Targeting Serotonin 5-HT1A and 5-HT7 Receptors to Prevent Audiogenic Seizures and Correct Translationally Valid EEG Phenotypes in a Juvenile Fmr1 Knock-Out Mouse Model of Fragile X Syndrome

癫痫发作发生在约12%的自闭症谱系障碍（ASD）患者中，约25%的自闭症谱系障碍（ASD）患者中。 脆性X综合征（FXS）-FMR 1突变导致FXS也是最常见的，已知的遗传风险 ASD的因素。儿童ASD和FXS的癫痫易感性高于成人，这意味着 神经发育窗口。用于FXS和ASD研究的幼年Fmr 1敲除小鼠表现出 强烈的感觉诱发的听源性癫痫发作，一种模拟感觉超敏反应和癫痫发作的表型， 个人FXS新的研究表明，FXS和Fmr 1基因敲除小鼠的个体也有 休息时脑电图（EEG）活动的扰动，例如，增加了大脑皮层的伽马能量 我们在此复制并报告初步数据。因此，病因学有效的Fmr 1敲除小鼠表现出两个 表型与FXS个体的翻译相关性。许多研究报告了 5-羟色胺（5-羟色胺，5-HT）系统在FXS和ASD，但知识的影响 FXS表型上的特异性5-HT受体（5-HTR）明显缺乏。我们现在报告的数据显示 一种新的5-HTR调节剂可以完全预防幼年Fmr 1基因敲除小鼠的听源性癫痫发作 具有5-HT 1AR和5-HT 7 R部分激动剂活性。在此，我们提出了明确的体内行为， 药理学和EEG实验以及离体受体药理学实验， 机械地探测5-HT 1AR和5-HT 7 R作为可以预防听源性癫痫发作的靶点（目标1）， 校正幼年Fmr 1基因敲除小鼠的EEG异常（目的2）。我们在目标1中假设选择性5-HT 1AR激活或选择性5-HT 7 R失活将减弱听源性癫痫发作，并且结合5-HT 1AR 激活/5-HT 7 R失活将防止幼年Fmr 1敲除小鼠中的听源性癫痫发作。我们扩展了 在目标2中检验该假设，检验该药效学效应以纠正EEG表型，例如 纠正幼年Fmr 1听觉皮层和躯体感觉皮层中异常高的伽马波段功率 基因敲除小鼠5-HT 1AR和5-HT 7 R在海马中密集表达，海马是一个神经系统， 在Fmr 1基因敲除小鼠和FXS患者中，癫痫发作阈值较低。在目标3中，选择性 放射性配体将用于饱和结合实验，以评价5-HT 1AR和5-HT 7 R在 将进行[35 S]GTPγS测定以确定5-HT 1AR和5-HT 7 R在海马中的功能。 与野生型小鼠相比，幼年Fmr 1基因敲除小鼠的海马。5-HT 1AR和5-HT 7 R偶联 以相反的方式调节腺苷酸环化酶活性，即，5-HT 1ARs刺激Gαi，而5-HT 7 Rs 刺激Gαs信号。由于已知cAMP在FXS和ASD中发生改变，因此该项目的结果将 提供关于5-HTR调节cAMP的推定影响的知识，以建立关键基础设施 诊断有效的FXS和ASD表型。结果还将提供有关以下方面的重要信息： 5-HT 1AR和/或5-HT 7 R是否是FXS或ASD的可行药理学靶点。

Seizures occur in ~12% of individuals with autism spectrum disorder (ASD) and in ~25% of individuals with fragile X syndrome (FXS)—mutations in FMR1 that cause FXS are also the most common, known genetic risk factor for ASD. Seizure susceptibility in ASD and FXS is higher in children than in adults, inferring a critical neurodevelopmental window. Juvenile Fmr1 knock-out mice, useful for the study of FXS and ASD, exhibit robust sensory-evoked, audiogenic seizures, a phenotype that models sensory hypersensitivity and seizures in individuals with FXS. New studies show that individuals with FXS and Fmr1 knock-out mice also have perturbations in electroencephalogram (EEG) activity at rest, e.g., increased gamma power in the cortex, which we reproduced and report here as preliminary data. Thus, etiologically-valid, Fmr1 knock-out mice exhibit two phenotypes with translational relevance to individuals with FXS. Numerous studies have reported alterations in the serotonin (5-hydroxytryptamine, 5-HT) system in FXS and ASD, yet knowledge regarding the impact of specific 5-HT receptors (5-HTRs) on FXS phenotypes are conspicuously lacking. We now report data showing that audiogenic seizures in juvenile Fmr1 knock-out mice are entirely prevented by a novel 5-HTR modulator with 5-HT1AR and 5-HT7R partial agonist activity. Herein, we propose definitive in vivo behavioral pharmacology and EEG experiments together with ex vivo receptor pharmacology experiments to mechanistically probe 5-HT1ARs and 5-HT7Rs as targets that can prevent audiogenic seizures (Aim 1) and correct EEG abnormalities (Aim 2) in juvenile Fmr1 knock-out mice. We hypothesize in Aim 1 that selective 5-HT1AR activation or selective 5-HT7R inactivation will attenuate audiogenic seizures and combined 5-HT1AR activation/5-HT7R inactivation will prevent audiogenic seizures in juvenile Fmr1 knock-out mice. We extend the test of this hypothesis in Aim 2, testing this pharmacodynamic effect to correct EEG phenotypes, e.g. to correct abnormally high gamma band power in the auditory cortex and somatosensory cortex of juvenile Fmr1 knock-out mice. 5-HT1ARs and 5-HT7Rs are densely expressed in the hippocampus, a neural system with a low seizure threshold that is altered in Fmr1 knock-out mice and in individuals with FXS. In Aim 3, selective radioligands will be used for saturation binding experiments to evaluate 5-HT1AR and 5-HT7R expression in the hippocampus, and [35S]GTPγS assays will be conducted to determine the function of 5-HT1ARs and 5-HT7Rs in the hippocampus of juvenile Fmr1 knock-out mice compared to wild-type mice. 5-HT1ARs and 5-HT7Rs couple to regulate adenylate cyclase activity in opposing ways, i.e., 5-HT1ARs stimulate Gαi, whereas 5-HT7Rs stimulate Gαs signaling. Since cAMP is known to be altered in FXS and ASD, outcomes from this project will provide knowledge to build a critical infrastructure regarding the putative impact of 5-HTR regulation of cAMP on translationally-valid FXS and ASD phenotypes. Results will also provide important information regarding whether 5-HT1ARs and/or 5-HT7Rs are viable pharmacotherapeutic targets for FXS or ASD.