Mapping the escape from inhibition.

描述（由申请人提供）：反馈抑制是主神经元的活动刺激中间神经元释放抑制性神经递质GABA到活跃的主神经元以防止失控兴奋的过程。因此，反馈抑制的失败是大多数癫痫发病理论的关键因素。然而，反馈抑制在正常大脑和癫痫灶中的功能解剖尚不清楚。光遗传学和多光子显微镜的最新发展使直接解决这个问题成为可能。本研究提出将通道视紫红质介导的靶锥体细胞的光激活与表达钙荧光黄色变色龙3.6的中间神经元反应的高速多光子成像结合起来。这些技术将使我们能够确定被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产生得到补偿。我们的计算机模型表明，这种萌芽所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的影响，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由反馈抑制解剖结构的特征变化来定义：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，因此局部反馈回路的解剖复杂性将在癫痫灶中增加。我们将用新的光遗传学和显微镜工具直接测试这一假设，在体外使用慢性癫痫的器官型切片培养，在体内使用慢性癫痫的动物。这些数据将为癫痫的病理生理学提供重要的新见解，尽管我们进行了非常详细的电生理学和经典解剖学研究，但我们仍无法获得这些新见解。对癫痫的特征性神经回路改变的测试将使新类型的治疗干预成为可能，包括对活动依赖性抑郁症的药理学操作，以及对关键神经回路元件的先发制人的激活。

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.