Intracellular Ca2+ and N-methyl-D-aspartate-stimulated neuritogenesis in rat cerebellar granule cell cultures.
Week-old rat cerebellar granule cells were grown in the presence of the cell-permeable calcium chelating agent BAPTA-acetoxy methyl ester (BAPTA-AM) for the first 8 h in vitro. There was a dose-dependent inhibition of process outgrowth with an IC50 of approximately 5 microM. Neurite outgrowth could be partially recovered by the addition of N-methyl-D-aspartate (NMDA; 50 microM) to BAPTA-AM-treated cells. Phorbol ester stimulation of treated cells evoked a profound inhibition of neuritogenesis compared to a stimulatory effect on control cultures. The inhibition of growth caused by phorbol esters could not be reversed by NMDA co-addition. Neurites extended by BAPTA-AM-treated granule cells were thinner than in control cultures and did not form elaborate growth cones even when growth was stimulated by NMDA. The distribution of tyrosinated and acetylated alpha-tubulin in the processes of BAPTA-AM-treated cells appeared similar to that in controls. However, rhodamine-phalloidin labelling of microfilaments in the cell cultures emphasised the loss of an elaborate actin-rich growth cone in BAPTA-AM-treated cells even when neurite formation was partially recovered. These results indicate the importance of [Ca2+]i in the production of neurites from cerebellar granule cells in vitro.
Cambray-Deakin
M A
MA
Department of Biomedical Science, University of Sheffield, UK.
Burgoyne
R D
RD
D008841:Actin Cytoskeleton / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology* / Q000187:drug effects; D003599:Cytoskeleton / Q000187:drug effects; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005456:Fluorescent Dyes; D008870:Microtubules / Q000737:chemistry; D016202:N-Methylaspartate / Q000502:physiology*; D016501:Neurites / Q000187:drug effects / Q000502:physiology*; D051381:Rats
D005456:Fluorescent Dyes; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D016202:N-Methylaspartate; D002118:Calcium