Functional reciprocity between Na⁺ channel Na_v1.6 and β1 subunits in the coordinated regulation of excitability and neurite outgrowth

Abstract

Voltage-gated Na⁺ channel (VGSC) β1 subunits regulate cell–cell adhesion and channel activity in vitro. We previously showed that β1 promotes neurite outgrowth in cerebellar granule neurons (CGNs) via homophilic cell adhesion, fyn kinase, and contactin. Here we demonstrate that β1-mediated neurite outgrowth requires Na⁺ current (I_Na) mediated by Na_v1.6. In addition, β1 is required for high-frequency action potential firing. Transient I_Na is unchanged in Scn1b (β1) null CGNs; however, the resurgent I_Na, thought to underlie high-frequency firing in Na_v1.6-expressing cerebellar neurons, is reduced. The proportion of axon initial segments (AIS) expressing Na_v1.6 is reduced in Scn1b null cerebellar neurons. In place of Na_v1.6 at the AIS, we observed an increase in Na_v1.1, whereas Na_v1.2 was unchanged. This indicates that β1 is required for normal localization of Na_v1.6 at the AIS during the postnatal developmental switch to Na_v1.6-mediated high-frequency firing. In agreement with this, β1 is normally expressed with α subunits at the AIS of P14 CGNs. We propose reciprocity of function between β1 and Na_v1.6 such that β1-mediated neurite outgrowth requires Na_v1.6-mediated I_Na, and Na_v1.6 localization and consequent high-frequency firing require β1. We conclude that VGSC subunits function in macromolecular signaling complexes regulating both neuronal excitability and migration during cerebellar development.

Voltage-gated Na⁺ channels (VGSCs), composed of one pore-forming α subunit and two β subunits (1), are responsible for initiation and conduction of action potentials (APs) (2). Of the nine α subunits (3), Na_v1.1, Na_v1.2, and Na_v1.6 are found in the postnatal CNS (4, 5) where they display developmentally regulated expression patterns in specialized neuronal subcellular domains. For example, Na_v1.1 and Na_v1.2 are replaced during postnatal development at the axon initial segment (AIS) and nodes of Ranvier by Na_v1.6. Scn8a null mice display motor dysfunction, ataxia, and lethality by postnatal day (P) 21, suggesting that this developmental switch to Na_v1.6 expression in brain is critical (6, 7). Scn8a null retinal ganglion neurons display impaired excitability, demonstrating that Na_v1.6 is vital for high-frequency firing (8 –10). Thus, VGSC-driven neuronal activity is important for proper CNS development, although the underlying mechanism(s) are not well understood.

VGSC β1 subunits are multifunctional molecules that modulate channel kinetics and gating, regulate channel cell surface expression, and participate in cell–cell adhesion in vitro (11). Scn1b (β1) null mice are ataxic, experience spontaneous seizures, and exhibit a prolonged cardiac QT interval, demonstrating that β1 modulates electrical excitability in vivo (12, 13). Consistent with this, human mutations in SCN1B result in epilepsy and arrhythmia (14 –21). As a member of the Ig superfamily of cell adhesion molecules (CAMs), β1 mediates cellular aggregation, cytoskeletal recruitment, and extracellular matrix interactions in vitro (11). β1 also promotes neurite outgrowth in cerebellar granule neurons (CGNs) via a lipid raft mechanism involving trans homophilic cell adhesion, fyn kinase, and contactin (22, 23). Axonal path finding and fasciculation are disrupted in Scn1b null mice and zebrafish scn1bb morphants (22, 24). Thus, β1 functions as a CAM to regulate neuronal migration, axon guidance, and fasciculation during CNS development. Disruptions in these critical processes likely contribute to disease in patients with SCN1B mutations. The next step in understanding the physiological role of β1 in vivo is to investigate whether β1-mediated modulation of I_Na and β1-mediated cell–cell adhesion are interrelated.

The aims of the present study were to assess the role of I_Na in β1-mediated neurite outgrowth and to investigate the effect(s) of β1 on I_Na and neuronal excitability. We demonstrate that β1 and Na_v1.6 have reciprocal functions in vivo, such that β1-mediated neurite outgrowth requires Na_v1.6-mediated I_Na, and Na_v1.6 localization and consequent high-frequency firing require β1. Our results demonstrate a requirement for partnering of specific VGSC α and β subunits during CNS development in vivo.

Results and Discussion

Tetrodotoxin and the Scn8a Null Mutation Inhibit β1-Mediated Neurite Outgrowth.

Electrical activity mediated by VGSCs regulates neuronal migration and development (25). The possible role of I_Na in β1-mediated neurite outgrowth has not yet been studied, however. Similarly, the potential involvement of β1 in activity-dependent neuronal development is unknown. To investigate whether I_Na is involved in β1-mediated neurite outgrowth, we isolated CGNs from P14 wild-type (WT) mice and cultured them on monolayers of control Chinese hamster lung (CHL) cells, which do not express β subunits, or on CHL cells stably expressing β1. β1–β1 trans-homophilic adhesion increased the average neurite length by 1.7-fold, as reported previously (P < 0.001) (23). Inclusion of tetrodotoxin (TTX) during the assay inhibited β1-mediated neurite outgrowth; this occurred across the entire neurite length distribution (Fig. 1 A and B). TTX had no effect on fibroblast growth factor (FGF; 20 ng/mL)-mediated neurite outgrowth, which is β1-independent (P < 0.001; Fig. 1 C and D) (22). Furthermore, FGF had no additive effect on the neurite outgrowth of CGNs plated on β1-expressing monolayers, suggesting that FGF-mediated and β1-mediated neurite outgrowth mechanisms ultimately activate a common signaling pathway (P = 0.57; n = 300). Thus, I_Na is specifically required for β1-mediated, but not FGF-mediated, neurite outgrowth.

Fig. 1.

β1-mediated neurite outgrowth is inhibited by TTX and the Scn8a null mutation. (A) Neurite length of WT CGNs grown on CHL or CHL-β1 monolayers and treated with/without TTX (10 μM) for 48 h (n = 20). (B) Neurite distribution (%) plotted against neurite length for CGNs in A. (C) Neurite length of WT CGNs grown on CHL monolayers and treated with/without TTX and/or FGF (20 ng/mL) for 48 h (n = 300). (D) Neurite distribution (%) plotted against neurite length for CGNs in C. (E) Neurite lengths of WT and Scn8a null CGNs grown on CHL or CHL-β1 monolayers (n = 300). (F) Neurite distribution (%) plotted against neurite length for CGNs in E. (G) Neurite lengths of WT and Scn8a null CGNs grown on CHL monolayers and treated with/without FGF for 48 h (n = 300). (H) Neurite distribution (%) plotted against neurite length for CGNs in G. Data are mean ± SEM. ***P < 0.001, ANOVA with Tukey’s post hoc test. (I) Western blot of WT and Scn8a null cerebellar membrane protein, using anti-β1. Anti-α-tubulin was used as a loading control.

We next used neurons isolated from Scn8a null mice to test whether Na_v1.6 is required for I_Na-dependent, β1-mediated neurite outgrowth. β1 in the monolayer increased the neurite length of WT CGNs by 1.7-fold after 24 h (P < 0.001). In contrast, no increase in neurite length was seen in Scn8a null CGNs grown on β1-expressing monolayers (Fig. 1 E and F). FGF-dependent neurite outgrowth was not affected by the Scn8a null mutation (Fig. 1G). The neurite length distribution was increased by FGF in both WT and Scn8a null CGNs (Fig. 1H), demonstrating that, similar to Scn1b neurons (22), Scn8a null neurons are not deficient in downstream mechanisms of neurite outgrowth that converge between lipid raft-mediated and non–raft-mediated pathways (26). β1 protein expression was unchanged in the cerebella of Scn8a null mice, suggesting that the lack of response of Scn8a null CGNs to the β1 monolayer was not due to a reduction in β1 in the CGNs (Fig. 1I). These findings suggest that β1-mediated, but not FGF-mediated, neurite outgrowth requires I_Na and Na_v1.6 expression.

Scn1b Null CGNs Have Impaired Excitability.

Given that Scn1b mutations result in channelopathies in vivo (12 –21), and that β1-mediated neurite outgrowth in CGNs requires I_Na and Na_v1.6, we postulated that β1 might regulate electrical excitability in the cerebellum. To test this, we recorded APs in P12-13 WT and Scn1b null CGNs in cerebellar slices by whole-cell patch clamping. WT and Scn1b null CGNs typically had small membrane capacitance (Table S1) and lacked spontaneous firing. Both WT and Scn1b null CGNs displayed normal inward and outward currents (Fig. 2A and Table S1). Resting potential, input resistance, and AP parameters were similar in the WT and Scn1b null CGNs (Table S1).

Fig. 2.

Electrical excitability is impaired in Scn1b null CGNs in vivo. (A) Voltage (V)-gated currents in a (i) WT and (ii) Scn1b null CGN following depolarization to 0 mV. (B) Membrane V recordings from (i) WT and (ii) Scn1b null CGNs following current (I) injection. Upper traces represent CGNs that sustained repetitive firing during 500 ms; lower traces represent CGNs that failed. (C) AP firing rate plotted as a function of I, normalized to AP threshold for WT (filled circles) and Scn1b null (open circles) CGNs. Data are mean ± SEM (n ≥ 15). *P < 0.05; **P < 0.01; ***P < 0.001, t test. (D) Distribution of APs across the 500-ms I injection for WT (dark bars) and Scn1b null (light bars) CGNs. For each CGN, the number of APs was counted in each 100-ms interval for the sweep giving maximum firing frequency. Data are mean ± SEM (n ≥ 16). P < 0.001 between WT and Scn1b null (two-way ANOVA). (E) Instantaneous firing frequency (Inst. freq., in Hz), calculated as the reciprocal of the interval between two adjacent APs, for WT (filled circles) and Scn1b null (open circles) CGNs, plotted for the first 10 AP intervals. Data are expressed as mean ± SEM (n ≥ 11). P < 0.001 between WT and Scn1b null (two-way ANOVA).

Both WT and Scn1b null CGNs were capable of sustained repetitive firing, although some failed within the 500-ms pulse (Fig. 2B). The number of spikes escalated in response to increasing current, although the firing rate was consistently reduced in Scn1b null CGNs at all input intensities (Fig. 2C). Accordingly, the maximum firing rate across the 500-ms current pulse was reduced in Scn1b null CGNs (P < 0.05), as was the mean number of spikes fired before failure (P < 0.01) (Table S1). In addition, the maximum number of APs fired within each 100 ms interval was smaller in Scn1b null CGNs (P <.001; Fig. 2D), and the maximum instantaneous firing frequency (inverse of the interspike interval) was significantly reduced in Scn1b null CGNs (P < 0.001; Fig. 2E).

These findings suggest that β1 regulates CGN excitability in situ. CGNs lacking β1 were less able to sustain prolonged high-frequency firing over a range of input intensities. Furthermore, CGNs lacking β1 were less efficient in generating APs at short interspike intervals. We postulated that the impaired excitability of Scn1b null CGNs might be due to altered I_Na, altered VGSC expression, or both. We then tested these possibilities.

Resurgent, but not Transient, I_Na Is Reduced in Scn1b Null CGNs.

We recorded I_Na in P14 dissociated CGNs under whole-cell voltage clamp 24 h after plating. Both WT and Scn1b null dissociated CGNs expressed robust I_Na (Fig. S1A). The absence of β1 had no effect on peak I_Na density, voltage dependence or kinetics (Fig. S1 B–D and Table S2). Contactin, which is required for β1-mediated neurite outgrowth, interacts with the β1 Ig loop and increases VGSC cell surface expression (22, 27). Cntn null mice have significantly reduced brain levels of β1, are ataxic, and survive only to P21, suggesting that contactin is required for normal cerebellar excitability (22, 28). However, here the Cntn null mutation had no effect on transient I_Na in P12 dissociated CGNs (Fig. S2 and Table S3).

One problem encountered when recording I_Na in acutely dissociated neurons is that processes present in vivo are lost during the extraction (29). This may hinder the ability to detect any alteration in axonal VGSC expression in Scn1b null CGNs in vivo that might underlie their impaired excitability. To address this issue, we recorded I_Na in CGNs cultured for 14 DIV with axonal processes intact (30, 31). As in dissociated CGNs, transient I_Na was unchanged in 14 DIV Scn1b null CGNs (Table S4). Persistent I_Na also was unchanged in 14 DIV Scn1b null CGNs (Fig. 3 A and C).

Fig. 3.

Resurgent Na⁺ current (I_Na) is reduced in Scn1b null CGNs cultured for 14 days in vitro (DIV). (A) Whole-cell I_Na from WT (black), and Scn1b null (gray) CGNs elicited by 60-ms depolarizing voltage (V) pulses to −40 mV normalized to peak transient I (%). Inset, I persisting for 50–55 ms after onset of depolarization. (B) Resurgent I from WT (black) and Scn1b null (gray) CGNs elicited by repolarization to −30 mV following a depolarizing pulse to +30 mV for 20 ms, normalized to peak transient I at +30 mV (%). (C) Persistent I measured as the mean I at 50–55 ms after onset of depolarization (Left) and resurgent I (Right). Data are presented as mean ± SEM (n ≥ 19). *P = 0.01 (t test). (D) Western blots of WT and Scn1b null cerebellar membrane protein using anti-β4 and anti-β2. Anti–α-tubulin was used as a loading control.

In CGNs, resurgent I_Na flows after membrane repolarization, due to the release of an endogenous open-channel blocker, which rapidly terminates the transient I_Na during strong depolarizations (32). This resurgent I_Na is proposed to promote repetitive firing by rapidly restoring VGSC availability (32). We found that resurgent I_Na was reduced in 14 DIV Scn1b null CGNs (P = 0.01; Fig. 3 B and C). A peptide corresponding to the cytoplasmic domain of β4 mediates resurgent current in Purkinje neurons in vitro, implicating β4 in this mechanism (33). β4 protein levels were unchanged in Scn1b null cerebella, suggesting that the reduced resurgent I_Na in Scn1b null CGNs was not due to altered β4 expression (Fig. 3D), although the absence of β1 may change the conformation of α–β4 interactions (34). The level of β2 protein was reduced by 39% in Scn1b null cerebella (P < 0.05; Fig. 3D), although its subcellular distribution remained unchanged (Fig. S3). In summary, reduced resurgent I_Na in Scn1b null CGNs is consistent with reduced repetitive firing and supports the hypothesis that the impaired excitability of Scn1b null CGNs in situ is caused by altered I_Na.

Na_v1.6 Is Reduced at the AIS of Scn1b Null CGNs.

We next tested the idea that impaired excitability of Scn1b null CGNs may be caused by altered VGSC expression. Given that Na_v1.6 is essential for high-frequency repetitive firing and carries resurgent I_Na in Purkinje neurons (8, 10), we hypothesized that axonal localization of Na_v1.6 might be altered in Scn1b null CGNs. Consistent with previous work, ankyrin G (Ank_G) and Na_v1.6 were strongly coexpressed at the AIS in 14 DIV WT CGNs (Fig. 4A) (31, 35). In contrast, the proportion of Ank_G-positive AIS expressing Na_v1.6 was reduced in Scn1b null CGNs (P < 0.001; Fig. 4 A and E). Our data cannot rule out the possibility that Na_v1.6 may still be weakly expressed at the AIS of Scn1b null CGNs, albeit at levels below the limit of detection. Nonetheless, our results suggest that β1 is required for efficient localization of Na_v1.6 at the AIS. We propose that β1 may provide an additional link between Ank_G and Na_v1.6 during AIS maturation. In Scn8a null mice, there is a neuron-specific compensatory increase in Na_v1.1 and Na_v1.2 at the AIS (8). We investigated whether a similar up-regulation occurs in Scn1b null CGNs. Both WT and Scn1b null CGNs had strong pan-α subunit immunoreactivity at the AIS (Fig. 4 B and E). In addition, Na_v1.2 was robustly expressed at the AIS of both WT and Scn1b null CGNs (Fig. 4 C and E). In contrast, there was an up-regulation of Na_v1.1-positive AIS in Scn1b null CGNs (P < 0.001; Fig. 4 D and E). We concluded that Na_v1.1, but not Na_v1.2, compensated for the loss of Na_v1.6 at the AIS of Scn1b null CGNs.

Fig. 4.

Na_v1.6 expression is reduced at the AIS of Scn1b null CGNs. (A–D) 100× Z-series confocal projections of WT (i) and Scn1b null (ii) 14 DIV CGNs labeled with anti-ankyrinG (Ank_G) (red) and α subunit antibodies (green): Na_v1.6 (A), pan-α subunit (B), Na_v1.2 (C), and Na_v1.1 (D). (Scale bar: 20 μm.) Arrows point to AIS expressing Ank_G. (E) Proportion of Ank_G-expressing CGN AIS that also express α subunits for WT (dark bars) and Scn1b null (light bars) (n = 60 fields of view taken from three mice of each genotype). Data are mean ± SEM. ***P <.001, Mann-Whitney test.

We next investigated Na_v1.6 expression in cerebellar cryosections from P14 WT and Scn1b null mice. Although Na_v1.6 was expressed in the inner granular layer of both WT and Scn1b null sections, it was not possible to resolve the localization of Na_v1.6 to specific compartments due to the tight compaction of CGNs within this region (Fig. 5A). It was possible to determine Na_v1.6 expression in the soma and at the AIS of Purkinje neurons, however (Fig. 5A) (36). Consistent with 14 DIV CGNs, the proportion of Scn1b null Purkinje neurons expressing Na_v1.6 at the AIS was significantly reduced (P < 0.001; Fig. 5 A and B). These defects in Na_v1.6 expression were observed in all three Scn1b null mice studied compared with WT littermates. β1 was robustly expressed at the AIS of WT calbindin-positive Purkinje neurons in cerebellar slices and colocalized at the AIS of 14 DIV CGNs with α subunits (Fig. 5 C and D). Thus, Na_v1.6 expression at the AIS may require clustering of β1 in this region, similar to the CAMs neurofascin-186 and NrCAM, which also are β1-binding partners (37 –39).

Fig. 5.

Na_v1.6 expression is reduced at the AIS of Scn1b null Purkinje neurons. (A) 100× Z-series confocal projections of WT (i) and Scn1b null (ii) P14 cerebellum. AIS are labeled with anti-Ank_G (red) and anti-Na_v1.6 (green). Insets, 3× zoom of white boxes, showing colocalization of Ank_G and Na_v1.6 at Purkinje neuron AIS in WT, but not in Scn1b null. Arrows indicate Scn1b null Purkinje neuron AIS expressing both Ank_G and Na_v1.6. (B) Proportion of Ank_G-expressing Purkinje neuron AIS that also express Na_v1.6 for WT (dark bars) and Scn1b null (light bars) littermates (n = 60 fields of view from three mice of each genotype). Data are mean ± SEM. ***P < 0.001, t test. (C) 100× Z-series confocal projection of WT Purkinje neuron in situ labeled with anti-calbindin (red) and anti-β1 (green). The arrow shows β1 at AIS. (D) 100× Z-series confocal projection of WT 14 DIV CGN labeled with anti-pan VGSC α subunit (red) and anti-β1 (green). The arrow indicates β1 at AIS. (Scale bars: 20 μm.)

Although cerebellar neurons express TTX-sensitive VGSCs, TTX-resistant (TTX-R) Na_v1.8 is up-regulated under pathophysiological conditions (40 –42). TTX-R Na_v1.5 channels also are up-regulated in Scn1b null ventricular myocytes (12). Consequently, we investigated whether TTX-R channels are up-regulated in CGNs in the absence of β1. TTX (500 nM) completely blocked the I_Na in both WT and Scn1b null CGNs in cerebellar slices (n = 5 each), indicating no up-regulation of TTX-R channels in the absence of β1.

In summary, the proportion of AIS expressing Na_v1.6 was significantly reduced in the absence of β1. In addition, there was an increase in the proportion of AIS expressing Na_v1.1 in Scn1b null CGNs with no changes in Na_v1.2. Our electrophysiological data, together with the observation that β1 is expressed at the AIS of WT CGNs and Purkinje neurons, support the hypothesis that impaired excitability of Scn1b null CGNs is caused by both altered I_Na and altered VGSC expression.

A Model for Activity-Dependent Neurite Outgrowth Regulated by β1 and Na_v1.6.

The present results, combined with previous work, show that β1-mediated neurite outgrowth requires fyn kinase, contactin, Na_v1.6, and I_Na (22). We hypothesized that a complex containing these proteins may be present at the growth cone. We found that whereas VGSC α subunits and β1 were most highly expressed at the AIS of 14 DIV WT CGNs, they also were expressed in the soma, along the neurite, and at the growth cone, defined by phalloidin labeling of F-actin (Fig. 6A) (22). Similar to β1, contactin was localized to the soma, along the neurite, and at the growth cone (Fig. 6B). Interestingly, whereas α subunit immunoreactivity was clearly highest at the AIS, contactin was fairly constant along the axon (Fig. 6B). The arrangement of α subunits, β1, and contactin at the AIS, along the axon, and at the growth cone is consistent with their functions within complexes in these regions that regulate electrical activity on the one hand and neurite outgrowth on the other hand.

Fig. 6.

α subunits, β1, and contactin are present at the growth cone. 100× Z-series confocal projections of WT 14 DIV CGNs labeled with pan-α subunit (green), anti-β1 (A; red) or anti-contactin (B; red), and Alexa 594–conjugated phalloidin (magenta). (Scale bar: 10 μm.) he panels on the right show 4× digital zoom views highlighting growth cone (arrows). These distributions were observed in all three mice studied.

We propose that in immature CGNs, including those used in our neurite outgrowth experiments, Na_v1.6, β1, contactin, fyn kinase and Ank_G are present along the neurite and at the growth cone to promote β1-mediated neurite extension and migration (Fig. 7A) (11, 22, 25). At this stage, the AIS is not yet formed, and Na_v1.6 is proposed to conduct Na⁺ into microdomains surrounding channel complexes in response to localized membrane depolarizations during neurite extension. In our model, this localized Na⁺ influx is specifically required for β1-mediated neurite outgrowth (Fig. 7A). Furthermore, in this model VGSC complexes are present along the entire neurite, permitting adhesion and β1-dependent fasciculation in vivo (22). β1-independent, FGF-mediated neurite outgrowth also would occur.

Fig. 7.

A model for I_Na involvement in β1-mediated neurite outgrowth. (A) In immature CGNs lacking AIS, complexes containing Na_v1.6, β1, and contactin are present throughout the neuronal membrane in the soma, neurite, and growth cone. Localized Na⁺ influx is necessary for β1-mediated neurite extension and migration (11, 22, 25). VGSC complexes along the neurite are proposed to participate in cell–cell adhesion and fasciculation (22). (B) In 14 DIV CGNs, β1 is also required for Na_v1.6 expression at the AIS, and subsequent high-frequency AP firing through modulation of resurgent INa (8, 10, 45). Electrical activity may further promote β1-mediated neurite outgrowth at or near the growth cone in vivo (8, 10, 45). Thus, the developmental functions of β1 and Na_v1.6 are complementary, such that Na⁺ influx carried by Na_v1.6 is required for β1-mediated neurite outgrowth and β1 is required for normal expression/activity of Na_v1.6 at the AIS. Fyn kinase and Ank_G also are likely present in all complexes (11), but they are only shown once in each panel for clarity. The FGF-mediated, β1-independent neurite outgrowth pathway is shown as well. Other CAMs that regulate neurite outgrowth and also may interact with β1 in this system (11, 26), have been omitted for clarity.

β1 is required for normal localization of Na_v1.6 at the AIS, for resurgent I_Na, and for repetitive firing. We postulate that reciprocity of function occurs between β1 and Na_v1.6, in which β1 is required for Na_v1.6-dependent high-frequency firing on the one hand, and Na_v1.6 is required for β1-mediated neurite outgrowth on the other hand. Given that both Scn1b and Scn8a null mice die shortly after the transition to high-frequency firing should it occur, this unique reciprocal relationship between β1 and Na_v1.6 likely is critical to postnatal development (8, 13). Thus, we propose a second, more complex scenario in CGNs in vivo in which the AIS has formed, represented by the 14 DIV CGNs. In this model, complexes of α subunits, β1, contactin, fyn kinase, and Ank_G are expressed along the entire axon, but are concentrated at the AIS and the growth cone to promote β1-mediated neurite extension and migration (Fig. 7B). As in the immature CGNs, here Na_v1.6 permits localized Na⁺ influx required for β1-mediated neurite outgrowth. In addition, electrical activity generated at the AIS may provide a depolarizing signal to open Na_v1.6 channels at the growth cone, further promoting β1-mediated neurite outgrowth (22). Interestingly, CGNs do not display spontaneous APs (43), and thus synaptic input likely would be required for activity-dependent β1-mediated neurite outgrowth in vivo.

In conclusion, our findings indicate that β1 is required for high-frequency firing in CGNs, by regulating localization of Na_v1.6 to the AIS and contributing to resurgent I_Na. We propose that localized Na⁺ influx carried by Na_v1.6 is required for β1-mediated neurite outgrowth. In conclusion, β1 and Na_v1.6 perform complementary roles, interacting from within distinct regions in the neuron to regulate excitability and neurite extension in a coordinated fashion. As a result, VGSCs function in macromolecular complexes that participate in signaling on multiple time scales to regulate excitability, adhesion, neurite outgrowth, and migration in the developing CNS.

Materials and Methods

Animals.

Mice were maintained in accordance with the guidelines of the University of Michigan Committee on the Use and Care of Animals. Details of mouse strains are given in SI Materials and Methods.

Neurite Outgrowth.

Neurite outgrowth assays were conducted as described previously (22). Additional details are provided in SI Materials and Methods.

Cerebellar Slice Recording.

Cerebellar slices were prepared and electrophysiological recordings performed and analyzed as described previously (12, 30, 44). Additional details are provided in SI Materials and Methods.

Whole-Cell Recording of Na⁺ Currents in CGNs in Vitro.

Cerebellar tissue from P12–14 mice was dissociated as described previously (22). Voltage clamp recordings were performed using standard methods. Additional details are given in SI Materials and Methods.

Immunocytochemistry and Immunohistochemistry.

Procedures for antibody labeling and confocal microscropy have been described previously (22). Additional details are given in SI Materials and Methods and Figs. S4 and S5.

Western Blot Analysis.

SDS/PAGE and transfer to nitrocellulose were performed as described previously (22). Additional details are provided in SI Materials and Methods.