GABA: EXCITATORY TRANSMITTER IN DEVELOPING HYPOTHALAMUS

描述（由申请人提供）：氨基酸GABA作为成人大脑中的主要抑制性递质。相反，在发育中的下丘脑中，GABA可以通过使膜电位去极化、提高胞质钙和诱发动作电位而兴奋。目前的建议集中在GABA在发育中的下丘脑神经元的兴奋作用。聚合方法利用fura-2钙数字成像，基因芯片，免疫细胞化学，电子显微镜，和全细胞膜片钳记录与常规和短杆菌肽访问。每组实验都使用培养的小鼠下丘脑神经元和下丘脑切片来测试关于GABA早期兴奋作用的特定假设。将使用含有外侧下丘脑/穹窿周围区的下丘脑切片检查GABA的早期兴奋作用，并研究与去极化兴奋或分流相关的计时事件。GABA的尖峰依赖性释放将通过诱发反应的持久增加来加强发育中的GABA能突触的假设将在体外单个自适应神经元的模型系统中进行测试，集中于合成肽MCH的单一类型的GABA神经元，并通过用MCH启动子驱动的dsRed或GFP报告基因转染来鉴定。假设GABA的突触作用，兴奋时，增加神经元的生长将在下丘脑MCH神经元，使用延时成像进行研究。基因阵列将用于测试以下假设：GABA的兴奋性突触作用增强发育中但不成熟的下丘脑神经元中编码突触蛋白、营养因子和转录因子以及Cl-转运体的特定基因的表达;以及神经元创伤抑制向外Cl-转运体表达并重现GABA对基因表达的兴奋性作用。下丘脑控制体温、内分泌系统、昼夜节律、自主神经系统、性别分化、能量稳态和水平衡，并且参与这些关键功能的许多突触释放GABA。GABA在发育过程中的兴奋作用遍布整个大脑。因此，我们从下丘脑神经元的实验中所学到的东西应该对其他CNS神经元具有普遍适用性。

DESCRIPTION (provided by applicant): 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.