FMRP regulation of local and long-range neocortical circuits in the mouse: Links with EEG phenotypes

项目1（P1）的研究人员已经确定了脆性X综合征（FXS）患者的皮层EEG表型， 例如与临床结果相关的增强的静息状态伽马功率。值得注意的是，许多人类 EEG表型在FXS小鼠模型中是保守的，Fmr 1敲除（KO），如项目 2名（P2）调查员。EEG表型的跨物种保守性强烈表明， 小鼠和人类的功能障碍性皮层回路，并丧失了Fmr 1的功能。因此，我们建议的研究， 理解和纠正Fmr 1 KO小鼠的电路功能障碍可能与人类高度相关， FXS。我们已经确定了两个主要的功能电路缺陷，在初级感觉皮层的Fmr 1 KO 可能导致Fmr 1 KO小鼠和患有FXS - 1的人类中特定EEG表型的小鼠） 皮质区域内“局部”回路的过度兴奋和2）功能性兴奋性长- 皮层区域之间的联系在离体脑中观察到局部回路的超兴奋性 具有2个关键指标的切片：1）新皮层回路的延长回路激活（PCA），自发或 响应于感官刺激和2）增强的伽马功率。我们假设， 局部新皮层微回路是静息状态γ功率增加和 在Fmr 1 KO小鼠和FXS患者中观察到EEG的感觉诱发γ夹带。我们 在老鼠身上通过机械保守来验证这一假设。使用Fmr 1 KO小鼠的离体新皮质切片 我们已经发现了3种突触微回路的变化可能引起局部回路的过度兴奋; 1） 锥体神经元之间兴奋性突触的超连通性2）减少兴奋性突触驱动到 小清蛋白阳性（PV）抑制性神经元3）增强内源性大麻素对抑制性突触的抑制作用， 驱动-可能来自胆囊收缩素阳性（CCK）神经元。在目标1和2中，我们与方案2密切合作， 将使用光遗传学、化学遗传学和药理学方法来操纵PV和CCK抑制剂， 为了确定它们的功能障碍如何导致局部皮层回路的过度兴奋， 异常EEG表型和相关行为。除了过激励局部电路， 我们的新发现揭示了Fmr 1基因敲除小鼠大脑皮层区域之间的弱兴奋性连接， 在同伦对侧皮质区之间（胼胝体连接）。功能障碍性长程突触 连接性可能有助于皮层区域之间的异常长距离功能耦合， 在患有FXS的人中通过EEG观察到）。在目标3和4中，我们提出实验来确定潜在的 突触和分子机制，以及功能电路水平的后果，这些弱长- 范围兴奋性连接。项目3（P3）的结果预计将确定特定的功能障碍细胞- 可能介导FXS中人类相关EEG表型的类型、微电路和生化机制 并为特定的、有针对性的治疗策略提供了理论基础。

Project 1 (P1) investigators have identified cortical EEG phenotypes in humans with Fragile X Syndrome (FXS), such as enhanced resting state gamma power, that correlate with clinical outcomes. Remarkably, many human EEG phenotypes are conserved in the mouse model of FXS, the Fmr1 knockout (KO), as discovered by Project 2 (P2) investigators. The across-species conservation of EEG phenotypes strongly suggest similarly dysfunctional cortical circuits in mice and humans with loss of function of Fmr1. Thus, our proposed studies to understand and correct circuit dysfunction in the Fmr1 KO mouse will likely be highly relevant to humans with FXS. We have identified two major functional circuit defects in primary sensory cortex of the Fmr1 KO mouse that likely contribute to specific EEG phenotypes in Fmr1 KO mice and humans with FXS – 1) hyperexcitability of “local” circuits within a cortical region and 2) reduced functional excitatory long- range connections between cortical regions. Hyperexcitability of local circuits is observed ex vivo in brain slices with 2 key measures: 1) Prolonged Circuit Activation (PCA) of neocortical circuits, either spontaneously or in response to sensory stimulation and 2) enhanced gamma power. We hypothesize that hyperexcitability of local neocortical microcircuits underlies the increased resting state gamma power and alterations in sensory-evoked gamma entrainment of the EEG observed in Fmr1 KO mice and humans with FXS. We test this hypothesis in mice through mechanistic conservation. Using ex vivo neocortical slices of Fmr1 KO mice we have discovered 3 synaptic microcircuit changes likely to give rise to hyperexcitable local circuits; 1) Hyperconnectivity of excitatory synapses between pyramidal neurons 2) Reduced excitatory synaptic drive onto Parvalbumin-positive (PV) inhibitory neurons 3) enhanced endocannabinoid suppression of inhibitory synaptic drive – likely from Cholecystokinin-positive (CCK) neurons. In Aims 1 and 2, in close collaboration with P2, we will use optogenetic, chemogenetic, and pharmacological approaches to manipulate PV and CCK inhibitory circuits in order to determine how their dysfunction contributes to hyperexcitability of local cortical circuits, abnormal EEG phenotypes, and related behaviors in Fmr1 KO mice. In addition to hyperexcitable local circuits, our new findings reveal weak excitatory connectivity between cortical regions in Fmr1 KO mice - specifically between homotopic contralateral cortical areas (callosal connections). Dysfunctional long-range synaptic connectivity likely contributes to the abnormal long-range functional coupling between cortical areas as observed by EEG in humans with FXS). In Aims 3 and 4, we propose experiments to determine the underlying synaptic and molecular mechanisms as well as the functional circuit level consequences of these weak long- range excitatory connections. Results of Project 3 (P3) are expected to determine the specific dysfunctional cell- types, microcircuits, and biochemical mechanisms that likely mediate human relevant EEG phenotypes in FXS and provide a rationale for specific, targeted therapeutic strategies.