Altered functions of ion channels, membrane receptors and neuronal networks associated with thalamocortical dysrhythmia syndrome.

丘脑异常在神经和精神疾病(如失神癫痫、可卡因滥用)中很常见。基于觉醒时慢的增量和爆裂的theta范围的存在，它们被称为丘脑皮质节律紊乱综合征。丘脑皮质(TC)神经元的超极化膜电位被认为是异常爆发活动的基础。神经元超极化可通过过度抑制、去传入或阻断NMDA受体而发生。然而，感觉剥夺和K+通道功能增强，都可能导致膜超极化，目前还没有被考虑到。K2P通道家族的成员在神经元超极化静息膜电位的产生中起着至关重要的作用。为了能够充分阐明K2P通道在丘脑皮质节律失常中的可能作用，有必要对不同类型丘脑细胞中调节这些通道的细胞信号通路进行表征。关于这一方面，关于GABA能神经元(局部中间神经元；网状丘脑核神经元，NRT)的信息完全缺失。本项目的目的是：(1)确定GABA能神经元中K2P通道的功能。(2)测定精神刺激剂和K2P通道激活剂/抑制剂对细胞和网络活动的影响。由于精神刺激剂通过增加单胺的可获得性发挥作用，尤其是多巴胺，我们将分析多巴胺能途径。(3)探讨K2P通道在感觉剥夺中的作用。实验方法为：(1)用聚合酶链式反应和免疫组织化学方法检测离子通道和膜受体的表达和定位。(2)采用全细胞膜片钳记录技术，研究不同类型丘脑细胞离子通道和膜受体的电生理和药理学特性。在谷氨酸脱羧酶67启动子(GAD67-EGFP)的控制下，GABA能神经元将直接靶向表达增强型绿色荧光蛋白(EGFP)的敲除小鼠。(3)将通过在躯体感觉系统的水平丘脑切片和丘脑皮质切片(即，包含腹-基底丘脑复合体；NRT；初级体感皮质)中进行局部场电位(LFP)记录来处理网络活动。(4)将体感系统中的LFP/单单位记录与活体行为分析相结合，确定系统功能。(5)采用K2P通道敲除小鼠和修剪胡须单侧感觉剥夺小鼠作为实验动物模型，将有助于确定K2P通道在丘脑皮质节律紊乱综合征中的新的功能作用和可能的病理影响及其治疗潜力。

Thalamic abnormalities are common in neurological and psychiatric diseases (e. g., absence epilepsy, cocaine abuse). Based on the presence of slow delta and theta range bursting during wakefulness they are referred to as thalamocortical dysrhythmia syndromes. A hyperpolarized membrane potential of thalamocortical (TC) neurons is suggested to be the basis for aberrant burst activity. Neuronal hyperpolarization may occur by excess inhibition, deafferentation or block of NMDA receptors. However sensory deprivation and enhanced function of K+ channels, both potentially inducing membrane hyperpolarizations, have not been considered yet. Members of the K2P channel family crucially contribute to the generation of the hyperpolarized resting membrane potential in neurons. In order to be able to fully address the possible contribution of K2P channels to thalamocortical dysrhythmia, it is necessary to characterize the cellular signaling pathways modulating these channels in different thalamic cell types. Concerning this aspect, information is completely missing for GABAergic thalamic neurons (local interneurons; neurons of the reticular thalamic nucleus, NRT).The aims of the project are: (1) To determine the function of K2P channels in GABAergic thalamic neurons. (2) To determine the influence of psychostimulants and K2P channel activators / inhibitors on cellular and network activity. Since psychostimulants act via the increased availability of monoamines, especially dopamine, we will analyze dopaminergic pathways. (3) To determine the role of K2P channels in sensory deprivation as a new model of thalamocortical dysrhythmia based on deafferentation.The experimental approach will be: (1) PCR and immunohistochemical staining will be used to determine the expression and location of ion channels and membrane receptors. (2) Whole-cell patch-clamp recordings will be performed to determine electrophysiological and pharmacological properties of ion channels and membrane receptors in different thalamic cell types. GABAergic neurons will be directly targeted in knock-in mice expressing enhanced green fluorescent protein (EGFP) under the control of the glutamate decarboxylase 67 promotor (GAD67-EGFP). (3) Network activity will be addressed by performing local field potential (LFP) recordings in horizontal thalamic slices and thalamocortical slices of the somatosensory system (i.e., containing the ventrobasal thalamic complex; NRT; primary somatosensory cortex). (4) System function will be determined by combining LFP / single unit recordings in the somatosensory system and behavioral analysis in vivo. (5) As experimental animal models K2P channel knock out mice and unilateral sensory deprivation in mice by trimming of whiskers will be used.The results of the proposed study will help to determine new functional roles and possible pathological influences of K2P channels and their therapeutic potential in thalamocortical dysrhythmia syndromes.