本文關鍵詞：癲癇腦片中及左乙拉西坦作用下多巴胺能神經(jīng)元電生理特性的研究　出處：《吉林大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 膜片鉗技術 中腦腹側(cè)被蓋區(qū) 多巴胺能神經(jīng)元 癲癇腦片模型 左乙拉西坦

【摘要】：研究背景:癲癇相關的精神障礙(psychosis of epilepsy,POE)是指癲癇患者伴發(fā)精神障礙,如焦躁、抑郁、易激惹等癥狀,其在癲癇患者中的發(fā)病率要遠遠高于普通人群。近年來也越來越受到社會關注。既往研究表明,POE與癲癇狀態(tài)下中腦邊緣多巴胺系統(tǒng)中多巴胺(dopamine,DA)神經(jīng)元超敏性有關。中腦腹側(cè)被蓋區(qū)(ventral tegmental area,VTA)是DA能神經(jīng)元聚集的地方,參與自然獎賞與厭惡、藥物成癮以及一些精神疾病等。另外癲癇動物模型在體胞外記錄發(fā)現(xiàn)癲癇狀態(tài)下DA能神經(jīng)元自發(fā)放電增多,而且可能是由腹側(cè)下腳鉤回-伏隔核-腹側(cè)蒼白球-VTA通路介導,但具體的機制尚不清楚。除了癲癇發(fā)作,服用抗癲癇藥物(antiepileptic drugs,AEDs)也可以出現(xiàn)POE,臨床常見的是左乙拉西坦(levetiracetam,LEV)。它是一種新型廣譜抗癲癇藥,主要作用于中樞神經(jīng)系統(tǒng)中的突觸囊泡蛋白2A(synaptic vesicle protein2A,SV2A),但臨床上部分癲癇患者在服用LEV后會出現(xiàn)精神心理變化。而關于LEV的精神方面副作用,據(jù)報道與DA含量有關。但LEV對DA能神經(jīng)元電生理的作用既往罕見報道。研究目的:本研究利用膜片鉗技術,記錄癲癇腦片模型中及加入LEV后VTA區(qū)DA能神經(jīng)元自發(fā)放電、細胞內(nèi)在興奮性以及外部興奮與抑制性突觸輸入等的改變來探索癲癇發(fā)作以及抗癲癇藥物LEV導致POE的機制,為尋找新的治療靶點提供新的思路和方向。研究方法:1.選用TH-GFP轉(zhuǎn)基因型小鼠制備離體皮層-中腦VTA腦片。同時利用無鎂離子人工腦脊液(artificial cerebrospinal fluid,ACSF)孵育腦片誘導皮層出現(xiàn)癲癇樣放電制作離體癲癇模型(epileptic model,EP-model)。2.利用細胞貼附式、全細胞式膜片鉗技術記錄正常狀態(tài)下VTA區(qū)神經(jīng)元自發(fā)動作電位以及激發(fā)動作電位的特點,并通過Avidin以及免疫組化染色識別VTA區(qū)神經(jīng)元的種類。同時將DA能神經(jīng)元放電模式進行分類。3.癲癇腦片模型中,DA能神經(jīng)元自發(fā)放電頻率的改變以及與內(nèi)在興奮性的關系。記錄正常狀態(tài)下以及EP-model中,DA能神經(jīng)元自發(fā)放電頻率、膜輸入阻抗、細胞膜電容、動作電位閾值、動作電位半寬、后超級化電位、基強度、導數(shù)最大值、導數(shù)最小值、發(fā)放頻率曲線等的改變。4.癲癇腦片模型中,DA能神經(jīng)元自發(fā)放電頻率的改變與外在突觸輸入的關系。利用加入銫離子(Cs+)和QX-314的電極內(nèi)液記錄正常以及EP-model下DA能神經(jīng)元自發(fā)興奮性輸入突觸后電流(spontaneous excitatory postsynaptic currents,s EPSC)和抑制性突觸后電流(spontaneous inhibitory postsynaptic currents,s IPSC),并分析其反應-反應間隔(inter-event interval IEI)、頻率以及幅度的改變。給予局部胞外金屬電極刺激,記錄正常以及EP-model下DA能神經(jīng)元激發(fā)后興奮性突觸后電流(evoked excitatory postsynaptic currents,e EPSC)和激發(fā)后抑制性突觸后電流(evoked inhibitory postsynaptic currents,e IPSC),通過計算成對脈沖比(paired-pulse-ratio,PPR)了解癲癇狀態(tài)下突觸前遞質(zhì)釋放概率的改變。5.癲癇腦片模型下,DA能神經(jīng)元興奮性改變與外部突觸輸入的關系的進一步驗證。利用細胞貼附式技術記錄正常ACSF、EP-model、EP-model加入興奮性突觸受體阻斷劑CNQX(AMPA受體拮抗劑)和AP5(NMDA受體拮抗劑)后自發(fā)動作電位的改變進一步驗證興奮性突觸輸入在癲癇狀態(tài)下對DA能神經(jīng)元的作用。6.LEV對DA能神經(jīng)元外部突觸輸入的作用。利用含有加入Cs+和QX-314的電極內(nèi)液,分別記錄正常ACSF以及100μM、200μM、400μM濃度LEV灌流液下DA能神經(jīng)元s EPSC、s IPSC的IEI、頻率及幅度的改變。另外通過加入TTX(電壓依賴鈉通道阻斷劑)+PTX(GABAA受體阻斷劑)+AP5記錄正常及200μM LEV下的微小興奮性突觸后電流(minature excitatory postsynaptic currents,m EPSC)。通過加入TTX+CNQX+AP5記錄正常及200μM LEV下微小抑制性突觸后電流(minature inhibitory postsynaptic currents,m IPSC),并分析m EPSC和m IPSC的IEI、頻率和幅度的改變。7.LEV對DA能神經(jīng)元內(nèi)在興奮性的作用。利用全細胞式膜片鉗技術,記錄正常及200μM LEV灌流下DA能神經(jīng)元內(nèi)在興奮性的改變,記錄內(nèi)容同方法3。8.LEV對DA能神經(jīng)元自發(fā)放電的作用。利用細胞貼附式膜片鉗技術,記錄正常及LEV灌流下DA能神經(jīng)元自發(fā)放電頻率的改變。研究結(jié)果:1.TH-GFP小鼠中在中腦VTA區(qū)域,根據(jù)動作電位發(fā)放特點,結(jié)合免疫組化染色,其神經(jīng)元主要可分為兩大類,DA能神經(jīng)元和GABA能神經(jīng)元。前者有典型的起搏式放電特點,與GABA能神經(jīng)元相比,其動作電位寬度相對較寬,發(fā)放頻率相對較慢。根據(jù)其動作電位發(fā)放特點,可以分為三型,I型占37.5%;II型47.9%;III型14.6%。2.在癲癇腦片模型中,用貼附式膜片鉗技術記錄DA能神經(jīng)元的自發(fā)放電頻率顯著增加,伴動作電位寬度顯著變窄。全細胞膜片鉗技術記錄自發(fā)動作電位的結(jié)果與其一致。3.在癲癇腦片模型中,DA能神經(jīng)元內(nèi)在興奮性顯著增加,發(fā)放頻率曲線在癲癇狀態(tài)下左移,在0-280 p A下激發(fā)動作電位發(fā)放頻率顯著高于對照組。同時動作電位半寬變窄,動作電位導數(shù)最小值及面積均變小,而且有統(tǒng)計學差異。但膜輸入阻抗及細胞膜電容未明顯改變。4.在癲癇腦片模型中,DA能神經(jīng)元s EPSC的IEI累積分布曲線左移伴頻率顯著增加,而s EPSC的幅度無明顯改變。而s IPSC的頻率和幅度未明顯改變。利用胞外電刺激誘發(fā)出e EPSC和e IPSC,對比正常狀態(tài)下發(fā)現(xiàn)e EPSC PPR和e IPSC PPR雖然沒有統(tǒng)計學差異,但均有增加趨勢。5.在癲癇腦片模型中,加入興奮性谷氨酸受體拮抗劑CNQX和AP5之后,癲癇狀態(tài)下增加的自發(fā)放電頻率能夠被抑制,而且介于正常及癲癇狀態(tài)之間。6.加入不同濃度LEV后,發(fā)現(xiàn)在200μM LEV以及400μM LEV作用下,DA能神經(jīng)元s EPSC IEI累積分布曲線較對照組左移,在400μM LEV下有統(tǒng)計學差異,但頻率均值無統(tǒng)計學差異。另外s IPSC、m EPSC以及m IPSC的IEI、頻率及幅度也未發(fā)生明顯改變。7.LEV在一定范圍內(nèi)可以抑制DA能神經(jīng)元的內(nèi)在興奮性。發(fā)放頻率曲線在200μM LEV作用下右移,并在40-100 p A以及140、160 p A處激發(fā)動作電位發(fā)放頻率顯著低于對照組。同時伴細胞膜電容變大,細胞膜阻抗變小,且均有統(tǒng)計學差異。8.在200μM LEV及400μM LEV作用下DA能神經(jīng)元的自發(fā)放電頻率較對照組降低,但沒有統(tǒng)計學差異。研究結(jié)論:1.利用無鎂離子ACSF孵育30分鐘可成功誘導皮層出現(xiàn)癲癇樣放電。2.在TH-GFP品系小鼠中,可以根據(jù)動作電位發(fā)放特點及動作電位半寬來鑒定中腦VTA區(qū)域中DA能神經(jīng)元。動作電位寬度大于1 ms的神經(jīng)元為DA能神經(jīng)元。根據(jù)斜坡的存在情況,DA能神經(jīng)元的動作電位發(fā)放模式可分為三型。3.在癲癇腦片模型下,DA能神經(jīng)元自發(fā)放電數(shù)目增多,與內(nèi)在興奮性的增加及外在接受的興奮性突觸后電流增加均有關系。而且癲癇狀態(tài)下,DA能神經(jīng)元動作電位半寬變窄,但突觸前釋放概率并不改變。4.LEV對DA能神經(jīng)元的自發(fā)動作電位的頻率沒有明顯調(diào)節(jié)作用,但是可以在一定范圍內(nèi)抑制DA能神經(jīng)元的內(nèi)在興奮性。
[Abstract]:Background: mental disorders related to epilepsy (psychosis of, epilepsy, POE) refers to mental disorders, such as epilepsy patients with anxiety, depression, irritability and other symptoms, the incidence of epilepsy patients is much higher than the general population. In recent years, more and more social attention. Previous studies showed that dopamine the mesolimbic dopamine system and epilepsy in the state of POE (dopamine, DA) neurons hypersensitivity. The ventral tegmental area (ventral tegmental, area, VTA) is DA neurons gathering place, participate in natural reward and aversion, drug addiction and psychiatric problems. In addition the epilepsy animal model in vivo extracellular recording under the condition of DA can be found in epileptic spontaneous discharges of neurons increased, and may be caused by the ventral subiculum uncinate nucleus accumbens ventral pallidum mediated by -VTA pathway, but the mechanism is not clear. In addition to seizures, antiepileptic Drug (antiepileptic drugs, AEDs) can also appear POE, clinical common is levetiracetam (levetiracetam, LEV). It is a new broad-spectrum antiepileptic drug, the main role of synaptic vesicle in the central nervous system in the global 2A (synaptic vesicle protein protein2A, SV2A), but the clinical part of epilepsy patients will appear spirit psychological changes after taking LEV and LEV on the mental side effects, according to reports related with DA content. But LEV neurons function rarely reported on DA. Previous research objective: This study using patch clamp technique, recorded epileptic brain slices in the model after accession to the LEV and VTA DA to the spontaneous discharge of neurons cells, the intrinsic excitability and external excitatory and inhibitory synaptic input to explore the change of epilepsy and the anti epilepsy drug LEV to POE mechanism, provides new ideas and direction for the search for new treatment targets. Research methods: 1. selected TH-GFP transgenic mice were prepared in vitro midbrain cortex brain slices of VTA. At the same time using magnesium free ACSF (artificial cerebrospinal, fluid, ACSF) incubating brain slices induced cortex epileptiform discharges in vitro model of epilepsy (epileptic model, EP-model.2.) by using cell attached, whole cell patch clamp technique to record characteristics of VTA neurons in the normal state of spontaneous action potentials and excitation action potentials, and by Avidin and immunohistochemical staining of neurons in VTA region of species identification. At the same time, DA can classify the firing pattern of neurons.3. epileptic brain slice model, DA can change the spontaneous discharge rate of neurons and inner excitement the record under normal condition and EP-model, DA neurons spontaneous discharge frequency, membrane input impedance, membrane capacitance, action potential threshold, action potential half width, Afterhyperpolarization, medium intensity, the maximum of the derivative derivative, the minimum value, the change of firing frequency curve.4. epileptic brain slice model, DA can change the spontaneous discharge rate of neurons and synaptic input. By adding external cesium ion (Cs+) electrode and QX-314 fluid in the normal and EP-model DA record neurons spontaneous excitatory postsynaptic current input (spontaneous excitatory postsynaptic currents, s EPSC) and inhibitory postsynaptic currents (spontaneous inhibitory postsynaptic currents, s IPSC), and to analyze the response response interval (inter-event interval IEI), the frequency and amplitude changes. Local cell stimulation and recording of normal metal electrode. EP-model DA neurons excited by excitatory postsynaptic currents (evoked excitatory postsynaptic currents, e EPSC) inhibitory postsynaptic currents and excitation (evoked inhibit Ory postsynaptic currents, e IPSC), by calculating the paired pulse ratio (paired-pulse-ratio, PPR) of epileptic state presynaptic neurotransmitter release probability change.5. epilepsy brain slice model, DA relationship between neuronal excitability changes and external synaptic input to further verify. Using cell attached recording technique of normal ACSF, EP-model. EP-model joined the excitatory synaptic receptor antagonist CNQX (AMPA receptor antagonist) and AP5 (NMDA receptor antagonist) after spontaneous action potential changes to further verify the role of excitatory synaptic inputs to.6.LEV neurons of DA in epileptic state neurons outside the synaptic input effect on DA. By adding Cs+ and QX-314 containing electrode in the liquid, were recorded in normal ACSF and 100 M, 200 M, 400 M concentration of LEV perfusate DA neurons s EPSC, IPSC s IEI, the frequency and amplitude changes. In addition by adding TT X (voltage dependent sodium channel blocker +PTX (GABAA) receptor antagonist) +AP5 record normal and 200 M LEV miniature excitatory postsynaptic currents (minature excitatory postsynaptic currents, m EPSC). By adding the TTX+CNQX+AP5 record and 200 M LEV under normal miniature inhibitory postsynaptic currents (minature inhibitory postsynaptic currents m, IPSC, EPSC and m) and analysis of M IPSC IEI, frequency and amplitude changes of.7.LEV neurons intrinsic excitatory effect on DA. By using the whole cell patch clamp technique, log normal and 200 M LEV perfusion DA neurons intrinsic excitability changes, with the method of DA 3.8.LEV records effect of spontaneous discharges of neurons. Using cell attached patch clamp technique, normal and LEV perfusion DA can record the spontaneous discharge rate of neurons change. Results: in VTA region in 1.TH-GFP mice, according to the dynamic Potential distribution characteristics, combined with immunohistochemical staining, the neurons can be divided into two categories, DA neurons and GABA neurons. The former has the typical characteristics of pacing type discharge, compared with GABA neurons, the action potential width is relatively wide, relatively slow release frequency. According to the characteristics of action potentials. Can be divided into three types, I type accounted for 37.5%; II 47.9%; III 14.6%.2. in epileptic brain slice model, with the spontaneous discharge frequency attached patch recording technique of DA neurons increased significantly, with action potential width is significantly narrowed. The whole cell patch clamp technique to record the spontaneous action potential in agreement with the.3. in the epileptic brain slice model, DA significantly increased the intrinsic excitability of neurons, firing frequency curve in epileptic state left, spike frequency was significantly higher than the control group at 0-280 P excitation A. At the same time the half width of the action potential Narrow, the minimum value and derivative action potential area was smaller, but there were significant differences. But the membrane input resistance and membrane capacitance of.4. did not change significantly in epileptic brain slice model, s EPSC DA neurons IEI cumulative distribution curve to the left with a significant increase in the frequency, and no significant changes in the amplitude of EPSC and S. The frequency and amplitude of s IPSC did not change significantly. The extracellular evoked by electrical stimulation of E EPSC and E IPSC, compared with normal state EPSC PPR and E IPSC e PPR although not statistically significant, but the increasing trend of.5. in the epileptic brain slice model, after adding excitatory glutamate receptor antagonists CNQX and AP5. Spontaneous discharge increased frequency of epileptic state can be reduced, and between normal and epileptic.6. with different concentrations of LEV, found in 200 M LEV and 400 M LEV, s EPSC IEI DA neurons cumulative distribution curve Compared with the control group, there were significant differences in the left, 400 M LEV, but no significant difference between the mean frequency. In addition, s IPSC, m EPSC and m IPSC IEI, frequency and amplitude are not significantly change the intrinsic excitability of.7.LEV can inhibit DA neurons in a certain range of firing frequency curve to the right in the 200. M LEV and 40-100 P under A P and 140160 A excitation spike frequency was significantly lower than the control group. At the same time with the membrane capacitance change, cell membrane impedance becomes small, and there were significant differences in.8. at 200 M LEV and 400 M LEV function DA neural element self release the electric frequency is lower than control group, but the difference was not statistically significant. Conclusions: 1. the magnesium ion ACSF were incubated for 30 minutes can successfully induce cortex epileptiform discharges in.2. TH-GFP mice, according to the characteristics of action potentials and action potentials in half width Set in the VTA region of midbrain DA neurons. The action potential width of more than 1 ms neurons DA neurons. According to the existing situation of the slope can be divided into three types of.3. in epileptic brain slice model under the action potential firing pattern of DA neurons, DA neurons firing number, current increased between acceptance and the increase in intrinsic excitability and extrinsic excitatory postsynaptic and epileptic state, DA neurons action potential half width narrowed, but does not change the probability of presynaptic release of.4.LEV spontaneous action potentials of the DA neurons frequency has no obvious regulating effect, but can inhibit the intrinsic excitability of DA neurons in a certain range.

【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R742.1

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