Somatic Ca2+ signaling in cerebellar Purkinje neurons.

Activity-driven Ca(2+) signaling plays an important role in a number of neuronal functions, including neuronal growth, differentiation, and plasticity. Both cytosolic and nuclear Ca(2+) has been implicated in these functions. In the current study, we investigated membrane-to-nucleus Ca(2+) signaling in cerebellar Purkinje neurons in culture to gain insight into the pathways and mechanisms that can initiate nuclear Ca(2+) signaling in this neuronal type. Purkinje neurons are known to express an abundance of Ca(2+) signaling molecules such as voltage-gated Ca(2+) channels, ryanodine receptors, and IP3 receptors. Results show that membrane depolarization evoked by brief stimulation with K(+) saline elicits a prominent Ca(2+) signal in the cytosol and nucleus of the Purkinje neurons. Ca(2+) influx through P/Q- and L-type voltage-gated Ca(2+) channels and Ca(2+)-induced Ca(2+) release (CICR) from intracellular stores contributed to the Ca(2+) signal, which spread from the plasma membrane to the nucleus. At strong K(+) stimulations, the amplitude of the nuclear Ca(2+) signal exceeded that of the cytosolic Ca(2+) signal, suggesting the involvement of a nuclear amplification mechanism and/or differences in Ca(2+) buffering in these two cellular compartments. An enhanced nuclear Ca(2+) signal was more prominent for Ca(2+) signals elicited by membrane depolarization than for Ca(2+) signals elicited by activation of the metabotropic glutamate receptor pathway (mGluR1), which is linked to Ca(2+) release from intracellular stores controlled by the IP3 receptor.

L-Type calcium channels mediate calcium oscillations in early postnatal Purkinje neurons.

Ca(2+) signaling is important in many fundamental neuronal processes including neurotransmission, synaptic plasticity, neuronal development, and gene expression. In cerebellar Purkinje neurons, Ca(2+) signaling has been studied primarily in the dendritic region where increases in local Ca(2+) have been shown to occur with both synaptic events and spontaneous electrical activity involving P-type voltage-gated Ca(2+) channels (VGCCs), the predominant VGCC expressed by Purkinje neurons. Here we show that Ca(2+) signaling is also a prominent feature of immature Purkinje neurons at developmental stages that precede expression of dendritic structure and involves L-type rather than P-type VGCCs. Immature Purkinje neurons acutely dissociated from postnatal day 4-7 rat pups exhibit spontaneous cytoplasmic Ca(2+) oscillations. The Ca(2+) oscillations require entry of extracellular Ca(2+), are blocked by tetrodotoxin, are communicated to the nucleus, and correlate closely with patterns of endogenously generated spontaneous and evoked electrical activity recorded in the neurons. Immunocytochemistry showed that L-, N-, and P/Q-types of VGCCs are present on the somata of the Purkinje neurons at this age. However, only the L-type VGCC antagonist nimodipine effectively antagonized the Ca(2+) oscillations; inhibitors of P/Q and N-type VGCCs were relatively ineffective. Release of Ca(2+) from intracellular Ca(2+) stores significantly amplified the Ca(2+) signals of external origin. These results show that a somatic signaling pathway that generates intracellular Ca(2+) oscillations and involves L-type VGCCs and intracellular Ca(2+) stores plays a prominent role in the Ca(2+) dynamics of early developing Purkinje neurons and may play an important role in communicating developmental cues to the nucleus.

Cannabinoids enhance NMDA-elicited Ca2+ signals in cerebellar granule neurons in culture.

A physiological role for cannabinoids in the CNS is indicated by the presence of endogenous cannabinoids and cannabinoid receptors. However, the cellular mechanisms of cannabinoid actions in the CNS have yet to be fully defined. In the current study, we identified a novel action of cannabinoids to enhance intracellular Ca2+ responses in CNS neurons. Acute application of the cannabinoid receptor agonists R(+)-methanandamide, R(+)-WIN, and HU-210 (1-50 nM) dose-dependently enhanced the peak amplitude of the Ca2+ response elicited by stimulation of the NMDA subtype of glutamate receptors (NMDARs) in cerebellar granule neurons. The cannabinoid effect was blocked by the cannabinoid receptor antagonist SR141716A and the Gi/Go protein inhibitor pertussis toxin but was not mimicked by the inactive cannabinoid analog S(-)-WIN, indicating the involvement of cannabinoid receptors. In current-clamp studies neither R(+)-WIN nor R(+)-methanandamide altered the membrane response to NMDA or passive membrane properties of granule neurons, suggesting that NMDARs are not the primary sites of cannabinoid action. Additional Ca2+ imaging studies showed that cannabinoid enhancement of the Ca2+ signal to NMDA did not involve N-, P-, or L-type Ca2+ channels but was dependent on Ca2+ release from intracellular stores. Moreover, the phospholipase C inhibitor U-73122 and the inositol 1,4,5-trisphosphate (IP3) receptor antagonist xestospongin C blocked the cannabinoid effect, suggesting that the cannabinoid enhancement of NMDA-evoked Ca2+ signals results from enhanced release from IP3-sensitive Ca2+ stores. These data suggest that the CNS cannabinoid system could serve a critical modulatory role in CNS neurons through the regulation of intracellular Ca2+ signaling.

Opioid enhancement of calcium oscillations and burst events involving NMDA receptors and L-type calcium channels in cultured hippocampal neurons.

Opioid receptor agonists are known to alter the activity of membrane ionic conductances and receptor-activated channels in CNS neurons and, via these mechanisms, to modulate neuronal excitability and synaptic transmission. In neuronal-like cell lines opioids also have been reported to induce intracellular Ca(2+) signals and to alter Ca(2+) signals evoked by membrane depolarization; these effects on intracellular Ca(2+) may provide an additional mechanism through which opioids modulate neuronal activity. However, opioid effects on resting or stimulated intracellular Ca(2+) levels have not been demonstrated in native CNS neurons. Thus, we investigated opioid effects on intracellular Ca(2+) in cultured rat hippocampal neurons by using fura-2-based microscopic Ca(2+) imaging. The opioid receptor agonist D-Ala(2)-N-Me-Phe(4),Gly-ol(5)-enkephalin (DAMGO; 1 microM) dramatically increased the amplitude of spontaneous intracellular Ca(2+) oscillations in the hippocampal neurons, with synchronization of the Ca(2+) oscillations across neurons in a given field. The effects of DAMGO were blocked by the opioid receptor antagonist naloxone (1 microM) and were dependent on functional NMDA receptors and L-type Ca(2+) channels. In parallel whole-cell recordings, DAMGO enhanced spontaneous, synaptically driven NMDA receptor-mediated burst events, depolarizing responses to exogenous NMDA and current-evoked Ca(2+) spikes. These results show that the activation of opioid receptors can augment several components of neuronal Ca(2+) signaling pathways significantly and, as a consequence, enhance intracellular Ca(2+) signals. These results provide evidence of a novel neuronal mechanism of opioid action on CNS neuronal networks that may contribute to both short- and long-term effects of opioids.

Pentobarbital augments serotonin-mediated inhibition of cerebellar Purkinje cells.

The ability of pentobarbital to modify the direct effects of iontophoretically ejected serotonin on the firing rates of cerebellar Purkinje cells was examined. Serotonin elicited inhibition, excitation, or a biphasic effect on cerebellar Purkinje cells. With continuous application of iontophoretic pentobarbital at currents found to potentiate GABA-induced inhibition, serotonin-mediated inhibitions were also augmented consistently. When application of serotonin elicited excitation, including a late component of biphasic responses, iontophoretic pentobarbital converted the effect to, primarily, inhibition. Besides increasing the magnitude of serotonin-mediated inhibition, iontophoretic pentobarbital increased the duration of this effect. In another series of experiments using pentobarbital rather than urethan as the anesthetic, serotonin-mediated inhibition was significantly augmented for all ejection currents tested. The GABA antagonists bicuculline, pentylenetetrazole and picrotoxin attenuated pentobarbital augmentation of serotonin-elicited inhibition. We conclude that serotonin-mediated inhibition of Purkinje cells is modifiable by pentobarbital and this effect bears a strong semblance to the actions of barbiturates on GABAergic neurotransmission.

Chronic ethanol exposure enhances AMPA-elicited Ca2+ signals in the somatic and dendritic regions of cerebellar Purkinje neurons.

Intracellular Ca2+ signals produced by the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA; 5 microM) were measured in the somatic and dendritic regions of cerebellar Purkinje neurons in mature cerebellar control cultures (> or = 20 days in vitro) and cultures chronically treated with 32 mM ethanol (146 mg%; 8-11 days). Recordings were made in physiological saline without ethanol. The mean peak amplitude of the Ca2+ signal elicited by AMPA (applied by brief 1-s microperfusion) in the somatic region was enhanced 38% in chronic ethanol-treated Purkinje neurons compared with control neurons. In contrast, Ca2+ signals evoked by AMPA in the dendritic region were similar in magnitude between control and chronic ethanol-treated Purkinje neurons. When tetrodotoxin (TTX; 500 nM) was included in the bath saline to block spike activity and synaptically-generated events, the mean peak amplitude of the Ca2+ signal elicited by AMPA was enhanced 60% in both the somatic and dendritic regions of chronic ethanol-treated Purkinje neurons compared with control neurons. Thus, TTX-sensitive mechanisms (i.e., spike or synaptic activity) appear to play a role in normalizing neuronal functions involved in Ca2+ signaling in the chronic ethanol-treated neurons. In parallel current clamp experiments, the resting membrane potential of chronic ethanol-treated neurons was slightly depolarized compared with control neurons. However, no differences were found between control and chronic ethanol-treated Purkinje neurons in input resistance or the peak amplitude or duration of the depolarizations or hyperpolarizations elicited by AMPA. AMPA receptors mediate fast excitatory neurotransmission in the majority of neurons in the central nervous system (CNS) and Ca2+ signals in response to AMPA receptor activation contribute to synaptic function. Thus, our results suggest that modulation of Ca2+ signals to AMPA receptor activation (or other cellular inputs) may provide an important mechanism contributing to the actions of prolonged ethanol exposure in the CNS.

Modulatory effects of acute ethanol on metabotropic glutamate responses in cultured Purkinje neurons.

Ethanol has been shown to affect several transmitter- and voltage-gated channels in the brain, although little attention has focused on potential interactions between ethanol and metabotropic glutamate receptors (mGluRs). This is of interest as mGluRs are now recognized to be important components of synaptically mediated responses, including short- and long-term changes in the efficacy of neurotransmission. Cerebellar Purkinje neurons are sensitive to the effects of ethanol and express high levels of mGluRs. We made extracellular recordings from cerebellar Purkinje neurons at 21-37 days in culture to examine the effect of ethanol on mGluR-mediated responses. mGluRs were activated by pressure ejection of 300 microM (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), a selective agonist of mGluRs, or 5 microM quisqualate (Quis). As Quis activates both ionotropic and metabotropic glutamate receptors, 50 microM 6,7-dinitroquinoxaline-2,3-dione (DNQX) was used to block the ionotropic component of Quis-mediated responses. Both ACPD and Quis produced biphasic changes in firing rates consisting of an initial brief excitatory phase (5-20 s) followed by a prolonged inhibitory phase (10 s to 2.5 min), and induced the generation of bursts. Addition of 33 mM (150 mg%) ethanol to the recording medium had little effect on ACPD-mediated responses. In the presence of 66 mM (300 mg%) ethanol, however, ACPD-mediated responses exhibited an increase in the total response duration, with no change in the percent excitation or the induction of bursts as compared to controls. On the other hand, 66 mM ethanol decreased Quis-induced burst activity, while having no effect on the percent excitation or the total response duration.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Serotonin depresses excitatory amino acid-induced excitation of cerebellar Purkinje cells in the adult rat in vivo.

The modulatory effects of serotonin (5-HT) on excitatory amino acid (EAA)-induced excitations of Purkinje cells (PCs) were examined in urethane-anesthetized adult male rats using microiontophoresis and extracellular recordings. Application of 5-HT had minimal effects on the spontaneous firing rates of PCs but depressed excitations elicited by glutamate (Glu), aspartate (Asp), kainate (KA), and quisqualate (QA), and to a lesser extent those of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). Excitations induced by the metabotropic EAA agonist, (+-)-1-aminocyclopentane-trans-1,3-dicarboxylate (t-ACPD), were unaffected by 5-HT. In summary, 5-HT depressed EAA-mediated excitations with the following rank order of effectiveness: Glu = Asp = KA = QA > AMPA >> t-ACPD. These findings suggest that 5-HT shows some selectivity in its modulation of EAA-mediated excitations of PCs and thus may serve an important neuromodulatory role in the cerebellum.

L-type Ca2+ channels contribute to current-evoked spike firing and associated Ca2+ signals in cerebellar Purkinje neurons.

The physiological properties of Purkinje neurons play a central role in their ability to regulate information transfer through the cerebellum. A number of ion channels contribute to Purkinje neuron physiology including an abundance of P-type Ca2+ channels, particularly in the dendritic region. Purkinje neurons also express L-type Ca2+ channels both during development and in the mature state. However, a role for L-type channels in Purkinje neuron physiology has yet to be fully defined. In the current study we used physiological recordings from cultured Purkinje neurons and the L-type Ca2+ channel agonist S-(-)-Bay K to assess a potential role for L-type Ca2+ channels in spike firing. Results show that Bay K alters current-evoked spike firing in young, immature Purkinje neurons without dendritic structure and in older, more mature Purkinje neurons with dendritic structure. Bay K also enhanced Ca2+ signals associated with the current-evoked spike firing. The effect of Bay K was more prominent in the young Purkinje neurons than in the older Purkinje neurons, suggesting that L-type Ca2+ channels may be more important in the Purkinje neuron physiology during the early stages of development rather than at mature stages. In the older Purkinje neurons, immunohistochemical studies using antibodies to L-type Ca2+ channels showed more intense immunolabeling in the somatic region than in the dendritic region. This result suggests that L-type Ca2+ channels may play a more important role in somatic physiology than dendritic physiology, whereas P-type channels may play a more important role in dendritic physiology.

Developmental changes in Ca2+-regulated functions of early postnatal Purkinje neurons.

Ca(2+) influx through L-type Ca(2+) channels regulates several different cellular processes in developing Purkinje neurons, including activation of transcription factors and expression of cellular proteins. In the current studies, we examined the age dependence of these actions of Ca(2+) during the early developmental period. Purkinje neurons acutely isolated from postnatal day 4-8 rat pups were studied. We also examined the sensitivity of the Ca(2+)-regulated processes to a toxic environmental factor (ethanol) known to show age-dependent actions on developing Purkinje neurons. Results show that Ca(2+) activation of the transcription factor cAMP-responsive element binding protein (CREB) and Ca(2+)-induced alterations in the level of the apoptotic enzyme caspase 3 show both dose and age dependence in the early-developing Purkinje neurons. Interestingly, the age dependence was opposite for the two proteins. Ca(2+) regulation of calbindin, a Ca(2+) binding protein, was dose dependent but showed little age dependence. Exposure to ethanol altered Ca(2+) activation of pCREB in an age-dependent manner but did not alter Ca(2+) regulation of caspase 3 or calbindin levels. Taken together, these results show that the downstream effects of Ca(2+) signaling have age-dependent components during early Purkinje neuron development. This age dependence may play an important role in the normal developmental program and could contribute to the critical window of sensitivity observed for certain toxic agents during early development.

Contribution of L-type channels to Ca2+ regulation of neuronal properties in early developing purkinje neurons.

Activity driven Ca2+ signaling is an important regulator of neuronal development. Early developing Purkinje neurons (postnatal day 5-7) prior to the stage of dendritic development express a somatic Ca2+ signaling pathway that is electrically driven and communicates information from the cell membrane to the cytosol and nucleus. In the current studies, we examined the properties and potential functional role of this pathway using acutely isolated Purkinje neurons from postnatal day 5-7 rat pups and brief K+ stimulation to activate the pathway. Results show that the amplitude of the nuclear Ca2+ signal increases as a function of the cytosolic Ca2+ signal but is larger than the cytosolic Ca2+ signal at strong K+ stimulations. Both L-type and P-type Ca2+ channels contribute to the Ca2+ signal. We also show using semiquantitative immunohistochemical methods that activation of this Ca2+ signaling pathway results in activation the transcription factor CREB and that L-type Ca2+ channels play a prominent role in this effect. The level of cfos, a transcription factor whose expression is regulated by CREB, was also increased by K+ stimulation. K+ stimulation also altered the level of the Ca2+ binding protein calbindin, an effect that involved L-type Ca2+ channels. The relationship between increases in Ca2+ and calbindin expression was bell-shaped, with high levels of Ca2+ decreasing calbindin expression. The level of the transmitter GABA was also increased by K+ stimulation but this effect was not dependent on L-type Ca2+ channels. Taken together, these results support a role for L-type channels in the phenotypic expression of Purkinje neuron properties during early development and suggest that the different activity patterns of early developing Purkinje neurons could be one mechanism for signaling the induction of specific genes through differences in cytosolic or nuclear Ca2+.

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.

Ca2+ signaling pathways linked to glutamate receptor activation in the somatic and dendritic regions of cultured cerebellar purkinje neurons.

1. Ca2+ signaling elicited by ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate (iGluR) and metabotropic (mGluR) glutamate receptor agonists was studied in the somatic and dendritic regions of cultured cerebellar Purkinje neurons using microscopic video imaging and the Ca2+ sensitive dye fura-2. 2. iGluR and mGluR agonists and K+ depolarization applied by brief micropressure pulses evoked Ca2+ signals in both the somatic and dendritic regions of all Purkinje neurons studied. The Ca2+ signals were generated simultaneously in both cellular regions. The Ca+ signals to these stimulants were similar in general form, consisting of an initial peak and slow recovery phase, but differed in details of amplitude, time course, and complexity. 3. Removal of extracellular Ca2+ abolished the Ca2+ signal to the iGluR agonist AMPA, indicating that Ca2+ influx was essential to the generation of Ca2+ signals by iGluR agonists. The Ca2+ channel blocker lanthanum almost completely eliminated the Ca2+ signals to AMPA, indicating that Ca2+ influx through voltage-sensitive Ca2+ channels was the main pathway for Ca2+ influx. Omega-agatoxin IVA, a P-type Ca2+ channel blocker, significantly reduced the Ca2+ signals to AMPA suggesting that Ca2+ influx was predominately through P-type Ca2+ channels. 4. Pharmacological manipulation of intracellular Ca2+ stores significantly reduced the Ca2+ signals to AMPA, indicating that release of Ca2+ from intracellular Ca2+ stores also plays a prominent role in the generation of the Ca2+ signals to iGluR agonists. These manipulations included blocking Ca2+ release from intracellular stores with dantrolene, an antagonist at the ryanodine receptor that controls Ca2+ release from one pool of intracellular Ca2+ stores, and depletion of intracellular Ca2+ stores with caffeine or ryanodine. 5. Ca2+ influx through voltage-sensitive Ca2+ channels did not appear to be involved in the Ca2+ signals to mGluR activation, because neither lanthanum nor omega-agatoxin IVA altered Ca2+ signals to mGluR agonists. Manipulation of intracellular stores with Ca(2+)-ATPase inhibitors and dantrolene significantly reduced the Ca2+ signal to mGluR agonists, indicating that Ca2+ signals were derived from both the inositol trisphosphate (IP3) and the ryanodine receptor-controlled intracellular Ca2+ stores. 6. Ca2+ signals to the iGluR agonist AMPA correlated temporally with the prolonged, multiphasic membrane responses elicited by similar agonist application in parallel electrophysiological studies. Pharmacological manipulation of Ca2+ influx and release of Ca2+ from intracellular stores significantly influenced components of the membrane response to AMPA, indicating a potential modulator or mediator role for Ca2+ in the membrane response to iGluR activation.

Chronic interleukin-6 alters NMDA receptor-mediated membrane responses and enhances neurotoxicity in developing CNS neurons.

Recent studies show that the cytokine interleukin-6 (IL-6) is expressed at elevated levels in the CNS in several disease states and contributes to the neuropathological process. The mechanisms through which IL-6 exerts its CNS effects are primarily unknown. We have investigated the pathophysiological effects of IL-6 on developing CNS neurons using a culture model system and a chronic treatment paradigm. Here, we show, using current- and voltage-clamp recordings, that chronic IL-6 treatment of developing cerebellar granule neurons increases the membrane and current response to NMDA and that these effects are the primary mechanism through which IL-6 produces an enhanced calcium signal to NMDA. We also show that calcium influx through voltage-sensitive calcium channels contributes to the enhanced calcium signal to NMDA in the IL-6-treated neurons in a developmentally regulated manner and that the membrane depolarization to NMDA is more sensitive to the NMDA receptor antagonist ifenprodil in the IL-6-treated neurons compared with control neurons at a late developmental stage, consistent with a larger proportion of NMDA receptors containing the NMDAR2B subunit in the IL-6-treated neurons. Additional studies show that IL-6 treatment reduces the number of granule neurons in culture and enhances neurotoxicity involving NMDA receptors. These results support a pathological role for IL-6 in the CNS and indicate that NMDA receptor-mediated functions are likely to play a critical role in neuropathological changes observed in CNS diseases associated with elevated CNS levels of IL-6.

Metabotropic glutamate receptor agonists alter neuronal excitability and Ca2+ levels via the phospholipase C transduction pathway in cultured Purkinje neurons.

Selective agonists for metabotropic glutamate receptor (mGluR) subtypes were tested on mature, cultured rat cerebellar Purkinje neurons (> or = 21 days in vitro) to identify functionally relevant mGluRs expressed by these neurons and to investigate the transduction pathways associated with mGluR-mediated changes in membrane excitability. Current-clamp recordings (nystatin/perforated-patch method) were used to measure the membrane response of Purkinje neurons to brief microperfusion pulses (1.5 s) of the group I (mGluR1/mGluR5) agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (300 microM), quisqualate (5 microM), and (R,S)-3,5-dihydroxyphenylglycine (50-500 microM). All group I mGluR agonists elicited biphasic membrane responses and burst activity in the Purkinje neurons. In addition, the group I mGluR agonists produced alterations in the active membrane properties of the Purkinje neurons and depressed the OFF response after hyperpolarizing current injection. In parallel microscopic Ca2+ imaging experiments, application of the group I mGluR agonists to fura-2-loaded cells elicited increases in intracellular Ca2+ in both the somatic and dendritic regions. The group II (mGluR2/mGluR3) agonist (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine (10 microM) and the group III (mGluR4/mGluR6/mGluR7/mGluR8) agonists L(+)-2-amino-4-phosphonobutyric acid (1 mM) and O-phospho-L-serine (200 microM) had no effect on the membrane potential or intracellular Ca2+ levels of the Purkinje neurons. The cultured Purkinje neurons, but not granule neurons or interneurons, showed immunostaining for mGluR1alpha in both the somatic and dendritic regions. All effects of the group I mGluR agonists were blocked by (+)-alpha-methyl-4-carboxyphenylglycine (1 mM), an mGluR antagonist. Furthermore, the phospholipase C inhibitor 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H -pyrrole-2,5-dione (2 microM) blocked the group I mGluR agonist-mediated electrophysiological response and greatly attenuated the Ca2+ signal elicited by group I mGluR agonists, particularly in the dendrites. The inactive analogue 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2, 5-pyrrolidine-dione (2 microM) was relatively ineffective against the electrophysiological response and Ca2+ signal. These results indicate that functional group I mGluRs (but not group II or III mGluRs) can be activated on mature Purkinje neurons in culture and result in changes in neuronal excitability and intracellular Ca2+ mediated through phospholipase C. These data obtained from a defined neuronal type, the Purkinje neuron, confirm biochemical and molecular studies on the transduction mechanisms of group I mGluRs and show that this transduction pathway is linked to neuronal excitability and intracellular Ca2+ release in the Purkinje neurons.