title: Pharmacological and immunocytochemical characterization of metabotropic glutamate receptors in cultured Purkinje cells.
abstract: Metabotropic glutamate receptor (mGluR) is highly expressed in cerebellar Purkinje cells. The purpose of this study was pharmacological and immunocytochemical characterization of the mGluR in single cerebellar neurons, especially Purkinje cells. Ca2+ imaging with fura-2 in cultured cerebellar neurons, identified immunocytochemically, was used to record the direct effects of drugs in stable conditions. In addition, the expression of mGluR was examined, and expression of the intracellular receptor for inositol trisphosphate (IP3) produced by mGluR activation was studied immunocytochemically with specific antibodies. Purkinje neurons and some other neurons showed Ca(2+)-mobilizing responses to mGluR agonists. These responses were mediated by mGluR because they were not blocked by ionotropic GluR antagonists, were independent of the caffeine-sensitive Ca2+ pool, and were blocked by inhibitors of IP3-induced Ca2+ release. This is the first pharmacological characterization of mGluR at single Purkinje cells. The results differed as follows from those in earlier studies in which phosphoinositide turnover of the entire population of cerebellar cells was monitored: (1) the mGluR responses were not blocked by pertussis toxin or D,L-2-amino-3-phosphonopropionic acid; (2) glutamate was a potent agonist, whereas L-aspartate was ineffective; and (3) the dose-response relationship showed an all-or-none tendency. The metaboltropic response of Purkinje cells changed markedly during development, with a sharp peak after day 4 of culture, whereas mGluR and IP3 receptor proteins increased steadily during maturation. This apparent desensitization of mGluR was not blocked by inhibitors of protein kinase C (PKC) or ADP-ribosyltransferase. The metabotropic responses were mainly localized to the center of the somata of Purkinje cells even on day 4, whereas both receptor proteins were expressed throughout the cell. These results suggest that the function of mGluR is spatially and developmentally controlled by a posttranslational mechanism involving a mechanism other than phosphorylation by PKC or ADP-ribosylation.
authors.affiliation: Division of Behavior and Neurobiology, National Institute for Basic Biology, Aichi, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Yuzaki
authors.forename: K
authors.initials: K
authors.lastname: Mikoshiba
mesh_terms: D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels; D002478:Cells, Cultured; D016922:Cellular Senescence; D004305:Dose-Response Relationship, Drug; D016257:Fura-2; D019204:GTP-Binding Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007425:Intracellular Membranes / Q000378:metabolism; D051379:Mice; D037342:Pertussis Toxin; D011689:Purkinje Cells / Q000378:metabolism*; D016318:Quisqualic Acid / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear; D017470:Receptors, Glutamate / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D014018:Tissue Distribution; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D000596:Amino Acids; D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D017470:Receptors, Glutamate; D010566:Virulence Factors, Bordetella; D016318:Quisqualic Acid; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D002118:Calcium; D016257:Fura-2