Adrenoceptors were among the first neurotransmitter receptors identified in glial cells, but it is not known whether these receptors meditate glial responses during neuronal activity. We show that repetitive nerve activity evoked a rise of intracellular calcium in Bergmann glia and neighboring Purkinje neurons of cerebellar slices of mice. The glial but not the neuronal calcium transient persisted during block of ionotropic and metabotropic glutamate receptors. In contrast, the glial calcium response was abolished by cyclopiazonic acid and prazosin; however, prazosin affected neither the inward current nor the resulting depolarization that accompanied the stimulus-induced glial calcium transients. The glial depolarization was attenuated by 38% by the mixture of glutamate receptor blockers, which abolished the evoked neuronal depolarization and afterhyperpolarization. Ba(2+) reduced the glial currents by 66% without affecting the concomitant calcium transients. In the presence of Ba(2+), the mixture of glutamate receptor blockers exerted no effect on the glial inward current or calcium rise. Furthermore, Ba(2+) greatly potentiated both the activity-related Purkinje cell inward current and the accompanying neuronal calcium rises. The results indicate that release of noradrenaline from afferent fibers activates a glial alpha(1) adrenoceptor that promotes calcium release from intracellular stores. Glial calcium rises are known to stimulate a diversity of processes such as transmitter release, energy metabolism, or proliferation. Thus the adrenoceptor-mediated mechanism described here is well suited for feedback modulation of neuronal function that is independent of glutamate.

Using in situ hybridisation histochemistry in combination with patch‐clamp recordings and specific pharmacological tools, the molecular nature of the channels underlying Ca2+‐dependent K+ currents was determined in dorsal vagal neurones (DVNs) of rat brainstem slices. In situ hybridisation analysis at cellular resolution revealed the presence of ‘big’‐conductance Ca2+‐ and voltage‐activated K+ (BK) channel α‐subunit mRNA, and of only one ‘small’‐conductance Ca2+‐activated K+ (SK) channel subunit transcript, SK3, at very high levels in DVNs. By contrast, SK1 and SK2 mRNAs were below the threshold limit of detection. The SK channel‐mediated after‐hyperpolarising current (IAHP) was blocked by apamin with a half‐maximal inhibitory concentration of ∼2.2 nm. This is consistent with homomultimeric SK3 channels mediating IAHP in DVNs. IAHP was also blocked by scyllatoxin (20–30 nm) and curare (100–200 μm). Application of apamin (100 nm) or scyllatoxin (20 nm) invariably caused a substantial increase to 146.1 ± 10.4 and 181.8 ± 12.9 % of control, respectively, in the spontaneous firing rate of DVNs. Action potential duration was not affected by these SK channel blockers. The selective BK channel blocker iberiotoxin (50 nm) increased action potential duration by 22.5 ± 7.3 %, as did low concentrations of tetraethylammonium (0.5 mm; 99.3 ± 16.4 %) and the Ca2+ channel blocker Cd2+ (100 μm; 49.5 ± 20.9 %). BK channel blockade did not significantly affect the firing rate of DVNs. These results allow us to establish a tight correlation between the properties of cloned and native BK and SK channels, and to achieve an understanding, at the molecular level, of their role in regulating the spontaneous firing frequency and in shaping single action potentials of central neurones.

Whole cell recordings of fura-2 dialyzed vagal neurons of brain stem slices were used to monitor interstitial glutamate accumulation within the dorsal vagal complex. Anoxia produced a sustained outward current (60 pA) and a moderate [Ca(2+)](i) rise (40 nM). These responses were neither mimicked by [1S,3R]-1-aminocyclo-pentane-1, 3-dicarboxylic acid nor affected by Ca(2+)-free solution, 6-cyano-7-nitroquino-xaline-2,3-dione (CNQX), 2-amino-5-phosphonovalerate (APV), or tetrodotoxin. Anoxia or cyanide in glucose-free saline (in vitro ischemia) as well as ouabain or iodoacetate elicited an initial anoxia-like [Ca(2+)](i) increase that turned after several minutes into a prominent Ca(2+) transient (0.9 microM) and inward current (-1.8 nA). APV plus CNQX (plus methoxyverapamil) inhibited this inward current as well as accompanying spontaneous synaptic activity, and reduced the secondary [Ca(2+)](i) rise to values similar to those during anoxia. Each of the latter drugs delayed onset of both ischemic current and prominent [Ca(2+)](i) rise by several minutes and attenuated their magnitudes by up to 40%. Ca(2+)-free solution induced a twofold delay of the ischemic inward current and suppressed the prominent Ca(2+) increase but not the initial moderate [Ca(2+)](i) rise. Cyclopiazonic acid or arachidonic acid in Ca(2+)-free saline delayed further the ischemic current, whereas neither inhibitors of glutamate uptake (dihydrokainate, D,L-threo-beta-hydroxyaspartate, L-transpyrrolidone-2,4-dicarboxylate) nor the Cl(-) channel blocker 5-nitro-2-(3-phenylpropyl-amino) benzoic acid had any effect. In summary, the response to metabolic arrest is due to activation of ionotropic glutamate receptors causing Ca(2+) entry via N-methyl-D-aspartate receptors and voltage-activated Ca(2+) channels. An early Ca(2+)-dependent exocytotic phase of ischemic glutamate release is followed by nonvesicular release, not mediated by reversed glutamate uptake or Cl(-) channels. The results also show that glycolysis prevents glutamate release during anoxia.

Ca2ϩ imaging and (perforated) patch recording were used to analyze the mechanism of GABA-and glycine-induced depolarizations in lumbar motoneurons of spinal cord slices from fetal rats. In fura-2 ester-loaded cells, the agonist-induced depolarizations increased [Ca 2ϩ ] i by up to 100 nM. The GABA-and glycine-evoked [Ca 2ϩ ] i transients were suppressed by bicuculline and strychnine, respectively. Their magnitude decreased by ϳ50% between embryonic days 15.5 and 19.5. Ϫ efflux has a major contribution to depolarizations mediated by GABA A and glycine receptor-coupled anion channels in prenatal neurons. We hypothesize that the HCO 3 Ϫ -dependent depolarizing component, which is likely to produce an intracellular acidosis, might play an important role during the early postnatal period when the Cl Ϫ -dependent component gradually shifts to hyperpolarization. Key words: bicarbonate; calcium; chloride pump; development; imaging; motor neurons; neuronal maturationThe principal hyperpolarizing and thus inhibitory neurotransmitters GABA and glycine exert a depolarizing action during development of neuronal structures (Ben-Ari et al., 1989;Obrietan and van den Pol, 1995). In the immature hippocampus, depolarizing GABAergic IPSPs inhibit synaptic responses of CA3 pyramidal neurons (Psarropoulou and Descombes, 1999;Palva et al., 2000), but periodic GABA release produces a "giant" neuronal depolarization and action potential discharge (Ben-Ari et al., 1989). The rise in the concentration of free intracellular Ca 2ϩ ([Ca 2ϩ ] i ) associated with the GABA-induced depolarization (Leinekugel et al., 1995;Garaschuk et al., 1998) might be implicated in trophic or hebbian modulation of developing synapses and activity-dependent formation of the hippocampal network Leinekugel et al., 1999).In motoneurons that are among the earliest neurons to differentiate within the brain, activity-related rises of [Ca 2ϩ ] i are also supposed to have a trophic effect. Suppression of neurite outgrowth in developing motoneurons (Owen and Bird, 1997; Metzger et al., 1998) appears to be causally related with an increase of [Ca 2ϩ ] i attributable to activation of Ca 2ϩ -permeable glutamate receptors (Metzger et al., 2000). Accordingly, maturation of motoneurons of cultured lumbar spinal cord is retarded upon blockade of glutamatergic neurotransmission (Xie and Ziskind-Conhaim, 1995). One week before birth, the isolated spinal cord of rats generates rhythmic nerve activity that is impaired by blockers of glycine and GABA A receptors (Nishimaru et al., 1996). It was found previously that GABA and glycine depolarize lumbar motoneurons that provide the output of this rhythmically active network in the fetus (Obata et al., 1978;Wu et al., 1992;Gao and Ziskind-Conhaim, 1995). The latter studies indicated a major role of Cl Ϫ ions in this depolarization. However, it is yet not clear whether efflux of HCO 3 Ϫ through the receptor-coupled anion pore (Bormann et al., 1987;Fatima-Shad and Barry, 1993) contributes to the GABA-and glycine-evoked r...

Intracellular Ca(2+) ([Ca(2+)](i)) was fluorometrically measured with fura-2 in lumbar motoneurons of acutely isolated spinal cord slices from embryonic rats. In ester-loaded cells, bath-applied glutamate (3 microM to 1 mM) evoked a [Ca(2+)](i) increase by up to 250 nM that was abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) plus 2-amino-5-phosphonovalerate (APV). CNQX or APV alone reduced the response by 82 and 25%, respectively. The glutamatergic agonists kainate (KA), quisqualate (QUI), and S-alpha-amino-3-hydroxy-5-methyl-4-isoxalone (S-AMPA) evoked a similar [Ca(2+)](i) transient as glutamate. N-methyl-D-aspartate (NMDA) was only effective to increase [Ca(2+)](i) in Mg(2+)-free saline, whereas [1S,3R]-1-aminocyclopentane-1,3-dicarboxylic acid ([1S,3R]-ACPD) had no effect. The glutamate-induced [Ca(2+)](i) rise was suppressed in Ca(2+)-free superfusate. Depletion of Ca(2+) stores with cyclopiazonic acid (CPA) did not affect the response. Thirty-six percent of the [Ca(2+)](i) increase in response to membrane depolarization induced by a 50 mM K(+) solution persisted on combined application of the voltage-gated Ca(2+) channel blockers nifedipine, omega-conotoxin-GVIA and omega-agatoxin-IVA. In fura-2 dialyzed motoneurons, the glutamate-induced [Ca(2+)](i) increase was attenuated by approximately 70% after changing from current to voltage clamp. Forty percent of the remaining [Ca(2+)](i) transient and 20% of the concomitant inward current of 0.3 nA were blocked by Joro spider toxin-3 (JSTX). The results show that voltage-gated Ca(2+) channels, including a major portion of R-type channels, constitute the predominant component of glutamate-induced [Ca(2+)](i) rises. NMDA and Ca(2+)-permeable KA/AMPA receptors contribute about equally to the remaining component of the Ca(2+) rise. The results substantiate previous assumptions that Ca(2+) influx through JSTX-sensitive KA/AMPA receptors is involved in (trophic) signaling in developing motoneurons.