PATCH CLAMP STUDY OF GASTROINTESTINAL NEURONS

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This is a proposal for a patch clamp study of ionic currents in neurons of the myenteric plexus from the guinea-pig small intestine. The study has four specific aims directed to improving understanding of the ionic basis of excitability changes during synaptic or paracrine signaling. Protocols of the initial aim are designed to identify and characterize the ionic currents that are activated by changes in transmembrane voltage, elevation of intraneuronal calcium levels and ligand binding to receptors in AH/Type 2 and S/Type 1 myenteric neurons. The second aim will test the hypothesis that elevation of intraneuronal cAMP in AH/Type 2 neurons enhances excitability by suppressing: 1) conductance in calcium channels open at rest; 2) voltage activation of high threshold N-type calcium channels; 3) conduction in calcium-activated potassium channels; 4) voltage activation of A-type and delayed rectifier potassium channels. Experiments of Aim 3 are organized to determine the action of chemical mediators of slow synaptic excitation (substance P, 5-HT and histamine) on sodium, potassium and calcium currents in AH/Type 2 myenteric neurons. This phase of the study will also test the hypothesis that these mimetics of slow synaptic excitation will behave like elevation of cAMP in their actions on the ionic currents underlying slow synaptic excitatory behavior. The final aim is to determine the action of chemical mediators of slow synaptic inhibition (galanin, metenkephalin and adenosine) on sodium, potassium and calcium currents in AH/Type 2 myenteric neurons. This phase of the work will also test the hypothesis that these mimetics of slow synaptic inhibition act through G-proteins coupled to opening of one or more types of potassium channels in their actions on the ionic currents underlying slow synaptic inhibition in AH/Type 2 and S/Type 1 myenteric neurons. The work to accomplish each aim will be done on acutely dissociated myenteric ganglia from adult intestine and on myenteric neuronal cultures derived from adult intestine. The focus of the work is on adult ganglia that develop into a replica of the myenteric plexus when cultured. Observations to be made in acutely dissociated ganglia are controls for any changes in phenotypic expression of channels or receptors in the culture conditions. Other than preliminary data from the proponents' laboratory, no results of patch clamp studies on adult myenteric neurons have been reported. Consequently, application of patch clamp methodology is needed to acquire the information necessary for filling existing voids in understanding of the electrical and synaptic behavior of the neurons that make up the integrated circuits of the enteric nervous system.