The genetic architecture of the CSF-contacting nucleus in mice and the screening of its key target via CSF pathway for mediating neuropathic pain

Abstract

The cerebrospinal fluid (CSF)-contacting nucleus has been well characterized in rats and macaques but not yet precisely defined in mice. This study aims to provide a comprehensive description of the location, adjacent structures, cell morphology, spatial imaging, and genetic architecture of the mouse CSF-contacting nucleus; Previous work has shown that knockout of the rat CSF-contacting nucleus elevates pain sensitivity; accordingly, we further verify certain key molecular substances within the mouse CSF-contacting nucleus may mediate neuropathic pain (NP) via the CSF pathway. Cholera toxin subunit B, Alexa Fluor™ 488/594 Conjugate (CB-AF488/594) or recombinant adeno-associated virus (rAAV) was unilaterally injected into the lateral ventricle (LV) of C57BL/6J mice for specific labeling of the CSF-contacting nucleus. Three-dimensional imaging of the nucleus was obtained using fMOST technology, followed by accurate isolation via laser capture microdissection (LCM) and gene expression profiling using Smart-seq2. Differentially expressed genes (DEGs) between normal mice and NP model mice were subjected to GO and KEGG functional enrichment analysis, and key regulatory genes were identified via bioinformatics and molecular techniques (immunofluorescence, qPCR, Western blot). The mouse CSF-contacting nucleus extends continuously from the ventral periaqueductal gray (vPAG) of the inferior midbrain to the central gray of the fourth ventricle (4 V) floor in the superior pons. fMOST reveals a rivet-like shape. In NP mice, 284 shared-genes were detected between the dorsal root ganglion (DRG) and the CSF-contacting nucleus, 190 of which showed strong interactions. Among seven highly interconnected clusters, chemokine C-C receptor 5 ( Ccr5 ) in cluster 2 had the highest MCODE score. Molecular assays confirmed its significant upregulation in the nucleus versus controls, and intranuclear injection of maraviroc (a CCR5 inhibitor) markedly attenuated NP-induced nociceptive behaviors. Unilateral injection of CB-AF488/594 or rAAV into the LV specifically labels the mouse CSF-contacting nucleus, whose anatomy and cell biology are nearly identical to those of rats and macaques. The mouse CSF-contacting nucleus possesses a unique genetic profile, and CCR5 is a key target for its pain regulation, inhibiting CCR5 may be a potential strategy for pain treatment via CSF pathway.