The amino acid GABA acts as the primary inhibitory transmitter in the adult brain. In contrast, in the developing hypothalamus GABA can be excitatory by depolarizing the membrane potential, raising cytosolic calcium, and evoking action potentials. The present proposal focuses on the excitatory actions of GABA in developing hypothalamic neurons. Converging approaches utilize fura-2 calcium digital imaging, gene chips, immunocytochemistry, electron microscopy, and whole cell patch clamp recording with conventional and gramicidin access. Each set of experiments tests a specific hypothesis regarding GABA's early excitatory role, using both cultured hypothalamic neurons and hypothalamic slices from mice. Hypothalamic slices containing the lateral hypothalamus/perifornical area will be used to examine early excitatory actions of GABA, and to study timing events related to depolarizing excitation or shunting. The hypothesis that spike-dependent release of GABA will strengthen developing GABAergic synapses by a long-lasting increase in the evoked response will be tested in a model system of a single autaptic neuron in vitro, focusing on a single type of GABA neuron that synthesizes the peptide MCH, and is identified by transfection with dsRed or GFP reporter genes driven by the MCH promoter. The hypothesis that synaptic actions of GABA, when excitatory, increase neuronal growth will be studied in hypothalamic MCH neurons, using time-lapse imaging. Gene arrays will be used to test the hypothesis that excitatory synaptic actions of GABA enhance the expression of specific genes coding for synaptic proteins, trophic and transcription factors, and CI- transporters in developing, but not mature, hypothalamic neurons; and that neuron trauma depresses outward CI- transporter expression and recapitulates the excitatory actions of GABA on gene expression. The hypothalamus controls body temperature, the endocrine system, circadian rhythms, the autonomic nervous system, gender differentiation, energy homeostasis, and water balance, and many of the synapses involved in these critical functions release GABA. GABA's excitatory actions during development are widespread throughout the brain. Thus, what we learn from our experiments on hypothalamic neurons should have general applicability to other CNS neurons.
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