Developmental aspects of benzodiazepine receptors and GABA-gated chloride channels in primary cultures of spinal cord neurons.

The developmental aspects of gamma-aminobutyric acid (GABA)-benzodiazepine receptor ionophore complex in mouse cultured spinal cord neurons was investigated in vitro. We examined benzodiazepine binding, GABA enhancement of benzodiazepine binding and GABA activation of chloride channels to intact cultured neurons during the development (days 1-14). Specific benzodiazepine binding increased with time from day 3 (Bmax = 546 fmol/mg protein) to day 14 (Bmax = 1746 fmol/mg protein). There was no significant difference in the affinity (Kd) of [3H]flunitrazepam for its receptor sites from day 3 to 14. Specific binding of [3H]flunitrazepam to the intact cells was also increased in the presence of GABA during all stages of development. The EC50 value of the GABA concentration (14-16 microM) required to enhance [3H]flunitrazepam binding did not change from day 3 to 14. GABA receptor-activated chloride channels, studied by 36Cl-influx was also observed at all ages in culture, and indicated an early development (as early as day 3) of these GABA-gated channels. These studies indicate that GABA-benzodiazepine-ionophore complex develop early in cultured spinal cord neurons, and these neurons provide an ideal system to study GABAergic events.