Calcretinin (CR) is a calcium binding protein that is mostly found in subpopulations of brain neurons such as the substantia nigra (SN) and the ventral tegmental area (VTA). We have previously shown that some of the SN/VTA dopamine- containing cells colocalize with CR. A further study of intact rat embryo brains revealed that colocalization begins at E16. We observed many CR immunoreactive (IR) neurons in addition to tyrosine hydroxylase (TH) immunoreactive cells in tissue culture, but we have not found CR+TH colocalization in embryonic cells (E14) grown for up to 12 days in culture, It appears that some factor is required which is present in brain but not dissociated cultures. We also studied the effects of excitatory amino acids (EAA) on the expression of CR and TH in SN cultures. CR and TH neurons were differentially affected by 24 hour incubation with kainic acid (KA), NMDA or AMPA (100-500 muM)> In general, TH-IR neurons exhibited greater change than CR-IR neurons after EAA treatment, and both types of cells were affected in a decreasing order of severity: KA greater than AMPA greater than NMDA. Degeneration of neuronal cells and processes were less frequently observed in CR-IR cells after KA (47%) and NMDA (22%) compared with TH-IR cells (KA, 57%; NMDA and AMPA, 35%). Both surviving CR and TH cells exhibited increased fiber ramifications, but the cells' appearances differed. Whereas surviving CR cell processes extended for very long distances, TH cells had increased numbers of spoke-like processes emerging directly from cell bodies, many of which had swollen varicosities. The dramatic appearance of long ramifying varicose processes following EAA, especially KA, focuses our attention on whether sprouting of new fibers occurs within 24 h following the EAA, or on the possibility that there is an increased turnover and/or axoplasmic flow of CR to existing fibers that were not visible under control conditions. A radioimmunoassay to quantify CR revealed a loss of CR protein after KA (42%), and AMPA (17%), but no effect in NMDA-treated cells. A lysate ribonuclease protection assay revealed a significant loss of mRNA for both CR and TH in the KA-treated group (CR:81%, TH:76%) and a lesser decrease in the AMPA-treated (CR:62%, TH:43%) and NMDA-treated cells (CR:58% TH:36%). These data do not definitively reveal the significance of the increased plexus of fibers following EAA, as CR-IR suggests a major movement of protein from cell bodies into norway gnawer or sprouting in some cells while a reduction in both the protein and mRNA suggests degeneration of a subset of cells. Whether this is a reflection of differences between SN compacta (A9) or VTA (A10) cells remains to be shown. Such a distinction was previously uncovered following treatment in monkeys with the SN neurotoxin, MPTP, which destroyed A9 cells but not the A10 subpopulation. Since colocalization of CR within TH cells in culture was not revealed, we cannot conclude from these experiments that CR-containing dopamine cells are protected from neurotoxic injury by EAA.
