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

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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.