REGULATION OF EXCITABILITY OF ENTERIC NEURONS BY CALCIUM

钙对肠神经元兴奋性的调节

• 批准号: 6448975
• 负责人: Terence Keith Smith
• 金额: $ 18.5万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6448975
• 关键词: action potentials; calcium channel; calcium flux; colon; electrophysiology; fluorescent dye /probe; gastrointestinal motility /pressure; guinea pigs; immunocytochemistry; laboratory mouse; membrane channels; microelectrodes; molecular cloning; neurons; neurotransmitters; smooth muscle; synapses; voltage /patch clamp

Colonic motility is controlled by the complex circuitry of the enteric nervous system which regulates the contractility of the longitudinal and circular muscle layers. This circuitry, which consists of sensory neurons, interneurons and motor neurons with diverse firing patterns, is responsible for the complex patterns of motility in the large intestine. Abnormal motility patterns result from alteration, degeneration or disruption of enteric neurons. In the CNS, increases in intracellular calcium concentration ([CA2+]I) are important in regulating the excitability of neurons. The role of [CA2+]I in controlling the excitability of enteric neurons, however, is not well understood. The aims of this study are, therefore, to investigate how [CA2+]I regulates the activity of functionally identified neurons in the myenteric plexus of the guinea-pig and mouse colon. We will use intracellular microelectrode recordings and patch clamp techniques combined with simultaneous fura-2 imaging of calcium to determine how the excitability of these neurones is regulated by changes in [Ca2+]I, produced by action potentials and synaptic activity. This will involve an examination of the role of calcium influx through various voltage gated calcium channels, the contribution of calcium release from intracellular calcium store and the participation of calcium dependent ion channels in the regulation of neuronal excitability. In the intracellular microelectrode studies neurones will be identified by either intracellular injection of neurobiotin and immunohistochemistry, or by Di-I labeling specific functional classes of neurones (sensory neurones and motor neurones to the circular muscle). In the patch clamp studies we will examine the regulation of the calcium dependent ion channels in freshly dispersed sensory neurons and motor neurons identified by the Di-I-retrograde labeling technique. Finally, we will also examine the diversity of calcium dependent ion channels in these particular neurones using molecular cloning techniques and immunohistochemistry. These studies will further establish that the physiology of functionally different neurones is determined by differences in calcium dependent mechanisms controlling the excitability of neurons which express unique combinations of ion channels. Such knowledge will lead to a greater understanding and more specific design of drugs designed to treat and control motility disorders.

结肠运动由肠神经系统的复杂电路控制，肠神经系统调节纵向和环形肌肉层的收缩性。该回路由具有不同放电模式的感觉神经元、中间神经元和运动神经元组成，负责大肠中复杂的运动模式。异常的运动模式是由肠神经元的改变、退化或破坏引起的。在中枢神经系统中，细胞内钙浓度 ([CA2+]I) 的增加对于调节神经元的兴奋性非常重要。然而，[CA2+]I 在控制肠神经元兴奋性中的作用尚不清楚。因此，本研究的目的是研究 [CA2+]I 如何调节豚鼠和小鼠结肠肌间神经丛中功能确定的神经元的活动。我们将使用细胞内微电极记录和膜片钳技术结合同时进行的 fura-2 钙成像来确定这些神经元的兴奋性如何通过动作电位和突触活动产生的 [Ca2+]I 的变化进行调节。这将涉及检查通过各种电压门控钙通道的钙流入的作用、细胞内钙储存的钙释放的贡献以及钙依赖性离子通道在神经元兴奋性调节中的参与。在细胞内微电极研究中，通过细胞内注射神经生物素和免疫组织化学，或通过 Di-I 标记神经元的特定功能类别（环肌的感觉神经元和运动神经元）来识别神经元。在膜片钳研究中，我们将检查通过 Di-I 逆行标记技术识别的新鲜分散的感觉神经元和运动神经元中钙依赖性离子通道的调节。最后，我们还将使用分子克隆技术和免疫组织化学检查这些特定神经元中钙依赖性离子通道的多样性。这些研究将进一步证实，功能不同的神经元的生理学是由控制表达独特离子通道组合的神经元兴奋性的钙依赖性机制的差异决定的。这些知识将导致对旨在治疗和控制运动障碍的药物有更深入的了解和更具体的设计。