Studying nicotinic AChRs in histaminergic neurons

描述(申请人提供)：结节乳头(TM)下丘脑核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，抗组胺药物引起的镇静证明了这一点。α-银环蛇毒素敏感的烟碱型乙酰胆碱受体(NAChRs)是TM神经元中唯一高密度表达的nAChRs，其动力学和药理学与高钙通透性同源A7nAChRs相似。我们的实验表明，在没有电压门控钙通道(VGCC)的情况下，TM nAChRs的激活不会增加TM胞浆中的钙浓度([Ca+]i)。因此，A7样nAChRs的动力学以及A7样nAChRs与其他TM钙来源之间的功能联系成为决定A7样nAChR激活对[Ca~(2+)]i影响的关键因素。这一意义是重要的，因为A7 nAChRs的生理作用尚不清楚，可能涉及内源性和治疗性尼古丁类药物对TM组胺能功能的细胞保护和调节。目的1验证TMA7样nAChRs是异构体(涉及非A7亚基)的假说，其对钙离子的通透性低于同聚体A7nAChRs。因此，目标2将检验这一假设，即A7样nAChRs通过与关键的钙来源：VGCC、钙储存和Na+-钙交换器的功能链接来间接调节TM[钙]i。我们将使用钙离子成像和膜片钳技术、逆转录聚合酶链式反应、Western印迹和免疫组织化学方法来确定A7样nAChRs的性质，[Ca2+]i的调节机制，以及A7样nAChRs与TM神经元中其他关键钙来源之间的联系。这项拟议的研究将提供有关组胺能TM神经元中A7样nAChRs的性质的重要信息；以及A7样nAChR驱动的钙稳态调节机制，涉及连接胆碱能和组胺能脑系统的细胞保护和组胺能功能调节。有趣的是，尼古丁和组胺对行为和认知的影响是相似的：这两种物质都具有抗伤害性、提高注意力、提高警觉性和唤醒能力以及抑制食物摄入的作用。尼古丁和治疗性尼古丁制剂的部分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.