GABA-EXCITATORY TRANSMITTER IN DEVELOPING HYPOTHALAMUS

下丘脑发育中的 GABA 兴奋性递质

• 批准号: 6529184
• 负责人: ANTHONY N VAN DEN POL
• 金额: $ 28.67万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6529184
• 关键词: age difference; brain injury; calcium flux; calcium indicator; developmental neurobiology; disease /disorder model; gamma aminobutyrate; gene expression; genetically modified animals; glutamates; green fluorescent proteins; growth cones; hypothalamus; immunocytochemistry; laboratory mouse; laboratory rat; neurogenetics; neurotrophic factors; newborn animals; synapsins; synaptogenesis; syntaxin; tissue /cell culture; transcription factor; voltage /patch clamp

DESCRIPTION (Verbatim from the Applicant's Abstract): 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.

描述（来自申请人摘要的逐字）：在成年神经元中，GABA 作为主要的抑制性递质。相反，在发展中国家， 下丘脑GABA可通过使膜电位去极化而兴奋， 提高胞质钙离子浓度，并诱发动作电位。现时的建议 专注于GABA能神经元的早期突触形成。趋同办法 利用Fura-2数字钙显像、免疫细胞化学、北方印迹 mRNA分析和用gramididin的全细胞膜片钳记录 穿孔解决了五个假设。每一组实验测试一个特定的 关于GABA的早期兴奋作用的假设，使用两种培养的 来自大鼠或小鼠的下丘脑神经元和下丘脑切片。第一组 实验证实了GABA从轴突释放的假设 在突触形成之前，生长锥，这种释放是由 其他的递质受体在生长的轴突上。我们检验了一个假设， 营养因子，特别是NT-3和BDNF，发挥快速的生理作用， 在培养和切片中观察GABA发育中的突触。这一快速行动将 在GABA兴奋期间增强GABA释放，但这 增强效应在较老的神经元中将消失。的假设 活动相关的GABA释放将加强发育中的GABA能突触 通过诱发反应的长期持续增加将在培养的 神经元; GABA能神经元的鉴定将通过使用 转基因小鼠，表达水母基因的绿色荧光蛋白在培养 下丘脑GABA能细胞突触释放GABA的假说 增强编码突触蛋白和转录的基因的表达 发育中的因素，但不成熟，突触耦合神经元将被测试 用北方印迹分析。最后一组实验继续我们先前的实验， 研究表明，GABA从其成人抑制作用恢复到 在培养的神经元损伤后的兴奋性一。这将扩展到测试 假设直接对大脑的损伤会导致去极化 GABA的作用。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，以及许多 参与这些功能的突触释放GABA。GABA的兴奋作用 在发展过程中，并不局限于下丘脑，而是 遍布大脑因此，我们从实验中了解到， 下丘脑神经元对其他CNS神经元具有普遍适用性。