Role of alpha3-containing nicotinic receptors in mediating central nicotine effec

描述(申请人提供)：这项提案的长期目标是了解尼古丁成瘾特性的分子和细胞基础，并阐明神经性尼古丁乙酰胆碱受体(NAChRs)在尼古丁的许多中枢调节的行为和生理效应中的功能作用。这一建议集中于含有α3亚单位的nAChRs，它高密度地存在于内侧缰核，并与腹侧被盖区(VTA)有关，腹侧被盖区是奖赏通路中公认的组成部分。内侧缰核是进化上保守的大脑区域，已被证明对尼古丁的神经毒性效应非常敏感。此外，有人提出，精神分裂症患者的一些认知缺陷可能与内侧缰核的潜在病理有关。这项研究中使用的主要工具是一种新型的敲入小鼠，其中尼古丁α3亚基被一个含有5个氨基酸取代的亚基取代，这些氨基酸替换使小鼠对α-银环蛇毒素(BGTX)的药理阻断具有敏感性。这种敲入小鼠能够使用经典的药理拮抗剂BGTX来探索含有α3的nAChRs在内侧缰核和VTA中的功能和行为作用。首先，重要的是确认BGTX敏感的、含有Alpha3的神经元nAChRs在从目标alpha3/alpha1[5]突变(+/tm1.1)杂合的小鼠脑冰冻切片中的区域表达。放射性-BGTX结合部位的放射自显影将用于此目的。在立体定向插管后，将BGTX微量注射到(+/tm1.1)小鼠的内侧缰核和VTA，以确定药物阻断含α-nAChRs对三种尼古丁相关行为的影响。这些包括：1)急性、低剂量全身尼古丁的运动减速效应；2)对尼古丁的口服偏好；以及3)尼古丁诱发的癫痫发作。相关性：尼古丁是一种极易上瘾的毒品，占西方世界所有可预防的死亡人数的20%。尼古丁还显著提高认知能力，一些遗传性癫痫涉及尼古丁受体。此外，胆碱能神经元的丧失与阿尔茨海默病有关，这是一种目前缺乏有效治疗的疾病。因此，了解尼古丁与中枢神经系统中尼古丁受体的分子相互作用对人类健康具有重要的潜在意义。

DESCRIPTION (provided by applicant): The long-term goals of this proposal are to understand the molecular and cellular basis for nicotine's addictive properties and to elucidate the functional role of neuronal nicotinic acetylcholine receptors (nAChRs) in the many centrally mediated behavioral and physiological effects of nicotine. This proposal focuses on alpha3-subunit-containing nAChRs which are found at high density in the medial habenula and which also have been implicated in the ventral tegmental area (VTA), a well recognized component of the reward pathway. The medial habenula is an evolutionarily conserved brain region that has been shown to be very sensitive to the neurotoxic effects of nicotine. Furthermore, it has been suggested that some of the cognitive deficits seen in schizophrenia may be linked to underlying pathology in the medial habenula. The main tool to be utilized in this study is a novel knock-in mouse in which the nicotinic alpha3-subunit has been replaced with one containing five amino acid substitutions that impart sensitivity to pharmacological blockade by alpha-bungarotoxin (Bgtx). This knock-in mouse enables the use of the classic pharmacological antagonist, Bgtx, to probe the functional and behavioral role of alpha3-containing nAChRs in the medial habenula and VTA. First, it will be important to confirm the regional expression of Bgtx-sensitive, alpha3- containing neuronal nAChRs in frozen sections of brains isolated from mice heterozygous for the targeted alpha3/alpha1[5] mutation (+/tm1.1). Autoradiography of radioactive-Bgtx binding sites will be used for this purpose. Following stereotaxic cranial cannulation, Bgtx will be microinjected into the medial habenula and VTA of (+/tm1.1) mice to determine the effect of pharmacological blockade of alphas-containing nAChRs on three nicotine-associated behaviors. These include: 1) the hypolocomotor effect of acute, low-dose systemic nicotine; 2) entrained oral preference for nicotine; and 3) nicotine-induced seizures. Relevance: Nicotine is an extremely addictive drug responsible for up to 20% of all preventable mortality in the western world. Nicotine also significantly enhances cognitive performance, and some inherited forms of epilepsy involve nicotinic receptors. In addition, a loss of cholinergic neurons is implicated in Alzheimer's disease, a disorder currently lacking effective treatment. Understanding the molecular interactions of nicotine with its receptors in the central nervous system therefore has significant potential to benefit human health.