Development of gamma-aminobutyric acid (GABA)ergic neurons in cerebral cortical neurons in primary culture.

The developmental patterns of gamma-aminobutyric acid (GABA)ergic neurons in primary culture obtained from the neopallium of 15-day-old fetus of mouse were investigated in terms of morphological features, GABA metabolism and GABA receptor binding. Morphological investigations revealed that these cells possessed typical features of neurons and the formation of synapses was detected at 10 days after the inoculation. During neuronal growth on polylysine surfaces, GABA contents and activity of GABA transaminase (GABA-T) showed a progressive increase in the time of culture. Similarly, L-glutamic acid decarboxylase (GAD) showed a progressive elevation during neuronal development in vitro, which corresponded well with the change in immunoreactivity to anti-GAD examined immunohistochemically. In addition, the high K+-evoked release of [3H]GABA also showed an enhancement during the growth in vitro. The numbers of binding sites (Bmax) for [3H]muscimol and [3H]flunitrazepam (FLN) also showed increases with the time of incubation, although affinity (Kd) to the labeled ligands did not show any noticeable changes. Moreover, it was observed that [3H]FLN binding was enhanced by GABA even in neurons cultured for 7 days. These results indicate that cerebral cortical neurons in primary culture possess GABA biosynthesizing and degrading systems including a high-affinity uptake mechanism for GABA. The present results also indicate that these cells possess synaptic contacts as well as GABAA receptors coupled with benzodiazepine receptor from a relatively early stage of cellular development.