利用單細胞RT-PCR與電生理方法研究分析大鼠之迷走神經元與A7核區兒茶酚胺神經元的突觸外γ-胺基丁酸受體

Abstract

越來越多的研究成果顯示受γ-胺基丁酸(GABA)活化的突觸外圍之γ-胺基丁酸受體(GABAARs)會對所位在的神經元有持續性抑制的作用，並控制此神經元的興奮性。位在疑核(NA)的心臟迷走神經元(CVNs)是控制心跳的主要動作神經元；而分泌正腎上腺素的A7核區兒茶酚胺神經元(A7 neurons)會透過在脊索神經背角釋放正腎上腺素(NA)來調節痛覺的敏感度，綜多研究也顯示出GABA在控制CVNs (來自孤束核(NTS))及A7 neurons (來自分泌GABA之周邊中間神經元)的興奮性上是扮演重要的角色的，且這些神經元是接受非常強烈之釋放GABA的神經傳入影響。在CVNs和A7 neurons上除了有負責調控即效姓抑制的GABAARs與甘胺酸受體(GlyRs)之外，還有處於突觸外圍負責調控持續性抑制的GABAARs存在，這本篇論文中，我們想要更進一步辨認並比較在CVNs和A7 neurons上到底是由哪些GABAARs次單元組成來調節持續性抑制作用的。首先我們把逆向螢光追蹤染劑施打在大鼠(7~10天大)的圍心膜內側，經過約48小時的術後回復後就把腦幹做冠狀切面，然後在螢光顯微鏡下找到這些被螢光標訂的CVNs，做完電生理實驗後就用玻璃電極把細胞吸起，而後將其溶解並使用Nanoprep Kit來抽取它們的mRNA，經歷過標準的單細胞逆轉錄聚合酶鏈反應(single-cell RT-PCR)程序後即可分析它們GABAARs次單元表現情況。A7 neurons的實驗程序也是跟CVNs一樣的，它們坐落在大鼠(7~10天大)腦幹矢狀切面之三叉動作神經核(Mo5)約200 μm的前方並擁有蠻大的細胞本體(直徑約20 μm)。先前就有研究指出有功能的突觸外圍GABAARs可能是由α1、α5、γ1、γ2、δ或ε次單元所組成，所以我們就檢驗這些次單元在兩種神經元上的表現情形，並進一步分析收集到的總共77個被螢光標記的CVNs及33個A7 neurons的GABAARs次單元組成，其中只有35個CVNs與27個A7 neurons各別同時表現了膽鹼乙醯轉移酶(CAT)和多巴胺β羥化酶(DBH)兩種細胞標誌蛋白。在所有CVNs裡，α1次單元是被偵測到最多的，α5、γ1、γ2和ε次單元的表現量次之，而δ次單元的量是最稀少的；這結果與A7 neurons有些相似，不過其γ2次單元的表現量也非常高。以上結果同樣地與我們的電生理實驗數據相符，GABAARs在兩種神經元上所調控的持續姓抑制電流都可被zolpidem (α1、γ次單元的協同劑)強化，並皆能被picrotoxin (GABAARs抑制劑)所阻礙，而在受α5、δ次單元協同劑的影響下電流並沒有顯著的變化，總而言之，這些結果說明了α1次單元在調節CVNs與A7 neurons之GABAARs的持續姓抑制作用上扮演了一關鍵性的角色。

A growing number of reports have demonstrated that GABA acting on extrasynaptic GABAA receptors (GABAARs) exerts strong tonic inhibition in the target neurons, thereby controls the neuronal excitability. The cardiac vagal neurons (CVNs) in nucleus ambiguus (NA) is the principal motor neurons that control hart rate, and the noradrenergic (NAergic) A7 neurons are involved in modulating nociception by releasing noradrenaline in the dorsal spinal cord. It is proposed that GABA plays an important role in control excitability of CVNs (from NTS) and A7 neurons (from local GABAergic interneurons), these neurons receive very strong GABAergic inputs. In addition to phasic inhibitions mediated by GABAA and glycine receptors, evidences for existence of extrasynaptic GABAAR-mediated tonic inhibition in CVNs and A7 neurons have been provided. In this study, I wish to further characterize and compare what subunits of GABAARs are responsible to tonic GABAARs mediated current in these two nucleus. CVNs were retrograde-labeled by fluorescent tracer applied to pericardiac cavity in rats (P7-10 days). After surgery, the animals were allowed to survive for 48 hours and the transverse brainstem slices were cut. The fluorescent labeled CVNs were searched under fluorescent microscope and picked up by a grass pipette after electrophysiological experiments. They were lysed and the mRNA was extracted using Nanoprep Kit. Standard RT-PCR procedures were employed for analysis of expression profile of GABAAR subunits in CVNs. The procedure of A7 neurons were same as CVNs. They had a large somata diameter (~20 μm) located ~200 μm rostral to the trigeminal motor nucleus (the presumed A7 area) in sagittal brainstem slices in rats (P7-10 days). Since it has been reported that functional extrasynaptic GABAARs may consist of α1, α5, δ, γ1, γ2, and ε subunit, expression of these subunits was examined in both neurons. A total number of 77 fluorescent labeled CVNs and 33 A7 neurons were collected and subjected to GABAARs analysis. Only 35 of CVNs and 27 of A7 neurons expressed cholineacetyltransferase (ChAT) and dopamine beta-hydroxylase (DBH). In all of the CVNs, α1 subunits were detected mostly, α5, γ1, γ2, ε but less the δ subunits were also detected. The results of A7 neurons were similar as CVNs but the expression of γ2 subunits were also abundant. These results are consistent with our previously electrophysiological results, in which picrotoxin sensitive tonic GABAAR current was enhanced by zolpidem, an α1 and γ subunit agonist, and not by drug acting at α5 or δ subunits. Taken together, the present results suggest a role for α1 subunit in mediating tonic GABAAR-mediated inhibition in CVNs and A7 neurons.