Neuroprotective effects of the antifungal drug clotrimazole

Pretreatment with 10 microM of the antifungal drug clotrimazole potently reduced the death of cultured rat cerebellar granule cells induced by oxygen/glucose deprivation, and the excitotoxic effect of glutamate on cultured hippocampal neurons and cerebellar granule cells. In patch-clamped hippocampal pyramidal neurons, 10-50 microM clotrimazole caused a decrease in the amplitude of N-methyl-D-aspartate (NMDA) receptor-mediated currents. Glutamate induced intracellular Ca(2+) overload, as measured by Fluo-3 confocal fluorescence imaging, while clotrimazole reduced Ca(2+) overload and promoted the recovery of intracellular calcium homeostasis after glutamate treatment. Using tetramethylrhodamine ethyl ester fluorescence as a marker of mitochondrial membrane potential we found that clotrimazole prevented the glutamate-induced loss of mitochondrial membrane potential. Our data provide evidence that the protective effect of clotrimazole against oxygen/glucose deprivation and excitotoxicity is due to the ability of this drug to partially block NMDA receptor-gated channel, thus causing both reduced calcium overload and lower probability of the mitochondrial potential collapse.     

银杏内酯B对体外培养的视网膜神经细胞内钙离子浓度和线粒体功能的影响

目的： 观察银杏内酯B对体外培养的大鼠视网膜神经细胞内钙离子浓度和线粒体功能的影响。方法: 采用体外原代培养的大鼠视网膜神经细胞,建立谷氨酸损伤的视网膜神经细胞凋亡模型,与银杏内酯B共同培养,用激光扫描共聚焦显微镜检测对视网膜神经细胞内钙离子浓度和线粒体膜电位的影响。结果: 谷氨酸（8 mmol/L）作用后,视网膜神经细胞存活率降低,细胞凋亡增加,细胞内钙离子浓度增加,线粒体膜电位下降。GB干预后,钙离子浓度降低,线粒体膜电位显著升高,细胞凋亡明显减少。结论: GB能对抗谷氨酸兴奋性毒性, 保护视网膜神经细胞,这一作用可能是通过降低细胞内钙离子浓度和升高线粒体膜电位来实现的。     

Role of Thrombin in Activation of Neurons in Rat Hippocampus

The effect of thrombin, an agonist of proteinase-activated receptor (PAR) family, was studied on cultured rat hippocampal neurons. Thrombin in a concentration range of 1 pM - 10 nM induced a transitory dose-dependent increase in intracellular free calcium concentration. Involvement of PAR1 in neural response to thrombin was corroborated in experiments with TFLLRN, a selective synthetic peptide agonist of these receptors. In a calcium-free medium and after treatment with cyclopiazonic acid (inhibitor of Ca(2+)-ATPase in the endoplasmic reticulum) activation of PAR not only mobilized Ca(2+) from intracellular stores, but also induced Ca(2+) entry into the cells. Thrombin decreased Ca(2+) signal triggered by activation of NMDA-subtype glutamate receptors.     

Cellular mechanisms by which lipoic acid confers protection during the early stages of cerebral ischemia: a possible role for calcium

Lipoic acid (LA) is a naturally occurring compound and dietary supplement with powerful antioxidant properties. Although LA is neuroprotective in models of stroke, little is known about the cellular mechanisms by which it confers protection during the early stages of ischemia. Here, using a rat model of permanent middle cerebral artery occlusion (MCAO), we demonstrated that administration of LA 30 min prior to stroke, reduces infarct volume in a dose dependent manner. Whole-cell patch clamp techniques in rat brain slices were used to determine if LA causes any electrophysiological alterations in either healthy neurons or neurons exposed to oxygen and glucose deprivation (OGD). In healthy neurons, LA (0.005 mg/ml and 0.05 mg/ml) did not significantly change resting membrane potential, threshold or frequency of action potentials or synaptic transmission, as determined by amplitude of excitatory post synaptic currents (EPSCs). Similarly, in neurons exposed to OGD, LA did not alter the time course to loss of EPSCs. However, there was a significant delay the onset of anoxic depolarization as well as in the time course of the depolarization. Next, intracellular calcium (Ca(2+)) levels were monitored in isolated neurons using fura-2. Pretreatment with 0.005 mg/ml and 0.05 mg/ml LA for 30 min and 6 h did not significantly alter resting Ca(2+) levels or Ca(2+) response to glutamate (250 μM). However, pretreatment with 0.5 mg/ml LA for 6 h significantly increased resting Ca(2+) levels and significantly decreased the Ca(2+) response to glutamate. In summary, these findings suggest that LA does not affect neuronal physiology under normal conditions, but can protect cells from an ischemic event.     

Involvement of Na+-Ca2+ exchanger on metabotropic glutamate receptor 1-mediated [Ca2+]i transients in rat cerebellar Purkinje neurons

Cerebellar Purkinje neurons have intracellular regulatory systems including Ca2+-binding proteins, intracellular Ca2+ stores, Ca2+-ATPase and Na+-Ca2+ exchanger (NCX) that keep intracellular Ca2+ concentration ([Ca2+]i) in physiological range. Among these, NCX interacts with AMPA receptors, activation of which induces cerebellar synaptic plasticity. And the activation of metabotropic glutamate receptor 1 (mGluR1) is also involved in the induction of cerebellar long-term depression. The interaction of NCX with mGluR1 is not known yet. Thus, in this study, the functional relationship between NCX and mGluR1 in modulating the [Ca2+]i in rat Purkinje neurons was investigated. The interaction between NCX and mGluR1 in Purkinje neurons was studied by measuring intracellular Ca2+ transients induced by an agonist of group I mGluRs, 3,5-dihydroxyphenylglycine (DHPG). The DHPG-induced Ca2+ transient was significantly reduced by treatments of NCX inhibitors, bepridil and KB-R7943. When cells were pretreated with antisense oligodeoxynucleotides of NCX, the DHPG-induced Ca2+ transient was also inhibited. These results suggest that NCX modulates the activity of mGluR1 in cerebellar Purkinje neurons. Therefore, NCX appears to play an important role in the physiological function of cerebellar Purkinje neurons such as synaptic plasticity.     

Capsaicin-induced depolarisation of mitochondria in dorsal root ganglion neurons is enhanced by vanilloid receptors

Capsaicin, a pungent ingredient of hot chilli peppers, triggered Ca(2+) influx in dorsal root ganglion (DRG) neurons, which express specific vanilloid receptors of type 1, with ED(50)<100 nM. An increase in capsaicin concentration to 10 microM inhibited Ca(2+) clearance from the cytosol, but did not affect the amplitude of intracellular Ca(2+) elevation. In DRG neurons, 10 microM capsaicin also produced a significant drop in mitochondrial membrane potential (Deltapsi), as measured with the mitochondria-specific potentiometric fluorescent dye JC-1. Similar loss of mitochondrial potential upon application of capsaicin was observed in non-neuronal primary (human lymphocytes) and transformed (human myeloid leukaemia cell line, HL-60) cells. The EC(50) values for capsaicin-induced mitochondrial depolarisation were 6.9 microM (DRG neurons), 200 microM (human lymphocytes) and 150 microM (HL-60 cells). Removal of extracellular Ca(2+) or an application of the antioxidant trolox attenuated capsaicin-induced dissipation of Deltapsi in DRG neurons, but not in human lymphocytes and HL-60 cells. Rotenone, an inhibitor of complex I of the mitochondrial respiratory chain, and oligomycin, an inhibitor of F(0)F(1)-ATPase, significantly enhanced the mitochondrial depolarisation produced by capsaicin in DRG neurons. In human lymphocytes and HL-60 cells, only oligomycin potentiated the effect of capsaicin. From our results, we suggest that, in DRG neurons and non-neuronal cells, capsaicin dissipates Deltapsi, possibly due to a direct inhibition of complex I of the mitochondrial respiratory chain. The presence of vanilloid receptor-1 in DRG neurons makes their mitochondria 20-30-fold more sensitive to the depolarising effect of capsaicin compared with non-neuronal cells lacking vanilloid receptor-1. The higher sensitivity of DRG neurons to capsaicin may underlie a selective neurotoxicity of capsaicin towards sensory neurons.     

BDNF rapidly induces aspartate release from cultured CNS neurons

We investigated the effects of brain-derived neurotrophic factor (BDNF) on aspartate release from cultured cerebellar neurons. This release occurred within 1 min after the addition of 100 ng/ml BDNF. The amount of aspartate released was less than that of glutamate. Aspartate release induced by BDNF was rapid and transient, as in the case of glutamate. Although high potassium evoked the release of both excitatory (glutamate and aspartate) and inhibitory (GABA and glycine) amino acid transmitters, BDNF only induced glutamate and aspartate release. BDNF-induced aspartate release was completely blocked by pretreatment with K252a or TrkB-IgG. The aspartate release induced by BDNF was not dependent on the extracellular Ca(2+), but required intracellular Ca(2+) mobilization. These results showed that BDNF may be involved in excitatory transmission using aspartate as well as glutamate through TrkB-mediated signaling in cerebellum.     

BDNF induces calcium elevations associated with IBDNF, a nonselective cationic current mediated by TRPC channels

Brain-derived neurotrophic factor (BDNF) has potent actions on hippocampal neurons, but the mechanisms that initiate its effects are poorly understood. We report here that localized BDNF application to apical dendrites of CA1 pyramidal neurons evoked transient elevations in intracellular Ca(2+) concentration, which are independent of membrane depolarization and activation of N-methyl-d-aspartate receptors (NMDAR). These Ca(2+) signals were always associated with I(BDNF), a slow and sustained nonselective cationic current mediated by transient receptor potential canonical (TRPC3) channels. BDNF-induced Ca(2+) elevations required functional Trk and inositol-tris-phosphate (IP(3)) receptors, full intracellular Ca(2+) stores as well as extracellular Ca(2+), suggesting the involvement of TRPC channels. Indeed, the TRPC channel inhibitor SKF-96365 prevented BDNF-induced Ca(2+) elevations and the associated I(BDNF). Thus TRPC channels emerge as novel mediators of BDNF-induced intracellular Ca(2+) elevations associated with sustained cationic membrane currents in hippocampal pyramidal neurons.     

Calcium-dependent regulation of cholinergic cell phenotype in the hypothalamus in vitro

Glutamate is a major fast excitatory neurotransmitter in the CNS including the hypothalamus. Our previous experiments in hypothalamic neuronal cultures showed that a long-term decrease in glutamate excitation upregulates ACh excitatory transmission. Data suggested that in the absence of glutamate activity in the hypothalamus in vitro, ACh becomes the major excitatory neurotransmitter and supports the excitation/inhibition balance. Here, using neuronal cultures, fura-2 Ca(2+) digital imaging, and immunocytochemistry, we studied the mechanisms of regulation of cholinergic properties in hypothalamic neurons. No ACh-dependent activity and a low number (0.5%) of cholinergic neurons were detected in control hypothalamic cultures. A chronic (2 wk) inactivation of N-methyl-D-aspartate (NMDA) ionotropic glutamate receptors, L-type voltage-gated Ca(2+) channels, calmodulin, Ca(2+)/calmodulin-dependent protein kinases II/IV (CaMK II/IV), or protein kinase C (PKC) increased the number of cholinergic neurons (to 15-24%) and induced ACh activity (in 40-60% of cells). Additionally, ACh activity and an increased number of cholinergic neurons were detected in hypothalamic cultures 2 wk after a short-term (30 min) pretreatment with bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid tetrakis(acetoxy-methyl) ester (BAPTA AM; 2.5 microM), a membrane permeable Ca(2+)-chelating agent that blocks cytoplasmic Ca(2+) fluctuations. An increase in the number of cholinergic neurons following a chronic NMDA receptor blockade was likely due to the induction of cholinergic phenotypic properties in postmitotic noncholinergic neurons, as determined using 5-bromo-2'-deoxyuridine (BrdU) labeling. In contrast, a chronic inactivation of non-NMDA glutamate receptors or cGMP-dependent protein kinase had little effect on the expression of ACh properties. The data suggest that Ca(2+), at normal intracellular concentrations, tonically suppresses the development of cholinergic properties in hypothalamic neurons. However, a decrease in Ca(2+) influx into cells (through NMDA receptors or L-type Ca(2+) channels), inactivation of intracellular Ca(2+) fluctuations, or downregulation of Ca(2+)-dependent signal transduction pathways (CaMK II/IV and PKC) remove the tonic inhibition and trigger the development of cholinergic phenotype in some hypothalamic neurons. An increase in excitatory ACh transmission may represent a novel form of neuronal plasticity that regulates the activity and excitability of neurons during a decrease in glutamate excitation. This type of plasticity has apparent region-specific character and is not expressed in the cortex in vitro; neither increase in ACh activity nor change in the number of cholinergic neurons were detected in cortical cultures under all experimental conditions.     

Increase in intracellular Ca(2+) and calcitonin gene-related peptide release through metabotropic P2Y receptors in rat dorsal root ganglion neurons

We examined the effects of the activation of metabotropic P2Y receptors on the intracellular Ca(2+) concentration and the release of neuropeptide calcitonin gene-related peptide (CGRP) in isolated adult rat dorsal root ganglion neurons. In small-sized dorsal root ganglion neurons (soma diameter<30 microm) loaded with fura-2, a bath application of ATP (100 microM) evoked an increase in intracellular Ca(2+) concentration, while the removal of extracellular Ca(2+) partly depressed the response to ATP, thus suggesting that the ATP-induced increase in intracellular Ca(2+) concentration is due to both the release of Ca(2+) from intracellular stores and the influx of extracellular Ca(2+). Bath application of uridine 5'-triphosphate (UTP; 100 microM) also caused an increase in intracellular Ca(2+) concentration in small-sized dorsal root ganglion neurons and the P2 receptor antagonists suramin (100 microM) and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 10 microM) virtually abolished the response, indicating that the intracellular Ca(2+) elevation in response to UTP is mediated through metabotropic P2Y receptors. This intracellular Ca(2+) increase was abolished by pretreating the neurons with thapsigargin (100 nM), suggesting that the UTP-induced increase in intracellular Ca(2+) is primarily due to the release of Ca(2+) from endoplasmic reticulum Ca(2+) stores. An enzyme-linked immunosorbent assay showed that an application of UTP (100 microM) significantly stimulated the release of CGRP and that suramin (100 microM) totally abolished the response, suggesting that P2Y receptor-mediated increase in intracellular Ca(2+) is accompanied by CGRP release from dorsal root ganglion neurons.These results suggest that metabotropic P2Y receptors contribute to extracellular ATP-induced increase in intracellular Ca(2+) concentration and subsequent release of neuropeptide CGRP in rat dorsal root ganglion neurons.     

Na(+) and Ca(2+) homeostasis pathways, cell death and protection after oxygen-glucose-deprivation in organotypic hippocampal slice cultures

Intracellular ATP supply and ion homeostasis determine neuronal survival and degeneration after ischemic stroke. The present study provides a systematic investigation in organotypic hippocampal slice cultures of the influence of experimental ischemia, induced by oxygen-glucose-deprivation (OGD). The pathways controlling intracellular Na(+) and Ca(2+) concentration ([Na(+)](i) and [Ca(2+)](i)) and their inhibition were correlated with delayed cell death or protection. OGD induced a marked decrease in the ATP level and a transient elevation of [Ca(2+)](i) and [Na(+)](i) in cell soma of pyramidal neurons. ATP level, [Na(+)](i) and [Ca(2+)](i) rapidly recovered after reintroduction of oxygen and glucose. Pharmacological analysis showed that the OGD-induced [Ca(2+)](i) elevation in neuronal cell soma resulted from activation of both N-methyl-d-aspartate (NMDA)-glutamate receptors and Na(+)/Ca(2+) exchangers, while the abnormal [Na(+)](i) elevation during OGD was due to Na(+) influx through voltage-dependent Na(+) channels. In hippocampal slices, cellular degeneration occurring 24 h after OGD, selectively affected the pyramidal cell population through apoptotic and non-apoptotic cell death. OGD-induced cell loss was mediated by activation of ionotropic glutamate receptors, voltage-dependent Na(+) channels, and both plasma membrane and mitochondrial Na(+)/Ca(2+) exchangers. Thus, we show that neuroprotection induced by blockade of NMDA receptors and plasma membrane Na(+)/Ca(2+) exchangers is mediated by reduction of Ca(2+) entry into neuronal soma, whereas neuroprotection induced by blockade of AMPA/kainate receptors and mitochondrial Na(+)/Ca(2+) exchangers might result from reduced Na(+) entry at dendrites level.     

A hyperpolarizing response induced by glutamate in mouse cerebellar Purkinje cells.

In the vertebrate nervous system, glutamate (Glu) receptors are generally known to cause depolarizing responses. We report here a novel type of Glu response in Purkinje neurons of mouse cerebellar slices, namely glutamate-induced hyperpolarization (GH). This response is not due to activation of inhibitory interneurons, because application of tetrodotoxin (TTX), bicuculline, or strychnine did not abolish GH. In addition, GH persisted in a Ca(2+)-free or a low-Cl- solution, which rules out the involvement of gK(Ca) or GABAA mechanisms. Quisqualate (Quis) and trans-1-amino-1,3-cyclopentanedicarboxylic acid (tACPD), which are potent and selective agonists, respectively, for the metabotropic Glu receptor (mGluR), failed to induce GH. L-2-Amino-4-phosphonobutyric acid (L-AP4) was also ineffective. Simultaneous recording of electrical activity and intracellular Ca2+ concentration ([Ca2+]i) showed that GH was not accompanied by [Ca2+]i changes. Voltage clamp experiments showed that GH is due to reduction of a tonically active conductance with a reversal potential around 0 mV. Two possible mechanisms are suggested for GH: (1) changes in the desensitized steady state of ionotropic Glu receptors, or (2) a novel Glu-mediated mechanism.     

Substrates for coincidence detection and calcium signaling for induction of synaptic potentiation in the neonatal visual cortex

Regulation of the efficacy of synaptic transmission by activity-dependent processes has been implicated in learning and memory as well as in developmental processes. We previously described transient potentiation of excitatory synapses onto layer 2/3 pyramidal neurons in the visual cortex that is induced by coincident presynaptic stimulation and postsynaptic depolarization. In the adult visual cortex, activation of N-methyl-d-aspartate (NMDA) glutamate receptors is necessary to induce this plasticity. These receptors act as coincidence detectors, sensing presynaptic glutamate release and postsynaptic depolarization, and cause an influx of Ca(2+) that is necessary for the potentiation. In the neurons of the neonatal visual cortex, on the other hand, coincident presynaptic stimulation and postsynaptic depolarization induce stable long-term potentiation (LTP). In addition, reduced but significant LTP can be induced in many neurons in the presence of the NMDA receptor (NMDAR) antagonist, 2-amino-5-phosphonovaleric acid despite the Ca(2+) requirement. Therefore there must be an alternative postsynaptic Ca(2+) source and coincidence detection mechanism linked to the LTP induction mechanism in the neonatal cortex operating in addition to NMDARs. In this study, we find that in layer 2/3 pyramidal neurons, release of Ca(2+) from inositol trisphosphate (InsP(3)) receptor-mediated intracellular stores and influx through voltage-gated Ca(2+) channels (VGCCs) provide alternative postsynaptic Ca(2+) sources. We hypothesize that InsP(3)Rs are coincidence detectors, sensing presynaptic glutamate release through linkage with group I metabotropic glutamate receptors (mGluRs), and depolarization, through VGCCs. We also find that the downstream protein kinases, PKA and PKC, have a role in potentiation in layer 2/3 pyramidal neurons of the neonatal visual cortex.     

N-Methyl-D-aspartate receptor activation inhibits protein synthesis in cortical neurons independently of its ionic permeability properties

Transient cerebral ischemia, which is accompanied by a sustained release of glutamate, strongly depresses protein synthesis. We have previously demonstrated in cortical neurons that a glutamate-induced increase in intracellular Ca(2+) is likely responsible for the blockade of the elongation step of protein synthesis. In this study, we provide evidence indicating that NMDA mobilizes a thapsigargin-sensitive pool of intracellular Ca(2+). Exposure of cortical neurons to NMDA, in the absence of external Ca(2+), produced a transient rise in intracellular Ca(2+) that was suppressed by pretreatment with thapsigargin. This rise in intracellular Ca(2+) did not result from an influx of Na(+) via reversal of the mitochondrial Na(+)/Ca(2+) exchanger since it persisted in a Na(+)-free medium or in the presence of CGP 37157, an inhibitor of the exchanger. Moreover, the NMDA-induced increase in intracellular Ca(2+) required the presence of D-serine, was blocked by D(-)-2-amino-5-phosphonopentanoic acid, but was not reduced in the presence of external Mg(2+). This unexpected non-ionotropic effect of NMDA was associated with an inhibition of protein synthesis that was also insensitive to the absence of external Ca(2+) or Na(+), or presence of Mg(2+). NMDA treatment resulted in an increase in the phosphorylation of eEF-2 in the absence or presence of external Ca(2+). The initiation step of protein synthesis was not blocked by NMDA since the phosphorylation of initiation factor eIF-2alpha subunit was not altered by NMDA treatment. In conclusion, we provide evidence indicating that NMDA can inhibit protein synthesis in cortical neurons through a process that involves the mobilization of intracellular Ca(2+) stores via a mechanism that is not linked to the ionic properties of NMDA receptors.     

Role of protein kinase C in Ca2+ homeostasis disorders in cultured rat neurons during hyperstimulation of glutamate receptors

The primary culture of rat cerebellar neurons was used to study protein kinase C activity, intracellular variations in calcium concentration ([Ca²⁺]_i), changes in the mitochondrial potential, and neuronal death during hyperstimulation of glutamate receptors and after 24-h incubation with phorbol ester. Prolonged exposure of neurons to glutamate (100 μM, 45 min) was followed by the development of delayed calcium dysregulation. Protein kinase C activity depended on the time of cell incubation with glutamate. Protein kinase C activity increased in response to application of glutamate for 15 min. However, protein kinase C activity decreased after 45-min exposure to glutamate and development of delayed calcium dysregulation. Protein kinase C activity was nearly undetected after 24-h preincubation of neurons with phorbol ester. Under these conditions, delayed calcium dysregulation developed more slowly and was observed in a smaller number of neurons. Neuronal death decreased to 2±1%. Our results suggest that protein kinase C plays an important role in death of neurons, which exhibit delayed calcium dysregulation during glutamate treatment. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 5, pp. 533–537, May, 2008     

Glutamine-mediated protection from neuronal cell death depends on mitochondrial activity

The specific aim of this study was to elucidate the role of mitochondria in a neuronal death caused by different metabolic effectors and possible role of intracellular calcium ions ([Ca²⁺]_i) and glutamine in mitochondria- and non-mitochondria-mediated cell death. Inhibition of mitochondrial complex I by rotenone was found to cause intensive death of cultured cerebellar granule neurons (CGNs) that was preceded by an increase in intracellular calcium concentration ([Ca²⁺]_i). The neuronal death induced by rotenone was significantly potentiated by glutamine. In addition, inhibition of Na/K-ATPase by ouabain also caused [Ca²⁺]_i increase, but it induced neuronal cell death only in the absence of glucose. Treatment with glutamine prevented the toxic effect of ouabain and decreased [Ca²⁺]_i. Blockade of ionotropic glutamate receptors prevented neuronal death and significantly decreased [Ca²⁺]_i, demonstrating that toxicity of rotenone and ouabain was at least partially mediated by activation of these receptors. Activation of glutamate receptors by NMDA increased [Ca²⁺]_i and decreased mitochondrial membrane potential leading to markedly decreased neuronal survival under glucose deprivation. Glutamine treatment under these conditions prevented cell death and significantly decreased the disturbances of [Ca²⁺]_i and changes in mitochondrial membrane potential caused by NMDA during hypoglycemia. Our results indicate that glutamine stimulates glutamate-dependent neuronal damage when mitochondrial respiration is impaired. However, when mitochondria are functionally active, glutamine can be used by mitochondria as an alternative substrate to maintain cellular energy levels and promote cell survival.     

Synapse-specific plasticity and compartmentalized signaling in cerebellar stellate cells

Here we demonstrate that cerebellar stellate cells diffusionally isolate synaptically evoked signals in dendrites and are capable of input-specific synaptic plasticity. Sustained activity of parallel fibers induces a form of long-term depression that requires opening of calcium (Ca(2+))-permeable AMPA-type glutamate receptors (CP-AMPARs) and signaling through class 1 metabotropic glutamate receptors (mGluR1) and CB1 receptors. This depression is induced by postsynaptic increases in Ca(2+) concentration ([Ca(2+)]) and is limited to activated synapses. To understand how synapse-specific plasticity is induced by diffusible second messengers in aspiny dendrites, we examined diffusion of Ca(2+) and small molecules within stellate cell dendrites. Activation of a single parallel fiber opened CP-AMPARs, generating long-lived Ca(2+) transients that were confined to submicron dendritic stretches. The diffusion of Ca(2+) was severely retarded due to interactions with parvalbumin and a general restriction of small molecule mobility. Thus stellate cell dendrites spatially restrict signaling cascades that lead from CP-AMPAR activation to endocannabinoid production and trigger the selective regulation of active synapses.     

N-Acetylcysteine and ebselen but not nifedipine protected cerebellar granule neurons against 4-hydroxynonenal-induced neuronal death

4-Hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, has been shown to induce neurotoxicity in various types of neurons. To clarify the mechanisms underlying HNE-induced neurotoxicity, the effects of antioxidants (N-acetylcysteine (NAC) and ebselen with or without NAC pretreatment) and Ca(2+)-related reagents were examined in cerebellar granule neurons. The decreases in neuronal survival and mitochondrial membrane potential induced by HNE were suppressed by pretreatment with NAC at concentrations of 500 and 1000 microM. HNE-induced protein modification and reactive oxygen species generation were also suppressed by pretreatment with NAC at 1000 microM. Although simultaneous application of ebselen (10 microM) did not protect against HNE-induced neurotoxicity, it completely suppressed HNE-induced injury after pretreatment with NAC at 300 microM. HNE increased [Ca(2+)](i) levels, and this increase was significantly attenuated by simultaneous application of nifedipine (10 microM) or EGTA (1000 microM), but not by MK-801 or CNQX. However, none of these Ca(2+)-related reagents was able to prevent HNE-induced neuronal death or mitochondrial injury. These results suggest that pretreatment with a low concentration of NAC dramatically potentiates the neuroprotective activity of ebselen, and that HNE-induced increase in [Ca(2+)](i) is not involved in HNE-induced neuronal death in cerebellar granule neurons.     

D1/D5 dopamine receptors stimulate intracellular calcium release in primary cultures of neocortical and hippocampal neurons

D1/D5 dopamine receptors in basal ganglia, hippocampus, and cerebral cortex modulate motor, reward, and cognitive behavior. Previous work with recombinant proteins revealed that in cells primed with heterologous G(q/11)-coupled G-protein-coupled receptor (GPCR) agonists, the typically G(s)-linked D1/D5 receptors can stimulate robust release of calcium from internal stores when coexpressed with calcyon. To learn more about the intracellular signaling mechanisms underlying these D1/D5 receptor regulated behaviors, we explored the possibility that endogenous receptors stimulate internal release of calcium in neurons. We have identified a population of neurons in primary cultures of hippocampus and neocortex that respond to D1/D5 dopamine receptor agonists with a marked increase in intracellular calcium (Ca) levels. The D1/D5 receptor stimulated responses occurred in the absence of extracellular Ca(2+) indicating the rises in Ca involve release from internal stores. In addition, the responses were blocked by D1/D5 receptor antagonists. Further, the D1/D5 agonist-evoked responses were state dependent, requiring priming with agonists of G(q/11)-coupled glutamate, serotonin, muscarinic, and adrenergic receptors or with high external K(+) solution. In contrast, D1/D5 receptor agonist-evoked Ca(2+) responses were not detected in neurons derived from striatum. However, D1/D5 agonists elevated cAMP levels in striatal cultures as effectively as in neocortical and hippocampal cultures. Further, neither forskolin nor 8-Br-cAMP stimulation following priming was able to mimic the D1/D5 agonist-evoked Ca(2+) response in neocortical neurons indicating that increased cAMP levels are not sufficient to stimulate Ca release. Our data suggest that D1-like dopamine receptors likely modulate neocortical and hippocampal neuronal excitability and synaptic function via Ca(2+) as well as cAMP-dependent signaling.     

Glutamate-induced Ca2+ influx in third-order neurons of salamander retina is regulated by the actin cytoskeleton

Ligand-gated ion channels (ionotropic receptors) link to the cortical cytoskeleton via specialized scaffold proteins and thereby to appropriate signal transduction pathways in the cell. We studied the role of filamentous actin in the regulation of Ca influx through glutamate receptor-activated channels in third-order neurons of salamander retina. Staining by Alexa-Fluor 488-phalloidin, to visualize polymerized actin, we show localization of filamentous actin in neurites, and the membrane surrounding the cell soma. With Ca(2+) imaging we found that in dissociated neurons, depolymerization of filamentous actin by latrunculin A, or cytochalasin D significantly reduced glutamate-induced intracellular Ca(2+) accumulation to 53+/-7% of control value. Jasplakinolide, a stabilizer of filamentous actin, by itself slightly increased the glutamate-induced Ca(2+) signal and completely attenuated the inhibitory effect when applied in combination with actin depolymerizing agents. These results indicate that in salamander retinal neurons the actin cytoskeleton regulates Ca(2+) influx through ionotropic glutamate receptor-activated channels, suggesting regulatory roles for filamentous actin in a number of Ca(2+)-dependent physiological and pathological processes.