Studying nicotinic AChRs in histaminergic neurons

描述（由申请人提供）：下丘脑结节乳头核（TM）的神经元代表大脑中组胺的主要来源，并将其轴突投射到大多数大脑区域以控制注意力和唤醒，抗组胺引起的镇静证明了这一点。α -班加罗毒素敏感型尼古丁乙酰胆碱受体（nAChRs）是唯一在TM神经元中高密度表达的nAChRs，其动力学和药理学与高Ca2+渗透性的同源物a7 nAChRs相似。我们的实验表明，在没有电压门控Ca2+通道（VGCCs）的情况下，TM nAChRs的激活不会提高TM体细胞内的胞质Ca2+浓度（[Ca2+]i）。因此，a7-like nAChR的动力学以及a7-like nAChR与其他TM Ca2+源之间的功能联系成为决定a7-like nAChR活化对[Ca2+]i影响的关键因素。这一发现很重要，因为a7 nachr的生理作用尚不清楚，可能涉及内源性和治疗性尼古丁剂（如胆碱和尼古丁）对TM组胺能功能的细胞保护和调节。Aim 1将验证TM a7样nAChRs是异质（涉及非a7亚基）的假设，其对Ca2+离子的通透性低于同质a7 nAChRs。因此，Aim 2将验证a7样nAChRs通过与关键Ca2+来源的功能联系间接调节TM [Ca2+]i的假设：vgcc；Ca2 +商店;和Na+-Ca2+交换剂。我们将使用Ca2+成像和膜片钳；逆转录聚合酶链反应；Western blot和免疫组化检测a7样nachr的性质；TM神经元中[Ca2+]i调控的机制，以及a7样nAChRs与其他关键Ca2+来源之间的联系。该研究将提供有关组胺能TM神经元中a7样nachr性质的重要信息；以及a7样nachr驱动的Ca2+稳态调节机制，涉及细胞保护和组胺能功能调节，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：两者都是抗伤害性的；改善注意力;提高警觉性和觉醒；并抑制食物摄入。尼古丁和治疗性尼古丁制剂的某些作用可能源于它们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.