大鼠疑核區內心臟迷走神經元上的γ-氨基丁酸A型受器組成單元之探討及戊巴比妥鈉對其抑制性神經傳導影響

Abstract

在中樞神經系統內，長效性抑制電流對於神經興奮性的調控扮演一個重要的角色。在阻斷麩胺酸的神經傳導下，以全細胞膜電鉗(whole cell patch-clamp recording)技術，可在疑核內的心臟迷走神經元中紀錄到大量自發性抑制性突觸後電流。這種自發性抑制性突觸後電位可以被A型 γ-胺基丁酸受體(GABAA受體)阻斷劑PTX和甘氨酸(glycine受體)拮抗劑strychnine所阻斷，代表這些自發性抑制性突觸後電流是由代表這些自發性抑制性突觸後電流是由GABAA受體和glycine受體所貢獻。同時發現到PTX可以降低原本維持紀錄電位的電流，但是給予strychnine並沒有這現象。這樣的結果可以指出心臟迷走神經元上的長效性抑制電流是由GABAA受體所貢獻。用另一種GABA拮抗劑gabazine及strychnine可以阻斷所有的自發性抑制性突觸後電流，但是並無法改變原本維持紀錄電位的電流。這種長效性抑制電流並不會被作用於d單元的GABAA受體協同劑酒精和THDOC所影響，這代表長效性抑制電流並非由含有d單元的GABAA受體所貢獻。γ單元的GABA協同劑zolpidem也會降低原本維持紀錄電位的電流，代表γ單元的GABA受器分佈在心臟迷走神經元上。另外我們研究petobarbital(Pento)這個廣泛用於試驗動物的麻醉藥，因為它對心血管功能方面有重大影響。目前已知Pento是透過GABAA受體去影響神經元。因此Pento對心血管方面的影響是否透過GABAA受體作用於疑核上心臟迷走神經元是我們想探討的。給予Pento於心臟迷走神經元後，所記錄到電流開始不穩，另外自發性抑制性突觸後電流衰減時間延長，同時有將近120微安培的電流流入細胞內，這些作用可以用PTX和GABAA受體拮抗劑Biculline所阻斷。給予突觸前動作電位阻斷劑TTX後，發現到自發性抑制性突觸後電流的產生頻率減少很多，代表小型的抑制性突觸後電位很少發生在心臟迷走神經元上。但是Pento所增加的電流不受TTX的影響，表示電流的增加不是由快速去敏感化的GABAA受體所疊加產生的變化。另外增加紀錄時的溫度，可以減少長效性抑制電流的影響，發現到一樣無法影響Pento所引發的電流。總結以上的結果，心臟迷走神經元的長效性抑制電流是由γ單元的GABAA受體所調控，而非d單元。另外我們推測Pento可以直接作用GABAA受體而增加效能。因此增加快速和長效性GABAA受體的作用會使心臟迷走神經元被抑制且降低心血管功能。

Tonic inhibition has emerged as a key regulator of neuronal excitability in the central nervous system. Under block of glutamatergic transmission, there was many spontaneous IPSC (sIPSC) activity could be recorded by whole cell patch-clamp recording in cardiac vagal neurons (CVNs) in nucleus ambiguous (NA). The sIPSC could only be completely blocked with GABAA receptor blocker picrotoxin (PTX) and glycine receptor antagonist strychnine (Stry), showing it was mediated by GABAA and glycine receptors. Interestingly, application of PTX was associated with a reduction in holding current (Ihold), which was not observed with Stry application. These results suggest that there is a tonic inhibitory current (Itonic) mediated by extrasynaptic GABAA receptors in CVNs. Application of GABAA receptor antagonist gabazine (GBZ) and Stry also completely blocked sIPSC but did not have effect on Ihold. The Itonic was insensitive to d subunit-containing GABAA receptor agonists ethanol and THODC, showing no role for d subunit in Itonic. Application γ subunit-containing GABAA receptor agonist zolpidem reduced the Ihold, implyingγ subunit-containing GABAA receptors were expressed in CVNs. Pentobarbital (Pento), a widely used anesthetic in experimental animals, has profound effect on cardiovascular functions. The best known effect of Pento on neurons is that it augments GABAA receptor function. To test whether this role also involved in cardiovascular effect of Pento, we examined the effect of Pento on CVNs in NA. Bath application of Pento resulted in significant increase in noise level and decay of sIPSC, and a large increase in Ihold (IPento) of ~120 pA, all of which were completely blocked by Ptx or GABAA receptor agonist bicuculline. Application of presynaptic action potential antagonist tetrodotoxin (TTX) reduced the frequency of sIPSC, showing a very low incident of miniature IPSC activity in CVN. The IPento was not affected by TTX, suggesting that it can not be ascribed to the increased chance of summation of phasic sIPSC. This argument is supported by the fact that IPento was insensitive to GBZ. Raising recording temperature which decrease tonic current also did not reduce IPento. In summary, it was γ subunit, not δ subunit, contributing to tonic current of GABAA receptor. The present results suggest that Pento can directly activate GABAA receptors located at synaptic and extrasynaptic sites in CVN. This increased phasic or tonic GABAA receptor conductance silences CVN and inhibits cardiovascular function.