Cell types and neuronal genetic architecture in the rat CSF-contacting nucleus and the role of 5-HT in this nucleus in mediating morphine addiction through the brain-CSF circuit.

Introduction: To elucidate the cellular composition of the cerebrospinal fluid-contacting nucleus, establish a comprehensive gene expression database for this nucleus, and investigate its potential functional roles. Methods: we used single-cell sequencing technology combined with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses to characterize the transcriptional architecture of the CSF-contacting nucleus and elucidate its potential biological functions. Additionally, Conditioned place preference (CPP) testing, chemogenetic techniques, ELISA, and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) were employed to examine the functional relationships between the CSF-contacting nucleus, cerebrospinal fluid, and morphine addiction-related behaviors. Results: Single-cell RNA sequencing revealed that the CSF-contacting nucleus had an average of 22,046 genes expressed per cell. Unsupervised clustering revealed 25 cellular subsets belonging to five canonical CNS cell types (neuron, astrocyte, oligodendrocyte, microglia, endothelial) as annotated against the Genomics RNAseq database. The raw sequencing data have been deposited in the China National Center for Bioinformation (CNCB) accession number: CRR790158. GO and KEGG enrichment analyses demonstrated that the CSF-contacting nucleus neurons were significantly enriched in calcium signaling pathways, neurotransmitter regulation, and addiction-related pathways (including morphine, cocaine, and other substances). Given prior reports of morphine-induced alterations in CSF composition and the unique anatomical features of the CSF-contacting nucleus, we performed additional experimental validation. Chemogenetic manipulation experiments demonstrated that inhibition of the CSF-contacting nucleus attenuated morphine-induced CPP, UPLC-MS and ELISA revealed a marked increase in 5-HT levels in the CSF of the morphine addiction group. Knock out and chemogenetic inhibition of the CSF-contacting nucleus led to a significant reduction in CSF 5-HT levels. These findings suggest that the CSF-contacting nucleus may facilitate morphine addiction through regulated 5-HT release into the CSF. This discovery provides new experimental evidence for understanding CSF-mediated neuromodulation mechanisms. Conclusion: The present study delineates the single-cell transcriptional architecture and cellular composition of the CSF-contacting nucleus. Bioinformatics analyses revealed the involvement of the CSF-contacting nucleus in multiple addiction-related pathways for various substances of abuse and neurodegenerative disorders. These findings suggest that the CSF-contacting nucleus plays multifaceted roles in neural circuitry, particularly serving as a crucial mediator in neuro-cerebrospinal fluid regulatory mechanisms.