In vivo recruitment of neocortical neurons in stargazer absence seizures

 描述（由申请人提供）：癫痫是一种典型的神经回路障碍，一年患病率约为7/1,000，社会成本高。癫痫的遗传或后天病因导致神经元回路超同步，表现为癫痫发作。癫痫的一个主要未解决的问题是在癫痫发作事件的演变过程中，单个单位如何在体内被招募。具体而言，尚不清楚神经元是否在每次癫痫发作事件中以刻板的模式或序列放电，这是否可靠地发生或是否/如何取决于细胞类型。重要的是确定锥体神经元是否显示出与各种类别的中间神经元不同的募集模式，以及是否存在可靠地参与的特殊（“中枢”）单元，并且因此在募集其他单元以癫痫发作事件中发挥重要作用。 stargazer小鼠是一种经过验证的人类失神癫痫实验模型。Stargazin蛋白的突变导致受损的AMPA受体膜运输，并且这被认为主要抑制投射在抑制性（小清蛋白+）中间神经元上的兴奋性输入（Maheshwari等人，Frontiers in Cellular Neuroscience，2013）。这种相对沉默的抑制被认为是解除抑制周围的微电路，促进超同步和癫痫发作。这种情况是否发生在体内以及它如何影响新皮层回路仍然未知。其他中间神经元类所起的作用也仍然不清楚。 我们将使用慢性双光子成像来绘制个体皮质神经元在观星者缺席癫痫发作事件期间如何在体内招募，并测量其时间活动概况和招募的可靠性。初步数据表明，募集不是随机的，而是可能取决于细胞类型，层状位置和神经元的“枢纽”状态。识别表现出高水平同步性的细胞群将揭示对癫痫发作表现重要的局部子网络。最后，将进行体内全细胞膜片钳实验，以1）验证双光子结果，和2）研究在锥体神经元与选择类别的GABA能中间神经元中的失神发作事件期间抑制性和兴奋性输入如何演变。小清蛋白+中间神经元活性水平的光遗传学操作将建立因果关系。对癫痫回路故障的深入了解可能会导致细胞靶向治疗干预的新策略。

 DESCRIPTION (provided by applicant): Epilepsy is a prototypical neural circuit disorder with a one-year prevalence of ~7/1,000 and high cost to society. Genetic or acquired etiologies of epilepsy lead to neuronal circuit hyper-synchrony that manifests as a seizure. A major unsolved question in epilepsy is how single units get recruited, in vivo, during the evolution of seizure events. Specifically it is not known whether neurons fire in a stereotyped pattern or sequence per seizure event, whether this happens reliably or whether/how it depends on cell type. It is important to determine whether pyramidal neurons show different patterns of recruitment than various classes of interneurons, and whether there exist special ("hub") units that are reliably engaged and may therefore play an important role in recruiting other units to seizure events. The stargazer mouse is a validated experimental model for human absence epilepsy. Mutation of the protein stargazin leads to impaired AMPA receptor membrane trafficking, and this is thought to suppress primarily excitatory inputs projecting on inhibitory (Parvalbumin+) interneurons (Maheshwari et al., Frontiers in Cellular Neuroscience, 2013). This relative silencing of inhibition is thought to disinhibit the surrounding microcircuit, promoting hyper-synchrony and seizures. Whether this happens in vivo and how it entrains neocortical circuits remains unknown. The role that other interneuronal classes play remains also obscure. We will use chronic two-photon imaging to map how individual cortical neurons are recruited in vivo during stargazer absence seizure events, and to measure their temporal activity profiles and reliability of recruitment. Preliminary data suggests that recruitment is not random, but potentially depends on cell type, laminar location, and the neuron's "hub" status. Identifying groups of cells that exhibit high levels of synchrony will reveal local sub-networks important for seizure manifestation. Finally, in vivo whole-cell patch clamp experiments will be performed to 1) validate the two photon results, and 2) to study how inhibitory and excitatory inputs evolve during absence seizure events in pyramidal neurons versus in select classes of GABA-ergic interneurons. Optogenetic manipulation of Parvalbumin+ interneuron activity levels will establish causality. Obtained insights into epileptic circuit malfunction will potentially lead to new strategies for cell-targeted therapeutic interventions.