Endogenous opioid signalling regulates spinal ependymal cell proliferation

After injury, mammalian spinal cords develop scars to confine the lesion and prevent further damage. However, excessive scarring can hinder neural regeneration and functional recovery. These competing actions underscore the importance of developing therapeutic strategies to dynamically modulate scar progression. Previous research on scarring has primarily focused on astrocytes, but recent evidence has suggested that ependymal cells also participate. Ependymal cells normally form the epithelial layer encasing the central canal, but they undergo massive proliferation and differentiation into astroglia following certain injuries, becoming a core scar component. However, the mechanisms regulating ependymal proliferation in vivo remain unclear. Here we uncover an endogenous ?-opioid signalling pathway that controls ependymal proliferation. Specifically, we detect expression of the ?-opioid receptor, OPRK1, in a functionally under-characterized cell type known as cerebrospinal fluid-contacting neuron (CSF-cN). We also discover a neighbouring cell population that expresses the cognate ligand prodynorphin (PDYN). Whereas ?-opioids are typically considered inhibitory, they excite CSF-cNs to inhibit ependymal proliferation. Systemic administration of a ?-antagonist enhances ependymal proliferation in uninjured spinal cords in a CSF-cN-dependent manner. Moreover, a ?-agonist impairs ependymal proliferation, scar formation and motor function following injury. Together, our data suggest a paracrine signalling pathway in which PDYN+ cells tonically release ?-opioids to stimulate CSF-cNs and suppress ependymal proliferation, revealing an endogenous mechanism and potential pharmacological strategy for modulating scarring after spinal cord injury. Overall design: We characterized the transcriptomic profile of cerebrospinal fluid-contacting neurons (CSF-cNs) in the mouse spinal cord to study their physiological role. We expressed tdTomato in CSF-cNs of 4 wildtype C57BL6/J mice by intracerebroventricular injection of AAV2-CAG-tdT. Sagittal slices of the spinal cord containing the ependymal region were enzymatically dissociated and tdTomato+ cells were captured by fluorescence-activated cell sorting (FACS). Each sample contains 50 cells collected from a single mouse.