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.     

Curcumin inhibits glutamate release in nerve terminals from rat prefrontal cortex: possible relevance to its antidepressant mechanism

There is abundant evidence suggesting the relevance of glutamate to depression and antidepressant mechanisms. Curcumin, a major active compound of Curcuma longa, has been reported to have the biological function of antidepressant. The aim of the present study was to investigate the effect of curcumin on endogenous glutamate release in nerve terminals of rat prefrontal cortex and the underlying mechanisms. The results showed that curcumin inhibited the release of glutamate that was evoked by exposing synaptosomes to the K⁺ channel blocker 4-aminopyridine (4-AP). This phenomenon was blocked by the chelating the extracellular Ca²⁺ ions, and by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-β-benzyl-oxyaspartate (DL-TBOA). Further experiments demonstrated that curcumin decreased depolarization-induced increase in [Ca²⁺]_C, whereas it did not alter the resting membrane potential or 4-AP-mediated depolarization. Furthermore, the inhibitory effect of curcumin on evoked glutamate release was prevented by blocking the Ca_v2.2 (N-type) and Ca_v2.1 (P/Q-type) channels, but not by blocking intracellular Ca²⁺ release or Na⁺/Ca²⁺ exchange. These results suggest that curcumin inhibits evoked glutamate release from rat prefrontocortical synaptosomes by the suppression of presynaptic Ca_v2.2 and Ca_v2.1 channels. Additionally, we also found that the inhibitory effect of curcumin on 4-AP-evoked glutamate release was completely abolished by the clinically effective antidepressant fluoxetine. This suggests that curcumin and fluoxetine use a common intracellular mechanism to inhibit glutamate release from rat prefrontal cortex nerve terminals.     

Facilitation of glutamate release from rat cerebrocortical glutamatergic nerve terminals (synaptosomes) by phosphatidylserine and phosphatidylcholine

Phosphatidylserine (PS) and phosphatidylcholine (PC) have been shown to enhance cognitive function. Considering that brain glutamatergic system is thought to participate in cognitive processing, our objective was to determine the effect of PS and PC on glutamate release from the nerve terminal (synaptosome) freshly isolated from rat cerebral cortex. Data showed that both PS and PC potently facilitate 4‐aminopyridine (4‐AP)‐evoked Ca²⁺‐dependent and Ca²⁺‐independent glutamate release. Facilitation of glutamate release by PS or PC was associated with an increase of 4‐AP‐evoked depolarization and downstream elevation of cytoplasmic free calcium concentration ([Ca²⁺]_c). In addition, glutamate release elicited by direct Ca²⁺‐entry with Ca²⁺‐ionophore (ionomycin) was also facilitated by PS or PC. Furthermore, PS‐ or PC‐mediated facilitation of 4‐AP‐evoked glutamate release was superseded or suppressed by protein kinase C (PKC) activator and inhibitor, respectively. Together, these results suggest that PS or PC effects a facilitation of glutamate exocytosis by increasing nerve terminal excitability and Ca²⁺ influx into cerebrocortical nerve terminals through a signaling cascade involving PKC. Synapse 63:215–223, 2009. © 2008 Wiley‐Liss, Inc.     

Inhibition of glutamate release by bupropion in rat cerebral cortex nerve terminals

Central glutamate neurotransmission has been postulated to play a role in pathophysiology of depression and in the mechanism of antidepressants. The present study was undertaken to elucidate the effect and the possible mechanism of bupropion, an atypical antidepressant, on endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes). Result showed that bupropion exhibited a dose-dependent inhibition of 4-aminopyridine (4-AP)-evoked release of glutamate. The effect of bupropion on the evoked glutamate release was prevented by the chelating the intrasynaptosomal Ca²⁺ ions, and by the vesicular transporter inhibitor, but was insensitive to the glutamate transporter inhibitor. Bupropion decreased depolarization-induced increase in [Ca²⁺]_C, whereas it did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. The effect of bupropion on evoked glutamate release was abolished by the N-, P- and Q-type Ca²⁺ channel blocker, but not by the ryanodine receptor blocker, or the mitochondrial Na⁺/Ca²⁺ exchanger blocker. In addition, the inhibitory effect of bupropion on evoked glutamate release was prevented by the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors. Western blot analyses showed that bupropion significantly decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), and this effect also was blocked by MEK inhibitor. These results are the first to suggest that, in rat cerebrocortical nerve terminals, bupropion suppresses voltage-dependent Ca²⁺ channel and MEK/ERK activity and in so doing inhibits evoked glutamate release. This finding may provide important information regarding the beneficial effects of bupropion in the brain.     

Quantifying the effect of light activated outer and inner retinal inhibitory pathways on glutamate release from mixed bipolar cells.

Inhibition mediated by horizontal and amacrine cells in the outer and inner retina, respectively, are fundamental components of visual processing. Here, our purpose was to determine how these different inhibitory processes affect glutamate release from ON bipolar cells when the retina is stimulated with full‐field light of various intensities. Light‐evoked membrane potential changes (ΔV_m) were recorded directly from axon terminals of intact bipolar cells receiving mixed rod and cone inputs (Mbs) in slices of dark‐adapted goldfish retina. Inner and outer retinal inhibition to Mbs was blocked with bath applied picrotoxin (PTX) and NBQX, respectively. Then, control and pharmacologically modified light responses were injected into axotomized Mb terminals as command potentials to induce voltage‐gated Ca²⁺ influx (Q^Ca) and consequent glutamate release. Stimulus‐evoked glutamate release was quantified by the increase in membrane capacitance (ΔC_m). Increasing depolarization of Mb terminals upon removal of inner and outer retinal inhibition enhanced the ΔV_m/Q^Ca ratio equally at a given light intensity and inhibition did not alter the overall relation between Q^Ca and ΔC_m. However, relative to control, light responses recorded in the presence of PTX and PTX + NBQX increased ΔC_m unevenly across different stimulus intensities: at dim stimulus intensities predominantly the inner retinal GABAergic inhibition controlled release from Mbs, whereas the inner and outer retinal inhibition affected release equally in response to bright stimuli. Furthermore, our results suggest that non‐linear relationship between Q^Ca and glutamate release can influence the efficacy of inner and outer retinal inhibitory pathways to mediate Mb output at different light intensities.     

Differential involvement of protein kinase C and protein kinase A in ghrelin-induced growth hormone and gonadotrophin release from goldfish (Carassius auratus) pituitary cells

Ghrelin (GRLN) and its receptor have been identified and characterised in goldfish brain and the pituitary, and recent evidence shows that goldfish (g)GRLN₁₉ induces both growth hormone (GH) and maturational gonadotrophin (LH) release through an extracellular Ca²⁺‐dependent mechanism in goldfish. To further understand the role of GRLN in hormone release, the present study examined the involvement of protein kinase C (PKC) and protein kinase A (PKA) in gGRLN₁₉‐induced GH and LH release and corresponding Ca²⁺ signals in primary cultures of goldfish pituitary cells. Treatments with PKC inhibitors, Bis‐II and Gö 6976, significantly reduced gGRLN₁₉‐induced GH and LH release and their corresponding intracellular Ca²⁺ signals in identified somatotrophs and gonadotrophs, respectively. gGRLN₁₉ was unable to further stimulate hormone release or Ca²⁺ signals when cells were pretreated with the PKC agonist, DiC8. PKA inhibitors, H‐89 and KT 5720, inhibited gGRLN₁₉‐induced LH release and Ca²⁺ signals in gonadotrophs but not GH release or Ca²⁺ signals in somatotrophs. Interestingly, pretreatment of pituitary cells with the adenylate cyclase activator forskolin potentiated gGRLN₁₉‐induced GH, but not LH, release, although it had no effect on intracellular Ca²⁺ signals in either cell type. Taken together, the results suggest that PKC is an important intracellular component in gGRLN₁₉‐induced GH and LH release, whereas PKA is involved in gGRLN₁₉‐elicited LH release. Furthermore, the PKA pathway potentiates gGRLN₁₉‐induced GH release via a Ca²⁺‐independent mechanism. Overall, the present study provides insight into the neuroendocrine regulation of GH and LH release by elucidating the mechanistic aspects of GRLN, a hormone involved in many critical physiological processes, including pituitary functions.     

Osthole or imperatorin‐mediated facilitation of glutamate release is associated with a synaptic vesicle mobilization in rat hippocampal glutamatergic nerve endings

Osthole and imperatorin, two active compounds of Cnidium monnieri (L.) Cusson, have previously been shown to facilitate depolarization‐evoked glutamate release from rat hippocampal nerve terminals by increasing voltage‐dependent Ca²⁺ entry. In this study, we further investigated whether osthole and imperatorin possess an action at the exocytotic machinery itself, downstream of a Ca²⁺ influx. Our data showed that ionomycin‐induced glutamate release and KCl‐evoked FM1‐43 release were facilitated by osthole and imperatorin, suggesting that some steps after Ca²⁺ entry are regulated by these two compounds. Consistent with this, osthole or imperatorin‐mediated facilitation of ionomycin‐induced glutamate release was occluded by cytochalasin D that inhibits actin polymerization, implying that the disassembly of cytoskeleton is involved. In addition, the facilitatory action of osthole or imperatorin on ionomycin‐induced glutamate release was attenuated by the Ca²⁺/calmodulin‐dependent kinase II (CaMKII) inhibitor KN62. Furthermore, Western blotting analysis further showed that osthole or imperatorin significantly increased ionomycin‐induced phosphorylation of CaMKII and synapsin I, the main presynaptic target of CaMKII. These results suggest, therefore, that osthole or imperatorin‐mediated facilitation of glutamate release involves modulation of downstream events controlling synaptic vesicle recruitment and exocytosis, possibly through an increase of CaMKII activation and synapsin I phosphorylation, thereby increasing synaptic vesicle availability for exocytosis. Synapse 64:390–396, 2010. © 2009 Wiley‐Liss, Inc.     

Ultralow concentrations of bupivacaine exert anti‐inflammatory effects on inflammation‐reactive astrocytes

Bupivacaine is a widely used, local anesthetic agent that blocks voltage‐gated Na⁺ channels when used for neuro‐axial blockades. Much lower concentrations of bupivacaine than in normal clinical use, < 10^?8 m , evoked Ca²⁺ transients in astrocytes from rat cerebral cortex, that were inositol trisphosphate receptor‐dependent. We investigated whether bupivacaine exerts an influence on the Ca²⁺ signaling and interleukin‐1β (IL‐1β) secretion in inflammation‐reactive astrocytes when used at ultralow concentrations, < 10^?8 m . Furthermore, we wanted to determine if bupivacaine interacts with the opioid‐, 5‐hydroxytryptamine‐ (5‐HT) and glutamate‐receptor systems. With respect to the μ‐opioid‐ and 5‐HT‐receptor systems, bupivacaine restored the inflammation‐reactive astrocytes to their normal non‐inflammatory levels. With respect to the glutamate‐receptor system, bupivacaine, in combination with an ultralow concentration of the μ‐opioid receptor antagonist naloxone and μ‐opioid receptor agonists, restored the inflammation‐reactive astrocytes to their normal non‐inflammatory levels. Ultralow concentrations of bupivacaine attenuated the inflammation‐induced upregulation of IL‐1β secretion. The results indicate that bupivacaine interacts with the opioid‐, 5‐HT‐ and glutamate‐receptor systems by affecting Ca²⁺ signaling and IL‐1β release in inflammation‐reactive astrocytes. These results suggest that bupivacaine may be used at ultralow concentrations as an anti‐inflammatory drug, either alone or in combination with opioid agonists and ultralow concentrations of an opioid antagonist.     

Calendar of events

Intramyofiber accumulation of β‐amyloid fragments (Aβ) is a pathologic hallmark of inclusion‐body myositis (IBM), a progressive skeletal muscle disorder. We investigated the temporal pattern of alterations in the resting cytoplasmic [Ca²⁺] ([Ca²⁺]_i) as well as the depolarization‐evoked Ca²⁺ release from the sarcoplasmic reticulum in skeletal muscle from transgenic mice expressing human βAPP (MCK‐βAPP). MCK‐βAPP mice show an age‐dependent increase in [Ca²⁺]_i along with a reduction in depolarization‐evoked Ca²⁺ release, which appear well before the other reported aspects of IBM, such as inclusion formation, inflammation, centralized nuclei, atrophy, and skeletal muscle weakness. In the young MCK‐βAPP animals the increase in resting [Ca²⁺]_i can be attributed largely to Ca²⁺ influx through nifedipine‐sensitive Ca²⁺ channels. In the adult MCK‐βAPP mice, in addition to the nifedipine‐sensitive pathway, there is also a substantial contribution by the intracellular compartments to the increase in [Ca²⁺]_i. These results suggest that β‐amyloid‐induced disuption of Ca²⁺ handling may represent an early event in the pathogenesis of IBM. Muscle Nerve, 2010     

Glutamate increases the [Ca]i but stimulates Ca-independent release of [H]GABA in cultured chick retina cells

The effect of glutamate of [Ca²⁺]_i and on [³H]γ-aminobutyric acid (GABA) release was studied on cultured chick embryonic retina cells. It was observed that glutamate (100 μM) increases the [Ca²⁺]_i by Ca²⁺ influx through Ca²⁺ channels sensitive to nitrendipine, but not to ω-conotoxin GVIA (ω-Cg Tx) (50%), and by other channels insensitive to either Ca²⁺ channel blocker. Mobilization of Ca²⁺ by glutamate required the presence of external Na⁺, suggesting that Na⁺ mobilization through the ionotropic glutamate receptors is necessary for the Ca²⁺ channels to open. The increase in [Ca²⁺]_i was not related to the release of [³H]GABA induced by glutamate, suggesting that the pathway for the entry of Ca²⁺ triggered by glutamate does not lead to exocytosis. In fact, the glutamate-induced release of [³H]GABA was significantly depressed by Ca_o²⁺, but it was dependent on Na_o⁺, just as was observed for the [³H]GABA release induced by veratridine (50 μM). The veratridine-induced release could be fully inhibited by TTX, but this toxin had no effect on the glutamate-induced [³H]GABA release. Both veratridine- and glutamate-induced [³H]GABA release were inhibited by 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid (NNC-711), a blocker of the GABA carrier. Blockade of the NMDA and non-NMDA glutamate receptors with MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, almost completely blocked the release of [³H]GABA evoked by glutamate. Continuous depolarization with 50 mM K⁺ induced maximal release of [³H]GABA of about 1.5%, which is much smaller than the release evoked by glutamate under the same conditions (6.0–6.5%). Glycine (3 μM) stimulated [³H]GABA release induced by 50 mM K⁺, and this effect was blocked by MK-801, suggesting that the effect of K⁺ on [³H]GABA release was partially mediated through the NMDA receptor which probably was stimulated by glutamate released by K⁺ depolarization. We conclude that glutamate induces Ca²⁺-independent release of [³H]GABA through reversal of the GABA carrier due to Na⁺ entry through the NMDA and non-NMDA, TTX-insensitive, channels. Furthermore the GABA carrier seems to be inhibited by Ca²⁺ entering by the pathways open by glutamate. This Ca²⁺ does not lead to exocytosis, probably because the Ca²⁺ channels used are located at sites far from the active zones.     

Deletion of Cav2.1(α1A) subunit of Ca2+‐channels impairs synaptic GABA and glutamate release in the mouse cerebellar cortex in cultured slices

Deletion of both alleles of the P/Q‐type Ca²⁺‐channel Ca_v2.1(α_1A) subunit gene in mouse leads to severe ataxia and early death. Using cerebellar slices obtained from 10 to 15 postnatal days mice and cultured for at least 3 weeks in vitro, we have analysed the synaptic alterations produced by genetically ablating the P/Q‐type Ca²⁺‐channels, and compared them with the effect of pharmacological inhibition of the P/Q‐ or N‐type channels on wild‐type littermate mice. Analysis of spontaneous synaptic currents recorded in Purkinje cells (PCs) indicated that the P/Q‐type channels play a prominent role at the inhibitory synapses afferent onto the PCs, with the effect of deleting Ca_v2.1(α_1A) partially compensated. At the granule cell (GC) to PC synapses, both N‐ and P/Q‐type Ca²⁺‐channels were found playing a role in glutamate exocytosis, but with no significant phenotypic compensation of the Ca_v2.1(α_1A) deletion. We also found that the P/Q‐ but not N‐type Ca²⁺‐channel is indispensable at the autaptic contacts between PCs. Tuning of the GC activity implicates both synaptic and sustained extrasynaptic γ‐aminobutyric acid (GABA) release, only the former was greatly impaired in the absence of P/Q‐type Ca²⁺‐channels. Overall, our data demonstrate that both P/Q‐ and N‐type Ca²⁺‐channels play a role in glutamate release, while the P/Q‐type is essential in GABA exocytosis in the cerebellum. Contrary to the other regions of the CNS, the effect of deleting the Ca_v2.1(α_1A) subunit is partially or not compensated at the inhibitory synapses. This may explain why cerebellar ataxia is observed at the mice lacking functional P/Q‐type channels.     

Glutamate release evoked by glutamate receptor agonists in cultured chick retina cells: Modulation by arachidonic acid

We studied the effect of ionotropic glutamate receptor agonists on the release of endogenous glutamate or of [³H]D -aspartate from reaggregate cultures (retinospheroids) or from monolayer cultures of chick retinal cells, respectively. Kainate increased the fluorescence ratio of the Na⁺ indicator SBFI and stimulated a dose-dependent release of glutamate in low (0.1 mM) Ca²⁺ medium, as measured using a fluorometric assay. Under the same experimental conditions, the release evoked by N-methyl-D -aspartate (NMDA; 400 μM) was about half of that evoked by the same kainate concentration; α-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid (AMPA; 400 μM) did not trigger a significant response. In the presence of 1 mM CaCl₂, all of the agonists increased the [Ca²⁺]_i, as determined with the fluorescence dye Indo-1, but the glutamate release evoked by NMDA and kainate was significantly lower than that measured in 0.1 mM CaCl₂ medium. Inhibition by Ca²⁺ of the kainate-stimulated release of glutamate was partially reversed by the phospholipase A₂ inhibitor oleiloxyethyl phosphorylcholine (OPC), suggesting that the effect was mediated by the release of arachidonic acid, which inhibits the glutamate carrier. Accordingly, kainate, NMDA, and AMPA stimulated a Ca²⁺-dependent release of [³H]arachidonic acid, and the direct addition of the exogenous fatty acid to the medium decreased the release of glutamate evoked by kainate in low (0.1 mM) CaCl₂ medium. In monolayer cultures, we showed that NMDA, kainate, and AMPA also stimulated the release of [³H]D -aspartate, but in this case release in the presence of 1 mM CaCl₂ was significantly higher than that evoked in media with no added Ca²⁺. The ranking order of efficacy for stimulation of Ca²⁺-dependent release of [³H]D -aspartate was NMDA ≪ kainate < AMPA. © 1996 Wiley-Liss, Inc.     

Facilitation of glutamate release from rat cerebral cortex nerve terminal by subanesthetic concentration propofol

Propofol is now the most commonly used intravenous anesthetic‐for general anesthesia and sedation because of its rapid onset and recovery. Besides the well‐known adverse effects of cardiovascular and respiratory depression, recent studies indicate that propofol may cause excitatory phenomena such as myoclonus, opisthotonus, and even seizure. However, the mechanisms of these excitatory effects of propofol have not been elucidated. Considering glutamate as the principle excitatory neurotransmitter in the central nervous system and excessive glutamatergic synaptic transmission can cause seizure, we examined the effect of propofol on the release of glutamate from rat cerebral cortex nerve terminals (synaptosomes). Results showed that subanesthetic concentration propofol facilitated 4‐aminopyridine (4‐AP), but not KCl‐ or ionomycin‐evoked glutamate release from nerve terminals. The facilitation of 4‐AP‐evoked glutamate release by propofol also occurred in the calcium chelation and significantly attenuated by glutamate transporter inhibitors, DL ‐threo‐β‐benzyloxyaspartic acid (DL ‐TBOA) and L ‐trans‐pyrrolidine‐2,4‐dicarboxylic acid (L ‐trans‐PDC). In addition, propofol increased 4‐AP‐evoked depolarization of the plasma membrane potential. Furthermore, protein kinase C (PKC) inhibition suppressed propofol‐mediated facilitation of glutamate release. These results suggest that subanesthetic concentration propofol facilitates glutamate release from rat cerebrocortical glutamatergic terminals by increasing nerve terminal excitability, likely through the activation of PKC pathway. This finding may provide an explanation for propofol‐induced excitatory phenomena. Synapse 63:773–781, 2009. © 2009 Wiley‐Liss, Inc.     

Calcium dependence of spontaneous neurotransmitter release

Spontaneous release of neurotransmitters is regulated by extracellular [Ca²⁺] and intracellular [Ca²⁺]. Curiously, some of the mechanisms of Ca²⁺ signaling at central synapses are different at excitatory and inhibitory synapses. While the stochastic activity of voltage‐activated Ca²⁺ channels triggers a majority of spontaneous release at inhibitory synapses, this is not the case at excitatory nerve terminals. Ca²⁺ release from intracellular stores regulates spontaneous release at excitatory and inhibitory terminals, as do agonists of the Ca²⁺‐sensing receptor. Molecular machinery triggering spontaneous vesicle fusion may differ from that underlying evoked release and may be one of the sources of heterogeneity in release mechanisms.     

Neuronal input triggers Ca2+ influx through AMPA receptors and voltage-gated Ca2+ channels in oligodendrocytes

Communication between neurons and developing oligodendrocytes (OLs) leading to OL Ca²⁺ rise is critical for axon myelination and OL development. Here, we investigate signaling factors and sources of Ca²⁺ rise in OLs in the mouse brainstem. Glutamate puff or axon fiber stimulation induces a Ca²⁺ rise in pre-myelinating OLs, which is primarily mediated by Ca²⁺-permeable AMPA receptors. During glutamate application, inward currents via AMPA receptors and elevated extracellular K⁺ caused by increased neuronal activity collectively lead to OL depolarization, triggering Ca²⁺ influx via P/Q- and L-type voltage-gated Ca²⁺ (Ca_v) channels. Thus, glutamate is a key signaling factor in dynamic communication between neurons and OLs that triggers Ca²⁺ transients via AMPARs and Ca_v channels in developing OLs. The results provide a mechanism for OL Ca²⁺ dynamics in response to neuronal input, which has implications for OL development and myelination.     

Protein kinase C modulates field-evoked transmitter release from cultured rat cerebellar granule cells via a dendrotoxin-sensitive K+ channel

The role of protein kinase C (PKC) in the control of neurotransmitter release from cultured rat cerebellar granule cells was investigated. Release of preloaded [³H]-d -aspartate which is incorporated into synaptic vesicles in this preparation was evoked by electrical field stimulation or elevated KCl. PKC activation by phorbol esters resulted in a large facilitation of field-evoked Ca²⁺-dependent [³H]-d -aspartate release and a lesser enhancement of KCl-stimulated release. Inhibition of PKC by Ro 31-8220 or staurosporine virtually abolished field-evoked release but had no effect on KCl-evoked release. Field-evoked, but not KCl-evoked, synaptic vesicle exocytosis monitored by the fluorescent vesicle probe FM2-10 was inhibited by staurosporine. PKC was not directly modulating neurite Ca²⁺ channels coupled to release, as Ro 31-8220 did not inhibit these channels. Activation or inhibition of PKC modulated field-evoked plasma membrane depolarization, but had no effect on KCl-evoked depolarization, consistent with a regulation of Na⁺ or K⁺ channels activated by field stimulation. No modulation of field-evoked neurite Na⁺ influx was seen using phorbol esters. Phorbol ester-induced facilitation of field-evoked [³H]-d -aspartate release and neurite Ca²⁺ entry was non-additive with that produced by the specific K⁺ channel antagonist dendrotoxin-1, suggesting that PKC modulates transmitter release from field-stimulated cerebellar granule cells by inhibiting a dendrotoxin-1-sensitive K⁺ channel.     

Ionotropic and Metabotropic Glutamate Receptor Agonist-Induced [Ca2+]i Increase in Isolated Rat Supraoptic Neurons

In the present study, the effects of glutamate and of agonists for ionotropic and metabotropic glutamate receptors on intracellular Ca²⁺ concentration ([Ca²⁺]_i) were investigated in neurons of the rat supraoptic nucleus (SON). We used the intracellular Ca²⁺ imaging technique with fura-2, in single magnocellular neurons dissociated from the SON of rats. Glutamate (10^?6?10^?4 M) evoked a dose-dependent increase in [Ca²⁺]_i. The glutamate agonists exerted similar effects, although with some differences in the characteristics of their responses. The [Ca²⁺]_i response to NMDA was smaller than those of glutamate or the non-NMDA receptor agonists, AMPA and kainate, but was significantly enhanced by the removal of extracellular Mg²⁺. Glutamate, as well as quisqualate, an agonist for both ionotropic and metabotropic glutamate receptors, evoked a [Ca²⁺]_i increase in a Ca²⁺-free condition, suggesting Ca²⁺ release from intracellular Ca²⁺ stores. This was further evidenced by [Ca²⁺]_i increases in response to a more selective metabotropic glutamate receptor agonist, t-ACPD, in the absence of extracellular Ca²⁺. Furthermore, the quisqualate-induced Ca²⁺ release was abolished by the selective metabotropic glutamate receptor antagonist, (S)-4-carboxyphenylglycine. The results suggest that metabotropic glutamate receptors as well as non-NMDA and NMDA receptors are present in the SON neurons, and that activation of the first leads to Ca²⁺ release from intracellular Ca²⁺ stores and the activation of the latter two types induces Ca²⁺ entry. These dual mechanisms of Ca²⁺ signalling may play a role in the regulation of SON neurosecretory cells by glutamate.     

P2X7R‐mediated Ca2+‐independent d‐serine release via pannexin‐1 of the P2X7R‐pannexin‐1 complex in astrocytes

d ‐serine is a coagonist of N‐methyl‐d ‐aspartate (NMDA) subtype of glutamate receptor and plays a role in regulating activity‐dependent synaptic plasticity. In this study, we examined the mechanism by which extracellular ATP triggers the release of d ‐serine from astrocytes and discovered a novel Ca²⁺‐independent release mechanism mediated by P2X₇ receptors (P2X₇R). Using [³H] d ‐serine, which was loaded into astrocytes via the neutral amino acid transporter 2 (ASCT2), we observed that ATP and a potent P2X₇R agonist, 2′(3′)‐O‐(4‐benzoylbenzoyl)adenosine‐5′‐triphosphate (BzATP), stimulated [³H]D‐serine release and that were abolished by P2X₇R selective antagonists and by shRNAs, whereas enhanced by removal of intracellular or extracellular Ca²⁺. The P2X₇R‐mediated d ‐serine release was inhibited by pannexin‐1 antagonists, such as carbenoxolone (CBX), probenecid (PBN), and ¹⁰Panx‐1 peptide, and shRNAs, and stimulation of P2X₇R induced P2X₇R‐pannexin‐1 complex formation. Simply incubating astrocytes in Ca²⁺/Mg²⁺‐free buffer also induced the complex formation, and that enhanced basal d ‐serine release through pannexin‐1. The P2X₇R‐mediated d ‐serine release assayed in Ca²⁺/Mg²⁺‐free buffer was enhanced as well, and that was inhibited by CBX. Treating astrocytes with general protein kinase C (PKC) inhibitors, such as chelerythrine, GF109203X, and staurosporine, but not Ca²⁺‐dependent PKC inhibitor, Gö6976, inhibited the P2X₇R‐mediated d ‐serine release. Thus, we conclude that in astrocytes, P2X₇R‐pannexin‐1 complex formation is crucial for P2X₇R‐mediated d ‐serine release through pannexin‐1 hemichannel. The release is Ca²⁺‐independent and regulates by a Ca²⁺‐independent PKC. The activated P2X₇R per se is also functioned as a permeation channel to release d ‐serine in part. This P2X₇R‐mediated d ‐serine release represents an important mechanism for activity‐dependent neuron‐glia interaction. GLIA 2015;63:877–893     

Galanin inhibition of voltage‐dependent Ca2+ influx in rat cultured myenteric neurons is mediated by galanin receptor 1

Galanin activates three receptors, the galanin receptor 1 (GalR1), GalR2, and GalR3. In the gastrointestinal tract, GalR1 mediates the galanin inhibition of cholinergic transmission to the longitudinal muscle and reduction of peristalsis efficiency in the small intestine. Galanin has also been shown to inhibit depolarization‐evoked Ca²⁺ increases in cultured myenteric neurons. Because GalR1 immunoreactivity is localized to cholinergic myenteric neurons, we hypothesized that this inhibitory action of galanin on myenteric neurons is mediated by GalR1. We investigated the effect of galanin 1‐16, which has high affinity for GalR1 and GalR2, in the presence or absence of the selective GalR1 antagonist, RWJ‐57408, and of galanin 2‐11, which has high affinity for GalR2 and GalR3, on Ca²⁺ influx through voltage‐dependent Ca²⁺ channels in cultured myenteric neurons. Myenteric neurons were loaded with fluo‐4 and depolarized by high K⁺ concentration to activate voltage‐dependent Ca²⁺ channels. Intracellular Ca²⁺ levels were quantified with confocal microscopy. Galanin 1‐16 (0.01–1 μM) inhibited the depolarization‐evoked Ca²⁺ increase in a dose‐dependent manner with an EC₅₀ of 0.172 μM. The selective GalR1 antagonist, RWJ‐57408 (10 μM), blocked the galanin 1‐16 (1 μM)‐mediated inhibition of voltage‐dependent Ca²⁺ channel. By contrast, the GalR2/GalR3 agonist, galanin 2‐11 did not affect the K⁺‐evoked Ca²⁺ influx in myenteric neurons. GalR1 immunoreactivity was localized solely to myenteric neurons in culture, as previously observed in intact tissue. These findings indicate that the inhibition of depolarization‐evoked Ca²⁺ influx in myenteric neurons in culture is mediated by GalR1 and confirm the presence of functional GalR1 in the myenteric plexus. This is consonant with the hypothesis that GalR1 mediates galanin inhibition of transmitter release from myenteric neurons. © 2008 Wiley‐Liss, Inc.     

Glutamate‐induced calcium increase mediates magnesium release from mitochondria in rat hippocampal neurons

Excess administration of glutamate is known to induce Ca²⁺ overload in neurons, which is the first step in excitotoxicity. Although some reports have suggested a role for Mg²⁺ in the excitotoxicity, little is known about its actual contribution. To investigate the role of Mg²⁺ in the excitotoxicity, we simultaneously measured intracellular Ca²⁺ and Mg²⁺, using fluorescent dyes, Fura red, a fluorescent Ca²⁺ probe, and KMG‐104, a highly selective fluorescent Mg²⁺ probe developed by our group, respectively. Administration of 100 μM glutamate supplemented with 10 μM glycine to rat hippocampal neurons induced an increase in intracellular Mg²⁺ concentration ([Mg²⁺]_i). Extracellular Mg²⁺ was not required for this glutamate‐induced increase in [Mg²⁺]_i, and no increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) or [Mg²⁺]_i was observed in neurons in nominally Ca²⁺‐free medium. Application of 5 μM carbonyl cyanide p‐(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of mitochondrial inner membrane potential, also elicited increases in [Ca²⁺]_i and [Mg²⁺]_i. Subsequent administration of glutamate and glycine following FCCP treatment did not induce a further increase in [Mg²⁺]_i but did induce an additive increase in [Ca²⁺]_i. Moreover, the glutamate‐induced increase in [Mg²⁺]_i was observed only in mitochondria localized areas. These results support the idea that glutamate is able to induced Mg²⁺ efflux from mitochondria to the cytosol. Furthermore, pretreatment with Ru360, an inhibitor of the mitochondrial Ca²⁺ uniporter, prevented this [Mg²⁺]_i increase. These results indicate that glutamate‐induced increases in [Mg²⁺]_i result from the Mg²⁺ release from mitochondria and that Ca²⁺ accumulation in the mitochondria is required for this Mg²⁺ release. © 2010 Wiley‐Liss, Inc.