大鼠心臟迷走節前神經元活性的延腦調控機轉之探討

本計劃以雄性Wistar 大鼠為實驗動物，採 用正向及逆向神經路徑追蹤技術(anterograde and retrograde tract-tracing methods)，來探討延 腦的孤獨核(nucleus tractus solitarius, NTS)與 心臟迷走節前神經元之間是否有直接之連 結； 並以電生理記錄法在疑核區(nucleus ambiguus, NA)搜尋心臟迷走神經元，分析其電 生理特性。解剖的實驗中，以雙管玻璃微電極 在大鼠延腦閂門(obex)附近的外側NTS，以微 量注射麩胺酸 (L-glutamate, 10 mM, 5-10 nl)的 方式進行化學性刺激，找到明顯血壓下降的反 應點後，再將另一玻璃管中的正向追蹤劑10% biotin-conjugated dextran (BD, 15 nl)注入此反 應點。經過7 天的存活期後，將逆向追蹤劑 2.5% WGA- HRP(Sigma,10 l)注入同一隻大鼠 之心臟。再經過48-72 小時之存活期後，將動 物灌流固定，取出腦幹及上段頸髓，進行冷凍 切片(50 m) ， 以組織化學反應將BD 與 WGA-HRP 分別呈色。結果顯示在延腦兩側NA 區發現許多來自NTS 的BD標定神經纖維之分 布，在NA 中的HRP 標定心臟迷走節前神經細 胞上亦可見BD 標定之神經末梢及終鈕。電生 理實驗中，則以自行研發、能同時注射藥物和 記錄神經元活性之電極，進行大鼠延腦NA 區 神經元活性之記錄與特性分析，以及抑心作用 之評估。結果顯示可在閂門附近NA 腹外側區 中，記錄到能在呼氣時放電、具心週期節律之 迷走抑心神經元活性，此神經元活性能被血壓 上升所激發或增加；少量麩胺酸(~50 pmol)刺 激即能引發強烈抑心作用(-63 4%)。本研究顯 示NTS 之神經元可能藉由直接投射至NA區之 心臟迷走節前神經元而調控抑心之功能。In the present study, we investigate the medullary neural connection and electrophysiological characteristics of the cardiac vagal preganglionic neurons in male Wistar rats. In the anatomical experiment, we examine the projections of nucleus tractus solitarius (NTS) to the cardioinhibitory vagal neurons in the nucleus ambiguus (NA) by anterograde and retrograde tract-tracing methods. Two-barrel glass micropipettes were constructed. Biotin-conjugated dextran (BD, Molecular Probes, 10%, 15-30 nl) was injected into the NTS, where profound depressor response was induced by microinjection of glutamate solution (Glu, 10 mM, 5-10 nl). Seven days later, WGA-HRP (Sigma, 2.5%, 10l) was injected into the heart. After 48-72 hours survival, the animal was reanesthetized, perfused and fixed. The brainstem was sectioned and processed with immunohistochemistry. We found many BD labeled fiber terminals, which projected from NTS, in cardioinhibitory region of NA, bilaterally. We also observed that BD-labeled fiber terminals and buttons on HRP-labelled NA neurons. In the electrophysiological experiment, we recorded the activity of the cardiac vagal neuron in the NA by using our recent invention of a recording /injection microelectrode. We found that the cardiac vagal neuron displayed cardiac cycle related rhythm, discharged during the expiratory period, and increased their activity after i.v. injection of phenylephrine despite their low spontaneous activity. Profound bradycardia (-63 4%) was elicited with picomoles of Glu (~50 pmol). The data suggested that the NTS may play an important role in controlling the cardioinhibitory function by direct projecting to the cardiac vagal preganglionic neurons in NA