Cerebrospinal fluid-contacting neurons (CSF-cNs) represent a distinct group of interneurons characterized by their prominent apical globular protrusions penetrating the spinal cord\'s central canal and their basal axons extending towards adjacent cells. Identified nearly a century back, the specific roles and attributes of CSF-cNs have just started to emerge due to the historical lack of definitive markers. Recent findings have confirmed that CSF-cNs expressing PKD2L1 possess attributes of neural stem cells, suggesting a critical function in the regeneration processes following spinal cord injuries. This review aims to elucidate the molecular markers of CSF-cNs as potential neural stem cells during spinal cord development and assess their roles post-spinal cord injury, with an emphasis on their potential therapeutic implications for spinal cord repair.

Spinal cerebrospinal fluid-contacting neurons (CSF-cNs) form an evolutionary conserved bipolar cell population localized around the central canal of all vertebrates. CSF-cNs were shown to express molecular markers of neuronal immaturity into adulthood; however, the impact of their incomplete maturation on the chloride (Cl-) homeostasis as well as GABAergic signaling remains unknown. Using adult mice from both sexes, in situ hybridization revealed that a proportion of spinal CSF-cNs (18.3%) express the Na+-K+-Cl- cotransporter 1 (NKCC1) allowing intracellular Cl- accumulation. However, we did not find expression of the K+-Cl- cotransporter 2 (KCC2) responsible for Cl- efflux in any CSF-cNs. The lack of KCC2 expression results in low Cl- extrusion capacity in CSF-cNs under high Cl- load in whole-cell patch clamp. Using cell-attached patch clamp allowing recordings with intact intracellular Cl- concentration, we found that the activation of ionotropic GABAA receptors (GABAA-Rs) induced both depolarizing and hyperpolarizing responses in CSF-cNs. Moreover, depolarizing GABA responses can drive action potentials as well as intracellular calcium elevations by activating voltage-gated calcium channels. Blocking NKCC1 with bumetanide inhibited the GABA-induced calcium transients in CSF-cNs. Finally, we show that metabotropic GABAB receptors have no hyperpolarizing action on spinal CSF-cNs as their activation with baclofen did not mediate outward K+ currents, presumably due to the lack of expression of G-protein-coupled inwardly rectifying potassium (GIRK) channels. Together, these findings outline subpopulations of spinal CSF-cNs expressing inhibitory or excitatory GABAA-R signaling. Excitatory GABA may promote the maturation and integration of young CSF-cNs into the existing spinal circuit.

The Reissner fiber (RF) is an acellular thread positioned in the midline of the central canal that aggregates thanks to the beating of numerous cilia from ependymal radial glial cells (ERGs) generating flow in the central canal of the spinal cord. RF together with cerebrospinal fluid (CSF)-contacting neurons (CSF-cNs) form an axial sensory system detecting curvature. How RF, CSF-cNs and the multitude of motile cilia from ERGs interact in vivo appears critical for maintenance of RF and sensory functions of CSF-cNs to keep a straight body axis, but is not well-understood. Using in vivo imaging in larval zebrafish, we show that RF is under tension and resonates dorsoventrally. Focal RF ablations trigger retraction and relaxation of the fiber\'s cut ends, with larger retraction speeds for rostral ablations. We built a mechanical model that estimates RF stress diffusion coefficient D at 5 mm2/s and reveals that tension builds up rostrally along the fiber. After RF ablation, spontaneous CSF-cN activity decreased and ciliary motility changed, suggesting physical interactions between RF and cilia projecting into the central canal. We observed that motile cilia were caudally-tilted and frequently interacted with RF. We propose that the numerous ependymal motile monocilia contribute to RF\'s heterogenous tension via weak interactions. Our work demonstrates that under tension, the Reissner fiber dynamically interacts with motile cilia generating CSF flow and spinal sensory neurons.

Cerebrospinal fluid-contacting neurons (CSF-cNs) act crucial role in chemosensory and mechanosensory function in spinal cord. Recently, CSF-cNs were found to be an immature neuron and may be involved in spinal cord injury recovery. But how to culture it and explore its function in vitro are not reported in previous research. Here, we first reported culture and identification of CSF-cNs in vitro. We first established a protocol for in vitro culture of CSF-cNs from the cervical spinal cord of mice within 24 h after birth. Polycystic kidney disease 2-like 1 (PKD2L1)+ cells were isolated by fluorescence-activated cell sorting and expressed the neuron marker β-tubulin III and CSF-cNs marker GABA. Intriguingly, PKD2L1+ cells formed neurosphere and expressed neural stem cell markers Nestin, Sox2 and GFAP. Thus, our research provided culture and isolation of CSF-cNs and this facilitate the investigation the CSF-cNs function in vitro.

Sensory neurons previously shown to optimize speed and balance in fish by providing information about the curvature of the spine show similar morphology and connectivity in mice.

The neural stem cells (NSCs) in the ventricular-subventricular zone of the adult mammalian spinal cord may be of great benefit for repairing spinal cord injuries. However, the sources of NSCs remain unclear. Previously, we have confirmed that cerebrospinal fluid-contacting neurons (CSF-cNs) have NSC potential in vitro. In this study, we verified the NSC properties of CSF-cNs in vivo. In mouse spinal cords, Pkd2l1+ CSF-cNs localized around the central canal express NSC markers. In vitro, Pkd2l1+ CSF-cNs form a neurosphere and express NSC markers. Activation and proliferation of CSF-cNs can be induced by injection of the neurotrophic factors basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) into the lateral ventricle. Spinal cord injury (SCI) also induces NSC activation and proliferation of CSF-cNs. Collectively, our results demonstrate that Pkd2l1+ CSF-cNs have NSC properties in vivo and may be involved in SCI recovery.

From swimming to walking and flying, animals have evolved specific locomotor strategies to thrive in different habitats. All types of locomotion depend on the integration of motor commands and sensory information to generate precisely coordinated movements. Cerebrospinal-fluid-contacting neurons (CSF-cN) constitute a vertebrate sensory system that monitors CSF composition and flow. In fish, CSF-cN modulate swimming activity in response to changes in pH and bending of the spinal cord; however, their role in mammals remains unknown. We used mouse genetics to study their function in quadrupedal locomotion. We found that CSF-cN are directly integrated into spinal motor circuits. The perturbation of CSF-cN function does not affect general motor activity nor the generation of locomotor rhythm and pattern but results in specific defects in skilled movements. These results identify a role for mouse CSF-cN in adaptive motor control and indicate that this sensory system evolved a novel function to accommodate the biomechanical requirements of limb-based locomotion.

In the spinal cord, cerebrospinal fluid-contacting neurons (CSF-cNs) are GABAergic interoceptive sensory neurons that detect spinal curvature via a functional coupling with the Reissner fiber. This mechanosensory system has recently been found to be involved in spine morphogenesis and postural control but the underlying mechanisms are not fully understood. In zebrafish, CSF-cNs project an ascending and ipsilateral axon reaching two to six segments away. Rostralmost CSF-cNs send their axons ipsilaterally into the hindbrain, a brain region containing motor nuclei and reticulospinal neurons (RSNs), which send descending motor commands to spinal circuits. Until now, the synaptic connectivity of CSF-cNs has only been investigated in the spinal cord, where they synapse onto motor neurons and premotor excitatory interneurons. The identity of CSF-cN targets in the hindbrain and the behavioral relevance of these sensory projections from the spinal cord to the hindbrain are unknown. Here, we provide anatomical and molecular evidence that rostralmost CSF-cNs synapse onto the axons of large RSNs including Mauthner cells and V2a neurons. Functional anatomy and optogenetically assisted mapping reveal that rostral CSF-cNs also synapse onto the soma and dendrites of cranial motor neurons innervating hypobranchial muscles. During acousto-vestibular evoked escape responses, ablation of rostralmost CSF-cNs results in a weaker escape response with a decreased C-bend amplitude, lower speed, and deficient postural control. Our study demonstrates that spinal sensory feedback enhances speed and stabilizes posture, and reveals a novel spinal gating mechanism acting on the output of descending commands sent from the hindbrain to the spinal cord.

Neurons in contact with the cerebrospinal fluid (CSF) are found around the medullo-spinal central canal (CC) in adult mice. These neurons (CSF-cNs), located within or below the ependymal cell layer, known as the stem cell niche, present a characteristic morphology with a dendrite projecting to the CC and ending with a protrusion. They are GABAergic, present an intermediate neuronal maturity and selectively express PKD2L1, a member of the transient receptor potential channel superfamily with sensory properties. Using immunohistological and electrophysiological recording techniques in mice, we characterize the properties of a new population of PKD2L1 positive cells that is distant from the CC in a zone enriched with astrocytes and ependymal fibers of the ventro-medial spinal cord and medulla. They appear around embryonic day 16 and their number increases up to early postnatal days. With development and the reorganization of the CC region, they progressively become more distant from the CC, suggesting some migratory capabilities. These neurons share functional and phenotypical properties with CSF-cNs but appear subdivided in two groups. One group, present along the midline, has a bipolar morphology and extends a long dendrite along ependymal fibers and towards the CC. The second group, localized in more ventro-lateral regions, has a multipolar morphology and no apparent projection to the CC. Altogether, we describe a novel population of PKD2L1+ neurons distant from the CC but with properties similar to CSF-cNs that might serve to sense modification in the composition of either CSF or interstitial liquid, a function that will need to be confirmed.

Cerebrospinal fluid-touching neurons (CSF-cNs) exist in the region surrounding the central canal of the spinal cord, which locate in the adult neurogenic niche. Previous research showed that CSF-cNs expressed the molecular markers of immature neural cells in vivo. Here, we explored the potential of CSF-cNs as neural stem cell in intro. We first found that PKD2L1+ CSF-cNs, isolating by FACS using the molecular marker PKD2L1 of CSF-cNs, expressed neural stem cells markers like Nestin, Sox2, and GFAP by immunofluorescence staining. PKD2L1+ CSF-cNs were able to form neurospheres and passaged in vitro. Immunofluorescence staining showed that the neurospheres forming by PKD2L1+ CSF-cNs also expressed neural stem cell markers Nestin, Sox2 and GFAP. The neurospheres expressed proliferation markers Ki67 and PCNA by immunofluorescence staining, indicating that the neurospheres forming by PKD2L1+ CSF-cNs were proliferative. The neurospheres, forming by CSF-cNs, had the ability of differentiation into neurons, astrocytes, and oligodendrocytes. Collectively, our data suggested that PKD2L1+ CSF-cNs have the properties of neural stem cells in vitro and may provide a promising approach for the repair of spinal cord injury.