In adult neurons, GABA acts as the primary inhibitory transmitter. 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 early synapse formation in GABAergic neurons. Converging approaches utilizing fura-2 digital calcium imaging, immunocytochemistry, Northern blot mRNA analysis, and whole cell patch clamp recording with gramididin perforations address five hypotheses. Each set of experiments tests a specific hypothesis regarding GABA's early excitatory role, using both cultured hypothalamic neurons and hypothalamic slices from rats or mice. The first set of experiments addresses the hypothesis that GABA is released from axonal growth cones prior to synapse formation, and that this release is modulated by other transmitters receptors on the growing axon. We test the hypothesis that trophic factors, specifically NT-3 and BDNF, exert rapid physiological effects at the GABA developing synapse in culture and slice. This rapid action will enhance GABA release during the period when GABA is excitatory, but this enhancing effect will disappear in older neurons. The hypothesis that activity-related release of GABA will strengthen developing GABAergic synapses by a long-lasting increase in the evoked response will be tested in cultured neurons; identification of GABAergic neurons will be aided by the use of transgenic mice that express the jellyfish gene for GFP in cultured hypothalamic GABAergic cells. The hypothesis that synaptic release of GABA enhances the expression of genes coding for synaptic proteins and transcription factors in developing, but not mature, synaptic coupled neurons will be tested with Northern blot analysis. The final set of experiments pursues our earlier work showing that GABA reverts from its adult inhibitory action to an excitatory one after neuronal injury in culture. This will be extended to test the hypothesis that injury directly to the brain will result in depolarizing actions of GABA. 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 functions release GABA. GABA's excitatory actions during development are not restricted to the hypothalamus, but rather 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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