Pkd2l1 is required for mechanoception in cerebrospinal fluid-contacting neurons and maintenance of spine curvature

Defects in cerebrospinal fluid (CSF) flow may contribute to idiopathic scoliosis. However, the mechanisms underlying detection of CSF flow in the central canal of the spinal cord are unknown. Here we demonstrate that CSF flows bidirectionally along the antero-posterior axis in the central canal of zebrafish embryos. In the cfap298tm304 mutant, reduction of cilia motility slows transport posteriorly down the central canal and abolishes spontaneous activity of CSF-contacting neurons (CSF-cNs). Loss of the sensory Pkd2l1 channel nearly abolishes CSF-cN calcium activity and single channel opening. Recording from isolated CSFcNs in vitro, we show that CSF-cNs are mechanosensory and require Pkd2l1 to respond to pressure. Additionally, adult pkd2l1 mutant zebrafish develop an exaggerated spine curvature, reminiscent of kyphosis in humans. These results indicate that CSF-cNs are mechanosensory cells whose Pkd2l1-driven spontaneous activity reflects CSF flow in vivo. Furthermore, Pkd2l1 in CSF-cNs contributes to maintenance of natural curvature of the spine

State-Dependent Modulation of Locomotion by GABAergic Spinal Sensory Neurons

International audienceThe cerebrospinal fluid (CSF) constitutes an interface through which chemical cues can reach and modulate the activity of neurons located at the epithelial boundary within the entire nervous system. Here, we investigate the role and functional connectivity of a class of GABAergic sensory neurons contacting the CSF in the vertebrate spinal cord and referred to as CSF-cNs. The remote activation of CSF-cNs was shown to trigger delayed slow locomotion in the zebrafish larva, suggesting that these cells modulate components of locomotor central pattern generators (CPGs). Combining anatomy, electrophysiology, and optogenetics in vivo, we show that CSF-cNs form active GABAergic synapses onto V0-v glutamatergic interneurons, an essential component of locomotor CPGs. We confirmed that activating CSF-cNs at rest induced delayed slow locomotion in the fictive preparation. In contrast, the activation of CSF-cNs promptly inhibited ongoing slow locomotion. Moreover, selective activation of rostral CSF-cNs during ongoing activity disrupted rostrocaudal propagation of descending excitation along the spinal cord, indicating that CSF-cNs primarily act at the premotor level. Altogether, our results demonstrate how a spinal GABAergic sensory neuron can tune the excitability of locomotor CPGs in a state-dependent manner by projecting onto essential components of the excitatory premotor pool

State-Dependent Modulation of Locomotion by GABAergic Spinal Sensory Neurons

International audienceThe cerebrospinal fluid (CSF) constitutes an interface through which chemical cues can reach and modulate the activity of neurons located at the epithelial boundary within the entire nervous system. Here, we investigate the role and functional connectivity of a class of GABAergic sensory neurons contacting the CSF in the vertebrate spinal cord and referred to as CSF-cNs. The remote activation of CSF-cNs was shown to trigger delayed slow locomotion in the zebrafish larva, suggesting that these cells modulate components of locomotor central pattern generators (CPGs). Combining anatomy, electrophysiology, and optogenetics in vivo, we show that CSF-cNs form active GABAergic synapses onto V0-v glutamatergic interneurons, an essential component of locomotor CPGs. We confirmed that activating CSF-cNs at rest induced delayed slow locomotion in the fictive preparation. In contrast, the activation of CSF-cNs promptly inhibited ongoing slow locomotion. Moreover, selective activation of rostral CSF-cNs during ongoing activity disrupted rostrocaudal propagation of descending excitation along the spinal cord, indicating that CSF-cNs primarily act at the premotor level. Altogether, our results demonstrate how a spinal GABAergic sensory neuron can tune the excitability of locomotor CPGs in a state-dependent manner by projecting onto essential components of the excitatory premotor pool

Comparative Distribution and In Vitro Activities of the Urotensin II-Related Peptides URP1 and URP2 in Zebrafish: Evidence for Their Colocalization in Spinal Cerebrospinal Fluid-Contacting Neurons

International audienceUrotensin II (UII) is an evolutionarily conserved neuropeptide initially isolated from teleost fish on the basis of its smooth muscle-contracting activity. Subsequent studies have demonstrated the occurrence of several UII-related peptides (URPs), such that the UII family is now known to include four paralogue genes called UII, URP, URP1 and URP2. These genes probably arose through the two rounds of whole genome duplication that occurred during early vertebrate evolution. URP has been identified both in tetrapods and teleosts. In contrast, URP1 and URP2 have only been observed in ray-finned and cartilaginous fishes, suggesting that both genes were lost in the tetrapod lineage. In the present study, the distribution of urp1 mRNA compared to urp2 mRNA is reported in the central nervous system of zebrafish. In the spinal cord, urp1 and urp2 mRNAs were mainly colocalized in the same cells. These cells were also shown to be GABAergic and express the gene encoding the polycystic kidney disease 2-like 1 (pkd2l1) channel, indicating that they likely correspond to cerebrospinal fluid-contacting neurons. In the hindbrain, urp1-expressing cells were found in the intermediate reticular formation and the glossopharyngeal-vagal motor nerve nuclei. We also showed that synthetic URP1 and URP2 were able to induce intracellular calcium mobilization in human UII receptor (hUT)-transfected CHO cells with similar potencies (pEC50=7.99 and 7.52, respectively) albeit at slightly lower potencies than human UII and mammalian URP (pEC50=9.44 and 8.61, respectively). The functional redundancy of URP1 and URP2 as well as the colocalization of their mRNAs in the spinal cord suggest the robustness of this peptidic system and its physiological importance in zebrafish

Investigation of spinal cerebrospinal fluid-contacting neurons expressing PKD2L1: evidence for a conserved system from fish to primates

International audienceOver 90 years ago, Kolmer and Agduhr identified spinal cerebrospinal fluid-contacting neurons (CSF-cNs) based on their morphology and location within the spinal cord. In more than 200 vertebrate species, they observed ciliated neurons around the central canal that extended a brush of microvilli into the cerebrospinal fluid (CSF). Although their morphology is suggestive of a primitive sensory cell, their function within the vertebrate spinal cord remains unknown. The identification of specific molecular markers for these neurons in vertebrates would benefit the investigation of their physiological roles. PKD2L1, a transient receptor potential channel that could play a role as a sensory receptor, has been found in cells contacting the central canal in mouse. In this study, we demonstrate that PKD2L1 is a specific marker for CSF-cNs in the spinal cord of mouse (Mus musculus), macaque (Macaca fascicularis) and zebrafish (Danio rerio). In these species, the somata of spinal PKD2L1 + CSF-cNs were located below or within the ependymal layer and extended an apical bulbous extension into the central canal. We found GABAergic PKD2L1-expressing CSF-cNs in all three species. We took advantage of the zebrafish embryo for its transparency and rapid development to identify the progenitor domains from which pkd2l1 + CSF-cNs originate. pkd2l1 + CSF-cNs were all GABAergic and organized in two rows—one ventral and one dorsal to the central canal. Their location and marker expression is consistent with previously described Kolmer–Agduhr cells. Accordingly, pkd2l1 + CSF-cNs were derived from the progenitor domains p3 and pMN defined by the expression of nkx2.2a and olig2 transcription factors, respectively. Altogether our results suggest that a system of CSF-cNs expressing the PKD2L1 channel is conserved in the spinal cord across bony vertebrate species

<i>urp1</i> mRNA occurs in cells located along the ventral edge of the central canal of spinal cord in adult zebrafish.

<p>Expression of <i>urp1</i> revealed by ISH (BM purple, violet) on free-floating sections of adult spinal cord. <i>urp1</i>⁺ cells form a quasi-continuous line at the ventral edge of the central canal <b>(A)</b>. <i>urp1</i>⁺ cells are in close contact to the lumen of the central canal (arrowhead) <b>(B)</b>. <b>A1</b> and <b>A2</b>, lateral sections with dorsal up; <b>B</b>, coronal section with dorsal up. <i>urp1</i>⁺ cells boxed in <b>A1</b> are shown in <b>A2</b> at higher magnification. M, melanocytes. Scale bars: 50 μm.</p

Both <i>urp1</i>⁺ and <i>urp2</i>⁺ cells are GABAergic neurons in the zebrafish embryo.

<p><i>urp1</i><b>(A)</b> and <i>urp2</i><b>(B)</b> expression revealed by fluorescent ISH (FITC, green) on 24 hpf-embryo, together with a fluorescent immunostaining for GAD_65/67 (Alexa Fluor 546, red). Both <i>urp1</i>⁺ and <i>urp2</i>⁺ cells are GAD⁺ (arrows). Note that only ventral KA (KA”) cells are doubly stained. In contrast, dorsal KA (KA’) cells are GAD⁺ but do not express <i>urp1</i> (arrowhead). The white dash line indicates the central canal. <b>A</b> and <b>B</b>, coronal sections with dorsal up. Scale bars: 15 μm.</p

<i>urp1</i> and <i>urp2</i> mRNAs are exclusively detected in the brain and spinal cord in adult zebrafish.

<p>Tissue distribution of <i>urp1</i> and <i>urp2</i> mRNAs assessed by RT-PCR. Parallel amplification of zebrafish β-actin mRNA served as internal control. NTC, non-template control.</p

URP1 and URP2 are equipotent to induce intracellular calcium mobilization in a <i>h</i>UT-transfected CHO cell line.

<p>Representative dose-response curves of <i>h</i>UII (●), <i>m</i>URP (■), URP1 (▲) and URP2 (▼) on the intracellular calcium mobilization <b>(A)</b>. The values are expressed as percentages of the baseline and each point is the mean (± S.E.M.) of 3 replicates. Experimental data were fitted using a four-parameter logistic equation. The potencies of 7–13 independent experiments for each peptides were plotted as—Log(EC₅₀) with box and whiskers <b>(B)</b>. Values were considered as statistically different as assessed by analysis of variance followed by Tukey’s post-test, n.s., not significant, *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001, ****<i>p</i> < 0.0001.</p

<i>urp1</i> mRNA is found in the caudal part of the hindbrain in adult zebrafish.

<p>Expression of <i>urp1</i> revealed by fluorescent ISH (FITC, green) on coronal sections of adult brain <b>(A)</b>. <i>urp1</i> mRNA is visible in neurons located in the intermediate reticular formation <b>(A1</b>) and the region of the glossopharyngeal-vagal motor nerve nuclei (<b>A2–A3</b>). More caudally, at the level of the junction between hindbrain and spinal cord, <i>urp1</i> mRNA occurs at the ventral edge of the central canal <b>(A4)</b>. Schematic sagittal view of an adult zebrafish brain depicting the distribution of <i>urp1</i> mRNA (red dots). Levels of sections shown in A are indicated. The anatomical structures are designated according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119290#pone.0119290.ref038" target="_blank">38</a>] <b>(B)</b>. CC, cerebellar crest; C, central canal; CCe, corpus cerebelli; DON, dorsal octavolateralis nucleus; EW, Edinger-Westphal nucleus; FLo, facial lobe; Ha, habenula; H, hypothalamus; IMRF, intermediate reticular formation; MO, medulla oblongata; NC, commissural nucleus of Cajal; nIX-X, glossopharyngeal-vagal motor nerve nuclei; OB, olfactive bulbs; OC, optic chiasma; P, pallium; PN, preopic nucleus; RV, rhombencephalic ventricle; SCsm, spinal cord somatomotor neurons; SP, subpallium; T, thalamus; TO, tectum opticum; TL, torus longitudinalis; TP, posterior tuberculum; TS, torus semicircularis; VLo, vagal lobe. Scale bars: 100 μm.</p