Effects of anesthetics on thalamic excitability

项目摘要 丘脑是感觉信息从外周流向皮层的主要通道， 丘脑皮质连接的破坏可能是催眠作用的一个基本共同特征， 许多全身麻醉剂（GAs）。此外，最近的研究已经确定了中央的重要作用， 丘脑的内侧核（CeM）和腹基底核（VB）控制觉醒和自然睡眠。 虽然丘脑T型钙通道（T通道）在自然睡眠中的作用相当不错， 虽然这些通道已经建立，但它们在麻醉中的作用仍然知之甚少。此外，过去 我们确定，暴露于临床相关GA的大鼠幼崽会导致T- 丘脑网状核（nRT）中的电流密度，这反过来又有助于持久的过度兴奋 和丘脑皮质网络的病理振荡。这些神经元的变化 通过选择性拮抗T通道逆转功能。 我们推测，使用靶向T通道的药物抑制剂可能会促进GA诱导的损失。 因此可以减少手术所需的强效挥发性GA的使用。整体 本申请的目的是检验中心假设，即在细胞中T通道的CaV3.1同种型 丘脑CeM和VB核在麻醉诱导催眠中起重要作用。 为了验证这一假设，我们将使用体外膜片钳记录从急性脑切片和重组 细胞在体外和脑电图（EEG）记录在体内从CeM和VB核，选择性 药理学T通道抑制剂，以及具有全局和区域特异性沉默的小鼠遗传学， CaV3.1通道在丘脑追求以下具体目标： 目的1：探讨重组和天然CaV3.1通道的生物物理和分子机制 异氟醚（ISO）和七氟醚（SEVO）两种常见GAs体外抑制作用及其对神经细胞的影响 CeM的神经兴奋性。我们还将使用ISO（AZI-ISO）光亲和配体的新方法， SEVO（AZI-SEVO）用于鉴定CaV3.1通道上挥发性GA的特异性分子结合位点。 目的2：确定CeM中CaV 3.1通道是否参与丘脑皮质的改变 在使用挥发性GAs和给予选择性T通道的镇静/催眠期间的网络功能 抑制剂TTA-P2，如通过体内EEG记录评估的。 目的3：确定CeM和VB丘脑中CaV3.1通道的区域特异性沉默是否会导致 对ISO和SEVO在体内诱导的镇静/催眠和丘脑皮层振荡的不同作用。 这项工作的创新之处在于，麻醉诱导的意识丧失的新机制将被发现。 表征了它具有医学意义，因为它描述了靶向电压门控的药物的重要性。 钙离子通道的潜在发展更安全的做法在临床麻醉。

Project Summary The thalamus is the major gateway for the flow of sensory information from the periphery to the cortex and the disruption of thalamocortical connectivity may be an essential common feature of the hypnotic effects of many general anesthetic (GAs). Furthermore, recent studies have identified important roles of the central medial nucleus (CeM) and ventrobasal (VB) nucleus of the thalamus in control of arousal and natural sleep. Although the role of thalamic T-type calcium channels (T-channels) in natural sleep is reasonably well established, the role of these channels in anesthesia remains poorly understood. In addition, in the past cycle of this grant we established that exposure of rat pups to clinically-relevant GAs causes increased T- current densities in the reticular thalamic nucleus (nRT), which in turn contributes to lasting hyperexcitabil- ity and pathological oscillations in thalamocortical networks in vitro and in vivo. These changes in neuronal function were reversed by selective antagonism of T-channels. We postulate that the use of pharmacological inhibitors that target T-channels may facilitate GA-induced loss of consciousness and hence may reduce usage of potent volatile GAs needed for surgery. The overall objective of this application, is to test the central hypothesis that the CaV3.1 isoform of T-channels in the CeM and VB thalamic nuclei is important for anesthetic-induced hypnosis. To test this hypothesis, we will use in vitro patch-clamp recordings from acute brain slices and recombinant cells in vitro and electroencephalographic (EEG) recordings in vivo from the CeM and VB nuclei, selective pharmacological T-channel inhibitors, and mouse genetics with global and region-specific silencing of CaV3.1 channels in the thalamus to pursue the following specific aims: Aim 1: To determine the biophysical and molecular mechanism of recombinant and native CaV3.1 channel inhibition in vitro by two common GAs, isoflurane (ISO) and sevoflurane (SEVO), and their effect on neu- ronal excitability of CeM. We will also use new approach with photoaffinity ligands of ISO (AZI-ISO) and SEVO (AZI-SEVO) to identify specific molecular binding sites on CaV3.1 channels for volatile GAs. Aim 2: To determine whether CaV3.1 channels in the CeM contribute to the alterations in thalamocortical network function during sedation/hypnosis with volatile GAs and administration of the selective T-channel inhibitor TTA-P2, as assessed by in vivo EEG recordings. Aim 3: To determine if region-specific silencing of CaV3.1 channels in the CeM and VB thalamus will have differential effects on sedation/hypnosis and thalamocortical oscillations in vivo induced by ISO and SEVO. The proposed work is innovative in that new mechanisms of anesthetic-induced loss of consciousness will be characterized. It is medically significant because it describes the importance of drugs that target voltage- gated calcium channels for potential development of safer practices in clinical anesthesia.