Studies conducted in this laboratory have clearly demonstrated that exposure to the widely used anxiolytic drug diazepam (DZ) during late gestation in the rat induces neural and behavioral changes that (1) are apparent long after the drug can be detected, (2) often are not apparent until after the onset of puberty, and (3) include altered responses to stressors and altered sensitivity to drugs and environmental stimuli. Several effects of early DZ exposure are sexually dimorphic. Research now proposed will test mechanisms that could underlie these characteristics of early DZ exposure. The target of DZ action on the brain is the GABAA receptor, and processing of information via this receptor appears to be critical for integrated responses to stressors. Furthermore, the subunit composition of this receptor complex may dictate pharmacologic sensitivity and functional responsiveness of the complex. We propose to evaluate the effect of early DZ exposure on expression of mRNAs for various subunits of the GABAA receptor. Since responsiveness of the complex changes over normal adolescent development, we will measure mRNA levels over this time period in control as well as in prenatally exposed rats. To determine that any changes in mRNA expression correspond to changes in the actual composition of the receptor complex, we will identify the presence of specific peptide subunits in the brain using both immunoprecipitation analysis and immunocytochemical analysis of the reaction to specific antipeptides. To address the characteristic of sexual dimorphism of early DZ exposure, we will test the hypothesis that such effects relate to the in utero interaction between DZ and specific reduced steroid hormones at the level of the GABAA receptor. We will determine whether the presence of a specific benzodiazepine antagonist given concurrently with DZ will prevent the effect of DZ exposure on environment-specific social interaction in adult males and females. To link the effect of DZ to an effect of sex steroids in utero, some animals exposed to DZ over gestational days 14 through 20 will be exposed to the antagonist only over gestational days 19 and 20, the period of a known surge in testosterone levels in male fetuses. We will also determine whether the antagonist, given over either the prolonged or limited gestational period, alters the effect of prenatal DZ exposure on expression of mRNA for GABAA receptor subunits. The interaction between DZ and reduced steroids will also be evaluated using in vitro analysis of GABAA receptor function. To address the latent appearance of the effects of in utero DZ exposure, we will determine changes that take place over normal adolescent development in identified targets of the early exposure, the GABAA receptor and norepinephrine (NE) projections, in the hypothalamus: this region is known to be critical to integrated stress responses. We have shown that destruction of NE terminals in this region in naive adult male rats alters responsiveness of the GABAA receptor in the cerebral cortex to stressors. These studies together will identify mechanisms whereby early psychoactive drug exposures can lead to behavioral disorders that appear in late adolescence.
