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.

Characterization of the axon initial segment (AIS) of motor neurons and identification of a para-AIS and a juxtapara-AIS, organized by protein 4.1B.

BACKGROUND: The axon initial segment (AIS) plays a crucial role: it is the site where neurons initiate their electrical outputs. Its composition in terms of voltage-gated sodium (Nav) and voltage-gated potassium (Kv) channels, as well as its length and localization determine the neuron's spiking properties. Some neurons are able to modulate their AIS length or distance from the soma in order to adapt their excitability properties to their activity level. It is therefore crucial to characterize all these parameters and determine where the myelin sheath begins in order to assess a neuron's excitability properties and ability to display such plasticity mechanisms. If the myelin sheath starts immediately after the AIS, another question then arises as to how would the axon be organized at its first myelin attachment site; since AISs are different from nodes of Ranvier, would this particular axonal region resemble a hemi-node of Ranvier? RESULTS: We have characterized the AIS of mouse somatic motor neurons. In addition to constant determinants of excitability properties, we found heterogeneities, in terms of AIS localization and Nav composition. We also identified in all α motor neurons a hemi-node-type organization, with a contactin-associated protein (Caspr)+ paranode-type, as well as a Caspr2+ and Kv1+ juxtaparanode-type compartment, referred to as a para-AIS and a juxtapara (JXP)-AIS, adjacent to the AIS, where the myelin sheath begins. We found that Kv1 channels appear in the AIS, para-AIS and JXP-AIS concomitantly with myelination and are progressively excluded from the para-AIS. Their expression in the AIS and JXP-AIS is independent from transient axonal glycoprotein-1 (TAG-1)/Caspr2, in contrast to juxtaparanodes, and independent from PSD-93. Data from mice lacking the cytoskeletal linker protein 4.1B show that this protein is necessary to form the Caspr+ para-AIS barrier, ensuring the compartmentalization of Kv1 channels and the segregation of the AIS, para-AIS and JXP-AIS. CONCLUSIONS: α Motor neurons have heterogeneous AISs, which underlie different spiking properties. However, they all have a para-AIS and a JXP-AIS contiguous to their AIS, where the myelin sheath begins, which might limit some AIS plasticity. Protein 4.1B plays a key role in ensuring the proper molecular compartmentalization of this hemi-node-type region.

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.

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.

Expression of Nav1.6 sodium channels by Schwann cells at neuromuscular junctions: role in the motor endplate disease phenotype.

In addition to their role in action potential generation and fast synaptic transmission in neurons, voltage-dependent sodium channels can also be active in glia. Terminal Schwann cells (TSCs) wrap around the nerve terminal arborization at the neuromuscular junction, which they contribute to shape during development and in the postdenervation processes. Using fluorescent in situ hybridization (FISH), immunofluorescence, and confocal microscopy, we detected the neuronal Nav1.6 sodium channel transcripts and proteins in TSCs in normal adult rats and mice. Nav1.6 protein co-localized with the Schwann cell marker S-100 but was not detected in the SV2-positive nerve terminals. The med phenotype in mice is due to a mutation in the SCN8A gene resulting in loss of Nav1.6 expression. It leads to early onset in postnatal life of defects in neuromuscular transmission with minimal alteration of axonal conduction. Strikingly, in mutant mice, the nonmyelinated pre-terminal region of axons showed abundant sprouting at neuromuscular junctions, and most of the alpha-bungarotoxin-labeled endplates were devoid of S-100- or GFAP-positive TSCs. Using specific antibodies against the Nav1.2 and Nav1.6 sodium channels, ankyrin G and Caspr 1, and a pan sodium channel antibody, we found that a similar proportion of ankyrin G-positive nodes of Ranvier express sodium channels in mutant and wild-type animals and that nodal expression of Nav1.2 persists in med mice. Our data supports the hypothesis that the lack of expression of Nav1.6 in Schwann cells at neuromuscular junctions might play a role in the med phenotype.

Differential targeting and functional specialization of sodium channels in cultured cerebellar granule cells.

The ion channel dynamics that underlie the complex firing patterns of cerebellar granule (CG) cells are still largely unknown. Here, we have characterized the subcellular localization and functional properties of Na+ channels that regulate the excitability of CG cells in culture. As evidenced by RT-PCR and immunocytochemical analysis, morphologically differentiated CG cells expressed Nav1.2 and Nav1.6, though both subunits appeared to be differentially regulated. Nav1.2 was localized at most axon initial segments (AIS) of CG cells from 8 days in vitro DIV 8 to DIV 15. At DIV 8, Nav1.6 was found uniformly throughout somata, dendrites and axons with occasional clustering in a subset of AIS. Accumulation of Nav1.6 at most AIS was evident by DIV 13-14, suggesting it is developmentally regulated at AIS. The specific contribution of these differentially distributed Na+ channels has been assessed using a combination of methods that allowed discrimination between functionally compartmentalized Na+ currents. In agreement with immunolocalization, we found that fast activating-fully inactivating Na+ currents predominate at the AIS membrane and in the somatic plasma membrane.

Molecular determinants of emerging excitability in rat embryonic motoneurons.

Molecular determinants of excitability were studied in pure cultures of rat embryonic motoneurons. Using RT-PCR, we have shown here that the spike-generating Na(+) current is supported by Nav1.2 and/or Nav1.3 alpha-subunits. Nav1.1 and Nav1.6 transcripts were also identified. We have demonstrated that alternatively spliced isoforms of Nav1.1 and Nav1.6, resulting in truncated proteins, were predominant during the first week in culture. However, Nav1.6 protein could be detected after 12 days in vitro. The Nav beta 2.1 transcript was not detected, whereas the Nav beta 1.1 transcript was present. Even in the absence of Nav beta 2.1, alpha-subunits were correctly inserted into the initial segment. RT-PCR (at semi-quantitative and single-cell levels) and immunocytochemistry showed that transient K(+) currents result from the expression of Kv4.2 and Kv4.3 subunits. This is the first identification of subunits responsible for a transient K(+) current in spinal motoneurons. The blockage of Kv4.2/Kv4.3 using a specific toxin modified the shape of the action potential demonstrating the involvement of these conductance channels in regulating spike repolarization and the discharge frequency. Among the other Kv alpha-subunits (Kv1.3, 1.4, 1.6, 2.1, 3.1 and 3.3), we showed that the Kv1.6 subunit was partly responsible for the sustained K(+) current. In conclusion, this study has established the first correlation between the molecular nature of voltage-dependent Na(+) and K(+) channels expressed in embryonic rat motoneurons in culture and their electrophysiological characteristics in the period when excitability appears.

Voltage-gated Na+ channel activation induces both action potentials in utricular hair cells and brain-derived neurotrophic factor release in the rat utricle during a restricted period of development.

The mammalian utricular sensory receptors are commonly believed to be non-spiking cells with electrical activity limited to graded membrane potential changes. Here we provide evidence that during the first post-natal week, the sensory hair cells of the rat utricle express a tetrodotoxin (TTX)-sensitive voltage-gated Na+ current that displays most of the biophysical and pharmacological characteristics of neuronal Na+ current. Single-cell RT-PCR reveals that several alpha-subunit isoforms of the Na+ channels are co-expressed within a single hair cell, with a major expression of Nav1.2 and Nav1.6 subunits. In neonatal hair cells, 30 % of the Na+ channels are available for activation at the resting potential. Depolarizing current injections in the range of the transduction currents are able to trigger TTX-sensitive action potentials. We also provide evidence of a TTX-sensitive activity-dependent brain-derived neurotrophic factor (BDNF) release by early post-natal utricle explants. Developmental analysis shows that Na+ currents decrease dramatically from post-natal day 0 (P0) to P8 and become almost undetectable at P21. Concomitantly, depolarizing stimuli fail to induce both action potential and BDNF release at P20. The present findings reveal that vestibular hair cells express neuronal-like TTX-sensitive Na+ channels able to generate Na+-driven action potentials only during the early post-natal period of development. During the same period an activity-dependent BDNF secretion by utricular explants has been demonstrated. This could be an important mechanism involved in vestibular sensory system differentiation and synaptogenesis.

Interaction of the Nav1.2a subunit of the voltage-dependent sodium channel with nodal ankyrinG. In vitro mapping of the interacting domains and association in synaptosomes.

Voltage-dependant sodium channels at the axon initial segment and nodes of Ranvier colocalize with the nodal isoforms of ankyrin(G) (Ank(G) node). Using fusion proteins derived from the intracellular regions of the Nav1.2a subunit and the Ank repeat domain of Ank(G) node, we mapped a major interaction site in the intracellular loop separating alpha subunit domains I-II. This 57-amino acid region binds the Ank repeat region with a K(D) value of 69 nm. We identified another site in intracellular loop III-IV, and we mapped both Nav1.2a binding sites on the ankyrin repeat domain to the region encompassing repeats 12-22. The ankyrin repeat domain did not bind the beta(1) and beta(2) subunit cytoplasmic regions. We showed that in cultured embryonic motoneurons, expression of the beta(2) subunit is not necessary for the colocalization of Ank(G) node with functional sodium channels at the axon initial segment. Antibodies directed against the beta(1) subunit intracellular region, alpha subunit loop III-IV, and Ank(G) node could not co-immunoprecipitate Ank(G) node and sodium channels from Triton X-100 solubilisates of rat brain synaptosomes. Co-immunoprecipitation of sodium channel alpha subunit and of the 270- and 480-kDa AnkG node isoforms was obtained when solubilization conditions that maximize membrane protein extraction were used. However, we could not find conditions that allowed for co-immunoprecipitation of ankyrin with the sodium channel beta(1) subunit.