{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,10]],"date-time":"2026-11-10T12:13:15Z","timestamp":1794312795825,"version":"4.9.0"},"reference-count":38,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,6,16]],"date-time":"2025-06-16T00:00:00Z","timestamp":1750032000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Neurosci."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>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: <jats:ext-link>CRR790158<\/jats:ext-link>. 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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>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.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fnins.2025.1603486","type":"journal-article","created":{"date-parts":[[2025,6,16]],"date-time":"2025-06-16T06:17:04Z","timestamp":1750054624000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":2,"title":["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\u2013CSF circuit"],"prefix":"10.3389","volume":"19","author":[{"given":"Ying","family":"Li","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bin","family":"Gui","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yijun","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Licai","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,6,16]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"707","DOI":"10.1042\/EBC20190030","article-title":"Enhancer DNA methylation: Implications for gene regulation.","volume":"63","author":"Angeloni","year":"2019","journal-title":"Essays Biochem."},{"key":"B2","doi-asserted-by":"publisher","first-page":"11","DOI":"10.1016\/j.neuron.2020.12.010","article-title":"Single-cell sequencing of brain cell transcriptomes and epigenomes.","volume":"109","author":"Armand","year":"2021","journal-title":"Neuron"},{"key":"B3","doi-asserted-by":"publisher","first-page":"eabq5745","DOI":"10.1126\/sciadv.abq5745","article-title":"An orbitofrontal cortex-anterior insular cortex circuit gates compulsive cocaine use.","volume":"8","author":"Chen","year":"2022","journal-title":"Sci. Adv."},{"key":"B4","doi-asserted-by":"publisher","first-page":"203","DOI":"10.1016\/j.neubiorev.2014.08.008","article-title":"Endocannabinoid signaling and food addiction.","volume":"47","author":"D\u2019Addario","year":"2014","journal-title":"Neurosci. Biobehav. Rev."},{"key":"B5","doi-asserted-by":"publisher","first-page":"105","DOI":"10.1182\/blood.V90.1.105","article-title":"Phenotypic consequences of mutations in the Fanconi anemia FAC gene: An international fanconi anemia registry study.","volume":"90","author":"Gillio","year":"1997","journal-title":"Blood"},{"key":"B6","doi-asserted-by":"publisher","first-page":"R93062","DOI":"10.7554\/eLife.93062","article-title":"The role of heterochronic gene expression and regulatory architecture in early developmental divergence.","volume":"13","author":"Harry","year":"2024","journal-title":"Elife"},{"key":"B7","doi-asserted-by":"publisher","first-page":"11510","DOI":"10.1073\/pnas.1201547109","article-title":"Brain dopamine and serotonin differ in regulation and its consequences.","volume":"109","author":"Hashemi","year":"2012","journal-title":"Proc. Natl. Acad. Sci. U S A."},{"key":"B8","doi-asserted-by":"publisher","first-page":"874","DOI":"10.1186\/1471-2164-15-874","article-title":"Whole-genome expression profile in zebrafish embryos after chronic exposure to morphine: Identification of new genes associated with neuronal function and mu opioid receptor expression.","volume":"15","author":"Herrero-Turri\u00f3n","year":"2014","journal-title":"BMC Genomics"},{"key":"B9","doi-asserted-by":"publisher","first-page":"20","DOI":"10.1016\/j.coph.2004.08.011","article-title":"Synaptic plasticity and drug addiction.","volume":"5","author":"Jones","year":"2005","journal-title":"Curr. Opin. Pharmacol."},{"key":"B10","doi-asserted-by":"publisher","first-page":"28","DOI":"10.1002\/pro.3711","article-title":"KEGG Mapper for inferring cellular functions from protein sequences.","volume":"29","author":"Kanehisa","year":"2020","journal-title":"Protein Sci."},{"key":"B11","doi-asserted-by":"publisher","first-page":"D480","DOI":"10.1093\/nar\/gkm882","article-title":"KEGG for linking genomes to life and the environment.","volume":"36","author":"Kanehisa","year":"2008","journal-title":"Nucleic Acids Res."},{"key":"B12","doi-asserted-by":"publisher","first-page":"940","DOI":"10.1038\/npp.2010.232","article-title":"Effects of direct- and indirect-acting serotonin receptor agonists on the antinociceptive and discriminative stimulus effects of morphine in rhesus monkeys.","volume":"36","author":"Li","year":"2011","journal-title":"Neuropsychopharmacology"},{"key":"B13","doi-asserted-by":"publisher","first-page":"9598","DOI":"10.1080\/21655979.2021.1999549","article-title":"Identification of hub genes associated with cognition in the hippocampus of Alzheimer\u2019s Disease.","volume":"12","author":"Liu","year":"2021","journal-title":"Bioengineered"},{"key":"B14","doi-asserted-by":"publisher","first-page":"188","DOI":"10.1016\/j.neulet.2010.11.026","article-title":"Substance P in the cerebrospinal fluid-contacting nucleus contributes to morphine physical dependence in rats","volume":"488","author":"Lu","year":"2011","journal-title":"Neurosci Lett"},{"key":"B15","doi-asserted-by":"publisher","first-page":"188","DOI":"10.1016\/j.neulet.2010.11.026","article-title":"Studies on the roles and mechanisms of targeted lesion of the cerebrospinal fluid-contacting nucleusin pathological pain, morphine dependence and withdrawal behaviors in rats.","volume":"488","author":"Lu","year":"2011","journal-title":"Neurosci. Lett."},{"key":"B16","doi-asserted-by":"publisher","first-page":"503","DOI":"10.1523\/JNEUROSCI.0537-18.2018","article-title":"Chemogenetic manipulations of ventral tegmental area dopamine neurons reveal multifaceted roles in cocaine abuse.","volume":"39","author":"Mahler","year":"2019","journal-title":"J. Neurosci."},{"key":"B17","doi-asserted-by":"publisher","first-page":"1331","DOI":"10.3390\/nu14071331","article-title":"Investigation of the effect of curcumin on protein targets in NAFLD using bioinformatic analysis.","volume":"14","author":"Mahmoudi","year":"2022","journal-title":"Nutrients"},{"key":"B18","doi-asserted-by":"publisher","first-page":"e175616","DOI":"10.1172\/JCI175616","article-title":"Meningeal lymphatic CGRP signaling governs pain via cerebrospinal fluid efflux and neuroinflammation in migraine models.","volume":"134","author":"Nelson-Maney","year":"2024","journal-title":"J. Clin. Invest."},{"key":"B19","doi-asserted-by":"publisher","first-page":"210","DOI":"10.1016\/j.bbi.2021.08.006","article-title":"Cerebrospinal fluid test results and associations with subsequent mental disorders, neurological diseases, and CNS infections: A population-based cohort study.","volume":"98","author":"Orlovska-Waast","year":"2021","journal-title":"Brain Behav. Immun."},{"key":"B20","doi-asserted-by":"publisher","first-page":"1131","DOI":"10.1038\/nn.4366","article-title":"Disentangling neural cell diversity using single-cell transcriptomics.","volume":"19","author":"Poulin","year":"2016","journal-title":"Nat. Neurosci."},{"key":"B21","doi-asserted-by":"publisher","first-page":"1326","DOI":"10.3321\/j.issn:1001-1978.2007.10.016","article-title":"The nNOS expression in rat distal CSF contacting neurons and its relationship with morphine withdrawal.","volume":"23","author":"Qing","year":"2007","journal-title":"Chinese Pharmacol. Bull."},{"key":"B22","doi-asserted-by":"publisher","first-page":"7839","DOI":"10.3390\/ijms21217839","article-title":"Comparative analysis of proteomes of a number of nosocomial pathogens by KEGG modules and KEGG pathways.","volume":"21","author":"Slizen","year":"2020","journal-title":"Int. J. Mol. Sci."},{"key":"B23","doi-asserted-by":"publisher","first-page":"22","DOI":"10.3389\/fnana.2018.00022","article-title":"The establishment of a CSF-contacting nucleus;Knockout.","volume":"12","author":"Song","year":"2018","journal-title":"Model Animal. Front. Neuroanat."},{"key":"B24","doi-asserted-by":"publisher","first-page":"47","DOI":"10.3389\/fnana.2019.00047","article-title":"Stereotaxic coordinates and morphological characterization of a unique nucleus (CSF-contacting nucleus) in rat.","volume":"13","author":"Song","year":"2019","journal-title":"Front. Neuroanat."},{"key":"B25","doi-asserted-by":"publisher","first-page":"7","DOI":"10.3389\/fnana.2020.00007","article-title":"Monosynaptic input mapping of diencephalic projections to the cerebrospinal fluid-contacting nucleus in the rat.","volume":"14","author":"Song","year":"","journal-title":"Front. Neuroanat."},{"key":"B26","doi-asserted-by":"publisher","first-page":"11","DOI":"10.3389\/fncir.2020.00011","article-title":"Connection input mapping and 3D reconstruction of the brainstem and spinal cord projections to the CSF-contacting nucleus.","volume":"14","author":"Song","year":"","journal-title":"Front. Neural Circuits"},{"key":"B27","doi-asserted-by":"publisher","first-page":"1638","DOI":"10.1007\/s12264-023-01084-z","article-title":"Genetic architecture and functional implications of the CSF-contacting nucleus.","volume":"39","author":"Song","year":"2023","journal-title":"Neurosci. Bull."},{"key":"B28","doi-asserted-by":"publisher","first-page":"600555","DOI":"10.3389\/fnana.2020.600555","article-title":"The CSF-contacting nucleus receives anatomical inputs from the cerebral cortex: A Combination of retrograde tracing and 3D reconstruction study in rat.","volume":"14","author":"Song","year":"","journal-title":"Front. Neuroanat."},{"key":"B29","doi-asserted-by":"publisher","first-page":"57","DOI":"10.3389\/fnana.2020.00057","article-title":"Novel Projections to the cerebrospinal fluid-contacting nucleus from the subcortex and limbic system in rat.","volume":"14","author":"Song","year":"","journal-title":"Front. Neuroanat."},{"key":"B30","doi-asserted-by":"publisher","first-page":"53","DOI":"10.3389\/fnana.2020.00053","article-title":"A special cranial nucleus (CSF-Contacting nucleus) in primates.","volume":"14","author":"Song","year":"","journal-title":"Front. Neuroanat."},{"key":"B31","doi-asserted-by":"publisher","first-page":"377","DOI":"10.1038\/nmeth.1315","article-title":"mRNA-Seq whole-transcriptome analysis of a single cell.","volume":"6","author":"Tang","year":"2009","journal-title":"Nat. Methods"},{"key":"B32","doi-asserted-by":"publisher","first-page":"108350","DOI":"10.1016\/j.neuropharm.2020.108350","article-title":"NPY alterations induced by chronic morphine exposure affect the maintenance and reinstatement of morphine conditioned place preference.","volume":"181","author":"Wang","year":"2020","journal-title":"Neuropharmacology"},{"key":"B33","doi-asserted-by":"publisher","first-page":"1979","DOI":"10.1093\/brain\/awad031","article-title":"Cerebrospinal fluid immunoglobulins in primary progressive multiple sclerosis are pathogenic.","volume":"146","author":"Wong","year":"2023","journal-title":"Brain"},{"key":"B34","doi-asserted-by":"publisher","first-page":"291","DOI":"10.1016\/j.physbeh.2015.04.034","article-title":"Role of cerebrospinal fluid-contacting nucleus in sodium sensing and sodium appetite.","volume":"147","author":"Xing","year":"2015","journal-title":"Physiol. Behav."},{"key":"B35","doi-asserted-by":"publisher","first-page":"148168","DOI":"10.1016\/j.brainres.2022.148168","article-title":"Cerebrospinal fluid contacting nucleus and its 5-HT: A new insight into the regulation mechanism of general intravenous anesthesia.","volume":"1798","author":"Zhai","year":"2023","journal-title":"Brain Res."},{"key":"B36","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/s0006-8993(03)03123-8","article-title":"The distributions and signaling directions of the cerebrospinal fluid contacting neurons in the parenchyma of a rat brain.","volume":"989","author":"Zhang","year":"2003","journal-title":"Brain Res."},{"key":"B37","doi-asserted-by":"publisher","first-page":"811585","DOI":"10.3389\/fcell.2021.811585","article-title":"Identification of diagnostic markers for breast cancer based on differential gene expression and pathway network.","volume":"9","author":"Zhang","year":"2021","journal-title":"Front. Cell. Dev. Biol."},{"key":"B38","doi-asserted-by":"publisher","first-page":"576","DOI":"10.1007\/s12264-013-1332-0","article-title":"Evaluation of three tracers for labeling distal cerebrospinal fluid-contacting neurons","volume":"29","author":"Zhou","year":"2013","journal-title":"Neurosci Bull"}],"container-title":["Frontiers in Neuroscience"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fnins.2025.1603486\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,6,16]],"date-time":"2025-06-16T06:17:06Z","timestamp":1750054626000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fnins.2025.1603486\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,6,16]]},"references-count":38,"alternative-id":["10.3389\/fnins.2025.1603486"],"URL":"https:\/\/doi.org\/10.3389\/fnins.2025.1603486","relation":{"has-part":[{"id-type":"doi","id":"https:\/\/doi.org\/10.3389\/fnins.2025.1603486.s001","asserted-by":"subject"}]},"ISSN":["1662-453X"],"issn-type":[{"value":"1662-453X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,6,16]]},"article-number":"1603486"}}