GABA-EXCITATORY TRANSMITTER IN DEVELOPING HYPOTHALAMUS

下丘脑发育中的 GABA 兴奋性递质

• 批准号: 2860911
• 负责人: ANTHONY N VAN DEN POL
• 金额: $ 26.47万
• 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/2860911
• 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（逐字摘自申请人摘要）：在成体神经元中，GABA 充当主要抑制性递质。相比之下，在发展中 下丘脑 GABA 可通过膜电位去极化而兴奋， 提高细胞质钙并激发动作电位。目前的建议 专注于 GABA 能神经元的早期突触形成。趋同的方法 利用 fura-2 数字钙成像、免疫细胞化学、Northern 印迹 mRNA 分析和用短杆菌肽进行全细胞膜片钳记录 穿孔解决了五个假设。每组实验测试特定的 关于 GABA 早期兴奋作用的假设，使用培养的 来自大鼠或小鼠的下丘脑神经元和下丘脑切片。第一组 实验提出了 GABA 从轴突释放的假设 突触形成之前的生长锥，并且这种释放受到调节 生长中的轴突上的其他递质受体。我们检验以下假设： 营养因子，特别是 NT-3 和 BDNF，发挥快速的生理作用 在培养物和切片中 GABA 正在发育突触。这一迅速的行动将 当GABA兴奋时，增强GABA的释放，但这 在较老的神经元中，增强作用将会消失。假设 GABA 的活动相关释放将加强 GABA 能突触的发育 通过诱发反应的持久增加将在培养中进行测试 神经元； GABA能神经元的鉴定将通过使用 在培养中表达水母 GFP 基因的转基因小鼠 下丘脑 GABA 能细胞。假设突触释放 GABA 增强编码突触蛋白和转录的基因表达 将测试发育中但不成熟的突触耦合神经元的因素 与 Northern 印迹分析。最后一组实验延续了我们之前的实验 研究表明 GABA 从成体抑制作用恢复到成体抑制作用 培养中神经元损伤后的兴奋性。这将扩展到测试 直接伤害大脑会导致去极化的假设 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会释放 GABA。 GABA的兴奋作用 在发育过程中不限于下丘脑，而是 广泛分布于整个大脑。因此，我们从实验中学到的 下丘脑神经元应该对其他中枢神经系统神经元具有普遍的适用性。