Studying nicotinic AChRs in histaminergic neurons

描述（由申请人提供）：下丘脑结节乳头核（TM）的神经元代表脑中组胺的主要来源，并将其轴突投射到大多数脑区域以控制注意力和觉醒，如抗组胺药引起的镇静所证明的。α-银环蛇毒素敏感的烟碱乙酰胆碱受体（nAChR）是TM神经元中唯一高密度表达的nAChR，其动力学和药理学与高Ca 2+渗透性的同聚体α 7 nAChR相似。我们的实验已经表明，在电压门控Ca 2+通道（VGCC）不存在的情况下，TM nAChRs的激活不会升高TM胞体中的胞质Ca 2+浓度（[Ca 2 +]i）。因此，α 7-like nAChR的动力学以及α 7-like nAChR与其他TM Ca 2+源之间的功能联系成为决定α 7-like nAChR激活对[Ca 2 +]i影响的关键因素。这一意义是重要的，因为α 7 nAChR的生理作用仍然未知，并且可能涉及通过内源性和治疗性烟碱剂（如胆碱和尼古丁）的细胞保护和TM组胺能功能的调节。目的1将检验TM α 7样nAChR是异聚体（涉及非α 7亚基）的假设，其对Ca 2+离子的渗透性低于同聚体α 7 nAChR。因此，目的2将检验α 7样nAChR通过与关键Ca 2+来源的功能联系间接调节TM [Ca 2 +]i的假设：VGCC; Ca 2+储存;和Na+-Ca 2+交换剂。我们将使用Ca 2+成像和膜片钳;逆转录聚合酶链反应;蛋白质印迹和免疫组织化学来确定α 7样nAChRs的性质;[Ca 2 +]i调节的机制，以及α 7样nAChRs和TM神经元中其他关键Ca 2+来源之间的联系。拟议的研究将提供关于组胺能TM神经元中α 7样nAChR的性质的重要信息;以及α 7样nAChR驱动的涉及细胞保护和组胺能功能调节的Ca 2+稳态调节机制，其连接胆碱能和组胺能脑系统。有趣的是，尼古丁和组胺对行为和认知的影响是相似的：两种药物都是抗伤害性的;提高注意力;促进警觉性和唤醒;抑制食物摄入。尼古丁和治疗性烟碱类药物的某些作用可能是由于其调节组胺能功能的能力。

DESCRIPTION (provided by applicant): Neurons of the tuberomammillary (TM) hypothalamic nucleus represent the major source of histamine in the brain and project their axons to most brain regions to control attention and arousal, as evidenced by the sedation caused by anti-histamines. Alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors (nAChRs), whose kinetics and pharmacology are similar to those of highly Ca2+-permeable homomeric a7 nAChRs, are the only nAChRs expressed in TM neurons in high densities. Our experiments have shown that activation of TM nAChRs does not elevate cytosolic Ca2+ concentrations ([Ca2+]i) in TM somata in the absence of voltage-gated Ca2+ channels (VGCCs). Therefore, the kinetics of a7-like nAChRs and functional links among a7-like nAChRs and other TM Ca2+ sources become the key factors determining the impact of a7-like nAChR activation on [Ca2+]i. This implication is important because the physiological role of a7 nAChRs remains unknown, and may involve cytoprotection and regulation of TM histaminergic function by endogenous and therapeutic nicotinic agents, such as choline and nicotine. Aim 1 will test the hypothesis that TM a7-like nAChRs are heteromeric (involve non-a7 subunits), whose permeability to Ca2+ ions is lower than that of homomeric a7 nAChRs. Accordingly, Aim 2 will test the hypothesis that a7-like nAChRs regulate TM [Ca2+]i indirectly, via functional links to key Ca2+ sources: VGCCs; Ca2+ stores; and Na+-Ca2+ exchangers. We will use Ca2+ imaging and patch-clamp; reverse transcription polymerase chain reaction; Western blot and immunohistochemistry to determine the nature of a7-like nAChRs; mechanisms of [Ca2+]i regulation, and links among a7-like nAChRs and other key Ca2+ sources in TM neurons. The proposed study will provide important information regarding the nature of a7-like nAChRs in histaminergic TM neurons; and a7-like nAChR-driven mechanisms of regulation of Ca2+ homeostasis implicated in cytoprotection and regulation of histaminergic function, linking cholinergic and histaminergic brain systems. Intriguingly, the behavioral and cognitive effects of nicotine and histamine are similar: both agents are anti-nociceptive; improve attention; promote alertness and arousal; and inhibit food intake. Some of these effects of nicotine and therapeutic nicotinic agents may arise from their ability to modulate histaminergic function.