The hypothalamic paraventricular nucleus (PVN) is a highly complex brain region that is crucial for homeostatic regulation through neuroendocrine signaling, outflow of the autonomic nervous system, and projections to other brain areas. In the past years, single-cell datasets of the hypothalamus have contributed immensely to the current understanding of the diverse hypothalamic cellular composition. While the PVN has been adequately classified functionally, its molecular classification is currently still insufficient. To address this, we created a detailed atlas of PVN transcriptomic cell types by integrating various PVN single-cell datasets into a recently published hypothalamus single-cell transcriptome atlas. Furthermore, we functionally profiled transcriptomic cell types, based on relevant literature, existing retrograde tracing data, and existing single-cell data of a PVN-projection target region. Finally, we validated our findings with immunofluorescent stainings. In our PVN atlas dataset, we identify the well-known different neuropeptide types, each composed of multiple novel subtypes. We identify Avp-Tac1, Avp-Th, Oxt-Foxp1, Crh-Nr3c1, and Trh-Nfib as the most important neuroendocrine subtypes based on markers described in literature. To characterize the preautonomic functional population, we integrated a single-cell retrograde tracing study of spinally projecting preautonomic neurons into our PVN atlas. We identify these (presympathetic) neurons to cocluster with the Adarb2⁺ clusters in our dataset. Further, we identify the expression of receptors for Crh, Oxt, Penk, Sst, and Trh in the dorsal motor nucleus of the vagus, a key region that the pre-parasympathetic PVN neurons project to. Finally, we identify Trh-Ucn3 and Brs3-Adarb2 as some centrally projecting populations. In conclusion, our study presents a detailed overview of the transcriptomic cell types of the murine PVN and provides a first attempt to resolve functionality for the identified populations.

Introduction: Cushing's disease (CD) is a rare and severe endocrine disease characterized by hypercortisolemia. Previous studies have found structural brain alterations in remitted CD patients compared to healthy controls, specifically in the anterior cingulate cortex (ACC). However, potential mechanisms through which these persistent alterations may have occurred are currently unknown. Methods: Structural 3T MRI's from 25 remitted CD patients were linked with gene expression data from neurotypical donors, derived from the Allen Human Brain Atlas. Differences in gene expression between the ACC and an unaffected control cortical region were examined, followed by a Gene Ontology (GO) enrichment analysis. A cell type enrichment analysis was conducted on the differentially expressed genes, and a disease association enrichment analysis was conducted to determine possible associations between differentially expressed genes and specific diseases. Subsequently, cortisol sensitivity of these genes in existing datasets was examined. Results: The gene expression analysis identified 300 differentially expressed genes in the ACC compared to the cortical control region. GO analyses found underexpressed genes to represent immune function. The cell type specificity analysis indicated that underexpressed genes were enriched for deactivated microglia and oligodendrocytes. Neither significant associations with diseases, nor evidence of cortisol sensitivity with the differentially expressed genes were found. Discussion: Underexpressed genes in the ACC, the area vulnerable to permanent changes in remitted CD patients, were often associated with immune functioning. The specific lack of deactivated microglia and oligodendrocytes implicates protective effects of these cell types against the long-term effects of cortisol overexposure.