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和帕金森氏症之间负相关的机制基础。该项目测试了长期接触尼古丁是否会上调基底神经节中功能性烟碱型乙酰胆碱受体(NAChRs)的表达，从而改变细胞和回路功能。“细胞特异性α4上调”假说认为，关键的上调受体对尼古丁高度敏感(HS)，因此主要是α4β2*，也可能是α6*亚型；细胞特异性α4*nAChR(S)的增加发生在长期接触尼古丁的过程中；这种细胞特异性上调为神经元活动的基于电路的改变提供了基础。目的1用含基底节的小鼠脑片检测α4*受体的分布，进而测定CSAUR。在适当的情况下，这些研究将使用nAChR功能的定量电生理评估、改良nAChRs的小鼠品系、细胞标记和免疫组织化学技术。将对黑质致密部(SNC)进行研究，以确认这些神经元中没有上调。这一假说将得到检验，即慢性尼古丁通过阻断GABAA受体或激活NMDA受体，使SNC神经元对突发性放电不那么敏感。将对丘脑底核(STN)进行研究，以证实初步数据表明它表达a*受体。如果在STN中发生CSAUR，则将研究突发发射。将对丘脑背侧中棘神经元(MSN)进行研究，以确定慢性尼古丁是否改变DA终末的α4*功能。如果是，将使用DA释放的电化学检测。GABA能中间神经元(INS)将接受α4*受体测试，然后进行α4*上调。如果检测到CSAUR，将通过解剖学鉴定α4*表达的INS；并将识别特定亚类被抑制的MSN。在目标2中，将监测完整动物的基底节中单个DA和GABA能细胞，以评估当神经元回路完整时揭示的慢性尼古丁的影响。在Aim3中，将在表达全功能荧光α4*受体的敲入小鼠中测量荧光。这种荧光的上调将在特定类型的细胞中进行测量。用双光子显微镜进行的高分辨率亚细胞研究将确定alpha4*表达和上调的亚细胞特异性。了解慢性尼古丁在基底节引起的细胞和回路变化可能会导致帕金森病的新疗法。公共卫生意义：帕金森氏病是由黑质神经细胞退化引起的。这些实验旨在揭示强负相关的机制基础。吸烟和帕金森氏症之间的关系。了解慢性尼古丁在基底节引起的细胞和回路变化可能会导致新的治疗方法。

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.