Evidence for two subpopulations of cerebrospinal-fluid contacting neurons with opposite GABAergic signaling in adult mouse spinal cord

Spinal cerebrospinal fluid-contacting neurons (CSF-cNs) form an evolutionary conserved bipolar cells 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 remain 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 chloride concentration, we found that activation of ionotropic GABA A receptors 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 GABA B 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 GABA A receptors signaling. Excitatory GABA may promote maturation and integration of young CSF-cNs into the existing spinal circuit. Significant Statement Spinal CSF-contacting neurons (CSF-cNs) form a heterogeneous neural population with distinct maturation states in adult mice, but whether this reflects CSF-cNs with different GABAergic signaling remains unknown. Herein, we show that activation of GABA A receptors generates depolarization or hyperpolarization of CSF-cNs membrane potential in adult mouse spinal cord. Depolarizing GABA can trigger intracellular Ca 2+ elevations through the activation of voltage-gated Ca 2+ channels. Our results highlight a subpopulation of CSF-cNs in adult mice with depolarizing GABA that may promote their maturation and integration into the spinal cord.