本文關(guān)鍵詞： 黑質(zhì)致密部 多巴胺神經(jīng)元 振蕩 共振 超級化激活的陽離子流 小電導鈣依賴性鉀通道 全細胞膜片鉗　出處：《第四軍醫(yī)大學》2012年博士論文　論文類型：學位論文

【摘要】：神經(jīng)振蕩性電活動是指在生理狀態(tài)下核團內(nèi)及神經(jīng)網(wǎng)絡(luò)中的神經(jīng)元的電生理活動特點。振蕩性電活動是電流/電壓的周期性變化，分為同步振蕩和異步振蕩兩種情況。其中，能夠保持同步而穩(wěn)定運行的振蕩稱為同步振蕩。膜共振是用來描述神經(jīng)元對輸入信號的頻率選擇性能力的物理量，同時也是神經(jīng)元振蕩活動的內(nèi)在機制之一。頻率選擇性是神經(jīng)元膜共振活動所具有的一種內(nèi)在特性。神經(jīng)元的膜共振特性和頻率選擇性在調(diào)節(jié)腦內(nèi)神經(jīng)網(wǎng)絡(luò)的節(jié)律性活動中發(fā)揮重要作用，其調(diào)控作用尤其體現(xiàn)在神經(jīng)元相互之間進行信息傳遞的過程中。不同的神經(jīng)元具有不同的膜共振特性是神經(jīng)網(wǎng)絡(luò)共振的基礎(chǔ)。多巴胺能神經(jīng)元是黑質(zhì)致密部中的主要神經(jīng)元。了解多巴胺能神經(jīng)元的膜共振特性，將有助于增進我們對基底節(jié)區(qū)神經(jīng)元細胞對信息處理過程的認識。黑質(zhì)致密部的多巴胺神經(jīng)元輸出的信息涉及運動、學習和記憶等生理功能。這些信息的輸出，對我們認知黑質(zhì)致密部DA神經(jīng)元及其損害后引起的神經(jīng)系統(tǒng)疾病，特別是帕金森病，具有重要的意義。在前期研究中，已發(fā)現(xiàn)帕金森病患者基底節(jié)區(qū)存在病理性同步振蕩現(xiàn)象，其主要表現(xiàn)是過度的同步振蕩。一般認為，同步振蕩與神經(jīng)系統(tǒng)信息的檢測、處理及整合密切相關(guān)，因此，過度的同步振蕩也可能成為帕金森病感覺、運動信息紊亂并導致出現(xiàn)不同臨床癥狀的內(nèi)在機制之一�！罢袷幠Ｐ汀笔切陆岢龅囊环N帕金森病的發(fā)病機制，目前仍有很多機制尚不明確。此外，既往臨床和基礎(chǔ)研究已經(jīng)表明，黑質(zhì)致密部的多巴胺神經(jīng)元的病理性的退行性改變是PD、抑郁癥等多種疾病的發(fā)生和發(fā)展的重要特征。故研究探索PD的DA（dopamine）神經(jīng)元的膜共振特性及機制將有助于我們從一個新的角度去認識PD的發(fā)病機制。 本課題分成二個部分，其一是在大鼠腦片水平上利用紅外線可視全細胞膜片鉗技術(shù)觀察記錄多巴胺神經(jīng)元的電活動及其膜共振性質(zhì)；其二是對膜共振特性潛在的離子機制進行更深入的探討，以期在正常大鼠腦片水平上，了解多巴胺能神經(jīng)元的膜共振特性，為進一步研究帕金森大鼠腦片水平的多巴胺神經(jīng)元膜共振特性的改變，提供堅實的理論依據(jù)。一、黑質(zhì)致密部多巴胺神經(jīng)元的膜共振 神經(jīng)元的振蕩是神經(jīng)系統(tǒng)電活動一個重要的生物節(jié)律，無論單個神經(jīng)元的振蕩，還是神經(jīng)網(wǎng)絡(luò)的振蕩，對腦的功能都具有重要意義。神經(jīng)元的膜共振是用來描述神經(jīng)元對輸入信號的頻率選擇性，是神經(jīng)元網(wǎng)絡(luò)振蕩的內(nèi)在機制之一。目前已在海馬CA1區(qū)錐體神經(jīng)元、海馬下托錐體神經(jīng)元及內(nèi)嗅皮質(zhì)Ⅱ?qū)有切渭毎炔煌愋蜕窠?jīng)元中檢測到θ膜共振。多巴胺神經(jīng)元作為在黑質(zhì)致密部結(jié)構(gòu)中最主要的神經(jīng)元，對神經(jīng)網(wǎng)絡(luò)的作用是通過直接、間接通路與其它核團神經(jīng)元的相互作用而產(chǎn)生的結(jié)果，然而對于黑質(zhì)致密部DA神經(jīng)元是否也存在膜共振，尚未見報道。因此，本課題的第一部分實驗內(nèi)容就是給予黑質(zhì)致密部DA神經(jīng)元一個隨時間增加頻率連續(xù)變化的正弦電流（ZAP，0Hz～16Hz，20s）作為刺激電流，觀察并檢測出DA神經(jīng)元的是否存在膜共振，及其膜共振的頻率范圍，并對其結(jié)果進行進一步的分析討論。 主要結(jié)果如下： 1、使用紅外線可視全細胞膜片鉗技術(shù)在黑質(zhì)致密部冠狀腦片水平上記錄到多巴胺神經(jīng)元電生理活動，同時用TH染色證實DA神經(jīng)元其形態(tài)為梭形神經(jīng)元。 2、我們發(fā)現(xiàn)在給予SNc的DA神經(jīng)元去極化電流刺激時，記錄到的SNc的DA神經(jīng)元放電模式為規(guī)則的低頻的放電模式。實驗還發(fā)現(xiàn)，我們利用電生理方法可以明確鑒定DA神經(jīng)元，在給予ZAP電流刺激時，在-55mV到-85mV范圍內(nèi)DA神經(jīng)元表現(xiàn)出膜共振反應(yīng)。 3、DA神經(jīng)元的膜共振頻率具有溫度依賴性，即膜共振頻率隨溫度的升高而升高，在33℃～38℃為2Hz～4Hz左右，證實其頻率值處在θ頻率范圍內(nèi)。 4、DA神經(jīng)元的膜共振頻率具有電壓依賴性，表現(xiàn)為當鉗制在不同的超極化膜電位水平，其膜共振頻率有所不同。 二、黑質(zhì)致密部多巴胺神經(jīng)元的膜共振特性的離子機制 DA神經(jīng)元的離子通道主要有K~+、Na~+、Ca~(2+)離子通道。Ih電流是由HCN通道所介導一種混合性的陽離子流，由Na~+、K~+及其它陽離子共同組成，是一種緩慢激活的陽離子電流。目前已經(jīng)證實這種電流對神經(jīng)元細胞膜的興奮性和節(jié)律性具有重要的調(diào)節(jié)作用，這在各個腦區(qū)的中樞神經(jīng)元系統(tǒng)已經(jīng)得到了充分驗證。研究結(jié)果顯示，Ih電流對神經(jīng)元的調(diào)節(jié)，主要是通過對突觸的傳遞的調(diào)節(jié)，而去調(diào)節(jié)神經(jīng)元細胞膜的興奮性及節(jié)律性，并且證實其參與了膜共振的形成。這在海馬神經(jīng)元、皮層等神經(jīng)元業(yè)已被證實，，其作為主動電流參與形成閾下膜共振。但其是否作為主動電流也參與形成DA神經(jīng)元閾下膜共振，尚不明確。同時是否有其它離子通道也參與或調(diào)節(jié)了DA神經(jīng)元的閾下膜共振，目前也不明確。另外，有文獻報道，小電導鈣依賴性鉀通道（the small-conductance calcium-dependent potassiumchannel, SK）和Ca~(2+)通道參與了膜振蕩的形成，其中SK通道的阻斷和激活能夠顯著影響DA神經(jīng)元的節(jié)律和興奮性。但是能否證實其在膜共振方面的作用，還有待進一步研究。 主要結(jié)果如下: 1、超極化激活的陽離子流（Hyperpolarization-activated cation current，Ih）是DA神經(jīng)元產(chǎn)生θ頻率膜共振的主動電流成分，作用范圍大概在-65mV～-85mV之間。 2、鈣依賴性鉀通道（SK）也是DA神經(jīng)元產(chǎn)生θ頻率膜共振的主動電流成分，其作用范圍大概在-60mV～-70mV之間。 3、持續(xù)性鈉電流（Persistent sodium currents，INap）可以水平放大膜共振。 主要結(jié)論 1、使用紅外線可視全細胞膜片鉗技術(shù)在大鼠黑質(zhì)致密部冠狀腦片水平上記錄到梭形神經(jīng)元，存在神經(jīng)元膜共振。 2、大鼠黑質(zhì)致密部DA神經(jīng)元的膜共振具有溫度依賴性和電壓依賴性。 3、證實了大鼠黑質(zhì)致密部多巴胺神經(jīng)元在-65mV左右，產(chǎn)生膜共振的主動電流是SK通道電流。 4、證實了大鼠黑質(zhì)致密部多巴胺神經(jīng)元在-75mV左右，產(chǎn)生膜共振的主動電流是Ih電流。 5、證實了I_(Nap)可以增強膜共振峰，但對膜共振頻率沒有影響。 6、鈣離子參與了膜共振的產(chǎn)生，具體機制尚需進一步研究。 我們的研究表明黑質(zhì)致密部多巴胺神經(jīng)元具有θ頻率膜共振，其離子機制具有一定的特殊性，這種獨特的離子機制，對于我們進一步了解黑質(zhì)致密部DA神經(jīng)元及其在腦內(nèi)神經(jīng)網(wǎng)絡(luò)中的地位具有重要的作用，并且為進一步研究PD患者的DA電生理功能特性提供了基礎(chǔ)。
[Abstract]:The electrical activity of the oscillation of the neural electrophysiological activity refers to the characteristics in the physiological condition and the nuclei of neurons in the neural network. The oscillatory electrical activity is periodic change of voltage / current, divided into synchronous oscillation and asynchronous oscillation in two cases. Among them, can keep the stable operation of the oscillation is called synchronous oscillation film. Resonance is a physical quantity to describe the frequency selective ability of neurons to the input signal, but also one of the internal mechanism of neuronal oscillatory activity. The frequency selectivity is an intrinsic property of neurons. The membrane resonance characteristics and frequency selective neurons play an important role in the regulation of neural networks in the brain of rhythmic activity in its regulatory role especially in the process of information transmission between neurons. The neurons with different membrane resonance characteristics of different neural network The resonance of the foundation. The dopaminergic neurons in the substantia nigra neurons is mainly in the membrane. Understand the resonance characteristics of dopaminergic neurons, will help to enhance our understanding of the neurons in basal ganglia of information processing. The substantia nigra dopamine neuron output information relates to sports, such as learning and memory function the output of these information, the nervous system disease caused by our perception of the substantia nigra DA neurons and damage, especially Parkinson's disease, is of great significance. In previous studies, have been found to have pathological synchronous oscillation Parkinson patients in the basal ganglia, the main performance is the excessive synchronous oscillation. Generally, detection of synchronous oscillation and nervous system information processing and integration, are closely related, therefore, synchronous oscillation over Parkinson's disease may also become a feeling of movement informationdisorder One of the internal mechanism and lead to different clinical symptoms. The "oscillation model" is a kind of pathogenesis of Parkinson's disease recently proposed, there are still a lot of mechanism is not clear. In addition, previous clinical and basic studies have shown that degenerative pathological dopaminergic neurons of the substantia nigra pars compacta is the change of PD occurred and the development of an important feature of depression and other diseases. So the research of PD DA (dopamine) film resonant characteristics and mechanism of neurons will contribute to the pathogenesis of us from a new perspective to understand PD.
This topic is divided into two parts, one is at the level of rat brain slices using infrared visual whole cell patch clamp technique to observe and record the dopamine neurons electrical activity and membrane resonance properties; the ionic mechanism of membrane potential resonance characteristics are more in-depth study, in normal rat brain slices on the level of film to understand the resonance characteristics of dopaminergic neurons, dopamine neurons membrane resonance characteristics of further research on brain slice level Parkinson rats changes, provide a solid theoretical basis. A membrane resonance, substantia nigra dopaminergic neurons
Neuronal electrical activity in nerve system oscillation is an important biological rhythm, whether single neurons or neural network oscillation, oscillation, plays an important role in brain function. The neurons membrane resonance is used to describe the frequency selectivity of neurons of the input signal, is one of the intrinsic mechanism of neuronal network oscillations. Now in cone neurons of the CA1 hippocampus, subiculum and entorhinal cortex pyramidal neurons in layer II stellate cells of different types of neurons were detected in the membrane. Dopamine neurons as theta resonance in the substantia nigra in the structure of the main neurons, the neural network is generated by direct and indirect interactions with other nuclei pathway the results for neurons, however the substantia nigra DA neurons have membrane resonance, it has not been reported. Therefore, the subject of the first part of the experiment content is Is to give the substantia nigra DA neurons in a continuously changing frequency increases with time sinusoidal current (ZAP, 0Hz ~ 16Hz, 20s) as the stimulation current, observe and detect the existence of DA neuron membrane resonance, and the resonance frequency range of membrane, and the results of the analysis and further discussion.
The main results are as follows:
1, we used infrared visible whole cell patch clamp technique to record the electrophysiological activity of dopaminergic neurons at the level of the coronal slices of substantia nigra. Meanwhile, TH staining showed that DA neurons were spindle shaped neurons.
2, we found that the depolarizing currents of DA neurons in SNc stimulation, low frequency discharge patterns of DA neurons recorded SNc rules. Experimental results also show that we used electrophysiological methods can clearly identify the DA neurons in ZAP current stimulation, in the range of -85mV -55mV to DA neurons the film resonance reaction.
3, the resonant frequency of DA neurons is temperature dependent, that is, the resonant frequency of the membrane increases with the increase of temperature. It is 2Hz ~ 4Hz at 33 ~ 38 C, which confirms that its frequency is in theta frequency range.
4, the frequency of membrane resonance of DA neurons is voltage dependent, which shows that the frequency of membrane resonance is different when it is clamped at different hyperpolarized membrane potential levels.
Two, the ionic mechanism of the membrane resonance characteristics of dopamine neurons in the dense mass of the substantia nigra
The ion channels of DA neurons were mainly K~+, Na~+, Ca~ (2+).Ih ion channel current by HCN channel mediated by a cationic mixed flow, by Na~+, K~+ and other cations together, is a kind of cationic current slow activation. It has been confirmed that plays an important role in the regulation of the current the neuronal membrane excitability and rhythm, which in various brain regions of central neuron system has been fully verified. The results showed that the Ih current regulation of neurons, mainly through the transfer of the regulation of synaptic excitability, and rhythm to regulate neuronal cell membrane, and confirmed their participation in the formation of the film resonance. This in hippocampal neurons of cortex neurons have been confirmed as actively involved in the formation of membrane current resonance. But whether subliminal as active current is also involved in the formation of membrane of DA neurons in subthreshold resonance, Is not clear. At the same time, whether there are other ion channels are involved in regulating the membrane or resonance of DA neurons under the threshold, at present is not clear. In addition, there are reports of small conductance calcium dependent potassium channel (the small-conductance calcium-dependent potassiumchannel, SK) and Ca~ (2+) formation channel is involved in the film oscillation, including SK channel blocking and activation can significantly affect the rhythm and excitability of DA neurons. But can prove the membrane resonance of the role, and further research is needed.
The main results are as follows:
1, the hyperpolarized activated cation flow (Hyperpolarization-activated cation current, Ih) is the active current component of DA neurons producing theta frequency membrane resonance, and its range of action is between -65mV and -85mV.
2, the calcium dependent potassium channel (SK) is also an active component of the DA neurons that produce theta frequency membrane resonance, whose scope of action is probably between -60mV and -70mV.
3, the continuous sodium current (Persistent sodium currents, INap) can amplify the membrane resonance horizontally.
Main conclusions
1, the infrared visual whole cell patch clamp technique was used to record the spindle neurons at the level of the coronal brain slices of the substantia nigra of the rat, and there was a neuronal membrane resonance.
2, the membrane resonance of the DA neurons in the dense part of the substantia nigra of the rat has a temperature dependence and voltage dependence.
3, it is confirmed that the dopamine neurons in the dense part of the rat substantia nigra are around -65mV, and the active current of the membrane resonance is the current of the SK channel.
4, it is confirmed that the dopamine neurons in the dense part of the rat substantia nigra are around -75mV, and the active current of the membrane resonance is the Ih current.
5, it is proved that I_ (Nap) can enhance the membrane resonance peak, but it has no effect on the resonance frequency of the membrane.
6, calcium ions are involved in the production of membrane resonance, and the specific mechanism needs to be further studied.
Our study shows that dopaminergic neurons of substantia nigra pars compacta with theta frequency resonance film, its ionic mechanism has some particularity, the ionic mechanism of this unique, for us to further understand the substantia nigra DA neurons and its role in brain neural network plays an important role, and to provide a basis for DA electrophysiology further study the functional properties of PD patients.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R312

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相關(guān)期刊論文 前2條

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