RESUMEN
The type 1 metabotropic glutamate receptor (mGluR1) plays an import role in the synaptic physiology and development of cerebellar Purkinje neurons. mGluR1 expression occurs early in the developmental program of Purkinje neurons, at an age that precedes expression of the dendritic structure. Few studies have investigated the physiological response produced by mGluR1 activation in early-developing Purkinje neurons. To address this question, simultaneous recording of membrane potential and intracellular Ca(2+) was performed in immature cultured Purkinje neurons coupled with exogenous application of mGluR1 agonists. Membrane potential was measured using the perforated patch method of whole-cell recording, and intracellular Ca(2+) was measured using fura-2-based Ca(2+) imaging. Brief, 1-sec micropressure application of the group I mGluR-selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) evoked a prominent Ca(2+) signal and coincident fast hyperpolarization in the immature neurons. The mGluR1-selective antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester blocked the Ca(2+) signal and fast hyperpolarization, confirming the involvement of mGluR1s. Amplitude of the fast hyperpolarization varied as a function of membrane potential and intracellular Ca(2+) and was blocked by apamin, an antagonist of the small-conductance Ca(2+)-activated K(+) channel (SK), identifying this K(+) channel as an underlying mechanism. In similar experiments with mature cultured Purkinje neurons, DHPG elicited a Ca(2+) signal, but fast membrane hyperpolarization was not evident. These results suggest that mGluR1 activation and the resulting release of Ca(2+) from intracellular stores and activation of SK channels may be a mechanism through which mGluR1 can modulate neuronal excitability of Purkinje neurons during early development.
Asunto(s)
Señalización del Calcio/fisiología , Potenciales de la Membrana/fisiología , Canales de Potasio/metabolismo , Células de Purkinje/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Apamina/farmacología , Membrana Celular/metabolismo , Células Cultivadas , Potenciales de la Membrana/efectos de los fármacos , Metoxihidroxifenilglicol/análogos & derivados , Metoxihidroxifenilglicol/farmacología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/efectos de los fármacosRESUMEN
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.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Glicina/análogos & derivados , Interleucina-6/metabolismo , Células de Purkinje/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Animales , Encefalopatías/inmunología , Encefalopatías/metabolismo , Encefalopatías/fisiopatología , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Esquema de Medicación , Inhibidores Enzimáticos/farmacología , Agonistas de Aminoácidos Excitadores/farmacología , Glicina/farmacología , Interleucina-6/inmunología , Interleucina-6/farmacología , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potasio/farmacología , Células de Purkinje/efectos de los fármacos , Células de Purkinje/inmunología , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/efectos de los fármacos , Resorcinoles/farmacología , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
CNS levels of the cytokine interleukin-6 (IL-6) are elevated during CNS injury and disease, but it is unclear if IL-6 contributes to the pathologic process. Our studies show that in a well-characterized CNS developmental model system, primary cultures of rodent cerebellar granule neurons, chronic exposure to IL-6 during neuronal development can result in cell damage and death in a subpopulation of developing granule neurons. Chronic exposure to IL-6 also increased the susceptibility of the granule neurons to a toxic insult produced by excessive activation of NMDA receptors. These results are consistent with a role for IL-6 in the neuropathology observed in the developing CNS during injury and disease.
Asunto(s)
Corteza Cerebelosa/inmunología , Interleucina-6/toxicidad , Neuronas/efectos de los fármacos , Animales , Animales Recién Nacidos , Muerte Celular/efectos de los fármacos , Muerte Celular/inmunología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebelosa/citología , Interacciones Farmacológicas/fisiología , Interleucina-6/inmunología , N-Metilaspartato/toxicidad , Neuronas/inmunología , Neurotoxinas/inmunología , Neurotoxinas/toxicidad , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/inmunologíaRESUMEN
BACKGROUND: Alcohol exposure during human fetal development can result in fetal alcohol syndrome, a condition characterized by central nervous system dysfunction. Detailed studies in animal models of fetal alcohol syndrome show that the cerebellar region is particularly sensitive to alcohol exposure during early development; however, the cellular mechanisms underlying the alcohol sensitivity of the immature cerebellum are poorly understood. METHODS: Primary neuronal cultures of cerebellar cells were prepared from embryonic day 20 rat pups. Cultures were exposed to ethanol (33 mM; 150 mg/100 ml) during the main period of morphological development of the Purkinje neurons, from 6 to 17 days in vitro. After the ethanol treatment, the response of Purkinje neurons to the selective metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD; 300 microM) was examined in parallel fura-2 Ca2+ imaging and current-clamp experiments. In an additional set of experiments, ethanol-treated cultures were allowed to withdraw from ethanol overnight before recordings were performed. RESULTS: In Ca2+ imaging studies, the mean peak amplitude of ACPD-evoked Ca2+ signals was depressed in the dendritic region of chronic ethanol-treated Purkinje neurons compared with control neurons (p < 0.05, unpaired t test), whereas there was no apparent difference in the somatic region. In contrast, peak ACPD-evoked Ca2+ signals were enhanced in both the somatic and dendritic regions of withdrawn Purkinje neurons compared with control neurons. Parallel current-clamp studies showed no consistent effect of chronic ethanol treatment or ethanol withdrawal on the membrane response to ACPD. CONCLUSIONS: These results show that prolonged ethanol exposure and early withdrawal lead to alterations in mGluR-evoked Ca2+ signaling in cerebellar Purkinje neurons. Metabotropic GluRs in the Purkinje neuron play important roles in cerebellar development and function, suggesting that alterations of mGluR signaling pathways by ethanol may play a key role in the actions of ethanol on the developing cerebellum.