The developing brain is bipotential, capable of being either masculinized by gonadal steroid hormones or remaining feminized if not exposed to steroids during a sensitive perinatal period. Testosterone produced by the embryonic and neonatal testis is converted to estrogen in the brain to mediate this differentiation process. Neuroanatomical endpoints include changes in synaptic patterning, cell death and fiber tract thickness. These result in sexually dimorphic physiology and behavior in adulthood. From a mechanistic standpoint, the process of steroid-mediated differentiation remains poorly understood. We have formulated a hypothesis that increased neuronal excitation induced by estrogen mediates the process of masculinization. Central to this hypothesis is the observation that GABA is an excitatory neurotransmitter during the perinatal sensitive period for steroid-mediated differentiation and that GABA levels are twice as high in males compared to females. There are also higher levels of glutamate binding to the non-NMDA receptor in the hypothalamus of males during the same time period. The functional importance of increased neuronal excitation is the consequent increased in intracellular calcium, which then regulates a variety of cellular processes including but not limited to cell death, neurite extension and synapse formation. The appropriate buffering of cytosolic calcium is crucial to cell functioning and survival. We have found that two calcium buffering proteins, calbindin and calretinin, are up to 2-fold higher in male brains at the same time as increased excitatory GABA and glutamate binding.
Three specific aims will focus on the further characterization of the hormonal modulation of excitatory GABA, glutamate and calcium binding proteins. Experimental approaches include in situ hybridization and in vitro receptor autoradiography to quantify cellular parameters related to GABA and glutamate action. Dispersed hypothalamic cultures and acute slices will be employed to assess hormonal modulation of calcium influx induced by GABA glutamate. The relation between these amino acid transmitters and sex differences in calbindin and calretinin will also be elucidated. Most importantly, however, a cause and effect relationship between dimorphisms in excitatory GABA action, glutamate receptor binding and levels of calcium binding proteins and the process of steroid-mediated differentiation will be determined with the use of antisense oligonucleotides targeted against select mRNAs. This approach reduces or eliminates sex differences in specific parameters during the sensitive period, after which animals are be raised to adulthood and tested for sex specific behavior and brains assessed for sexually dimorphic morphometry. Establishing the mechanisms by which steroids influence neuronal differentiation increases our understanding of basic growth processes and furthers our understanding of sex differences in neurological disorders, particularly those associated with development which tend to be more prevalent in males.
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