Differential effects of excitatory amino acids on mesencephalic neurons expressing either calretinin or tyrosine hydroxylase in primary cultures

In mesencephalic primary cultures derived from E14 rat embryos, calretinin- and tyrosine hydroxylase-immunoreactive neurons comprised 2% and 5% of the total cell population, respectively, at 6–7 days in vitro. The number of calretinin-immunoreactive neurons was unchanged after a 12- or 24-h exposure to 500 μM kainic acid (KA), but a 50% cell loss was detected after a 48-h exposure to KA. Tyrosine hydroxylase-immunoreactive neurons demonstrated a 50% and 67% cell loss at 24- and 48-h exposures to 500 μM KA. A 500 μM N-methyl- d-aspartic acid (NMDA) incubation for 24 h had no effect on calretinin-immunoreactive cell number, but did significantly reduce tyrosine hydroxylase-immunoreactive cell numbers by 26%. In tyrosine hydroxylase-immunoreactive cells, exposure to KA appeared to stimulate the retraction of the neuritic tree and to cause somatic swelling. In contrast, calretinin-immunoreactive neurons developed larger and more complex neuritic trees after a 24-h exposure to 500 μM KA but not NMDA. Immunohistochemical colocalization studies revealed that all tyrosine hydroxylase-immunoreactive and the majority of calretinin-immunoreactive neurons expressed the glutamate receptor subunits GluR2-R3. Very low levels of NMDAR1 receptor subunits were detected on cells in this culture and GluR4 receptor subunits were not detectable. Our experiments showed that glutamate receptors present in both calretinin- and tyrosine hydroxylase-immunoreactive cells were functional, since phosphorylated cAMP/Ca 2+ response element-binding protein levels were increased in both cell types after 10 or 30 min exposures to 500 μM KA. The present results indicate that in the mesencephalic cultures tyrosine hydroxylase-immunoreactive cells are more vulnerable to KA excitotoxicity than calretinin-immunoreactive neurons.