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The Journal of Neuroscience, October 15, 2002, 22(20):9086-9098
Spinal Neurons that Possess the Substance P Receptor Are Required
for the Development of Central Sensitization
Sergey G.
Khasabov1, 2,
Scott D.
Rogers1,
Joseph R.
Ghilardi1,
Christopher M.
Peters1,
Patrick W.
Mantyh1, 3, 4, and
Donald A.
Simone2, 3, 4
Departments of 1 Preventive Sciences,
2 Oral Science, 3 Psychiatry, and
4 Neuroscience, University of Minnesota, Minneapolis,
Minnesota 55455
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ABSTRACT |
In previous studies, we have shown that loss of spinal neurons that
possess the substance P receptor (SPR) attenuated pain and hyperalgesia
produced by capsaicin, inflammation, and nerve injury. To determine the
role of SPR-expressing neurons in modulating pain and hyperalgesia,
responses of superficial and deep lumbar spinal dorsal horn neurons
evoked by mechanical and heat stimuli and by capsaicin were made after
ablation of SPR-expressing neurons using the selective cytotoxin
conjugate substance P-saporin (SP-SAP). Morphological analysis and
electrophysiological recordings were made after intrathecal infusion of
vehicle, saporin alone, or SP-SAP. SP-SAP, but not vehicle or SAP
alone, produced an ~62% decrease in SPR-expressing neurons in the
dorsal horn. Loss of SPR-expressing neurons diminished the responses of
remaining neurons to intraplantar injection of capsaicin. Peak
responses to 10 µg of capsaicin were ~65% lower in animals
pretreated with SP-SAP compared with controls. Additionally,
sensitization to mechanical and heat stimuli that normally follows
capsaicin was rarely observed. Importantly, responses to mechanical and
heat stimuli in the absence of capsaicin were not altered after SP-SAP
treatment. In addition, nociceptive neurons did not exhibit windup in
the SP-SAP-treated group. These results demonstrate that SPR-expressing
neurons located in the dorsal horn are a pivotal component of the
spinal circuits involved in triggering central sensitization and
hyperalgesia. It appears that this relatively small population of
neurons can regulate the physiological properties of other nociceptive
neurons and drive central sensitization.
Key words:
hyperalgesia; capsaicin; electrophysiology; spinal cord; substance P-saporin; windup
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INTRODUCTION |
Chronic pain and hyperalgesia are
symptoms associated with tissue injury and inflammation. The neural
mechanisms underlying persistent pain and hyperalgesia are not fully
understood but are known to involve sensitization of primary afferent
nociceptors and central sensitization. Previous studies have shown that
excitability of nociceptive dorsal horn neurons is enhanced after
tissue injury, and responses evoked by both innocuous and noxious
stimuli are increased after injury and are believed to contribute to
allodynia and hyperalgesia (Treede et al., 1992 ; Millan, 1999; Mannion
and Woolf, 2000).
It is well established that substance P (SP) participates in
nociceptive transmission in the spinal cord. SP is synthesized in
small-caliber afferent fibers (McCarthy and Lawson, 1989) and released
into the spinal cord after noxious stimulation (Duggan et al., 1987;
Schaible et al., 1990), excites nociceptive dorsal horn neurons
(Radhakrishnan and Henry, 1991), and contributes to the development of
hyperalgesia (Moochhala and Sawynok, 1984). When released into the
spinal cord, SP interacts with the substance P receptor (SPR), also
referred to as the neurokinin-1 (NK-1) receptor, to produce its
postsynaptic effects. Although <10% of lamina I neurons possess the
SPR (Brown et al., 1995; Littlewood et al., 1995), the majority belong
to the spinothalamic tract (STT) and spinoparabrachial tract and are
involved in the ascending transmission of nociceptive information. We
have demonstrated that SPR-expressing neurons have a unique role in
processing nociceptive information, and their intact function may be
critical for the development of chronic pain and hyperalgesia. Using
internalization of the SPR as a portal of entry to the cell,
intrathecal application of a conjugate of SP and the
ribosome-inactivating toxin saporin (SP-SAP) resulted in a dramatic
loss of lamina I SPR-expressing neurons and attenuated the nocifensive
behavior and hyperalgesia produced by capsaicin (Mantyh et al., 1997),
inflammation, and nerve injury (Nichols et al., 1999). Importantly,
basal pain reactivity was not affected. Although these studies showed
that SPR-expressing neurons are necessary for the development of
hyperalgesia, the exact role of these neurons in pain processing is
unclear. One possibility is that these are the neurons excited by and
sensitized after injury or inflammation. This is supported by studies
demonstrating that STT neurons, including those located in lamina I,
become sensitized after injury and contribute to hyperalgesia (Simone et al., 1991). To determine further the role of SPR-containing neurons
in nociceptive transmission, we examined the response properties and
sensitization of superficial and deep dorsal horn neurons in rats
pretreated intrathecally with SP-SAP. Our results suggest that
SPR-possessing neurons in the dorsal horn are an integral component of
a spinal and/or supraspinal circuit that is crucial for the development
of central sensitization after capsaicin.
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MATERIALS AND METHODS |
Subjects
Seventy-two adult male Sprague Dawley rats (Harlan Industries,
Indianapolis, IN) weighing 290-470 gm have been housed and used under
approval of the Animal Care Committee at the University of Minnesota.
Experiments were conducted according to the guidelines set forth by the
International Association for the Study of Pain.
Intrathecal injection
Rats were anesthetized by intramuscular injection of ketamine
(100 mg/kg) and acepromazine (45 mg/kg) and were placed into a
stereotaxic frame. An incision was made in the atlanto-occipital membrane, and a polyethylene catheter (Intramedic, Sparks, MD) (inner diameter, 0.28 mm; outer diameter, 0.61 mm) was inserted into
the intrathecal space to the area of lumbar enlargement. Animals were
given one intrathecal injection of normal saline (n = 27), 5 × 10 5
M saporin (n = 12), or 5 × 105 M SP-SAP
(n = 33). All injections were given in a volume of 10 µl followed by a 5 µl flush with saline. After injection, the catheter was removed, and the incision was closed by suture.
Experiments were performed 10 or 30 d after injection.
Immunohistochemistry and quantification
Animals pretreated with an intrathecal injection of saline
vehicle, SAP, or SP-SAP (n = 5 per group) were deeply
anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and perfused
intracardially with 12 ml of 0.1 M PBS followed
by 25 ml of 4% formaldehyde in 0.1 M PBS. Spinal
cord segments L1-S2 were removed, postfixed for 16 hr in the perfusion
fixative, and cryoprotected for 24 hr in 30% sucrose in 0.1 M PBS. Serial frozen spinal cord sections, 60 µm thick, were cut on a sliding microtome, collected in PBS, and
processed as free-floating sections. Tissue sections were incubated for
30 min at room temperature in a blocking solution of 1% normal donkey
or goat serum in PBS with 0.3% Triton X-100 and then incubated
overnight at room temperature in the primary antiserum for the
substance P receptor (rabbit anti-SPR, 1:5000; raised in our
laboratory). After incubation, tissue sections were washed three times
for 10 min in PBS and incubated in the secondary antibody solution for
2 hr at room temperature. Secondary antibodies conjugated to the
fluorescent marker Cy3 (Jackson ImmunoResearch, West Grove, PA) were
used at 1:600. Finally, the sections were washed three times for 10 min
in PBS, mounted on gelatin-coated slides, air dried, dehydrated via an
alcohol gradient (70, 90, and 100%), cleared in xylene, and
coverslipped. To confirm the specificity of the primary antibody,
controls included preabsorption with the corresponding synthetic
peptide or omission of the primary antibody.
Slides were viewed through a 1 cm2
eyepiece grid, which was divided into 100 1 × 1 mm units, and the
total number of immunofluorescent cell bodies per unit area was
counted. The mean numbers of SPR-immunoreactive (SPR-IR) cell
bodies located in the superficial (laminas I and II) and deep (laminas
III-V) dorsal horn were obtained from three to eight sections per animal.
Electrophysiological recordings
Rats were anesthetized by intramuscular injection of ketamine
(100 mg/kg) and acepromazine (45 mg/kg). The trachea was cannulated to
provide unobstructed ventilation, and a catheter was inserted into the
external jugular vein for supplemental anesthesia with sodium
pentobarbital (10 mg · kg1 · hr1).
Areflexia was maintained by monitoring the corneal reflex at frequent
intervals throughout the experiment. The carotid artery was cannulated,
and mean blood pressure was monitored continuously with a pressure
transducer (World Precision Instruments, Sarasota, FL).
Experiments were terminated if mean pressure dropped below 60 mmHg. The
lumbar enlargement was exposed by laminectomy, and the animal was
secured in a spinal frame. The spinal cord was continually bathed in a
pool of warm (37°C) mineral oil. Core body temperature was maintained
at 37°C by a feedback-controlled heating pad.
Extracellular recordings of single dorsal horn neurons with receptive
fields (RFs) located on the plantar surface of the hindpaw were
obtained using stainless steel microelectrodes (Frederick Haer and Co.,
Brunswick, ME) (10 m ). Recording electrodes were lowered into the
spinal cord at the L4 and L5 segments using an electronic
micromanipulator (Burleigh Instruments, Fishers, NY) in 5 µm steps.
Recordings were made only from single neurons whose amplitude could be
easily discriminated. Electrophysiological activity was amplified using
an alternating current amplifier (model DAM80; World Precision
Instruments), audio monitored (Grass AM8 audiomonitor; Grass
Instruments, West Warwick, RI), and displayed on a storage oscilloscope
before being sent to a computer for data collection using a customized
version of Lab View (National Instruments, Austin, TX) software that
enabled storage of raw data, discriminated impulses, and stimulus
temperature. In most experiments, recordings were obtained from two
neurons, one on each side of the spinal cord.
Functional classification of spinal neurons. Search stimuli
consisted of mechanical stimulation (stroking the skin and mild pinching with the experimenter's fingers) of the rat hindpaw. The RFs
of isolated neurons were mapped with a suprathreshold von Frey
monofilament. Each spinal neuron was characterized based on its
response to graded intensities of mechanical stimulation applied to the
RF. Innocuous stimuli consisted of stroking the skin with a cotton
swab. Noxious stimulation included mild pinching with the
experimenter's fingers and with serrated forceps, but this latter
stimulus was applied sparingly to avoid neuronal sensitization. Neurons
were classed functionally according to responses evoked by mechanical
stimuli as: (1) low threshold if they were excited maximally by
innocuous stimulation, (2) wide dynamic range (WDR) if they responded
in a graded manner to increasing intensity of stimulation, and (3) high
threshold (HT) if responses were evoked by noxious stimulation only.
Only WDR and HT neurons were studied.
Evoked response measures and experimental design. After
identification and general functional characterization of a neuron as
WDR or HT, the RF was mapped by stroking and mildly pinching with
forceps and outlined on the skin with a felt-tip pen. Mechanical threshold (in milliNewtons) was determined using calibrated von Frey monofilaments applied to the most sensitive area of the RF. To
obtain responses evoked by mechanical stimuli before and after capsaicin, four test sites within the RF were marked on the skin and
stimulated with a von Frey monofilament (178 mN bending force applied
for 2 sec). Each test site was stimulated three times with a 10 sec
interval between stimuli. To determine response evoked by heat, stimuli
of 35-51°C were applied in ascending order of 2°C increments from
a base temperature of 32°C using a Peltier thermode (contact area of
1 cm2). Stimuli were of 5 sec duration and
were delivered at a ramp rate of 18°C/sec with an interstimulus
interval of 60 sec. Capsaicin (10 or 100 µg in 10 µl) was injected
intradermally into the middle of the RF. Responses evoked by capsaicin
were recorded for 5 min, and responses evoked by mechanical and heat
stimuli were again determined as described above. At the end of the
experiment, the recording site was marked by passing current (10 µA
for 20 sec) through the recording electrode.
In separate experiments, we determined whether nociceptive neurons in
vehicle-treated animals (n = 8) and SP-SAP-treated
animals (n = 8) exhibited windup. Neurons were
activated by 12 successive electrical stimuli applied to the RF via
fine needle electrodes. Stimuli of 1 msec duration were applied at the
rate of 0.5 Hz and at a current intensity that was 150% of threshold
intensity that produced a long latency (110-450 msec) C-fiber-evoked response.
Histological localization of recording sites. Animals were
perfused with normal saline followed by 10% formalin containing 1%
potassium ferrocyanide. Serial transverse sections (50 µm) were cut
using a vibratome and stained with neutral red. Recording sites were
identified by Prussian Blue marks or small lesions.
Data analyses
The numbers of SPR-IR cell bodies, impulses evoked by capsaicin,
and impulses evoked by mechanical and heat stimuli before and after
capsaicin were compared between groups using ANOVA and Bonferroni
post hoc comparisons. Evoked responses were determined by
subtracting the spontaneous discharge rate from the response that
occurred during the stimulus. The proportion of neurons that were
classed as HT and WDR neurons was compared between the groups using the
 2 test. The number of SPR-expressing
neurons after SAP and SP-SAP was normalized to the number of neurons
found in vehicle-treated animals. For all statistical tests, a
p value of <0.05 was considered significant.
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RESULTS |
Ablation of SPR-expressing neurons after intrathecal SP-SAP:
morphological characteristics
In animals pretreated with vehicle or SAP, SPR immunoreactivity is
observed on cell bodies and dendrites located primarily in lamina I;
however, distinct SPR immunoreactivity was also found on neurons
located in the deep dorsal horn. No differences occurred in the number
of SPR-expressing neurons in animals pretreated with vehicle or with
SAP alone (Fig. 1). Animals pretreated
with SAP alone exhibited 80 ± 16% and 79 ± 13% of SPR-IR
neurons in lamina I/II and laminas III-V, respectively, compared with
animals pretreated with vehicle. In contrast, a significant reduction in the number of SPR-IR neurons was observed in animals pretreated with
SP-SAP. These animals exhibited only 32 ± 13% and 42 ± 9.9% of SPR-IR neurons in lamina I/II and in laminas III-V,
respectively, compared with the vehicle-treated group. Thus, animals
pretreated with SP-SAP exhibited a decrease in SPR-expressing neurons
in the superficial and deep dorsal horn of 65 and 58%,
respectively.
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Figure 1.
Loss of SPR-expressing neurons after intrathecal
infusion of SP-SAP. A, Confocal images showing
representative examples of SPR-IR in animals pretreated intrathecally
with vehicle, SAP alone, or SP-SAP. A dramatic reduction in SPR-IR is
evident after SP-SAP. B, Mean ± SEM percentage of
neurons that express the SPR after intrathecal vehicle, SAP, or SP-SAP.
The number of SPR-expressing cells was obtained from individual
animals, and a mean ± SEM was calculated. This mean value was
designated as 100%, and the SEM was proportionately adjusted (as a
percentage) to provide a measure of variability. Data for SAP- and
SP-SAP-treated groups represent the percentage of cells compared with
the vehicle-treated group. *Significant differences from
vehicle.
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Ablation of SPR-expressing neurons after intrathecal SP-SAP:
electrophysiological responses
General response properties
Seventy-two rats were pretreated intrathecally with saline
(vehicle; n = 27), SAP alone (n = 12),
or SP-SAP (n = 33). Electrophysiological responses were
obtained from a total of 104 nociceptive dorsal horn neurons. Forty-two
cells (27 WDR and 15 HT) were studied in animals pretreated with
vehicle, 20 cells (13 WDR and 7 HT) were studied in animals pretreated
with SAP, and 42 cells (39 WDR and 3 HT) were studied after SP-SAP
treatment. Within each group, no differences were observed in responses
of neurons studied 10 or 30 d after treatment, and data from these
time points were therefore combined. Receptive fields of all neurons
included the plantar surface of the hindpaw. Recording sites were
recovered for 53 neurons and were found to be located in the
superficial and deep dorsal horn. Recording sites of WDR and HT neurons
in vehicle-, SAP-, and SP-SAP-treated groups are illustrated in Figure 2, which shows that recording sites were
distributed throughout the dorsal horn in all treatment groups.
Similarly, the mean recording depth from the spinal cord surface for
all neurons did not differ between groups and was 547 ± 63, 502 ± 77, and 438 ± 44 µm for the vehicle, SAP, and
SP-SAP groups, respectively.
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Figure 2.
Location of recording sites for all dorsal horn
neurons. Neurons studied in rats pretreated with vehicle, SAP, and
SP-SAP were distributed in the superficial and deep dorsal horn in all
groups.
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The proportion of WDR and HT neurons studied in each treatment group,
as well as their general response characteristics (mean mechanical
threshold, mean number of impulses evoked by a suprathreshold von Frey
monofilament with a bending force of 178 mN applied for 2 sec, and mean
heat threshold) before capsaicin injection, are provided in Table
1. Responses of WDR and HT neurons showed
quantitative differences in their responses to mechanical stimuli in
that HT neurons exhibited a higher response threshold and were less
responsive to the suprathreshold von Frey monofilament. However,
responses of WDR and HT neurons to mechanical stimuli before capsaicin
did not differ between treatment groups. Additionally, no differences were found between WDR and HT neurons in their responses to heat before
capsaicin. As shown in Figure 3, mean
response functions of nociceptive neurons for heat stimuli of
35-51°C before capsaicin did not differ between groups. The mean
cumulative numbers of impulses evoked across all heat stimuli were
759.3 ± 124.1, 726.9 ± 179.7, and 805.5 ± 172.1 after
vehicle, SAP, and SP-SAP, respectively.
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Table 1.
General response properties of nociceptive neurons follow
intrathecal injection of vehicle, SAP, or SP-SAP
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Figure 3.
The mean ± SEM number of impulses evoked by
heat after pretreatment with vehicle, SAP, and SP-SAP. Responses evoked
by heat stimuli of 35-51°C did not differ between groups before
capsaicin injection.
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Although the response properties of nociceptive neurons did not differ
among the groups, a difference was found in the proportion of WDR and
HT neurons encountered between groups. Neurons classed as HT were 36 and 35% in vehicle- and SAP-treated animals, respectively, whereas the
number of HT neurons encountered in SP-SAP-treated animals was
significantly lower. Only three neurons (7%) in SP-SAP-treated rats
were classed as HT (p < 0.003) (Table 1).
Responses to capsaicin and central sensitization
Responses evoked by intraplantar capsaicin injections were
decreased in animals pretreated with SP-SAP. Injection of 10 µg of
capsaicin (in 10 µl) produced a vigorous and long-lasting discharge in all WDR and HT neurons recorded from animals pretreated with vehicle
(n = 34) or SAP alone (n = 20).
Discharge rates were highest soon after injection, decreased to a
moderate level within ~1 min after injection, and typically persisted
for >3 min (Fig. 4A).
Responses of WDR and HT neurons were similar and did not differ between
vehicle- and SAP-treated groups. In contrast, capsaicin-evoked responses of WDR and HT neurons (n = 29) were much
weaker in animals pretreated with SP-SAP, and the duration of response
was often shorter. Before injection, mean discharge rates of
spontaneous activity did not differ between the groups. Because no
differences in responses occurred between WDR and HT neurons within
each group, responses of these neurons were combined. The mean number
of impulses evoked during the first 15 sec after capsaicin was 855 ± 101 and 757 ± 102 for the vehicle- and SAP-treated groups,
respectively, but was only 303 ± 50 impulses in animals treated
with SP-SAP (p < 0.01) (Fig.
4B). Thus, the peak response to capsaicin was ~65%
less in animals pretreated with SP-SAP compared with vehicle. Although
the mean number of impulses that occurred in each consecutive 15 sec
interval after capsaicin was similar for the vehicle- and SAP-treated
groups, capsaicin-evoked discharges were less in SP-SAP-treated rats
during the entire 3 min period after capsaicin injection.
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Figure 4.
Responses evoked by 10 µg of capsaicin are
diminished in animals pretreated with SP-SAP. A,
Responses of single WDR neurons to intradermal injection of capsaicin
from vehicle-, SAP-, and SP-SAP-treated rats. Bin size is 500 msec.
B, Mean ± SEM number of impulses per 15 sec
interval after capsaicin. Arrows indicate the time of
injection. *Significant difference between vehicle and SP-SAP groups.
Responses to capsaicin were weaker in animals that received SP-SAP
compared with those that received vehicle or SAP alone.
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Before and at 10 min after injection of capsaicin, responses evoked by
a suprathreshold von Frey monofilament (178 mN bending force) were
determined at selected sites within the RF that were  2 mm away from
the capsaicin injection. In animals pretreated with vehicle or with SAP
alone, all WDR and HT neurons located in the superficial or deep dorsal
horn exhibited an increase in mechanically evoked responses after
capsaicin. Responses to mechanical stimuli were increased similarly
throughout the RF. In vehicle-treated animals, the mean number of
impulses evoked by the von Frey monofilament increased from 24.6 ± 6.4 to 54.2 ± 12.1 (or 120%; p < 0.001) after capsaicin. A similar increase of 98% in evoked responses was
found in the SAP-treated group; the mean number of impulses increased
from 27.3 ± 4.3 to 54.2 ± 8.2 (p < 0.001). In contrast, none of the WDR or HT neurons in SP-SAP-treated
rats exhibited sensitization to mechanical stimuli after capsaicin
injection (Fig. 5). The mean number of
impulses evoked by the monofilament was 34.6 ± 11.5 before
capsaicin and 35.3 ± 12.4 after capsaicin.
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Figure 5.
Sensitization of nociceptive neurons to mechanical
stimuli (178 mN bending force) after 10 µg of capsaicin does not
occur in animals pretreated with SP-SAP. Left panels,
Representative examples illustrating responses of WDR neurons to
mechanical stimuli before and after capsaicin in vehicle-, SAP-, and
SP-SAP-treated groups. RFs are indicated by the stippled
area, and test sites for mechanical stimulation are indicted by
the dots within the RF. Arrows point to
specific test sites at which pairs of responses (before and after
capsaicin) were obtained. The capsaicin injection is indicated by the
×. Horizontal bars denote time of stimulation (2 sec). Right panels, Mean ± SEM number of impulses
evoked by a single mechanical stimulus before and after capsaicin in
animals pretreated with vehicle, SAP, or SP-SAP. *Significant
difference after capsaicin compared with before capsaicin.
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Pretreatment with SP-SAP also prevented the sensitization to heat
stimuli that normally occurs after capsaicin. Before capsaicin, heat
thresholds and responses evoked by suprathreshold heat stimuli did not
differ between WDR and HT neurons, and no differences in responses of
these neurons were observed between treatment groups. In animals
pretreated with vehicle, WDR and HT neurons exhibited sensitization to
heat after capsaicin as indicated by a mean decrease of 5.3°C in
response threshold (from 43.9 ± 0.9°C to 38.6 ± 0.6°C;
p < 0.001) and increased responses to suprathreshold stimuli (Fig. 6). The mean cumulative
number of impulses evoked by all heat stimuli increased from 663 ± 96 before capsaicin to 1297 ± 158 after capsaicin (96%
increase; p < 0.001). A similar degree of
sensitization to heat was observed for WDR and HT neurons in
SAP-treated animals (Fig. 7). The mean
response threshold decreased 5.7°C (from 44.1 ± 0.8°C to
38.4 ± 0.8°C after capsaicin; p < 0.001), and
the mean cumulative number of impulses increased from 727 ± 180 to 1441 ± 277 (98% increase; p < 0.001). In
contrast, sensitization to heat after capsaicin injection did not occur in SP-SAP-treated animals (Fig. 8). Mean
heat thresholds were unchanged after capsaicin (43.8 ± 0.7°C
before capsaicin and 43.7 ± 0.6°C after capsaicin), and the
mean, cumulative number of impulses evoked by all heat stimuli
decreased from 749 ± 217 before capsaicin to 397 ± 127 after capsaicin (47% decrease; p < 0.01). The
decrease in the cumulative responses to heat was attributed to a
decrease in responses evoked by the higher stimulus temperatures (Fig. 8B).
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Figure 6.
Sensitization to heat produced by 10 µg of
capsaicin in animals pretreated with intrathecal infusion of vehicle.
A, Responses of an HT neuron to heat stimuli of 41, 47, and 51°C before and after capsaicin. Also shown is the localization
of the recording site for these neurons and its RF (stippled
area). The arrow points to location of capsaicin
injection. B, Mean ± SEM number of impulses evoked
by heat stimuli before and after capsaicin for all neurons. Mean
responses to heat increased after capsaicin. C,
Mean ± SEM heat threshold for all neurons before and after
capsaicin. Response threshold decreased after capsaicin. *Significant
difference between mean values obtained before and after
capsaicin.
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Figure 7.
Sensitization to heat in SAP-treated animals after
10 µg of capsaicin. A, Responses of a WDR neuron to
heat stimuli before and after capsaicin. Also shown are the recording
site and the RF (stippled area) for this neuron. The
arrow points to location of capsaicin injection.
B, Mean ± SEM number of impulses evoked by heat
stimuli before and after capsaicin for all neurons. Responses to heat
increased after capsaicin. C, Mean ± SEM heat
threshold for all neurons before and after capsaicin. Response
threshold decreased after capsaicin. *Significant difference after
capsaicin.
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Figure 8.
Lack of capsaicin-evoked sensitization to heat in
animals pretreated with SP-SAP. A, Responses of a WDR
neuron to heat stimuli before and after capsaicin. The recording site
and the RF (stippled area) are shown for this neuron.
The arrow points to location of capsaicin (10 µg)
injection. B, Mean ± SEM number of impulses evoked
by heat stimuli before and after capsaicin for all neurons. There was a
tendency for heat-evoked responses to decrease after capsaicin.
C, Mean ± SEM heat threshold for all neurons
before and after capsaicin.
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Because the excitation of nociceptive neurons evoked by 10 µg of
capsaicin was weak in animals pretreated with SP-SAP, it was unclear
whether central sensitization failed to occur because the
capsaicin-evoked response was not strong enough to induce sensitization
or whether there was a disruption of the mechanisms that drive central
sensitization. To address this issue, we repeated the experiments above
but used a capsaicin dose of 100 µg to increase the capsaicin-evoked
response in SP-SAP-treated animals. In vehicle-treated animals,
intraplantar injection of 100 µg of capsaicin produced a strong and
long-lasting discharge of eight neurons (seven WDR and one HT). The
mean number of impulses during the first 15 sec after capsaicin was
898 ± 82 and was similar to that produced by the 10 µg dose of
capsaicin in control animals. A similar level of excitation was
produced in 13 neurons (12 WDR and 1 HT) of animals pretreated with
SP-SAP (Fig. 9). The mean number of
impulses evoked during the first 15 sec after 100 µg of capsaicin was
836 ± 118 and was significantly greater than the 303 ± 50 impulses evoked during the first 15 sec after 10 µg of capsaicin.
Thus, mean responses of nociceptive neurons evoked by 100 µg of
capsaicin in vehicle- and SP-SAP-treated animals were similar to
responses produced by injection of 10 µg of capsaicin in control
animals.
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Figure 9.
Neuronal discharges evoked by 100 µg of
capsaicin do not differ between groups pretreated with vehicle or with
SP-SAP. The mean number of impulses and the temporal profile of the
capsaicin-evoked responses were nearly identical for both groups.
Responses shown are the mean ± SEM number of impulses evoked
during each consecutive 15 sec interval after capsaicin. The
arrow indicates the time of injection.
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Although nociceptive neurons in animals pretreated with vehicle and
SP-SAP exhibited a similar degree of capsaicin-evoked excitation,
sensitization did not occur in the SP-SAP group. As illustrated in
Figure 10, the mean number of impulses
evoked by the suprathreshold von Frey monofilament increased from
31.1 ± 9 to 57.1 ± 11.3 (or 83%; p < 0.001) in animals pretreated with vehicle. However, responses of
neurons in SP-SAP-treated animals were unchanged after capsaicin
(30 ± 6.8 impulses before capsaicin and 33.8 ± 8.8 impulses
after capsaicin). In addition, neurons in vehicle-treated animals, but
not in SP-SAP-treated animals, exhibited sensitization to heat after
capsaicin. In animals pretreated with vehicle, capsaicin decreased the
mean heat threshold from 43.8 ± 1.1°C to 39.3 ± 0.9°C
(p < 0.001), and the mean cumulative number of
impulses evoked by all heat stimuli increased from 755 ± 228 to
1444 ± 204 (an increase of 91%; p < 0.05). In
contrast, response thresholds for heat in animals pretreated with
SP-SAP remained unchanged after capsaicin (42.3 ± 1°C before
injection and 43 ± 1.6°C after injection), and the mean
cumulative number of impulses evoked by all heat stimuli after
capsaicin decreased from 892 ± 244 to 499 ± 208 (or 44%;
p < 0.01).
View larger version (33K):
[in this window]
[in a new window]
|
Figure 10.
Capsaicin (100 µg) produces sensitization after
pretreatment with vehicle, but this sensitization fails to occur after
SP-SAP pretreatment. Top row, Data from animals
pretreated with vehicle. A, Mean ± SEM number of
impulses evoked by a von Frey monofilament (178 mN) before and after
capsaicin. B, Mean ± SEM number of impulses evoked
by heat stimuli before and after capsaicin. C, Mean ± SEM heat response threshold before and after capsaicin.
Bottom row, Responses to mechanical
(D) and heat (E,
F) stimuli before and after capsaicin in animals
pretreated with SP-SAP. *Significant difference after
capsaicin.
|
|
Effect of SP-SAP on windup
We also examined the ability of nociceptive neurons in vehicle-
and SP-SAP-treated animals to develop windup. Responses to consecutive
electrical stimuli were obtained for 13 WDR neurons in the
vehicle-treated group and 10 WDR neurons in the SP-SAP-treated group.
Electrical stimulation of the RF at a frequency of 0.5 Hz induced a
significant increase in the C-fiber-evoked response of 187 ± 21%
(p < 0.01) by the 12th stimulus compared with
the response evoked by the first stimulus. Windup failed to occur in
animals pretreated with SP-SAP, as shown in Figure
11.
View larger version (29K):
[in this window]
[in a new window]
|
Figure 11.
Nociceptive spinal neurons in SP-SAP-treated
animals do not exhibit windup. A, Responses to
electrical stimulation of a WDR neuron recorded from a vehicle-
(top) and an SP-SAP (bottom)-pretreated
animal. Responses are shown for the 1st, 6th, and 12th electrical
stimulus. The time frame of the C-fiber component is within the
dashed line. The C-fiber response was facilitated in the
vehicle-treated animal on the 6th and 12th trial but not in the
SP-SAP-treated animal. Arrows indicate time of
electrical stimulation. B, Mean ± SEM normalized
C-fiber responses evoked by 12 successive electrical stimuli in rats
pretreated with vehicle or SP-SAP. Responses were normalized to the
response evoked by the first stimulus.
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|
| |
DISCUSSION |
Elimination of spinal neurons that possess the SPR using SP-SAP
offers the unique opportunity to determine the role of SPR-expressing neurons, as well as other neurons, in pain processing. SP-SAP induced
specific degeneration of neurons expressing SPR receptors. This
observation was made previously (Mantyh et al., 1997; Nichols et al.,
1999) and is supported by present data. An interesting finding of the
present study was the proportional change in the functional
classification of spinal neurons encountered in animals pretreated with
SP-SAP. The proportion of HT neurons encountered in control animals was
~36%, whereas only 7% of the neurons identified in SP-SAP-treated
animals were HT. This suggests that SP-SAP targeted primarily HT
neurons. It has been shown in cats that HT neurons possess more SPRs
than WDR neurons (Ma et al., 1996, 1997), suggesting that HT neurons
are more vulnerable to SP-SAP because of the greater number of SPRs on
these neurons.
The absence of sensitization and windup after SP-SAP results from
elimination of SPR-expressing spinal neurons within the area of
intrathecal application for the following reasons. First, SPR
immunoreactivity was not altered in thoracic or cervical segments after
application of SP-SAP to the lumbar spinal cord, and its administration
did not disrupt daily behavior patterns or locomotion (Nichols et al.,
1999). These data indicate that the toxic effect of SP-SAP is localized
to treated segments. Second, cell loss was not observed in the DRG
(Mantyh et al., 1997; Nichols et al., 1999) after intrathecal SP-SAP,
indicating that the effects of SP-SAP on central sensitization are not
attributed to a presynaptic effect on primary afferent terminals.
It is particularly intriguing that a relatively small population of
neurons can have such a dramatic effect on the behavioral and
physiological responses of nociceptive neurons located throughout the
dorsal horn. Our previous studies demonstrated that SPR-expressing neurons were necessary for the full expression of hyperalgesia after
capsaicin, inflammation, and nerve injury (Mantyh et al., 1997; Nichols
et al., 1999). One explanation to account for these changes is that
neurons that express the SPR are the neurons that undergo central
sensitization and transmit nociceptive information rostrally to account
for hyperalgesia. This has been proposed to account for decreased
hyperalgesia after ablation of SPR-expressing neurons using
SP-diphtheria toxin conjugates (Benoliel et al., 1999). In the present
study, we found that loss of ~60% of SPR-expressing neurons, which
constitute a small population of ascending tract neurons located
primarily in the superficial dorsal horn, resulted in the inability of
remaining neurons to respond vigorously to capsaicin and to exhibit
sensitization and windup. This percentage of neuron loss may represent
a threshold effect related to the concentration of SP-SAP, suggesting
that relatively few SPR-containing neurons are required for the
development of central sensitization. Remarkably, neurons located in
the deep as well as the superficial dorsal horn underwent this change
in their capacity to become sensitized. This indicates that
SPR-expressing neurons, either directly or indirectly, modulate
excitability of other superficial as well as deep dorsal horn neurons.
It is possible that only SPR-expressing neurons become sensitized after
capsaicin, and that the apparent sensitization of other neurons results
from direct or indirect synaptic connections with these neurons. This is supported by our findings that increasing capsaicin-evoked neuronal
activity in SP-SAP-treated animals to that evoked in control animals,
by using a higher dose of capsaicin, failed to induce sensitization.
This hypothesis can be tested by using iontophoretically applied
SP and determining whether only SP-responsive neurons become
sensitized. The absence of central sensitization and windup recorded
electromyographically from hindlimb muscles (De Felipe et al., 1998) or
from single spinal neurons (Weng et al., 2001) in mice lacking the SPR
also supports this concept. Regarding their role in transmission of
acute pain, SPR-expressing neurons may have a minor function, because
loss of these neurons in the present study, or deletion of the SPR
(Weng et al., 2001), did not alter responses of nociceptive neurons to
acute noxious stimuli. These findings are consistent with behavioral
studies showing that antagonists of the SPR (Garces et al., 1993),
knock-out of the SPR (De Felipe et al., 1998; Mansikka et al., 1999;
Weng et al., 2001), or ablation of SPR-IR neurons by SP-SAP (Mantyh et al., 1997; Nichols et al., 1999) did not alter withdrawal responses to
acute stimuli. Rather, it appears that neurons possessing the SPR play
a pivotal role in central sensitization in that they are capable of
driving the sensitization of other nociceptive neurons after injury.
Unfortunately, clinical studies have shown that SPR (NK-1) antagonists
were not very effective in reducing hyperalgesia (Hill, 2000; Urban and
Fox, 2000). One possibility to account for this disappointing finding
is that the SPR may be important for the development but not the
maintenance of central sensitization (Ma and Woolf, 1995). Activation
of the SPR may play a pivotal role in initiating sensitization of
SPR-expressing neurons, which would influence excitability of other
neurons. However, once central sensitization is developed, it is
maintained by circuitry involving sensitized SPR-expressing neurons.
The inability of sensitization to occur after deletion of the SPR (De
Felipe et al., 1998; Weng et al., 2001) or ablation of SPR-expressing neurons by SP-SAP (Nichols et al., 1999) supports this concept.
The precise mechanisms by which the small population of SPR-expressing
neurons contributes to the development of central sensitization are
unclear. One possibility is that these neurons are part of a
supraspinal circuit that activates descending facilitation mechanisms to increase the excitability of nociceptive dorsal horn neurons. Accumulating evidence shows that in addition to the well known descending antinociceptive pathways (Basbaum and Fields, 1978; Fields
and Basbaum, 1978), other descending pathways exist that facilitate
spinal nociceptive responses (Urban and Gebhart, 1999; Porreca et al.,
2001; Vanderah et al., 2001a,b). Pronociceptive actions have been
proposed for descending noradrenergic projections (Martin et al., 1999)
and serotoninergic neurons (Calejesan et al., 1998) using spinal
5-HT1A (Millan and Colpaert, 1991a,b) and
possibly 5-HT3 (Ali et al., 1996) receptors.
Interestingly, descending facilitation and inhibition could be produced
from the rostral ventromedial medulla (RVM) depending on intensity of
activation. Low and high levels of RVM stimulation correspondingly produced pronociceptive and antinociceptive effects on spinal neurons
(Smith et al., 1997; Urban and Gebhart, 1997; Zhuo and Gebhart, 1997;
Calejesan et al., 1998). Moreover, lesion of the RVM (Urban et al.,
1996, 1999) or injection of lidocaine into the RVM (Mansikka and
Pertovaara, 1997; Kauppila et al., 1998; Pertovaara, 1998)
eliminated descending facilitation and inhibited certain
types of hyperalgesia. Importantly, neurons in the superficial spinal
laminas project to the periaqueductal gray matter (PAG) (Keay et al.,
1997), a large proportion of which possesses the SPR (Todd et al.,
2000), and from the PAG to RVM (Li et al., 1990; Zeng et al.,
1991). Thus, activation of ascending SPR-expressing neurons may
activate those structures involved in descending facilitation. Interestingly, it is likely that SPR-expressing neurons do not play a
prominent role in activation of descending tonic inhibition from the
RVM (Li et al., 1998), because no differences were observed in the
neuronal responses evoked by mechanical and heat stimuli between SP-SAP
and control animals before capsaicin. If SPR-expressing neurons
contributed to tonic activation of descending inhibitory pathways,
responses of spinal neurons would be expected to increase in
SP-SAP-treated animals. Blockade of descending inhibitory pathways has
been shown to increase the spontaneous and evoked activity of
nociceptive dorsal horn neurons (Vanegas et al., 1997; Budai et al.,
1998; Li et al., 1998) and to increase receptive field area (Pubols et
al., 1991).
A second possible mechanism by which SPR-expressing neurons promote
central sensitization is through segmental processes whereby these
neurons can modulate the excitability of neighboring neurons. Central
sensitization can occur through local processes and without bulbo-spinal influences, as demonstrated using an isolated rat spinal
cord (Woolf, 1992; Baba et al., 1999; Nakatsuka et al., 1999).
Anatomical studies have shown that many deep dorsal horn neurons
possess dorsally directed dendrites that receive information from
lamina I neurons (Honore et al., 1999; Nichols et al., 1999). Thus,
activation and sensitization of SPR-expressing neurons may provide at
least part of the input for other ascending tract cells. We also found
that in SP-SAP-treated rats, responses to the most intense heat stimuli
(47-51°C) tended to decrease after capsaicin. This may be caused by
the disruption of segmental neuronal circuits normally driven by
SPR-expressing neurons and is supported by studies demonstrating a
unique function for SP in transmission of moderate to intense pain (Cao
et al., 1998; Mansikka et al., 1999).
In conclusion, dorsal horn neurons that possess the SPR play a pivotal
role in the development of central sensitization and hyperalgesia.
Activity of these neurons is capable of modifying response properties
of remaining nociceptive neurons through either direct or indirect
circuitry. Understanding the mechanisms by which SPR-expressing neurons
can influence excitability of other neurons, as well as the molecular
changes that occur in these cells after their activation, may identify
new targets for treating chronic pain and hyperalgesia.
| |
FOOTNOTES |
Received May 20, 2002; revised July 29, 2002; accepted Aug. 5, 2002.
This work was supported by National Institutes of Health Grant DA11986.
Saporin and SP-SAP were obtained from Advanced Targeting Systems.
Correspondence should be addressed to Dr. Donald A. Simone, Department
of Oral Science, University of Minnesota, 515 Delaware Street
Southeast, 17-252 Moos, Minneapolis, MN 55455. E-mail: simon003{at}umn.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
Compound via MeSH
