Hervé Kalaimakan

Guillaume Arulkandarajah

Agathe Osterstock

Hervé Lafont

Nathalie Le Corronc

Silvia Escalas

Barbara Corsini

Linda Le Bras

Juliette Chenane

Antonny Boeri

Czarnecki

Kalaimakan Hervé Arulkandarajah

Guillaume Osterstock

Agathe Lafont

Hervé Le Corronc

Nathalie Escalas

Silvia Corsini

Barbara Le Bras

Linda Chenane

Juliette Boeri

Antonny Czarnecki

Christine Mouffle

Erika Bullier

Elim Hong

Cathy Soula

Pascal Legendre

PhD Jean-Marie Mangin
email: jean-marie.mangin@inserm.fr

Neuroepithelial progenitors generate and propagate non-neuronal action potentials across the spinal cord

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Spontaneous neural activity (SNA) is a hallmark of developing neural networks and has been shown to regulate various developmental processes such as neuronal differentiation, migration, axon guidance and synapse formation [START_REF] Moody | Ion channel development, spontaneous activity, and activity-dependent development in nerve and muscle cells[END_REF][START_REF] Spitzer | Electrical activity in early neuronal development[END_REF][START_REF] Blankenship | Mechanisms underlying spontaneous patterned activity in developing neural circuits[END_REF][START_REF] Kirischuk | Modulation of neocortical development by early neuronal activity: Physiology and pathophysiology[END_REF] . Until now, the electrical signals generated during SNA have been found to originate exclusively from differentiating neurons as they acquire the ability to fire action potentials and form their first functional synapses. By contrast, neuroepithelial progenitors (NEPs) -the stem cells of the central nervous systemhave never been reported to actively generate or propagate any form of electrical signals, although they can be passively depolarized and/or exhibit calcium events in response to neurotransmitters released by developing neurons [START_REF] Kirischuk | Modulation of neocortical development by early neuronal activity: Physiology and pathophysiology[END_REF][START_REF] Corlew | Spontaneous, synchronous electrical activity in neonatal mouse cortical neurones[END_REF][START_REF] Vitali | Progenitor Hyperpolarization Regulates the Sequential Generation of Neuronal Subtypes in the Developing Neocortex[END_REF][START_REF] Loturco | GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis[END_REF][START_REF] Noctor | Dividing precursor cells of the embryonic cortical ventricular zone have morphological and molecular characteristics of radial glia[END_REF][START_REF] Weissman | Calcium waves propagate through radial glial cells and modulate proliferation in the developing neocortex[END_REF][START_REF] Rosa | Neuron-glia signaling in developing retina mediated by neurotransmitter spillover[END_REF][START_REF] Wang | Characterization of Rhythmic Ca2+ Transients in Early Embryonic Chick Motoneurons: Ca2+ Sources and Effects of Altered Activation of Transmitter Receptors[END_REF] .

SNA was initially described in the embryonic spinal cord where it triggers the first motor rhythmic behavior [START_REF] Hamburger | Observations and experiments on spontaneous rhythmical behavior in the chick embryo[END_REF][START_REF] Suzue | Highly reproducible spatiotemporal patterns of mammalian embryonic movements at the developmental stage of the earliest spontaneous motility[END_REF] , necessary for the proper development of the neuro-muscular and musculo-skeletal systems of vertebrates [START_REF] Landmesser | General principles of spinal motor circuit development: early contributions from research on avian embryos[END_REF][START_REF] Shwartz | One load to rule them all: Mechanical control of the musculoskeletal system in development and aging[END_REF][START_REF] Hanson | Spontaneous rhythmic activity in early chick spinal cord influences distinct motor axon pathfinding decisions[END_REF] . Spinal SNA starts at midgestation stagesaround the 12 th day of embryonic development in mice (E12.5) -and is generated autonomously by the ventral spinal cord, before the establishment of sensory inputs from the periphery and descending inputs from the brain. Therefore, SNA is fully preserved in the isolated fetal spinal cord [START_REF] Hanson | Characterization of the circuits that generate spontaneous episodes of activity in the early embryonic mouse spinal cord[END_REF][START_REF] Czarnecki | Acetylcholine controls GABA-, glutamate-, and glycine-dependent giant depolarizing potentials that govern spontaneous motoneuron activity at the onset of synaptogenesis in the mouse embryonic spinal cord[END_REF] . Although its underlying molecular and cellular mechanisms have yet to be fully resolved, spinal SNA has been shown to involve spontaneous waves of electrical activity depolarizing cholinergic motoneurons as well as GABAergic and glutamatergic interneurons along the length of the ventral spinal cord [START_REF] Hanson | Spontaneous rhythmic activity in early chick spinal cord influences distinct motor axon pathfinding decisions[END_REF][START_REF] Hanson | Characterization of the circuits that generate spontaneous episodes of activity in the early embryonic mouse spinal cord[END_REF][START_REF] Czarnecki | Acetylcholine controls GABA-, glutamate-, and glycine-dependent giant depolarizing potentials that govern spontaneous motoneuron activity at the onset of synaptogenesis in the mouse embryonic spinal cord[END_REF] . By contrast, the electrophysiological behavior of ventral NEPs remains poorly characterized and they have been assumed to remain electrically passive, as shown in other brain structures. Previous reports indicate that ventral NEPs can exhibit calcium transients during episodes of SNA [START_REF] Wang | Characterization of Rhythmic Ca2+ Transients in Early Embryonic Chick Motoneurons: Ca2+ Sources and Effects of Altered Activation of Transmitter Receptors[END_REF] and that the ventral-most NEPs located in the floor-plate display T-type-like voltage-gated calcium currents [START_REF] Frischknecht | Voltage-and ligand-gated ion channels in floor plate neuroepithelia of the rat[END_REF] . This suggests that spinal NEPs located in the ventral spinal cord could generate active electrical signals during SNA. To investigate this possibility, we used patchclamp, optogenetic stimulation and calcium imaging to elucidate the electrophysiological behavior of ventral NEPs during spinal SNA.

5

RESULTS

We first examined the electrical activity of ventral NEPs using patch-clamp recordings on a preparation of whole spinal cord from E12.5 mouse embryos in the open-book configuration [START_REF] Czarnecki | Acetylcholine controls GABA-, glutamate-, and glycine-dependent giant depolarizing potentials that govern spontaneous motoneuron activity at the onset of synaptogenesis in the mouse embryonic spinal cord[END_REF][START_REF] Osterstock | Axoglial synapses are formed onto pioneer oligodendrocyte precursor cells at the onset of spinal cord gliogenesis[END_REF] . This preparation gave us access to all NEPs lining the central canal while preserving SNA generation (Figure 1A). Five distinct bilateral domains of NEPs are classically identified along the central canal of the ventral spinal cord: p0, p1, p2, pMN and p3 domains (Figure 1A). A sixth unique domain called the floor-plate is located at the base of the spinal cord, joining both sides of the neuroepithelium at the ventral midline. Patch-clamp recordings first revealed that ventral NEPs exhibit spontaneous and rhythmic depolarizations (Figure 1B & 1G) at an interval of 183 ± 38 s (N = 32 cells from 23 embryos), similar to the interval previously reported in cholinergic motoneurons during spinal SNA [START_REF] Czarnecki | Acetylcholine controls GABA-, glutamate-, and glycine-dependent giant depolarizing potentials that govern spontaneous motoneuron activity at the onset of synaptogenesis in the mouse embryonic spinal cord[END_REF] . Strikingly, the amplitude of NEP spontaneous depolarization was not uniform but was inversely correlated to the distance of the recorded NEPs from the ventral midline (Figure 1C-E), with the largest depolarization observed in floor-plate NEPs. To determine whether NEP depolarizations were temporally correlated between these different regions, we performed paired recordings between the floor-plate and the neighboring p3 domain. Some of the recorded pairs were also filled with neurobiotin and their molecular identity assessed by post-hoc immunostaining against the transcription factors FOXA2 and NKX2.2 (Figure 1F), which are respectively expressed in floor-plate NEPs and in the adjacent p3 neuroepithelial progenitor cells [START_REF] Lupo | Mechanisms of ventral patterning in the vertebrate nervous system[END_REF] 1I) when compared to the floor-plate. The results suggested that these rhythmic depolarizations propagated from the floor-plate to other NEP domains through direct electrical coupling. Indeed, neuroepithelial progenitors are known to be extensively coupled by gap-junctions at this developmental stage [START_REF] Bittman | Patterns of cell-cell coupling in embryonic spinal cord studied via ballistic delivery of gap-junctionpermeable dyes[END_REF] . We found that neurobiotin filling via patch pipette often labeled several adjacent cells in the p3 domain (14.7 ± 6.3 cells in p3, N= 6 cells from 6 embryos; Figure 1F) as well as in the floor-plate (6.6 ± 2.9 cells in FP, N = 10 cells from 10 embryos; Figure 1F and2C). Depolarizing and hyperpolarizing current steps injected in the floor-plate NEP were also systematically associated with a proportional, albeit much smaller, depolarization or hyperpolarization in the NEP located in the p3 domain (Ratio [p3/FP] = 4.2 ± 1.5 %, N=14 pairs from 14 embryos), showing that they are electrically coupled (Figure 1J). Conversely, injecting currents in the NEP located in the p3 domain led to depolarization of the floor-plate cell (Ratio [FP/p3] = 4.6 ± 1.7 %, N = 18 pairs from 18 embryos). Electrical coupling was lost after application of the connexin blocker β18-GA (50µM, paired Wilcoxon test, p<0.05; Control Ratio [p3/FP] = 2.9 ± 0.6 % vs β18-GA Ratio [p3/FP] = 0.18 ± 0.07 %, N = 5 pairs from 5 embryos; Figure 1J and 1K) [START_REF] Rozental | How to close a gap junction channel. Efficacies and potencies of uncoupling agents[END_REF] . Taken together, our results indicate that NEP depolarizations observed during episodes of SNA originate from the floor-plate and propagate dorsally into the rest of the neuroepithelium, most likely through gap-junctions.

To understand how floor-plate NEPs could exhibit such large and rhythmic depolarizations, we investigated their electrophysiological properties in more detail. Floorplate cells are easily discriminated from surrounding NEPs by their location at the midline, their thin apical processes and their higher translucency. Identity of the recorded cells were also confirmed by post-hoc immunostaining against the transcription factors FoxA2 and Nkx2.2. All floor-plate cells filled by neurobiotin were found to be FoxA2 + and Nkx2.2 -(N= 10/10 cells from 10 embryos; Figure 1F and2C). Unlike other NEPs that sequentially generate neurons and glial cells during embryonic development [START_REF] Rowitch | Glial specification in the vertebrate neural tube[END_REF] , floor-plate NEPs display glial features at an early embryonic stage and have only been shown to generate ependymocytes [START_REF] Deneen | The Transcription Factor NFIA Controls the Onset of Gliogenesis in the Developing Spinal Cord[END_REF][START_REF] Barry | Differentiation of radial glia from radial precursor cells and transformation into astrocytes in the developing rat spinal cord[END_REF][START_REF] Cañizares | Multiple steps characterise ventricular layer attrition to form the ependymal cell lining of the adult mouse spinal cord central canal[END_REF][START_REF] Khazanov | Floor plate descendants in the ependyma of the adult mouse Central Nervous System[END_REF][START_REF] Mirzadeh | Bi-and uniciliated ependymal cells define continuous floor-plate-derived tanycytic territories[END_REF] . Indeed, we found that all FoxA2 + /Nkx2.2 -cells co-expressed the glutamate astrocyte transporter GLAST, a marker for glial progenitors (Figure S1A-H exhibited a complex and stereotypical time course (Figure 2E). These spontaneous events comprised a small initial depolarization (amplitude = 6.9 ± 1.0 mV; half-width = 2.8 ± 1.0 s) followed by an AP exhibiting a biphasic time course comprising first a slow component (amplitude = 18.9 ± 5.5 mV; half-width = 274 ± 57 ms) followed by a fast component (amplitude = 42.4 ± 7.2 mV; half-width = 11.8 ± 3.2 ms; Figure 2E). Identical events could be evoked using electrical stimulation (32 mA, 200 µs) placed on the midline, 2 mm rostral to the site of recording (Figure 2F). Application of the T-type calcium channel blocker TTA-P2 was sufficient to fully block the biphasic action potential, while the small initial depolarization remained unaffected (N=8/8 cells from 8 embryos). To confirm and further characterize the expression of T-type calcium channels in the floor-plate, we performed in situ hybridization for the 3 known T-type calcium channels (cacna1g, cacna1h, cacna1i). We found that mRNA coding for T-type calcium channels were all highly and predominantly expressed in the floor-plate (Figure 2G &2H).

Since SNA is dependent on acetylcholine released from motoneurons [START_REF] Hanson | Characterization of the circuits that generate spontaneous episodes of activity in the early embryonic mouse spinal cord[END_REF][START_REF] Czarnecki | Acetylcholine controls GABA-, glutamate-, and glycine-dependent giant depolarizing potentials that govern spontaneous motoneuron activity at the onset of synaptogenesis in the mouse embryonic spinal cord[END_REF] , we investigated whether rhythmic floor-plate action potentials were triggered by this neurotransmitter. First, we found that spontaneous rhythmic depolarizations disappeared (N=5/5 cells from 5 embryos; Figure 3A) in the presence of the nicotinic acetylcholine receptor (nAChRs) antagonists mecamylamine (50 µM) and d-Tubocurarine (5µM). Evoked floor-plate depolarizations were also completely blocked by the same nAChR antagonists (paired Wilcoxon test, p<0.01; N=12 cells from 12 embryos; Figure 3B &3C) as well as by the nAChR antagonist DHβE (5µM; N=4 cells from 4 embryos). By contrast, they remained unaffected by antagonists against GABAA receptors (Gabazine, 3 µM) and glutamate ionotropic receptors (DL-APV 200 µM and CNQX 20µM), two other neurotransmitters released by spinal interneurons during SNA [START_REF] Hanson | Characterization of the circuits that generate spontaneous episodes of activity in the early embryonic mouse spinal cord[END_REF][START_REF] Czarnecki | Acetylcholine controls GABA-, glutamate-, and glycine-dependent giant depolarizing potentials that govern spontaneous motoneuron activity at the onset of synaptogenesis in the mouse embryonic spinal cord[END_REF] (N=6 cells from 6 embryos; Figure 3B &3C). Conversely, exogenous application of acetylcholine (30 µM) was sufficient to depolarize floor-plate cells (Amplitude = 14.0 ± 2.9 mV; N=9 cells from 9 embryos) and trigger biphasic action potentials (amplitude = 59 ± 7 mV) in a nAChR dependent manner (paired Wilcoxon test, p<0.05; N=6 cells from 6 embryos; Figure 3D &3E). Application of the sodium channel blocker TTX together with antagonists of GABA, glutamate and glycine ionotropic receptors had no effect on the depolarization induced by acetylcholine, suggesting that acetylcholine acts directly onto nAChR expressed by floor-plate cells (Figure 3D &3E). We found that TTX suppressed the fast component of the biphasic action potential triggered by acetylcholine (paired Wilcoxon test, p<0.05; N=6 cells from 6 embryos; Figure S2), demonstrating that it resulted from the activation of voltage-gated sodium channels in floor-plate cells. The slow component was blocked by TTA-P2 indicating that this resulted from the activation of T-type calcium channels (paired Wilcoxon test, p<0.05; N=6 cells from 6 embryos; Figure S2). To confirm that ACh released from motoneurons triggers floor-plate action potentials, we performed optogenetic stimulation using a transgenic mouse model (ChAT:ChR2-YFP) where channelrhodopsin 2 is expressed in cholinergic motoneurons (Figure 3F-G). Optogenetic stimulation of cholinergic neurons was sufficient to depolarize and trigger an action potential in floor-plate cells, which could be fully blocked by nAChR antagonists (paired Wilcoxon test, p<0.05; N = 5 cells from 5 embryos ; Figure 3H &3I). To investigate whether cholinergic axons from motoneurons directly contact the floor-plate, we performed immunostaining against vAChT and FoxA2 in coronal sections from E12 HB9:GFP transgenic mice, where GFP is expressed under the promoter for HB9, a homeodomain transcription factor specific of motoneurons at this developmental stage [START_REF] Wichterle | Directed differentiation of embryonic stem cells into motor neurons[END_REF][START_REF] Bras | In vivo assembly of the axon initial segment in motor neurons[END_REF] . We found the presence of a few HB9:GFP + /vAChT + axons crossing into the ventral midline (Figure S3) where floor-plate basal processes are located. Taken together, our results show that floor-plate action potentials are triggered by the activation of nAChR, most likely in response to acetylcholine released by cholinergic motoneurons during SNA.

Finally, we examined whether and how floor-plate activity could propagate between floor-plate cells along the rostro-caudal axis of the spinal cord. We expressed the genetically encoded calcium indicator GCaMP6f in floor-plate cells using a conditional transgenic approach (GCaMP6floxP x GLASTCreERT2; see Methods). This was achieved by taking advantage of the fact that floor-plate cells are the first neuroepithelial cells to express the glutamate aspartate transporter (GLAST) during spinal cord development [START_REF] Deneen | The Transcription Factor NFIA Controls the Onset of Gliogenesis in the Developing Spinal Cord[END_REF][START_REF] Barry | Differentiation of radial glia from radial precursor cells and transformation into astrocytes in the developing rat spinal cord[END_REF][START_REF] Shibata | Glutamate transporter GLAST is expressed in the radial glia-astrocyte lineage of developing mouse spinal cord[END_REF] (Figure 4A and Figure S1A-H). Using this approach, we found that midline floor-plate cells expressing GCaMP6f (Figure 4A However, rostral to caudal waves initiated in the cervical area were more frequent (67%, 41/61 waves from 10 embryos) than caudal to rostral ones (33%, 20/61 waves) (Figure S4 and Video S2). Average propagation speed of spontaneous rostro-caudal (2.4 ± 0.9 mm/s) and caudo-rostral floor-plate calcium waves (2.1 ± 1.3 mm/s) were not significantly different (Mann-Whitney test, p=0.12) and their speed was consistent with the propagation speed estimated for SNA waves in motoneurons [START_REF] Hanson | Characterization of the circuits that generate spontaneous episodes of activity in the early embryonic mouse spinal cord[END_REF][START_REF] Scain | Glycine release from radial cells modulates the spontaneous activity and its propagation during early spinal cord development[END_REF] .

As observed for floor-plate action potentials, calcium waves could also be consistently triggered using electrical stimulation (32 mA, 200 µs, Figure 4G, H, J, K andVideo S3).

Application of the T-type calcium channel blocker TTA-P2 was sufficient to fully block evoked calcium waves (Mann-Whitney test, p<0.001, N = 24 waves in 8 embryos; Figure S5A, S5B & Video S4), even after increasing the duration of stimulation (32 mA, 1200-1600 µs). The initiation and propagation of evoked calcium waves was also blocked by the application of nAChR antagonists (paired Wilcoxon test, p<0.001, N = 6 embryos; Figure 4G & Video S5), as observed with floor-plate action potentials (Figure 3B). However, increasing stimulation duration (32 mA, 1200-1600 µs) allowed the recovery of floor-plate calcium waves, albeit with a significantly decreased propagation speed of 0.8 ± 0.5 mm/s (paired Wilcoxon test, p<0.001, N =31 waves in 6 embryos; Figure 4G &H, Video S5). Similarly, patch-clamp experiments showed that after increasing the duration of stimulation in the presence of a cocktail of antagonists to nAChR, GABAaR, GluR and GlyR, the electrical stimulus triggered a delayed biphasic AP without the small initial cholinergic depolarization (N = 4 cells in 4 embryos; Figure 4I). This result suggests that, although the initiation and subsequent propagation of floor-plate APs and calcium waves strongly depend on cholinergic transmission, gap-junction coupling between floor-plate cells could also participates in this propagation (Figure 2C). We tested this hypothesis by applying the connexin blocker β18-GA. We found that it either reduced or blocked the propagation of floor-plate calcium waves confirming that connexins are necessary for calcium wave propagation. The effect of β18-GA could result from the blockade of gap-junction and/or connexin hemichannels [START_REF]Inhibitors of connexin and pannexin channels as potential therapeutics[END_REF] .

It has previously been shown that the propagation of slow calcium waves between cortical progenitors occurs via ATP signaling acting through high-affinity and slow-acting metabotropic P2YR [START_REF] Weissman | Calcium waves propagate through radial glial cells and modulate proliferation in the developing neocortex[END_REF] . By contrast, we found here that the propagation speed of floor plate cells is at least a hundred times faster and primarily rely on nAChRs, T-type calcium channels and connexins. However, there is evidence that ependymal cells derived from the floor-plate express low-affinity but fast-acting ionotropic P2X7 receptors to ATP [START_REF] Marichal | Purinergic signalling in a latent stem cell niche of the rat spinal cord[END_REF] . We found that floor plate cells only responded to ATP at concentrations above 1 mM, suggesting that they only expressed low-affinity P2X7R (Figure 5A &5B). Indeed, ATP responses were blocked by the competitive antagonist A438079, specific to the low-affinity P2X7R [START_REF] Donnelly-Roberts | Discovery of P2X 7 receptorselective antagonists offers new insights into P2X 7 receptor function and indicates a role in chronic pain states[END_REF] (Figure 5C &D).

We thus investigated whether P2X7R could actively participate in the propagation of floor plate calcium waves. We found that A438079 significantly reduced the propagation speed of evoked floor-plate calcium waves (Figure 5E &F). Since P2X7R have been found to be predominantly expressed by ependymal cells in the postnatal nervous system [START_REF] Collo | Tissue distribution of the P2X7 receptor[END_REF][START_REF] Genzen | Ependymal cells along the lateral ventricle express functional P2X7 receptors[END_REF] , our results are consistent with the ependymal nature of floor plate cells.

Altogether, our results show that the floor-plate of the neuroepithelium possesses the intrinsic ability to generate and propagate electrical signals along the embryonic spinal cord

DISCUSSION

Although it has been shown previously that a subpopulation of glial progenitors expressing the proteoglycan NG2 can be induced to generate TTX-sensitive sodium spikes in the postnatal brain [START_REF] Chittajallu | NG2-positive cells in the mouse white and grey matter display distinct physiological properties[END_REF][START_REF] Káradóttir | Spiking and nonspiking classes of oligodendrocyte precursor glia in CNS white matter[END_REF] , the neuroepithelial floor-plate is the first example of a non-neuronal structure able to both spontaneously generate and propagate action potentials in the developing central nervous system. Until now, these abilities were considered to be exclusive features of differentiating neurons. Accordingly, the ability to generate and propagate action potentials has been routinely used as a functional assay to confirm the neuronal phenotype of cells during development. In this context, although Ca 2+ /Na + biphasic action potentials relying on T-type calcium channels are not commonly observed in neurons, their previous discovery in neural cells in the Xenopus embryonic spinal cord [START_REF] Gu | Spontaneous neuronal calcium spikes and waves during early differentiation[END_REF] and in the mouse fetal hindbrain [START_REF] Moruzzi | Differential expression of membrane conductances underlies spontaneous event initiation by rostral midline neurons in the embryonic mouse hindbrain[END_REF] has been interpreted as an early and transient form of excitability in developing neurons. Instead, we found here that Ca 2+ /Na + biphasic action potentials are generated in the mouse spinal cord by gliogenic neuroepithelial progenitors located in the floor-plate and that they propagate dorsally in other neuroepithelial cells also engaged into a gliogenic program at this stage [START_REF] Deneen | The Transcription Factor NFIA Controls the Onset of Gliogenesis in the Developing Spinal Cord[END_REF] .

It has previously be shown that T-type voltage-gated currents can be elicited in floor plate cells at later embryonic and perinatal stages in rats [START_REF] Frischknecht | Voltage-and ligand-gated ion channels in floor plate neuroepithelia of the rat[END_REF] . However, only modest depolarizations (10-15 mV) could be elicited in this study when triggering T-type calcium channels by current injection and it was not reported that floor-plate cells could actually generate action potentials. This discrepancy may be explained by the use of coronal slices and/or the absence of voltage-gated sodium channels in floor-plate cells of rats. Indeed, our experiments suggest that the generation of full-fledged action potentials involves the simultaneous activation of numerous cells along the rostro-caudal axis of the floor-plate, as observed during our calcium imaging experiments. The use of coronal sections may thus have greatly impeded the ability of floor-plate cells to coordinate their activation via gap-junction coupling. The absence of voltage-gated sodium channels would also have impeded the ability of floor-plate cells to generate action potentials in rats. This lack of voltage-gated sodium channels could be the result of the differences in species and/or developmental stages [START_REF] Placzek | The floor plate: Multiple cells, multiple signals[END_REF] .

We found that spontaneous floor-plate calcium waves are initiated at different rostrocaudal location and can propagate in both directions. 2 thirds of the waves are initiated in the cervical area from where they propagate both caudally into the rest of the spinal cord and rostrally towards the brainstem. This latter observation suggests that floor-plate calcium waves may not be restricted to the spinal cord. The last third of the waves propagate in a caudal-to-rostral direction -both at cervical and thoracic level -originating from a yet to be determined caudal location(s). This proportion of waves with a cervical and caudal origin is similar to that previously reported for waves of muscle contractions observed in vivo in mouse embryos at the same developmental stage [START_REF] Suzue | Highly reproducible spatiotemporal patterns of mammalian embryonic movements at the developmental stage of the earliest spontaneous motility[END_REF] . This suggest that floor-plate activity is likely to be tightly correlated with the first pattern of motoneuron activity and muscle contraction observed in vivo.

From an evolutionary point of view, the discovery of an epithelial electrical conduction system in the vertebrate fetal CNS should not come as a complete surprise. Indeed, epithelial and syncytial conduction systems are commonly observed in non-bilaterian animals exhibiting either a "primitive" nervous system, like jellyfish and comb jellies, or in those lacking a nervous system like the glass sponges [START_REF] Meech | Electrogenesis in the lower Metazoa and implications for neuronal integration[END_REF][START_REF] Mackie | Neuroid conduction and the evolution of conducting tissues[END_REF][START_REF] Anderson | Epithelial conduction: Its properties and functions[END_REF][START_REF] Leys | The Biology of Glass Sponges[END_REF] . It has been proposed that the modern neuron-based nervous systems of bilaterians using axonal/chemical synaptic transmission evolved from such epithelial conduction systems coupled electrically by gapjunctions [START_REF] Castelfranco | Evolution of rapid nerve conduction[END_REF] . This evolution would also have been accompanied by a switch from slow voltagegated calcium channels -inherited from our protist ancestors and still found in the modern paramecium [START_REF] Eckert | Sensory mechanisms in Paramecium[END_REF] -to fast voltage-gated sodium channels [START_REF] Castelfranco | Evolution of rapid nerve conduction[END_REF] . Our discovery of a "primitive" electrical conduction system in the neuroepithelium that gives rise to the "modern" nervous system of vertebrates during fetal development could therefore be interpreted as strong evidence supporting this evolutionary scenario.

One may also notice the functional similitude existing between the floor-plate of His and the heart electrical conduction system known as the bundle of His. The bundle of His was discovered by the anatomist and cardiologist Wilhem His, Jr. in 1893 [START_REF] Bast | Wilhelm His, Jr. and the Bundle of His[END_REF] , five year after his father had described the floor-plate [START_REF] Kingsbury | The extent of the floor-plate of his and its significance[END_REF] . Both structures act as electrical conduction systems connected by gap junctions able to propagate electrical signals across their respective organs.

By showing that these structures not only share the same name but also share similar functional properties, our work reveals that the nervous and cardiac physiologies of vertebrates is more deeply related than previously thought. Further work will be needed to decipher what function(s) are fulfilled by this nonneuronal electrical conduction system. Although the electrical excitability of floor-plate cells could only be a vestigial property inherited from non-bilaterian ancestors, it is more likely playing a significant role in the development of the spinal cord. Indeed, the energetic cost of generating and propagating electrical signals [START_REF] Attwell | An energy budget for signaling in the grey matter of the brain[END_REF] as well as the redundancy of T-type channel subtypes expressed in the floor-plate point towards having a conserved and significant function. One hypothesis is that floor-plate excitability regulates and coordinates the release of the morphogen Sonic Hedgehog (Shh), required for neural tube patterning, or the expression of axon guidance molecules such as Slit and Netrin. Indeed, recent studies have shown unexpected interactions between early calcium electrical excitability and Shh signalling in the Xenopus spinal cord [START_REF] Belgacem | Inversion of Sonic hedgehog action on its canonical pathway by electrical activity[END_REF][START_REF] Borodinsky | Crosstalk among electrical activity, trophic factors and morphogenetic proteins in the regulation of neurotransmitter phenotype specification[END_REF] . Moreover, T-type channel mutants have recently been identified in a forward genetic screen for neural tube defects in Ciona intestinalis [START_REF] Abdul-Wajid | T-type Calcium Channel Regulation of Neural Tube Closure and EphrinA/EPHA Expression[END_REF] , suggesting that the electrical gradient established by the floor-plate in the ventral neuroepithelium could represent a novel morphogenetic mechanism. An additional and nonexclusive hypothesis is that floor-plate action potentials initiated by acetylcholine released by motoneurons during SNA act as a feedback signal to promote the switch from neurogenesis to gliogenesis in ventral neuroepithelial progenitors [START_REF] Deneen | The Transcription Factor NFIA Controls the Onset of Gliogenesis in the Developing Spinal Cord[END_REF][START_REF] Barry | Differentiation of radial glia from radial precursor cells and transformation into astrocytes in the developing rat spinal cord[END_REF] since both events occur around the same developmental stage.

In conclusion, our discovery of the floor-plate of His acting as a non-neuronal electrical conduction system could profoundly change how we conceive of the origin, extent and role of electrical signals during the development of the central nervous system of vertebrates. breeding male mice expressing a BAC transgene containing a Channelrhodopsin2-EYFP sequence driven by a Choline Acetyl Transferase (ChAT) promoter (ChAT-ChR2-EYFP, The Jackson Laboratory, USA, https://www.jax.org/strain/014546) [START_REF] Zhao | Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function[END_REF] . We used C57BL/6 mice expressing a Hb9-GFP transgene [START_REF] Wichterle | Directed differentiation of embryonic stem cells into motor neurons[END_REF] to visualize motoneuron axons crossing into the floorplate. For calcium imaging of floor-plate cells, we used C57BL/6 breeding male mice expressing a genetically encoded floxed Calcium indicator GCaMP6f (Ai95, The Jackson Laboratory, USA, https://www.jax.org/strain/028865), crossed with Swiss female mice expressing a tamoxifen-inducible Cre recombinase under the control of the glutamate aspartate transporter promoter (GLASTCreERT2, gift from Dr Frank Pfrieger) 57 . Cre recombination was induced by oral gavage of pregnant mice with 5 mg of tamoxifen (Sigma-Aldrich, France, T-5648) at embryonic days E10.5 and E11.5 58 . Tamoxifen was dissolved in corn oil at 37°C for several hours to prepare 20 mg/mL solution 59 .

MAIN-TEXT

Method details

In situ RNA hybridization staining

Mouse embryos were fixed in 3.7% paraformaldehyde (PFA) in PBS overnight at 4°C. Tissues were then sectioned at 60-80 µm using a vibratome (Microm, France). In all experiments, sections were performed at the cervico/brachial level. In situ hybridization (ISH) was performed on vibratome sections automatically (InsituPro, Intavis, Germany) using the whole-mount ISH as previously reported 60 . Digoxigenin (DIG)-labeled antisense RNA probes were synthesized using T3 polymerase from the following I.M.A.G.E. Consortium cDNA Clones 61 : cacna1g, (ID 6410519); cacna1h (ID 6820407) and cacna1i (ID 6825802). RNA labeled probes were detected by an alkaline-phosphatase coupled antibody (Roche Diagnostics), and NBT/BCIP (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) were used as a chromogenic substrate for the alkaline phosphatase (Boehringer Mannheim, Germany). Stained sections were digitized and analyzed using Zeiss acquisition software (Zeiss, Germany).

Isolated embryonic spinal cord preparation

E12.5 embryonic mouse spinal cords were dissected as previously described [START_REF] Czarnecki | Acetylcholine controls GABA-, glutamate-, and glycine-dependent giant depolarizing potentials that govern spontaneous motoneuron activity at the onset of synaptogenesis in the mouse embryonic spinal cord[END_REF][START_REF] Osterstock | Axoglial synapses are formed onto pioneer oligodendrocyte precursor cells at the onset of spinal cord gliogenesis[END_REF] .

Briefly, pregnant mice were killed using a lethal dose of CO2 followed by cervical dislocation. Mice embryos of both sexes were dissected and whole spinal cord were left to recover for at least 30 min at 32˚C in artificial cerebrospinal fluid (ACSF) containing 125 mM NaCl, 25mM NaHCO3, 11 mM Glucose, 3 mM KCl, 1 mM MgCl2, 2 mM CaCl2, and 1 mM NaH2PO4 (pH 7.3; 307 mOsm) continuously bubbled with 95% O2 -5% CO2 gas, before being recorded or imaged. For ChAT-ChR2-EYFP and GCaMP6floxP x GLASTCreERT2 transgenic embryos, their tails were cut and observed under a fluorescent stereo-microscope (Leica MZ FLIII) to identify positive embryos for EYFP and GCaMP6, respectively.

Patch-clamp recordings

Isolated spinal cords were placed in open-book configuration in a recording chamber under a nylon holding grid and continuously perfused (2 ml/min) at room temperature with the oxygenated ACSF described above. In contrast to previous studies [START_REF] Czarnecki | Acetylcholine controls GABA-, glutamate-, and glycine-dependent giant depolarizing potentials that govern spontaneous motoneuron activity at the onset of synaptogenesis in the mouse embryonic spinal cord[END_REF][START_REF] Osterstock | Axoglial synapses are formed onto pioneer oligodendrocyte precursor cells at the onset of spinal cord gliogenesis[END_REF] , the orientation of open-book spinal cord was inverted so that the neuroepithelial layer was facing upward.

Whole-cell voltage-clamp recordings of floor-plate cells and other neuroepithelial progenitors were performed under direct visualization using an infrared-sensitive CCD video camera. In addition to their location at the midline, floor-plate cells were distinguished from surrounding neuroepithelial progenitors based on their smaller apical process and their higher translucency when using brightfield microscopy. To confirm their identity, some recorded cells were filled with neurobiotin (1 mg/ml, Vector labs, USA) and revealed in combination with FoxA2 and Nkx2.2 immunostaining. Whole-cell patch-clamp electrodes were pulled from thick-wall borosilicate glass using a Brown-Flaming puller (P97, Sutter Instrument, USA). The tip of the electrode was fire-polished using a microforge (MF-830, Narishige, Japan). Patch-clamp electrodes had tip resistances comprised between 4 and 7 MΩ. Electrodes were filled with an intracellular solution containing either (in mM): 96.4 K + -methanesulfonate, 33.6 KCl, 4 MgCl2, 4 Na2ATP, 0.3 Na3GTP, 10 EGTA, and 10 HEPES or 100 K + -gluconate, 34 KCl, 0.5 EGTA, 10 HEPES, 4 MgCl2, 4 Na2ATP (pH 7.2; 290 mOsm). In both solutions, the theoretical equilibrium potential for chloride ions (ECl) was -30 mV, while cation reversal potential (Ecat) was 0 mV. Signals were recorded with a low-pass filtered at 4 kHz using a Multiclamp 700B amplifier (Molecular Devices, USA) and digitized at 20 kHz using a Digidata 1440A interface coupled to pClamp 10.5 software (Molecular Devices, USA) running on a PC computer. Cells exhibiting an average resting membrane potential above -50 mV (I=0 pA) were discarded from further analysis.

Electrical and optogenetic stimulation

For electrical stimulations experiments, we used bipolar platinum electrodes (FHC, USA) placed 2 mm rostrally to the recording site onto the midline. For optogenetic stimulation of ChAT-ChR2 we used a 400 µm diameter optical fiber coupled with a 470 LED (Thorlabs, Germany) and centered onto the recording site. Electrical and optogenetic stimulations were performed at a low frequency (0.005 Hz, 1 stimulation every 180 s) in order to evoke a stable cholinergic response.

Calcium imaging

Isolated spinal cords were placed in open-book configuration under a nylon holding grid in a recording chamber and continuously perfused (2mL/min) with the same oxygenated following pharmacological agents were used: Acetylcholine chloride (30 µM, Sigma-Aldrich, Germany), TTX (1 µM, Alomone Labs, Israel), gabazine (3 µM, Tocris, USA), CNQX Disodium (20 µM, Tocris, USA), DL-APV (200 µM, Tocris, USA), Mecamylamine hydrochloride (100 µM, Tocris, USA), D-tubocurarine chloride (10 µM, Tocris, USA), 18β glycyrrhetinic acid (50 µM, Sigma-Aldrich, Germany), TTA-P2 (3µM, Alomone, Israel), A438079 (30 µM, Tocris, USA) and DHβE (5 µM, Tocris, USA). All drugs were dissolved at final concentration in ACSF.

Immunohistochemistry

E12.5 embryos or whole embryonic spinal cord were immersion-fixed in phosphate buffer saline (PBS) with 4% PFA freshly prepared in PBS (pH 7.4) for 1 h at 4°C. Embryos were then rinsed with PBS and cryoprotected in PBS-15% sucrose at 4°C for 24 h and then in PBS-30% sucrose at 4°C for 24 h. Embryos were embedded in OCT medium (VWR, USA) and quickly frozen. Serial sections 20 µm thick were collected onto slides using a cryostat (Leica, Wetzlar, Germany) and stored at -80°C until immunohistochemical studies. For immunostaining, slides and whole spinal cord were washed in PBS, incubated in NH4Cl (50 mM) diluted in PBS for 20 min and then permeabilized for 30 min in a blocking solution (10% goat serum in PBS) with 0.2% Triton X-100. They were incubated overnight at 4°C with the primary antibodies, which were diluted in the 0.2% Triton X-100 blocking solution.

Slides or whole spinal cords were then washed in PBS and incubated for 2h at room temperature in the appropriate secondary antibodies (diluted at 1/1000 in the 0.2% Triton X-100 blocking solution). The following primary antibodies were used: a mouse monoclonal anti-HNF-3β/FoxA2 (H-4) antibody sc-374376 (1:300, Santa Cruz Biotechnology, USA), a mouse monoclonal anti-Nkx2.2 antibody 74.5A5 (1:600, Developmental Studies Hybridoma Bank, USA), a guinea-pig polyclonal anti-EAAT1/GLAST antibody 250 114 (1:1000,

  . The average distance between pairs of FP and p3 NEPs filled with neurobiotin was 22 ± 7 µm (N= 6 pairs in 6 embryos). Simultaneous pair recordings demonstrated that rhythmic depolarizations were systematically correlated between FP and p3 NEPs (16/16 events; 5 pairs from 5 embryos; Figure 1G), although depolarizations in the p3 domain exhibited significantly smaller amplitudes (Amplitudep3 = 22.2 ± 5.5 mV vs AmplitudeFP = 55.3 ± 6.8 mV; Figure 1G and 1H) with a slight delay (Delay [FP-p3] = 61.2 ± 16.4 ms; Figure 1G and

)

  together with the transcription factor Sox2, a marker for neuroepithelial progenitors (FigureS1I-L). Floor-plate NEPs had an average membrane resistance of 480 ± 410 MΩ, an average membrane capacitance of 18.3 ± 6.6 pF and an average resting membrane potential of -64.8 ± 9.7 mV (N= 24 cells from 24 embryos). More importantly, we found that floor-plate NEPs had the unique ability to generate action potentials (AP) in response to membrane depolarization (threshold = -37 ± 3 mV; amplitude = 43.8 ± 14 mV; N= 15 cells from 15 embryos; Figure 2A). Unlike most neuronal APs which only rely on voltage-gated sodium channels, floor-plate APs could only be blocked by a combination of TTA-P2 (3 µM), a specific T-type calcium channel blocker, and TTX, a sodium channel blocker (Figure 2A & 2B). By contrast, NEPs recorded in the adjacent p3 domain did not exhibit any voltagedependent responses (N= 24 cells from 24 embryos). During SNA, floor-plate rhythmic depolarizations (amplitude = 54 ± 15 mV; N = 22 cells from 22 embryos; Figure 1G & 2D)

  ) generated spontaneous calcium waves at an interval of 176 ± 67 s (N = 31 waves from 10 embryos; Figure 4B & C and Video S1), similar to the interval observed for spontaneous floor-plate action potentials. Spontaneous floor-plate calcium waves propagated along the length of the spinal cord and could propagate in either direction.

(

  paired Wilcoxon test, p<0.05, N=18 waves in 6 embryos; Figure 4J & K, Video S5) and action potentials (N = 4 cells in 4 embryos; Figure 4L) that were evoked in the presence of nAChR antagonists and other neurotransmitter antagonists. Floor-plate calcium waves were also blocked when applying the connexin blocker β18-GA alone (Figure S5C & D),

Figure 1 .

 1 Figure 1. Neuroepithelial progenitors are spontaneously depolarized during SNA

Figure 2 .F,

 2 Figure 2. Floor-plate neuroepithelial progenitors generate rhythmic action potentials

Figure 3 .

 3 Figure 3. Floor-plate biphasic action potentials are triggered by the activation of

Figure 4 .

 4 Figure 4. Floor-plate action potentials are associated with calcium waves and propagate

Figure 5 .

 5 Figure 5. P2X7R are expressed by floor-plate cells and participate to calcium wave

F

  .W.(2007). Transgenic mice for conditional gene manipulation in astroglial cells.Glia 55, 1565-1576. 58. Eon, J.P., Sun, X., Nichol, P., Saijoh, Y., Martin, J.F., and Moon, A.M.(2008). System for tamoxifen-inducible expression of Cre-recombinase from the Foxa2 locus in mice. Dev. Dyn. 237, 447-453. 59. Zervas, M., Millet, S., Ahn, S., and Joyner, A.L. (2004). Cell behaviors and genetic lineages of the mesencephalon and rhombomere 1. Neuron 43, 345-357. 60. Touahri, Y., Escalas, N., Benazeraf, B., Cochard, P., Danesin, C., and Soula, C. (2012). Sulfatase 1 promotes the motor neuron-to-oligodendrocyte fate switch by activating Shh signaling in Olig2 progenitors of the embryonic ventral spinal cord. J. Neurosci. 32, 18018-18034. 61. Lennon, G., Auffray, C., Polymeropoulos, M., and Soares, M.B. (1996). The I.M.A.G.E consortium: An integrated molecular analysis of genomes and their expression. Genomics 33, 151-152.

ACKNOWLEDGEMENTS

We thank Isabelle Dusart, Marine Robouam, Odile Favière, Goran Fort and Maryse Dardenne for their help in managing mouse colonies; Susanne Bolte, Jean-François Gilles and France Lam for assistance with confocal imaging. We thank Frank Pfrieger for the GLASTCreERT2 mouse line. This study was financially supported by Institut de Recherche sur la Moelle et l'encéphale (JMM), AFM-Telethon (JMM), Agence Nationale de la Recherche (JMM) and Fondation pour la Recherche Médicale (JMM and PL). Each protocol was triplicated in each cell (patch-clamp experiments) and embryos (calcium imaging experiments). Statistical comparisons for electrophysiological recordings and calcium imaging parameters were conducted using GraphPad Prism 8 (GraphPad Software, USA) by performing non-parametric Wilcoxon-Mann-Whitney U tests (calcium imaging experiments) and Wilcoxon signed rank test for paired samples (patch-clamp experiments).

We determined that statistical significance was obtained when the one-tailed p-value was <0.05.

Materials Availability

This study did not generate new unique reagents.

Data and code availability

• This study did not generate any unique dataset.

• This study did not generate any original code.

• All data needed to evaluate the conclusions are present in this paper and/or the supplemental information. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

AUTHOR CONTRIBUTIONS

DECLARATION OF INTERESTS

The authors declare no competing interests.

STAR METHODS

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, jean-Marie Mangin (jean-marie.mangin@inserm.fr)

Experimental models and subject details

All experiments were performed in accordance with European Community guiding principles on the care and use of animals (86/609/CEE, CE Off J no. L358, 18 December, 1986), French decree no. 97/748 of October 19, 1987 (J Off République Française, 20 October, 1987, pp. 12245-12248) and recommendations from the CNRS. All efforts were made to minimize the suffering of animals. In order to maximize litter size, experiments were performed on E12.5 mouse embryos obtained by crossing C57BL/6 males with females from a Swiss strain. For optogenetic stimulation of cholinergic motoneurons, we used C57BL/6 ACSF described above. Recombined fluorescent cells were observed under an epifluorescence microscope BX51W1 (Olympus) with a 470-515nm fluorescent excitation light to confirm GCaMP6f expression in the floor-plate. Time-lapse imaging was performed using either an Orca Flash4.0 (Hamamatsu, Japan) or Orca 03G (Hamamatsu, Japan) camera and recorded using the HC Image Live software (Hamamatsu, Japan) at an acquisition frequency of 5 or 10Hz with an exposure time of 200 ms and 100 ms, respectively. Images were encoded using a 256x256 or 512x512 pixel resolution and 16-bit grey scale. Time-lapse images were analyzed using the ImageJ software (National Institute of Health, USA). Signals were either extracted by subtracting each frame from the one following it (Figure 4g & 4i) or by subtracting an average of 10 frames preceding the onset of an event (Figure 4d, Supplementary Figure 3 and Supplementary videos 1-5). To measure the propagation speed, we pinpointed three regions of interest on the midline and measured the delays necessary to reach half-peak between each region of interest and averaged them. Intensity of signals (dF/F) were measured by dividing the peak amplitude of the events by the baseline value in raw images.

Pharmacological agents

In most experiments, drugs were applied via the bath perfusion system. Typically, the effect of drugs reached equilibrium within 3 to 5 minutes. For acetylcholine application, we used a 0.5 mm diameter quartz tubing positioned 50 µm away from the recording area under direct visual control. Typically, response to acetylcholine reached equilibrium a few seconds (6 sec) after solution switch. The quartz tubing was connected using a manifold to six solenoid valves linked with six reservoirs. Solutions were gravity-fed into the quartz tubing.

Drug application was controlled using a VC-8 valve controller (Warner Instruments, USA).

At least 3 events were recorded in each condition after waiting 5 min for drugs to act. The Synaptic System, Germany), a mouse monoclonal anti-Sox2 (E-4) antibody sc-365823 (1:500, Santa Cruz Biotechnology, USA), a rabbit polyclonal anti-vAChT antibody 139 103 (1:1000, Synaptic System, Germany) and a rabbit polyclonal anti-GFP antibody A-11122 (1:1000, Thermo Fisher Scientific, USA). Alexa Fluor 405-, 488-, or 594-and 647-conjugated secondary antibodies (1/1000; Invitrogen, USA) were used to detect mouse monoclonal, guinea pig and rabbit polyclonal primary antibodies. After washing in PBS, slides or whole spinal cords were dried and mounted in Mowiol medium (Millipore, USA).

Confocal microscopy

Once mounted, immunostained sections were imaged using a SP5 confocal microscope (Leica, Germany) using a 20X oil-immersion objective with a numerical aperture of 1.25, as well as with a 63X oil-immersion objective with a numerical aperture of 1.32 and a 1X digital zoom magnification. Serial optical sections were acquired with a Z-step of 3 µm (20X) and 0.78 µm (63X). Images (1024 X 1024; 8 to16 bit grayscale) were acquired using Leica software LAS-AF and analyzed using ImageJ (National Institutes of Health, USA).

Colocalization with neurobiotin staining was assessed in three axes using a single confocal slice and x and y orthogonal views of the stack (ImageJ 1.5). To quantify the number of cells filled by neurobiotin after patch-clamp recording in the floor-plate and/or p3 domain, neurobiotin staining was combined with antibody staining against FoxA2, Nkx2.2 and DAPI.

Quantification and statistical analysis