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.

脊髓脑脊液接触神经元（CSF-cN）形成了位于所有脊椎动物中央管周围的进化保守的双极细胞群。 CSF-cNs 显示出表达神经元不成熟至成年的分子标记；然而，它们的不完全成熟对氯 (Cl ^– ) 稳态以及 GABA 信号传导的影响仍然未知。使用来自两性的成年小鼠，原位杂交显示，一定比例的脊髓 CSF-cN (18.3%) 表达 Na ⁺ -K ⁺ -Cl ^-协同转运蛋白 1 (NKCC1)，允许细胞内 Cl ^-积累。然而，我们没有在任何 CSF-cN 中发现负责 Cl ^-流出的 K ⁺ -Cl ^-协同转运蛋白 2 (KCC2) 的表达。 KCC2 表达的缺乏导致全细胞膜片钳中高^Cl-负载下 CSF-cN 中^Cl-挤出能力低。使用细胞贴附的膜片钳可以记录完整的细胞内 Cl ^-浓度，我们发现离子型 GABA _A受体 (GABA _A -R) 的激活会诱导 CSF-cN 中的去极化和超极化反应。此外，去极化 GABA 反应可以通过激活电压门控钙通道来驱动动作电位以及细胞内钙升高。用布美他尼阻断 NKCC1 可抑制 CSF-cN 中 GABA 诱导的钙瞬变。 最后，我们发现代谢型 GABA _B受体对脊髓 CSF-cN 没有超极化作用，因为巴氯芬激活它们不会介导外向 K ⁺电流，可能是由于缺乏 G 蛋白偶联内向整流钾 (GIRK) 的表达渠道。总之，这些发现概述了表达抑制性或兴奋性 GABA _A -R 信号传导的脊髓 CSF-cN 亚群。兴奋性 GABA 可能促进年轻 CSF-cN 的成熟并整合到现有的脊髓回路中。