Acceleration of conduction velocity linked to clustering of nodal components precedes myelination

High-density accumulation of voltage-gated sodium (Na_v) channels at nodes of Ranvier ensures rapid saltatory conduction along myelinated axons. To gain insight into mechanisms of node assembly in the CNS, we focused on early steps of nodal protein clustering. We show in hippocampal cultures that prenodes (i.e., clusters of Na_v channels colocalizing with the scaffold protein ankyrinG and nodal cell adhesion molecules) are detected before myelin deposition along axons. These clusters can be induced on purified neurons by addition of oligodendroglial-secreted factor(s), whereas ankyrinG silencing prevents their formation. The Na_v isoforms Na_v1.1, Na_v1.2, and Na_v1.6 are detected at prenodes, with Na_v1.6 progressively replacing Na_v1.2 over time in hippocampal neurons cultured with oligodendrocytes and astrocytes. However, the oligodendrocyte-secreted factor(s) can induce the clustering of Na_v1.1 and Na_v1.2 but not of Na_v1.6 on purified neurons. We observed that prenodes are restricted to GABAergic neurons, whereas clustering of nodal proteins only occurs concomitantly with myelin ensheathment on pyramidal neurons, implying separate mechanisms of assembly among different neuronal subpopulations. To address the functional significance of these early clusters, we used single-axon electrophysiological recordings in vitro and showed that prenode formation is sufficient to accelerate the speed of axonal conduction before myelination. Finally, we provide evidence that prenodal clusters are also detected in vivo before myelination, further strengthening their physiological relevance.Voltage-gated sodium (Na_v) channels are highly enriched at the axon initial segment (AIS) and the node of Ranvier, allowing generation and rapid propagation of action potentials by saltatory conduction in myelinated fibers. These axonal domains also contain cell adhesion molecules e.g., neurofascin 186 (Nfasc186)] and the scaffolding proteins ankyrinG (AnkG) and βIV spectrin, which provide a potential link with the actin cytoskeleton (1). Flanking the nodes are the paranodes, where axoglial junctions between paranodal myelin loops and the axon are formed through interactions between axonal contactin-associated protein (Caspr)/contactin and glial Nfasc155 (2, 3). Although the mechanisms of nodal assembly are best characterized in the peripheral nervous system (4–9), less is known about the cellular and molecular mechanisms underlying node assembly in the CNS. ECM proteins, adhesion molecules, such as Nfasc186, and also, axoglial paranodal junctions have been shown to trigger CNS nodal clustering, although their respective roles remain uncertain (10–19). Moreover, axonal clustering of Na_v channels before myelin deposition and oligodendroglial contact has been shown to occur in retinal ganglion cell (RGC) cultures, where these clusters were induced by oligodendroglial-secreted factor(s) (20, 21).Here, we have investigated the cellular and molecular mechanisms underlying nodal protein assembly in hippocampal neuron cultures. We first showed that evenly spaced clusters of Na_v channels, colocalizing with Nfasc186 and AnkG, are detected along axons before myelination. Strikingly, this prenode assembly is restricted to GABAergic interneurons, suggesting the existence of different mechanisms of nodal assembly. The prenodal clustering can be induced on purified neurons by the addition of oligodendroglial-secreted factor(s) and also depends on intrinsic cues, such as AnkG. Furthermore, we also provide evidence that these clusters are detected in vivo before myelination on hippocampal tissue sections. Finally, to gain insight into their functional significance, we performed in vitro simultaneous somatic and axonal recordings and showed that the presence of prenodes increases the speed of action potential propagation along axons before myelination.