Synaptic Dissection of Cell Adhesion Molecule Function within Subicular Circuits

描述（由申请人提供）：越来越多的证据表明突触传递异常可能是一些神经精神疾病的关键机制。突触特性如何被指定和维持？突触细胞粘附分子（SCAMs）是调节突触功能的主要候选分子。不足为奇的是，基因组研究发现了神经素-3 （Nrx3）的突变，这是一种典型的突触前诈骗，是神经素/神经素复合物的一部分。与精神分裂症（SZ）和寻求奖励行为有关。这些疾病与巨大的社会和经济负担有关，并有一个共同的病理生理基础——伏隔亚核（NAc）壳回路内腹侧下带（vSub）突触传递异常导致多巴胺系统失调。两种不同的丘下投射神经元群作为海马的主要输出，接受来自CA1的输入并投射到皮层和皮层下区域，包括NAc壳中两种不同的中棘中间神经元（MSN）细胞类型。关于Nrx3如何在涉及DA失调的两个棘下突触（CA1输入到vSub和棘下输出到NAc壳）中塑造细胞类型特异性突触特性的基本理解尚未探索；因此，在这种疾病回路中，解剖细胞类型特异性的隐下神经元突触前和突触后功能可能为治疗策略开辟新的途径。为此，我们生成了一个Nrx3小鼠，其中剪接位点4 （SS4）是组成性包括的（SS4+），但可以有条件地排除（SS4-），剪接位点决定了与几乎所有神经蛋白配体的结合。我们观察到在Nrx3SS4+小鼠的背托下，由于表面AMPAR稳定性降低，AMPAR突触传递选择性减少。Nrx3SS4小鼠跨突触相互作用的改变可能模仿疾病相关的Nrx3突变，因为大多数突变影响表面暴露的残基和选择性剪接，很少导致完全的蛋白质丢失。这项资助的总体目标是在CA1-vSub突触和vSub-NAc壳突触的不同细胞类型中解剖突触前nrx3ss4依赖的功能。指导的目标是：1)在WT和Nrx3SS4动物中，通过立体定向注射逆行病毒和cree -重组酶，评估投射到CA1-vSub突触NAc壳上的vSub神经元的细胞类型特异性突触反应；2)通过立体定向注射通道视紫质，选择性地招募腹侧棘下纤维，检测vSub投射到纹状体回路中两个不同群体的NAc壳msn。对于该资助的R00阶段，将1.)利用在K99阶段获得的技术，通过解剖背侧Sub-NAc核心电路，扩大突触特异性纹状体电路的研究；2.)通过下一代单细胞RNA测序，确定新的SCAMs，长期目标是评估其在疾病相关电路中的突触功能。我预计，这一建议将揭示新的见解，细胞类型特异性突触特性在丘脑下纹状体电路和创建一个平台，为未来的独立研究如何其他scam在丘脑下和纹状体电路的功能。

DESCRIPTION (provided by applicant): Increasing evidence suggests that abnormalities in synaptic transmission may be a key mechanism underlying some neuropsychiatric disorders. How might synaptic properties be specified an maintained? Synaptic cell adhesion molecules (SCAMs) are primary candidates that play a critical role regulating synapse function. Not surprisingly, genomic studies have found mutations in neurexin-3 (Nrx3) a prototypical presynaptic SCAM that is part of the neurexin/neuroligin complex. that are linked to Schizophrenia (SZ) and reward-seeking behavior. These disorders are associated with an enormous social and economic burden and share a common pathophysiological basis - dopamine system dysregulation due to abnormalities in synaptic transmission in the ventral subiculum (vSub) within the vSub-nucleus accumbens (NAc) shell circuit. Two distinct populations of subicular projection neurons serve as the major output of the hippocampus, receive input from CA1 and project to cortical and subcortical regions, including to two distinct medium spiny interneuron (MSN) cell- types in the NAc shell. A fundamental understanding how Nrx3 shapes cell-type specific synaptic properties at two subicular synapses involved in DA dysregulation - CA1 input to the vSub and subicular output to the NAc shell - is unexplored; thus, the dissection of cell-type specific pre- and post-synaptic functions of subicular neurons within this disease circuit may open new avenues for treatment strategies. To this end, we generated a Nrx3 mouse where splice site 4 (SS4), the splice site that dictates binding to nearly all neurexin ligands, is constitutively included (SS4+) but can be conditionally excluded (SS4-). We observed a selective reduction in AMPAR synaptic transmission, due to reduced surface AMPAR stability, in the dorsal subiculum of Nrx3SS4+ mice. Altered transsynaptic interactions in the Nrx3SS4 mouse may imitate disease-related Nrx3 mutations because most mutations affect surface exposed residues and alternative splicing - rarely resulting in complete protein loss. The overarching goal of this grant is to dissect presynaptic Nrx3SS4-dependent functions in distinct cell-types at the CA1-vSub synapse and the vSub-NAc shell synapse. The mentored aims will 1.) assess cell-type specific synaptic responses in vSub neurons that project to the NAc shell at the CA1-vSub synapse using stereotactic co-injection of retrograde virus and cre-recombinase in WT and Nrx3SS4 animals and 2.) assay vSub projections to two distinct populations of NAc shell MSNs in the striatal circuit by utilizing stereotactic injection of channelrhodopsin to selectively recruit ventral subicular fibers. For the R00 phase of this grant, will 1.) expand the study of synapse-specific striatal circuitry by dissecting the dorsal Sub-NAc core circuit using techniques acquired during the K99 phase and 2.) identify novel SCAMs by next-generation single-cell RNA sequencing of subicular neurons with the goal long-term of assessing their synaptic function in disease-relevant circuits. I anticipate that this proposal wil uncover new insights into cell-type specific synaptic properties in the subicular-striatal circuit and create a platform for future independent investigations into how other SCAMs function in subicular and striatal circuitry.