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.

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.

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.

Neurotoxic effects of feline immunodeficiency virus, FIV-PPR.

FIV is a lentivirus of domestic cats that causes neurologic disorders which are remarkably similar to those found in HIV-1 infected people. Using feline neuron cultures, we investigated the potential of both FIV virus and FIV-Env protein to cause neuronal damage through the excitotoxicity mechanism. The neuron swelling and lactate dehydrogenase (LDH) release assays were used as measures of cellular damage. The effects of FIV Env protein on glutamate receptor mediated increases in intracellular calcium were also examined. We found that FIV virus and FIV-Env protein significantly increased LDH release from the neuron cultures. Additionally, an increase in neuron size was detected in the cultures exposed to the virus, while swelling did not occur with exposure to either saline, denatured virus, or FIV-Env by itself. However, when both 20 microM glutamate and the FIV-PPR Env protein were added to the culture, a significant increase in neuron cell size was observed. The NMDA calcium signals were similar in general form between the control and FIV-PPR Env exposed cultures. However, the FIV - PPR Env protein treated cultures resulted in significant enhancement of the NMDA induced calcium signal. Our results indicate that FIV Env protein (either within the virion or baculovirus expressed) induced neurotoxicity as measured by neuron swelling and LDH release assays and that exposure of feline neurons to FIV Env protein alters the handling of intracellular calcium. These findings help to validate the FIV/cat system as a potential animal model for evaluating therapeutic approaches that target the excitotoxicity mechanisms of lentivirus induced CNS disease.

Neonatal alcohol exposure reduces NMDA induced Ca2+ signaling in developing cerebellar granule neurons.

Glutamatergic neurotransmission through NMDA receptors is critical for both neurogenesis and mature function of the central nervous system (CNS), and is thought to be one target for developmentally-induced damage by alcohol to brain function. In the current study we examined Ca2+ signaling linked to NMDA receptor activation as a potential site for alcohol's detrimental effects on the developing nervous system. We compared Ca2+ signals to NMDA in granule neurons cultured from cerebella of rat neonates exposed to alcohol (ethanol) during development with responses to NMDA recorded in separated control groups. Alcohol exposure was by the vapor chamber method on postnatal days 4-7. An intermittent exposure paradigm was used where the pups were exposed to alcohol vapor for 2. 5 h/day to produce peak BALs of approximately 320 mg%. Control pups were placed in an alcohol-free chamber for a similar time period or remained with their mother. After culture under alcohol-free conditions for up to 9 days, Ca2+ signaling in response to NMDA was measured using fura-2 Ca2+ imaging. Results show that the peak amplitude of the Ca2+ signal to NMDA was significantly smaller in cultured granule neurons obtained from alcohol-treated pups compared to granule neurons from control pups. In contrast, the Ca2+ signal to K+ depolarization was not depressed by the alcohol treatment. Resting Ca2+ levels were also altered by the alcohol treatment. These results show that intermittent alcohol exposure during development in vivo can induce long-term changes in CNS neurons that affect the Ca2+ signaling pathway linked to NMDA receptors and resting Ca2+ levels. Such changes could play an important role in the CNS dysfunction associated with alcohol exposure during CNS development.

Acute ethanol alters calcium signals elicited by glutamate receptor agonists and K+ depolarization in cultured cerebellar Purkinje neurons.

The effect of acute ethanol on Ca2+ signals evoked by ionotropic (iGluR) and metabotropic (mGluR) glutamate receptor (GluR) activation and K+ depolarization was examined in cultured rat cerebellar Purkinje neurons to assess the ethanol sensitivity of these Ca2+ signaling pathways. Mature Purkinje neurons approximately 3 weeks in vitro were studied. iGluRs were activated by (RS)-alpha-amino-3-hydroxyl-5 methyl-4-isoxazolepropionic acid (AMPA; 1 and 5 microM) and domoate (5 microM). mGluRs were activated by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD; 300 microM) and (R,S)-3,5-dihydroxyphenylglycine (DHPG; 200 microM). These agents and K+ (150 mM) were applied from micropipettes by brief (1 s) microperfusion pulses. Ca2+ levels were monitored at 2-3 s intervals during pre- and post-stimulus periods using microscopic digital imaging and the Ca2+ sensitive dye fura-2. iGluR and mGluR agonists and K+ produced abrupt increases in intracellular Ca2+ that slowly recovered to baseline resting levels. Acute exposure to ethanol at 33 mM (150 mg%) and 66 mM (300 mg%) significantly reduced the amplitude of the Ca2+ signals to iGluR agonists and K+ with little or no effect on Ca2+ signals to mGluR agonists. In contrast, acute ethanol at 10 mM (45 mg%) had no effect on the Ca2+ signals to the iGluR agonist AMPA but significantly enhanced the Ca2+ signals to the mGluR agonist DHPG. These results show that ethanol modulates Ca2+ signaling linked to GluR activation in a receptor subtype specific manner, and suggest that Ca2+ signaling pathways linked to GluR activation and membrane depolarization may be important mechanisms by which ethanol alters the transduction of excitatory synaptic signals at glutamatergic synapses and thereby affects intercellular and intracellular communication in the CNS.

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.

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 alcohol reduces calcium signaling elicited by glutamate receptor stimulation in developing cerebellar neurons.

The effect of chronic alcohol (33 mM ethanol) on Ca2+ signals elicited by glutamate receptor agonists (quisqualate and NMDA) was examined in developing cerebellar Purkinje and granule neurons in culture. The neurons were exposed to alcohol during the second week in culture, the main period of morphological and physiological development. The Ca2+ signals were measured with fura-2 based microscopic video imaging. Chronic exposure to alcohol during development significantly reduced the peak amplitude of the Ca2+ signals to quisqualate (1 microM; Quis) in both the somatic and dendritic regions of the Purkinje neurons. The dendritic region was affected to a greater extent than the somatic region. Granule neurons also showed a reduced somatic Ca2+ signal to Quis (dendrites not measured) in the alcohol-treated cultures, indicating that the effect was not limited to Purkinje neurons. In addition to the effects on in the response to Quis, the peak amplitude of the Ca2+ signals to NMDA (100 microM) was reduced by chronic alcohol exposure during development in both the cultured Purkinje and granule neurons. Resting Ca2+ levels were not consistently affected by alcohol treatment in either neuronal type. These results indicate that Ca2+ signaling linked to glutamate receptor activation is an important target of alcohol in the developing nervous system and could be a contributing factor in the altered CNS function and development observed in animal models of fetal alcohol syndrome.

Interleukin-6 selectively enhances the intracellular calcium response to NMDA in developing CNS neurons.

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.

Chronic exposure to alcohol during development alters the calcium currents of cultured cerebellar Purkinje neurons.

The effect of chronic exposure to alcohol during development on the calcium currents of rat cerebellar Purkinje neurons was studied in a culture model system using voltage clamp techniques. The neurons were exposed to 30 mM alcohol (ethanol) during the main period of morphological and physiological development. The calcium currents were measured at the end of the treatment period, which lasted for 8-10 days. The currents were evoked by a series of depolarizing test commands from holding potentials of -62 mV and -90 mV. The evoked currents were qualitatively similar in control and alcohol-treated neurons and were comprised of a high threshold slowly inactivating calcium current and a low threshold rapidly inactivating calcium current. The low threshold current could be observed in isolation at test potentials ranging from -50 to -30 mV. The mean peak amplitude of this current was significantly smaller in the alcohol-treated neurons compared to controls. At more depolarized test potentials, the high threshold current dominated the current response, which was characterized by an initial peak that slowly declined to a smaller relatively sustained level. The mean amplitude of the high threshold current at both peak and sustained levels was significantly larger in the alcohol-treated neurons compared to controls. Measurement of cell size indicated that alcohol-treated neurons were approximately 25% smaller than control neurons, a difference that could contribute to the smaller low threshold current observed in these neurons. These data show that chronic exposure to alcohol during the development can significantly influence the amplitude of calcium currents of the cultured Purkinje neurons.(ABSTRACT TRUNCATED AT 250 WORDS)