrocytes.     

IP3 mobilization and diffusion determine the timing window of Ca2+ release by synaptic stimulation and a spike in rat CA1 pyramidal cells

Synaptically activated calcium release from internal stores in CA1 pyramidal neurons is generated via metabotropic glutamate receptors by mobilizing IP₃. Ca²⁺ release spreads as a large amplitude wave in a restricted region of the apical dendrites of these cells. These Ca²⁺ waves have been shown to induce certain forms of synaptic potentiation and have been hypothesized to affect other forms of plasticity. Pairing a single backpropagating action potential (bAP) with repetitive synaptic stimulation evokes Ca²⁺ release when synaptic stimulation alone is subthreshold for generating release. We examined the timing window for this synergistic effect under conditions favoring Ca²⁺ release. The window, measured from the end of the train, lasted 250–500 ms depending on the duration of stimulation tetanus. The window appears to correspond to the time when both IP₃ concentration and [Ca²⁺]_i are elevated at the site of the IP₃ receptor. Detailed analysis of the mechanisms determining the duration of the window, including experiments using different forms of caged IP₃ instead of synaptic stimulation, suggest that the most significant processes are the time for IP₃ to diffuse away from the site of generation and the time course of IP₃ production initiated by activation of mGluRs. IP₃ breakdown, desensitization of the IP₃ receptor, and the kinetics of IP₃ unbinding from the receptor may affect the duration of the window but are less significant. The timing window is short but does not appear to be short enough to suggest that this form of coincidence detection contributes to conventional spike timing‐dependent synaptic plasticity in these cells. © 2009 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.     

Muscarinic and purinergic Ca mobilizations in the neural retina of early embryonic chick

Acetylcholine and adenosine triphosphate (ATP) raise intracellular Ca²⁺ concentration via muscarinic receptors and P_2U purinoceptors by releasing Ca²⁺ from intracellular Ca²⁺ stores in the neural retina of early embryonic chick. The signal transduction mechanisms for the muscarinic and purinergic Ca²⁺ responses with fura-2 fluorescence measurements were studied. Li⁺ (1 mM), which inhibits phosphatidylinositol metabolism, enhanced both the Ca²⁺ rises to carbamylcholine (CCh, 30 μM), a muscarinic agonist, and ATP (200 μM). Thapsigargin (250 nM), an inhibitor of Ca²⁺-ATPase of inositol trisphosphate (IP₃)-sensitive Ca²⁺ stores, abolished both the Ca²⁺ rises to CCh (100 μM) and ATP (500 μM). U-73122 (2 μM), an inhibitor of phospholipase C_b, suppressed the Ca²⁺ rise to ATP (500 μM), but its analog U-73343 (2 μM) did not suppress it. In contrast, both U-73122 and U-73343 suppressed the Ca²⁺ rise to CCh (100 μM). Pertussis toxin (250 ng/ml) suppressed the ATP-induced Ca²⁺ rise at least partly, whereas no inhibition was observed on the CCh-induced Ca²⁺ rise. Cross-talk occurred between the muscarinic and purinergic Ca²⁺ mobilizations but they were not occlusive. This study suggests that the muscarinic and purinergic Ca²⁺ mobilizations utilize IP₃-sensitive Ca²⁺ stores, but different signal transduction pathways are involved in between the muscarinic and purinergic Ca²⁺ responses.     

Ca2+ transients in astrocyte fine processes occur via Ca2+ influx in the adult mouse hippocampus

Astrocytes display complex morphologies with an array of fine extensions extending from the soma and the primary thick processes. Until the use of genetically encoded calcium indicators (GECIs) selectively expressed in astrocytes, Ca²⁺ signaling was only examined in soma and thick primary processes of astrocytes where Ca²⁺‐sensitive fluorescent dyes could be imaged. GECI imaging in astrocytes revealed a previously unsuspected pattern of spontaneous Ca²⁺ transients in fine processes that has not been observed without chronic expression of GECIs, raising potential concerns about the effects of GECI expression. Here, we perform two‐photon imaging of Ca²⁺ transients in adult CA1 hippocampal astrocytes using a new single‐cell patch‐loading strategy to image Ca²⁺‐sensitive fluorescent dyes in the cytoplasm of fine processes. We observed that astrocyte fine processes exhibited a high frequency of spontaneous Ca²⁺ transients whereas astrocyte soma rarely showed spontaneous Ca²⁺ oscillations similar to previous reports using GECIs. We exploited this new approach to show these signals were independent of neuronal spiking, metabotropic glutamate receptor (mGluR) activity, TRPA1 channels, and L‐ or T‐type voltage‐gated calcium channels. Removal of extracellular Ca²⁺ almost completely and reversibly abolished the spontaneous signals while IP₃R2 KO mice also exhibited spontaneous and compartmentalized signals, suggesting they rely on influx of extracellular Ca²⁺. The Ca²⁺ influx dependency of the spontaneous signals in patch‐loaded astrocytes was also observed in astrocytes expressing GCaMP3, further highlighting the presence of Ca²⁺ influx pathways in astrocytes. The mechanisms underlying these localized Ca²⁺ signals are critical for understanding how astrocytes regulate important functions in the adult brain. GLIA 2016;64:2093–2103     

A muscarinic receptor agonist mobilizes Ca from caffeine and inositol-1,4,5-trisphosphate-sensitive Ca stores in cat adrenal chromaffin cells

To gain some understanding of the characteristics of intracellular Ca²⁺ stores of cat adrenal chromaffin cells, we investigated the effects of ryanodine, a blocker of Ca²⁺-induced Ca²⁺ release channels in muscle, on both cytosolic Ca²⁺ concentration and catecholamine secretion induced by caffeine or methacholine. The results suggest that Ca²⁺ stores consist of at least two compartments, one which is sensitive to both caffeine and inositol-1,4,5-trisphosphate (IP₃), and the other which is sensitive to IP₃ alone.     

Bacterial endotoxin induces [Ca2+]i transients and changes the organization of actin in microglia

We have employed amoeboid microglia purified from primary cultures of neonatal rat brain to examine the effect of bacterial lipopolysaccharide (LPS), a potent activator of immune cells, on intracellular calcium concentration ([Ca²⁺]i) in brain macrophages. In single brain macrophages loaded with indo 1, pulse administration of LPS elicited a rapid and transient increase in [Ca²⁺]i From a total of 70 cells examined, all responded to LPS with a similar [Ca²⁺]i transient, indicating a good homogeneity of the cell population with regard to the LPS response. It was concluded that the rise of cytosolic [Ca²⁺]i originated from intracellular stores because the response to LPS occured similarly in the presence or in the absence of extracellular Ca²⁺. A second administration of LPS to the same cells resulted in a second but reduced [Ca²⁺]i transient. In contrast to the first response to LPS, this second response was totally dependent on the presence of Ca²⁺ in the extracellular medium. The first response to LPS was strongly inhibited by ruthenium red and could be suppressed in a reversible manner by pre incubating the cells with caffeine in the absence of Ca²⁺ in the extracellular medium. These results indicate that caffeinesensitive intracellular Ca²⁺ stores may be the major source of Ca²⁺ in the response of brain macrophages to LPS. The possible release of Ca²⁺ from phosphatidylinositol(3,4,5)-trisphosphate (IP₃)-sensitive stores in brain macrophages was also evaluated by stimulating cells with the IP₃-mobilizing agonist histamine. Brain macrophages were heterogeneous with regard to the histamine response since histamine induced a [Ca²⁺]i rise in only 30% of cells examined. The increase in [Ca²⁺]i triggered by LPS may in turn activate several intracellular events involved in the transition from one microglial functional state to another. We examined the effect of LPS on the state of organization of actin, which is an essential component of chemoattractant signal transduction. Immunofluorescence staining with anti-actin antibody which recognizes actin in both filamentous and nonfilamentous configurations, indicated that LPS produced drastic changes in the actin organization. LPS-treated cells appeared more intensely fluorescent than untreated cells due to the concentration of diffuse fluorescence near the center of the cell. In addition, prominent fluorescent dots were present in the subplasmalemmal region in cells stimulated with LPS. This specific LPS-induced reorganization of actin was also observed in cells preincubated in the external medium without Ca²⁺. Thus, it is likely that the LPS-induced mobilization of Ca²⁺ from intracellular stores may be responsible for the changes observed in the actin organization. © 1994 Wiley-Liss, Inc.     

Leukocyte opioid receptors mediate analgesia via Ca2+-regulated release of opioid peptides

Opioids are the most powerful analgesics. As pain is driven by sensory transmission and opioid receptors couple to inhibitory G proteins, according to the classical concept, opioids alleviate pain by activating receptors on neurons and blocking the release of excitatory mediators (e.g., substance P). Here we show that analgesia can be mediated by opioid receptors in immune cells. We propose that activation of leukocyte opioid receptors leads to the secretion of opioid peptides Met-enkephalin, β-endorphin and dynorphin A (1–17), which subsequently act at local neuronal receptors, to relieve pain. In a mouse model of neuropathic pain induced by a chronic constriction injury of the sciatic nerve, exogenous agonists of δ-, μ- and κ-opioid receptors injected at the damaged nerve infiltrated by opioid peptide- and receptor-expressing leukocytes, produced analgesia, as assessed with von Frey filaments. The analgesia was attenuated by pharmacological or genetic inactivation of opioid peptides, and by leukocyte depletion. This decrease in analgesia was restored by the transfer of wild-type, but not opioid receptor-lacking leukocytes. Ex vivo, exogenous opioids triggered secretion of opioid peptides from wild-type immune cells isolated from damaged nerves, which was diminished by blockade of Gαi/o or Gβγ (but not Gαs) proteins, by chelator of intracellular (but not extracellular) Ca²⁺, by blockers of phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP₃) receptors, and was partially attenuated by protein kinase C inhibitor. Similarly, the leukocyte depletion-induced decrease in exogenous opioid analgesia was re-established by transfer of immune cells ex vivo pretreated with extracellular Ca²⁺ chelator, but was unaltered by leukocytes pretreated with intracellular Ca²⁺ chelator or blockers of Gαi/o and Gβγ proteins. Thus, both ex vivo opioid peptide release and in vivo analgesia were mediated by leukocyte opioid receptors coupled to the Gαi/o–Gβγ protein–PLC–IP₃ receptors–intracellular Ca²⁺ pathway. Our findings suggest that opioid receptors in immune cells are important targets for the control of pathological pain.     

Effect of dantrolene on KCl- or NMDA-induced intracellular Ca2+ changes and spontaneous Ca2+ oscillation in cultured rat frontal cortical neurons

Summary Dantrolene has been known to affect intracellular Ca²⁺ concentration ([Ca²⁺]_i) by inhibiting Ca²⁺ release from intracellular stores in cultured neurons. We were interested in examining this property of dantrolene in influencing the [Ca²⁺]_i affected by the NMDA receptor ligands, KCl, L-type Ca²⁺ channel blocker nifedipine, and two other intracellular Ca²⁺-mobilizing agents caffeine and bradykinin. Effect of dantrolene on the spontaneous oscillation of [Ca²⁺]_i was also examined. Dantrolene in M concentrations dose-dependently inhibited the increase in [Ca²⁺]_i elicited by NMDA and KCl. AP-5, MK-801 (NMDA antagonists), and nifedipine respectively reduced the NMDA and KCl-induced increase in [Ca²⁺]_i. Dantrolene, added to the buffer solution together with the antagonists or nifedipine, caused a further reduction in [Ca²⁺]_i to a degree similar to that seen with dantrolene alone inhibiting the increase in [Ca₂₊]_i caused by NMDA or KCl. At 30 M, dantrolene partially inhibited caffeine-induced increase in [Ca²⁺]_i whereas it has no effect on the bradykinin-induced change in [Ca²⁺]_i. The spontaneous oscillation of [Ca²⁺]_i in frontal cortical neurons was reduced both in amplitude and in base line concentration in the presence of 10 M dantrolene. Our results indicate that dantrolene's mobilizing effects on intracellular Ca²⁺ stores operate independently from the influxed Ca²⁺ and that a component of the apparent increase in [Ca²⁺]_i elicited by NMDA or KCl represents a dantrolene-sensitive Ca₂₊ release from intracellular stores. Results also suggest that dantrolene does not affect the IP₃-gated release of intracellular Ca²⁺ and that the spontaneous Ca²⁺ oscillation is, at least partially, under the control of Ca²⁺ mobilization from internal stores.Abbreviations AP-5 (±)-2-amino-5-phosphonopentanoic acid - AMPA amino-3-hydroxy-5-methyl-isoxazole-4-propionate - BSS balanced salt solution - CNS central nervous system - CICR Ca²⁺-induced Ca²⁺ release - DCKA 5,7-dichlorokynurenate - DNasel deoxyribonuclease I - DMEM Dulbecco's Modified Eagle's Medium - EGTA ethylene glycol-bis(-aminoethyl ether)N,N,N,N,-tetraacetic acid - FCS fetal calf serum - fura-2-AM 1-(2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy-2-ethane-N,N,N,N-te-traacetic acid, pentaacetoxymethyl ester - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulfonic acid] - [Ca ²⁺] _i intracellular free Ca²⁺ concentration - LTP long-term potantiation - MK-801 (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]-cyclohepten-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate