Optical Tools to Dissect Synaptic Changes Underlying Epilepsy

描述(申请人提供)：光学工具解剖癫痫背后的突触变化癫痫的一种流行假说认为，神经回路变得过度兴奋是因为突触兴奋和抑制之间的病理性失衡。然而，关于这是否是癫痫样活动的主要原理，以及这种失衡是因为兴奋性突触得到加强，还是因为抑制性突触被削弱，或者两者兼而有之，仍然存在许多问题。这些问题很难解决，部分原因是传统的网络活动记录不允许容易地解决或剖析个别类型的突触输入的强度。为了克服这些困难，我们致力于开发新的方法，使用遗传编码的光学指示器来跟踪不同类型的突触前终末的贡献。我们已经构建了一种新的囊泡融合光学探针，称为sypHTomato，当突触小泡融合并释放神经递质时，它发出红色的荧光。我们目前正在培育一种小鼠，在基因靶向酶Cre重组酶的控制下，在特定类型的神经元中表达sypHTomato。SypHTomato可以与现有的绿色探针一起使用，如synaptopHluorin或GCaMP3。这将允许对多种类型的突触进行独立和同时的监测，无论这些突触是兴奋性的、一般性的抑制性神经元还是特定亚类的抑制性神经元；它还将允许跟踪突触活动以及动作电位的激发。我们将使用日益复杂和与癫痫相关的神经网络来验证和优化这一双色系统。光学记录将与实验室内目前使用的先进的电子记录方法一起进行。用于监测突触活动的探针将与通道视紫红质-2和卤视紫红质等光敏蛋白共同表达，以允许在监测输出的同时操纵选定的突触输入到电路。通过这种方式，特定突触的活性可以在脑片发作间期和发作活动的发展过程中进行评估，这种活性可以通过适当的光照进一步增强或关闭，以测试它们的起因作用。作为原则证明，我们将阐明在选定的癫痫实验模型中，有利于或抑制癫痫样发作发生的突触输入的变化。我们的分子试剂、动物和技术方法将免费提供给癫痫研究人员和整个科学界。记者策略可以很容易地与作为人类癫痫动物模型的现有小鼠品系相结合。因此，强大的光学方法来阐明癫痫样活动的基础可以很容易地在广泛的突变和实验环境中使用，从而利用癫痫遗传学的最新进展。

DESCRIPTION (provided by applicant): Optical tools to dissect synaptic changes underlying epilepsy A prevailing hypothesis about epilepsy contends that neural circuits become overexcitable because of a pathological imbalance between synaptic excitation and inhibition. However, many questions remain about whether this is the dominant principle of epileptiform activity, and whether the imbalance comes about because excitatory synapses are bolstered, because inhibitory synapses are weakened, or both. These issues are challenging to approach, in part because conventional recordings of network activity do not allow the strength of individual types of synaptic input to be readily resolved or dissected. To overcome such difficulties, we are engaged in developing new approaches that use genetically encoded optical indicators to track the contributions of different kinds of presynaptic terminal. We have constructed a new optical probe for vesicle fusion, called sypHTomato, which fluoresces in the red when synaptic vesicles fuse and release neurotransmitter. We are currently generating a mouse that will express sypHTomato within specific types of neurons under control of genetically targetable enzyme, Cre recombinase. SypHTomato can be used in conjunction with existing green probes such as synaptopHluorin or GCaMP3. This will enable independent and simultaneous monitoring at multiple types of synapses, be they excitatory, generically inhibitory, or inhibitory neurons of a particular subclass; it will also allow synaptic activity to be tracked along with action potential firing. We will validate and optimize this two-color system, using neural networks of increasing complexity and relevance to epilepsy. Optical recordings will be performed in conjunction with advanced methods for electrical recording currently in use within the lab. Probes for monitoring synaptic activity will be co-expressed in conjunction with light-sensitive proteins such as Channelrhodopsin-2 and Halorhodopsin to allow manipulation of selected synaptic inputs to a circuit while monitoring the output. In this way, the activity of specific synapses can be assessed during the development of interictal and ictal activity in brain slices and that activity can be further enhanced or turned off by appropriate illumination as tests of their causative role. As proof-of-principle, we will clarify the changes in synaptic input that favor or restrain the genesis of epileptiform bursts in select experimental models of epilepsy. Our molecular reagents, animals and technical approaches will be freely available to epilepsy investigators and to the scientific community at large. The reporter strategy can be easily integrated with existing lines of mice that serve as animal models of human epilepsy. Thus, powerful optical approaches to elucidate the underpinnings of epileptiform activity can be readily put to use in a wide range of mutational and experimental settings, thereby leveraging recent advances in the genetics of epilepsy.