Spontaneous network activity (SNA) emerges in the spinal cord (SC) before the formation of peripheral sensory inputs and central descending inputs. SNA is characterized by recurrent giant depolarizing potentials (GDPs). Because GDPs in motoneurons (MNs) are mainly evoked by prolonged release of GABA, they likely necessitate sustained firing of interneurons. To address this issue we analyzed, as a model, embryonic Renshaw cell (V1) activity at the onset of SNA (E12.5) in the embryonic mouse SC (both sexes). V1 are one of the interneurons known to contact MNs, which are generated early in the embryonic SC. Here, we show that V1 already produce GABA in E12.5 embryo, and that V1 make synaptic-like contacts with MNs and have putative extrasynaptic release sites, while paracrine release of GABA occurs at this developmental stage. In addition, we discovered that V1 are spontaneously active during SNA and can already generate several intrinsic activity patterns including repetitive-spiking and sodium-dependent plateau potential that rely on the presence of persistent sodium currents (). This is the first demonstration that is present in the embryonic SC and that this current can control intrinsic activation properties of newborn interneurons in the SC of mammalian embryos. Finally, we found that 5 μm riluzole, which is known to block, altered SNA by reducing episode duration and increasing inter-episode interval. Because SNA is essential for neuronal maturation, axon pathfinding, and synaptogenesis, the presence of in embryonic SC neurons may play a role in the early development of mammalian locomotor networks. The developing spinal cord (SC) exhibits spontaneous network activity (SNA) involved in the building of nascent locomotor circuits in the embryo. Many studies suggest that SNA depends on the rhythmic release of GABA, yet intracellular recordings of GABAergic neurons have never been performed at the onset of SNA in the SC. We first discovered that embryonic Renshaw cells (V1) are GABAergic at E12.5 and spontaneously active during SNA. We uncover a new role for persistent sodium currents () in driving plateau potential in V1 and in SNA patterning in the embryonic SC. Our study thus sheds light on a role for NaP in the excitability of V1 and the developing SC.

Renshaw cells (V1R) are excitable as soon as they reach their final location next to the spinal motoneurons and are functionally heterogeneous. Using multiple experimental approaches, in combination with biophysical modeling and dynamical systems theory, we analyzed, for the first time, the mechanisms underlying the electrophysiological properties of V1R during early embryonic development of the mouse spinal cord locomotor networks (E11.5-E16.5). We found that these interneurons are subdivided into several functional clusters from E11.5 and then display an unexpected transitory involution process during which they lose their ability to sustain tonic firing. We demonstrated that the essential factor controlling the diversity of the discharge pattern of embryonic V1R is the ratio of a persistent sodium conductance to a delayed rectifier potassium conductance. Taken together, our results reveal how a simple mechanism, based on the synergy of two voltage-dependent conductances that are ubiquitous in neurons, can produce functional diversity in embryonic V1R and control their early developmental trajectory.

Highlights d Neuroepithelial progenitors (NEPs) are depolarized by spontaneous neural activity d NEPs form a single electrical syncytium connected by gap junctions d Floor-plate NEPs generate Na + /Ca 2+ action potentials in response to acetylcholine d Neuroepithelial action potentials propagate across the entire spinal cord

In the developing central nervous system of vertebrates, the emergence of electrical signals is thought to result exclusively from the differentiation of neurons 1-3 . Accordingly, the neuro-epithelial progenitors generating neurons and glial cells have been assumed to remain electrically passive. Here, we show that the floor plate of His -a neuro-epithelial organizer located at the ventral midline of the fetal spinal cord 4 -has the unexpected ability to generate large biphasic action potentials resulting from the combined activity of voltage-gated T-type calcium channels and sodium channels. Floor plate action potentials are recurrently triggered by the neurotransmitter acetylcholine released from cholinergic motor neurons during early episodes of spontaneous neural activity. Moreover, we found that floor plate cells are connected by gap junctions, form a functional syncytium with other neuro-epithelial progenitor domains and establish a ventral to dorsal depolarization gradient in the neuroepithelium. Finally, we show that floor plate action potentials are associated with calcium waves and propagate through gap junctions along the length of the fetal spinal cord. Our present work demonstrates that the floor plate of His acts as a unique neuro-epithelial electrical conduction system, sharing functional similarities with the myo-epithelial electrical conduction system of the heart known as the bundle of His 5 . This discovery profoundly change how we conceive the development, origin, evolution and extent of electrical signals in the central nervous system of vertebrates.

A remarkable feature of the developing central nervous system is the generation of spontaneous network activity (SNA) at the onset of synaptogenesis. In the spinal cord (SC) of the mouse embryo, SNA occurs around the 12 th embryonic day (E12.5) and involves a recurrent functional loop between motoneurons (MNs) and GABAergic interneurons. We previously showed that Renshaw cells (V1 R ), which constitute a functionally homogeneous population in the adult, are the first neurons enriched with GABA in the mouse embryonic lumbar SC. V1 R make synaptic-like contacts with MNs and likely participate to the genesis of SNA. However, how electrophysiological intrinsic properties of V1 R evolve during early development remains poorly understood. Here, using patch-clamp recordings, cluster analysis and biophysical modeling, we analyzed the mechanisms underlying the firing patterns of V1 R between E11.5 and E16.5. This developmental period covers the initial phase of development of SC activity (E11.5-E14.5) when SNA is present, and a pivotal period (E14.5-E16.5) when locomotor-like activity emerges. We discovered that V1 R can be subdivided into different clusters before E13.5 and that many cells are capable of sustaining repetitive firing or plateau potentials. These V1 R then lose transiently this firing ability, which is later recovered at E16.5. This is in contrast to the classical developmental scheme, i.e. an increase of firing capability with time. Combining pharmacology and computational modeling, we showed that the firing patterns of embryonic V1 R rely on the synergy between two opposing voltage-dependent currents, namely the slowly inactivating persistent sodium current and the delayed rectifier potassium current. These two currents are responsible for the repetitive firing of action potentials in a vast majority of neurons. Such a synergy is sufficient to explain the clustering of embryonic V1 R and determines the developmental trajectories of the electrical phenotype. Taken together our findings reveal a simple mechanism that is at the core of the heterogeneity of firing patterns. Such a mechanism must be tuned later on to eventually achieve firing pattern homogeneity. Author summary 61A remarkable feature of the developing central nervous system is the generation, at the onset 62 of synaptogenesis, of a spontaneous network activity that is essential for the correct 63 development of neuronal networks. Although the role of this early spontaneous network 64 activity was extensively studied, little is known about the electrophysiological intrinsic 65properties of the key neurons involved in the genesis of this network activity. In the 66 embryonic spinal cord, this activity involves a recurrent functional loop between motoneurons 67 and GABAergic interneurons. We previously showed that embryonic Renshaw cells, known 68 to control motoneurons firing in the mature spinal cord, are the first spinal neurons to produce 69 GABA and likely participate to the genesis of the early spontaneous network activity. Using 70 multip...

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