Shank3自闭症小鼠刻板行为的皮质-纹状体通路和丘脑-纹状体通路调控机制

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders which present two types of symptoms: deficits in social communication / interaction and restricted / repetitive behaviors. Comparing to social communication/interaction deficits, repetitive behaviors often get overlooked. However, repetitive behaviors usually are the first sign of ASD patients and can keep in a lifetime. It also aggravate the social communication/interaction deficits of ASD patients and also the anxiety and burden of whole family. Recent genetic studies have revealed that some genes coding proteins important for synaptic development and function may be involved in ASD. Shank3 (SH3 and multiple ankyrin repeat domains 3) is a member of the Shank family of proteins (SHANK1-3) which interact with various postsynaptic density (PSD) proteins. It shows an important role in dynamically regulating excitatory synaptic development, function and structure. Genetic studies have strongly implicated that SHANK3 gene disruption or mutation may be a monogenic cause of ASD. Our previous work found Shank3 mutant mice exhibited ASD-like behavioral including repetitive grooming behaviors and social interaction deficits. Thus, the excitatory synapse deficit produced by Shank3 mutant may be particpated in neural mechanisms underlying ASD relevant behaviors. Recently we demonstrated that Shank3 deletion preferentially affected synapses onto striatal indirect pathway projection medium spiny neurons (Striatopallidal MSNs). Striatopallidal MSNs showed severe defects including synaptic transmission, synaptic plasticity and spine density. Importantly, the repetitive grooming behavior was rescued by selectively enhancing the striatopallidal MSNs activity using Designer Receptors Exclusively Activated by Designer Drugs (DREADDs-hM3Dq) technology. However, the striatum receives two excitatory synapses from cortex and thalamus. The excitatory synapses of cortico-striatal pathway (CSP) and thalamo-striatal pathway (TSP) show different synaptic location and transimission properties. It is not clear whether the glutamatergic synapse of CSP and TSP are differentially affected in Shank3 mutant mice. We are planning do serial works to research excitatory synaptic function and structure of CSP and TSP by specific targeting or modulateing CSP/TSP synpase on D1 and D2 MSNs in Shank3 KO mice with virus labeling, optogenetics, transgenic mice such as D1-Tdtomato, D2-GFP, D1-Cre and D2-Cre mice. We are also planning to test the relationship between CSP/TSP and repetitive behavior. We hope we can find a working flow of CSP and TSP modulating repetitive behavior and help to find the target for the diagnosis and therapies of repetitive behavior of ASDs.

孤独谱系障碍（Autism spectrum disorder，ASD）作为儿童神经发育性疾病，其沟通交流障碍症状广为关注，而刻板行为相对忽视。刻板行为是ASD病人最早症状，常加重患者交流障碍和家人的负担，神经基础尚不清楚。我们以往发现，Shank3缺失导致纹状体兴奋性突触异常可能参与ASD行为。我们新近证实，Shank3敲除导致小鼠纹状体间接通路兴奋性突触受损，直接通路相对正常。化学遗传学提高间接通路活动可改善刻板行为。然而，纹状体接收皮质、丘脑双重谷氨酸能支配，两条通路突触结构功能存在区别，是否在刻板行为中作用不同，尚不清楚。本研究拟在前期基础上，利用病毒标记技术、光遗传技术、荧光小鼠和Cre小鼠等工具鼠，选择性记录或调控皮质-纹状体通路和丘脑-纹状体通路谷氨酸突触，系统研究两条通路谷氨酸能突触功能结构改变及在ASD刻板行为中可能作用，探讨刻板行为的病理生理机制，寻找可能的诊疗策略。

刻板行为是孤独症核心症状之一，也是最早出现的症状，可加重孤独症社交障碍。我们前期发现背外侧纹状体（DLS）是Shank3敲除孤独症小鼠模型刻板理毛行为发生的关键脑区。因此，我们对DLS在刻板理毛行为中的环路机制进行了研究。发现，光遗传兴奋DLS直接通路可促进理毛行为，兴奋DLS间接通路抑制理毛行为，证实DLS内直接通路和间接通路平衡共同调节理毛行为。通过病毒环路标记，我们发现了DLS上游中背内侧丘脑（MD）、基底杏仁核（BLA）在刻板理毛行为调节中具有重要作用：兴奋MD促进理毛行为的发生，抑制MD则抑制理毛行为。而BLA对理毛行为调控刚好与MD相反。由此，我们发现了DLS的输入端的双相调控核团，这对于理解孤独症刻板行为发生机制提供了重要线索。此外，我们还发现Shank3敲除小鼠刻板理毛行为增加表现为易于启动、躯干清理时间延长，同时伴有焦虑的成分。抗焦虑药可缓解孤独症小鼠理毛次数，但对理毛序列异常没有作用。这表明孤独症小鼠刻板行为是涉及到异常焦虑情绪和运动调控异常两种因素。我们进而也对该小鼠模型的运动障碍进行研究，发现Shank3敲除小鼠步态异常，表现为类似小幅高频步态。这种步态异常与小脑浦肯野细胞层面积减少、细胞胞体面积降低、动作电位兴奋性降低有关。在研究过程中，我们还开发和改进了技术方法。我们设计一种全自动记录小鼠跳跃的装置，该装置能够自动、高效和准确地记录小鼠跳跃状态，并可以实时将小鼠跳跃的时刻信息通过TTL信号发送给外部设备，用于比如光纤记录设备的事件标记或者触发光遗传设备等。为了更好地标记特定类型神经元突起及树突棘等微小结构。我们前期利用法尼基化（Farnesylated）可以促使蛋白更易于与细胞膜结合并锚定在细胞膜上，通过将EGFP改造成法尼基化EGFP，用来标记神经元突起及树突棘微小结构。本项目中将其改造成Cre dependent版本，实现对特定类型神经元的选择性稀疏标记。这些技术改进应用到本项目中，提高了工作效率，也可为其它相关研究提供帮助。