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

摘要 前扣带皮层（ACC）与外侧前额叶皮层（LPFC）和边缘系统紧密相连， 杏仁核（AMY）等结构，形成认知和情感处理的重要途径。 人类神经影像学研究表明，ACC表现出LPFC和AMY的差异控制， 用反相关激活模式调节不同的边缘系统和认知功能静息状态网络。 控制觉醒和压力的脑干神经调节通路影响神经化学物质 环境和活动，通过调节内在的兴奋性和突触 在特定区域、层和细胞类型中的信号传导。了解迄今未知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 网络目标-在灵长类动物ACC，一个关键的皮质边缘中枢，将使我们能够识别新的生物标志物， 神经化学失衡和细胞损伤导致易受情绪压力影响。近期 单细胞RNA测序（scRNAseq）的进展已经允许揭示分子定义的神经元 小鼠皮层中的亚类。然而，在我们对层特定性如何理解方面存在很大的知识差距。 神经元的分子特性影响电生理特性和神经元互连的形成 在灵长类动物和人类身上。这一建议的总体假设是，板层特异性ACC CAPACITY和 ACC → LPFC投射神经元具有不同的分子特征，这些特征是其神经调节信号的基础 兴奋性和突触连接的特性和调节。我们建议确定基因表达 单个板层特异性ACC细胞类型和投射神经元的概况，侧重于差异表达 与兴奋和压力相关的受体和信号通路。我们将联合收割机神经束追踪与 和scRNAseq和体外电生理技术在成年恒河猴（Macaca mulatta）中的应用 目前资助的赠款（R 01 MH 116008）。我们将使用尖端的Patch-Seq方法进行转录组学研究， 单个电生理学表征的层特异性神经元和靶特异性神经元的分析。差异 通过单细胞转录组学鉴定的表达基因，将使用RNAscope进行验证， 免疫组织化学（IHC）分析，并将与不同ACC的特定功能特性相关 细胞类型。我们将具体阐明ACC β-AMY和ACC β-LPFC的分子和生理特性 投射神经元参与调节认知、唤醒和情绪压力。Findings from the 拟议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 这些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。这项拟议的研究将揭开层状和功能性的分子基础， ACC回路的多样性介导情感行为，这对理解具有广泛的治疗意义。 压力相关疾病的易感性

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.