Role of Na v 1.9 in activity-dependent axon growth in motoneurons

Spontaneous neural activity promotes axon growth in many types of developing neurons, including motoneurons. In motoneurons from a mouse model of spinal muscular atrophy (SMA), defects in axonal growth and presynaptic function correlate with a reduced frequency of spontaneous Ca 2 1 transients in axons which are mediated by N-type Ca 2 1 channels. To characterize the mechanisms that initiate spontaneous Ca 2 1 transients, we investigated the role of voltage-gated sodium channels (VGSCs). We found that low concentrations of the VGSC inhibitors tetrodotoxin (TTX) and saxitoxin (STX) reduce the rate of axon growth in cultured embryonic mouse motoneurons without affecting their survival. STX was 5- to 10-fold more potent than TTX and Ca 2 1 imaging conﬁrmed that low concentrations of STX strongly reduce the frequency of spontaneous Ca 2 1 transients in somatic and axonal regions. These ﬁndings suggest that the Na V 1.9, a VGSC that opens at low thresholds, could act upstream of spontaneous Ca 2 1 transients. qPCR from cultured and laser-microdissected spinal cord motoneurons revealed abundant expression of Na V 1.9. Na V 1.9 protein is preferentially localized in axons and growth cones. Suppression of Na V 1.9 expression reduced axon elongation. Motoneurons from Na V 1.9 2 / 2 mice showed the reduced axon growth in combination with reduced spontaneous Ca 2 1 transients in the soma and axon terminals. Thus, Na V 1.9 function appears to be essential for ac-tivity-dependent axon growth, acting upstream of spontaneous Ca 2 1 elevation through voltage-gated calcium channels (VGCCs). Na V 1.9 activation could therefore serve as a target for modulating axonal regeneration in motoneuron diseases such as SMA in which presynaptic activity of VGCCs is reduced.


INTRODUCTION
Axons of developing motoneurons grow long distances before they make synaptic contacts with their target tissue, the skeletal muscle (1). During this period, motoneurons depend on neurotrophic factors for their survival (2 -4). Cultured embryonic motoneurons also need neurotrophic factors for survival and neurite growth, thus allowing the analysis of signaling pathways for axon elongation and differentiation of presynaptic structures within axonal growth cones (1,5 -7). Embryonic motoneurons exhibit spontaneous activity at early stages, before they make synaptic contact with skeletal muscles (8 -12). Spontaneous Ca 2+ elevation is an evolutionary conserved phenomenon in growth and differentiation of neurons (10,11,13 -16) and is also observed in cultured mouse motoneurons (17). In motoneurons, spontaneous Ca 2+ transients in axons and axonal growth cones contribute to axon extension and presynaptic differentiation (17). These characteristics are also pathophysiologically relevant. Spontaneous Ca 2+ transients are reduced in motoneurons from a mouse model of spinal muscular atrophy (SMA) (17), the predominant form of motoneuron disease in children and young adults. The reduced frequency of Ca 2+ transients correlates with a reduced axon elongation and defective presynaptic differentiation in vitro (17), and disturbed synaptic transmission in vivo (18)(19)(20).
Survival and axon elongation of cultured motoneurons are strongly influenced by the availability of extracellular matrix proteins (1,21). Laminin-111 supports axon elongation in cultured motoneurons, while laminin-211/221 preparations reduce axon elongation (17). The synapse-specific b2-chain in laminin-221 mediates differentiation of presynaptic active zones by the direct interaction with Ca V 2.2, an N-type voltagegated calcium channel (VGCC) (22). In Smn -/-/SMN2 mice, defects in the clustering of the Ca V 2.2 are observed in axon terminals (17).
We investigated the role of voltage-gated sodium channels (VGSCs) in activity-dependent axon elongation in cultured motoneurons and found that TTX and STX, specific poreblockers of VGSCs, reduce spontaneous Ca 2+ transients and also axon growth. High sensitivity to STX indicated that Na V 1.9 might contribute to spontaneous excitability in embryonic motoneurons. Indeed, motoneurons from Na V 1.9 knockout mice have shorter axons, and exhibit a reduced frequency of spontaneous Ca 2+ transients. Taken together, these data indicate that Na V 1.9 plays a central role for spontaneous excitability that regulates the axonal growth in developing motoneurons.

VGSCs modulate axon growth in cultured motoneurons
To investigate whether VGSCs are involved in activitydependent axon elongation, we tested the effects of the sodium-channel inhibitors tetrodotoxin (TTX) and saxitoxin (STX) in cultures of isolated spinal motoneurons. TTX binds all channels of the Na V 1-family with high affinity, except the TTX-insensitive channels (23). The TTX-insensitive channels Na V 1.8 and Na V 1.9 carry a serine (S) residue, whereas Na V 1.5 carries a cysteine residue in a critical TTX affinity motif, which is close to the selectivity determining (inner ring residues Asp, Glu, Lys, and Ala) motif of VGSCs (23 -25). All other Na V 1-family members carry a phenylalanine (F) or tyrosine (Y) residue at the homologous site of domain DI, SS2-segment of the a-subunit (23,24). The determination of the equilibrium binding free energy of TTX and STX in dependence of these critical residues suggested that nonaromatic residues at the outer pore binding site of the channel shift the affinity of TTX-binding to lower values, while STX affinity is less affected (25).
Motoneurons were isolated from lumbar spinal cord of E14 C57/BL6 mice and plated at low density on laminin-111. These cell culture conditions allow minimal cell -cell contact and maximal axon extension of the cultured motoneurons (26). At DIV 7, motoneurons were stained with a-Tau antibody ( Fig. 1A and B) and the axon length was measured ( Fig. 1C and D). Motoneurons treated with 1 -10 nM STX showed a significant decrease in the axon length ( Fig. 1A and C). In contrast, TTX was not effective at the same low concentrations. The reduced axon length was observed only at TTX concentration of 50 nM ( Fig. 1B and D) and higher (data not shown). Neither STX nor TTX had an influence on motoneuron survival at the same concentrations that led to the reduced axon growth ( Fig. 1E and F). The number and length of motoneuron dendrites (Fig. 1G) were not affected when motoneurons were treated with 10 nM TTX or 10 nM STX ( Fig. 1H and I). Dendritic length: control, n ¼ 1182; 10 nM TTX, n ¼ 976; 10 nM STX, n ¼ 501; number of dendrites, control, n ¼ 230; 10 nM TTX, n ¼ 205; 10 nM STX, n ¼ 81. Results represent the mean + SEM of pooled data from three independent experiments, n, number of motoneurons that were scored in total from control or toxin treated motoneurons. * * * P , 0.0001; * * P , 0.01 tested by one-way ANOVA, Bonferroni post hoc test.

VGSC trigger spontaneous Ca 21 transients in cultured motoneurons
In cultured motoneurons, the axon growth is slow in a first phase until DIV 3, reaching about 100-150 mm of length. After DIV 3, axons grow fast, and they reach an average length of 600-800 mm at DIV 7. The frequency of local Ca 2+ transients in axonal growth cones correlates with the speed of axon elongation during these different time periods in culture (17). To analyze the role of VGSCs in spontaneous excitability of motoneurons, we measured spontaneous Ca 2+ transients with the calcium indicator dye Oregon-green BAPTA-1-AM (K d : 130 nM) between DIV 3 and DIV 4, when the speed of axon elongation is highest. Initial experiments at DIV 3 showed that motoneurons were heterogeneous with respect to spontaneous activity, some showing low activity with rare Ca 2+ transients and others with four or even more spontaneous transients per minute (not shown). Motoneurons can switch between such activity states (not shown). As suggested previously (15,27), we term broad spontaneous activity that results in synchronous transients in all parts of the cell as 'global activity', while locally restricted Ca 2+ transients are termed 'local activity'. An example for such global activity is presented in Figure 2A and Supplementary Material, Movie S1, showing synchronous transients in three regions of interest (roi), namely the axon initiation segment (roi1), the axon (roi2) and the axonal growth cone (roi3). Figure 2B and Supplementary Material, movie S2 show an example for a motoneuron exhibiting spontaneous, local Ca 2+ transients in the axon (roi2-4). The somatodendritic region was silent (roi1) when these Ca 2+ transients were measured in the same cell. From previous studies (17) we know that spontaneous Ca 2+ influx to motoneurons is blocked by V-conotoxin. In accordance with this, 30 nM V-conotoxin MVIIa fully blocked spontaneous Ca 2+ transients ( Fig. 2C1 and C2). V-Conotoxin MVIIa has a high affinity to N-type VGCCs, while much higher concentrations lead to a block of other VGCCs (28). We then tested the sodium-channel pore blockers TTX and STX to figure out whether the opening of VGSCs triggers Ca 2+ influx through VGCCs in motoneurons ( Fig. 2D and E, Fig. 3). Low concentrations of STX (10 nM) had a strong inhibitory function on spontaneous Ca 2+ influx (Figs 2D and 3B). TTX was less effective at 10 nM (Figs 2E and 3C), but showed a strong block of spontaneous activity when used at 100 nM ( Fig. 3A and D). For quantitative analysis of VGSC function in triggering spontaneous Ca 2+ transients, we analyzed motoneurons in the active state and monitored Ca 2+ transients during a period of 40 -60 min (long-term example is shown in Fig. 3A). Then, we analyzed transients in intervals (i) before the sodium-channel blocker treatment ( Fig. 3A; spontaneous), (ii) in the presence of either STX or TTX, (iii) during the wash out of the channel blockers and (iv) 10 min after STX or TTX treatment, when activity recovers (spontaneous recovery) from toxin treatment (Fig. 3A). Spontaneous Ca 2+ transients (Fig. 3B) were blocked by 10 nM STX in all cellular regions (soma, distal axon and growth cone), whereas TTX was less efficient to block these transients at the same concentration (Fig. 3C), in particular in the axon and axonal growth cone. The concentration of TTX necessary for an almost complete blockage of spontaneous Ca 2+ transients was 100 nM ( Fig. 3A and D). The effect was reversible when the inhibitors were washed out by a 10-fold artificial cerebrospinal fluid (ACSF) exchange (volume/min) over a period of 10 min. The experiments show that sodium-channel inhibitors reduce global and local Ca 2+ transients in all cellular regions of motoneurons. Thus, the observation that low concentrations of STX inhibit axon elongation ( Fig. 1) correlates with a reduced rate of Ca 2+ transients under acute STX application.

Na V 1.9 is expressed in cultured motoneurons
To identify the VGSCs that are responsible for triggering Ca 2+ transients in developing motoneurons, we concentrated on Na V 1.9. Several lines of evidence point to Na V 1.9 as the most likely candidate for initiating VGSC-dependent spontaneous Ca 2+ fluxes through VGCCs. Na V 1.9 has a low activation threshold and is able to mediate spontaneous excitation at resting potential levels, as shown previously in dorsal root ganglia (DRGs) and myenteric sensory neurons (29 -32).
We therefore amplified transcripts encoding the TTX-resistant VGSCs Na V 1.5, Na V 1.8, and Na V 1.9 by efficiency-controlled quantitative RT-PCR (qRT -PCR) and determined the number of Na V 1.9 transcripts in the developing spinal cord, in DRGs and cultured motoneurons ( Fig. 4A-D). In Fig. 4, representative amplification products and real-time PCR amplification curves are shown. For Na V 1.9 amplification, external standard dilution curves with E18 spinal cord RNA, 2 ng-100 pg, served as reference. Na V 1.5 is prominent for its function in triggering action potentials in the heart (24). Therefore, the heart RNA was used to produce Na V 1.5-specific external standard curves (10 ng-500 pg heart RNA). Na V 1.9 transcripts were detectable at E14 in spinal cord (8 copies/ 10 ng RNA) and DRG (70 copies/10 ng RNA). Na V 1.9 expression increased continuously between E14 and E18 (E18; spinal cord: 163 copies/10 ng RNA; DRG: 11 580 copies/10 ng RNA; Table 1; n ¼ 3 independent experiments). In motoneuron cultures, substantial expression of Na V 1.9 (Fig. 4B, right panel) and Na V 1.5 transcripts (Fig. 4B, left panel) was found at DIV 7 whereas Na V 1.8 was not detectable (Fig. 4B). As the amplification efficiency of both gene-specific qRT -PCR protocols was almost identical (eff Nav1.8 ¼ 1.92; eff Nav1.9 ¼ 1.95), we conclude that Na V 1.9 mRNA is expressed at relatively high levels in motoneurons in comparison to Na V 1.8, which was below the detection limit. Interestingly, relative expression of Na V 1.5 was at least 27-fold higher than the expression of Na V 1.9. However, the activation threshold of Na V 1.9 is lower than that of Na V 1.5 (24), thus making it less likely that Na V 1.5 is upstream of Na V 1.9 in spontaneous excitation of motoneurons.
To reveal expression of Na V 1.9 in spinal motoneurons in situ, we microdissected motoneurons from spinal cord by laser capture microscopy at postnatal day 2 (P2), a time point that corresponds to the age of motoneurons that had been isolated at E14 and maintained in culture for 7 days   Table 1). (B) At DIV 7, transcripts encoding Na V 1.9 (right panel) 26 copies per 10 ng RNA; 0.11% of the amount of GAPDH and Na V 1.5 (left panel) are expressed in cultured motoneurons. Na V 1.8 was not detected (left panel). (C and D) Real-time monitoring of the fluorescence emission of Sybr Green1 during PCR amplification of Na V 1.5 (C) and Na V 1.9 (D). (E and F) Na V 1.9 expression in motoneurons and DRG neurons in situ. Real-time amplification curves of Na V 1.9 cDNA from laser-dissected motoneurons of the spinal cord (E) or DRG neurons (G) and corresponding Na V 1.9-specific amplification products (F and H). In Na V 1.9 amplification, serial dilutions of spinal cord RNA served as external control (black lines in D, E, G); for Na V 1.5, heart RNA was used as control (black lines, C).

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Human Molecular Genetics, 2012, Vol. 21, No. 16 until they were analyzed. In addition, motoneurons were analyzed at P4. DRG neurons served as reference and GAPDH as denominator in all samples. As shown in Figure 4E and F, the spinal cord motoneurons express Na V 1.9 transcript at P2. In P2 motoneurons in situ, the number of Na V 1.9 transcripts is 0.44% of GAPDH transcripts, while in DRG neurons, 35 copies of Na V 1.9 correspond to 100 copies of GAPDH. Expression in motoneurons then decreases until P4 (Na V 1.9 versus GAPDH; 0.13%; Fig. 4E). At that stage, the relative expression of Na V 1.9 molecules versus GAPDH molecules in DRG neurons ( Fig. 4G and H) is much higher than that in motoneurons.
Anti-Na V 1.9 antibody and identification of mouse Na V 1.9 protein in tissue To detect Na V 1.9 protein in cultured motoneurons, anti-mouse Na V 1.9 antibodies were raised in rabbits. The C-terminal end of mouse Na V 1.9 was used for immunization ( Fig. 5A). For control experiments, full-length mouse Na V 1.9 was cloned into a modified pcDNA3 vector and served as control.
The rabbit antiserum [Na V 1.9 (71n)] that was raised against the C-terminus of the protein gave high titer antibodies and labeled recombinant human and mouse Na V 1.9 in western blots (Fig. 5B). Recombinant Na V 1.9 appeared in two bands with a relative molecular weight (M r ) of 180 and 280 kDa. The human Na V 1.9 protein was preferentially detected at 280 kDa, as described earlier (33). When GFP and Na V 1.9 were co-expressed in HEK293 cells, Na V 1.9 immunoreactivity (ImmR) was specific to GFP+ cells (Fig. 5C). By increasing amounts of peptide used for immunization, Na V 1.9 (71n) ImmR was suppressed (Fig. 5C). Next, we established a stable HEK293T cell line expressing mouse Na V 1.9. Here, Na V 1.9 (71n) ImmR showed a distinct overlap with ImmR obtained with a pan-VGSC antibody (pan Na V 1.x) (Fig. 5D). Because Na V 1.9 is highly expressed in DRG neurons (Fig. 4), we used these neurons to detect endogenous Na V 1.9 protein. Endogenous mNa V 1.9 also showed an M r of 180 and 280 kDa in western blots of lumbar and thoracic DRGs, while posterior nerve roots were Na V 1.9 negative in this experiment (Fig. 5E). Next, we prepared protein lysates of DRGs from Na V 1.9 2/2 mice. In this specific Na V 1.9 2/2 mouse line, a frameshift mutation introduces stop codons within exon 6 (31). In western blots from Na V 1.9 2/2 mice, Na V 1.9 ImmR is lost at an M r of 180 and 280 kDa in DRGs from Na V 1.9 2/2 mice (Fig. 5F).

Na V 1.9 protein is localized in axons and axonal growth cones
To localize Na V 1.9 in cultured motoneurons, we used stimulated emission depletion (STED) microscopy and combined this technique with standard confocal laser scanning microscopy for the detection of F-Actin and a-Tubulin as structural markers. Na V 1.9 protein was not homogenously distributed in axons. When cultured motoneurons were stained with anti-Na V 1.9, ImmR was detectable along the axon but the staining appeared to be enriched at some sites (arrows in Fig. 6A and B). In axons and growth cones, Na V 1.9 ImmR was high in the distal axons and also found in small protrusions (right arrow in Fig. 6C). Single confocal STED planes revealed that small punctate anti-Na V 1.9 ImmR was lining along the cell surface of growth cones (arrows in Fig. 6D, upper and lower panel).

Na V 1.9 depletion reduces axon elongation
To test the role of Na V 1.9 for axon elongation, lentiviral shRNA expression vectors were designed and used to suppress the expression of this channel in motoneurons. The fluorescent protein Tandem-tomato (TDtomato) was co-expressed as infection control (Fig. 7). As a control for the efficacy of repression, shRNA-mediated Na V 1.9 knockdown was verified by western blot analysis of HEK293T cells expressing mouse Na V 1.9 ( Fig. 7A; sh 63; sh 3028). Na V 1.9 expression was not reduced in cells expressing empty lentiviral vectors or constructs expressing missense control shRNA (mis 63) in which four bases were exchanged ( Fig. 7A; sh vector, mis 63). Then, lentiviral vectors were used to infect motoneurons at DIV 1. Infected motoneurons were identified at DIV 7 and the axon length was analyzed. In sh 63-RNA-treated motoneurons, the axon length was reduced by 43% in comparison to uninfected motoneurons in the same cultures or control cultures treated with missense shRNA-expressing virus (Fig. 7B, red). A second set of lentivirally applied shRNA against the 3 ′ -untranslated region of Na V 1.9, with GFP as infection control, showed 46% reduction in axon elongation in motoneurons at DIV 7 (Fig. 7B, green). Figure 7C shows representative motoneurons infected with lentiviral shRNA expression vectors expressing TDtomato as infection control. To address the question whether sh 63 was also effective on stably expressed Na V 1.9, a stable Na V 1.9 expressing-HEK293T cell line was transfected with control vector, sh 63 vector and a mis 63 shRNA. As shown in Figure 7D, shRNA expression but not mismatch shRNA reduced Nav1.9 expression to 18% compared with the control situation (Fig. 7E).
Motoneurons from Na V 1.9 2/2 mice exhibit shorter axons and reduced frequency of Ca 21 transients We also isolated motoneurons from Na V 1.9 knockout mice (31) and investigated them in comparison to strain-matched wild-type controls. No anti-Na V 1.9 ImmR was observed in motoneurons at DIV 5 (Fig. 8A). Na V 1.9 2/2 motoneurons showed highly reduced rates of spontaneous Ca 2+ transients at DIV 3, in the soma (275%), distal axonal regions (282%) and growth cones (283%) (Fig. 8B), indicating that the loss of Na V 1.9 affects global and local Ca 2+ transients of spontaneous excitability. Dendritic elongation (Fig. 8C) and motoneuron survival (Fig. 8D) were unaffected. However, axon elongation of Na V 1.9 2/2 motoneurons was reduced by 38% compared with wild-type littermates (Fig. 8E). We then investigated whether the reduction of spontaneous Ca 2+ transients observed in motoneurons from a mouse model of SMA (Smn 2/2 /SMN2) (17) is also influenced by inhibition of Na V 1.9 and other VGSCs. In Smn 2/2 /SMN2 mice, spontaneous Ca 2+ transients are reduced because of defects in the clustering of the Ca V 2.2 in axon terminals thus leading to a reduced axonal elongation (17). When Smn 2/2 /SMN2 motoneurons were treated with 10 nM STX, axonal elongation remained unchanged, showing that function-blocking concentrations of STX have no additional growth inhibiting effect on Smn 2/2 / SMN2 motoneurons (Fig. 8F). This experiment raised the question whether Na V 1.9 expression and protein distribution has changed in Smn 2/2 /SMN2 motoneurons. Immunolocalization of Na V 1.9 in Smn 2/2 /SMN2 motoneurons revealed Na V 1.9 concentration in axons and axon terminals (Fig. 9A, four representative distal axons and growth cones are shown). Next, we performed qPCR on RNA samples harvested from four independent single-embryo motoneuron cultures of Smn 2/2 /SMN2 motoneurons. Expression levels of Na V 1.9 at DIV 7 in Smn 2/2 /SMN2 motoneurons are comparable in control and SMN-deficient motoneurons ( Fig. 9B and C).

DISCUSSION
Spontaneous activity plays an important role during development of the nervous system when neurons make connections and synaptic networks are shaped (10,11,13 -16). During development, motoneurons become spontaneously active long before they make synaptic contacts with skeletal muscle (9,16,34). In culture, embryonic mouse motoneurons display spontaneous Ca 2+ transients that are important for axon elongation and growth cone differentiation (17). Downstream Ca 2+dependent signaling pathways modulate microfilament and microtubule networks that mediate the effects of spontaneous excitability on the axon growth (35). Blockade of VGCC by V-conotoxin leads to 40% reduced axon elongation in cultured embryonic mouse motoneurons on laminin-111 (17), indicating that depolarization-induced Ca 2+ influx plays a major role in axon elongation.
Here, we provide evidence that the VGSC Na V 1.9 acts as an upstream trigger of spontaneous excitatory Ca 2+ influx in motoneurons and thus modulates the rate of axon elongation, without affecting motoneuron survival. Apparently, other members of the Na V family cannot compensate for the deficiency of Na V 1.9. This is not unexpected. Among the nine members of the Na V 1 family, Na V 1.9 exhibits a specific property in that it opens spontaneously when neurons are kept close to the resting potential (29)(30)(31)(32). The effect on axon elongation Figure 5. Anti-mouse Na V 1.9 antibody. (A) A peptide was deduced from the carboxyterminal end of mouse Na V 1.9 (red) and used for immunization of rabbits. The selected peptide sequence is specific for Na V 1.9. Differences to rat or human Na V 1.9 are marked (yellow). (B) Anti-Na V 1.9 (71n) recognizes recombinant human Na v 1.9 and mouse Na V 1.9 in western blot analysis. As vector control, GFP-expressing vectors were used. (C) Immunoreactivity of anti-Na V 1.9 (71n) on recombinant Na V 1.9 is blocked by the corresponding 71n immunization peptide. Bar: 100 mm (D) Stable expression of mNa V 1.9 in HEK293T cells reveals a pronounced overlap of anti-Na V 1.9 (71n) and anti-pan Na V 1.x immunoreactivity. Bar: 15 mm. (E) Endogenous mouse Na V 1.9 from lumbar and thoracic DRGs is displayed by two bands at M r 180 kDa and 280 kDa. (F) Endogenous mouse Na V 1.9 in wild-type DRGs and recombinant Na V 1.9 in the stable cell line 293-mNa V 1.9 are displayed by the typical doubleband pattern bands at M r 180 kDa and 280 kDa. Both bands are lost in Na V 1.9 2/2 mice (representative for n ¼ 4). Anti-Trk (tropomyosin-relatedkinase) antibodies served as loading control for DRG tissue.

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Human Molecular Genetics, 2012, Vol. 21, No. 16 observed in motoneurons from Na V 1.9 knockout mice and in motoneurons in which Na V 1.9 is acutely suppressed by shRNA was similar. This argues against the possibility that deficiency of Na V 1.9 in knockout mice leads to a developmental defect that indirectly impairs spontaneous activity. Furthermore, the blockage of voltage-gated Na + channels by STX and, at least at higher concentrations, that by TTX provokes a similar extent in the reduction of spontaneous activity and axon elongation. This appears remarkable as TTX and STX should act on a broad spectrum of VGSCs, and the reduction in spontaneous activity and axon elongation observed with these pharmacological inhibitors is not higher than after Na V 1.9 knockout or shRNA-mediated knockdown. This indicates that Na V 1.9 is the predominant VGSC that triggers spontaneous activity for axon elongation. Spontaneous Ca 2+ transients in embryonic motoneurons are either locally restricted or globally distributed over the whole cell. In Na V 1.9 2/2 motoneurons, Ca 2+ transients are massively reduced at DIV 3 -4, both global transients that are synchronously observed in all parts of the cell and local transients that are seen only in axons. Voltage-gated Ca 2+ influx modulates the axon growth in motoneurons that are cultured at low-density so that the cells cannot make synaptic contact with each other. Furthermore, during the monitoring of Ca 2+ influx, cells were kept under fast perfusion in an ACSF extracellular solution with 3 mM potassium. These characteristics would hardly allow motoneuron depolarization with ligand-dependent stimuli by synaptic input or excitatory neurotransmitters in the medium. Na V 1.9 is responsible for prolonged sodium influx into a variety of neurons (29,31,33,36,37) and thus can depolarize the cells so that Ca 2+ influx through VGCCs can occur with high efficacy. Moreover, Na V 1.9 is predominantly localized in axons and axonal growth cones so that opening of VGCCs by Na V 1.9 is facilitated even under conditions when opening of Na V 1.9 provokes only a local depolarization in axonal growth cones. In embryonic chick motoneurons, a persistent sodium current is involved in the generation of spontaneous excitatory bursts (13) and experiments by Kastenenka and Landmesser have demonstrated that spinal motor circuits are sensitive to the precise frequency and pattern of spontaneous activity (38). The molecular identity of the persistent sodium current which is involved in normal firing patterns of chick motoneurons has not been identified and it is tempting to speculate that Na V 1.9 plays a central role. As chick Na V 1.9 (NM_001192868) is 79% identical with mouse Na V 1.9 (NM_011887), it may be possible that Na V 1.9 is the upstream trigger of spontaneous Ca 2+ bursts during motoneuron pathfinding in the chick.
A critical issue is how the opening of Na V 1.9 is regulated. Studies on fast (milliseconds) excitatory transients after brain-derived neurotrophic factor (BDNF) application (39) suggested that Na V 1.9 can be opened in central neurons by BDNF via TrkB activation (33,40). However, the existence of such BDNF -induced fast Na + currents has become controversial (41). In particular, it has been noted that the kinetics of BDNF release conflict with the much faster activation kinetics of the Na V 1.9-mediated currents (42). The motoneurons used in our study were continuously cultured with BDNF to support their survival. Thus, it is possible that TrkB signaling is involved in the cascades that lead to opening of VGCCs (43) through Na V 1.9. It is unlikely that Ca 2+ elevation in motoneurons is caused by spontaneously released BDNF from cells in these cultures, given that BDNF is present at concentrations of 10 ng/ml that saturate TrkB receptors on the cell surface. However, it is possible that other signaling mechanisms such as transactivation of TrkB receptors (44) that are not exposed on the cell surface contributes to the opening probability of Na V 1.9 (31,45) and therefore might increase the local, growth-mediating excitability of motoneurons. It is unresolved whether Na V 1.9 is opened by direct interaction with TrkB or indirectly via other signaling pathways or TrkB-dependent rapid changes in ion fluxes that alter the resting potential in such a way that Na V 1.9 can open.

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Human Molecular Genetics, 2012, Vol. 21, No. 16 The apparent effect of Na V 1.9 on axon elongation in cultured embryonic motoneurons appears to be not reflected by the relatively mild phenotype of Na V 1.9 knockout mice (31,46,47). These mice develop normally, and show only defects in pain perception, but no apparent functional defect that is caused by a reduced axon growth of motoneurons to reach their target, the skeletal muscles (46). This is not surprising when the effects of Na V 1.9 knockout are compared with models for SMA in which Smn expression is reduced. Isolated motoneurons from a mouse model of SMA (48) show severe defects in Ca 2+ transients in axons and axonal growth cones, and the reduced frequency of Ca 2+ transients also correlates with a reduced axon elongation (17). However, the axons of the Smn-deficient motoneurons reach skeletal muscle, and no reduction in neuromuscular endplate formation is observed in mouse embryos with Smn deficiency (49,50). However, when Smn (51) or Smn-interacting proteins such as hnRNPR (52) are knocked down by Morpholino technologies in zebrafish, the axons of motoneurons show severe defects in axon elongation and pathfinding. This indicates that animal models in which the motor axon growth occurs within a short time window suffer more from these genetic defects than developing mice in which axon elongation, pathfinding and synapse formation occur over a relatively prolonged time period. Thus, a failsafe mechanism seems to exist that compensates for the reduced speed of axon elongation and allows motoneurons to reach their target.
The signaling cascades leading to motoneuron differentiation and motoneuron axon elongation are also of central interest for the development of therapies for motoneuron diseases, in particular SMA in which a defect in synapse maintenance appears as a central pathophysiological mechanism (17,(48)(49)(50)53). Previous studies indicate that intracellular signaling pathways can activate persistent sodium currents by Na V 1.9 (31,45). This raises the hope that pharmacological activation to increase the opening probability of Na V 1.9 could be a way to stimulate axon regeneration and maintenance.

Animals
All experimental procedures were done in accordance with European Union guidelines, as approved by our institutional animal care and utilization committee. The following mouse lines were used in this study: C57BL/6J mice; Na V 1.9 2/2 mice on a C57BL/6J background (31) and Smn 2/2 /SMN2 mice (48) on a FVB/NCrl background.

Cell culture
Spinal motoneurons were isolated from 14-day-old C57/BL6/J mouse embryos of either sex, enriched by panning using an antibody against the p75 NTR receptor and plated at a density of 1500 cells/well on laminin-coated coverslips as described previously (26). Cells were grown in neurobasal medium containing B27 supplement, 10% heat inactivated horse serum, 500 mM Glutamax (Invitrogen), 10 ng/ml BDNF and 10 ng/ml ciliary neurotrophic factor. Medium was replaced after 24 h and then every second day. V-Conotoxins MVIIa (Sigma), tetrodotoxin (Sigma, Ascent Scientific) and saxitoxin (Sigma) was added at indicated concentrations. Motoneuron survival was determined after 7 days in culture as described earlier (54). Motoneurons from Na V 1.9 or Smn/SMN2 mouse models were prepared from single embryos. Genotyping was then performed from corresponding tissue samples.

Quantitative real-time reverse transcriptase -polymerase chain reaction
Reverse transcription, primer selection and qPCR were performed with minor modifications as described in earlier studies (55)(56)(57). RNA from mouse embryonic DRG and the spinal cord was isolated by standard protocols using RNeasy Plus-Mini-Kit (Qiagen), with the help of a silent crusher (Heidolph). RNA from single-embryo motoneuron cultures (Smn/ SMN2 RNA samples) was prepared with the RNeasy Micro Kit (Qiagen). qPCRs were run on a Lightcycler 1.5 (Roche) using FastStart DNA master SYBR green1 reagents, using kinetic PCR cycles. Offline analysis to calculate efficiencycontrolled relative expression levels or absolute copy numbers was carried out according to Rasmussen (58). Intron-spanning primers were selected with Oligo 6.0 software (MedProbe) and PCR conditions, primer concentration and MgCl 2 concentration were optimized as described (55). Reactions were performed in glass capillaries in a volume of 20 ml.

Laser capture microdissection
Laser capture microdissection (Leica DM6000B laser microdissection system) was used to isolate motoneurons from the spinal cord of 2-and 4-day-old postnatal mice. In addition, DRG neurons, which express high numbers of Na V 1.9 transcripts, were collected as reference. Spinal cord sections were embedded in optimum cutting temperature compound (Tissue-Tek) and immediately immersed in isopentane, chilled in liquid nitrogen for rapid freezing. Cross-sections of 15 mm thickness were prepared on a Leica cryostat, transferred to 0.9 mm POL membranes (Leica) and stained in Cresyl Violet solution. A total of 500 -1200 MN cell bodies were collected in RNA lysis buffer. Total RNA was purified from the samples (59) and real-time RT -PCR was performed (see above). To compare expression levels of Na V 1.9 in motoneurons versus DRG neurons, GAPDH expression served as denominator and relative expression was calculated.
Cloning of mouse Na V 1.9, shRNA expression vectors For stable propagation in E. coli, the rop-element of pBR322 was introduced to the backbone of pcDNA3 as described earlier (33). Next, mouse Na V 1.9 was cloned from mouse brain mRNA by fusing three partial cDNA PCR fragments. An artificial Kozak sequence (CCACCATG) was introduced before the start codon. The resulting construct was sequenced on both strands. Expression was tested by western blot analysis and immunocytochemistry using antibodies against Na V 1.9 (71n) and pan-Na V 1.X (Sigma, clone K58/35, 1-2 ng/ml). For lentiviral expression of shRNA targeted against Na V 1.9 transcripts and shRNA missense controls, two series of vectors were used. Sequences were selected (60) and cloned to the vector LL3.7 (61). In this case, shRNA was directed against the 3 ′ UTR of Na V 1.9 and expressed under U6 promoter. GFP expressed under cytomegalovirus (CMV) promoter served as infection control. In a second set basing on pSIH-H1 (System Biosciences), we replaced CMV-driven copGFP against TDTomato (62) and expressed shRNA against the coding region of Na V 1.9 and corresponding missense control shRNA under H1 promoter. To test the shRNA performance, mNa V 1.9 expression backbone (A.W., S.H. and R.B., in preparation) was mixed with shRNA expression vectors, transfected to HEK293T cells and co-expressed for 72 h. Then cells were lysed and Na V 1.9 protein was visualized by western blot analysis. In a second set of experiments, shRNA expression vectors were transfected to a cell with stable expression of mNa V 1.9 and quantitative analysis of shRNA efficiency was determined by western blot analysis.
Antibody production: anti-mouse Na V 1.9 (71n) First, pre-immune serum was tested by immunofluorescence and western blot analysis. Antibodies were raised in pre-tested

Lentivirus production
In HEK293T cells, pSIH-H1-based lentiviral vectors were packaged with pCMV-VSVG and pCMVDR8.91 (63) as described earlier (64). shRNA expressing pLL3.7 was packaged with pRSV Rev, pMDLg/pRRE and pMD.G (65,66). Vectors were transfected with Lipofectamine 2000 (Invitrogen) in OptiMEM medium with 10% fetal calf serum for 12-14 h and viral supernatants were harvested 72 h after transfection. Lentiviral particles were concentrated from cleared supernatants by two rounds of ultracentrifugation at 25 000 rpm in a Beckman SW28 rotor for 2 h at 48C. The virus pellet was soaked on ice for a minimum of 4 h in 200 ml tris-buffered saline buffer (in mM): 130 NaCl, 10 KCl, 5 MgCl 2 and 50 Tris-HCl, pH 7.8. Aliquots (10 ml) of the viral suspension were stored at 2808C. Titering was performed on HeLa cells and 10 5 infectious particles were used to infect 5 000 freshly prepared E14 motoneurons in suspension before plating.

STED microscopy
STED microscopy was performed on a Leica SP5 confocal laser scanning microscope equipped with a Mai-Tai multi photon laser (Spectra-Physics). mNa V 1.9 (71n) immunoreactivity (ImmR) was detected with anti-rabbit Atto647N secondary antibodies. A Leica HCX PL Apo CS ×100/1.4 oil objective was used for the STED detection. Serial scanning of anti-Tubulin and phalloidin ImmR was performed by standard confocal imaging. Images were taken at 12-bit, in single confocal planes.

Axon length analysis
The axon length was determined by applying a morphometric system (Leica, Bensheim, Germany) on confocal image material.

Confocal Ca 21 imaging
A 5 mM stock solution of Oregon green-BAPTA1-AM (Invitrogen; O6807) was prepared in 8.9 ml of 20% Pluronic F-127 (Invitrogen) in dimethyl sulfoxide by means of a sonifier bath (Bandelin) for 2 min. Motoneurons were incubated with 5 mM Oregon green-BAPTA1-AM in ACSF solution. Motoneurons were loaded with dye-containing ACSF in a cell culture incubator (378C, 5% CO 2 ) for 10-15 min. Motoneurons were imaged at an inverted confocal microscope (Leica SP series) using a ×20/0.7 objective under continuous perfusion in a low chamber volume ( 200 ml) with high buffer exchange rate ( 10× chamber volume per min, in some experiments: 20× volume exchange/min). Ligands (Sigma, Ascent Scientific) were used at the following concentrations: tetrodotoxin (TTX; 100-10 nM) and saxitoxin (STX; 100-10 nM). In some cases, muscimol (10 -50 mM); or g-aminobutyric acid (GABA) (100 mM) were used as control stimulus to monitor the excitatory action of GABA on embryonic motoneurons. Time lapse monitoring (256 × 256 pixel) of Ca 2+ dynamics was performed at 2.0 Hz. Oregon green-BAPTA1-derived fluorescence was excited with a 488 nm laser line (emission detection: 507/ 565 nm). For pharmacological effect of VGSC inhibitors on spontaneous Ca 2+ transients in motoneurons, cells were analyzed in their active state, under conditions of activity block Human Molecular Genetics, 2012, Vol. 21,No. 16 3665