Steroid requirements for regulation of the alpha4 subunit of the GABA(A) receptor in an in vitro model.

The alpha4 subunit of the GABA(A) receptor (GABAR) has relatively low expression in the CNS, but is increased in vivo following 48 h administration of the GABA-modulatory steroid 3alpha-OH-5alpha[beta]-pregnan-20-one (THP or [allo]pregnanolone) to female rats. The purpose of the following study was to determine the optimal conditions for steroid-induced upregulation of alpha4 expression in an in vitro model. To this end, we used the IMR-32 cell, a neuroblastoma cell line, which normally expresses alpha4 mRNA at low levels. In undifferentiated IMR-32 cells, 48 h administration of THP increased alpha4 expression when ambient THP levels were reduced by the 5alpha-reductase blocker 4MA, suggesting that the background steroid milieu affects steroid regulation of this subunit. Following neuronal differentiation in serum-free medium, 48 h THP treatment significantly increased alpha4 expression two-fold following application of nerve growth factor (NGF) suggesting that development of neuronal processes facilitates this effect of the steroid. In the absence of NGF treatment, combined administration of 17beta-estradiol (E2) plus THP also increased alpha4 expression to a similar extent as THP following NGF treatment. In addition, E2 alone effectively increased alpha4 expression to maximal levels following NGF treatment. In contrast, neuronal differentiation in the absence of serum deprivation did not increase alpha4 levels. These results suggest that both THP and E2 can increase expression of the GABAR alpha4 subunit, but that this effect is dependent upon the background steroid milieu as well as the degree of neuronal development. These findings demonstrate optimal conditions for steroid-induced upregulation of the alpha4 subunit in an in vitro system.