REGULATION OF EXCITABILITY OF ENTERIC NEURONS BY CALCIUM

结肠运动是由肠神经系统的复杂电路控制的，它调节纵向和圆形肌肉层的收缩性。这种回路由感觉神经元、中间神经元和具有不同放电模式的运动神经元组成，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）的增加在调节神经元的兴奋性方面是重要的。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肠丛中功能性识别神经元的活性。我们将使用细胞内微电极记录和膜片钳技术结合钙的同时fura-2成像来确定这些神经元的兴奋性是如何被动作电位和突触活动产生的[Ca2+]I的变化所调节的。这将包括检查钙通过各种电压门控钙通道流入的作用，钙从细胞内钙储存释放的贡献以及钙依赖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，神经元将通过细胞内注射神经生物素和免疫组织化学来识别，或者通过对神经元的特定功能类别（感觉神经元和运动神经元到环形肌肉）进行i标记来识别。在膜片钳研究中，我们将研究通过di - i逆行标记技术鉴定的新鲜分散的感觉神经元和运动神经元中钙依赖离子通道的调节。最后，我们还将使用分子克隆技术和免疫组织化学来研究这些特定神经元中钙依赖离子通道的多样性。这些研究将进一步证实，功能不同的神经元的生理是由钙依赖机制的差异决定的，钙依赖机制控制神经元的兴奋性，表达独特的离子通道组合。这些知识将导致对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。

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.