Evidence for Two Subpopulations of Cerebrospinal Fluid-Contacting Neurons with Opposite GABAergic Signaling in Adult...

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 GABA_A receptors (GABA_A-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 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-R signaling. Excitatory GABA may promote the maturation and integration of young CSF-cNs into the existing spinal circuit.