Abstract
The differentiation and survival of heterozygous Lurcher (+/Lc) Purkinje cells in vitro was examined as a model system for studying how chronic ionic stress affects neuronal differentiation and survival. The Lurcher mutation in the delta2 glutamate receptor (GluRdelta2) converts an orphan receptor into a membrane channel that constitutively passes an inward cation current. In the GluRdelta2(+/Lc) mutant, Purkinje cell dendritic differentiation is disrupted and the cells degenerate following the first week of postnatal development. To determine if the GluRdelta2(+/Lc) Purkinje cell phenotype is recapitulated in vitro, +/+, and +/Lc Purkinje cells from postnatal Day 0 pups were grown in either isolated cell or cerebellar slice cultures. GluRdelta2(+/+) and GluRdelta2(+/Lc) Purkinje cells appeared to develop normally through the first 7 days in vitro (DIV), but by 11 DIV GluRdelta2(+/Lc) Purkinje cells exhibited a significantly higher cation leak current. By 14 DIV, GluRdelta2(+/Lc) Purkinje cell dendrites were stunted and the number of surviving GluRdelta2(+/Lc) Purkinje cells was reduced by 75% compared to controls. However, treatment of +/Lc cerebellar cultures with 1-naphthyl acetyl spermine increased +/Lc Purkinje cell survival to wild type levels. These results support the conclusion that the Lurcher mutation in GluRdelta2 induces cell autonomous defects in differentiation and survival. The establishment of a tissue culture system for studying cell injury and death mechanisms in a relatively simple system like GluRdelta2(+/Lc) Purkinje cells will provide a valuable model for studying how the induction of a chronic inward cation current in a single cell type affects neuronal differentiation and survival.