Chronic ethanol treatment alters AMPA-induced calcium signals in developing Purkinje neurons

Cerebellar Purkinje neurons developing in culture were treated chronically with 30 mM (140 mg%; 3-11 days in vitro) ethanol to study the actions of prolonged ethanol exposure on responses to exogenous application of AMPA, a selective agonist at the AMPA subtype of ionotropic glutamate receptors. There was no consistent difference between control and chronic ethanol-treated neurons in resting membrane potential, input resistance, or the amplitude or duration of the membrane responses to AMPA (1 or 5 microM applied by brief microperfusion) as measured using the nystatin patch method of whole cell recording. In additional studies, the Ca2+ signal to AMPA was examined using the Ca2+ sensitive dye fura-2. The mean peak Ca2+ signal elicited by 5 microM AMPA was enhanced in the dendritic region (but not the somatic region) of chronic ethanol-treated Purkinje neurons compared to control neurons. In contrast, there was no difference between control and chronic ethanol-treated neurons in the peak amplitude of the Ca2+ signal to 1 microM AMPA, whereas the recovery of the Ca2+ signals was more rapid in both somatic and dendritic regions of ethanol-treated neurons. Resting Ca2+ levels in the somatic and dendritic regions were similar between control and ethanol-treated neurons. These data show that the membrane and Ca2+ responses to AMPA in Purkinje neurons are differentially affected by prolonged ethanol exposure during development. Moreover, chronic ethanol exposure produces a selective enhancement of AMPA-evoked dendritic Ca2+ signals under conditions reflecting intense activation (i.e., 5 microM AMPA), whereas both somatic and dendritic Ca2+ signals are attenuated with smaller levels of activation (i.e., 1 microM AMPA). Because Ca2+ is an important regulator of numerous intracellular functions, chronic ethanol exposure during development could produce widespread changes in the development and function of the cerebellum.     

Acute ethanol alters the firing pattern and glutamate response of cerebellar Purkinje neurons in culture

Modified explant cultures derived from the cortical region of fetal rat cerebellum, and extracellular recording techniques were used to examine the sensitivity and response or cerebellar neurons, isolated from extracellular afferent input, to acute ethanol (EtOH) exposure. Recordings were made from Purkinje neurons (PNs) and granule cells maintained in culture for several weeks, with the emphasis on the PN. Both the PNs and granule cells exhibited spontaneous activity inculture, but, unlike the PNs, not all of the granule cells were spontaneously active. The majority of PNs studied exhibited a high frequency, regular simple spike firing pattern, previously shown to be endogenously generated by voltage-sensitive mechanisms intrinsic to the PN. The granule cells exhibited slow, irregular patterns of activity. EtOH at doses as low as 22 mM (100 mg%), a concentration that reflects blood levels during EtOH intoxication, altered the spontaneous activity of both neuronal types, demonstrating that EtOH has direct actions on cerebellar neurons. In the PNs, acute EtOH (20–80 mM) produced an increase in the regularity of the spontaneous activity and either a transient increase or no change in firing rate. Acute EtOH also significantly altered the response of PNs to the excitatory transmitter glutamate. In the granule cells, acute EtOH altered firing pattern with small and variable effects on firing rate. These data demonstrate that there are multiple sites of EtOH action in the cerebellum and that changes in PN activity with acute EtOH exposure may occur via direct actions on the PN and indirect actions via synaptically connected cerebellar neurons. The demonstration of EtOH-sensitive sites intrinsic to the cerebellum suggests that EtOH actions at these sites contribute to alterations in PN activity that occur in vivo after acute EhOH exposure.     

Development of spontaneous and glutamate-evoked activity is altered by chronic ethanol in cultured cerebellar Purkinje neurons

The effects of continuous exposure to ethanol on the cytological and physiological development of a central nervous system (CNS) neuron were studied using the cultured Purkinje neuron of the rat cerebellar cortex. Purkinje neurons in fetal rat brain cultures which are established at one day before birth show development comparable to that described in vivo in other studies. In culture, Purkinje neurons progress from immature rounded cells with fine neurites to mature neurons with a branched dendritic structure. These structural changes are accompanied by an increase in the duration and complexity of the excitatory response to glutamate, by transitions in the patterns of spontaneous activity, and by an increase in mean firing rate. Our results demonstrate that chronic exposure to a low concentration of ethanol (90 mg%; 19.5 mM) during development selectively alters the electrophysiological but not the morphological properties of Purkinje neurons. Specifically, ethanol treatment reduces the responsiveness of these neurons to glutamate, delays the expected developmental transitions in patterns of spontaneous activity, and induces increased spontaneous bursting activity, particularly at the stage of dendritic formation. Impairment of responsiveness to glutamate is significant in that it may reflect the compromise by ethanol of a major excitatory pathway in the cerebellar cortex, resulting from the decreased efficacy of glutamatergic input from parallel fibers. In contrast to the results of other studies using adult neurons as a model for the effects of ethanol, our work suggests that the developing CNS neurons does not become tolerant; that is, in the continuing presence of ethanol, it does not express physiological function equivalent to that of the control.     

Regulation of neurotransmitter enzyme by quisqualate subtype glutamate receptors in cultured cerebellar and hippocampal neurons

The possible involvement of ionotropic and metabotropic quisqualate (QA) receptors in neuronal plasticity was studied in cultured glutamtergic cerebellar or hippocampal cells in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When cerebellar of hippocampal neurons were treated with QA, it elevated the specific activity of glutaminase in a dose-dependent manner. The half-maximal effect was obtained at about 0.1 μM, the maximum increase was at about 1 μM, but levels higher than 10 μM QA produced progressive reduction in glutaminase activity. In contrast, QA had little effects on the activities of lactate dehydrogenase and aspartate aminotransferase and the amount of protein, indicating that the increase in glutaminase was relatively specific. The QA-mediated increase in glutaminase was mimicked by the ionotropic QA receptor agonist -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; EC₅₀, about 0.5 μM), but not by the metabotropic QA receptor agonist trans-(±)-1-aino-cyclopentyl-1,3,dicarboxyalte (t-ACPD; up to 0.5 mM). The specific ionotropic QA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) inhibited QA- and AMPA-mediated increases in glutaminase activity in a dose-dependent manner, whereas other glutamate receptor antagonists, -2-amino-5-phosphonovalerate, γ- -glutamyl aminomethyl sulphonic acid and γ- -glutamyl diethyl ester were ineffective. The elevation of neurotransmitter enzyme was Ca²⁺-dependent. The increase in Ca²⁺ influx essentially through the activation of L-type voltage-operated Ca²⁺ channels, and not the mobilization of internal Ca²⁺ stores, was responsible for these QA receptor-mediated long-term plastic changes in hippocampal and cerebellar neurons.     

The influence of calcium transients on intracellular pH in cortical neurons in primary culture

The objective of this study was to assess the influence of Ca²⁺ influx on intracellular pH (pH_i) of neocortical neurons in primary culture. Neurons were exposed to glutamate (100–500 μM) or KCl (50 mM), and pH_i was recorded with microspectroflurometric techniques. Additional experiments were carried out in which calcium influx was triggered by ionomycin (2 μM) or the calcium ionophore 4-Br-A23187 (2 μM). Glutamate exposure either caused no, or only a small decrease in pH_i (ΔpH ≈ 0.06 units). When a decrease was observed, a rebound rise in pH_i above control was observed upon termination of glutamate exposure. In about 20% of the cells, the acidification was more pronounced (ΔpH ≈ 0.20 units), but all these cells had high control pH_i values, and showed gradual acidification. Exposure of cells to 50 mM KCl consistently increased pH_i. Since this increase was similar in the presence and nominal absence of HCO₃⁻, it probably did not reflect influx of HCO₃⁻ via a Na⁺-HCO₃⁻ symporter. Furthermore, since it occurred in the absence of external Ca²⁺ (or a measurable rise in Ca_i²⁺) it seemed independent of Ca²⁺ influx. It is tentatively concluded that the rise in pH_i was due to reduced passive influx of H⁺ along the electrochemical gradient, which is reduced by depolarization. In Ca²⁺-containing solutions, depolarization led to a rebound increase in pH_i above control. This, and the rebound found after glutamate transients, may reflect Ca²⁺-triggered phosphorylation and upregulation of the Na⁺/H⁺ antiporter which extrudes H⁺ from the cell. Ionomycin and 4-Br-A23187 gave rise to a large rise in Ca_i²⁺ and to alkalinization of the cell (ΔpH ≈ 0.5). Since amiloride or removal of Na⁺ from the external solution did not alter the rise in pH_i, it was probably not due to accelerated H⁺ extrusion. However, removal of Ca²⁺ from extracellular fluid prevented the rise, suggesting that it was secondary to Ca²⁺/2H⁺ exchange across plasma membranes.     

Suppression of presynaptic calcium influx by metabotropic glutamate receptor agonists in neonatal rat hippocampus

Mechanisms of presynaptic inhibition by metabotropic glutamate receptor (mGluR) agonists were investigated in neonatal rat hippocampal CA1 region using the optical recording technique recently developed. Following selective loading of presynaptic terminals with a fluorescent Ca²⁺ indicator dye rhod-2 AM, changes in Ca²⁺ signals and the corresponding field excitatory postsynaptic potentials (EPSPs) induced by single electrical stimuli to the Schaffer collateral-commissural (SCC) pathway were recorded simultaneously. Application of a mGluR agonist, 1 S,3 R-1-aminocyclopentane-1,3-dicar?ylic acid (1 S,3 R-ACPD; 100 μM) or (±)-1-aminocyclopentane-trans-1,3-dicar?ylic acid (trans-ACPD; 100 μM), reversibly reduced both the field EPSP and the presynaptic Ca²⁺ transient, and the quantitative relationship between them was quite similar to that observed during application of Cd²⁺, a non-selective Ca²⁺ channel blocker, or in a Ca²⁺-free solution. Application of 4-aminopyridine (4-AP; 1 mM), a blocker of certain subtypes of voltage-dependent K⁺ channels, significantly inhibited the 1 S,3 R-ACPD effect. Application of DCG-IV, a novel mGluR2/mGluR3-selective agonist, suppressed field EPSPs only slightly even at a high dose (3 μM). These results suggest that activation of presynaptic mGluR different from mGluR2/mGluR3 suppresses the action potential-triggered Ca²⁺ influx, probably via 4-AP-sensitive mechanisms, and thereby reduces glutamate release in neonatal rat hippocampal CA1 region.     

Chronic interleukin-6 exposure alters metabotropic glutamate receptor-activated calcium signalling in cerebellar Purkinje neurons

Chronic central nervous system expression of the cytokine interleukin-6 (IL-6) is thought to contribute to the histopathological, pathophysiological, and cognitive deficits associated with various neurological disorders. However, the effects of chronic IL-6 expression on neuronal function are largely unknown. Previous studies have shown that chronic IL-6 exposure alters intrinsic electrophysiological properties and intracellular Ca2+ signalling evoked by ionotropic glutamate receptor activation in cerebellar Purkinje neurons. In the current study, using primary cultures of rat cerebellum, we investigated the effects of chronic IL-6 exposure on metabotropic glutamate receptor (mGluR)-activated Ca2+ signalling and release from intracellular Ca2+ stores. Chronic exposure (6-10 days) of Purkinje neurons to 500 units/mL IL-6 resulted in elevated resting Ca2+ levels and increased intracellular Ca2+ signals evoked by the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) compared to untreated control neurons. Chronic IL-6 treatment also augmented Ca2+ signals evoked by brief 100 mm K+ depolarization, although to a lesser degree than responses evoked by DHPG. Depleting intracellular Ca2+ stores with sarcoplasmic-endoplasmic reticulum ATPase inhibitors (thapsigargin or cyclopiazonic acid) or blocking ryanodine receptor-dependent release from intracellular stores (using ryanodine) resulted in a greater reduction of DHPG- and K+-evoked Ca2+ signals in chronic IL-6-treated neurons than in control neurons. The present data show that chronic exposure to elevated levels of IL-6, such as occurs in various neurological diseases, alters Ca2+ signalling involving release from intracellular stores. The results support the hypothesis that chronic IL-6 exposure disrupts neuronal function and thereby may contribute to the pathophysiology associated with many neurological diseases.     

Agmatine induces glutamate release and cell death in cultured rat cerebellar granule neurons

We investigated the effect of agmatine on cell viability of rat cerebellar granule neurons in a high-K+ (27.5 mM) medium. Exposure of cultured rat cerebellar granule neurons to agmatine (200-800 microM) resulted in a significant decrease in cell viability. Agmatine-induced neuronal death began to occur 6-12 h after addition, and gradually progressed. The agmatine neurotoxicity was attenuated by N-methyl-D-aspartate (NMDA) receptor antagonists and by enzymatic degradation of L-glutamate with glutamic pyruvic transaminase. Furthermore, a significant increase in extracellular L-glutamate concentration was detected before cell death occurred. In addition, agmatine-induced glutamate release and cell death were both blocked by pretreatment with botulinum toxin C, which is known to specifically inhibit the exocytosis. The agmatine neurotoxicity was not observed when extracellular K+ concentration was lower (10 mM). These results suggest that agmatine induces glutamate release through the exocytosis and thereby causes NMDA receptor-mediated neuronal death in conditions in which extracellular K+ concentrations are elevated.     

Metabotropic glutamate receptor antagonists and agonists: Potential neuroprotectors in diffuse brain injury

Our previous study has suggested that metabotropic glutamate receptors (mGluRs) were significantly involved in the secondary processes after diffuse brain injury (DBI) and that mGluRs antagonists or agonists may be used for the treatment of DBI. In the present study, the neuroprotective effects of antagonists or agonists of mGluRs on DBI were further investigated. Sprague-Dawly rats were randomized into the following six groups: (i) normal control; (ii) sham-operated control; (iii) DBI; (iv) DBI treated with normal saline (NS); (v) DBI treated with alpha-methyl-4-carboxy-phenylglycine (MCPG); and (vi) DBI treated with (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV). Animals were injected intracerebroventricularly (icv) with 10 microL MCPG (100mmol/L), DCG-IV (10nmol/L) or the equivalent volume of normal saline 1 h after injury. The neurological severity score (NSS), brain water content and the number of damaged neurons were determined 6, 12, 24, 72 and 168 h after injury. In rats with DBI, it was found that the NSS was improved and the water content in the frontal cortex and the number of damaged neurons in the parietal cortex were significantly reduced following icv injection of either MCPG or DCG-IV. This suggests that icv injection of the mGluR group I antagonist MCPG or the mGluR group II agonist DCG-IV may exert neuroprotective effects in the early stage after DBI.     

The glutamate receptor delta 2 in relation to cerebellar development and plasticity

Understanding what are the mechanisms that strengthen, stabilize and restrict synaptic innervation is a relevant topic in glutamate receptor delta 2 (GluRδ2)-related research. It also involves targeting and selection of afferent input during formation of the neuronal circuitry in the cerebellar cortex and its functioning. This review will focus on the role of GluRδ2, one of the main players in this field. Special emphasis will be placed on the processes that regulate the rapid translocation from climbing fibres to parallel fibres of GluRδ2 and the role of GluRδ2 in the reduction of supernumerary climbing fibre contacts on a single Purkinje cell. Furthermore, GluRδ2 knockout mice show ataxia and impaired motor coordination, suggesting that the presence of GluRδ2 plays an important role in controlling cerebellar functioning.     

Acute cerebellar ataxia and consecutive cerebellitis produced by glutamate receptor delta2 autoantibody

Acute cerebellar ataxia is usually a self-limited benign disease, which may develop in children after certain viral infections or vaccinations. There are several reports of acute cerebellar ataxia associated with autoantibodies. Glutamate receptor delta2, a member of the glutamate receptor family, is predominantly expressed in cerebellar Purkinje cells and plays a crucial role in cerebellar functions. To date anti-GluRdelta2 autoantibody was detected in a patient with chronic cerebellitis. Herein, an 18-month-old boy presented with cerebellar ataxia 9 days following a mild respiratory tract infection. Although cerebellar ataxia gradually improved, it worsened yet again following mumps and varicella virus infection. Cerebro-spinal fluid examination and magnetic resonance imaging of the brain demonstrated pleocytosis and meningeal enhancement, respectively. Furthermore, glutamate receptor delta2 autoantibody was detected in serum and cerebro-spinal fluid. Thus, we believe that the glutamate receptor delta2 autoantibody may play a role in cerebellar ataxia and consecutive cerebellitis.     

Sodium signals in cerebellar Purkinje neurons and Bergmann glial cells evoked by glutamatergic synaptic transmission

Glial cells express specific high-affinity transporters for glutamate that play a central role in glutamate clearance at excitatory synapses in the brain. These transporters are electrogenic and are mainly energized by the electrochemical gradient for sodium. In the present study, we combined somatic whole-cell patch-clamp recordings with quantitative Na+ imaging in fine cellular branches of cerebellar Bergmann glial cells and in dendrites of Purkinje neurons to analyze intracellular Na+ signals close to activated synapses. We demonstrate that pressure application of glutamate and glutamate agonists causes local Na+ signals in the mM range. Furthermore, we analyzed the pharmacological profile, as well as the time course and spatial distribution of Na+ signals following short synaptic burst stimulation of parallel or climbing fibers. While parallel fibers stimulation resulted in local sodium transients that were largest in processes close to the stimulation pipette, climbing fibers stimulation elicited global sodium transients throughout the entire cell. Glial sodium signals amounted to several mM, were mainly caused by sodium influx following inward transport of glutamate and persisted for tens of seconds. Sodium transients in dendrites of Purkinje neurons, in contrast, were mainly caused by activation of AMPA receptors and had much faster kinetics. By reducing the driving force for sodium-dependent glutamate uptake, intracellular sodium accumulation in glial cells upon repetitive activity might provide a negative feedback mechanism, promoting the diffusion of glutamate and the activation of extrasynaptic glutamate receptors at active synapses in the cerebellum.     

Mastoparan-induced apoptosis of cultured cerebellar granule neurons is initiated by calcium release from intracellular stores

We have recently reported that mastoparan, a peptide toxin isolated from wasp venom, induces apoptosis in cultured cerebellar granule neurons that can be blocked by cholera toxin, an activator of G_s. Measurements of intracellular free calcium concentration ([Ca²⁺]_i) reveal that mastoparan induces a dramatic elevation of [Ca²⁺]_i that is frequently followed by enhanced leakage of fura-2 out of the neurons, suggesting that this rise in [Ca²⁺]_i may be due to a more generalized change in membrane permeability. However, the mastoparan-induced initial elevation of [Ca²⁺]_i is maintained in the absence of extracellular Ca²⁺, suggesting that the rise of [Ca²⁺]_i is from intracellular stores. This conclusion is supported by the observation that depletion of [Ca²⁺]_i stores by pretreatment with either caffeine or thapsigargin attenuates both the rise in [Ca²⁺]_i and cell death induced by mastoparan. Phospholipase C (PLC) inhibitors, neomycin and U73122 block mastoparan-induced increases of [Ca²⁺]_i and protect against neuronal death. Pretreatment with cholera toxin, but not pertussis toxin, reduced the mastoparan-induced rise in [Ca²⁺]_i. Taken together, our data suggest that mastoparan initiates cell death in cerebellar granule neurons by inducing Ca²⁺ release from intracellular stores, probably via activation of PLC and IP₃. A secondary or parallel process results in disruption of plasma membrane integrity and may be ultimately responsible for the death of these neurons by mastoparan.     

Glutamate effects on calcium homeostasis in cerebellar granule cells in primary cultures grown under depolarizing and nondepolarizing conditions

Cerebellar granule cell culture are normally grown under partly depolarizing conditions (in a medium with ～25 mMK⁺), but these cultures can also be grown at a normal potassium concentration (5.4 mM k⁺), although some of their characteristics are altered. In this study, intracellular free calcium concentration and ⁴⁵Ca uptake were measured in cerebellar granule cell cultures grown at either 25 or 5.4 mM extracellular potassium in the presence of glutamate, and/or some of its subtype-specific agonists and antagonists. Granule cells in cultures grown at 25mM K⁺ responded to glutamate, but not to quisqualate, with an increase in free cytosolic calcium concentration and in ⁴⁵Ca uptake. This increase in free cytosolic calcium concentration was dependent on extracellular calcium and it was antagonized by AP5 and Ketamine, NMDA receptor antagonists. In contrast, granule cells in cultures grown at 5.4 mM K⁺ responded to both glutamate and quisqualate, and these responses were independent of extracellular calcium and not sensitive to AP5 and ketamine. In agreement with this, ⁴⁵Ca uptake was not affected by glutamate. Neither of the two culture types responded to kainate with an increase in calcium concentration or uptake. These observations indicate that calcium uptake in granule cells in cultures grown at 25mM K⁺ reflect NMDA activation of calcium influx, whereas the cells in cultures grown at 5 mM K⁺ increase cytosolic calcium concentration on account of intracellular release of bound calcium, caused by activation of the metabotropic receptor. The two types of cultures may constitute valuable experimental tools for investigation of physiological and pathological effects of calcium releas from intracellular stores induced by glutamate via the metabotropic receptors and of calcium uptake induced by NMDA. © 1993 Wiley-Liss, Inc.     

The chemokine CCL2 modulates Ca2+ dynamics and electrophysiological properties of cultured cerebellar Purkinje neurons

The chemokine CCL2 is produced at high levels in the central nervous system (CNS) during infection, injury, neuroinflammation and other pathological conditions. Cells of the CNS including neurons and glia express receptors for CCL2 and these receptors may contribute to a signaling system through which pathologic conditions in the CNS are communicated. However, our understanding of the consequences of activation of chemokine signaling in the CNS is limited, especially for neurons. In many cell types, chemokine signaling alters intracellular Ca(2+) dynamics. Therefore, we investigated the potential involvement of this mechanism in neuronal signaling activated by CCL2. In addition, we examined the effects of CCL2 on neuronal excitability. The studies focused on the rat cerebellar Purkinje neuron, an identified CNS neuronal type reported to express both CCL2 and its receptor, CCR2. Immunohistochemical studies of Purkinje neurons in situ confirmed that they express CCR2 and CCL2. The effect of exogenous application on Purkinje neurons was studied in a cerebellar culture preparation. CCL2 was tested by micropressure or bath application, at high concentrations (13-100 nm) to simulate conditions during a pathologic state. Results show that Purkinje neurons express receptors for CCL2 and that activation of these receptors alters several neuronal properties. CCL2 increased resting Ca(2+) levels, enhanced the Ca(2+) response evoked by activation of metabotropic glutamate receptor 1 and depressed action potential generation in the cultured Purkinje neurons. Passive membrane properties were unaltered. These modulatory effects of CCL2 on neuronal properties are likely to contribute to the altered CNS function associated with CNS disease and injury.     

Expression and subcellular distribution of glutamate receptor subunits 2/3 in the developing cerebellar cortex

The expression and subcellular location of glutamate receptor subunits 2&3 was investigated in the developing postnatal cerebellum. Immunoblotting revealed that glutamate receptor subunits 2/3 is expressed in an identical pattern of immunoreactive bands of approximately 108 kDa from postnatal day zero to adult animals. Light microscopy showed that within the cerebellar cortex, GluR 2/3 immunoreactivity was essentially confined to Purkinje neurons. Strong immunostaining could be observed at postnatal days 1–3 within Purkinje cell bodies and primary dendrites. With ongoing development, the cell body and an increasingly elaborate dendritic tree was outlined by immunoreaction product. In adult animals, staining of Purkinje cell dendrites was patchy, and staining intensity of the cell body, in particular, was greatly reduced. Ultrastructural analysis revealed that during early postnatal development, immunoreaction product was localized to the cell membrane, but was not confined to postsynaptic densities. From the second postnatal week, glutamate receptor subunits 2/3 immunoreactivity was largely restricted to postsynaptic densities. These observations reveal a developmentally regulated refinement of the subcellular distribution of defining subunits of the AMPA-type glutamate receptor. The presence of membrane bound receptors prior to the formation of synapses also provides a rationale for the known transmitter-mediated modulation of Purkinje cell dendritogenesis. © 1996 Wiley-Liss, Inc.     

Modulation of calcium entry and glutamate release in cultured cerebellar granule cells by palytoxin

A channel open on the membrane can be formed by palytoxin (PTX). Ten nanomolar PTX caused an irreversible increase in the cytosolic calcium concentration ([Ca(2+)](c)), which was abolished in the absence of external calcium. The increase was eliminated by saxitoxin (STX) and nifedipine (NIF). Calcium rise is secondary to the membrane depolarization. PTX effect on calcium was dependent on extracellular Na(+). Li(+) decreased the PTX-evoked rise in [Ca(2+)](c); replacement of Na(+) by N-methyl-D-glucamine (NMDG) abolished PTX-induced calcium increase. [Ca(2+)](c) increase by PTX was strongly reduced after inhibition of the reverse operation of the Na(+)/Ca(2+) exchanger, in the presence of antagonists of excitatory amino acid (EAA) receptors, and by inhibition of neurotransmitter release. PTX did not modify calcium extrusion by the plasma membrane Ca(2+)-ATPase (PMCA), because blockade of the calcium pump increased rather than decreased the PTX-induced calcium influx. Extracellular levels of glutamate and aspartate were measured by HPLC and exocytotic neurotransmitter release by determination of synaptic vesicle exocytosis using total internal reflection fluorescence microscopy (TIRFM). PTX caused a concentration-dependent increase in EAA release to the culture medium. Ten nanomolar PTX decreased cell viability by 30% within 5 min. PTX-induced calcium influx involves three pathways: Na(+)-dependent activation of voltage-dependent sodium channels (VDSC) and voltage-dependent calcium channels (VDCC), reverse operation of the Na(+)/Ca(2+) exchanger, and indirect activation of EAA receptors through glutamate release. The neuronal injury produced by the toxin could be partially mediated by the PTX-induced overactivation of EAA receptors, VDSC, VDCC and the glutamate efflux into the extracellular space.     

磷酸化C—JUN不参与谷氨酸诱导的小脑颗粒神经元凋亡

观察磷酸化 C- JUN与谷氨酸诱导的小脑颗粒神经元凋亡的关系。在培养的小脑颗粒神经元建立谷氨酸凋亡模型 ;采用 MTT法分析细胞存活率 ,相差显微镜观察形态学 ,DNA凝胶电泳法分析细胞凋亡和原位细胞荧光免疫组织化学法检测磷酸化 C- JUN。结果显示 ,谷氨酸诱导大鼠小脑颗粒神经元细胞体积缩小 ,突触断裂、消失 ,DNA电泳呈典型的“梯状”条带 ;谷氨酸处理 2 4 h后细胞存活率为 2 8.6%± 5.2 %。神经元在谷氨酸处理 5,30 min及 1 ,2 ,4,8,1 6和 2 4 h后均未检测到有磷酸化 C- JUN阳性细胞 ,与去极化组 ( 2 5mmol/L KCl)相同。而复极化组 ( 5mmol/L KCl)则在 30 min检测到大量的磷酸化 C- JUN阳性细胞 ,4h荧光最强并持续。处理 4h后 ,40 0倍荧光显微镜下 ,复极化组、去极化组和谷氨酸组的磷酸化 C- JUN阳性细胞数分别为 1 2 4± 1 7,8± 3,5± 3。上述结果提示 ,谷氨酸诱导小脑颗粒神经元凋亡 ,磷酸化 C- JUN不参与谷氨酸诱导的大鼠小脑颗粒神经元凋亡。     

Regulation of the cytosolic free calcium concentration by Na spikes in immature cerebellar neurons with N-methyl-d-aspartate receptors

N-Methyl-d-aspartate and glycine increased the cytosolic free calcium concentration ([Ca]_in) in medium-sized cerebellar neurons. Spontaneous changes in [Ca]_in were occasionally observed in NMDA-responsive cells, but large increases in [Ca]_in were triggered only through depolarizations by adding veratridine or K⁺ channel blockers in every cell examined. The [Ca]_in increase was suppressed by voltage-dependent Na⁺ and Ca²⁺ channel blockers and by an inhibitory transmitter (GABA), suggesting that the generation of Na⁺ spikes is involved in the increase in [Ca]_in.     

Attenuation of seizures induced by homocysteic acid in immature rats by metabotropic glutamate group II and group III receptor agonists

Previous studies demonstrated that selected agonists for metabotropic glutamate group II and group III receptors can provide protection against seizures in adult animals. The present study has examined the potential effect of some of these compounds on seizures induced in immature rats by intracerebroventricular infusion of DL-homocysteic acid (DL-HCA, 600 nmol/side). Rat pups were sacrificed during generalised clonic-tonic seizures, 50--60 min after infusion. Comparable time intervals were used for sacrificing the pups which had received the protective drugs. The anticonvulsant effect was evaluated according to the suppression of behavioural manifestations of seizures and the protection of energy metabolite changes which normally accompany these seizures (large decreases of glucose and glycogen, and approximately 7- to 10-fold accumulation of lactate). Partial protection was exhibited by group II mGluR agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV, 0.6 nmol) and this effect was abolished after pretreatment with an antagonist for group II mGluRs (RS)-alpha-methyl-4-tetrazolylphenylglycine (MTPG, 100 nmol). In high doses (5--100 nmol), however, DCG IV evoked seizures which were prevented by AP7, suggesting that the convulsant effect was mediated by interaction with NMDA receptors. A pronounced anticonvulsant effect against DL-HCA-induced seizures was achieved with low doses of a highly selective group II mGluR agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC, 0.6 nmol), group II agonist and group I mGluR antagonist (S)-4-carboxy-3-hydroxyphenylglycine ((S)-4-C3HPG, 0.6 nmol) and group III mGluR agonist (RS)-1-amino-3-(phosphonomethylene) cyclobutane-carboxylic acid (32 nmol). Generalised clonic--tonic seizures were completely suppressed and the metabolic changes were markedly ameliorated, there being only a 1.5-, 2- and 2.5-fold rise of lactate, respectively. Higher doses of (S)-4-C3HPG (1--100 nmol) were, however, less anticonvulsant than low doses. The present results have confirmed that mGluRs may be considered a potential target for treatment of epilepsy.