Modulation of intercellular calcium signaling in astrocytes by extracellular calcium and magnesium

The extracellular concentrations of Ca(2+) and Mg(2+) are well known to play important roles in the function of the central nervous system. We examined the effects of extracellular Ca(2+) and Mg(2+) on ATP release and intercellular signaling in astrocytes. The extent of propagation of intercellular Ca(2+) waves evoked by mechanical stimulation was increased by reduction of extracellular Ca(2+) ([Ca(2+)](o)) or Mg(2+) concentration ([Mg(2+)](o)) and was decreased by elevated [Mg(2+)](o). Reduction of extracellular Ca(2+) concentration ([Ca(2+)](o)) evokes intercellular Ca(2+) signaling in astrocytes; a similar effect was observed in response to change from 5 mM [Mg(2+)](o) to 0 [Mg(2+)](o). Release of low-molecular-weight dyes and ATP was also activated by low [Ca(2+)](o) or [Mg(2+)](o) and inhibited by high [Ca(2+)](o) or [Mg(2+)](o). Astrocytes showed low [Ca(2+)](o)-activated whole cell currents consistent with currents through connexin hemichannels. These currents were inhibited by extracellular Mg(2+). We conclude that extracellular divalent cations modulate intercellular Ca(2+) signaling in astrocytes by modulating the release of ATP, possibly via connexin hemichannels.     

Gap junctions equalize intracellular Na+ concentration in astrocytes

Gap junctions between glial cells allow intercellular exchange of ions and small molecules. We have investigated the influence of gap junction coupling on regulation of intracellular Na⁺ concentration ([Na⁺]_i) in cultured rat hippocampal astrocytes, using fluorescence ratio imaging with the Na⁺ indicator dye SBFI (sodium-binding benzofuran isophthalate). The [Na⁺]_i in neighboring astrocytes was very similar (12.0 ± 3.3 mM) and did not fluctuate under resting conditions. During uncoupling of gap junctions with octanol (0.5 mM), baseline [Na⁺]_i was unaltered in 24%, increased in 54%, and decreased in 22% of cells. Qualitatively similar results were obtained with two other uncoupling agents, heptanol and α-glycyrrhetinic acid (AGA). Octanol did not alter the recovery from intracellular Na⁺ load induced by removal of extracellular K⁺, indicating that octanol's effects on baseline [Na⁺]_i were not due to inhibition of Na⁺, K⁺-ATPase activity. Under control conditions, increasing [K⁺]_o from 3 to 8 mM caused similar decreases in [Na⁺]_i in groups of astrocytes, presumably by stimulating Na⁺, K⁺-ATPase. During octanol application, [K⁺]_o-induced [Na⁺]_i decreases were amplified in cells with increased baseline [Na⁺]_i, and reduced in cells with decreased baseline [Na⁺]_i. This suggests that baseline [Na⁺]_i in astrocytes “sets” the responsiveness of Na⁺, K⁺-ATPase to increases in [K⁺]_o. Our results indicate that individual hippocampal astrocytes in culture rapidly develop different levels of baseline [Na⁺]_i when they are isolated from one another by uncoupling agents. In astrocytes, therefore, an apparent function of coupling is the intercellular exchange of Na⁺ ions to equalize baseline [Na⁺]_i, which serves to coordinate physiological responses that depend on the intracellular concentration of this ion. GLIA 20:299–307, 1997. © 1997 Wiley-Liss, Inc.     

缝隙连接在癫痫中的作用(英文)

癫痫是最常见的神经系统疾病之一,临床主要表现为周期性和无法预见性的癫痫样发作。目前认为,癫痫的形成、同步化以及癫痫样放电的维持与细胞间的缝隙连接有关。大量的体内外实验研究表明,缝隙连接抑制剂具有较强的抗癫痫作用,提示缝隙连接可能成为开发新型抗癫痫药物的潜在靶点。此外,选择性药理学研究和基因敲除技术研究表明,神经元间与胶质细胞间的缝隙连接在癫痫中的作用需区别看待。随着条件性基因敲除技术的成熟、高选择性的新型缝隙连接调控药物的出现以及更详细的人类癫痫脑组织样本的研究,人们将更全面地了解缝隙连接在癫痫中的作用。本综述总结了目前关于缝隙连接在癫痫发病机制中作用的研究结果,并对临床和基础研究中癫痫发作导致的缝隙连接蛋白表达的变化进行讨论。     

Kir4.1 channels mediate a depolarization of hippocampal astrocytes under hyperammonemic conditions in situ

Increased ammonium (NH(4) (+) ) concentration in the brain is the prime candidate responsible for hepatic encephalopathy (HE), a serious neurological disorder caused by liver failure and characterized by disturbed glutamatergic neurotransmission and impaired glial function. We investigated the mechanisms of NH(4) (+) -induced depolarization of astrocytes in mouse hippocampal slices using whole-cell patch-clamp and potassium-selective microelectrodes. At postnatal days (P) 18-21, perfusion with 5 mM NH(4) (+) evoked a transient increase in the extracellular potassium concentration ([K(+) ](o) ) by about 1 mM. Astrocytes depolarized by on average 8 mV and then slowly repolarized to a plateau depolarization of 6 mV, which was maintained during NH(4) (+) perfusion. In voltage-clamped astrocytes, NH(4) (+) induced an inward current and a reduction in membrane resistance. Amplitudes of [K(+) ](o) transients and astrocyte depolarization/inward currents increased from P3-4 to P18-21. Perfusion with 100 μM Ba(2+) did not alter [K(+) ](o) transients but strongly reduced both astrocyte depolarization and inward currents. NH(4) (+) -induced depolarization and inward currents were also virtually absent in slices from Kir4.1 -/- mice, while [K(+) ](o) transients were unaltered. Blocking Na(+) /K(+) -ATPase with ouabain caused an immediate and complex increase in [K(+) ](o) . Taken together, our results are in agreement with the hypothesis that reduced uptake of K(+) by the Na(+) , K(+) -ATPase in the presence of NH(4) (+) disturbs the extracellular K(+) homeostasis. Furthermore, astrocytes depolarize in response to the increase in [K(+) ](o) and by influx of NH(4) (+) through Kir4.1 channels. The depolarization reduces the astrocytes' capacity for channel-mediated flux of K(+) and for uptake of glutamate and might hereby contribute to the pathology of HE.     

GFAP-expressing cells in the postnatal subventricular zone display a unique glial phenotype intermediate between radial glia and astrocytes

Neural stem cells in the adult subventricular zone (SVZ) derive from radial glia and express the astroglial marker glial fibrillary acidic protein (GFAP). Thus, they have been termed astrocytes. However, it remains unknown whether these GFAP-expressing cells express the functional features common to astrocytes. Using immunostaining and patch clamp recordings in acute slices from transgenic mice expressing green fluorescent protein (GFP) driven by the promoter of human GFAP, we show that GFAP-expressing cells in the postnatal SVZ display typical glial properties shared by astrocytes and prenatal radial glia such as lack of action potentials, hyperpolarized resting potentials, gap junction coupling, connexin 43 expression, hemichannels, a passive current profile, and functional glutamate transporters. GFAP-expressing cells express both GLAST and GLT-1 glutamate transporters but lack AMPA-type glutamate receptors as reported for dye-coupled astrocytes. However, they lack 100 microM Ba2+-sensitive inwardly rectifying K+ (K(IR)) currents expressed by astrocytes, but display delayed rectifying K+ currents and 1 mM Ba2+-sensitive K+ currents. These currents contribute to K+ transport at rest and maintain hyperpolarized resting potentials. GFAP-expressing cells stained positive for both K(IR)2.1 and K(IR)4.1 channels, two major K(IR) channels in astrocytes. Ependymal cells, which also derive from radial glia and express GFAP, display typical glial properties and K(IR) currents consistent with their postmitotic nature. Our results suggest that GFAP-expressing cells in concert with ependymal cells can perform typical astrocytic functions such as K+ and glutamate buffering in the postnatal SVZ but display a unique set of functional characteristics intermediate between astrocytes and radial glia.     

Endothelins regulate astrocyte gap junctions in rat hippocampal slices

Gap junctional communication (GJC) is a typical feature of astrocytes proposed to contribute to the role played by these glial cells in brain physiology and pathology. In acutely isolated hippocampal slices from rat (P11-P19), intercellular diffusion of biocytin through gap junction channels was shown to occur between hundreds of cells immuno-positive for astrocytic markers studied in the CA1/CA2 region. Single-cell RT-PCR demonstrated astrocytic mRNA expression of several connexin (Cx) subtypes, the molecular constituent of gap junction channels, whereas immunoblotting confirmed that Cx43 and Cx30 are the main gap junction proteins in hippocampal astrocytes. In the brain, astrocytes represent a major target for endothelins (Ets), a vasoactive family of peptides. Our results demonstrate that Ets decrease the expression of phosphorylated Cx43 forms and are potent inhibitors of GJC. The Et-induced effects were investigated using specific Et receptor agonists and antagonists, including Bosentan (Tracleer trade mark ), an EtA/B receptor antagonist, and using hippocampal slices and cultures from EtB-receptor-deficient rats. Interestingly, the pharmacological profile of Ets effects did not follow the classical profile established in cardiovascular systems. The present study therefore identifies Ets as potent endogenous inhibitory regulators of astrocyte networks. As such, the action of these peptides on astrocyte GJC might be involved in the contribution of astrocytes to neuroprotective processes and have a therapeutic potential in neuropathological situations.     

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.     

Alzheimer's amyloid beta-peptide enhances ATP/gap junction-mediated calcium-wave propagation in astrocytes

Alzheimer’s disease (AD) involves the progressive extracellular deposition of amyloid β-peptide (Aβ), a self-aggregating 40–42 amino acid protein that can damage neurons resulting in their dysfunction and death. Studies of neurons have shown that Aβ perturbs cellular-calcium homeostasis so that calcium responses to agonists that induce calcium influx or release from internal stores are increased. The recent discovery of intercellular calcium waves in astrocytes suggests intriguing roles for astrocytes in the long-range transfer of information in the nervous system. We now report that Aβ alters calcium-wave signaling in cultured rat cortical astrocytes. Exposure of astrocytes to Aβ1-42 resulted in an increase in the amplitude and velocity of evoked calcium waves, and increased the distance the waves traveled. Suramin decreased wave propagation in untreated astrocytes and abrogated the enhancing effect of Aβ on calcium-wave amplitude and velocity, indicating a requirement for extracellular ATP in wave propagation. Treatment of astrocytes with an uncoupler of gap junctions did not significantly reduce the amplitude, velocity, or distance of calcium waves in control cultures, but completely abolished the effects of Aβ on each of the three wave parameters. These findings reveal a novel action of Aβ on the propagation of intercellular calcium signals in astrocytes, and also suggests a role for altered astrocyte calcium-signaling in the pathogenesis of AD.     

Mild brain ischemia induces unique physiological properties in striatal astrocytes

We studied the properties of GFAP-expressing cells in adult mouse striatum using acute brain slices from transgenic animals expressing EGFP under GFAP promoter. Under physiological conditions, two distinct populations of GFAP-EGFP cells could be identified: (1) brightly fluorescent cells had bushy processes, passive membrane properties, glutamate transporter activity, and high gap junction coupling rate typical for classical astrocytes; (2) weakly fluorescent cells were characterized by thin, clearly distinguishable processes, voltage-gated currents, complex responses to kainate, and low coupling rate reminiscent of an astrocyte subtype recently described in the hippocampus. Mild focal cerebral ischemia confers delayed neuronal cell death and astrogliosis in the striatum. Following middle cerebral artery occlusion and reperfusion, brightly fluorescent cells were the dominant GFAP-EGFP population observed within the ischemic lesion. Interestingly, the majority of these cells expressed voltage-gated channels, showed complex responses to kainate, and a high coupling rate exceeding that of brightly fluorescent control cells. A minority of cells had passive membrane properties and was coupled less compared with passive control cells. We conclude that, in the adult striatum, astrocytes undergo distinct pathophysiological changes after ischemic insults. The dominant population in the ischemic lesion constitutes a novel physiological phenotype unlike any normal astrocyte and generates a large syncytium which might be a neuroprotective response of reactive astrocytes.     

高钾和（或）谷氨酸对培养的星形胶质细胞Connexin43表达的影响

研究高浓度钾和 (或 )谷氨酸对星形胶质细胞缝隙连接蛋白表达的影响。利用培养的星形胶质细胞 ,用免疫细胞化学方法 ,观察connexin4 3(Cx4 3)表达的变化。结果 :单独给予氯化钾 (2 5mmol/L ,× 10min)或谷氨酸 (4 0 0 μmol/L ,× 2 0min) ,刺激后 8hCx4 3的表达较对照组增高 (P <0 .0 1) ,2 4h恢复正常 ;同时给予氯化钾 (2 5mmol/L)和谷氨酸 (4 0 0 μmol/L ,× 10min) ,刺激后 8hCx4 3的表达开始较对照组增高(P <0 .0 1) ,且随时间逐渐增高 ,于 4 8h达高峰。以上结果提示 :细胞外高浓度钾和 (或 )谷氨酸在一定时间内可引起Cx4 3表达上调 ,且两者可能有协同作用     

Modulation of connexin expression and gap junction communication in astrocytes by the gram-positive bacterium S. aureus

Gap junctions establish direct intercellular conduits between adjacent cells and are formed by the hexameric organization of protein subunits called connexins (Cx). It is unknown whether the proinflammatory milieu that ensues during CNS infection with S. aureus, one of the main etiologic agents of brain abscess in humans, is capable of eliciting regional changes in astrocyte homocellular gap junction communication (GJC) and, by extension, influencing neuron homeostasis at sites distant from the primary focus of infection. Here we investigated the effects of S. aureus and its cell wall product peptidoglycan (PGN) on Cx43, Cx30, and Cx26 expression, the main Cx isoforms found in astrocytes. Both bacterial stimuli led to a time-dependent decrease in Cx43 and Cx30 expression; however, Cx26 levels were elevated following bacterial exposure. Functional examination of dye coupling, as revealed by single-cell microinjections of Lucifer yellow, demonstrated that both S. aureus and PGN inhibited astrocyte GJC. Inhibition of protein synthesis with cyclohexamide (CHX) revealed that S. aureus directly modulates, in part, Cx43 and Cx30 expression, whereas Cx26 levels appear to be regulated by a factor(s) that requires de novo protein production; however, CHX did not alter the inhibitory effects of S. aureus on astrocyte GJC. The p38 MAPK inhibitor SB202190 was capable of partially restoring the S. aureus-mediated decrease in astrocyte GJC to that of unstimulated cells, suggesting the involvement of p38 MAPK-dependent pathway(s). These findings could have important implications for limiting the long-term detrimental effects of abscess formation in the brain which may include seizures and cognitive deficits.     

Differential connexin expression,gap junction intercellular coupling,and hemichannel formation in NT2/D1 human neural progenitors and terminally differentiated hNT neurons

Connexin-mediated gap junctions and open hemichannels in nonjunctional membranes represent two biologically relevant mechanisms by which neural progenitors can coordinate their response to changes in the extracellular environment. NT2/D1 cells are a teratocarcinoma progenitor line that can be induced to differentiate terminally into functional hNT neurons and NT-G nonneuronal cells. Clinical transplants of hNT neurons and experimental grafts of NT2/D1 progenitors or hNT neurons have been used in cell-replacement therapy in vivo. Previous studies have shown that NT2/D1 cells express connexin 43 (Cx43) and that NT2/D1 progenitors are capable of dye transfer. To determine whether NT2/D1 progenitors and differentiated hNT cultures express other connexins, Cx26, Cx30, Cx32, Cx36, Cx37, Cx43, and Cx46.6 mRNA and protein were analyzed. NT2/D1 progenitors express Cx30, Cx36, Cx37, and Cx43. hNT/NT-G cultures express Cx36, Cx37, and de novo Cx46.6. Cx26 and Cx32 were not expressed in NT2/D1 or hNT/NT-G cells. NT2/D1 progenitors formed functional gap junctions as assessed by dye coupling as well as open hemichannels in nonjunctional membranes as assessed by dye-uptake studies. Dye coupling was inhibited by the gap junction blocker 18alpha-glycyrrhetinic acid. Hemichannel activity was inhibited by the dual-specificity chloride channel/connexin hemichannel inhibitor flufenamic acid but not by the chloride channel inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Both dye coupling and dye uptake were substantially reduced following differentiation of NT2/D1 progenitors. We conclude that the pattern of connexin expression in NT2/D1 cells changes over the course of differentiation corresponding with a reduction in biochemical coupling and hemichannel activity in differentiated cells.     

Downregulation of connexin36 in mouse spinal dorsal horn neurons leads to mechanical allodynia

Connexin36 (Cx36), a component of neuronal gap junctions, is crucial for interneuronal communication and regulation. Gap junction dysfunction underlies neurological disorders, including chronic pain. Following a peripheral nerve injury, Cx36 expression in the ipsilateral spinal dorsal horn was markedly decreased over time, which paralleled the time course of hind paw tactile allodynia. Intrathecal (i.t.) injection of Cx36 siRNA (1 and 5 pg) significantly reduced the expression of Cx36 protein in the lumbar spinal cord, peaking 3 days after the injection, which corresponded with the onset of hind paw tactile allodynia. It is possible that some of the tactile allodynia resulting from Cx36 downregulation could be mediated through excitatory neuromodulators, such as glutamate and substance P. The Cx36 knockdown‐evoked tactile allodynia was significantly attenuated by i.t. treatment with the N‐methyl‐D‐aspartate glutamate receptor antagonist MK‐801 but not the substance P receptor antagonist CP96345. Immunohistochemistry showed that Cx36 was colocalized with glycine transporter‐2, a marker for inhibitory glycinergic spinal interneurons, but not with glutamate decarboxylase 67, a marker for inhibitory GABAergic spinal interneurons. The results indicate that spinal inhibition through glycinergic interneurons is reduced, leading to increased glutamatergic neurotransmission, as a result of Cx36 downregulation. The current data suggest that gap junction dysfunction underlies neuropathic pain and further suggest a novel target for the development of analgesics. © 2014 Wiley Periodicals, Inc.     

Glucocorticoids—potent modulators of astrocytic calcium signaling

Glucocorticoids are the first line of choice in the treatment of cerebral edema associated with brain tumors. High‐dose glucocorticoids reduce the extent of edema within hours, often relieving critical increases in intracranial pressure, but the mechanisms by which glucocorticoids modulate brain water content are not well‐understood. A possible target of action may be glucocorticoid receptor‐expressing astrocytes, which are the primary regulators of interstitial ion homeostasis in brain. In this study, we demonstrate that two glucocorticoids, methylprednisolone and dexamethasone, potentiate astrocytic signaling, via long‐range calcium waves. Glucocorticoid treatment increased both resting cytosolic calcium (Ca²⁺_i) level and the extent and amplitude of Ca²⁺ wave propagation two‐fold, compared to matched controls. RU‐486, a potent steroid receptor antagonist, inhibited the effects of methylprednisolone. The glucocorticoid‐associated potentiation of Ca²⁺ signaling may result from upregulation of the cellular ability to mobilize Ca²⁺ and release ATP, because both agonist‐induced Ca²⁺_i increments (via ATP and bradykinin) and ATP release were proportionally enhanced by glucocorticoids. In contrast, neither gap junction expression (as manifested connexin 43 immunoreactivity) nor functional coupling was significantly affected by methylprednisolone. Confocal microscopy revealed both the expression of glucocorticoid receptors and nuclear translocation of these receptors when exposed to methylprednisolone. We postulate that the edemolytic effects of glucocorticoids may result from enhanced astrocytic calcium signaling. GLIA 28:1–12, 1999. © 1999 Wiley‐Liss, Inc.     

The immunocytochemical localization of connexin 36 at rod and cone gap junctions in the guinea pig retina

Connexin 36 (Cx36) is a channel-forming protein found in the membranes of apposed cells, forming the hexameric hemichannels of intercellular gap junction channels. It localizes to certain neurons in various regions of the brain including the retina. We characterized the expression pattern of neuronal Cx36 in the guinea pig retina by immunocytochemistry using specific antisera against Cx36 and green/red cone opsin or recoverin. Strong Cx36 immunoreactivity was visible in the ON sublamina of the inner plexiform layer and in the outer plexiform layer, as punctate labelling patterns. Double-labelling experiments with antibody directed against Cx36 and green/red cone opsin or recoverin showed that strong clustered Cx36 immunoreactivity localized to the axon terminals of cone or close to rod photoreceptors. By electron microscopy, Cx36 immunoreactivity was visible in the gap junctions as well as in the cytoplasmic matrices of both sides of cone photoreceptors. In the gap junctions between cone and rod photoreceptors, Cx36 immunoreactivity was only visible in the cytoplasmic matrices of cone photoreceptors. These results clearly indicate that Cx36 forms homologous gap junctions between neighbouring cone photoreceptors, and forms heterologous gap junctions between cone and rod photoreceptors in guinea pig retina. This focal location of Cx36 at the terminals of the photoreceptor suggests that rod photoreceptors can transmit rod signals to the pedicle of a neighbouring cone photoreceptor via Cx36, and that the cone in turn signals to corresponding ganglion cells via ON and OFF cone bipolar cells.     

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 α-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. © 1996 Wiley-Liss, Inc.     

Gap junctions couple astrocytes but not neurons in dissociated cultures of rat suprachiasmatic nucleus

Individual neurons dissociated from rat suprachiasmatic nucleus can express independently phased circadian firing rhythms in culture. The phases of these rhythms are unperturbed by reversible blockade of neuronal firing lasting 2.5 days, indicating that multiple circadian clocks continue to operate in the absence of conventional synaptic transmission. The possibility remains, however, that these circadian rhythms might depend on some other form of intercellular communication. In the present study, a potential role for gap junctional coupling in SCN cultures was evaluated by introduction of the tracer molecule Neurobiotin into both neurons (n = 98) and astrocytes (n = 10), as well as by immunolabeling for specific connexins, the molecular components of gap junctions. Astrocytes were extensively coupled to each other by connexin43-positive gap junctions, but no evidence was found for coupling of neurons to each other or to astrocytes. These data support the hypothesis that neurons expressing independently phased circadian rhythms in SCN cultures (‘clock cells’) are autonomous, single cell circadian oscillators, but do not exclude a role for glia in synchronizing neuronal clock cells in vivo.     

ATP signaling is deficient in cultured Pannexin1-null mouse astrocytes

Pannexins (Panx1, 2, and 3) comprise a group of proteins expressed in vertebrates that share weak yet significant sequence homology with the invertebrate gap junction proteins, the innexins. In contrast to the other vertebrate gap junction protein family (connexin), pannexins do not form intercellular channels, but at least Panx1 forms nonjunctional plasma membrane channels. Panx1 is ubiquitously expressed and has been shown to form large conductance (500 pS) channels that are voltage dependent, mechanosensitive, and permeable to relatively large molecules such as ATP. Pharmacological and knockdown approaches have indicated that Panx1 is the molecular substrate for the so-called "hemichannel" originally attributed to connexin43 and that Panx1 is the pore-forming unit of the P2X(7) receptor. Here, we describe, for the first time, conductance and permeability properties of Panx1-null astrocytes. The electrophysiological and fluorescence imaging analyses performed on these cells fully support our previous pharmacological and Panx1 knockdown studies that showed profoundly lower dye uptake and ATP release than wild-type untreated astrocytes. As a consequence of decreased ATP paracrine signaling, intercellular calcium wave spread is altered in Panx1-null astrocytes. Moreover, we found that in astrocytes as in Panx1-expressing oocytes, elevated extracellular K(+) activates Panx1 channels independently of membrane potential. Thus, on the basis of our present findings and our previous report, we propose that Panx1 channels serve as K(+) sensors for changes in the extracellular milieu such as those occurring under pathological conditions.