N‐type calcium channels mediate a GABAB presynaptic modulation in the corticostriatal synapse in turtle's paleostriatum augmentatum

Spikes population evoked by a paired pulse protocol were used to assess the influence of GABA_A and GABA_B receptors agonists and antagonists on the synaptic potentials and in the S₂/S₁ ratio in a paired pulse (PP) protocol in the cortico‐paleostriatum augmentatum synapses of the turtle. GABA_A agonist, muscimol, decreased the amplitude of synaptic responses whereas the facilitation produced with the PP protocol did not change, suggesting a postsynaptic action for GABA_A receptors. GABA_B agonist, baclofen, enhanced paired pulse ratio indicating a presynaptic modulation through the GABA_B receptor. Selective antagonists for N‐ and P/Q‐type Ca²⁺‐channels also enhanced paired pulse ratio, suggesting that any of these channel types may be involved in neurotransmitter release. However, the strong paired pulse facilitation produced by baclofen was occluded by blocking the N‐type Ca²⁺ channels with ω‐conotoxin GVIA (1 μM), but not by the blockage of P/Q‐type Ca²⁺ channels with ω‐agatoxin TK (400 nM). These data suggest that N and P/Q channels participate in the neurotransmitter release, whereas only N‐type Ca²⁺ channels are involved in the presynaptic modulation of GABA_B in the corticostriatal synapse of the turtle. Synapse 63:855–862, 2009. © 2009 Wiley‐Liss, Inc.     

Enhanced Synaptic Inhibition Disrupts the Efferent Code of Cerebellar Purkinje Neurons in Leaner Cav2.1 Ca2+ Channel Mutant Mice

Cerebellar Purkinje cells (PCs) encode afferent information in the rate and temporal structure of their spike trains. Both spontaneous firing in these neurons and its modulation by synaptic inputs depend on Ca²⁺ current carried by Ca_v2.1 (P/Q) type channels. Previous studies have described how loss-of-function Ca_v2.1 mutations affect intrinsic excitability and excitatory transmission in PCs. This study examines the effects of the leaner mutation on fast GABAergic transmission and its modulation of spontaneous firing in PCs. The leaner mutation enhances spontaneous synaptic inhibition of PCs, leading to transitory reductions in PC firing rate and increased spike rate variability. Enhanced inhibition is paralleled by an increase in the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) measured under voltage clamp. These differences are abolished by tetrodotoxin, implicating effects of the mutation on spike-induced GABA release. Elevated sIPSC frequency in leaner PCs is not accompanied by increased mean firing rate in molecular layer interneurons, but IPSCs evoked in PCs by direct stimulation of these neurons exhibit larger amplitude, slower decay rate, and a higher burst probability compared to wild-type PCs. Ca²⁺ release from internal stores appears to be required for enhanced inhibition since differences in sIPSC frequency and amplitude in leaner and wild-type PCs are abolished by thapsigargin, an ER Ca²⁺ pump inhibitor. These findings represent the first account of the functional consequences of a loss-of-function P/Q channel mutation on PC firing properties through altered GABAergic transmission. Gain in synaptic inhibition shown here would compromise the fidelity of information coding in these neurons and may contribute to impaired cerebellar function resulting from loss-of function mutations in the Ca_V2.1 channel gene.     

Roscovitine differentially facilitates cerebellar glutamatergic and GABAergic neurotransmission by enhancing Cav2.1 channel‐mediated multivesicular release

Synaptic vesicle exocytosis is triggered by Ca²⁺ influx through several subtypes of voltage‐gated calcium channels in the presynaptic terminal. We previously reported that paired‐pulse stimulation at brief intervals increases Ca_v2.1 (P/Q‐type) channel‐mediated multivesicular release (MVR) at glutamatergic synapses between granule cells (GCs) and molecular layer interneurons (MLIs) in rat cerebellar slices. However, it has yet to be determined how Ca_v2 channel subtypes take part in MVR in single axon terminal. This study therefore aimed at examining the effects of roscovitine on different types of cerebellar synapses that make contacts with Purkinje cells (PCs), because this compound has been shown to enhance Ca_v2.1 channel‐mediated MVR at GC‐MLI synapses. Bath application of roscovitine profoundly increased the amplitude of excitatory postsynaptic currents (EPSCs) at GC‐PC synapses by a presynaptic mechanism as previously observed at GC‐MLI synapses, whereas it caused a marginal effect on climbing fiber‐mediated EPSCs in PCs. At MLI‐PC synapses, roscovitine increased both the amplitude and decay time of inhibitory postsynaptic currents (IPSCs) by enhancing multivesicular GABA release. When extracellular Ca²⁺ concentration ([Ca²⁺]_e) decreased, roscovitine became less effective in increasing GC‐PC EPSCs. By contrast, roscovitine was able to augment MLI‐PC IPSCs in the low [Ca²⁺]_e. The Ca_v2.1 channel blocker ω‐agatoxin IVA suppressed the roscovitine‐induced facilitatory actions on both GC‐PC EPSCs and MLI‐PC IPSCs. These results demonstrate that roscovitine enhances MVR at the GC‐PC excitatory synapses in a manner dependent on the driving force of Ca_v2.1 channel‐mediated Ca²⁺ influx into the nerve terminal, while it also facilitates MLI‐PC inhibitory transmission via Ca²⁺‐insensitive mechanisms.     

Involvement of R‐type Ca2+ channels in neurotransmitter release from spinal dorsolateral funiculus terminals synapsing motoneurons

Molecular studies have revealed the presence of R‐type voltage‐gated Ca²⁺ channels at pre‐ and postsynaptic regions; however, no evidence for the participation of these channels in transmitter release has been presented for the spinal cord. Here we characterize the effects of SNX‐482, a selective R channel blocker, on the monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in motoneurons by stimulation of dorsolateral funiculus (DLF) terminals in a slice preparation from the adult turtle spinal cord. SNX‐482 inhibited neurotransmission in a dose‐dependent manner, with an IC₅₀ of ～9 ± 1 nM. The EPSP time course and membrane time constant of the motoneurons were not altered, suggesting a presynaptic mechanism. The toxin inhibited the residual component of the EPSPs recorded in the presence of N‐ and P/Q‐type Ca²⁺ channel blockers, strongly suggesting a role for the R channels in neurotransmission at the spinal cord DLF terminals. Consistently with this, RT‐PCR analysis of turtle spinal cord segments revealed the expression of the Ca_V2.3 pore‐forming (α_1E) subunit of R channels, whereas the use of anti‐α_1E‐specific antibodies resulted in its localization in the DLF fibers as demonstrated by immunohistochemistry coupled with laser confocal microscopy. J. Comp. Neurol. 513:188–196, 2009. © 2009 Wiley‐Liss, Inc.     

The Ataxic Cacna1a-Mutant Mouse Rolling Nagoya: An Overview of Neuromorphological and Electrophysiological Findings

Homozygous rolling Nagoya natural mutant mice display a severe ataxic gait and frequently roll over to their side or back. The causative mutation resides in the Cacna1a gene, encoding the pore-forming α₁ subunit of Ca_v2.1 type voltage-gated Ca²⁺ channels. These channels are crucially involved in neuronal Ca²⁺ signaling and in neurotransmitter release at many central synapses and, in the periphery, at the neuromuscular junction. We here review the behavioral, histological, biochemical, and neurophysiological studies on this mouse mutant and discuss its usefulness as a model of human neurological diseases associated with Ca_v2.1 dysfunction.     

Serotonin 5‐HT1B receptor‐mediated calcium influx‐independent presynaptic inhibition of GABA release onto rat basal forebrain cholinergic neurons

Modulatory roles of serotonin (5‐HT) in GABAergic transmission onto basal forebrain cholinergic neurons were investigated, using whole‐cell patch‐clamp technique in the rat brain slices. GABA_A receptor‐mediated inhibitory postsynaptic currents (IPSCs) were evoked by focal stimulation. Bath application of 5‐HT (0.1–300 μm ) reversibly suppressed the amplitude of evoked IPSCs in a concentration‐dependent manner. Application of a 5‐HT_1B receptor agonist, CP93129, also suppressed the evoked IPSCs, whereas a 5‐HT_1A receptor agonist, 8‐OH‐DPAT had little effect on the evoked IPSCs amplitude. In the presence of NAS‐181, a 5‐HT_1B receptor antagonist, 5‐HT‐induced suppression of evoked IPSCs was antagonised, whereas NAN‐190, a 5‐HT_1A receptor antagonist did not antagonise the 5‐HT‐induced suppression of evoked IPSCs. Bath application of 5‐HT reduced the frequency of spontaneous miniature IPSCs without changing their amplitude distribution. The effect of 5‐HT on miniature IPSCs remained unchanged when extracellular Ca²⁺ was replaced by Mg²⁺. The paired‐pulse ratio was increased by CP93129. In the presence of ω‐CgTX, the N‐type Ca²⁺ channel blocker, ω‐Aga‐TK, the P/Q‐type Ca²⁺ channel blocker, or SNX‐482, the R‐type Ca²⁺ channel blocker, 5‐HT could still inhibit the evoked IPSCs. 4‐AP, a K⁺ channel blocker, enhanced the evoked IPSCs, and CP93129 had no longer inhibitory effect in the presence of 4‐AP. CP93129 increased the number of action potentials elicited by depolarising current pulses. These results suggest that activation of presynaptic 5‐HT_1B receptors on the terminals of GABAergic afferents to basal forebrain cholinergic neurons inhibits GABA release in Ca²⁺ influx‐independent manner by modulation of K⁺ channels, leading to enhancement of neuronal activities.     

Subcellular distribution of low‐voltage activated T‐type Ca2+ channel subunits (Cav3.1 and Cav3.3) in reticular thalamic neurons of the cat

Low‐voltage‐activated (LVA) Ca²⁺ channels play a critical role in the generation of burst firing in the thalamus. Recently, three LVA Ca²⁺ channel isoforms (Ca_v3.1, Ca_v3.2, Ca_v3.3) have been identified in the reticular thalamic nucleus (RE). Previous electrophysiological and modelling studies have suggested that kinetically different T‐type channels might be expressed in a compartmentalized manner in RE cells. However, their precise subcellular distribution has not been fully elucidated. Using light and electron microscopic (EM) immunocytochemistry, we investigated the subcellular expression pattern of Ca_v3.1 and Ca_v3.3 channel subunits in RE neurons of the cat. Fluorescent and peroxidase labelling demonstrated the presence of Ca_v3.1 channel predominantly on the somata and proximal dendrites and Ca_v3.3 channels on cell bodies. Quantitative immunogold localization disclosed that Ca_v3.1 and Ca_v3.3 isoforms showed 5.8‐ and 8.7‐fold higher density, respectively, in the cytoplasm compared with somatic plasma membrane. Density of Ca_v3.1 isoform in the somatic plasma membrane was 2.21‐fold higher compared with Ca_v3.3 subunit. In the dendritic plasma membrane, Ca_v3.1 channel isoform was expressed throughout the entire dendritic tree. In contrast, Ca_v3.3 isoform was absent from large‐caliber, presumably proximal dendritic segments. Quantitative comparison showed that the relative density of immunogold particles compared with dendritic surface was 8.9‐ and 14.8‐fold higher for Ca_v3.1 and Ca_v3.3, respectively, in small‐diameter dendrites than in large proximal dendritic segments or somata. Our results demonstrate a higher density of low‐threshold Ca²⁺ channels in distal dendrites and provide further evidence of the role of RE neuron dendrites in the generation of prolonged, low‐threshold spike bursts. © 2009 Wiley‐Liss, Inc.     

The Expression of Voltage-Gated Ca2+ Channels in Pituicytes and the Up-Regulation of L-Type Ca2+ Channels During Water Deprivation

The primary components of the neurohypophysis are the neuroendocrine terminals that release vasopressin and oxytocin, and pituicytes, which are astrocytes that normally surround and envelop these terminals. Pituicytes regulate neurohormone release by secreting the inhibitory modulator taurine in an osmotically‐regulated fashion and undergo a marked structural reorganisation in response to dehydration as well as during lactation and parturition. Because of these unique functions, and the possibility that Ca²⁺ influx could regulate their activity, we tested for the expression of voltage‐gated Ca²⁺ channel α1 subunits in pituicytes both in situ and in primary culture. Colocalisation studies in neurohypophysial slices show that pituicytes (identified by their expression of the glial marker S100β), are immunoreactive for antibodies directed against Ca²⁺ channel α1 subunits Ca_V2.2 and Ca_V2.3, which mediate N‐ and R‐type Ca²⁺ currents, respectively. Pituicytes in primary culture express immunoreactivity for Ca_V1.2, Ca_V2.1, Ca_V2.2, Ca_V2.3 and Ca_V3.1 (which mediate L‐, P/Q‐, N‐, R‐ and T‐type currents, respectively) and immunoblotting studies confirmed the expression of these Ca²⁺ channel α1 subunits. This increase in Ca²⁺ channel expression may occur only in pituicytes in culture, or may reflect an inherent capability of pituicytes to initiate the expression of multiple types of Ca²⁺ channels when stimulated to do so. We therefore performed immunohistochemistry studies on pituitaries obtained from rats that had been deprived of water for 24 h. Pituicytes in these preparations showed a significantly increased immunoreactivity to Ca_V1.2, suggesting that expression of these channels is up‐regulated during the adaptation to long‐lasting dehydration. Our results suggest that Ca²⁺ channels may play important roles in pituicyte function, including a contribution to the adaptation that occurs in pituicytes when the need for hormone release is elevated.     

Activation of ryanodine receptors is required for PKA‐mediated downregulation of A‐type K+ channels in rat hippocampal neurons

A‐type K⁺ channels (I_A channels) contribute to learning and memory mechanisms by regulating neuronal excitabilities in the CNS, and their expression level is targeted by Ca²⁺ influx via synaptic NMDA receptors (NMDARs) during long‐term potentiation (LTP). However, it is not clear how local synaptic Ca²⁺ changes induce I_A downregulation throughout the neuron, extending from the active synapse to the soma. In this study, we tested if two major receptors of endoplasmic reticulum (ER), ryanodine (RyRs), and IP₃ (IP₃R) receptors, are involved in Ca²⁺‐mediated I_A downregulation in cultured hippocampal neurons of rats. The downregulation of I_A channels was induced by doubling the Ca²⁺ concentration in culture media (3.6 mM for 24 hrs) or treating with glycine (200 μM for 3 min) to induce chemical LTP (cLTP), and the changes in I_A peaks were measured electrophysiologically by a whole‐cell patch. We confirmed that Ca²⁺ or glycine treatment significantly reduced I_A peaks and that their effects were abolished by blocking NMDARs or voltage‐dependent Ca²⁺ channels (VDCCs). In this cellular processing, blocking RyRs (by ryanodine, 10 μM) but not IP₃Rs (by 2APB, 100 μM) completely abolished I_A downregulation, and the LTP observed in hippocampal slices was more diminished by ryanodine rather than 2APB. Furthermore, blocking RyRs also reduced Ca²⁺‐mediated PKA activation, indicating that sequential signaling cascades, including the ER and PKA, are involved in regulating I_A downregulation. These results strongly suggest a possibility that RyR contribution and mediated I_A downregulation are required to regulate membrane excitability as well as synaptic plasticity in CA3‐CA1 connections of the hippocampus. © 2017 Wiley Periodicals, Inc.     

Upregulation of L‐type Cav1 channels in the development of psychological dependence

Although L‐type voltage‐dependent Ca²⁺ channels regulate activity‐dependent processes including synaptic plasticity and synapse formation, there are few data on the changes of Ca_v1 channel expression in psychological dependence. This study investigated the role of L‐type Ca_v1 channel expression in the brain of mouse that was psychologically dependent on methamphetamine (2 mg/kg, subcutaneous injection [s.c.]), cocaine (10 mg/kg, s.c.), and morphine (5 mg/kg, s.c.) with the conditioned place preference paradigm. Intracerebroventricular administration of nifedipine (3, 10, and 30 nmol/mouse) dose‐dependently reduced the development of methamphetamine‐, cocaine‐, and morphine‐induced rewarding effect. Under such conditions, protein levels of both Ca_v1.2 and Ca_v1.3 in the frontal cortex and the limbic forebrain were significantly increased on methamphetamine‐, cocaine‐, and morphine‐induced psychologically dependent mice. These findings suggest that the upregulation of Ca_v1.2 and Ca_v1.3 participated in the development of psychological dependence. Synapse 64:440–444, 2010. © 2010 Wiley‐Liss, Inc.     

Involvement of PKA and ERK pathways in ghrelin‐induced long‐lasting potentiation of excitatory synaptic transmission in the CA1 area of rat hippocampus

Acute effects of ghrelin on excitatory synaptic transmission were evaluated on hippocampal CA1 synapses. Ghrelin triggered an enduring enhancement of synaptic transmission independently of NMDA receptor activation and probably via postsynaptic modifications. This ghrelin‐mediated potentiation resulted from the activation of GHS‐R1a receptors as it was mimicked by the selective agonist JMV1843 and blocked by the selective antagonist JMV2959. This potentiation also required the activation of PKA and ERK pathways to occur as it was inhibited by KT5720 and U0126, respectively. Moreover it most probably involved Ca²⁺ influxes as both ghrelin and JMV1843 elicited intracellular Ca²⁺ increases, which were dependent on the presence of extracellular Ca²⁺ and mediated by L‐type Ca²⁺ channels opening. In addition, ghrelin potentiated AMPA receptor‐mediated [Ca²⁺]i increases while decreasing NMDA receptor‐mediated ones. Thus the potentiation of synaptic transmission by GHS‐R1a at hippocampal CA1 excitatory synapses probably results from postsynaptic mechanisms involving PKA and ERK activation, which are producing long‐lasting enhancement of AMPA receptor‐mediated responses.     

Neurosteroid allopregnanolone inhibits glutamate release from rat cerebrocortical nerve terminals

Allopregnanolone, an active metabolite of progesterone, has been reported to exhibit neuroprotective activity in several preclinical models. Considering that the excitotoxicity caused by excessive glutamate is implicated in many brain disorders, the effect of allopregnanolone on glutamate release in rat cerebrocortical nerve terminals and possible underlying mechanism were investigated. We observed that allopregnanolone inhibited 4‐aminopyridine (4‐AP)‐evoked glutamate release, and this inhibition was prevented by chelating the extracellular Ca²⁺ ions and the vesicular transporter inhibitor. Allopregnanolone reduced the elevation of 4‐AP‐evoked intrasynaptosomal Ca²⁺ levels, but did not affect the synaptosomal membrane potential. In the presence of N‐, P/Q‐, and R‐type channel blockers, allopregnanolone‐mediated inhibition of 4‐AP‐evoked glutamate release was markedly reduced; however, the intracellular Ca²⁺‐release inhibitors did not affect the allopregnanolone effect. Furthermore, allopregnanolone‐mediated inhibition of 4‐AP‐evoked glutamate release was completely abolished in the synaptosomes pretreated with inhibitors of Ca²⁺/calmodulin, adenylate cyclase, and protein kinase A (PKA), namely calmidazolium, MDL12330A, and H89, respectively. Additionally, the allopregnanolone effect on evoked glutamate release was antagonized by the GABA_A receptor antagonist SR95531. Our data are the first to suggest that allopregnanolone reduce the Ca²⁺ influx through N‐, P/Q‐, and R‐type Ca²⁺ channels, through the activation of GABA_A receptors present on cerebrocortical nerve terminals, subsequently suppressing the Ca²⁺‐calmodulin/PKA cascade and decreasing 4‐AP‐evoked glutamate release.     

Local anesthetics inhibit glutamate release from rat cerebral cortex synaptosomes

Local anesthetics have been widely used for regional anesthesia and the treatment of cardiac arrhythmias. Recent studies have also demonstrated that low‐dose systemic local anesthetic infusion has neuroprotective properties. Considering the fact that excessive glutamate release can cause neuronal excitotoxicity, we investigated whether local anesthetics might influence glutamate release from rat cerebral cortex nerve terminals (synaptosomes). Results showed that two commonly used local anesthetics, lidocaine and bupivacaine, exhibited a dose‐dependent inhibition of 4‐AP‐evoked release of glutamate. The effects of lidocaine or bupivacaine on the evoked glutamate release were prevented by the chelation of extracellular Ca²⁺ ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl ‐threo‐beta‐benzyl‐oxyaspartate did not have any effect on the action of lidocaine or bupivacaine. Both lidocaine and bupivacaine reduced the depolarization‐induced increase in [Ca²⁺]_C but did not alter 4‐AP‐mediated depolarization. Furthermore, the inhibitory effect of lidocaine or bupivacaine on evoked glutamate release was prevented by blocking the Ca_v2.2 (N‐type) and Ca_v2.1 (P/Q‐type) channels, but it was not affected by blocking of the ryanodine receptors or the mitochondrial Na⁺/Ca²⁺ exchange. Inhibition of protein kinase C (PKC) and protein kinase A (PKA) also prevented the action of lidocaine or bupivacaine. These results show that local anesthetics inhibit glutamate release from rat cortical nerve terminals. This effect is linked to a decrease in [Ca²⁺]_C caused by Ca²⁺ entry through presynaptic voltage‐dependent Ca²⁺ channels and the suppression of the PKA and PKC signaling cascades. Synapse 67:568–579, 2013 . © 2013 Wiley Periodicals, Inc.     

Multiple Ca2+ Channel‐Dependent Components in Growth Hormone Secretion from Rat Anterior Pituitary Somatotrophs

The involvement of L‐type Ca²⁺ channels in both ‘basal’ and ‘stimulated’ growth hormone (GH) secretion is well established; however, knowledge regarding the involvement of non‐L‐type Ca²⁺ channels is lacking. We investigated whether non‐L‐type Ca²⁺ channels regulate GH secretion from anterior pituitary (AP) cells. To this end, GH secretion was monitored from dissociated AP cells, which were incubated for 15 min with 2 mm K⁺ (‘basal’ secretion) or 60 mm K⁺ (‘stimulated’ secretion). The role of non‐L‐type Ca²⁺ influx was investigated using specific channel blockers, including ω‐agatoxin‐IVA, ω‐conotoxin GVIA or SNX‐482, to block P/Q‐, N‐ or R‐type Ca²⁺ channels, respectively. Our results demonstrate that P/Q‐, N‐ and R‐type Ca²⁺ channels contributed 21.2 ± 1.9%, 20.2 ± 7.6% and 11.4 ± 1.8%, respectively, to ‘basal’ GH secretion and 18.3 ± 1.0%, 24.4 ± 5.4% and 14.2 ± 4.8%, respectively, to ‘stimulated’ GH secretion. After treatment with a ‘cocktail’ that comprised the previously described non‐L‐type blockers, non‐L‐type Ca²⁺ channels contributed 50.9 ± 0.4% and 45.5 ± 2.0% to ‘basal’ and ‘stimulated’ GH secretion, respectively. Similarly, based on the effects of nifedipine (10 μM), L‐type Ca²⁺ channels contributed 34.2 ± 3.7% and 54.7 ± 4.1% to ‘basal’ and ‘stimulated’ GH secretion, respectively. Interestingly, the relative contributions of L‐type/non‐L‐type Ca²⁺ channels to ‘stimulated’ GH secretion were well correlated with the relative contributions of L‐type/non‐L‐type Ca²⁺ channels to voltage‐gated Ca²⁺ influx in AP cells. Finally, we demonstrated that compartmentalisation of Ca²⁺ channels is important for GH secretion. Lipid raft disruption (methyl‐β‐cyclodextrin, 10 mm ) abrogated the compartmentalisation of Ca²⁺ channels and substantially reduced ‘basal’ and ‘stimulated’ GH secretion by 43.2 ± 3.4% and 58.4 ± 4.0%, respectively. In summary, we have demonstrated that multiple Ca²⁺ channel‐dependent pathways regulate GH secretion. The proper function of these pathways depends on their compartmentalisation within AP cell membranes.     

Sphingosine‐1‐phosphate induces Ca2+ signaling and CXCL1 release via TRPC6 channel in astrocytes

A biologically active lipid, sphingosine‐1‐phosphate (S1P) is highly abundant in blood, and plays an important role in regulating the growth, survival, and migration of many cells. Binding of the endogenous ligand S1P results in activation of various signaling pathways via G protein‐coupled receptors, some of which generates Ca²⁺ mobilization. In astrocytes, S1P is reported to evoke Ca²⁺ signaling, proliferation, and migration; however, the precise mechanisms underlying such responses in astrocytes remain to be elucidated. Transient receptor potential canonical (TRPC) channels are Ca²⁺‐permeable cation channels expressed in astrocytes and involved in Ca²⁺ influx after receptor stimulation. In this study, we investigated the involvement of TRPC channels in S1P‐induced cellular responses. In Ca²⁺ imaging experiments, S1P at 1 μM elicited a transient increase in intracellular Ca²⁺ in astrocytes, followed by sustained elevation. The sustained Ca²⁺ response was markedly suppressed by S1P₂ receptor antagonist JTE013, S1P₃ receptor antagonist CAY10444, or non‐selective TRPC channel inhibitor Pyr2. Additionally, S1P increased chemokine CXCL1 mRNA expression and release, which were suppressed by TRPC inhibitor, inhibition of Ca²⁺ mobilization, MAPK pathway inhibitors, or knockdown of the TRPC channel isoform TRPC6. Taken together, these results demonstrate that S1P induces Ca²⁺ signaling in astrocytes via G_q‐coupled receptors S1P₂ and S1P₃, followed by Ca²⁺ influx through TRPC6 that could activate MAPK signaling, which leads to increased secretion of the proinflammatory or neuroprotective chemokine CXCL1.     

Synaptic Multivesicular Release in the Cerebellar Cortex: Its Mechanism and Role in Neural Encoding and Processing

The number of synaptic vesicles released during fast release plays a major role in determining the strength of postsynaptic response. However, it remains unresolved how the number of vesicles released in response to action potentials is controlled at a single synapse. Recent findings suggest that the Ca_v2.1 subtype (P/Q-type) of voltage-gated calcium channels is responsible for inducing presynaptic multivesicular release (MVR) at rat cerebellar glutamatergic synapses from granule cells to molecular layer interneurons. The topographical distance from Ca_v2.1 channels to exocytotic Ca²⁺ sensors is a critical determinant of MVR. In physiological trains of presynaptic neurons, MVR significantly impacts the excitability of postsynaptic neurons, not only by increasing peak amplitude but also by prolonging decay time of the postsynaptic currents. Therefore, MVR contributes additional complexity to neural encoding and processing in the cerebellar cortex.     

R‐type Ca2+ channels contribute to fast synaptic excitation and action potentials in subsets of myenteric neurons in the guinea pig intestine

Background R‐type Ca²⁺ channels are expressed by myenteric neurons in the guinea pig ileum but the specific function of these channels is unknown. Methods In the present study, we used intracellular electrophysiological techniques to determine the function of R‐type Ca²⁺ channels in myenteric neurons in the acutely isolated longitudinal muscle‐myenteric plexus. We used immunohistochemical methods to localize the Ca_V2.3 subunit of the R‐type Ca²⁺ channel in myenteric neurons. We also studied the effects of the non‐selective Ca²⁺ channel antagonist, CdCl₂ (100 μmol L^?1), the R‐type Ca²⁺ channel blockers NiCl₂ (50 μmol L^?1) and SNX‐482 (0.1 μmol L^?1), and the N‐type Ca²⁺ channel blocker ω‐conotoxin GVIA (CTX 0.1 μmol L^?1) on action potentials and fast and slow excitatory postsynaptic potentials (fEPSPs and sEPSPs) in S and AH neurons in vitro. Key Results Ca_V2.3 co‐localized with calretinin and calbindin in myenteric neurons. NiCl₂ and SNX‐482 reduced the duration and amplitude of action potentials in AH but not S neurons. NiCl₂ inhibited the afterhyperpolarization in AH neurons. ω‐conotoxin GVIA, but not NiCl₂, blocked sEPSPs in AH neurons. NiCl₂ and SNX‐482 inhibited cholinergic, but not cholinergic/purinergic, fEPSPs in S neurons. Conclusions and Inferences These data show that R‐type Ca²⁺ channels contribute to action potentials, but not slow synaptic transmission, in AH neurons. R‐type Ca²⁺ channels contribute to release of acetylcholine as the mediator of fEPSPs in some S neurons. These data indicate that R‐type Ca²⁺ channels may be a target for drugs that selectively modulate activity of AH neurons or could alter fast synaptic excitation in specific pathways in the myenteric plexus.     

γ‐Aminobutyric acid‐mediated regulation of the activity‐dependent olfactory bulb dopaminergic phenotype

γ‐Aminobutyric acid (GABA) regulates the proliferation and migration of olfactory bulb (OB) interneuron progenitors derived from the subventricular zone (SVZ), but the role of GABA in the differentiation of these progenitors has been largely unexplored. This study examines the role of GABA in the differentiation of OB dopaminergic interneurons using neonatal forebrain organotypic slice cultures prepared from transgenic mice expressing green fluorescent protein (GFP) under the control of the tyrosine hydroxylase (Th) gene promoter (ThGFP). KCl‐mediated depolarization of the slices induced ThGFP expression. The addition of GABA to the depolarized slices further increased GFP fluorescence by inducing ThGFP expression in an additional set of periglomerular cells. These findings show that GABA promoted differentiation of SVZ‐derived OB dopaminergic interneurons and suggest that GABA indirectly regulated Th expression and OB dopaminergic neuron differentiation through an acceleration of the maturation rate for the dopaminergic progenitors. Additional studies revealed that the effect of GABA on ThGFP expression required activation of L‐ and P/Q‐type Ca²⁺ channels as well as GABA_A and GABA_B receptors. These voltage‐gated Ca²⁺ channels and GABA receptors have previously been shown to be required for the coexpressed GABAergic phenotype in the OB interneurons. Together, these findings suggest that Th expression and the differentiation of OB dopaminergic interneurons are coupled to the coexpressed GABAergic phenotype and demonstrate a novel role for GABA in neurogenesis. © 2009 Wiley‐Liss, Inc.     

Developmental increase in D1‐like dopamine receptor‐mediated inhibition of glutamatergic transmission through P/Q‐type channel regulation in the basal forebrain of rats

Whole‐cell patch‐clamp recordings of non‐N‐methyl‐d ‐aspartate glutamatergic excitatory postsynaptic currents (EPSCs) were carried out from cholinergic neurons in slices of basal forebrain (BF) of developing rats aged 21–42 postnatal days to elucidate postnatal developmental change in Ca²⁺ channel subtypes involved in the transmission as well as that in dopamine D₁‐like receptor‐mediated presynaptic inhibition. The amplitude of EPSCs was inhibited by bath application of ω‐conotoxin GVIA (ω‐CgTX; 3 μm ) or ω‐agatoxin‐TK (ω‐Aga‐TK; 200 nm ) throughout the age range examined, suggesting that multiple types of Ca²⁺ channel are involved in the transmission. The EPSC fraction reduced by ω‐CgTX decreased with age, whereas that reduced by ω‐Aga‐TK increased. Inhibition of the EPSCs by a D₁‐like receptor agonist, SKF 81297 (SKF; 30 μm ) increased with age in parallel with the increase in ω‐Aga‐TK‐induced inhibition. An activator of the adenylyl cyclase (AC) pathway, forskolin (FK; 10 μm ) inhibited the EPSCs, and FK‐induced inhibition also increased with age in parallel with the increase in SKF‐induced inhibition. Throughout the age range examined, SKF showed no further inhibitory effect on the EPSCs after ω‐Aga‐TK‐ or FK‐induced effect had reached steady‐state. These findings suggest that D₁‐like receptor‐mediated presynaptic inhibition of glutamate release onto cholinergic BF neurons increases with age, and that the change is coupled with a developmental increase in the contribution of P/Q‐type Ca²⁺ channels as well as a developmental increase in AC pathway contribution.