REGULATION OF EXCITABILITY OF ENTERIC NEURONS BY CALCIUM

结肠运动由肠道神经系统的复杂回路控制，该回路调节纵向和环状肌层的收缩能力。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，负责大肠运动的复杂模式。肠道神经元的改变、退化或破坏导致了异常的运动模式。在中枢神经系统中，细胞内钙离子浓度([Ca~(2+)]i)的增加在调节神经元的兴奋性方面起着重要作用。然而，[Ca~(2+)]i在肠神经细胞兴奋性调控中的作用还不是很清楚。因此，本研究的目的是研究[Ca~(2+)]i如何调节豚鼠和小鼠结肠肌间神经丛中功能识别的神经元的活动。我们将使用细胞内微电极记录和膜片钳技术，结合同步的Fura-2钙成像来确定这些神经元的兴奋性如何受到动作电位和突触活动产生的[Ca+]i变化的调节。这将涉及通过各种电压门控钙通道的钙内流的作用，细胞内钙库钙释放的贡献，以及钙依赖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，神经元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(感觉神经元和环肌运动神经元)来识别。在膜片钳研究中，我们将研究Di-I逆行标记技术鉴定的新分散感觉神经元和运动神经元中钙依赖离子通道的调节。最后，我们还将利用分子克隆技术和免疫组织化学技术研究这些特定神经元中钙依赖离子通道的多样性。这些研究将进一步确定，不同功能的神经元的生理学是由控制神经元兴奋性的钙依赖机制的不同决定的，这些机制表达独特的离子通道组合。这些知识将有助于更好地理解和更具体地设计用于治疗和控制运动障碍的药物。

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.