EXPERIMENTAL STUDIES IN SENSORY PATHWAYS

正常的基底节功能高度依赖于黑质多巴胺(DA)能神经元和SNR/EP的GABA能神经元的活动。然而，影响多巴胺和GABA投射靶点的多巴胺和GABA释放的SNC和SNR神经活动的许多控制机制仍不清楚。最近，人们越来越关注从被盖脚脑桥核(PPN)和丘脑底核(STN)向SNc DA和SNR GABA神经元的汇聚输入。尽管对这些信息的功能知之甚少，但我们以前的研究表明，来自PPN的乙酰胆碱(ACh)通过增加阳离子内流和减少K+外流来去极化DA和GABA神经元的静息膜电位，并在起搏器样慢去极化(PLSD)过程中减少Ca~(2+)内流，从而降低超极化后随后峰的幅度和持续时间。因此，我们假设(1)ACh的作用增加了SNc DA和Snr GABA能神经元对来自STN和PPN的谷氨酸(Glu)的反应而表现出爆发式放电的倾向；(2)除了来自PPN和STN的Glu和ACh兴奋性投射外，还有GABA能抑制投射到SN(通过对SNr神经元的相性抑制而导致GABA系统的去抑制)正在调节SNc DA和Snr GABA能神经元的放电行为。为了验证这些假说，我们将利用最新开发的由PPN、SN、STH和STR组成的体外器官型培养制剂来进行形态和电生理研究。形态学研究将涉及光镜和电子显微镜，以(1)确定ACh、Glu和GABA能投射模式及其在SNc DA和Snr GABA神经元上的终末位置，(2a)用联合生物细胞素胞内标记法确定ACh和Glu投射到SNc DA和Snr GABA能神经元的核来源，(2b)确定与SNC DA和Snr GABA神经元突触的细胞内标记的生物细胞素终末的位置，以及(3)确定ACh、Glu和GABA受体在SNc DA和Snr GABA神经元上的位置。电生理学研究将包括使用全细胞(电流)钳或细胞内尖电极记录来描述(1)在PLSD和AHP过程中胆碱能受体激活对钙内流的影响，(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 GABA能神经元的放电特性和爆发的影响。对于运动和行为功能以及帕金森氏病等临床疾病，了解多个输入在控制和/或调节影响其靶结构(即纹状体和丘脑)的SNC和SNR细胞活动中所起的作用具有重要意义。

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.