化学遗传学调控高血压大鼠室旁核促肾上腺皮质激素释放因子神经元对前交感神经元兴奋性的影响

Effect of chemogenetic manipulation of PVN corticotropin-releasing factor-expressing neurons on excitability of presympathetic neurons in SHR

目的观察下丘脑室旁核(paraventricular nucleus,PVN)促肾上腺皮质激素释放因子(corticotropin-releasing factor,CRF)神经元对正常血压Wistar Kyoto(WKY)大鼠或自发性高血压大鼠(spontaneously hypertensive rats,SHR)前交感神经元兴奋性的影响,阐明交感中枢过度兴奋的可能神经环路机制。方法本研究使用成年雄性WKY和SHR。通过蛋白印迹实验明确WKY与SHR PVN CRF蛋白表达水平。同时,借助免疫荧光实验观察WKY与SHR PVN CRF神经元与前交感神经元。通过将CRF启动子特异性Cre依赖的腺相关病毒(adeno-associated viruses,AAV)与化学遗传学AAV共同注入PVN,使得CRF神经元特异性表达仅由设计性药物激活的设计性受体(designer receptors exclusively activated by designer drugs,DREADDs),即WKY室旁核CRF神经元特异性表达与Gq亚基偶联的人M3毒蕈碱型DREADD(hM3Dq),SHR室旁核CRF神经元特异性表达与Gi亚基偶联的人M4毒蕈碱型DREADD(hM4Di),而叠氮平-N-氧化物(clozapine-N-oxide,CNO)作为配体可结合兴奋性hM3Dq或抑制性hM4Di,进而调控PVN CRF神经元的兴奋性。而后向脊髓中间外侧柱(intermediolateral column,IML)微量注入荧光示踪剂逆行标记室旁核前交感神经元。最后,我们应用全细胞膜片钳记录CNO(10μmol·L^(-1))对室旁核前交感神经元自发兴奋性突触后电流(spontaneous excitatory postsynaptic currents,sEPSCs)和诱发动作电位的影响。结果SHR室旁核CRF蛋白表达水平明显高于WKY大鼠,SHR室旁核CRF神经元活动增强、数量增多。化学遗传学DREADDs可成功表达在PVN CRF神经元,同时,逆行荧光示踪剂可成功标记在向脊髓投射的PVN前交感神经元。用含CNO的人工脑脊液灌流预先在PVN CRF神经元表达hM4Di-mCherry的SHR脑片,可以引起PVN前交感神经元sEPSCs频率明显降低,但不影响其幅度。此外,用含CNO的人工脑脊液灌流预先在PVN CRF神经元表达hM3Dq-eGFP的WKY大鼠脑片,PVN前交感神经元sEPSCs的频率和幅度均无明显差异。PVN CRF神经元表达有hM3Dq-eGFP的WKY大鼠脑片或表达有hM4Di-mCherry的SHR脑片用CNO灌流给药,PVN前交感神经元电流诱发动作电位均无明显变化。结论自发性高血压大鼠PVN CRF神经元活动增强、数量增多,增强前交感神经元sEPSCs,室旁核CRF神经元与前交感神经元之间存在神经调控微环路。

Aim To observe the effect of corticotropin-releasing factor(CRF)-expressing neurons on presympathetic neurons in hypothalamic paraventricular nucleus(PVN)of normotensive Wistar Kyoto(WKY)rats or spontaneously hypertensive rats(SHR),and to elucidate the underlying neuronal circuit mechanism of central sympathetic hyperexcitability.Methods The expression levels of CRF protein in WKY rats and SHR PVN were determined by Western blot.Meanwhile,the WKY and SHR PVN CRF-expressing neurons and presympathetic neurons were observed by immunofluorescent staining.Adult WKY rats and SHR were used in this study.By microinjection of Cre-dependent adeno-associated viruses(AAV)that specifically recognized the CRF promoter and AAV of chemogenetics into the PVN,CRF-expressing neurons expressed designer receptors exclusively activated by designer drugs(DREADDs).Human M3 muscarinic DREADD coupled to Gq receptor(hM3Dq)was specifically expressed in PVN CRF-expressing neurons in WKY rats,while human M4 muscarinic DREADD coupled to Gi receptor(hM4Di)was specifically expressed in PVN CRF-expressing neurons in SHR.Clozapine-N-oxide(CNO),as a designer ligand,would couple to excitatory hM3Dq or inhibitory hM4Di to regulate the excitability of PVN CRF-expressing neurons.Then the PVN presympathetic neurons were retrogradely labeled by microinjection of fluosecent tracer into the intermediolateral column(IML)of spinal cord.Lastly,whole cell patch clamp was used to determine the effect of CNO(10μmol·L^(-1))on spontaneous excitatory postsynaptic currents(sEPSCs)and current-evoked firing of PVN presympathtic neurons of WKY rats and SHR.Results The expression of CRF protein in the PVN of SHR was significantly higher than that of WKY rats,and the activity and number of CRF-expressing neurons in the PVN of SHR were increased.PVN CRF-expressing neurons were expressed with chemogenetic DREADDs and PVN presympathetic neurons were retrogradely labeled with fluorescent tracer in WKY rats and SHR.In SHR expressed with chemogenetic inhibitory hM4Di-mCherry of PVN CRF-expressing neurons,bath application of CNO to the brain slices resulted in a significant decrease in sEPSCs frequency,but no change in their amplitude of labeled PVN presympathetic neurons.In contrast,in WKY rats expressed with excitatory hM3Dq-eGFP of PVN CRF-expressing neurons,CNO had no obvious effect on the sEPSCs frequency and amplitude in PVN presympathetic neurons.Furthermore,bath application of CNO had no significant effect on current-evoked firing of PVN presympathetic neurons of either WKY rats with hM3Dq-eGFP expression in CRF neurons or SHR with hM4Di-mCherry expression in CRF neurons.Conclusions The activity and number of PVN CRF-expressing neurons are increased in SHR,and CRF-expressing neurons enhance the excitability of presympathetic neurons,which acts as a regulatory neuronal microcircuit between CRF neurons and presympathetic neurons in the PVN.