小鼠癫痫模型中功能异常神经环路光遗传解析

发布时间：2018-03-27 20:13

本文选题：癫痫　切入点：捕食者恐惧　出处：《中国科学院大学(中国科学院深圳先进技术研究院)》2017年博士论文

【摘要】：目前全球神经精神疾病的治疗负担占全部疾病的13%,居所有疾病首位。癫痫是常见的一种神经系统疾病,因其间歇性不可预知的发作、高比例的共患病、过早死亡率升高等特点而具有极大危害。而更严峻的是由于癫痫发生的病理机制不清楚,缺乏对其深刻、全面的理解导致癫痫的治疗效果并不理想。颞叶癫痫是成人患者中最常见的一种类型,其典型特征是包括海马、内嗅皮层在内的颞叶有明显的病理性改变,其许多症状包括癫痫发作的发生与传播、癫痫导致的大脑功能障碍如认知功能等,可能都与此有关。研究认为癫痫的发作与神经活动的兴奋/抑制性的平衡有关,而且癫痫发作过程中神经元的同步化活动异常升高。此外,目前人们已经认识到癫痫是一种神经环路水平的疾病,癫痫发作不但传播到广泛的皮层区域引起抽搐等行为,还影响到丘脑、脑干等深部脑区引起失去意识等问题。已有的研究认为痫波在海马-内嗅皮层中以一种回路(loop)的方式传播,但因基于脑切片与摘取的完整大脑研究的结果存在争议,人们已经认识到已有的实验技术存在其局限性。随着神经环路研究相关技术的发展,我们得以在体水平解析痫波在海马-内嗅皮层结构中传播的神经环路机制以及癫痫大脑功能障碍相关的神经环路机制。为了更好地实现癫痫相关神经环路的解析,我们改进和完善了一些相关的电生理技术、方法。主要包括:1)麻醉条件下进行电生理记录和光遗传调控的同时实现了精确定量给药的功能电极,以及三个脑区的同步光刺激/电生理记录的实验方法;2)实现了自由活动的小鼠上,八个脑区的局部场电位记录,其中一个脑区能给予同步的光刺激;以及可步进的四个脑区神经元放电(spike)和局部场电位(local field potential,LFP)的记录;3)针对慢性电极因炎性反应造成神经元死亡、胶质包囊形成导致的电极记录效果下降的问题,我们在电极材料表面修饰了新的生物相容性导电材料——PVA/PAA水凝胶-PEDOT/PSS导电聚合物-NGF/DEX生物活性分子,通过原代神经元培养及动物植入实验的评估发现该修饰材料能够促进神经元存活、减少胶质细胞聚集。在此基础上我们对痫波在海马-内嗅皮层中的传播环路以及癫痫导致天生恐惧相关的海马神经环路进行了精确解析,具体如下:(1)首次采用基于光遗传和多脑区电生理记录技术,在动物活体水平上通过给药电极注射KA诱导癫痫发作后,对海马齿状回(dentate gyrus/hilus,DGH)和内嗅皮层(medial entorhinal cortex,MEC)以及初级运动皮层(primary motor cortex,M1)中神经元放电和局部场电位的同步记录,并进行锋电位触发的叠加平均信号(spike-triggered-average of LFP,STA)、叠加锋电位分析(multi-unit analysis),再在电生理记录的同时给予DGH或MEC的γ-氨基丁酸(GABA)能神经元光刺激,然后进行局部场电位交叉相关(LFP cross-correlation)、局部场电位相干分析(LFP coherence)等电生理信号分析,结果发现:癫痫发作主要是发作区域兴奋性神经元的高度同步化放电引起痫波,痫波传播至环路下游引起更广泛的异常同步化放电活动;此外不同于目前文献中报道的颞叶癫痫中痫波的主要传播方向“内嗅皮层—海马—内嗅皮层”环路,而是“海马—内嗅皮层”。最后,本文还发现利用光遗传调控能选择性地兴奋海马中间神经元阻断痫波传播并挽救小鼠的发作行为,证实海马中间神经元可能是重要干预靶点,为颞叶癫痫的临床治疗提供了新的指导思路。(2)以捕食者为非条件性恐惧刺激测试癫痫模型小鼠的防御行为,发现其恐惧下降。为解析其中环路机制,我们首先通过组织学方法确认癫痫小鼠的海马结构发生病理性改变;c-Fos蛋白表达结果显示海马参与捕食者恐惧反应,而癫痫小鼠的海马神经元在捕食者恐惧测试中激活异常。接着通过麻醉电生理/光刺激以及多脑区局部场电位记录/行为测试的方法,证实海马通过隔核到下丘脑具有功能连接,且该环路可能通过θ波参与捕食者恐惧行为。最后,癫痫小鼠的海马-下丘脑环路中θ波在捕食者恐惧测试中存在异常,提示我们该神经环路可能与癫痫小鼠天生的异常恐惧有关。综上所述,新的电生理/神经调控相关技术的发展有利于促进神经环路解析的研究,而颞叶癫痫发作传播相关的环路机制研究以及海马相关环路在癫痫本能恐惧反应异常中的发生与调控机制研究将不仅有助于更全面深入地理解癫痫大脑功能障碍及其早死率升高的问题,为全面改善癫痫患者治疗效果提供一些可能的新策略,还能从神经环路角度深入理解大脑海马相关神经网络的工作机制,理解其他相关精神疾病贡献新思路。
[Abstract]:The current global neuropsychiatric disease burden of disease treatment accounted for 13%, ranking the first of all diseases. Epilepsy is a common neurological disease, due to the intermittent paroxysmal unpredictable, prevalence of high rates of premature mortality in high and has great harm. But more serious is the pathological mechanism of epilepsy there is not clear, the lack of its profound and comprehensive understanding of the cause of epilepsy treatment is not satisfactory. Temporal lobe epilepsy is the most common type of adult patients, typically including hippocampus, entorhinal cortex, temporal lobe had obvious pathologic changes, many of the symptoms including seizures occurred with the spread of epileptic brain dysfunction, such as cognitive function, may be related. The research results indicate that the onset of epilepsy with neural activity in the excitatory / inhibitory balance, and seizures in the process The synchronization of neurons abnormal. In addition, at present, people have already realized that epilepsy is a neural circuit level of disease, seizures not only spread to the cortical regions widely cause convulsions and other acts, but also affects the thalamus, problems caused by unconscious brain areas of deep brain stem. According to the studies of epileptic waves in the hippocampus - in the entorhinal cortex in a loop (loop) way, but based on the removal of the intact brain slices and the results are controversial, people have realized that the experimental technique has its limitations. With the development of neural circuits and related technologies, we can analysis in vivo in the hippocampus of epileptic waves in the olfactory cortex structure in the loop propagation mechanism and epileptic brain dysfunction related to neural loop mechanism. In order to realize the analysis of related epilepsy neural loop, we modified and finished Good some electrophysiological techniques. Methods mainly include: 1) and electrophysiological recording and light anesthesia under genetic control to achieve a precise quantitative function to electrode drugs, and stimulation / experimental method of electrophysiological recording of synchrotron light in three brain regions; 2) to achieve a free activity in mice on the local field potentials recorded in eight brain regions, including an area of the brain can give synchronized light stimuli; and step four neurons brain discharge (spike) and local field potential (local field, potential, LFP) records; 3) for chronic inflammatory reaction caused by electrode neurons death, glial cyst formation decreased due to the effect of electrode recording problems, we modified the compatibility of conductive material -- PVA/PAA -PEDOT/PSS hydrogel polymer -NGF/DEX new bioactive molecules in biological electrode material surface, through the cultivation of primary neurons Evaluation and animal experiment showed that the modified material can promote neuronal survival, reduce glial cell aggregation. On the basis of our innate fear related hippocampal neural circuits in the hippocampus of epileptic waves in the entorhinal cortex - the spread of epilepsy were loop and accurate analytical details are as follows: (1), for the first time by genetic and light brain electrophysiological recording technique based on the electrode through the administration of injection of KA induced seizures in animal in vivo, the dentate gyrus (dentate, gyrus/hilus, DGH) and entorhinal cortex (medial entorhinal, cortex, MEC) and primary motor cortex (primary motor, cortex, M1) in neurons and local field potentials the synchronous recording and averaging signal spike triggered (spike-triggered-average of LFP, STA), superposition analysis of spike (multi-unit analysis), and then in the electrophysiological recording at the same time To DGH or MEC gamma amino butyric acid (GABA) neurons light stimulation, then the local field potential cross correlation (LFP cross-correlation), local field potential (LFP coherence) coherent analysis of electrophysiological signal analysis results showed that: epilepsy is mainly the highly synchronized discharge area of excitatory neurons caused by seizures of epileptic waves downstream, epileptic wave propagating to the loop caused by abnormal synchronized firing activities more widely; in addition different from the main propagation direction of epileptic wave "temporal lobe epilepsy reported in the current literature in the entorhinal cortex, hippocampus and entorhinal cortex" loop "- but the hippocampal entorhinal cortex. Finally, this paper also finds that the use of regulation light can be selectively excited genetic hippocampus interneurons blocking epileptic wave propagation behavior and save the mice attack, confirmed the hippocampal interneurons may be an important target for intervention, for the clinical treatment of temporal lobe epilepsy. For the new guideline. (2) the predator defense behavior of non fear conditioned stimulation test in mice model of epilepsy, the fear of falling. For the analysis of loop mechanism, we first confirmed by the hippocampus of epileptic mice pathological changes of histological method; the expression of c-Fos protein in the hippocampus showed predator fear response, the epileptic mice hippocampal neurons in fear of predators test. Then through the activation of abnormal electrophysiological / light stimulation and anesthesia in brain regions of the local field potential recording / behavior test method, confirmed the hippocampus through the septum to the hypothalamus has the function of connection, and the loop by theta in predator fear behavior. Finally, in the hippocampus of epilepsy mouse hypothalamus loop theta in predator fear test in the presence of abnormal neural circuits may prompt us to the epileptic mice born with abnormal fear Fear about. To sum up, the new development of neural electrophysiology / regulation technology is conducive to the promotion of neural circuit analysis, and temporal lobe epilepsy research and control system research and related mechanism of loop loop propagation in hippocampus associated abnormal fear response in epilepsy instinct will not only contribute to a more comprehensive understanding of the rise of epileptic brain dysfunction and death rate, and provide some new strategies possible for an overall improvement in patients with epilepsy treatment effect, but also from the perspective of understanding the neural circuits related neural network brain hippocampus work mechanism, understand new ideas with other mental disorders.

【学位授予单位】：中国科学院大学(中国科学院深圳先进技术研究院)
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R742.1;R-332

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