REGULATION OF EXCITABILITY OF ENTERIC NEURONS BY CALCIUM

结肠运动性由肠神经系统的复杂回路控制，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，负责大肠复杂的运动模式。异常运动模式是由肠神经元的改变、变性或破坏引起的。在CNS中，细胞内钙浓度（[CA 2 +]I）的增加在调节神经元的兴奋性中是重要的。然而，[CA 2 +]I在控制肠神经元兴奋性中的作用尚不清楚。本研究的目的是，因此，调查如何[CA 2 +]I调节功能鉴定的神经元的活动，在豚鼠和小鼠结肠肌间神经丛。我们将使用细胞内微电极记录和膜片钳技术结合同时fura-2钙成像，以确定这些神经元的兴奋性是如何通过[Ca 2 +]I的变化来调节的，这些变化是由动作电位和突触活动产生的。这将涉及检查通过各种电压门控钙通道的钙内流的作用，从细胞内钙库中释放钙的贡献以及钙依赖性离子通道在调节神经元兴奋性中的参与。在细胞内微电极研究中，将通过细胞内注射神经生物素和免疫组织化学，或通过Di-I标记特定功能类别的神经元（感觉神经元和运动神经元到环形肌）来鉴定神经元。在膜片钳研究中，我们将研究新鲜分散的感觉神经元和运动神经元的钙依赖性离子通道的调节，通过Di-I-逆行标记技术鉴定。最后，我们也将研究钙依赖离子通道的多样性，在这些特定的神经元使用分子克隆技术和免疫组织化学。这些研究将进一步确定功能不同的神经元的生理学是由控制表达离子通道的独特组合的神经元的兴奋性的钙依赖性机制的差异决定的。这些知识将导致更好的理解和更具体的设计药物设计来治疗和控制动力障碍。

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.