A critical role for glutamate in the organizational effects of estradiol on the developing hypothalamus

Abstract

Male and female brains exhibit fundamental morphological differences thought to underlie the differences in physiology and behavior between the sexes. In rodents, these sex differences are determined during a critical period of development, when testosterone secreted from the developing testes is converted to estradiol in neurons via the aromatase enzyme. This early estradiol exposure permanently differentiates the male from the female rodent brain by establishing sex differences in synaptic patterning, neuronal morphology and sex-typic behaviors. The medial basal hypothalamus (MBH) is a sexually dimorphic brain region that is responsive to estradiol. In newborn rats, males have twice the number of dendritic spines on hypothalamic neurons as females. Treatment of females with estradiol can rapidly increase the number of dendritic spines to levels seen in males. We determined that the estradiol-induced increase in dendritic spines in this region requires the activation of both AMPA and NMDA glutamate receptors. How does estradiol positively effect glutamatergic transmission in developing hypothalamic neurons? We hypothesized that estradiol enhances release of glutamate from presynaptic terminals. To test this hypothesis, cultured hypothalamic neurons were imaged with the fluorescent dye, FM4-64, which loads and unloads from presynaptic terminals upon depolarization. We found that estradiol significantly enhances neurotransmitter release from glutamatergic hypothalamic neurons in a receptor dependent manner, without the synthesis of new proteins, but requiring activation of PI3 kinase. Downstream of the glutamate receptor we further determined that MAP kinase activation is a necessary component of dendritic spine formation during this critical period. Based on these findings, our current working model for sexual differentiation of the MBH suggests that estradiol evokes glutamate release from developing hypothalamic terminals, activating both AMPA and NMDA receptors on the post-synaptic cell to stimulate MAP kinase and increase dendritic spines. We next predicted that these effects induce permanent defeminization of not only the brain, but also behavior. Blocking NMDA glutamate receptors during the critical period of sexual differentiation completely blocked estradiol-induced defeminization of behavior while having no effect on estradiol-induced masculinization of behavior. Surprisingly, females treated with NMDA postnatally showed not only behavioral defeminization but also masculinization during separate behavioral tests. We conclude that estradiol, via trans-synaptic NMDA receptor activation, permanently defeminizes adult sex behavior via its neonatal effects on neuronal morphology in the developing hypothalamus. These results are the first instance of a trans-synaptic mechanism by which estradiol permanently changes both the morphology and the function of neurons underlying a motivated behavior.