尿皮素Ⅱ对大鼠下丘脑室旁核小细胞分泌神经元活动的影响

发布时间：2018-06-05 10:05

  本文选题：尿皮素Ⅱ + 下丘脑室旁核　； 参考：《延边大学》2016年硕士论文

【摘要】：[目的]利用急性脑片全细胞膜片钳记录、神经药理学和组织化学染色技术,研究尿皮素II (UCN II)对幼年大鼠PVN分泌型小细胞自发性放电和膜电位的影响机制。[材料与方法]本研究使用生后为15-26天(Postnatal day 15-26, P15-P26)的Wistar系大鼠,用异氟烷吸入麻醉后,迅速断头取脑,然后用振动切片机来制备含PVN的下丘脑切片,切片厚度为250微米。在室温下(24-25℃),将含有下丘脑室旁核的脑切片放置于人工脑脊液(ACSF)中孵育60分钟以上,对ACSF持续充有95%02和5%CO2混合气体。ACSF的组成成分如下：118 mM NaCl,3 mM KC1, 1 mM MgCl2.6H20,1 mM NaH2PO4.2H2O,25mMNaHCO3,10 mM D-Glucose,2mMCaCl2。PH值为7.25-7.35,渗透压为295-300 mOsM。记录电极内灌装7微升电极内液,内液组成成分如下：120 mM potassium gluconate,10 mM HEPES,1 mM EGTA,5 mM KC1,3.5 mM MgCl2、4 mM NaCl,8 mM biocytin,4 mM Na2ATP,0.2 mM Na2GTP。用KOH将pH值调为7.3。电极阻抗为5-7 MΩ。PVN神经元电活动使用全细胞膜片钳系统记录,收集的数据存于电脑硬盘中,并将完整记录而且基线相对稳定的电生理数据用于最后的数据处理与统计学分析。电生理记录完成后,将下丘脑片固定于4%多聚甲醛中24小时以上,然后行DAB染色。在显微镜下,观察神经元的位置和形态学特点并拍照获取染色结果。电生理的实验数据采用Clampfit 10.4软件进行分析,数据的统计学分析采用的是SPSS软件,用配对T检验来比较给药前后的平均数,P0.05,记录结果认为有统计学差异。[结果]1.在电流钳下(current-clamp recording mode), PVN小细胞分泌神经元对去极化电流刺激敏感,但没有明显内向整流电流、低阈值放电(LTS)和超极化活化内向电流(Ih)。2.在电流钳下,UCN II (100 nM)可导致34.7%的PVN小细胞分泌神经元的放电频率明显降低,冲洗20分钟后恢复,UCN Ⅱ对自发性放电频率的影响随UCN Ⅱ浓度升高而减弱。3.UCN Ⅱ抑制自发性放电的同时,导致膜电位的超极化,UCN Ⅱ导致的膜电位超极化对河豚毒素(TTX)不敏感。4.在TTX存在条件下,UCN Ⅱ导致的PVN小细胞分泌神经元膜超极化具有剂量依存性,半数有效抑制浓度为37 nM。5.UCN Ⅱ明显抑制由去极化电流刺激诱发的动作电位的数量,并明显降低细胞膜的输入阻抗。6.电流-电压关系曲线分析表明,UCNII敏感电流的翻转电位与钾离子平衡电位相近。[结论]本项研究表明UCN II通过活化钾离子通道导致PVN小细胞分泌神经元亚群超极化和自发性放电频率降低,提示UCN Ⅱ与特异性受体结合直接影响PVN小细胞分泌神经元亚群的活动,参与调节该亚群的分泌功能。
[Abstract]:[objective] to study the effect of uroderin II (UCN II) on spontaneous discharge and membrane potential of PVN secretory small cells in young rats by using acute whole-cell patch clamp recording and neuropharmacological and histochemical staining techniques. [materials and methods] in this study, rats with postnatal day 15-26 (P15-P26) were anesthetized with isoflurane, then their heads were cut off quickly, and then the hypothalamus slices containing PVN were prepared by vibration slicing machine. The thickness of the slices was 250 渭 m. At room temperature, the brain sections containing the hypothalamic paraventricular nucleus were incubated in the artificial cerebrospinal fluid (ACSF) for more than 60 minutes. The composition of the mixture of 95 and 5 CO _ 2 for ACSF was as follows: 1: 118 mm NaCl-3 mm KC1, 1 mm MgCl _ 2 路6H _ (20) H _ 2O 1 mm NaH2PO _ (4) H _ (2) H _ (2) H _ (2) O _ (2) O _ (2) H _ (2) M NaHCO _ (3) O _ (3) 10 mm D-Glucose _ (2) MCaCl _ (2) 2.PH value was 7.25-7.35, and the osmotic pressure was 295-300 mOsM. The composition of the solution was as follows: 1 mm potassium gluconate 10 mm HEPESN 1 mm EGTA1 5 mm KC1 3. 5 mm NaCl 2 + 4 mm NaCl 8 mm M biocytinine 4 mm Na2ATP 0.2 mm Na 2GTP.The composition of the electrode was as follows: 1 mm potassium gluconate 10 mm HEPESN 1 mm EGTA 1 5 mm M MgCl 2 + 4 mm NaCl 8 mm M biocytinine 4 mm Na 2 ATP 0.2 mm Na 2GTP. The pH value was adjusted to 7.3 with Koh. The electrode impedance of 5-7 M 惟 .PVN neurons was recorded by a whole-cell patch clamp system, and the collected data were stored in the computer hard disk. The electrophysiological data, which were completely recorded and relatively stable at baseline, were used for the final data processing and statistical analysis. After electrophysiological recording was completed, the hypothalamus slices were fixed in 4% paraformaldehyde for more than 24 hours, then DAB staining was performed. Under microscope, the location and morphological characteristics of neurons were observed and the staining results were obtained. The experimental data of electrophysiology were analyzed by Clampfit 10.4 software, SPSS software was used to analyze the data, and the average of P0.05before and after administration was compared by paired T test. The results showed that there was statistical difference. [result] 1. In the presence of current-clamp recording modei, small cell secretory neurons were sensitive to depolarization current stimulation, but there was no obvious inward rectifier current, low threshold discharge current (LTSs) and hyperpolarization-activated inward current. The frequency of discharge of 34.7% of PVN small cell secretory neurons was significantly decreased under current clamp. The effect of recovery of UCN 鈪，

本文编号：1981605