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.

Increasing evidence supports a role for cytokines as chemical signals in the CNS, either under normal conditions or in the pathologic state. CNS levels of the cytokine interleukin-6 (IL-6) are known to be elevated in several diseases associated with developmental disorders and may contribute to the pathological state. To investigate the potential role of IL-6 in such disorders, neuronal effects of IL-6 were examined during development using an in vitro model system, cultured rat cerebellar granule neurons. The cultures were prepared from 8 d postnatal rat pups and exposed chronically to IL-6 (5 ng/ml) by addition to the culture medium. Neuronal effects of IL-6 were assessed by a comparison of calcium signals produced in control and IL-6 treated neurons by the glutamate receptor agonists NMDA and domoate and by K+ depolarization. IL-6 treatment significantly enhanced the response to NMDA and altered the developmental pattern of NMDA sensitivity, whereas only minor changes were observed for the response to domoate and K+. Reducing extracellular calcium and depleting intracellular stores significantly decreased the amplitude of the response to NMDA in control and IL-6 treated neurons. However, the IL-6 treated neurons were significantly more sensitive to these treatments than control neurons. These results suggest that elevated levels of IL-6 can significantly alter CNS neuron development and response to excitatory transmitters, and that IL-6 pretreatment selectively enhances the intracellular calcium responses to NMDA by altering the relative contribution of extracellular calcium influx and release of calcium from stores to the calcium signal.

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.

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.

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.