Abstracts

Seizure frequency in women often correlates with changes in gonadal steroid hormone levels during the menstrual cycle, but the underlying mechanisms are uncertain. Progesterone and its metabolite, allopregnanolone (5[alpha]-pregnan-3[alpha]-ol-20-one, ALLO), have anticonvulsant properties, while [beta]-estradiol is proconvulsant in several model systems. We hypothesized that fluctuations in steroid hormone levels could alter CNS inhibitory tone by changing the expression pattern of GABA[sub]A[/sub] receptor (GABAR) subunits, affecting the likelihood of seizures. We have examined the effects of these steroid hormones on GABAR subunit expression in a simplified [italic]in vitro[/italic] neuronal system, the NT2-N cell, derived from the human NTera2 teratocarcinoma cell line. Using RT-PCR, we have previously shown that 7 d exposure to progesterone increased [alpha]2 and [gamma]3 and decreased [alpha]5 subunit mRNAs, while [beta]-estradiol exposure increased [alpha]3, [beta]3 and [epsilon] subunit mRNAs, relative to actin. ALLO does not act via the progesterone receptor, but allosterically enhances GABAR function. We thus questioned whether ALLO also affects GABAR subunit expression during treatment or after withdrawal. NT2-N cells were maintained in culture for 5 weeks after 5 weeks retinoic acid (1 [mu]M) treatment, then exposed to ALLO (1 [mu]M) or vehicle (H[sub]2[/sub]O) for 2 d, and then harvested for RNA or withdrawn from ALLO for 1 d prior to RNA harvesting. After DNAse-I treatment, RNA was reacted for semiquantitative RT-PCR with 16 GABAR subunit-specific primer pairs, and analyzed by agarose gel electrophoresis with bands normalized to actin controls. ALLO treatment increased [epsilon] subunit mRNA 185 [plusmn] 26% (n=6, p[lt]0.05) without significantly changing the expression of other GABAR subunits. ALLO withdrawal increased [alpha]4 subunit expression 380 [plusmn] 69% (n=6, p[lt]0.01) and [alpha]2 expression 320 [plusmn] 82% (n=6, p[lt]0.05). Expression of [epsilon] remained elevated during ALLO withdrawal. Brief ALLO exposure and withdrawal altered the expression of specific GABAR subunits. Elevation of [alpha]4 subunit has been seen after ALLO withdrawal [italic]in vivo [/italic](Smith [italic]et al[/italic]., [underline]J Neurosci[/underline] 18:5275-84, 1998) and in primary neurons in culture (Follesa [italic]et al[/italic]., [underline]Brain Res. Reviews[/underline] 37:81-90,2001), suggesting that similar regulatory mechanisms are involved in this simplified neuronal system. GABARs containing the [alpha]4 subunit are insensitive to benzodiazepines but have increased sensitivity to ALLO (Whittemore [italic]et al[/italic]., [underline]Mol Pharmacol[/underline] 50:1364-75, 1996), hence changes in GABAR subunit composition may underlie increased seizure frequency and perimenstrual dysphoric states, possibly by altering interactions with endogenous GABAR modulators. These findings confirm the validity of NT2-N cells as a model of GABAR subunit regulation that may allow more detailed investigation of the mechanisms underlying neurosteroid control of GABAR composition. (Supported by The Myoclonus Research Foundation.)