GABAergic neurons in rat hippocampal culture

The experiments described here were designed to study biochemical and histological measures of gamma-aminobutyric acid (GABA) uptake and glutamic acid decarboxylase (GAD) in primary dissociated cell cultures prepared from 17-21-day fetal rat hippocampus. Preparations from all ages of animals, except 21-day fetuses, were enriched in GABAergic neurons, when compared to the adult hippocampus in situ. These cells comprise 30-50% of the large, phase-bright, process-bearing cells in hippocampal cultures as estimated by autoradiography of GABA uptake and GAD immunocytochemistry. Neurons concentrate GABA by a relatively slow but high-affinity process (Km = 2.6 microM) that has considerably higher maximum velocity than glial uptake (Vmax = 479 pmol/mg protein/min for neurons and 31 pmol/mg protein/min for glia). No low-affinity uptake process was noted in neurons or glia. GABA uptake into neurons was competitively inhibited by cis-4-OH-nipecotic acid (Ki = 39 +/- 11 microM). These cultures also possess considerable GAD activity, up to 6 nmol/mg protein/min in one-month-old cultures, which approximates that of the adult hippocampus. Both GABA uptake and GAD activity increased with time in culture. The enrichment of GABAergic markers indicates that this preparation may be useful for the detailed study of hippocampal GABAergic neurons.