PATCH CLAMP STUDY OF GASTROINTESTINAL NEURONS

这是一个建议，膜片钳研究的离子电流的神经元， 豚鼠小肠的肌间神经丛。 该研究 旨在提高对离子基础的理解的四个具体目标 在突触或旁分泌信号传导过程中的兴奋性变化。 协议 的最初目的是为了确定和表征离子 由跨膜电压变化激活的电流， AH/型神经元内钙水平和受体配体结合的研究 2和S/1型肌间神经元。 第二个目标将检验这一假设 AH/2型神经元中神经元内cAMP的升高增强了 通过抑制兴奋性：1）钙通道的电导率打开， 静息; 2）高阈值N型钙通道的电压激活; 3） 钙激活钾通道传导; 4）电压激活 A型和延迟整流钾通道。 目标3实验 被组织起来，以确定化学介质的行动， 突触兴奋（P物质、5-HT和组胺）对钠、钾 和AH/2型肌间神经元的钙电流。 这一阶段的 这项研究还将检验这一假设，即这些模拟慢突触的物质， 兴奋将表现得像cAMP的升高，在他们的行动上， 慢突触兴奋行为的离子电流。 最终 目的是确定化学介质的作用， 抑制（甘丙肽，甲硫氨酸和腺苷）对钠，钾和 AH/2型肌间神经元的钙电流。 本阶段工作 还将测试这些缓慢突触的模拟物的假设， 抑制作用通过G-蛋白偶联到一种或多种类型的开放 钾离子通道对离子电流的作用 AH/2型和S/1型肌间神经元中的缓慢突触抑制。 的 完成每个目标的工作将在急性分离的肌间 神经节从成人肠和肌间神经元培养物衍生 成人肠道 这项工作的重点是成人神经节， 在培养时发育成肌间神经丛的复制品。 在急性分离的神经节中进行的观察是对照， 细胞中通道或受体的表型表达的任何变化， 培养条件 除了提议者提供的初步数据外， 实验室，未对成人肌间神经元进行膜片钳研究 已被报道。 因此，应用膜片钳技术 需要获得填充现有空隙所需的信息 在理解神经元的电和突触行为方面 组成肠神经系统的集成电路。

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.