title: The role of depolarization in the survival and differentiation of cerebellar granule cells in culture.
abstract: Cultures greatly enriched in granule cells from early postnatal cerebellum (P8) were grown in a medium containing fetal calf serum. Under the conditions used, nerve cells died, usually within a week, unless the K+ concentration in the medium was greater than or equal to 20 mM. The requirement for elevated [K+]e was manifested by about 3 d in vitro, and after this time continuous exposure to high [K+]e was essential for the survival of the granule cells. The initial morphological and biochemical maturation of the granule cells was similar in the presence and the absence of elevated [K+]e, suggesting that the dependence on depolarizing conditions develops in parallel with the expression of the differentiated characteristics of the cells. The positive effect of elevated [K+]e on granule cell survival was not influenced by preventing bioelectric activity in the cultures with TTX and xylocaine. On the other hand, depolarization-induced transmembrane Ca2+ flux was essential in securing the maintenance of the granule cells. Depolarized nerve cells were compromised when Ca2+ entry was blocked by elevated Mg2+, EGTA, or organic Ca2+ antagonists, while dihydropyridine Ca2+ agonists [BAY K 8644, (+)-(S)-202 79 1 and CGP 28392] were potent agents preventing nerve cell loss in the presence of 15 mM [K+]e, which was ineffective on its own. Calmodulin inhibitors (1 microM trifluoperazine or calmidazolium) blocked the beneficial effect of K+-induced depolarization on granule cells. The comparison of the timing of the differentiation and innervation of the postmitotic granule cells in vivo with the development of the K+ dependence in vitro would indicate that depolarization of the granule neurons in culture mimics the influence of the physiological stimulation in vivo through excitatory amino acid receptors, including N-methyl-D-aspartate receptors, involving Ca2+ entry and the activation of a Ca2+/calmodulin-dependent protein kinase.
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules; D002452:Cell Count; D002454:Cell Differentiation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004247:DNA / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D008012:Lidocaine / Q000494:pharmacology; D008564:Membrane Potentials; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D013779:Tetrodotoxin / Q000494:pharmacology
chemical_list: D000954:Antigens, Surface; D002121:Calcium Channel Blockers; D015815:Cell Adhesion Molecules; D005971:Glutamates; D018698:Glutamic Acid; D013779:Tetrodotoxin; D004247:DNA; D008012:Lidocaine; D011188:Potassium; D002118:Calcium