1Department of Neurology, Yale University School of Medicine, New Haven, Connecticut 06510; 2Neuroscience Research Center, Veterans Administration Medical Center, West Haven, Connecticut 06516; and 3Department of Neuroscience, Karolinska Institute, SE-171 77 Stockholm, Sweden
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ABSTRACT |
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Fjell, Jenny,
Theodore R. Cummins,
Kaj Fried,
Joel A. Black, and
Stephen G. Waxman.
In vivo NGF deprivation reduces SNS expression and TTX-R sodium
currents in IB4-negative DRG neurons. Recent evidence suggests that changes in sodium channel expression and localization may be
involved in some pathological pain syndromes. SNS, a
tetrodotoxin-resistant (TTX-R) sodium channel, is preferentially
expressed in small dorsal root ganglion (DRG) neurons, many of which
are nociceptive. TTX-R sodium currents and SNS mRNA expression have
been shown to be modulated by nerve growth factor (NGF) in vitro and in
vivo. To determine whether SNS expression and TTX-R currents in DRG
neurons are affected by reduced levels of systemic NGF, we immunized
adult rats with NGF, which causes thermal hypoalgesia in rats with high antibody titers to NGF. DRG neurons cultured from rats with high antibody titers to NGF, which do not bind the isolectin IB4
(IB4) but do express TrkA, were studied with whole cell
patch-clamp and in situ hybridization. Mean TTX-R sodium current
density was decreased from 504 ± 77 pA/pF to 307 ± 61 pA/pF
in control versus NGF-deprived neurons, respectively. In comparison,
the mean TTX-sensitive sodium current density was not significantly
different between control and NGF-deprived neurons. Quantification of
SNS mRNA hybridization signal showed a significant decrease in the
signal in NGF-deprived neurons compared with the control neurons. The
data suggest that NGF has a major role in the maintenance of
steady-state levels of TTX-R sodium currents and SNS mRNA in
IB4
DRG neurons in adult rats in vivo.
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INTRODUCTION |
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Changes in sodium channel expression and
distribution in primary sensory neurons have been suggested to be
involved in several models of neuropathic and inflammatory pain,
including neuromas (Matzner and Devor 1994), axonal
transection (Dib-Hajj et al. 1996
), chronic constriction
of the sciatic nerve (Novakovic et al. 1998
) and
inflammatory pain (Tanaka et al. 1998
). Adult rat dorsal
root ganglion (DRG) neurons have been shown to express multiple sodium
channel mRNAs (Black et al. 1996
). One of the sodium
channel isoforms,
SNS/PN3, has received considerable attention with
respect to possible involvement in pain syndromes because it is
preferentially expressed in small-diameter DRG neurons, which include
neurons involved in nociception and thermoception. When expressed in
Xenopus oocytes,
SNS/PN3 produces a slowly inactivating,
tetrodotoxin-resistant (TTX-R) sodium current (Akopian et al.
1996
; Sangameswaran et al. 1996
). Sensory
neurons that express TTX-R sodium currents have broader action
potentials and characteristic inflections on the descending limb of the
action potentials; these characteristics are seen in virtually all
C-type neurons as well as in some A
and high-threshold A
neurons
(see Koerber and Mendell 1992
).
Recently, the expression of SNS/PN3 and TTX-R sodium currents was
examined in several models of neuropathic and inflammatory pain. After
transection of the sciatic nerve, there is a reduction in TTX-R
currents in small- and medium-diameter DRG neurons (Cummins and
Waxman 1997
; Rizzo et al. 1995
), and
concurrently there is a decrease in
SNS/PN3 mRNA in these neurons
(Dib-Hajj et al. 1996
). Tight ligature of L5/6 dorsal
roots is also accompanied by a substantial reduction in the expression
of
SNS/PN3 mRNA (Okuse et al. 1997
). In contrast to
the reduction in TTX-R currents and
SNS/PN3 mRNA expression after
axonal transection, TTX-R currents and
SNS/PN3 mRNA expression are
increased in small DRG neurons after carrageenan-induced inflammation
(Tanaka et al. 1998
). Although the mechanism for
alterations of
SNS/PN3 mRNA levels is not understood, it is
noteworthy that nerve growth factor (NGF) is thought to be a mediator
of inflammatory pain (Lewin and Mendell 1993
;
Lewin et al. 1993
, 1994
; Woolf et al. 1994
,
1996
).
Although the mechanism has not been clearly identified, NGF appears to
play an important role in the regulation of SNS/PN3 expression. In
an in vitro model of axotomy, supplementing the growth medium with NGF
inhibited a substantial reduction of
SNS/PN3 mRNA expression in
small DRG neurons (Black et al. 1997
). Moreover, exogenous delivery of NGF to the transected sciatic nerve stump resulted in an up-regulation of TTX-R sodium currents and
SNS/PN3 mRNA in small DRG neurons (Dib-Hajj et al. 1998
). NGF
has also been shown to be crucial for the maintenance of the TTX-R
component of the action potential in adult DRG neurons (Aguayo
and White 1992
; Lewin et al. 1992
;
Oyalese et al. 1997
; Ritter and Mendell 1992
). More recently, however, Wood and coworkers (Okuse
et al. 1997
) questioned the importance of NGF on the regulation
of SNS mRNA.
In adult animals, NGF is produced in small quantities in the epidermis
and is thought to act directly on some sensory neurons through the
neurotrophin receptors TrkA and p75. Despite the fact that most sensory
neurons are dependent on NGF during embryogenesis, less than one-half
of small DRG neurons in the adult have receptors for NGF
(McMahon et al. 1994). The NGF-responsive DRG neurons constitute a population of peptidergic nociceptors, which are characterized by their production of the neuropeptide CGRP. In contrast, nonpeptidergic small DRG neurons are characterized by their
ability to bind the lectin IB4 and are nonresponsive to NGF in the
adult (Molliver et al. 1997
; Petruska et al.
1997
; Plenderleith and Snow 1993
;
Plenderleith et al. 1988
). Thus most small neurons that
are not labeled by IB4 would be expected to be responsive to NGF.
Previous studies that examined the modulation of the expression of
SNS/PN3 mRNA and TTX-R sodium currents in DRG neurons by NGF
utilized models that involve axonal transection or inflammatory responses, making the role of NGF per se unclear. To study the role of
NGF on noninjured adult DRG neurons that have receptors for NGF, we
depleted adult rats of systemic NGF through autoimmunization (Chudler et al. 1997
; Doubleday and Robinson
1994
, 1995
; Gorin and Johnson 1979
, 1980
;
Otten et al. 1979
; Schwartz et al. 1982
) and examined the IB4-negative (IB4
) neurons. Our results
demonstrate that small, IB4
DRG neurons from NGF-depleted
rats exhibit a significant decrease in the expression of TTX-R sodium
currents and
SNS/PN3 mRNA. These results are consistent with a
direct action of NGF in the regulation of
SNS/PN3 in vivo.
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METHODS |
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Animals
Twenty adult (100-125 g) male Sprague-Dawley rats were immunized with 50 µg 7S mouse NGF in 100 µl normal saline solution emulsified with 100 µl complete Freund's adjuvant (Sigma). The animals were boosted with a second injection of NGF in incomplete Freund's adjuvant (Sigma) 2 wk later. Untreated animals served as controls. Animals were group housed and had access to food and water ad libidum. All immunized animals gained weight, and they did not show signs of discomfort. Their weights ranged from 350 to 405 g by the time of killing.
Two and 4 wk after the second immunization, peripheral blood was collected, and the antibody titers were analyzed with enzyme-linked immunosorbent assay. Multiwell plates (Nalge-Nunc) were coated with 2 µg/ml 7S-NGF in 0.1 M bicarbonate buffer (pH 9.3) and incubated at 4°C overnight. The plates were washed three times with 0.1 M phosphate-buffered saline (PBS) (pH 7.4) containing 0.05% Tween (PBS-Tween), and nonspecific binding was blocked with 2% bovine serum albumin (BSA) in PBS-Tween for 1 h at 37°C. The plates were washed three times in PBS-Tween, and serial dilutions from 1:4000 to 1:96000 of serum were made in PBS-Tween and applied to preblocked wells for 2 h at 37°C. After three washes with PBS-Tween, goat-anti-rat immunoglobulin G conjugated with alkaline phosphatase (Zymed) (1:1000) was applied for 1 h at 37°C. The plates were washed in PBS-Tween and incubated with p-nitrophenyl phosphate substrate for 30 min before optical density (OD) was measured at 405 nm. The endpoint titer was defined as the dilution at which the OD was twice the background.
Animals with high anti-NGF antibody titers (>1:48000) were selected for in situ hybridization and electrophysiology studies. In each experiment, one control and one high titer rat were deeply anesthetized with xylazine/ketamine (40/2.5 mg/kg ip) and decapitated. The L4 and L5 ganglia were quickly removed and desheathed in sterile complete saline solution (CSS) (pH 7.2). The DRGs were then digested with collagenase A (1 mg/ml) for 20 min at 37°C in CSS and then in collagenase D (1 mg/ml) containing papain (30 units/ml) for 15 min at 37°C in CSS. The DRGs were gently centrifuged (100 g for 3 min), and the pellet was dissolved and triturated in DRG media (DMEM:F12, 10% FCS) with 1 mg/ml BSA (Sigma, Fraction V) and 1 mg/ml trypsin inhibitor (Sigma). The cells were then plated on poly-ornithine laminin-coated glass coverslips and incubated at 37°C in a humidified 95% air-5% CO2 incubator.
Whole cell recordings
DRG neurons were recorded from in the whole cell patch-clamp
configuration 18-30 h after dissociation and plating. The cells were
incubated for 30-60 min with FITC-labeled isolectin B4 (40 µg/ml,
Sigma) just before being moved to the recording chamber. Cells that did
not exhibit IB4 fluorescence were chosen for recording. All recordings
were made with an EPC-9 amplifier, a Macintosh Quadra 950, and the
Pulse program (v. 7.52, HEKA Electronic, Germany). Recording electrodes
(0.8-1.5 M) were fabricated from 1.65-mm capillary glass (WPI) with
a Sutter P-87 puller. Cells were not considered for analysis if the
initial seal resistance was <2 G
or if they had high leakage
currents (holding current > 1 nA at
80 mV) or an access
resistance >5 M
. The average access resistance was 2.5 ± 0.9 M
(mean ± SD, n = 117). Voltage errors were
minimized with 70-80% series resistance compensation. Linear leak
subtraction and capacitance artifact cancellation were used for all
recordings. Membrane currents were filtered at 2.5 kHz and sampled at
10 kHz. The pipette solution contained (in mM) 140 CsF, 2 MgCl2, 1 ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid, and 10 Na-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES; pH 7.3). The standard extracellular solution contained (in
mM) 140 NaCl, 3 KCl, 2 MgCl2, 1 CaCl2, 0.1 CdCl2, and 10 HEPES (pH 7.3). Cadmium was included to block
calcium currents. The osmolarity of the solutions was adjusted to 310 mosm (Wescor 5550 osmometer). The liquid junction potential for these
solutions was <7 mV; data were not corrected to account for this
offset. The offset potential was zeroed before patching the cells and
checked after each recording for drift. All recordings were conducted
at room temperature (~21°C).
In situ hybridization
Before in situ hybridization, the culturing medium was
supplemented with biotin labeled-isolectin IB4 (Sigma) at 40 µg/ml and incubated for 30 min at 37°C. Coverslips with cells from
NGF-deprived and control animals were washed with CSS and then fixed
for 10 min in 4% formaldehyde in 0.14 M Sorensons buffer, pH 7.2. SNS probe construction and in situ hybridization were performed as previously described (Black et al. 1996) with minor
modifications. After hybridization and stringent washes, the coverslips
were incubated with 40 µg/ml streptavidin-CY2 (Amersham) and alkaline phosphatase conjugated anti-digoxigenin antibody (1:500)
(Boerhinger-Mannheim) in Tris-buffered blocking solution (1% BSA, 2%
normal sheep serum) at 4°C overnight. For each experiment, cells from
control and the NGF-deprived animals were incubated in the chromagen
solution for the same length of time, and the reaction stopped before
the NBT-reaction reached saturation.
Quantification
Coverslips were examined with a BioRad MRC-600 confocal
microscope equipped with brightfield and BHS filter. Cells were
randomly selected from six to eight coverslips from each DRG culture
for each condition and captured with COMOS image acquisition program. IB4 neurons were characterized by the lack of fluorescent
signal above background levels (e.g., Fig. 3). Quantification of the SNS hybridization signal was performed as previously described (Black et al. 1997
). Briefly, OD measurements of the
neurons were obtained with the NIH Image program. The brightfield gray
levels were linearly calibrated to OD (R2 > 0.99) with optical filters with OD = 0.1, 0.3, and 0.6. The OD of
randomly selected IB4
neurons was obtained by outlining
the cell body and then measuring average density and surface area.
Within each experiment, the mean OD of IB4
cells in the
NGF-immunized rat was compared with the mean OD of DRG neurons from
control animals with the Student's t-test. A total of 193 IB4
neurons was analyzed from 5 high antibody titer
animals and 4 age-matched controls.
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RESULTS |
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Animals
Adult male Sprague-Dawley rats were immunized to 7S NGF, and the
effect on TTX-R sodium current and SNS mRNA expression in IB4 DRG neurons was examined. The NGF autoimmunization
resulted in antibody titers ranging from 1:4000 to 1:96000. DRG neurons
from animals with the highest anti-NGF antibody titers (>1:48000;
n = 5) were used for whole cell patch-clamp and in situ
hybridization studies. Age-matched control rats showed no titers to
NGF.
Whole cell patch clamp
EFFECT OF NGF DEPRIVATION ON SODIUM CURRENT AMPLITUDE
Sodium currents were recorded in the whole cell patch-clamp
configuration from small (14- to 28-µm diam) DRG neurons that did not
exhibit IB4 fluorescence (IB4 neurons). We recorded 47 control cells (cultured from 4 control animals) and 70 NGF-deprived
cells (cultured from 5 NGF-deprived animals). For the majority of
cells, we used prepulse inactivation (Cummins and Waxman
1997
; McLean et al. 1988
; Roy and
Narahashi 1992
) to distinguish between the fast inactivating,
TTX-sensitive (TTX-S) and the slow inactivating, TTX-R sodium currents.
This allowed simultaneous measurement of both TTX-R and TTX-S current amplitudes in every cell. In a previous study, without IB4 labeling, we
observed that ~85% of small cells exhibited both TTX-R and TTX-S
currents (Cummins and Waxman 1997
). In our control
group, the average TTX-R current density in the IB4
cells
was 504 ± 77 pA/pF (mean ± SE, n = 47),
where current density is estimated by dividing the peak current
amplitude by the cell capacitance. Thirty-eight percent of the control
IB4
neurons expressed low levels (<200 pA/pF) of TTX-R
current. By contrast, the average TTX-R current density in the
IB4
cells from NGF-deprived animals was 307 ± 61 pA/pF (n = 70), and 67% of these cells exhibited low
levels of TTX-R currents (Fig. 1A). Both
the peak TTX-R current amplitude (measured with a test pulse to
10
mV) and the TTX-R current density were significantly (P < 0.05) lower for the NGF-deprived cells than for the control cells.
However, we did not find a significant difference between control and
NGF-deprived animals in terms of TTX-S peak current amplitude (Fig.
1B) or TTX-S current density (1,111 ± 131 pA/pF for
control cells; 1,312 ± 135 pA/pF for anti-NGF cells).
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TTX-R current properties in control and NGF-deprived cells
We examined the basic properties of TTX-R sodium currents in
IB4 neurons to determine if they were altered by NGF
deprivation. Figure 2A shows
TTX-R currents recorded from a typical control cell and a typical
NGF-deprived cell. The voltage dependence of activation and
steady-state inactivation were similar for neurons in both groups (Fig.
2, B and C). The mean midpoint of activation obtained by prepulse subtraction (Cummins and Waxman
1997
) was near
19 mV, and the mean midpoint of steady-state
inactivation (Vh) was near
30 mV for both
groups. Thus, although NGF deprivation decreases the amplitude of the
TTX-R current, it does not alter the properties of the TTX-R currents
in small IB4
DRG neurons.
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Rizzo and coworkers (1994) observed a large degree of interneuronal
variation in the properties of the TTX-R sodium current in small DRG
neurons. In our study, the amount of variability in the voltage
dependence of activation and steady-state inactivation of the TTX-R
currents was similar for control and NGF-deprivation groups. Recently,
Rush and Elliott (1997)
proposed the existence of two distinct
populations of TTX-R currents based on differences in drug sensitivity
and midpoints of steady-state inactivation. By their classification,
type I and type II TTX-R currents had average Vh
of
29 and
46 mV, respectively. If
37.5 mV is used as the dividing
value between type I and type II TTX-R currents, the majority of cells
in both our control and NGF-deprived IB4
groups would be
classified as displaying type I currents. Only two cells in each group
might be considered as displaying TTX-R type II currents.
In situ hybridization
The SNS hybridization signal in control and NGF-deprived
IB4 DRG neurons was quantified by microdensitometry. The
confocal laser microscope was operated in dual-channel mode
(brightfield and FITC fluorescence), which allowed simultaneous
visualization of neurons and their IB4-reactivity (Fig.
3). Images of DRG neurons were captured
and subsequently analyzed in terms of IB4 fluorescence, size (expressed
as surface area) and hybridization signal (OD) with NIH-Image software.
The neuronal size distributions were similar in control and autoimmune
animals (Fig. 4). The mean OD of
NGF-deprived, IB4
neurons with a surface area <1,200
µm2 (~39 µm in diameter) was 0.115 ± 0.70 compared with 0.22 ± 0.108 for control IB4
neurons;
the difference in hybridization signal between control and NGF-deprived
neurons was significant (P < 0.05) in four of five
separate experiments (Table 1). The
relative hybridization signal of IB4-reactive (IB4+) and
IB4
neurons from NGF-deprived rats is shown in Fig.
5. Although IB4
neurons
from NGF-deprived rats exhibit a significant decrease of hybridization
signal compared with control neurons, no significant change of the
hybridization signal is seen in IB4+ neurons. As a measure
of the number of IB4
neurons expressing significant
levels of SNS mRNA, the number of IB4
neurons with an OD
twofold greater than background in situ hybridization signal was
determined. With this threshold, 25 ± 20% of neurons from
NGF-deprived animals expressed significant levels of SNS mRNA (Table
2), compared with 57 ± 4.1% of
neurons from control animals. The difference between the number of
cells positive for SNS mRNA in the autoimmune (27/110) versus control
group (46/83) was significant at P < 0.0005 (odds
ratio = 0.26,
2 with Yates correction).
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DISCUSSION |
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To study the effect of NGF deprivation on the expression of sodium
currents and sodium channel SNS mRNA in noninjured DRG neurons in vivo,
we immunized adult Sprague-Dawley rats to NGF according to the protocol
of Chudler and coworkers (1997). With this immunization protocol,
approximately one-third of the immunized rats developed high antibody
titers (>1:48,000) to NGF, which are associated with decreased levels
of systemic NGF and thermal hypoalgesia (Chudler et al.
1997
). In the present study, IB4
DRG neurons
cultured from high titer rats exhibited a significant reduction in
TTX-R sodium currents and in the levels of SNS mRNA. In contrast, no
significant changes in the amplitude of TTX-R currents or in the levels
of SNS mRNA in IB4+ neurons were detected.
Adult DRG neurons are heterogeneous with respect to size, sensory
modalities, and expression of neurotrophin receptors (Averill et
al. 1995). Small-diameter neurons constitute 70-80% of
neurons in L4 and L5 DRG and are principally nociceptors and
thermoreceptors. Approximately one-half of these small DRG neurons
express neurotrophin receptors TrkA and p75 and synthesize
neuropeptides, whereas the other one-half do not express TrkA or p75
receptors but bind isolectin B4 from Griffonia simplicifolia
(Averill et al. 1995
; McMahon et al.
1994
; Molliver et al. 1995
; Wright and
Snider 1995
). IB4 provides an especially tractable label for
the discrimination between small neurons that express TrkA, and thus
respond to NGF, and those small neurons that lack TrkA receptors
because the expression of many other possible workers, such as CGRP, is
modulated by NGF and may be down-regulated in NGF-depleted neurons.
Because IB4 labeling predominately is found on neurons lacking NGF
receptors, it is unlikely that NGF depletion would affect the IB4
labeling. In support of this notion, our studies showed no difference
in the intensity or distribution of IB4
reactivity in
neurons from NGF-depleted versus control animals (data not shown).
Previous studies have implicated NGF in the up-regulation of TTX-R
sodium currents (Dib-Hajj et al. 1998; Oyalese et
al. 1997
) and
SNS/PN3 mRNA (Black et al.
1997
; Dib-Hajj et al. 1998
) in DRG neurons after
transection of their peripheral axons. However, little information has
been available concerning the role of NGF in the maintenance of TTX-R
sodium channels or
SNS/PN3 transcript levels in intact adult DRG
neurons. Here we show that TTX-R sodium currents and
SNS/PN3 mRNA
are decreased in IB4
neurons from NGF-autoimmunized rats
compared with age-matched adult control rats. Previous work has
demonstrated that rats immunized with NGF develop characteristics
indicative of systemic NGF deprivation, including loss of substance P
immunoreactivity (Schwartz et al. 1982
) and a reduction
of the size and protein content of adrenergic neurons in the superior
cervical ganglia (Otten et al. 1979
). Our observations
are consistent with an important modulatory role by NGF in the
expression of
SNS/PN3 mRNA and TTX-R sodium currents in the adult
nervous system. This hypothesis is supported by several recent studies.
A reduction in TTX-R sodium currents and
SNS/PN3 mRNA was observed
after sciatic nerve transection (Cummins and Waxman
1997
; Dib-Hajj et al. 1996
; Okuse et al.
1997
), and infusion of exogenous NGF to the transected nerve
stump partially rescued TTX-R sodium currents and expression of
SNS/PN3 mRNA (Dib-Hajj et al. 1998
). These
observations suggest that the loss of SNS mRNA and TTX-R currents in
small DRG neurons after axonal transection might, at least in part,
depend on a reduction of retrogradely transported NGF from the
periphery. Furthermore, although induction of inflammation with
Freund's adjuvant might not result in changes in the levels in SNS
mRNA (Okuse et al. 1997
), it has been shown that
experimentally induced inflammation of the rat hind paw by carrageenan
increases cutaneous levels of NGF (Donnerer et al. 1992
)
and, concomitantly, TTX-R sodium currents and
SNS/PN3 mRNA in small
DRG neurons (Tanaka et al. 1998
). These observations are
consistent with a direct role for peripherally derived NGF in the
regulation of
SNS/PN3 expression in small DRG neurons.
Rats that develop high antibody titers to NGF through autoimmunization
show thermal hypoalgesia (Chudler et al. 1997). Although the mechanism underlying this phenomenon is unknown, our results suggest that a reduction of TTX-R currents could contribute to this
hypoalgesia. Agents associated with hyperalgesia, such as serotonin and
prostaglandin E2, have been shown to increase TTX-R currents in small DRG neurons (Gold and Levine 1996
;
Gold et al. 1996
), possibly mediated through
phosphorylation of a TTX-R sodium channel (England et al.
1996
). Moreover, several studies suggest a role of TTX-R sodium
channels in nociception (Arbuckle and Docherty 1995
;
Jeftinija 1994
). Although the precise role that TTX-R
currents play in the firing patterns of DRG neurons is not known,
Elliott (1997)
proposed that slow sodium channel inactivation (as seen in
SNS/PN3) may be a major factor in the generation and/or
maintenance of spontaneous bursts of action potentials. Therefore a
decrease in
SNS/PN3 channels might be expected to affect the
excitability of DRG neurons.
Several alternatives might account for the thermal hypoalgesia
associated with NGF deprivation, in addition to a reduction of TTX-R
currents. First, NGF might have a general trophic effect that maintains
or enhances protein synthesis in a nonselective manner, although such
an effect seems unlikely because the amplitude and density of the TTX-S
currents were unaffected in the NGF-deprived animals. Second, although
we did not see a difference in somatic TTX-S sodium current density,
this does not rule out the possibility that NGF deprivation causes
hypoalgesia by altering TTX-S sodium currents. Mandel and coworkers
(Toledo-Aral et al. 1997) have shown that PN1 TTX-S
channels, which are modulated by NGF in PC12 cells, are preferentially
targeted to the neurite terminals of cultured DRG neurons. Moreover,
PN1 channels have been shown to respond to slow depolarizing inputs
close to resting potential, consistent with a role in signal
amplification or transduction close to sensory terminals
(Cummins et al. 1998
). Therefore NGF deprivation could
affect neuronal excitability by selectively influencing the levels of
TTX-S sodium channels at nerve terminals or by altering the properties
of TTX-S currents. Third, NGF deprivation through autoimmunization has
been shown to decrease the levels of the neuropeptide substance P, a
sensitizing mediator of acute inflammation, in sensory ganglia, spinal
cord, and hind paw skin (Schwartz et al. 1982
). However,
the role, if any, for substance P in noninflamed tissue is not well
known. Fourth, although Chudler and coworkers (1997)
did not detect any
antibodies to NGF in cerebrospinal fluid, the possibility that
peripheral NGF depletion might cause central desensitization through
indirect mechanisms cannot be excluded. Fifth, the sympathetic nervous
system, which has been suggested to be involved in some pain syndromes,
is dependent on NGF (Andreev et al. 1995
); however,
sympathectomy only produces a very transient reduction in inflammatory
hypersensitivity, and it is therefore unlikely that the sympathic
nervous system is of major importance in regulating long-term
sensitivity (Woolf 1996
). Sixth, mast cells play a key
role in skin inflammation and respond to NGF by releasing inflammatory
mediators such as serotonin that in turn might affect the sensitivity
of sensory neurons (Bruni et al. 1982
; Mazurek et
al. 1986
; Nilsson et al. 1997
). Mast cells also
produce and release NGF (Leon et al. 1994
), which
enables an autocrine amplification of the mast cell response. Experimental degranulation of mast cells reduces and delays thermal hyperalgesia after systemic NGF injection (Lewin et al.
1994
), and mast cells are therefore thought to be mediators of
inflammatory hyperalgesia. Although NGF can activate mast cells as part
of the inflammatory response, it is not clear if mast cells have a role
in maintaining the sensitivity of sensory neurons in noninflamed tissue. Arguing against this possibility, Lewin and coworkers (1994)
did not detect a significant difference in heat algesia between animals
whose mast cells had been degranulated and control animals before NGF
injection. This suggests that the thermal hypoalgesia seen in the
NGF-depletion model may not be explained solely by a reduction in mast
cell activity. Finally, the expression of bradykinin receptors was
found to be under NGF control and might be involved in the regulation
of sensitivity in NGF-deprived animals (Bennett et al.
1998
; Petersen et al. 1998
).
Our observations suggest that NGF is involved in the regulation of
SNS/PN3 sodium channel expression in the DRG of adult rats. Why is
there a need for tonic regulation of the electrical properties of
sensory neurons in the adult animal? One possible role of this phenomenon could be to recruit nociceptors after tissue-damaging injury. Several mechanisms, such as sensitization of peripheral nerves
through local mediators and central sensitization, play a role in
ensuring that inflamed and damaged tissue is guarded from further
injury. It has also been suggested that the activation of "silent
nociceptors" might be involved in hyperalgesia and allodynia. These
silent or "very-high-threshold" nociceptors were detected in
viscera, joint capsule, and skin and are characterized by being
evokable only in inflamed tissue (McMahon and Koltzenburg 1990; Michaelis et al. 1996
; Schmidt et
al. 1995
). The levels of NGF increase dramatically in
experimentally induced inflammation of the skin (Donnerer et al.
1992
; Woolf et al. 1994
), and high levels of NGF
are found in synovial fluid from rheumatic arthritis patients
(Aloe et al. 1992
). Furthermore, some of the algesic actions of NGF in inflammatory models have a delay of onset consistent with an effect on gene transcription (Woolf et al.
1994
). Thus the sensitivity of some nociceptors to NGF in the
adult animal may provide a mechanism for lowering the threshold of
silent nociceptors as an additional mechanism to ensure guarding
behavior to protect an inflamed limb or joint. Regardless of the
physiological role for tonic regulation of nociceptors, the
finding that NGF affects the electrical properties of uninjured small
sensory neurons suggests that NGF may play a role in pain associated
with chronic inflammatory conditions by modulating the sensitivity of
some nociceptive neurons.
Our findings support the conclusion that in adult rats, the expression
of SNS/PN3 is tonically regulated by NGF in IB4
DRG
neurons, many of which are nociceptive. These observations add to the
body of data indicating that NGF participates in maintaining the
phenotypic properties of some neuronal populations in the adult nervous
system and suggest that NGF can modulate the electrophysiological properties of DRG neurons, thereby affecting pain perception.
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ACKNOWLEDGMENTS |
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We thank Drs. P. Hjelmstrom and K. Ishiwawa for expert assistance.
This work was supported in part by the Medical Research Service, Department of Veterans Affairs, and the National Multiple Sclerosis Society and by grants from the Paralyzed Veterans of America and the Eastern Paralyzed Veterans Association. J. Fjell was partly supported by the Sweden-America 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, 333 Cedar St., 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 12 June 1998; accepted in final form 22 October 1998.
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REFERENCES |
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