In vivo recruitment of neocortical neurons in stargazer absence seizures

 描述(申请人提供)：癫痫是一种典型的神经回路疾病，一年内患病率约为7/1000，社会成本很高。癫痫的遗传或获得性病因导致神经元回路超同步，表现为癫痫发作。癫痫中一个尚未解决的主要问题是，在癫痫发作事件的演变过程中，单个单位是如何在体内被招募的。具体地说，还不知道神经元是否在每次癫痫发作事件中以刻板的模式或顺序放电，这是否可靠地发生，或者它是否/如何取决于细胞类型。重要的是要确定锥体神经元是否表现出与各种类型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”)单位，从而在招募其他单位参与癫痫发作事件中发挥重要作用。观星小鼠是人类失神癫痫的一个经过验证的实验模型。Stargazin蛋白的突变导致AMPA受体膜转运受损，这被认为主要抑制了投射到抑制性(小蛋白+)中间神经元的兴奋性输入(Mahehwari等人，《细胞神经科学前沿》，2013)。这种相对沉默的抑制被认为可以解除对周围微回路的抑制，促进超同步和癫痫发作。这是否在体内发生，以及它是如何进入新皮质回路的，目前尚不清楚。其他神经元间类所扮演的角色也仍不清楚。我们将使用慢性双光子成像来绘制单个皮质神经元在观星者缺席发作事件期间如何在体内招募，并测量它们的时间活动曲线和招募的可靠性。初步数据表明，招募不是随机的，而是潜在地取决于细胞类型、板层位置和神经元的“中枢”状态。识别表现出高度同步性的细胞组将揭示对癫痫发作表现重要的局部亚网络。最后，将进行在体全细胞膜片钳实验，以1)验证双光子结果，2)研究锥体神经元与特定类别的GABA能中间神经元在失神发作事件中抑制性和兴奋性输入的演变。对小白蛋白+神经元间活性水平的光遗传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针对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.