Excitatory versus inhibitory GABA as a divergence point in steroid-mediated sexual differentiation of the brain

Abstract

Whereas adult sex differences in brain morphology and behavior result from developmental exposure to steroid hormones, the mechanism by which steroids differentiate the brain is unknown. Studies to date have described subtle sex differences in levels of proteins and neurotransmitters during brain development, but these have lacked explanatory power for the profound sex differences induced by steroids. We report here a major divergence in the response to injection of the gamma-aminobutyric acid type A (GABA(A)) agonist, muscimol, in newborn male and female rats. In females, muscimol treatment primarily decreased the phosphorylation of cAMP response element binding protein (CREB) within the hypothalamus and the CA1 region of the hippocampus. In contrast, muscimol increased the phosphorylation of CREB in males within these same brain regions. Within the arcuate nucleus, muscimol treatment increased the phosphorylation of CREB in both females and males. Thus, the response to GABA can be excitatory or inhibitory on signal-transduction pathways that alter CREB phosphorylation depending on the sex and the region in developing brain. This divergence in response to GABA allows for a previously unknown form of steroid-mediated neuronal plasticity and may be an initial step in establishing sexually dimorphic signal-transduction pathways in developing brain.

Figure 1

Schematic drawing of areas in which the pCREB-immunoreactive cell number was quantified. Boxes indicate the area used for image analysis within a given region. PVP, paraventricular thalamic nucleus, posterior part; and Ctx, frontal cortex.

Figure 2

In females, GABA_A-receptor activation either increases or decreases the phosphorylation of CREB in sexually dimorphic brain regions. (a) Activation of GABA_A receptors by muscimol leads to a decrease in the number of cells expressing pCREB within the MPOA and the VMH of female rat brain. In contrast, GABA_A-receptor activation leads to an increase in the number of cells expressing pCREB within the Arc of female rat brain (*, P < 0.05). GABA_A-receptor activation by muscimol does not alter pCREB-immunoreactive cell number within the CTX or the PVP in female rat brain. (b) Representative photomicrographs of pCREB immunoreactivity within the VMH and the Arc in saline- or muscimol-treated rats. Note the opposite effects on the expression of pCREB immunoreactivity in the VMH and the Arc after GABA_A-receptor activation by muscimol. (c) Photomicrographs of pCREB immunoreactivity within the MPOA of saline- or muscimol-treated female rats. (Bar, 50 μm.)

Figure 3

In males, GABA_A-receptor activation increases the phosphorylation of CREB in sexually dimorphic brain regions. (a) Activation of GABA_A receptors by muscimol leads to an increase in the number of cells expressing pCREB within the MPOA, VMH, and the Arc of male rat brain (*, P < 0.05). GABA_A-receptor activation by muscimol does not change the pCREB-immunoreactive cell number within the CTX or the PVP of male brain. (b) Representative photomicrographs of pCREB immunoreactivity within the VMH and the Arc in saline- or muscimol-treated rats. Note that GABA_A-receptor activation by muscimol increases pCREB-immunoreactive cell number within the VMH and the Arc. (c) Photomicrographs of pCREB immunoreactivity within the MPOA of saline- or muscimol-treated male rats. (Bar, 50 μm.)

Figure 4

GABA_A-receptor activation alters immunoreactivity for phosphorylated CREB within the CA1 but not in the CA3. (a) In males (Left), muscimol treatment increases the pCREB-immunoreactive area within the CA1 region but not in the CA3 region (*, P < 0.05). (b) In females (Right), muscimol treatment decreases the pCREB-immunoreactive area within the CA1 region of the hippocampus but not in the CA3 region of the hippocampus. (c) Photomicrographs of pCREB immunoreactivity within the hippocampus of saline- (Left) or muscimol-treated males (Right).

Figure 5

Summary of the effects of GABA_A-receptor activation by muscimol on the phosphorylation of CREB. (Left) Effects of muscimol relative to saline on pCREB immunoreactivity in male rat brain. (Right) Effects of muscimol on pCREB immunoreactivity in female rat brain. Black, increased pCREB-immunoreactive cell number; gray, unchanged pCREB-immunoreactive cell number; white, decreased pCREB-immunoreactive cell number.