REGULATION OF EXCITABILITY OF ENTERIC NEURONS BY CALCIUM

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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.