TROUSLARD, J. | Search bioRxiv

bioRxiv2022

GABA excitatory actions in cerebrospinal-fluid contacting neurones of 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 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 GABAA 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 GABAB 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 pointsO_LICSF contacting neurones (CSF-cNs) are located around the central canal of spinal cord across all vertebrates. C_LIO_LICSF-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. C_LIO_LIHere, 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. C_LIO_LIAs a result of intracellular Cl- accumulation, GABA does not mediate inhibition in most CSF-cNs but rather excitation and intracellular Ca2+ elevations through the activation of voltage-gated Ca2+ channels. C_LIO_LIExcitatory GABAergic signalling associated with intracellular calcium modulation may underlie the maturation and integration of CSF-cNs into the spinal circuit of adult mice. C_LI

neuroscience↗

bioRxiv2021

Evidence for PKD2L1-positive neurons present distant from the central canal in the ventromedial spinal cord and Medulla of the adult mouse

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, characterized by an immature neuronal phenotype and selectively express PKD2L1, a channel member of the TRP channel superfamily with properties of sensory receptor. Using immunohistological techniques in mice, we characterize a new population of PKD2L1 positive cells that is observed around embryonic day 16 (E16), is present distant from the CC in a zone enriched with astrocytes and ependymal fibers of the ventro-medial spinal cord and medulla. With development, their number appears stable although smaller than that of CSF-cNs and they progressively become more distant from the CC with the reorganization of the CC region. These neurons share both functional and phenotypical properties with CSF-cNs, but they appear subdivided in two groups. One, present along the midline, has a bipolar morphology and extend 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.

neuroscience↗