1Department of Neurology and 2Paralyzed Veterans of America/Eastern Paralyzed Veterans Association Center for Neuroscience Research, Yale University School of Medicine, New Haven, Connecticut 06520; 3Rehabilitation Research Center, Veterans Affairs Connecticut, West Haven, Connecticut 06516; and 4Molecular Pharmacology Unit, Glaxo-Wellcome Research and Development, Medicines Research Centre, Stevenage, Hertfordshire SG1 2NY, United Kingdom
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ABSTRACT |
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Black, J. A.,
T. R. Cummins,
C. Plumpton,
Y. H. Chen,
W. Hormuzdiar,
J. J. Clare, and
S. G. Waxman.
Upregulation of a Silent Sodium Channel After Peripheral, but not
Central, Nerve Injury in DRG Neurons.
J. Neurophysiol. 82: 2776-2785, 1999.
After transection of
their axons within the sciatic nerve, DRG neurons become
hyperexcitable. Recent studies have demonstrated the emergence of a
rapidly repriming tetrodotoxin (TTX)-sensitive sodium current that may
account for this hyperexcitability in axotomized small (<27 µm diam)
DRG neurons, but its molecular basis has remained unexplained. It has
been shown previously that sciatic nerve transection leads to an
upregulation of sodium channel III transcripts, which normally are
present at very low levels in DRG neurons, in adult rats. We show here
that TTX-sensitive currents in small DRG neurons, after transection of
their peripheral axonal projections, reprime more rapidly than those in
control neurons throughout a voltage range of 140 to
60 mV, a
finding that suggests that these currents are produced by a different sodium channel. After transection of the central axonal projections (dorsal rhizotomy) of these small DRG neurons, in contrast, the repriming kinetics of TTX-sensitive sodium currents remain similar to
those of control (uninjured) neurons. We also demonstrate, with two
distinct antibodies directed against different regions of the type III
sodium channel, that small DRG neurons display increased brain type III
immunostaining when studied 7-12 days after transection of their
peripheral, but not central, projections. Type III sodium channel
immunoreactivity is present within somata and neurites of peripherally
axotomized, but not centrally axotomized, neurons studied after <24 h
in vitro. Peripherally axotomized DRG neurons in situ also exhibit
enhanced type III staining compared with control neurons, including an
accumulation of type III sodium channels in the distal portion of the
ligated and transected sciatic nerve, but these changes are not seen in
centrally axotomized neurons. These observations are consistent with a
contribution of type III sodium channels to the rapidly repriming
sodium currents observed in peripherally axotomized DRG neurons and
suggest that type III channels may at least partially account for the
hyperexcitibility of these neurons after injury.
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INTRODUCTION |
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It is well-established that after injury to their
axons within peripheral nerves, dorsal root ganglion (DRG) neurons and
their axons can become hyperexcitable, and this can contribute to
neuropathic pain (Devor 1994; Ochoa and Torebjork
1980
; Zhang et al. 1997
). The postinjury
hyperexcitability of small DRG neurons, which give rise predominantly
to C-type nociceptive fibers, appears to be due, at least in part, to a
change in sodium channel expression because it is accompanied by the
emergence of a fast, tetrodotoxin (TTX)-sensitive sodium current
characterized by rapid repriming (i.e., rapid recovery from
inactivation), together with an attenuation of slow, TTX-resistant
sodium currents (Cummins and Waxman 1997
). The
upregulation of a TTX-sensitive, rapidly repriming sodium current in
DRG neurons after axotomy is of special interest because rapid recovery
from inactivation is associated with a reduced refractory period that
can result in hyperexcitability (Chahine et al. 1994
;
Yang et al. 1994
).
The molecular identity of the channel(s) responsible for the rapidly
repriming TTX-sensitive sodium current after axonal injury in DRG
neurons has not been established. Immunocytochemical studies with
pan-sodium channel antibodies have demonstrated accumulations of sodium
channels in injured axonal endings within neuromas (Devor et al.
1989; England et al. 1994
, 1996
); however, the
identity of these channel(s) has not yet established. Although
accumulation of SNS/PN3 sodium channels has been reported at injured
axonal endings (Novakovic et al. 1998
), SNS/PN3 encodes
a TTX-resistant channel (Akopian et al. 1996
;
Sangameswaran et al. 1996
), leaving the channel
responsible for the increase in rapidly repriming TTX-sensitive current unidentified.
In situ hybridization and RT-PCR both demonstrate an upregulation in
the expression of the previously silent type III sodium channel gene,
which results in the production of mRNA for type III sodium channels in
DRG neurons after axonal injury (Dib-Hajj et al. 1996;
Waxman et al. 1994
). It has been suggested that the type
III sodium channel may provide a molecular basis for the rapidly
repriming sodium current in axotomized DRG neurons (Cummins and
Waxman 1997
). However, the upregulation of type III mRNA
expression is not necessarily accompanied by the deployment of type III
channel protein because translational regulation and posttranslational modulation, as well as transcriptional regulation, can contribute to
the control of ion channel expression within the cell membrane (Black et al. 1998
; Hales and Tyndale
1994
; Sharma et al. 1993
; Sucher et al.
1993
). Therefore we have examined the expression of type III
sodium channel protein in small DRG neurons after axotomy. In this
study, we used patch-clamp recording to study TTX-sensitive sodium
currents, together with immunocytochemical methods with
isoform-specific antibodies that are selective for type III sodium
channels, to ask whether rapidly repriming current/type III sodium
channels are deployed in DRG neurons after injury to their peripheral
or central axons and found accelerated repriming, accompanied by
increased type III sodium channel immunoreactivity, after peripheral
but not central axotomy. We also asked whether the newly deployed type
III channels are targeted to a particular part of the injured neurons,
and we report that they are present not only within somata but also
within distal parts of ganglion cell axons in vitro and in situ, close
to the region of transection.
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METHODS |
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In this study, peripheral projections of DRG neurons were
axotomized via ligation and transection of the sciatic nerve at the
mid-thigh level (Dib-Hajj et al. 1996) or central
projections of DRG neurons were axotomized by transection of the dorsal
root (dorsal rhizotomy) (Kenney and Kocsis 1997
). DRG
neurons were studied by patch clamp 7-12 days after peripheral or
central axotomy. Immunocytochemical methods also were used 7-12 days
after transection of either peripheral or central projections of DRG
neurons to study the deployment of brain type III sodium channel
protein within these cells. DRG neurons initially were studied after
short-term (<24 h) culture; this protocol was chosen to provide as
close a match as possible to earlier studies (Cummins and Waxman
1997
), which provided quantitative patch-clamp data on the
sodium currents in axotomized DRG neurons. Subsequently DRG neurons and
their peripheral projections in situ also were studied 7-12 days
postaxotomy, using similar immunocytochemical methods.
Surgery
For transection of the peripheral projections of the DRG
neurons, adult female Sprague-Dawley rats were anesthetized with ketamine/xylazine (40/2.5 mg/kg ip) and the right sciatic nerves were
exposed at the midthigh level, ligated with 4-0 silk sutures to prevent
regeneration to peripheral targets, transected and the proximal stumps
placed in silicon cuffs to prevent regeneration (Waxman et al.
1994). In some animals, hydroxystilbamine methanesulfonate (4%
w/vol; Molecular Probes, Eugene, OR), a retrogradely transported fluorescent label, was placed in the cuff before insertion of the nerve
stump. The fluorescent label clearly identified neurons in which their
axons were transected. The contralateral sciatic nerves served as controls.
For transection of the central projections of the DRG neurons, adult female Sprague-Dawley rats were anesthetized with pentobarbital sodium (50 mg/kg ip), and a L3 laminectomy was performed. An incision was made in the dura, and the L4 and L5 dorsal roots were identified and transected (dorsal rhizotomy) with iridectomy scissors. The lesion was packed with Gel-foam and the overlying muscles and skin were closed in layers with 4-0 silk sutures.
Seven- to 12 days after surgery, rats either were killed with an overdose of ketamine/xylazine and decapitated and the tissue was harvested for cell culture or were anesthetized with ketamine/xylazine, perfused with 4% paraformaldehyde in 0.14 M Sorensen's phosphate buffer and tissue obtained for immunocytochemical studies.
Cell culture
Cultures of DRG neurons were established as described previously
(Rizzo et al. 1994). Briefly, axotomized and control
(uninjured) lumbar ganglia (L4,
L5) were excised, freed from their connective tissue sheaths and incubated sequentially in enzyme solutions containing collagenase and then papain. The tissue was triturated in
culture medium containing 1:1 Dulbecco's modified Eagle medium (DMEM)
and Hanks' F12 medium and 10% fetal calf serum, 1.5 mg/ml trypsin
inhibitor, 1.5 mg/ml bovine serum albumin, 100 U/ml penicillin, and 0.1 mg/ml streptomycin and plated on polyornithine/laminin-coated coverslips. The cells were maintained at 37°C in a humidified 95%
air-5% CO2 incubator overnight and then used for
patch-clamp investigation or processed for immunocytochemical studies
as described previously (Black et al. 1998
;
Cummins and Waxman 1997
).
Electrophysiology
Sodium currents in small (18-27 µm diam) DRG neurons were
studied after short-term culture (12-24 h). Whole cell patch-clamp recordings were conducted at room temperature (~21°C) using an EPC-9 amplifier and the Pulse program (v 7.89). Fire-polished electrodes (0.8-1.5 M) were fabricated from 1.65-mm Corning 7052 capillary glass using a Sutter P-97 puller. The average access resistance was 2.2 ± 0.6 M
(mean ± SD, n = 38) for control cells and 2.1 ± 0.7 M
(n = 35) for axotomized cells. Voltage errors were minimized using 70-80%
series resistance compensation, and the capacitance artifact was
canceled using the computer-controlled circuitry of the patch-clamp
amplifier. Linear leak subtraction, based on resistance estimates from
four to five hyperpolarizing pulses applied before the depolarizing
test potential, was used for all voltage-clamp recordings. Membrane
currents were usually filtered at 2.5 kHz and sampled at 10 kHz. The
pipette solution contained (in mM) 140 CsF, 1 EGTA, 10 NaCl, and 10 HEPES, pH 7.3. The standard bathing solution was (in mM) 140 NaCl, 3 KCl, 1 MgCl2, 1 CaCl2, 0.1 CdCl2, and 10 HEPES, pH 7.3. Cadmium was included to block calcium currents. The osmolarity of all solutions was adjusted
to 310 osM.
Immunocytochemistry
ANTIBODIES. Two anti-brain type III sodium channel polyclonal antibodies were used in these experiments. Initial experiments were performed with antibody K175, which is directed against a polypeptide sequence in the C-terminus region of the sodium channel (see following text); subsequent experiments were performed with anti-type III sodium channel antibody (Alamone Labs, Jerusalem), which is directed against a polypeptide sequence within the intracellular loop joining domains I and II of the sodium channel. Both antibodies yielded similar results.
ANTIBODY K175 PRODUCTION. The 21-residue peptide QRLKNISSKYDKETIKGRIDC corresponding to amino acid residues 1869-1888+Cys of human brain III was synthesized on a Biosearch 9500 peptide synthesizer using solid-phase Fmoc chemistry. Cleaved peptide was purified by gel filtration and conjugated to a purified protein derivative of tuberculin (PPD) using sulfo-SMCC. Dutch rabbits, presensitized against BCG, were immunized with the resulting conjugate emulsified in incomplete Freund's adjuvant. The specific antibody response was followed by indirect ELISA using free synthetic peptide as antigen, and the antibody utilized was named K175. The antibody preparations used in this study were affinity-purified using immobilized cognate peptide.
IMMUNOBLOTTING. A HEK 293 cell line that stably expresses the human brain type III sodium channel, and a control HEK 293 cell line were grown to confluence at 37°C and 5% CO2 in Dulbecco's MOD Eagle medium, containing 10% fetal bovine serum, 1× nonessential amino acids (Life Technologies), and 2 mM L-glutamine. Cells were washed briefly in PBS, then lysed in buffer containing 1% Nonidet-P40, 150 mM NaCl, 50 mM Tris-HCl (pH8.0), 0.5% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, and a proteinase inhibitor cocktail (Boehringer Mannheim). The cell extract was spun at 1,400 g for 10 min at 4°C, the supernatant was recovered and the protein concentration was determined using Pierce BCA reagent before mixing with 2× SDS sample buffer (0.5 M Tris-HCl pH6.8, 10% SDS, 0.2% bromphenol blue, 2% DTT, and 20% glycerol). Eleven micrograms of each sample was loaded on a 6% Tris-glycine precast gel (Novex). Proteins were transferred to nitrocellulose paper using a semidry blotter (Pharmacia Biotech) for 1.5 h. The blot was incubated for 2 h at room temperature in PBS containing 5% fat-free milk powder, and probed with a human brain type III sodium channel specific antibody (K175) at 4°C overnight in PBS containing 5% fat-free milk powder, 0.1% Tween20. The blot was washed for 45 min in PBS containing 0.1% Tween20 with several changes and incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Sigma) for 1 h at room temperature. It then was washed for 45 min PBS containing 0.1% Tween20. Binding of antibody was binding detected using chemiluminescence with ECL reagent according to manufacture's instructions (Supersignal, Pierce).
ANTIBODY CHARACTERIZATION. Antibody K175 was produced by immunization of a rabbit with a polypeptide specific for human brain type III sodium channel, which is identical to the equivalent rat polypeptide. Alignment of the immunizing type III polypeptide sequence with the equivalent regions of the other sodium channels found in DRG shows that the immunizing sequence is specific for brain type III sodium channels (Table 1). Western blot analysis of antibody K175 showed that it reacted with a predominant species at ~260 kDa (Fig. 1).
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IMMUNOSTAINING. Coverslips with neurons derived from control or axotomized L4/L5 DRG and maintained in vitro for <24 h were processed for immunocytochemistry as follows: 1) complete saline solution, twice,1 min each; 2) 4% paraformaldehyde in 0.14 M Sorensen's phosphate buffer, 10 min; 3) PBS, three times, 3 min each; 4) PBS containing 20% normal goat serum, 1% bovine serum albumin, and 0.1% Triton X-100, 15 min; 5) primary antibody [rabbit K175, 1:100, or anti-type III antibody (Alamone), 1:100, in blocking solution without Triton], overnight at 4°C; 6) PBS, six times, 5 min each; 7) secondary antibody [goat anti-rabbit IgG-Cy3, 1: 3000]; and 8) PBS, six times, 5 min each.
Fourteen-micrometer cryosections of intact control and axotomized DRG and sciatic nerves were mounted on poly-l-lysine coated glass slides and processed for immunocytochemistry as described above with the following minor modifications: slides were incubated in 50 mM NH4Cl2 (20 min, room temperature) to reduce autofluorescence, the slides were not incubated in 4% paraformaldehyde, and slides were incubated in blocking solution for 30-45 min. After the immunocytochemical procedure, the slides were mounted with Aqua-poly mount and examined with a Leitz Aristoplan light microscope equipped with bright field, Nomarski ,and epifluorescence optics. Images were captured with a Dage DC-330T color camera and Scion CG-7 color PCI frame grabber. Digitized images were manipulated in Adobe Photoshop, with control and experimental tissue being processed in identical manners. Control experiments included incubation without primary antibody and preadsorption of the antibody with 100-500 M excess of immunizing peptide. Only background levels of fluorescence were detected in the control experiments. ![]() |
RESULTS |
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Sodium currents in DRG neurons
In a previous study, it was reported that the TTX-sensitive
current in peripherally axotomized small (18-27 µm diam) DRG neurons recovered from inactivation (reprime) more rapidly than the
TTX-sensitive currents in uninjured neurons (Cummins and Waxman
1997). We confirmed this observation in the present study:
whereas 86% of peripherally axotomized (identified by fluorescent
label backfill) DRG neurons (n = 35) recovered rapidly
from inactivation, <20% of control neurons did (n = 36). However, in our previous study, repriming kinetics were only
measured at
100 mV. In the present study, we therefore asked whether
TTX-sensitive sodium currents in axotomized small neurons also reprime
much faster than control neurons at voltages near the expected resting
potential for DRG neurons. Figure 2 shows
representative results at a holding potential of
80 mV and
demonstrates that repriming was more rapid in peripherally axotomized
neurons. We examined the time course for repriming at voltages ranging
from
140 to
50 mV. Figure 3 shows
that the TTX-sensitive currents in the axotomized small neurons reprime more rapidly than the TTX-sensitive current in control neurons throughout this voltage range. These results show that the distinct repriming kinetics for the TTX-sensitive current in peripherally axotomized neurons do not simply reflect a shift in the voltage dependence of recovery from inactivation; rather, they suggest that the
predominant TTX-sensitive channels in peripherally axotomized small
neurons are fundamentally different from those in uninjured neurons.
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We also asked whether transection of the central projections (dorsal
roots) of small DRG neurons elicited the emergence of a similar rapidly
repriming TTX-sensitive sodium current in these neurons. In contrast to
peripherally axotomized neurons, which exhibited a significant shift in
repriming kinetics, the repriming kinetics of centrally axotomized and
control DRG neurons were identical at 80 mV (Fig.
4; tau =145 ± 15, n = 25, and 147 ± 15, n = 25, respectively). Figure 4 also shows that the repriming kinetics of
the TTX-sensitive currents in centrally axotomized neurons was similar
at all voltages examined from
140 to
50 mV. These results indicate
that transection of the central projection of small DRG neurons does
not induce the appearance of a rapidly repriming TTX-sensitive sodium
current.
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Type III expression in DRG neurons
All experiments used two polyclonal antibodies generated against different regions of the type III sodium channel, which yielded similar results.
CULTURED NEURONS. We first studied DRG neurons that had been maintained overnight in culture to match the conditions used in the patch-clamp studies. Sodium channel type III protein was not detected above background levels in most control (nonaxotomized) small (<27 µm diam) DRG neurons (Fig. 5, a and e). Less than 20% of control neurons displayed type III immunostaining above background, and in most of these, the staining was at a low level. In contrast, most small neurons derived from peripherally transected DRG displayed substantial type III immunoreactivity (Fig. 5, b and f). Preadsorption of the antibodies with their respective immunizing peptides eliminated type III immunostaining in the axotomized neurons (Fig. 5, c and g).
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DRG NEURONS IN SITU. Within control DRG in situ, type III immunoreactivity was limited to low-to-moderate levels in ~10% of small DRG neurons (Fig. 8, a and e). In contrast, type III immunostaining was substantially increased in peripherally axotomized small DRG neurons (Fig. 8, b and f). Most axotomized small neurons exhibited at least a moderate level of type III staining, and many of these neurons showed high levels of immunofluorescence. Unlike transection of the sciatic nerve, dorsal root transection was not accompanied by an up-regulation of type III immunostaining in most small DRG neurons (Fig. 8, d and h).
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SCIATIC NERVE IN SITU. Control sciatic nerves did not exhibit detectable type III immunostaining at any point along their lengths (Fig. 9c). In contrast, there was marked type III immunoreactivity in the distal region of ligated and transected sciatic nerves; a collar of prominent type III immunostaining was present in the transected axons just proximal to the ligature (Fig. 9, a, b, and e). The region of sciatic nerve constricted by the ligature did not exhibit type III immunostaining nor did the portion of the sciatic nerve proximal to the immunostaining collar (Fig. 9, a and e). Within the region of accumulation of type III immunoreactivity, the distal parts of many individual axons could be observed to exhibit type III immunostaining (Fig. 9b). Preadsorption of the type III specific antibodies with their respective immunizing peptides completely eliminated immunostaining in the ligated sciatic nerve (9, d and f).
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DISCUSSION |
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Although hyperexcitability of DRG neurons has been implicated as
an important factor in the pathophysiology of neuropathic pain, its
molecular basis has not been elucidated. On the basis of
electrophysiological results in axotomized motor neurons
(Sernagor et al. 1986; Titmus and Faber
1986
) and of observations of increased sodium channel
immunoreactivity within axonal endings in neuromas (Devor et al.
1989
; England et al. 1994
, 1996
), postaxotomy
hyperexcitability has been attributed, at least in part, to a
pathological buildup of sodium channels. Previous studies have been
interpreted as suggesting that this is due to abnormal vectorial
transport resulting from loss of target membrane within the amputated
distal nodes of Ranvier and nerve endings (Devor 1994
;
Devor et al. 1989
; Titmus and Faber
1990
). The question, of whether different types of
sodium channels might be synthesized in DRG neurons after nerve injury, was raised by Waxman et al. (1994)
in a study that
demonstrated an upregulation of the previously silent type III sodium
channel gene in axotomized DRG neurons. Patch-clamp studies
subsequently demonstrated a change in the characteristics of
TTX-sensitive sodium currents in DRG neurons after sciatic nerve
transection with the emergence of a current displaying a fourfold
acceleration of recovery from inactivation that appears to contribute
to hyperexcitability (Cummins and Waxman 1997
). The data
shown in Figs. 2 and 3 extend our previous results by demonstrating
that, after peripheral axotomy within the sciatic nerve, the recovery
from inactivation of TTX-sensitive currents is also significantly
faster at voltages near the resting membrane potential for DRG neurons
(
80 to
60 mV). Axotomized DRG neurons with rapidly repriming
TTX-sensitive sodium currents therefore should to be able to sustain
higher firing frequencies, supporting the suggestion (Cummins
and Waxman 1997
) that expression of sodium channels with
distinct kinetic properties contributes to hyperexcitability in these cells.
When expressed in oocytes, the type III sodium channel is TTX sensitive
(Joho et al. 1990; Suzuki et al. 1988
).
Within the normal nervous system, type III sodium channel mRNA is
expressed in embryonic neurons but is strongly downregulated as
development proceeds (Beckh et al. 1989
; Black et
al. 1996
; Brysch et al. 1991
; Dib-Hajj et
al.1996
; Felts et al. 1997
). The appearance of
type III sodium channel mRNA (Dib-Hajj et al. 1996
;
Waxman et al. 1994
) and protein (this paper) in
axotomized DRG neurons therefore was unexpected. We observed similar
increases in type III protein in DRG neurons after peripheral axotomy
using two different antibodies directed against the type III channel.
This increase in type III protein could not have been predicted from the previous demonstration of type III mRNA because increased transcription of channel mRNA not always is accompanied by increased levels of functional proteins (Black et al. 1998
;
Hales and Tyndale 1994
; Sharma et al.
1993
; Sucher et al. 1993
). The upregulation of
the type III sodium channel, moreover, is not part of a global up-regulation of channel synthesis in axotomized DRG neurons because expression of the mRNAs for the TTX-resistant SNS/PN3 (Dib-Hajj et al. 1996
) and NaN (Dib-Hajj et al. 1998
)
sodium channels is significantly reduced after axotomy.
The data presented here support the hypothesis that the type III sodium
channel underlies the TTX-sensitive rapidly repriming sodium current in
peripherally injured small DRG neurons (Cummins and Waxman
1997). There is a good correspondence between the percentages of axotomized small neurons (identified by fluorescent backfill) that
show TTX-sensitive rapid repriming (86%) and those that have type III
immunoreactivity (79%). Moreover, patch-clamp and immunocytochemical studies reveal parallel changes in demonstrating a lack of rapid repriming and type III staining in small DRG neurons after dorsal rhizotomy 7-12 days previously (it should be noted that cultured cells
were studied after <24 h in vitro, so that any effect of shearing off
the cells' main stem axon was a short-term one). This latter
observation demonstrates that axonal injury in itself is not sufficient
to upregulate type III sodium channel protein in DRG neurons. The
neuronal response is dependent on whether central or peripheral
projections are injured. In agreement with our results, previous
studies have demonstrated pronounced electrophysiological changes in
sensory neurons after peripheral axotomy, whereas central axotomy
triggered much smaller electrophysiological alterations in these
neurons (Gallego et al. 1987
; Gurtu and Smith
1988
). Likewise, results have been reported for several other
molecules, including heat shock protein 27 (Costigan et al.
1998
) and c-Jun (Broude et al. 1997
;
Kenney and Kocsis 1997
), which show significantly altered levels after peripheral, but not central axotomy, presumably at
least in part due to deprivation from a peripheral source of trophic
molecules (see Dib-Hajj et al. 1998
)
The present results demonstrate increased levels of type III sodium
channel protein not only in cell bodies but also in the neurites of
axotomized DRG neurons. The apparent disparity between the in situ
results (type III expression at severed axon tips expression along
the proximal axon trunk) and the in vitro results (type III expression
along entire neurite) may reflect the shorter length of DRG neuron
axons in vitro, which does not permit the development of a
proximo-distal gradient of channels. Irrespective of this, our
observations of the presence of high levels of type III protein within
the tips of DRG axons in vitro and of the highest levels of type III
protein within the tips of transected axons in situ are consistent with
results showing that axons within neuromas are hyperexcitable and can
act as ectopic impulse generators (Burchiel 1984
;
Meyer et al. 1985
; Scadding 1981
;
Wall and Gutnick 1974a
,b
). The present findings provide
support for the hypothesis (Cummins and Waxman 1997
)
that the type III channel produces the rapidly repriming TTX-sensitive
sodium current that emerges in small DRG neurons after axotomy and
suggest that the activation of the previously quiescent type III sodium
channel gene contributes to abnormal hyperexcitability of injured
sensory neurons and/or their processes.
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ACKNOWLEDGMENTS |
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The authors thank B. Toftness for excellent technical assistance. We also thank Simon Tate, Ph. D., for a critical reading of the manuscript.
This work was supported in part by grants from the Medical Research Service and the Rehabilitation Research Service, Department of Veterans Affairs, National Multiple Sclerosis Society, and the Paralyzed Veterans of America/Eastern Paralyzed Veterans Association. T. R. Cummins was supported in part by fellowships from the Eastern Paralyzed Veterans Association and the Spinal Cord Research Foundation.
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FOOTNOTES |
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Address for reprint requests: S. G. Waxman, Dept. of Neurology, LCI 707, Yale University School of Medicine, P.O. Box 208018, New Haven, CT 06510.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 4 June 1999; accepted in final form 6 July 1999.
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REFERENCES |
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