We have investigated the effects of chronic treatment with the neurosteroid 5 alpha-pregnan-3 alpha-ol-20-one (5 alpha 3 alpha) on the gamma-aminobutyric acid (GABA)A receptor complex in cultured mammalian cortical neurons. Chronic 5 alpha 3 alpha treatment (up to 2 microM, 5 days) did not produce any changes in the morphological appearance or the cell protein content of cortical neurons. The basal binding of [3H]flunitrazepam, [3H]Ro15-1788, and [3H]Ro15-4513 was not altered after the chronic treatment. Chronic 5 alpha 3 alpha treatment did not alter the Kd or Bmax values of [3H]flunitrazepam binding to intact cortical neurons. However, chronic 5 alpha 3 alpha treatment produced uncoupling between GABA, barbiturate, and neurosteroid sites and the benzodiazepine site. The EC50 values of these ligands were not significantly altered; however, their Emax values were decreased after chronic 5 alpha 3 alpha treatment. The 5 alpha 3 alpha-induced uncoupling was time and concentration dependent. The binding of [3H]GABA and t-[35S]butylbicyclophosphorothionate was also decreased after chronic 5 alpha 3 alpha treatment. Chronic 5 alpha 3 alpha treatment decreased the Bmax of the low affinity GABAA receptor sites, without affecting the high affinity sites, and decreased the Bmax of t-butylbicyclophosphorothionate binding sites. The EC50 value for GABA-induced 36Cl- influx was not altered, whereas the Emax value was decreased after chronic 5 alpha 3 alpha treatment. Furthermore, the 5 alpha 3 alpha-induced uncoupling was reversed by concomitant exposure of the cortical neurons to 5 alpha-pregnan-3 beta-ol-20-one or R5135, suggesting an involvement of the neurosteroid and GABA recognition sites in the observed uncoupling. Taken together, these results suggest that chronic 5 alpha 3 alpha treatment produces heterologous uncoupling at the GABAA receptor complex.