Optical Tools to Dissect Synaptic Changes Underlying Epilepsy

描述（申请人提供）：用于解剖癫痫突触变化的光学工具关于癫痫的一个普遍假设认为，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不平衡。然而，关于这是否是癫痫样活动的主要原理，以及这种不平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，部分原因是网络活动的传统记录不允许轻易地解决或剖析单个类型的突触输入的强度。为了克服这些困难，我们正致力于开发新的方法，利用遗传编码的光学指示器来跟踪不同类型的突触前终端的贡献。我们已经构建了一种新的用于囊泡融合的光学探针，称为sypHTomato，当突触囊泡融合并释放神经递质时，它会发出红色荧光。我们目前正在培育一种小鼠，它将在基因靶向酶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（如synaptopHluorin或GCaMP3）结合使用。这将能够在多种类型的突触上进行独立和同时的监测，无论是兴奋性的，一般抑制性的还是特定亚类的抑制性神经元；它还可以跟踪突触活动和动作电位的激发。我们将验证和优化这个双色系统，使用日益复杂和与癫痫相关的神经网络。光学记录将与实验室目前使用的先进电记录方法一起进行。用于监测突触活动的探针将与光敏蛋白（如Channelrhodopsin-2和Halorhodopsin）一起共表达，以在监测输出的同时操纵选定的突触输入电路。通过这种方式，可以在大脑切片的间歇期和间歇期活动的发展过程中评估特定突触的活动，并且可以通过适当的照明进一步增强或关闭该活动，作为其致病作用的测试。作为原理证明，我们将在选定的癫痫实验模型中阐明突触输入的变化，这些变化有利于或抑制癫痫样爆发的发生。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。报告策略可以很容易地与作为人类癫痫动物模型的现有小鼠系相结合。因此，阐明癫痫样活动基础的强大光学方法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.