Chronic nicotine: cell-specific receptor and circuit alterations in basal ganglia

描述（由申请人提供）：这些实验的总体目标是揭示吸烟与帕金森病之间负相关的机制基础。该项目测试慢性暴露于尼古丁是否上调基底节区功能性尼古丁乙酰胆碱受体（nAChRs），改变细胞和电路功能。“细胞特异性alpha4上调”（cell-specific alpha4 up- regulation， CSAUR）假说认为，关键的上调受体对尼古丁具有高敏感性（HS），因此主要是alpha4beta2*亚型，也可能是alpha6*亚型；α 4* nAChR(s)的细胞特异性增加发生在慢性尼古丁暴露期间；这种细胞特异性的上调为神经元活动中基于回路的变化提供了基础。Aim 1利用含有基底节区的小鼠脑切片检测alpha4*受体的分布，进而确定CSAUR。在适当的情况下，研究将采用nAChR功能的定量电生理评估、修饰nAChR的小鼠品系、细胞标记和免疫组织化学技术。我们将对黑质致密部（SNc）进行研究，以证实这些神经元中不存在上调。该假设将被验证，即慢性尼古丁使SNc神经元对GABAA受体阻断或NMDA受体激活介导的突发放电不那么敏感。我们将对丘脑下核（STN）进行研究，以证实其表达a*受体的初步数据。如果在STN中发生CSAUR，将研究爆发射击。我们将研究丘脑背侧的中棘神经元（MSNs），以确定慢性尼古丁是否会改变DA末端的α 4*功能。如果是这样，将采用电化学检测DA释放。GABAergic interneuron （INs）将检测α 4*受体，然后检测α 4*上调。如果检测到CSAUR，将通过解剖鉴定表达alpha4*的INs；以及被抑制的msn的特定亚类。在Aim 2中，将监测完整动物基底神经节中的单个DA和gaba能细胞，以评估慢性尼古丁在神经元回路完整时所显示的影响。在Aim3中，将在表达全功能荧光α 4*受体的敲入小鼠中测量荧光。这种荧光的上调将在特定的细胞类型中被测量。高分辨率亚细胞研究与双光子显微镜将确定亚细胞特异性α 4*的表达和上调。了解慢性尼古丁在基底神经节引起的细胞和电路变化可能会导致帕金森病的新疗法。公共卫生相关性：帕金森病是由黑质神经细胞退化引起的。这些实验旨在揭示强逆相关的机制基础。吸烟和帕金森氏症之间的关系了解慢性尼古丁在基底神经节引起的细胞和电路变化可能会带来新的治疗方法。

DESCRIPTION (provided by applicant): The overall goal of these experiments is to reveal a mechanistic basis for the inverse correlation between smoking and Parkinson's disease. The project tests whether chronic exposure to nicotine up-regulates functional nicotinic acetylcholine receptors (nAChRs) in the basal ganglia, changing cell and circuit function. The "cell-specific alpha4 up- regulation" (CSAUR) hypothesis states that the key up-regulated receptors are characterized by a high sensitivity (HS) to nicotine and are therefore mostly of the alpha4beta2* and possibly of the alpha6* subtypes; that cell-specific increases in alpha4* nAChR(s) occur during chronic exposure to nicotine; and that this cell-specific upregulation provides the basis for circuit-based changes in neuronal activity. Aim 1 uses mouse brain slices containing basal ganglia to detect the distribution of alpha4* receptors, then to determine CSAUR. The studies will employ, where appropriate, quantitative electrophysiological assessment of nAChR function, mouse strains with modified nAChRs, cell labeling, and immunohistochemical techniques. Substantia nigra pars compacta (SNc) will be studied to confirm the absence of up-regulation in these neurons. The hypothesis will be tested that chronic nicotine renders SNc neurons less sensitive to burst firing mediated by GABAA receptor blockade or by NMDA receptor activation. Subthalamic nucleus (STN) will be studied to confirm preliminary data suggesting that it expresses a* receptors. If CSAUR occurs in STN, burst firing will be studied. Medium spiny neurons (MSNs) in dorsal thalamus will be studied to determine whether chronic nicotine changes alpha4* function at the DA terminals. If so, electrochemical detection of DA release will be used. GABAergic interneurons (INs) will be tested for alpha4* receptors, and then for alpha4* up-regulation. If CSAUR is detected, the alpha4*-expressing INs will be identified by anatomy; and the particular subclass of inhibited MSNs will be identified. In Aim 2, single DA and GABAergic cells will be monitored in basal ganglia of intact animals, to assess effects of chronic nicotine that are revealed when neuronal circuits are intact . In Aim3, fluorescence will be measured in knock-in mice that express fully functional fluorescent alpha4* receptors. Up-regulation of this fluorescence will be measured in specific cell types. High-resolution subcellular studies with 2-photon microscopy will determine the sub-cellular specificity of alpha4* expression and up-regulation. Understanding the cellular and circuit changes induced by chronic nicotine in basal ganglia could lead to new therapies for Parkinson's disease. PUBLIC HEALTH RELEVANCE: Parkinson's disease is caused by degeneration of nerve cells in the substantia nigra. These experiments are designed to reveal a mechanistic basis for the strong inverse correlation. between smoking and Parkinson's disease. Understanding the cellular and circuit changes induced by chronic nicotine in basal ganglia could lead to new therapies.