NERP-1对大鼠下丘脑室旁核MNCs活动的影响机制

Effect of NERP-1 on MNCs activity in paraventricular nucleus of hypothalamus in rats

目的分析神经内分泌调节肽(NERP)-1对大鼠下丘脑室旁核(PVN)神经分泌大细胞(MNCs)活动的影响及其对PVN MNCs活动调节的突触机制。方法实验动物选用出生2~3周龄的雄性Wistar系大鼠,异氟烷麻醉后立即断头取出脑,随后用振动切片机制备厚度为250μm的含PVN的下丘脑切片。在室温(24~25℃)条件下,将切片置于持续充有混合气体(95%O_(2)和5%CO_(2))的人工脑脊液中孵育1 h以上。电极的阻抗为5~7 MΩ。使用膜片钳放大器(Axopatch-200B)及Clampex数据采集软件记录PVN神经分泌细胞的电活动,NERP-1、河豚毒素(TTX)和犬尿喹啉酸(KYC)经蠕动泵灌流给药。在电生理记录结束后,用4%多聚甲醛溶液将脑片行固定24 h后,进行二氨基联苯胺(DAB)染色、显微照相,观察所记录神经分泌细胞的组织形态学特点,确认所记录细胞为PVN NMCs。使用Clampfit10.4和MiniAnalysis(6.0)软件进行电生理实验数据分析。结果在电流钳记录模式下,PVN MNCs对去极化电流刺激表现出明显的外向整流电流。给予NERP-1(100 nmol/L)可导致PVN神经分泌大细胞的放电频率显著降低并伴随膜电位超极化,冲洗后恢复到给药前水平。给予非选择性离子型谷氨酸受体拮抗剂犬KYC可阻断NERP-1引起的PVN MNCs放电频率降低和膜超极化作用。NERP-1对PVN MNCs的自发性放电活动的抑制作用不受GABAA受体阻断剂的影响。在电压依存性钠通道阻断剂TTX的存在下,NERP-1显著降低NMCs的微小兴奋性突触后电流(mEPSCs)的发生频率,但对mEPSCs的振幅没有显著影响。结论NERP-1下调下丘脑PVN MNCs兴奋性谷氨酸能传入效能,降低PVN MNCs的兴奋性,提示NERP-1通过间接方式调节PVN缩宫素和血管紧张素神经元的分泌活动。

Objective To study the effect of neuroendocrine regulatory peptide(NERP)-1 on the activity of hypothalamic paraventricular nucleus(PVN)magnocellular endocrine cells(MNCs),by whole-cell patch-clamp recording technique,neuropharmacology and immunohistochemistry methods,and to explore the synaptic mechanism of NERP-1 regulating the activity of PVN MNCs.Methods Male Wistar rats aged 2~3 weeks were used in the experiment.After deeply anesthetized with isoflurane,the head of rat was decapitated immediately.The whole brain was taken out and put into ice-cold artificial cerebral spinal fluid(ACSF)quickly.Then,the hypothalamus sections including PVN(250μm in thickness)were prepared with vibration microtome.The slices were incubated in ACSF filled with 95%O_(2) and 5%CO_(2) for more than 1 hour at room temperature(24~25℃).The recording pipette resistances were 5~7 MΩin the bath.The spontaneous spike firing of PVN neurons was recorded by patch-clamp amplifier(Axopatch-200B)and Clampex data acquisition software.NERP-1,tetrodotoxin(TTX)and kynurenic acid(KYC)were applied by peristaltic pump.At the end of electrophysiological recording,the brain slices were fixed with 4%paraformaldehyde solution for 24 hours.DAB staining and micrograph were performed to observe the histomorphological characteristics of the recorded cells and confirm that the cells were PVN NMCS.Clampfit 10.4 and MiniAnalysis(6.0)software were used for analyzing electrophysiological data.Results Under current-clamp recording conditions,PVN MNCs were sensitive to the injection of depolarization currents,which exhibited outwardly rectification currents.Application of NERP-1 induced a significant decrease in spike firing rate accompanied with a membrane hyperpolarization of PVN MCNs,which were recovery after washout.Non-selective ionotropic glutamate receptors blocker(kynurenic acid,KYC)could block the NERP-1-induced depression in frequency of spike firing rate and the membrane hyperpolarization of PVN MCNs.The NERP-1-induced inhibition in the spike firing activity and membrane hyperpolarization of MNCs was persisted in absence of GABAA receptor activity.In the presence of voltage dependent sodium channel blocker,TTX,NERP-1 significantly reduced the frequency of miniature postsynaptic currents(mEPSCs),but had no effect on the amplitude of mEPSCs.Conclusions NERP-1 could down regulate the excitatory glutamatergic afferent efficiency of PVN MNCs and decrease the excitability of PVN MNCs,which suggests that NERP-1 could indirectly regulate the secretion of oxytocin and angiotensin neurons in PVN of rats.