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

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 e 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, e 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.