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

项目1(P1)研究人员已经确定了脆性X综合征(FXS)患者的皮质脑电表型， 例如增强的静息状态伽马功率，与临床结果相关。值得注意的是，许多人类 Project发现，FXS小鼠模型Fmr1基因敲除(KO)的脑电表型是保守的 2名(P2)调查员。脑电表型的跨物种保护强烈地表明了类似的情况 Fmr1功能丧失的小鼠和人的皮层回路功能障碍。因此，我们建议的研究是为了 了解和纠正Fmr1 KO小鼠的电路功能障碍可能与人类高度相关 FXS。我们发现了Fmr1KO初级感觉皮层的两个主要功能回路缺陷 可能对Fmr1 KO小鼠和携带FXS-1的人的特定脑电表型有贡献的小鼠) 皮层区域内“局部”环路的超兴奋性和2)功能兴奋性长时间的减少。 大脑皮层区域之间的范围连接。在体外脑内观察到局部环路的超兴奋性 具有2个关键措施的脑片：1)新皮质电路的延长电路激活(PCA)，自发或 以响应感官刺激和2)增强的伽马功率。我们假设大脑的过度兴奋性 局部新皮质微电路是静息状态伽马功率增加和 在Fmr1KO小鼠和患有FXS的人类中观察到感觉诱发的脑电伽马夹带。我们 通过机械守恒在老鼠身上验证这一假说。Fmr1KO小鼠体外新皮质脑片的应用 我们发现了3个突触微回路的变化，可能会导致局部回路的过度兴奋；1) 锥体神经元之间兴奋性突触的高度连接2)减少了兴奋性突触对 小白蛋白阳性(PV)抑制神经元3)增强内源性大麻素抑制抑制性突触 驱动--可能来自CCK阳性神经元。在目标1和目标2中，我们与P2密切合作， 将使用光遗传、化学发生和药理学方法来操纵PV和CCK抑制 为了确定它们的功能障碍如何导致局部皮质回路的过度兴奋， Fmr1KO小鼠异常脑电表型及相关行为。除了超兴奋的本地电路之外， 我们的新发现揭示了Fmr1 KO小鼠大脑皮层区域之间的兴奋性连接很弱-特别是 同位对侧皮质区之间(胼胝体连接)。功能障碍的长程突触 连接性可能是大脑皮层区域之间异常的长程功能耦合的原因，因为 在患有FXS的人身上观察到的EEG)。在目标3和4中，我们建议进行实验以确定潜在的 突触和分子机制以及功能回路水平的后果这些弱的长- 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- FXS中可能介导人类相关脑电表型的类型、微电路和生化机制 并为具体的、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.