GABA excitatory actions in cerebrospinal-fluid contacting neurones of adult mouse spinal cord

Abstract 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 functional relevance of their incomplete maturation remains unknown. Neuronal maturation is classically associated with the expression of the K + -Cl - cotransporter 2 (KCC2), allowing chloride (Cl - ) extrusion and hyperpolarising GABA transmission. Here, we show no detectable expression of KCC2 in CSF-cNs of adult mouse spinal cord. Accordingly, 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 recordings, we found that activation of ionotropic GABA A receptors induced a dominant depolarising effect in 70% of CSF-cNs recorded with intact intracellular chloride concentration. Moreover, in these cells, depolarising GABA-responses can drive action potentials as well as intracellular calcium elevations by activating voltage-gated calcium channels. CSF-cNs express the Na + -K + -Cl - cotransporter 1 (NKCC1) involved in Cl - uptake and its inhibition by bumetanide blocked the GABA-induced calcium transients in CSF-cNs. Finally, we show that activation of metabotropic GABA B receptors did not mediate hyperpolarisation in spinal CSF-cNs, presumably due to the lack of expression of G protein-coupled inwardly rectifying potassium (GIRK) channels. Together, these findings outline CSF-cNs as a unique neuronal population in adult spinal cord with immature Cl - homeostasis and no hyperpolarising GABAergic signalling but rather generation of excitation and intracellular calcium modulation. GABA may therefore promote CSF-cNs maturation and integration into the existing spinal circuit. Key points CSF contacting neurones (CSF-cNs) are located around the central canal of spinal cord across all vertebrates. CSF-cNs express canonical markers of immature neurons during adulthood in mice but the impact of such persistent immaturity on their chloride (Cl - ) homeostasis as well as GABAergic signalling were not addressed yet. Here, we show that spinal CSF-cNs express the Na + -K + -Cl - cotransporter 1 (NKCC1) involved in Cl - uptake but not the K + -Cl - cotransporter 2 (KCC2) classically allowing Cl - extrusion. As a result of intracellular Cl - accumulation, GABA does not mediate inhibition in most CSF-cNs but rather excitation and intracellular Ca 2+ elevations through the activation of voltage-gated Ca 2+ channels. Excitatory GABAergic signalling associated with intracellular calcium modulation may underlie the maturation and integration of CSF-cNs into the spinal circuit of adult mice.