本文選題：尿皮素Ⅱ + 下丘腦室旁核��； 參考：《延邊大學(xué)》2016年碩士論文

【摘要】：[目的]利用急性腦片全細(xì)胞膜片鉗記錄、神經(jīng)藥理學(xué)和組織化學(xué)染色技術(shù),研究尿皮素II (UCN II)對(duì)幼年大鼠PVN分泌型小細(xì)胞自發(fā)性放電和膜電位的影響機(jī)制。[材料與方法]本研究使用生后為15-26天(Postnatal day 15-26, P15-P26)的Wistar系大鼠,用異氟烷吸入麻醉后,迅速斷頭取腦,然后用振動(dòng)切片機(jī)來制備含PVN的下丘腦切片,切片厚度為250微米。在室溫下(24-25℃),將含有下丘腦室旁核的腦切片放置于人工腦脊液(ACSF)中孵育60分鐘以上,對(duì)ACSF持續(xù)充有95%02和5%CO2混合氣體。ACSF的組成成分如下：118 mM NaCl,3 mM KC1, 1 mM MgCl2.6H20,1 mM NaH2PO4.2H2O,25mMNaHCO3,10 mM D-Glucose,2mMCaCl2。PH值為7.25-7.35,滲透壓為295-300 mOsM。記錄電極內(nèi)灌裝7微升電極內(nèi)液,內(nèi)液組成成分如下：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將pH值調(diào)為7.3。電極阻抗為5-7 MΩ。PVN神經(jīng)元電活動(dòng)使用全細(xì)胞膜片鉗系統(tǒng)記錄,收集的數(shù)據(jù)存于電腦硬盤中,并將完整記錄而且基線相對(duì)穩(wěn)定的電生理數(shù)據(jù)用于最后的數(shù)據(jù)處理與統(tǒng)計(jì)學(xué)分析。電生理記錄完成后,將下丘腦片固定于4%多聚甲醛中24小時(shí)以上,然后行DAB染色。在顯微鏡下,觀察神經(jīng)元的位置和形態(tài)學(xué)特點(diǎn)并拍照獲取染色結(jié)果。電生理的實(shí)驗(yàn)數(shù)據(jù)采用Clampfit 10.4軟件進(jìn)行分析,數(shù)據(jù)的統(tǒng)計(jì)學(xué)分析采用的是SPSS軟件,用配對(duì)T檢驗(yàn)來比較給藥前后的平均數(shù),P0.05,記錄結(jié)果認(rèn)為有統(tǒng)計(jì)學(xué)差異。[結(jié)果]1.在電流鉗下(current-clamp recording mode), PVN小細(xì)胞分泌神經(jīng)元對(duì)去極化電流刺激敏感,但沒有明顯內(nèi)向整流電流、低閾值放電(LTS)和超極化活化內(nèi)向電流(Ih)。2.在電流鉗下,UCN II (100 nM)可導(dǎo)致34.7%的PVN小細(xì)胞分泌神經(jīng)元的放電頻率明顯降低,沖洗20分鐘后恢復(fù),UCN Ⅱ?qū)ψ园l(fā)性放電頻率的影響隨UCN Ⅱ濃度升高而減弱。3.UCN Ⅱ抑制自發(fā)性放電的同時(shí),導(dǎo)致膜電位的超極化,UCN Ⅱ?qū)е碌哪る娢怀瑯O化對(duì)河豚毒素(TTX)不敏感。4.在TTX存在條件下,UCN Ⅱ?qū)е碌腜VN小細(xì)胞分泌神經(jīng)元膜超極化具有劑量依存性,半數(shù)有效抑制濃度為37 nM。5.UCN Ⅱ明顯抑制由去極化電流刺激誘發(fā)的動(dòng)作電位的數(shù)量,并明顯降低細(xì)胞膜的輸入阻抗。6.電流-電壓關(guān)系曲線分析表明,UCNII敏感電流的翻轉(zhuǎn)電位與鉀離子平衡電位相近。[結(jié)論]本項(xiàng)研究表明UCN II通過活化鉀離子通道導(dǎo)致PVN小細(xì)胞分泌神經(jīng)元亞群超極化和自發(fā)性放電頻率降低,提示UCN Ⅱ與特異性受體結(jié)合直接影響PVN小細(xì)胞分泌神經(jīng)元亞群的活動(dòng),參與調(diào)節(jié)該亞群的分泌功能。
[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，

本文編號(hào)：1981605