Biochemical and morphological studies on GABA neurons in reaggregate culture

Dissociated cells from the 14-day fetal mouse corpus striatum (CS), rostral mesencephalic tegmentum (RMT) and tectum were reaggregated in rotation-mediated cultures in the following combinations: CS alone, RMT-CS, RMT-tectum, and tectum alone. Reaggregates were cultured for 1-4 weeks. An even distribution of gamma-aminobutyric acid (GABA)-positive cells was observed in all the reaggregate combinations that were cultured for 1 and 2 weeks. With increasing time in culture, cellular staining was decreased while positive fiber staining increased. CS and RMT-CS co-cultures increased their capacity to take up [3H]GABA with time in culture. All reaggregates reached a maximum uptake/accumulation capacity of 30-40 pmol/mg protein/30 min, by 4 weeks in vitro. There were no significant differences between the various co-aggregate combinations in the accumulation capacity. [3H]GABA accumulation in the reaggregates was largely blocked by the putative neuronal GABA uptake inhibitor, diaminobutyric acid, and was inhibited to a much lesser degree by the putative glial uptake inhibitor beta-alanine. All reaggregates released [3H]GABA to 70 mM potassium depolarization, in a calcium-dependent manner. One-three week CS reaggregates released more [3H]GABA in response to the potassium-induced depolarization than RMT-CS co-cultures. Since nigral dopamine neurons within the RMT proliferate processes and actively release dopamine only when co-cultured with CS target cells, it is suggested that these dopamine neurons might chronically inhibit striatal GABA neurons in the RMT-CS co-cultures, thereby depressing stimulated release of [3H]GABA.