Experimental Neurology 224 (2010) 362–368

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Experimental Neurology

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A mutation linked to epilepsy increases ramp and persistent current of Nav1.3 and induces hyperexcitability in hippocampal neurons

Mark Estacion 1, Andreas Gasser 1, Sulayman D. Dib-Hajj, Stephen G. Waxman ⁎

Department of Neurology, Yale University School of Medicine, New Haven, CT 06510, USA Center for Neuroscience and Regeneration Research, Yale University School of Medicine, New Haven, CT 06510, USA Rehabilitation Research Center, Veterans Affairs Connecticut Healthcare System, West Haven, CT 06516, USA article info abstract

Article history: Voltage-gated sodium underlie many excitability disorders. SCN1A, SCN2A and Received 15 February 2010 SCN9A, which encode pore-forming α-subunits NaV1.1, NaV1.2 and NaV1.7, are clustered on human Revised 12 April 2010 2, and mutations in these genes have been shown to underlie epilepsy, migraine, and somatic Accepted 16 April 2010 pain disorders. SCN3A, the which encodes Na 1.3, is part of this cluster, but until recently was not Available online 24 April 2010 V associated with any mutation. A charge-neutralizing mutation, K345Q, in the NaV1.3 DI/S5-6 linker has recently been identified in a patient with cryptogenic partial epilepsy. Pathogenicity of the Na 1.3/K354Q Keywords: V Sodium channel mutation has been inferred from the conservation of this residue in all sodium channels and its absence from Epilepsy control alleles, but functional analysis has been limited to the corresponding substitution in the cardiac Hippocampal neuron muscle sodium channel NaV1.5. Since identical mutations may produce different effects within different

Voltage-clamp sodium channel isoforms, we assessed the K354Q mutation within its native NaV1.3 channel and studied the Current-clamp effect of the mutant NaV1.3/K354Q channels on hippocampal neuron excitability. We show here that the K354Q mutation enhances the persistent and ramp currents of NaV1.3, reduces current threshold and produces spontaneous firing and paroxysmal depolarizing shift-like complexes in hippocampal neurons. Our data provide a pathophysiological basis for the pathogenicity of the first epilepsy-linked mutation within

NaV1.3 channels and hippocampal neurons. © 2010 Elsevier Inc. All rights reserved.

Introduction neonatal rodent CNS, and is almost undetectable in the adult rodent CNS (Beckh et al., 1989; Black et al., 1996; Felts et al., 1997; Waxman Monogenic linkage of sodium channelopathies and disorders et al., 1994). However, relatively higher levels of this channel are of excitability is now well-documented in skeletal muscle (SCN4A/ retained in the adult human brain (Whitaker et al., 2001). Until

Nav1.4, Cannon 2006), sensory neurons (SCN9A/Nav1.7, Dib-Hajj recently, NaV1.3-related pathogenesis has been limited to pain, a role et al., 2007), cardiac myocytes (SCN5A/Nav1.5, George 2005); and for Nav1.3 which has been inferred from rodent models of nervous brain; (Meisler and Kearney, 2005). More than 300 sodium channel system injury (Waxman and Hains, 2006), and from the accumulation mutations, mostly in SCN1A which encodes sodium channel NaV1.1, of NaV1.3 at blind endings of axons from human painful neuromas together with a smaller number of mutations in SCN2A which encodes (Black et al., 2008). Recently, a charge-neutralizing K354Q mutation in sodium channel NaV 1.2 (Lossin 2009) have been associated with NaV1.3, which substitutes an evolutionarily conserved lysine located epilepsy (George 2004; Helbig et al., 2008; Noebels 2003; Sisodiya et within the channel pore region with a glutamine, has been reported in al., 2007). SCN3A (which encodes sodium channel Nav1.3) is located a patient with cryptogenic childhood epilepsy (Holland et al., 2008). A on the same chromosomal cluster as SCN1A and SCN2A, but there is genetic link of NaV1.3 mutation and epilepsy would be of special little evidence linking it with epilepsy or other inherited channelo- interest, because it would support a potential pathogenic role for this pathies. NaV1.3 is expressed predominantly in the embryonic and channel in the human CNS and would increase the repertoire of genetic loci that may underlie cryptogenic epilepsies.

Pathogenicity of the NaV1.3/K354Q mutation has been suggested on ⁎ Corresponding author. Neuroscience Research Center, Bldg 34, VA Connecticut the basis of conservation of this residue in all sodium channels, its Healthcare System (127A), 950 Campbell Avenue, West Haven, CT 06516. Fax: +1 203 absence from control alleles, and from functional analysis of the cor- 937 3801. responding substitution in the cardiac muscle sodium channel NaV1.5 E-mail addresses: [email protected] (M. Estacion), [email protected] (Holland et al., 2008). However, since identical mutations may produce (A. Gasser), [email protected] (S.D. Dib-Hajj), [email protected] (S.G. Waxman). different functional changes in different channel isoforms (Lampertetal., 1 These authors made equal contribution. 2006a; Lossin et al., 2002), a more direct demonstration of pathogenicity

0014-4886/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2010.04.012 M. Estacion et al. / Experimental Neurology 224 (2010) 362–368 363

requires functional analysis of the K354Q mutation within NaV1.3, and 140 NaCl, 3 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES, pH 7.3 with NaOH assessment of the contribution of the mutant NaV1.3/K354Q channels to (adjusted to 320 mOsm with dextrose). The pipette solution con- excitability of CNS neurons. Here we report gain-of-function properties tained (in mM): 135 Cs-Aspartate, 10 NaCl, 2 MgCl2, 0.1 CaCl2, 1.1 of NaV1.3/K354Q mutant channels and demonstrate that they produce EGTA (pCa=8), 10 HEPES, pH 7.2 with CsOH (adjusted to 310 mOsm epileptiform changes in transfected hippocampal neurons. with dextrose). Patch-pipettes had a resistance of 1–3MΩ when filled with pipette solution, and once whole-cell recording mode was Methods achieved the access resistance averaged 4–5MΩ.Thejunction potential of 16 mV (calculated by JPcalc, CLAMP software) was com- Plasmid and transient transfection pensated by setting holding potential during the seal test period to −16 mV. Once the seal had formed, these two solutions were

For comparison of biophysical properties of WT NaV1.3 and the no longer in contact and the applied potential was correct. Upon fi NaV1.3/K354Q mutant, we studied both by patch-clamp using a rat achieving whole-cell recording con guration, pipette and cell capac- NaV1.3 construct. The construct, converted to a tetrodotoxin-resistant itance were manually minimized using Axopatch 200B (Molecular form (TTX-R, rNaV1.3R) by the Y384S substitution, permits the current Devices, Union City, CA) compensation circuitry. To reduce voltage – produced by rNaV1.3 to be studied in isolation after expression in errors, 80 90% series resistance and prediction compensation were neuronal backgrounds when TTX is included in the bath solution, but applied. Cells were excluded from analysis if the predicted voltage does not alter the voltage-dependence or kinetics of the channel error exceeded 3 mV. Recorded currents were digitized using pCLAMP (Cummins et al., 2001; Herzog et al., 2003). The K354Q mutation was software (version 10) and a Digidata 1440A interface (Molecular introduced using QuickChange XL II site-directed mutagenesis Devices) at 50 kHz after passing through a low-pass Bessel filter (Stratagene, La Jolla, CA). HEK293 cells, grown under standard culture setting of 10 kHz. Linear leak and residual capacitance artifacts were conditions (5% CO2, 37 °C) in Dulbeccos's Modified Eagle's Medium subtracted out using the P/N method (Clampex software). Sodium supplemented with 10% fetal bovine serum, were transiently- current recordings were initiated after a 5 min equilibration period transfected with plasmids containing β1-IRES-CD4 and β2-IRES-GFP once whole-cell configuration was achieved. fi (Lossin et al., 2002) and either rNav1.3R (referred to as WT hereinafter) Data analysis was performed using Clamp t (Molecular Devices) or rNaV1.3R/K354Q using Lipofectamine2000 (Invitrogen, Carlsbad, CA) or Origin (Microcal Software, Northampton, MA). To generate with a stoichiometry of plasmids of 1:1:5 by mass. Since our HEK cells do activation curves, cells were held at −100 mV and stepped to −80 not express endogenous Na-current, no TTX was necessary for the to +40 mV in 5 mV increments for 100 ms. Peak inward currents characterization of heterologously expressed NaV1.3 channels. from activation protocols were converted to conductance values using the equation, G=I/(Vm −ENa), where G is the conductance, I is the Primary hippocampal neuron isolation and transfection peak inward current, Vm is the membrane potential step used to elicit the response and ENa is the sodium reversal potential (determined for Animal protocols complied with NIH guidelines, and were approved each cell using the x-axis intercept of a linear fit of peak inward by the VA Connecticut Healthcare System Animal Use Committee. current responses). Conductance data were normalized by maximum – Sprague Dawley rat pups (Harlan, Indianapolis, IN) between postnatal conductance and fit with a Boltzmann equation of the form G=Gmin + days 10 and 14 were anesthetized with ketamine/xylazine (100/ (Gmax −Gmin)/(1+exp[(V1/2 −Vm)/k)], where V1/2 is the activation 10 mg/kg, i.p.) and sacrificed by decapitation. Hippocampi were midpoint and k is the slope factor. To generate steady-state fast- dissected out in ice-cold HABG [HA medium (Brain Bits, Springfield, inactivation curves, cells were stepped to inactivating potentials of IL) supplemented with 2% B-27 (Invitrogen) and 0.5 mM Glutamax −180 to −20 mV for 500 ms followed by a 50 ms step to −10 mV. (Invitrogen)], sliced into 300 µm sections using a McIlwain tissue Peak inward currents obtained from steady-state fast inactivation chopper (Warner Instruments, Hamden, CT) and digested (30 min at protocol were normalized by maximum current amplitude and fitwitha

30 °C) with papain (30 U/ml; Worthington, Lakewood, NJ) in Boltzmann equation of the form I=Imin +(Imax −Imin)/(1+exp[(Vm − calcium-free HA medium (Brain Bits) with 0.5 mM Glutamax. After V1/2)/k)], where Vm represents the inactivating pre-pulse membrane digestion, tissue was resuspended in HABG and triturated with a 1 ml potential and V1/2 represents the midpoint of inactivation. Data are plastic pipette tip. After settling of remaining tissue, the supernatant was expressed as means±standard error (SEM). Statistical significance was filtered through 40 µm mesh and centrifuged at 1000×g (4 min, room determined by Student's t-test. temperature). For transfection, hippocampal neurons were resuspended in 100 μl of buffer (1.5×107cells/ml). 10 µl aliquots were transfected by Electrophysiology: Current-clamp electroporation (MicroPorator, Harvard Apparatus, Holliston, MA, 2 pulses, each 15 ms, of 1500 V) using 0.5 µg of plasmid DNA per transfection (0.4 μg Whole-cell current-clamp recordings were obtained using the WT or K354Q, and 0.1 μg EGFP). Following electroporation, cells were Axopatch 200B amplifier, digitized using the Digidata 1440A interface resuspended and incubated for 5 min in 45 µl calcium-free HABG medium and controlled using pCLAMP software. The bath solution for current- at room temperature. 45 µl of complete medium [Neurobasal medium, clamp recordings was the same as for voltage-clamp recordings supplemented with 2% B-27, 0.5 mM Glutamax, 10 µg/ml gentamycin, specified above with the exception that CaCl2 and MgCl2 were in- 5 ng/ml recombinant mouse FGF2, and 5 ng/ml recombinant mouse creased from 1 mM to 2 mM. The pipette solution contained (in mM): PDGFbb (Invitrogen)] was added. Cells were plated on 12 mm glass 122 K-methanesulfonate, 0.9 EGTA, 9 HEPES, and 1.8 MgCl2, 4 Mg- coverslips coated with poly-D-lysine and laminin (BD Biosciences, Bedford, ATP, 0.3 GTP (Tris salt) and 14 phosphocreatine (di-Tris salt) pH 7.4 MA), placed in a 24 well plate, and incubated at 37 °C for 1 h to allow with KOH (adjusted to 300 mOsm with dextrose). No junction attachment. Finally, 500 µl/well complete medium was added, and cells potential correction was applied for current-clamp experiments. wereincubatedat37°Cuntilusedforrecordingsat40–50h post- Recordings were obtained 40 h–50 h post-transfection from trans- transfection. At this time culture neurites are sparse, permitting isolated fected hippocampal pyramidal neurons with small diameter (10– pyramidal-shaped neurons to be studied by patch-clamp. 15 μm) triangular cell bodies that exhibited GFP fluorescence. While recordings from multiple cells could be obtained from the same Electrophysiology: Voltage-clamp coverslip, the coverslip was replaced if the time since transferring to the recording chamber exceeded an hour. Whole-cell configuration Whole-cell voltage-clamp recordings were obtained using the was obtained in voltage-clamp mode before proceeding to current- following solutions. The extracellular solution contained (in mM): clamp recording mode. After initiating current-clamp, a small (−2to 364 M. Estacion et al. / Experimental Neurology 224 (2010) 362–368

−50 pA) holding current was applied to bring the resting membrane Current-clamp recordings: Hippocampal neurons potential (RMP) to −80 mV. Spontaneous firing activity was captured using a parallel data acquisition system consisting of the two-channel Neurons were studied by current-clamp 40–50 hours post-transfec- Minidigi-1A (Molecular Devices) acquiring the membrane voltage tion. At this post-transfection time, neurites are sparse. Recordings were and membrane current continuously at 500 Hz. obtained from isolated pyramidal-shaped neurons displaying GFP- positive fluorescence which usually were not contacted by axons.

Results To assess the effect of the NaV 1.3/K354Q mutation on excitability, hippocampal neurons were transfected with either the WT or NaV 1.3/ Biophysical effects of the K354Q mutation on NaV1.3 channels K354Q constructs and current-clamp recording conditions were used to study firing properties in transfected pyramidal neurons. The aver-

We assessed the effects of the K354Q mutation on NaV1.3 by age capacitance of the recorded pyramidal neurons was 8.5±0.5 pF carrying out voltage-clamp recordings on HEK 293 cells expressing (n=20) for WT, and 7.5±0.5 pF (n=22) for NaV 1.3/K354Q trans- either wild-type NaV1.3 (WT) or NaV1.3/K354Q channels. The fected cells. The input resistance for these neurons was high at 2.9± activation properties of the sodium currents expressed by these 0.7 GΩ for WT and 2.7±0.4 GΩ for NaV 1.3/K354Q transfected cells. cells were measured by holding the cells at −100 mV and then The resting membrane potential started depolarized (−40±3 mV applying 100 ms test pulses to potentials between −80 and +40 mV for WT, −39±3 mV for NaV 1.3/K354Q transfected cells) but only in 5 mV increments. Inward currents recorded by this protocol are needed small hyperpolarizing holding currents to re-establish a shown in Fig. 1 for WT and K354Q (Figs. 1A,C) channels, as well as resting membrane potential near −80 mV. Only small currents were currents in response to the fast-inactivation protocol (Figs. 1B and D). required to reach threshold (36±10 pA for WT, 16±15 pA for NaV 1.3/K354Q transfected cells; p=0.025).

Voltage-dependence of activation and fast-inactivation A notable difference between neurons transfected with NaV 1.3/ K354Q compared to neurons transfected with WT channels was a

To assess the voltage-dependence of activation and fast-inactiva- population of neurons that exhibited spontaneous firing. The NaV 1.3/ tion, cells expressing WT and NaV1.3/K354Q channels were studied by K354Q transfected neurons showed spontaneous firing of action first constructing peak current versus voltage (I–V) curves and next potentials in 6 of 16 cells, while only 1 of 13 WT transfected neurons transforming them into conductance versus voltage (G–V) curves as fired spontaneously. The majority of the NaV 1.3/K354Q transfected described in Methods. Each G–V curve was fit to a Boltzmann function neurons that exhibited spontaneous activity also showed fluctuations which directly gives the voltage midpoint (V1/2) as well as the slope of resting membrane potential that sometimes elicited bursts of action factor (k) of the voltage-dependent response. The Boltzmann fits for potentials as illustrated in Fig. 3. Paroxysmal depolarizing shift-like both activation and fast-inactivation were derived for each cell complexes (arrows and panels D1,D2, and D3 in Fig. 3) were present in individually. The averages of the normalized G–V curves for activation 4 of 16 neurons expressing the NaV 1.3/K354Q mutation (including 3 for NaV1.3/K354Q and WT channels were not significantly different neurons, such as the one shown in Fig. 4, which were clearly isolated (WT n =12: V1/2 = −24.0±1.6 mV, k =7.6±0.3; NaV1.3/K354Q and not contacted by axons), but were not present in any of the 13 WT n=13: V1/2 =−21.6±1.3 mV, k=8.1±0.3) as illustrated in Fig. 1E. expressing neurons studied by current-clamp. The average of the fits for fast-inactivation revealed that K354Q channels were also not significantly different compared to WT channels Discussion

(WT n =7: V1/2 = −69.9±1.3 mV, k =6.4±0.2; NaV1.3/K354Q n=11: V1/2 =−70.5±1.1 mV, k=6.5±0.3)asshownin Fig. 1F. Although more than 300 epilepsy-linked sodium channel muta- tions have been reported, the great majority have been mutations of

Persistent current NaV1.1 and NaV1.2. Only one mutation, K354Q, has recently been reported in NaV1.3 in a patient with cryptogenic epilepsy (Holland et Both the WT and NaV 1.3/K354Q channels exhibited a fraction of al., 2008). This mutation was not examined physiologically within channels resistant to inactivation as illustrated by the insets of Figs. 1A NaV1.3 but, because of challenges associated with instability of NaV1.3 and C. When the persistent current was normalized to the maximal during cloning and mutagenesis, was functionally profiled within peak inward current, the average persistent current measured at a cardiac NaV1.5 sodium channels (Holland et al., 2008). The functional −15 mV test potential for K354Q channels (8.1±1.4%, n=15) was effects of single amino acid substitutions, however, can differ significantly increased compared to that for WT expressing cells (4.4± substantially depending on the channel isoform in which they occur 0.4%, n=13, p=0.03). The average peak current density was not (Lampert et al., 2006a; Lossin et al., 2002). In the present study, we significantly different (119±20 pA/pF for WT, 152±45 pA/pF for NaV expressed the K354Q mutation in its native NaV1.3 background, and 1.3/K354Q). Normalizing the persistent current for cell size, the have demonstrated by voltage-clamp that this mutation enhances the persistent current density for NaV 1.3/K354Q expressing cells (10.5± NaV1.3 persistent current nearly two-fold and ramp current more 2.3 pA/pF) remained significantly increased compared to WT expres- than two-fold. We demonstrate by current-clamp that transfection of sing cells (4.9±0.9 pA/pF, p=0.04). hippocampal pyramidal neurons with the mutant NaV1.3 channel reduces current threshold, and produces spontaneous firing and par- Ramp stimulus responses oxysmal depolarizing shift-like complexes. The present results build upon a previous demonstration of per-

We also evaluated the response of WT and NaV 1.3/K354Q chan- sistent sodium current in uninjured mammalian hippocampal neurons nels to small slow depolarizations using a slow-ramp protocol from a (French et al., 1990), which are known to express NaV1.1, NaV1.2, holding potential of −100 mV to +20 mV applied over 600 ms for a NaV1.3, and NaV1.6 isoforms of sodium channels (Whitaker et al., ramp rate of 0.2 mV/ms. To compare between cells, the response was 2001). Previous studies have demonstrated that NaV 1.3 can produce a normalized to the peak inward current recorded during the activation non-inactivating, persistent current (Lampert et al., 2006a,b; Sun et al.,

I–V protocol. The response of the NaV 1.3/K354Q mutant in com- 2007). A number of studies (e.g Alzheimer et al., 1993) indicate that parison to WT channels is shown in Fig. 2. The peak of the slow-ramp persistent current participates in the control of intrinsic neuronal response of NaV 1.3/K354Q channels was 2-fold higher than the peak excitability. There is, for example, also evidence indicating that per- for WT channels (WT, n=8:5.1±0.8%ofpeak;NaV 1.3/K354Q, n=10: sistent sodium currents can drive spontaneous neuronal firing in a 10.8±2.4% of peak, p=0.047). variety of cell types (Baker et al., 2003; Pennartz et al., 1997; Taddese M. Estacion et al. / Experimental Neurology 224 (2010) 362–368 365

Fig. 1. Voltage-clamp of WT and K354Q mutant channels. (A) Superimposed selected traces recorded from a HEK293 cell expressing WT channels in response to the activation stimulation protocol (100 ms duration pulses, see inset). To better illustrate the persistent current remaining at the end of the 100 ms pulses, portions of three of the traces are plotted on an expanded scale. Note that the unit of the vertical scale bar for the inset is in units of percent of maximal peak current. (B) Superimposed selected traces from the same cell as in (A) in response to the fast-inactivation pulse protocol (see inset). (C) Selected traces recorded from a HEK293 cell expressing K354Q channels are illustrated in the same format as in panel (A). (D) Superimposed selected traces from the same cell as in (C) in response to the fast-inactivation pulse protocol. (E) The peak currents for each cell are analyzed to obtain the conductance–voltage (G–V) relation as described in the Methods. The G–V curves for both WT (filled squares) and K354Q (open squares) cells are normalized and then averaged to obtain the plotted data. (F) The responses to the fast-inactivation protocol are analyzed to obtain the voltage-dependence of fast-inactivation as described in the Methods. Normalized and averaged data are shown for both WT (filled squares) and K354Q (open squares) channels. Error bars are standard error of the mean (SEM). 366 M. Estacion et al. / Experimental Neurology 224 (2010) 362–368

and Bean 2002) including hippocampal neurons (Azouz et al., 1996) and, in fact, large persistent currents tend to be present in subicular neurons isolated from patients with temporal lobe epilepsy (Vreug-

denhil et al., 2004). A number of mutations of NaV1.1 associated with epilepsy have been demonstrated to enhance persistent current (Escayg et al., 2000; Lossin et al., 2002, 2003; Rhodes et al., 2004). Interestingly, persistent sodium current tends to be reduced by the anti-convulsants phenytoin, carbamazepine, and topiramate (Lampl et al., 1998; Sun et al., 2007). Enhanced persistent current, as produced

by the NaV1.3/K354Q mutation, would be expected to lead to neuronal hyperexcitability. The evidence presented here thus establishes the

potential of the K354Q mutation, in NaV1.3, as pathogenic for epilepsy. While persistent sodium currents can amplify EPSPs (Llinas and Sugimori 1980; Stuart and Sakmann 1995; Lipowsky et al., 1996), the hippocampal neurons that we studied had been dissociated and cultured for less than 50 h prior to recording, suggesting that they had not developed robust synaptic connections. Consistent with the Fig. 2. K354Q channels exhibit an enhanced ramp response. The currents evoked during appearance of paroxysmal depolarizing shift-like complexes in a smoothly increasing voltage ramp from −100 mV to +20 mV over 600 ms are shown neurons expressing the NaV1.3/K354Q mutation in the absence of from a WT (gray line) and K354Q (black line) expressing cell. The traces have been synaptic events, similar complexes characterized by multiple action normalized to the maximal peak current recorded from that cell. The amplitude and corresponding voltage of the inward peak of the slow ramps were averaged and plotted potentials superimposed on prolonged depolarizations can be as the gray circle (WT) and black circle (K354Q). Error bars are standard error of the recorded from peripheral sensory axons following axotomy (Kocsis mean (SEM). and Waxman, 1983). The appearance of these complexes, which can

Fig. 3. K354Q-expressing hippocampal neurons exhibit spontaneous activity. Continuous segments of current-clamp recording from hippocampal pyramidal cells illustrate patterns of spontaneous firing seen in a subset of cells expressing the K354Q mutant channels. Cells were held at a resting potential of −80 mV. Traces A–D are two minute recordings from four different cells. Small regions of trace (D) are expanded and labeled D1–3 to illustrate action potentials riding on top of transient paroxysmal depolarizing shift- like depolarizations of the membrane potential, which are indicated by arrows in traces B and C. M. Estacion et al. / Experimental Neurology 224 (2010) 362–368 367

and provide strong support for the idea that NaV1.3 mutations can produce epilepsy in postnatal humans. These results more generally

suggest that NaV1.3 channels may be functional in the postnatal human brain, and indicate the need for careful assessment of CNS

function in studies on NaV1.3 blockade in human subjects.

Acknowledgments

We thank Lynda Tyrrell and Bart Toftness for excellent technical assistance. This work was supported by the Medical Research Service and Rehabilitation Research Service, Dept. of Veterans Affairs and by grants from the National Multiple Sclerosis Society and the Erythro- melalgia Association. The Center for Neuroscience and Regeneration Research is a Collaboration of the Paralyzed Veterans of America and the United Spinal Association with Yale University.

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