大鼠黑质致密部多巴胺神经元的膜共振现象及其机制研究

发布时间：2018-01-25 22:43

本文关键词： 黑质致密部多巴胺神经元振荡共振超级化激活的阳离子流小电导钙依赖性钾通道全细胞膜片钳　出处：《第四军医大学》2012年博士论文　论文类型：学位论文

【摘要】：神经振荡性电活动是指在生理状态下核团内及神经网络中的神经元的电生理活动特点。振荡性电活动是电流/电压的周期性变化，分为同步振荡和异步振荡两种情况。其中，能够保持同步而稳定运行的振荡称为同步振荡。膜共振是用来描述神经元对输入信号的频率选择性能力的物理量，同时也是神经元振荡活动的内在机制之一。频率选择性是神经元膜共振活动所具有的一种内在特性。神经元的膜共振特性和频率选择性在调节脑内神经网络的节律性活动中发挥重要作用，其调控作用尤其体现在神经元相互之间进行信息传递的过程中。不同的神经元具有不同的膜共振特性是神经网络共振的基础。多巴胺能神经元是黑质致密部中的主要神经元。了解多巴胺能神经元的膜共振特性，将有助于增进我们对基底节区神经元细胞对信息处理过程的认识。黑质致密部的多巴胺神经元输出的信息涉及运动、学习和记忆等生理功能。这些信息的输出，对我们认知黑质致密部DA神经元及其损害后引起的神经系统疾病，特别是帕金森病，具有重要的意义。在前期研究中，已发现帕金森病患者基底节区存在病理性同步振荡现象，其主要表现是过度的同步振荡。一般认为，同步振荡与神经系统信息的检测、处理及整合密切相关，因此，过度的同步振荡也可能成为帕金森病感觉、运动信息紊乱并导致出现不同临床症状的内在机制之一。“振荡模型”是新近提出的一种帕金森病的发病机制，目前仍有很多机制尚不明确。此外，既往临床和基础研究已经表明，黑质致密部的多巴胺神经元的病理性的退行性改变是PD、抑郁症等多种疾病的发生和发展的重要特征。故研究探索PD的DA（dopamine）神经元的膜共振特性及机制将有助于我们从一个新的角度去认识PD的发病机制。本课题分成二个部分，其一是在大鼠脑片水平上利用红外线可视全细胞膜片钳技术观察记录多巴胺神经元的电活动及其膜共振性质；其二是对膜共振特性潜在的离子机制进行更深入的探讨，以期在正常大鼠脑片水平上，了解多巴胺能神经元的膜共振特性，为进一步研究帕金森大鼠脑片水平的多巴胺神经元膜共振特性的改变，提供坚实的理论依据。一、黑质致密部多巴胺神经元的膜共振神经元的振荡是神经系统电活动一个重要的生物节律，无论单个神经元的振荡，还是神经网络的振荡，对脑的功能都具有重要意义。神经元的膜共振是用来描述神经元对输入信号的频率选择性，是神经元网络振荡的内在机制之一。目前已在海马CA1区锥体神经元、海马下托锥体神经元及内嗅皮质Ⅱ层星形细胞等不同类型神经元中检测到θ膜共振。多巴胺神经元作为在黑质致密部结构中最主要的神经元，对神经网络的作用是通过直接、间接通路与其它核团神经元的相互作用而产生的结果，然而对于黑质致密部DA神经元是否也存在膜共振，尚未见报道。因此，本课题的第一部分实验内容就是给予黑质致密部DA神经元一个随时间增加频率连续变化的正弦电流（ZAP，0Hz～16Hz，20s）作为刺激电流，观察并检测出DA神经元的是否存在膜共振，及其膜共振的频率范围，并对其结果进行进一步的分析讨论。主要结果如下： 1、使用红外线可视全细胞膜片钳技术在黑质致密部冠状脑片水平上记录到多巴胺神经元电生理活动，同时用TH染色证实DA神经元其形态为梭形神经元。 2、我们发现在给予SNc的DA神经元去极化电流刺激时，记录到的SNc的DA神经元放电模式为规则的低频的放电模式。实验还发现，我们利用电生理方法可以明确鉴定DA神经元，在给予ZAP电流刺激时，在-55mV到-85mV范围内DA神经元表现出膜共振反应。 3、DA神经元的膜共振频率具有温度依赖性，即膜共振频率随温度的升高而升高，在33℃～38℃为2Hz～4Hz左右，证实其频率值处在θ频率范围内。 4、DA神经元的膜共振频率具有电压依赖性，表现为当钳制在不同的超极化膜电位水平，其膜共振频率有所不同。二、黑质致密部多巴胺神经元的膜共振特性的离子机制 DA神经元的离子通道主要有K~+、Na~+、Ca~(2+)离子通道。Ih电流是由HCN通道所介导一种混合性的阳离子流，由Na~+、K~+及其它阳离子共同组成，是一种缓慢激活的阳离子电流。目前已经证实这种电流对神经元细胞膜的兴奋性和节律性具有重要的调节作用，这在各个脑区的中枢神经元系统已经得到了充分验证。研究结果显示，Ih电流对神经元的调节，主要是通过对突触的传递的调节，而去调节神经元细胞膜的兴奋性及节律性，并且证实其参与了膜共振的形成。这在海马神经元、皮层等神经元业已被证实，，其作为主动电流参与形成阈下膜共振。但其是否作为主动电流也参与形成DA神经元阈下膜共振，尚不明确。同时是否有其它离子通道也参与或调节了DA神经元的阈下膜共振，目前也不明确。另外，有文献报道，小电导钙依赖性钾通道（the small-conductance calcium-dependent potassiumchannel, SK）和Ca~(2+)通道参与了膜振荡的形成，其中SK通道的阻断和激活能够显著影响DA神经元的节律和兴奋性。但是能否证实其在膜共振方面的作用，还有待进一步研究。主要结果如下: 1、超极化激活的阳离子流（Hyperpolarization-activated cation current，Ih）是DA神经元产生θ频率膜共振的主动电流成分，作用范围大概在-65mV～-85mV之间。 2、钙依赖性钾通道（SK）也是DA神经元产生θ频率膜共振的主动电流成分，其作用范围大概在-60mV～-70mV之间。 3、持续性钠电流（Persistent sodium currents，INap）可以水平放大膜共振。主要结论 1、使用红外线可视全细胞膜片钳技术在大鼠黑质致密部冠状脑片水平上记录到梭形神经元，存在神经元膜共振。 2、大鼠黑质致密部DA神经元的膜共振具有温度依赖性和电压依赖性。 3、证实了大鼠黑质致密部多巴胺神经元在-65mV左右，产生膜共振的主动电流是SK通道电流。 4、证实了大鼠黑质致密部多巴胺神经元在-75mV左右，产生膜共振的主动电流是Ih电流。 5、证实了I_(Nap)可以增强膜共振峰，但对膜共振频率没有影响。 6、钙离子参与了膜共振的产生，具体机制尚需进一步研究。我们的研究表明黑质致密部多巴胺神经元具有θ频率膜共振，其离子机制具有一定的特殊性，这种独特的离子机制，对于我们进一步了解黑质致密部DA神经元及其在脑内神经网络中的地位具有重要的作用，并且为进一步研究PD患者的DA电生理功能特性提供了基础。
[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.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R312

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