Two functionally different glutamate receptors of the kainate subtype in embryonic rat mesencephalic cells

The amino acid glutamate is a widespread excitatory neurotransmitter in the brain. It activates cation-selective channels expressed by nearly every neuron and by glial cells; also various second messenger cascades. Little is known about the ontogeny of glutamate neurotransmission during neurogenesis. We have analyzed the development and differentiation of excitatory amino acid responses and Na+ channels in cells dissociated from embryonic rat ventral mesencephalon and striatum as well as cortex and cerebellum using fluorescent voltage-sensitive oxonol dyes and flow cytometry. Analysis of fluorescence distribution revealed complex profiles under resting conditions which changed in a characteristic manner over the period studied (Embryonic (E) Days 12-20). The response to the Na+ channel agonist veratridine appeared at E12/13 in the mesencephalon. At E13 L-glutamate and kainate evoked changes in membrane potential interpreted as cellular hyperpolarization. At E15 some cells still responded by hyperpolarizing but an equal number began to depolarize. By E18 most cells depolarized. Both hyper- and depolarizations were eliminated by a specific antagonist at kainate receptors (6-cyano-7-nitroquinoxyline-2,3-dione) and by resuspending the cells in Na(+)-free medium. Both responses exhibited a concentration dependency with higher doses evoking stronger effects. In contrast, there was little effect of veratridine in the striatum at E15-E16, and the response to kainate or L-glutamate was predominantly depolarizing during the same embryonic period, with little or no effect until E18. These data show that in the developing CNS, sodium channel responses as well as excitatory aminoacid neurotransmitter responses first become functional in the mesencephalon and subsequently in the striatum, thus suggesting an anatomical gradient of expression. Our results also show that glutamate receptor-coupled functions vary with embryonic age and with regional distribution, suggesting possible roles of glutamate in early CNS embryogenesis, as morphogens or modulators of synaptic plasticity.