Transcriptomic, physiological, and neurochemical profiling of cortico-limbic projection neurons in monkey anterior cingulate cortex

摘要 前扣带回皮质(ACC)与外侧前额叶皮质(LPFC)和边缘紧密相连 杏仁核(AMY)等结构，形成对认知和情感加工至关重要的通路。 人类神经成像研究表明，ACC对LPFC和AMY TO具有不同的控制 用反相关的激活模式调节不同的边缘和认知功能静息状态网络。 控制觉醒和应激的上行脑干神经调节通路影响神经化学 这些皮质-边缘网络中的环境和活动通过调节固有的兴奋性和突触 在特定区域、层和小区类型中发送信号。了解尚不清楚的细胞类型特异性分子 神经调节信号通路的特征--受体表达谱和下游基因 网络靶点-在灵长类ACC中，一个关键的皮质-边缘枢纽，将使我们能够识别新的生物标记物 神经化学失衡和细胞损伤使人容易受到情绪压力的影响。最近的 单细胞RNA测序(ScRNAseq)的进步使揭示分子定义的神经元成为可能 小鼠皮质中的亚类。然而，在我们对如何特定于层的理解中存在着很大的知识差距 分子神经元同一性影响电生理特性和神经元相互连接的形成 在灵长类动物和人类身上。这一提议的总体假设是，椎板特异的ACC和AMY AccLPFC投射神经元具有不同的分子信号，这些信号是其神经调节信号的基础 兴奋性和突触连接的性质和调节。我们建议确定基因的表达 单个板层特异性ACC细胞类型和投射神经元的图谱，侧重于差异表达 与唤醒和压力相关的受体和信号通路。我们将把神经束追踪和批量结合起来 和scRNAseq及体外电生理技术对成年恒河猴(Macaca Mulatta)的研究 目前资助的赠款(R01MH116008)。我们将使用尖端的Patch-Seq方法进行转录 单个电生理学特征的层和靶点特异性神经元的轮廓。差异化的 通过单细胞转录鉴定的表达基因将使用RNAScope和 免疫组织化学(IHC)分析，并将与不同的ACC的特定功能特性相关联 单元类型。我们将具体阐述Acc、AMY和AccLpFC的分子和生理特性 与调节认知、觉醒和情绪压力有关的投射神经元。研究结果： 拟议中的研究将形成一个更大的研究计划的基础，以调查这些神经信号如何 特性与神经化学失衡和对不利条件的反应有关，如氧化应激 和炎症。拟议中的研究将揭开层流和功能性的分子基础。 调节情感行为的ACC回路的多样性，这对理解具有广泛的治疗意义 应激相关障碍的易感性。

ABSTRACT The anterior cingulate cortex (ACC) is heavily interconnected with the lateral prefrontal cortex (LPFC) and limbic structures such as the amygdala (AMY), forming pathways important for cognitive and affective processing. Human neuroimaging studies have shown that the ACC exhibits differential control of LPFC and AMY to modulate distinct limbic and cognitive functional resting state networks with anti-correlated activation patterns. Ascending brainstem neuromodulatory pathways that control arousal and stress influence the neurochemical environment and activity in these cortico-limbic networks through modulating intrinsic excitability and synaptic signaling in specific areas, layers, and cell types. Understanding the as yet unknown cell type-specific molecular signatures of neuromodulatory signaling pathways -- the receptor expression profiles and downstream gene network targets– in the primate ACC, a key cortico-limbic hub, will allow us to identify novel biomarkers for neurochemical imbalance and cellular damage that confer vulnerability to emotional stress. The recent advancement in single-cell RNA sequencing (scRNAseq) has allowed to uncover molecularly-defined neuronal subclasses in mouse cortex. However, there is a big knowledge gap in our understanding of how layer-specific molecular neuronal identity affects electrophysiological properties and the formation of neuronal interconnections in primates and humans. The overall hypothesis of this proposal is that the lamina-specific ACCAMY and ACCLPFC projection neurons have distinct molecular signatures that underlie their neuromodulatory signaling properties and regulation of excitability and synaptic connections. We propose to determine the gene expression profile of individual lamina-specific ACC cell types and projection neurons, focusing on the differential expression of receptors and signaling pathways related to arousal and stress. We will combine neural tract-tracing with bulk and scRNAseq and in vitro electrophysiological techniques in adult rhesus monkeys (Macaca mulatta) from our currently funded grant (R01MH116008). We will use the cutting-edge Patch-Seq method for transcriptomic profiling of individual electrophysiologically-characterized layer- and target- specific neurons. Differentially expressed genes identified via single cell transcriptomics, will be validated using RNAscope and immunohistochemistry (IHC) analysis, and will be correlated with specific functional properties of distinct ACC cell types. We will specifically illuminate the molecular and physiological identity of ACCAMY and ACCLPFC projection neurons that are implicated in regulating cognition, arousal and emotional stress. Findings from the proposed study will form the basis of a larger program of studies to investigate how these neural signaling properties are linked to neurochemical imbalance and responses to adverse conditions, such as oxidative stress and inflammation. The proposed study will unravel the molecular underpinnings of laminar and functional diversity of ACC circuits mediating affective behavior, which has broad therapeutic implications for understanding susceptibility in stress-related disorders.