EXPERIMENTAL STUDIES IN SENSORY PATHWAYS

感觉通路的实验研究

• 批准号: 6629253
• 负责人: Mark D Bevan
• 金额: $ 15.76万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6629253
• 关键词: acetylcholine; action potentials; basal ganglia; biocytin; brain mapping; cell membrane; central neural pathway /tract; dopamine; electron microscopy; electrophysiology; fluorescent dye /probe; glutamates; histochemistry /cytochemistry; immunocytochemistry; laboratory rat; neuroanatomy; neurophysiology; sensory mechanism; single cell analysis; substantia nigra; tissue /cell culture; voltage /patch clamp

Normal basal ganglia function is highly dependent on the activities of dopaminergic (DA) neurons in the SNc and GABAergic neurons in SNr/EP. However, many aspects of the mechanisms controlling SNc and SNr neural activity which influence dopamine and GABA release at their projection targets are still not well understood. Recently there has been a growing interest in converging inputs from the tegmental pedunculopontine (PPN) and subthalamic nucleus (STN) to the SNc DA and SNr GABA neurons. Although the functional role of these inputs is poorly understood, our previous studies suggested that acetylcholine (ACh) inputs from PPN depolarize the resting membrane potential of DA and GABA neurons by a combination of increased cationic influx and decreased K+ efflux, and reduces Ca2+ entry during the pacemaker-like slow depolarization (PLSD), which decreases the amplitude and duration of the subsequent spike after hyperpolarization. We, therefore, hypothesize that (1) the action of ACh increases the tendency for SNc DA and SNr GABAergic neurons to exhibit burst firing in response to glutamatergic (glu) input from the STN and PPN and (2) in addition to glu and ACh excitatory projections from the PPN and STN, but also GABAergic inhibitory projections to SN (causing disinhibition of GABA system by phasic inhibition of the SNr neurons) are modulating SNc DA and SNr GABAergic neuronal firing behavior. To test these hypothesis, we will utilize the newly developed in vitro organotypic culture preparation consisting of PPN, SN, STH and STR to conduct morphological and electrophysiological studies. Morphological studies will involve light and electron microscopy to (1) identify ACh, glu and GABAergic projection patterns and their terminal sites on the SNc DA and SNr GABA neurons, (2a) identify the nuclear origin of ACh and glu projections to SNc DA and SNr GABAergic neurons with a combined biocytin intracellular labeling, and (2b) identify the locations of the intracellularly labeled biocytin terminals making synapses with SNc DA and SNr GABA neurons and (3) identify the location of ACh, glu and GABA receptors on the SNc DA and SNr GABA neurons. Electrophysiological studies will involve use of whole-cell (current) clamp or intracellular sharp electrode recording to delineate the effect of (1) cholinergic receptor activation on Ca2+ entry during the PLSD and AHP,(2) the effect of excitatory synaptic inputs from PPN and STN and GABAergic inhibitory inputs from the STR on firing properties and bursting of SNc DA and SNr GABAergic neurons. Understanding the role of multiple inputs in the control and/or modulation of SNc and SNr cellular activity which affect their target structures (i.e. striatum and thalamus) is of fundamental importance relative to motor and behavioral function as well as clinical entities such as Parkinson's disease.

正常的基底神经节功能高度依赖于 SNc 中的多巴胺能 (DA) 神经元和 SNr/EP 中的 GABA 能神经元的活动。 然而，控制 SNc 和 SNr 神经活动（影响多巴胺和 GABA 在其投射目标处释放）的机制的许多方面仍不清楚。 最近，人们越来越关注将被盖脚桥 (PPN) 和底丘脑核 (STN) 的输入汇聚到 SNc DA 和 SNr GABA 神经元。 尽管对这些输入的功能作用知之甚少，但我们之前的研究表明，PPN 的乙酰胆碱 (ACh) 输入通过增加阳离子流入和减少 K+ 流出相结合，使 DA 和 GABA 神经元的静息膜电位去极化，并在起搏器样缓慢去极化 (PLSD) 过程中减少 Ca2+ 进入，从而降低随后的振幅和持续时间。 超极化后尖峰。因此，我们假设（1）ACh 的作用增加了 SN​​c DA 和 SNr GABA 能神经元响应来自 STN 和 PPN 的谷氨酸（glu）输入而表现出爆发放电的趋势，（2）除了来自 PPN 和 STN 的 glu 和 ACh 兴奋性投射外，还有对 SN 的 GABA 能抑制性投射（导致 GABA 系统的去抑制） SNr 神经元的阶段性抑制）正在调节 SNc DA 和 SNr GABA 能神经元放电行为。 为了验证这些假设，我们将利用新开发的由PPN、SN、STH和STR组成的体外器官培养制剂来进行形态学和电生理学研究。 形态学研究将涉及光学和电子显微镜，以 (1) 识别 ACh、glu 和 GABA 能投射模式及其在 SNc DA 和 SNr GABA 神经元上的末端位点，(2a) 通过联合生物胞素细胞内标记识别 ACh 和 glu 投射到 SNc DA 和 SNr GABA 能神经元的核起源，以及 (2b) 识别细胞内 标记的生物胞素末端与 SNc DA 和 SNr GABA 神经元形成突触，并且 (3) 识别 SNc DA 和 SNr GABA 神经元上的 ACh、glu 和 GABA 受体的位置。 电生理学研究将涉及使用全细胞（电流）钳或细胞内尖锐电极记录来描绘（1）PLSD和AHP期间胆碱能受体激活对Ca2+进入的影响，（2）来自PPN和STN的兴奋性突触输入和来自STR的GABA能抑制输入对SNc DA和SNr的放电特性和爆发的影响 GABA能神经元。 了解多种输入在控制和/或调节 SNc 和 SNr 细胞活性（影响其目标结构（即纹状体和丘脑））中的作用对于运动和行为功能以及帕金森病等临床实体具有根本重要性。