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

In the developing central nervous system, electrical signaling is thought to rely exclusively on differentiating neurons as they acquire the ability to generate and propagate action potentials. Accordingly, neuroepithelial progenitors (NEPs), which give rise to all neurons and glial cells during development, have been reported to remain electrically passive. Here, we investigated the physiological properties of NEPs at the onset of spontaneous neural activity (SNA) initiating motor behavior in mouse embryonic spinal cord. Using patch-clamp recordings, we discovered that spinal NEPs exhibit spontaneous membrane depolarizations during episodes of SNA. These rhythmic depolarizations exhibited a ventral-to-dorsal gradient with the highest amplitude located in the floor plate, the ventral-most part of the neuroepithelium. Paired recordings revealed that NEPs are coupled via gap junctions and form an electrical syncytium. Although other NEPs were electrically passive, we discovered that floor-plate NEPs generated large Na+/Ca2+ action potentials. Unlike in neurons, floor-plate action potentials relied primarily on the activation of voltage-gated T-type calcium channels (TTCCs). In situ hybridization showed that all 3 known subtypes of TTCCs are predominantly expressed in the floor plate. During SNA, we found that acetylcholine released by motoneurons rhythmically triggers floor-plate action potentials by acting through nicotinic acetylcholine receptors. Finally, by expressing the genetically encoded calcium indicator GCaMP6f in the floor plate, we demonstrated that neuroepithelial action potentials are associated with calcium waves and propagate along the entire length of the spinal cord. Our work reveals a novel physiological mechanism to generate and propagate electrical signals across a neural structure independently from neurons.

Persistent Sodium Current Drives Excitability of Immature Renshaw Cells in Early Embryonic Spinal Networks.

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 (V1R) activity at the onset of SNA (E12.5) in the embryonic mouse SC (both sexes). V1R are one of the interneurons known to contact MNs, which are generated early in the embryonic SC. Here, we show that V1R already produce GABA in E12.5 embryo, and that V1R 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 V1R 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 (INap). This is the first demonstration that INap 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 INaP, 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 INaP in embryonic SC neurons may play a role in the early development of mammalian locomotor networks.SIGNIFICANCE STATEMENT 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 (V1R) are GABAergic at E12.5 and spontaneously active during SNA. We uncover a new role for persistent sodium currents (INaP) in driving plateau potential in V1R and in SNA patterning in the embryonic SC. Our study thus sheds light on a role for INaP in the excitability of V1R and the developing SC.

Axoglial synapses are formed onto pioneer oligodendrocyte precursor cells at the onset of spinal cord gliogenesis.

Virtually all oligodendrocyte precursors cells (OPCs) receive glutamatergic and/or GABAergic synapses that are lost upon their differentiation into oligodendrocytes in the postnatal and adult brain. Although OPCs are generated at mid-embryonic stages, several weeks before the onset of myelination, it remains unknown when and where OPCs receive their first synapses and become susceptible to the influence of neuronal activity. In the embryonic spinal cord, neuro-epithelial precursors in the pMN domain cease generating cholinergic motor neurons (MNs) to produce OPCs when the first synapses are formed in the ventral-lateral marginal zone. We discovered that when the first synapses form onto MNs, axoglial synapses also form onto the processes of neuro-epithelial precursors located in the marginal zone as they differentiate into OPCs. After leaving the neuro-epithelium, these pioneer OPCs preferentially accumulate in the marginal zone where they are contacted by functional glutamatergic and GABAergic synapses. Spontaneous activity of these axoglial synapses was significantly potentiated by cholinergic signaling acting through presynaptic nicotinic acetylcholine receptors. Moreover, we discovered that chronic nicotine treatment significantly increases early OPC proliferation and density in the marginal zone. Our results demonstrate that OPCs are contacted by functional synapses as soon as they emerge from their precursor domain and that embryonic spinal cord colonization by OPCs can be regulated by cholinergic signaling acting onto these axoglial synapses.

Age-specific function of α5ß1 integrin in microglial migration during early colonization of the developing mouse cortex.

Microglia, the immune cells of the central nervous system, take part in brain development and homeostasis. They derive from primitive myeloid progenitors that originate in the yolk sac and colonize the brain mainly through intensive migration. During development, microglial migration speed declines which suggests that their interaction with the microenvironment changes. However, the matrix-cell interactions allowing dispersion within the parenchyma are unknown. Therefore, we aimed to better characterize the migration behavior and to assess the role of matrix-integrin interactions during microglial migration in the embryonic brain ex vivo. We focused on microglia-fibronectin interactions mediated through the fibronectin receptor α5ß1 integrin because in vitro work indirectly suggested a role for this ligand-receptor pair. Using 2-photon time-lapse microscopy on acute ex vivo embryonic brain slices, we found that migration occurs in a saltatory pattern and is developmentally regulated. Most importantly, there is an age-specific function of the α5ß1 integrin during microglial cortex colonization. At embryonic day (E) 13.5, α5ß1 facilitates migration while from E15.5, it inhibits migration. These results indicate a developmentally regulated function of α5ß1 integrin in microglial migration during colonization of the embryonic brain.

Cooperative Interactions between 480 kDa Ankyrin-G and EB Proteins Assemble the Axon Initial Segment.

The axon initial segment (AIS) is required for generating action potentials and maintaining neuronal polarity. Significant progress has been made in deciphering the basic building blocks composing the AIS, but the underlying mechanisms required for AIS formation remains unclear. The scaffolding protein ankyrin-G is the master-organizer of the AIS. Microtubules and their interactors, particularly end-binding proteins (EBs), have emerged as potential key players in AIS formation. Here, we show that the longest isoform of ankyrin-G (480AnkG) selectively associates with EBs via its specific tail domain and that this interaction is crucial for AIS formation and neuronal polarity in cultured rodent hippocampal neurons. EBs are essential for 480AnkG localization and stabilization at the AIS, whereas 480AnkG is required for the specific accumulation of EBs in the proximal axon. Our findings thus provide a conceptual framework for understanding how the cooperative relationship between 480AnkG and EBs induces the assembly of microtubule-AIS structures in the proximal axon. SIGNIFICANCE STATEMENT: Neuronal polarity is crucial for the proper function of neurons. The assembly of the axon initial segment (AIS), which is the hallmark of early neuronal polarization, relies on the longest 480 kDa ankyrin-G isoform. The microtubule cytoskeleton and its interacting proteins were suggested to be early key players in the process of AIS formation. In this study, we show that the crosstalk between 480 kDa ankyrin-G and the microtubule plus-end tracking proteins, EBs, at the proximal axon is decisive for AIS assembly and neuronal polarity. Our work thus provides insight into the functional mechanisms used by 480 kDa ankyrin-G to drive the AIS formation and thereby to establish neuronal polarity.

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.

A remarkable feature of early neuronal networks is their endogenous ability to generate spontaneous rhythmic electrical activity independently of any external stimuli. In the mouse embryonic SC, this activity starts at an embryonic age of ∼ 12 d and is characterized by bursts of action potentials recurring every 2-3 min. Although these bursts have been extensively studied using extracellular recordings and are known to play an important role in motoneuron (MN) maturation, the mechanisms driving MN activity at the onset of synaptogenesis are still poorly understood. Because only cholinergic antagonists are known to abolish early spontaneous activity, it has long been assumed that spinal cord (SC) activity relies on a core network of MNs synchronized via direct cholinergic collaterals. Using a combination of whole-cell patch-clamp recordings and extracellular recordings in E12.5 isolated mouse SC preparations, we found that spontaneous MN activity is driven by recurrent giant depolarizing potentials. Our analysis reveals that these giant depolarizing potentials are mediated by the activation of GABA, glutamate, and glycine receptors. We did not detect direct nAChR activation evoked by ACh application on MNs, indicating that cholinergic inputs between MNs are not functional at this age. However, we obtained evidence that the cholinergic dependency of early SC activity reflects a presynaptic facilitation of GABA and glutamate synaptic release via nicotinic AChRs. Our study demonstrates that, even in its earliest form, the activity of spinal MNs relies on a refined poly-synaptic network and involves a tight presynaptic cholinergic regulation of both GABAergic and glutamatergic inputs.

In vivo assembly of the axon initial segment in motor neurons.

The axon initial segment (AIS) is responsible for both the modulation of action potentials and the maintenance of neuronal polarity. Yet, the molecular mechanisms controlling its assembly are incompletely understood. Our study in single electroporated motor neurons in mouse embryos revealed that AnkyrinG (AnkG), the AIS master organizer, is undetectable in bipolar migrating motor neurons, but is already expressed at the beginning of axonogenesis at E9.5 and initially distributed homogeneously along the entire growing axon. Then, from E11.5, a stage when AnkG is already apposed to the membrane, as observed by electron microscopy, the protein progressively becomes restricted to the proximal axon. Analysis on the global motor neurons population indicated that Neurofascin follows an identical spatio-temporal distribution, whereas sodium channels and ß4-spectrin only appear along AnkG(+) segments at E11.5. Early patch-clamp recordings of individual motor neurons indicated that at E12.5 these nascent AISs are already able to generate spikes. Using knock-out mice, we demonstrated that neither ß4-spectrin nor Neurofascin control the distal-to-proximal restriction of AnkG.

Nav1.1 is predominantly expressed in nodes of Ranvier and axon initial segments.

Aggregation of voltage-gated sodium (Nav) channels in the axon initial segment (AIS) and nodes of Ranvier is essential for the generation and propagation of action potentials. From the three Nav channel isoforms (Nav1.1, Nav1.2 and Nav1.6) expressed in the adult CNS, Nav1.1 appears to play an important function since numerous mutations in its coding sequence cause epileptic syndromes. Yet, its distribution is still controversial. Here we demonstrate for the first time that in the adult CNS Nav1.1 is expressed in nodes of Ranvier throughout the mouse spinal cord and in many brain regions. We identified three populations of nodes: expressing Nav1.1, Nav1.6 or both. We also found Nav1.1 expression concentrated in a proximal AIS subcompartment in spinal cord neurons including 80% of motor neurons and in multiple brain areas. This novel distribution suggests that Nav1.1 is involved in the control of action potential generation and propagation.

VEGF-C is a trophic factor for neural progenitors in the vertebrate embryonic brain.

Vascular endothelial growth factor C (VEGF-C) was first identified as a regulator of the vascular system, where it is required for the development of lymphatic vessels. Here we report actions of VEGF-C in the central nervous system. We detected the expression of the VEGF-C receptor VEGFR-3 in neural progenitor cells in Xenopus laevis and mouse embryos. In Xenopus tadpole VEGF-C knockdowns and in mice lacking Vegfc, the proliferation of neural progenitors expressing VEGFR-3 was severely reduced, in the absence of intracerebral blood vessel defects. In addition, Vegfc-deficient mouse embryos showed a selective loss of oligodendrocyte precursor cells (OPCs) in the embryonic optic nerve. In vitro, VEGF-C stimulated the proliferation of OPCs expressing VEGFR-3 and nestin-positive ventricular neural cells. VEGF-C thus has a new, evolutionary conserved function as a growth factor selectively required by neural progenitor cells expressing its receptor VEGFR-3.

Oligodendrocyte development in the embryonic brain: the contribution of the plp lineage.

Oligodendrocytes are the myelin forming cells of the central nervous system. Over the last decade, their development in the embryonic brain and spinal cord has been documented following the discovery of early oligodendroglial markers. This review highlights the fundamental results obtained on the specification and migration of oligodendroglial cells and illustrates our advances in the knowledge of the cell lineage expressing plp (proteolipid protein), one of the early oligodendroglial genes.

Directional guidance of oligodendroglial migration by class 3 semaphorins and netrin-1.

Oligodendrocytes, the myelin-forming cells of the CNS, are generated from multiple foci distributed along the developing neural tube. Little is known about the endogenous guidance cues controlling the migration of oligodendrocyte precursor cells (OPCs) from their site of emergence toward their final destination, mainly the future white matter tracts. During embryonic development, the optic nerve is populated by OPCs originating in the diencephalon that migrate from the chiasm toward the retina. Here we show that OPCs migrating into the embryonic optic nerve express the semaphorin receptors neuropilin-1 and -2, as well as deleted in colorectal cancer (DCC) and, to a lesser extend unc5H1, two of the netrin-1 receptors. Using a functional migration assay, we provide evidence that Sema 3A and netrin-1 exert opposite chemotactic effects, repulsive or attractive, respectively, on embryonic OPCs. In addition, we show that Sema 3F has a dual effect, chemoattractive and mitogenic on embryonic OPCs. The localization of cells expressing Sema 3A, Sema 3F, and netrin-1 is consistent with a role for these ligands in the migration of OPCs in the embryonic optic nerve. Altogether, our results suggest that the migration of OPCs in the embryonic optic nerve is modulated by a balance of effects mediated by members of the semaphorin and netrin families.