Mapping the escape from inhibition.

描述（由申请人提供）：反馈抑制是主神经元中的活动刺激中间神经元将抑制性神经递质GABA释放到活性主神经元上以防止失控兴奋的过程。因此，反馈抑制的失败是癫痫发作发病机制的大多数理论中的关键因素。然而，反馈抑制在正常大脑和癫痫病灶的功能解剖是未知的。光遗传学和多光子显微镜的最新发展使得直接解决这个问题成为可能。在这里，我们建议结合联合收割机通道视紫红质介导的光激活的目标锥体细胞的高速多光子成像的响应在中间神经元表达的钙荧光团黄色变色龙3.6。这些技术将使我们能够确定由目标锥体细胞激活的中间神经元的位置。脑损伤后，主要神经元和中间神经元的损失通过新突触连接的萌芽来补偿。我们的计算机模型表明，这种萌芽产生的电路复杂性使中间神经元容易受到活动诱导的突触抑制，从而导致失控的兴奋和癫痫发作。因此，我们假设癫痫回路将被定义为反馈抑制解剖结构的特征性变化：更多的主神经元将共享相同的中间神经元反馈网络，单个中间神经元将被更广泛的锥体细胞激活，因此癫痫灶中局部反馈回路的解剖复杂性将增加。我们将直接测试这一假设与新的光遗传学和显微镜工具在体外使用慢性癫痫器官型切片培养，并在体内使用慢性癫痫动物。这些数据将为癫痫的病理生理学提供一个重要的新见解，尽管有非常详细的电生理学和经典解剖学研究，我们还没有能够获得。测试癫痫的特征性电路改变将使新的治疗干预措施成为可能，包括活动依赖性抑郁症的药理学操作，以及关键电路元件的抢先激活。

DESCRIPTION (provided by applicant): Feedback inhibition is the process by which activity in principal neurons stimulates interneurons to release the inhibitory neurotransmitter GABA onto the active principal neurons to prevent runaway excitation. Failure of feedback inhibition is thus a critical element in most theories of the pathogenesis of seizures. However, the functional anatomy of feedback inhibition in the normal brain and epileptic focus is unknown. Recent developments in optogenetics and multiphoton microscopy have made it possible to address this question directly. Here we propose to combine channelrhodopsin-mediated photoactivation of targeted pyramidal cells with high-speed multiphoton imaging of the responses in interneurons expressing the calcium fluorophore yellow chameleon 3.6. These techniques will allow us to define the location of the interneurons that are activated by the target pyramidal cell. After brain injury, the loss of principal neurons and interneurons is compensated by sprouting of new synaptic connections. Our computer modeling suggests that the circuit complexity engendered by this sprouting leaves the interneurons vulnerable to activity-induced synaptic depression that permits runaway excitation and seizures. We hypothesize therefore that epileptic circuits will be defined by characteristic changes in the anatomy of feedback inhibition: more principal neurons will share the same interneuron feedback networks, and individual interneurons will be activated by a wider anatomical range of pyramidal cells, so that the anatomical complexity of local feedback circuits will be increased in epileptic foci. We will test this hypothesis directly with the new optogenetic and microscopy tools in vitro using chronically epileptic organotypic slice cultures, and in vivo using chronically epileptic animals. This data will provide a critical new insight into the pathophysiology of epilepsy that we have not been able to acquire despite wonderfully detailed electrophysiological and classical anatomical studies. Testing for characteristic circuit alterations in epilepsy will make possible new classes of therapeutic interventions including pharmacological manipulation of activity-dependent depression, as well as preemptive activation of critical circuit elements.