EXCITATORY ACTIONS OF GABA IN DEVELOPING BRAIN ARE MEDIATED BY L-TYPE Ca 2 CHANNELS AND DEPENDENT ON AGE, SEX, AND BRAIN REGION T. S. PERROT-SINAL,* A. P. AUGER AND M. M. MCCARTHY Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA Abstract—Although GABA is the major inhibitory neurotrans- mitter in adult brain, it exerts depolarizing actions in devel- oping neurons that include activation of voltage-gated cal- cium channels. The depolarizing actions of GABA serve an obvious trophic function, but the specific physiological sig- nificance of excitatory versus inhibitory GABA action has been largely ignored. We previously demonstrated that estra- diol enhances the magnitude and duration of calcium influx through L-type voltage-gated calcium channels following GABA A receptor activation in neonatal hypothalamic neu- rons. This has led us to propose that GABA action represents a major divergence point in steroid-mediated sexual differen- tiation of rat brain. Presently, we examined sex differences in phosphorylation of the calcium-regulated transcription fac- tor, cyclic AMP response element binding protein, following activation of the GABA A receptor with muscimol, in vivo. Muscimol given 30 min before killing significantly increased the number of neurons exhibiting phosphorylated cyclic AMP response element binding protein in newborn male hypothal- amus and CA1 hippocampus but decreased phosphorylated cyclic AMP response element binding protein in most brain regions in females. Muscimol-induced increases in phos- phorylated cyclic AMP response element binding protein in hypothalamus and hippocampus of newborn males were at- tenuated by pretreatment with the L-type voltage-gated cal- cium channel blocker, nimodipine, suggesting that calcium influx is involved in phosphorylation of cyclic AMP response element binding protein in neonate brain. Muscimol treat- ment had no effect on hypothalamic or hippocampal phos- phorylated cyclic AMP response element binding protein lev- els in juvenile males and females. These results are consis- tent with a divergence in male and female rat brain in the calcium-mediated cellular response to muscimol that is re- stricted to the early neonatal period, a time critical for estra- diol-mediated sexual differentiation. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: CREB, calcium influx, sexual differentiation, hypothalamus, hippocampus, steroid. The GABA A receptor is an ion channel that allows influx or efflux of chloride ions (Cl - ) depending on the Cl - trans- membrane concentration gradient. Because of a higher intracellular Cl - concentration in immature neurons, acti- vation of the GABA A receptor by GABA or the selective agonist, muscimol (MUS), causes efflux of Cl - and mem- brane depolarization (Ben-Ari et al., 1994; Owens et al., 1996). This depolarization is sufficient to open L-type volt- age-gated calcium channels (VGCC), leading to calcium influx (Obrietan and van den Pol, 1997). GABA and GABA- induced calcium influx have been linked to trophic actions important for developmental processes such as neuronal migration (Ma and Barker, 1995), neurite outgrowth (Barbin et al., 1993), neuronal survival (Ikeda et al., 1997; Obata, 1997) and differentiation (LoTurco et al., 1995). The excitatory effect consequent to GABA A receptor bind- ing gradually switches to inhibitory as development progresses. The timing of the shift from depolarizing to hyperpolarizing GABA varies across brain regions but is generally complete by the second week of life (Cherubini et al., 1990; Ikeda et al., 1997; Obata, 1997). A notable exception is the hypothalamus, where the switch appears to occur earlier than other brain regions (Obrietan and van den Pol, 1995) and falls within the critical period for steroid- mediated sexual differentiation of the brain. The sensitive period for brain sexual differentiation is characterized by release of a large amount of testosterone from the male testes in rats (Corbier et al., 1978) and humans (Corbier et al., 1990), which gains access to the nervous system where it is readily aromatized to estradiol. High levels of estradiol in neonatal male rat brain are responsible for masculinizing both brain and behavior by mediating processes such as neuronal survival, neurite branching, and synaptogenesis (Gerall et al., 1992; Mat- sumoto et al., 2000). Various parameters of the GABAergic system are higher in brain of neonate male rats relative to females only during the sensitive period (Davis et al., 1996a, 1999) and disrupting GABA in the neonate inter- feres with development of adult sexual behavior (Davis et al., 2000). Recent investigations of GABA’s role in sexual differ- entiation have focused on the downstream effectors of GABA A receptor activation, namely calcium influx and sub- sequent activation of transcription factors. We have dem- onstrated that cultured hypothalamic neurons pretreated *Correspondence to: T. Perrot-Sinal, Department of Psychology and Neuroscience Institute, Dalhousie University, Life Sciences Centre, 1355 Oxford Street, Halifax, Nova Scotia, Canada B3H 4J1. Tel: +1-902-494-1875; fax: +1-902-494-6585. E-mail address: tara.perrot-sinal@dal.ca (T. Perrot-Sinal). Abbreviations: ANOVA, analysis of variance; ARC, arcuate nucleus of hypothalamus; CA1, CA1 subfield of hippocampus; CTX, retrosplenial cortex; CREB, cyclic AMP response element binding protein; ir, immunoreactive; IHC, immunohistochemistry; MUS, muscimol; NGS, normal goat serum; NIM, nimodipine; pCREB, phosphorylated cyclic AMP response element binding protein; PBS, phosphate-buffered saline; POA, medial preoptic area; PVP, posterior part of the paraven- tricular thalamic nucleus; SAL, saline; TPBS, phosphate-buffered sa- line containing 0.4% Triton X-100; VEH, vehicle; VGCC, voltage-gated calcium channel; VMH, ventromedial hypothalamus. Neuroscience 116 (2003) 995–1003 0306-4522/03$30.00+0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(02)00794-7 995