J Neurophysiol 93: 998 –1006, 2005.
First published September 24, 2004; doi:10.1152/jn.01160.2003.
Spinal Neurons That Express NK-1 Receptors Modulate Descending Controls
That Project Through the Dorsolateral Funiculus
Sergey G. Khasabov,1 Joseph R. Ghilardi, Patrick W. Mantyh,3,4 and Donald A. Simone1,3,4
1
Departments of Oral Sciences, Preventive Sciences, 3Psychiatry, and 4Neuroscience, University of Minnesota, Minneapolis, Minnesota
Submitted 3 December 2003; accepted in final form 22 September 2004
Hyperalgesia occurs after tissue injury and inflammation
and is often difficult to manage. The mechanisms underlying
the development and maintenance of hyperalgesia are not
fully understood; however, it is known that hyperalgesia is
mediated, at least in part, by the sensitization of nociceptive
spinal neurons (Millan 1999; Treede et al. 1992), also
referred to as central sensitization (Baranauskas and Nistri
1998; Woolf 1992). It is well established that substance P
(SP) is involved in nociceptive processing. SP is released
from nociceptive afferent fibers after noxious stimulation
(Duggan 1995; Duggan et al. 1987; Schaible et al. 1990),
excites nociceptive dorsal horn neurons (Henry 1976;
Radhakrishnan and Henry 1995; Salter and Henry 1991),
and contributes to the development of hyperalgesia (Moochhala and Sawynok 1984). In the spinal cord, SP interacts
with the SP receptor, also referred to as the NK-1 receptor.
We have previously demonstrated that NK-1-expressing
neurons in the spinal cord have a unique role in nociceptive
processing in that the development of hyperalgesia and central
sensitization requires their intact function. Selective ablation of
NK-1 positive neurons using a conjugate of SP and the ribosome-inactivating toxin saporin (SP-SAP) produced a dramatic
decrease in hyperalgesia induced by capsaicin, inflammation,
and nerve injury (Mantyh et al. 1997; Nichols et al. 1999).
Similar results were obtained after ablation of NK-1-positive
neurons using a SP-diphtheria toxin conjugate (Benoliel et al.
1999). Moreover, deletion of these neurons after intrathecal
SP-SAP prevented central sensitization produced by capsaicin
and prevented windup in remaining nociceptive neurons
(Khasabov et al. 2002). The mechanisms by which NK-1expressing spinal neurons modulate the excitability of other
spinal neurons remain to be determined. Because ablation of
NK-1-possesing superficial lumbar spinal neurons by SP-SAP
reduces nocifensive escape behavior (Vierck et al. 2003), an
essential role of these neurons in supraspinal nociceptive processes is suggested. Because the majority of NK-1-positive
neurons are projection neurons and are part of the spinothalamic tract (STT) and spinoreticular tracts (Marshall et al.
1996; Todd et al. 2000, 2002), one possibility is that they are
part of an ascending-descending circuitry that modulates excitability of nociceptive spinal neurons.
Processing of spinal nociceptive information is under the
control of descending systems from a variety of areas in the
brain, including the brain stem (Millan 2002). Descending
modulation was initially described as inhibitory because activation of these pathways produced antinociception (Basbaum
and Fields 1978; Duggan and Morton 1988; Fields and Basbaum 1978), although descending facilitation has also been
described (Fields et al. 1991; Kovelowski et al. 2000; Urban
and Gebhart 1999; Vanderah et al. 2001a,b). Projections from
brain stem nuclei descend to the spinal cord via the dorsolateral
funiculus (DLF) and ventrolateral funiculus (VLF) (Lakke
1997; Tracey 1995).
The rostral ventromedial medulla (RVM) is a major source
of descending axons that travel through the DLF (Lakke 1997;
Watkins et al. 1980) and has been implicated in inhibition as
well as facilitation of nociceptive transmission. For example,
activation of neurons in the RVM attenuates acute pain and
hyperalgesia (Basbaum and Fields 1978, 1984; Yaksh and
Wilson 1979) and decreases responses of spinal neurons
evoked by noxious stimulation (Fields et al. 1977; Floeter and
Fields 1991). Although inhibition of STT neurons occurs after
stimulation of the RVM (Beall et al. 1976; McCreery et al.
1979), hyperalgesia is blocked by inactivation or lesions of the
Address for reprint requests and other correspondence: D. A. Simone, Dept.
of Oral Sciences, University of Minnesota, 515 Delaware St. SE, 17-252 Moos
Tower, Minneapolis, MN 55455 (E-mail: [email protected]).
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.
INTRODUCTION
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0022-3077/05 $8.00 Copyright © 2005 The American Physiological Society
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Khasabov, Sergey G., Joseph R. Ghilardi, Patrick W. Mantyh,
and Donald A. Simone. Spinal neurons that express NK-1 receptors
modulate descending controls that project through the dorsolateral
funiculus. J Neurophysiol 93: 998 –1006, 2005. First published September 24, 2004; doi:10.1152/jn.01160.2003. Selective ablation of
spinal neurons possessing substance P receptors (NK-1 receptors)
using the selective cytotoxin conjugate substance P-saporin (SP-SAP)
decreases hyperalgesia and central sensitization. The mechanisms by
which NK-1 expressing neurons modulate the excitability of other
dorsal horn neurons are unclear. Because the majority of NK-1
expressing spinal neurons project rostrally, it is possible that they are
part of a spinal-supraspinal circuitry that contributes to descending
modulation of excitability of spinal nociceptive neurons. We therefore
determined whether ablation of spinal neurons that possess the NK-1
receptor altered descending systems that travel via the dorsolateral
funiculus (DLF). Spontaneous activity and responses of dorsal horn
neurons evoked by mechanical (von Frey monofilaments) and heat
(35–51°C) stimuli were determined before and after transection of the
DLF and were compared in rats pretreated with intrathecal application
of vehicle or SP-SAP. In vehicle-treated rats, transection of the DLF
caused a 233% increase in mean spontaneous activity of neurons and
enhanced their responses to mechanical and heat stimuli, whereas
these increases in excitation were blocked in rats pretreated with
SP-SAP. Importantly, SP-SAP alone had no effect on spontaneous or
evoked activity in the absence of DLF transection. These results
demonstrate that spinal neurons expressing the NK-1 receptor appear
to play a pivotal role in regulating descending systems that modulate
activity of nociceptive dorsal horn neurons.
SPINAL NK-1 EXPRESSING NEURONS AND DESCENDING MODULATION
METHODS
Subjects
Forty-six male Sprague-Dawley rats (Harlan Industries, Indianapolis, IN) weighing 290 – 470 g were used. Animals were housed on a
12-h light-dark schedule and had access to food and water ad libitum.
Studies were approved by the Animal Care Committee at the University of Minnesota and were conducted according to the guidelines set
forth by the International Association for the Study of Pain.
Intrathecal injection
Animals were anesthetized by intramuscular injection of ketamine (100 mg/kg) and acepromazine (45 mg/kg). Rats were
placed into a stereotaxic frame, and an incision was made in
atlanto-occipital membrane. A polyethylene catheter (Intramedic;
0.28 mm ID and 0.61 mm OD) was inserted into the intrathecal
space to the area of lumbar enlargement for single injection. Rats
received a single intrathecal injection of either vehicle (0.9% NaCl,
10 ␮l; n ⫽ 24) or SP-SAP (5 ⫻ 10⫺6 M in 0.9% NaCl, 10 ␮l; n ⫽
22) followed by a 5-␮l flush with saline. After injection, the
catheter was removed, the incision was closed by suture, and rats
were returned to their cages for recovery.
Electrophysiological recordings
Experiments were performed 28 –30 days after rats received
intrathecal injection. The rats were anesthetized by intramuscular
injection of ketamine (100 mg/kg) and acepromazine (45 mg/kg).
The trachea was cannulated, and a catheter was placed in the
external jugular vein to provide supplemental anesthesia with
sodium pentobarbital (10 mg 䡠 kg⫺1 䡠 h⫺1). The carotid artery was
cannulated, and blood pressure was monitored continuously with a
pressure transducer (World Precision Instruments). Experiments
were terminated if mean pressure dropped ⬍60 mmHg. The lumbar
enlargement and upper thoracic segments were exposed by laminectomies, and animals were placed in a stereotaxic apparatus and
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 dorsal horn neurons were obtained using
stainless steel microelectrodes (Frederick Haer, Brunswick, ME; 10
m⍀). Recording electrodes were lowered into the spinal cord in 5-␮m
steps at the L4 and L5 segments using an electronic microdrive
(Burleigh). Recordings were made only from single neurons the
J Neurophysiol • VOL
amplitude of which was easily discriminated. Electrophysiological
activity was amplified using an AC amplifier (World Precision Instruments, Model No. DAM80), audio-monitored (Grass AM8 audiomonitor), and displayed on a storage oscilloscope before being sent to
a computer for data collection using a customized version of Lab
View software (National Instruments, Austin, TX) that enabled storage of raw data, discriminated impulses, and stimulus temperature.
Functional classification of spinal neurons
Nociceptive neurons the receptor fields (RFs) of which were located on the plantar surface of the hindpaw were studied. Search
stimuli consisted of mechanical stimulation (stroking the skin and
mild pinching with the experimenter’s fingers) of the hindpaw. The
RFs of isolated neurons were mapped with a supratheshold von Frey
monofilament. Each neuron was characterized functionally according
to its response to graded intensities of mechanical stimuli 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. Neurons were classed as
low threshold (LT) if they were excited maximally by innocuous
stimulation, wide dynamic range (WDR) if they responded in a graded
fashion to increasing intensities of stimulation, and high-threshold
(HT) if responses were evoked by noxious stimulation only. Also,
some neurons responded preferentially to the movement of paw joints
and deep tissues. In addition to mechanical stimuli, heating the skin to
45– 46°C for 5 s was used to determine whether the neuron was
sensitive to heat stimuli. Only WDR and HT neurons that responded
to both mechanical and heat stimuli were studied.
Response measures and experimental design
After identification and functional classification of a neuron, the RF
was mapped by stroking and mildly pinching with forceps and was
outlined on the skin with a felt-tip pen. Mechanical threshold (mN)
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 transection of the DLF, four test
sites within the RF were marked on the skin and stimulated with a
standard von Frey monofilament (178 mN bending force applied for
2 s). Each test site was stimulated three times with a 10-s interval
between stimuli.
After responses to mechanical stimulation were obtained, sensitivity to heat stimuli was assessed. Stimuli of 35–51°C were delivered
from a base temperature of 32°C using a Peltier thermode (contact
area of 1 cm2) and were applied in ascending order of 2°C increments
with an interstimulus interval of 60 s. Stimuli were of 5-s duration and
were delivered at a ramp rate of 18°C/s.
After the initial responses of the neuron were determined, the
DLF on the ipsilateral side to the recording cell was transected at
the upper thoracic level (⬃T2). The mineral oil bathing the thoracic
laminectomy was removed and transection was performed just
ventral to the entry zone of the thoracic dorsal root 1.5–2 mm deep
into the white matter using microsurgical scissors. To confirm that
the effects of transection were not due to mechanical stimulation of
descending axons, in four experiments, 4% lidocaine was applied
to the cut ends of the spinal cord of vehicle-treated rats for a period
of 20 min using filter paper. The test stimuli were repeated as
described above at 1 h after transection.
Histological localization of recording sites and
DLF transection
At the end of the experiment, the recording site was marked by
passing current (10 ␮A for 15 s) through the recording electrode.
Animals were perfused with normal saline followed by 10%
Formalin containing 1% potassium ferrocyanide. Serial transverse
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RVM (Morgan and Fields 1994; Watkins et al. 1998), and
stimulation of the RVM can increase responses of nociceptive
dorsal horn neurons (Zhuo and Gebhart 1997, 2002). These
studies indicate that ascending afferent input to the supraspinal
level is under the immediate control of descending brain stem
modulatory systems.
One approach to studying descending modulation of nociceptive transmission is to disrupt the DLF and assess the
changes in responses of nociceptive dorsal horn neurons.
Under normal conditions, transection of the DLF increases
spontaneous and evoked activity of nociceptive spinal neurons
(Li et al. 1998; Pubols et al. 1991), leading to a state of
facilitation. The present study investigated whether NK-1positive spinal neurons modulated descending systems that
travel in the DLF. We examined responses of dorsal horn
neurons before and after removal of descending influences
through the DLF in untreated animals and in those pretreated
with intrathecal SP-SAP.
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KHASABOV, GHILARDI, MANTYH, AND SIMONE
sections (50 ␮m) were cut using a freezing microtome and were
stained with neutral red. The localization of recording sites were
identified by Prussian Blue marks or by small lesions. Similarly,
the thoracic spinal cord that contained the DLF transection was
removed, cut into transverse sections, and stained with neutral red.
Sections that contained the transection were traced to determine
the spatial extent of the lesion.
Immunohistochemistry studies
General characteristics of nociceptive neurons in vehicleand SP-SAP-treated animals
Recordings were made from 27 neurons in vehicle-treated
animals and from 25 neurons in animals pretreated with SPSAP. The RF of all neurons included the plantar surface of the
hindpaw. The distribution of recording sites from both groups
of animals was similar and recording sites identified histologically were located in both the superficial and deep dorsal horn
(Fig. 2). Of the neurons studied in the vehicle-treated group,
78% (21 cells) were classified functionally as WDR, whereas
22% (6 neurons) were classed as HT. The proportion of WDR
and HT neurons encountered differed in animals that received
SP-SAP: 96% (24) were WDR neurons and only 4% (1 neuron)
were HT (␹2 test, P ⬍ 0.05). Mechanical thresholds of each
class of neurons were similar in vehicle and SP-SAP-treated
groups. Mean response thresholds of WDR neurons were
12.0 ⫾ 2.3 and 13.0 ⫾ 2.6 mN in vehicle and SP-SAP-treated
groups, respectively. For HT cells, mean thresholds in vehicle
and SP-SAP-treated animals were 116.1 ⫾ 18.3 and 97.8 mN,
respectively. The proportion of all neurons in vehicle and
Data analyses
The numbers of impulses evoked by heat and mechanical stimuli
before and after the DLF transection were compared between groups
using ANOVA and Bonferroni post hoc comparisons. Evoked responses were determined by subtracting spontaneous discharge rate
from the response that occurred during the stimulus. The proportion of
neurons that were classed as HT and WDR neurons were compared
between the groups using the ␹2 test. For all statistical tests, a
probability value ⬍0.05 was considered significant.
RESULTS
Effects of lumbar intrathecal injection of SP-SAP on NK-1
labeling in lumbar and upper thoracic spinal segments
The mean number of NK-1-positive neurons in the superficial dorsal horn of lumbar enlargement was decreased 28 –30
days after SP-SAP treatment. The proportion of NK-1 expressing cells in the superficial dorsal horn was only 15.8 ⫾ 6.1%
of that found in vehicle-treated animals (84.4% decrease, P ⬍
0.001). However, NK-1-positive neurons in deeper laminae
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FIG. 1. Effect of lumbar infusion of vehicle or substance P-saporin (SPSAP) on labeling of NK-1 receptors in the dorsal horn of lumbar and thoracic
segments of the spinal cord. A: NK-1 immunoreactivity in the lumbar spinal
cord 28 days after intrathecal infusion of vehicle or SP-SAP. Representative
images of NK-1 labeling in the spinal cord following vehicle and SP-SAP are
shown (left). Sections were cut in the sagittal plane. Right: the mean percentage
of NK-1 labeled neurons in the superficial and deep dorsal horn of the lumbar
spinal cord relative to the vehicle-treated group. SP-SAP produced a significant
decrease in NK-1 immunoreactivity and in the percentage of NK-1-labeled
neurons in the superficial dorsal horn. B: NK-1 immunoreactivity in the
thoracic spinal cord 28 days after intrathecal infusion of vehicle or SP-SAP
into the lumbar spinal cord. Format is the same as for A. There was no change
in NK-1 immunoreactivity or in the percentage of neurons labeled for the
NK-1 receptor. *, a significant difference from the vehicle group (P ⬍ 0.001).
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Rats received an intrathecal injection of vehicle or SP-SAP (n ⫽ 5
per group). At 28 –30 days later, they were deeply anesthetized with
pentobarbital sodium (50 mg/kg ip) and perfused intracardially with
50 ml of 0.1 M PBS followed by 400 ml of 4% paraformaldehyde in
0.1 M PBS. Spinal cord segments of the lumbar enlargement (L4 –5)
and upper thoracic level (T2–3) were removed, postfixed for 2 h, and
cryoprotected in 20% sucrose for 48 h. Serial frozen sections (60 ␮m)
were cut on a sliding microtome in sagittal plane and collected in 0.1
M PBS for processing as free-floating sections and washed three times
in PBS for 15 min. Tissue sections were incubated for 1 h at room
temperature in the blocking solution of 10% normal donkey serum in
PBS, containing 0.3% Triton X-100 and incubated overnight at room
temperature in the primary rabbit anti-NK-1 receptors antiserum,
raised in our laboratory. Primary antibodies were dissolved at 1:5,000
by PBS containing 1% normal donkey serum and 0.3% Triton X-100.
The next day, sections were washed three times for 10 min in PBS and
incubated for 2 h in donkey anti-rabbit secondary antibodies labeled
by the fluorescent marker Cy3 (Jackson ImmunoReserch, West
Grove, PA). Secondary antibodies were dissolved at 1:600 using the
same dissolving media as for primary antibodies. Finally, sections
were washed three times for 10 min in PBS, mounted on gelatincoated slides, air dried, dehydrated via an alcohol gradient (50, 70, 80,
96, and 100%), cleared in xylene, and coverslipped. To confirm the
immunohistochemical detection specificity, controls included preabsorbtion of the primary antibody with the corresponding specific
peptide or omission of the primary antibody.
Sections were viewed through a 1-cm2 eyepiece grid divided into
100 1⫻1-mm units. Total numbers of NK-1 immunopositive cell
bodies per unit area were counted for superficial (I and II) and deep
(III–V) laminae in lumbar and thoracic spinal segments of vehicle and
SP-SAP-treated groups. Results are expressed as mean percentages of
surviving neurons in superficial and deep laminae in SP-SAP-treated
rats as compared with mean numbers of NK-1-positive neurons in
corresponding laminae of vehicle-treated rats.
(III–V) were unaffected by SP-SAP (Fig. 1A). Lumbar infusion
of SP-SAP did not alter the labeling of NK-1 neurons at the
thoracic level of the spinal cord. In SP-SAP-treated animals,
the number of NK-1-expressing neurons in the thoracic spinal
cord was 98.6 ⫾ 4.8% (superficial dorsal horn) and 98.9 ⫾
4.1% (deep dorsal horn) of that found in vehicle-treated animals (Fig. 1B).
SPINAL NK-1 EXPRESSING NEURONS AND DESCENDING MODULATION
1001
SP-SAP group and did not differ significantly (t-test). Thus
although the proportion of HT and WDR neurons differed
between vehicle- and SP-SAP-treated animals, spontaneous
activity and responses to acute mechanical and heat stimuli of
remaining nociceptive neurons were not altered by pretreatment with SP-SAP.
Effect of DLF transection on spontaneous activity
FIG.
2. Histologically identified recording sites in the spinal cord of animals pretreated with vehicle and SP-SAP. Neurons recorded from both groups
of animals were distributed in the superficial and deep laminae.
FIG. 3. The means ⫾ SE number of impulses evoked by heat stimuli in
vehicle and SP-SAP-treated groups prior to transection of the dorsolateral
funiculus (DLF). Responses evoked by stimulus intensities of 35- 51°C did not
differ between the groups.
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FIG. 4. Effect of DLF transection on spontaneous activity of dorsal horn
neurons in animals pretreated with vehicle and SP-SAP. A: representative
example of spontaneous activity for 1 cell after vehicle-pretreatment before
and at 1 h after transection. This cell was not spontaneously active prior to
transection of the DLF but developed spontaneous after transection. Also
illustrated is the maximal extent of the DLF lesion (left) in this animal and the
recording site in the spinal cord for this neuron (right). B: example of
spontaneous activity for 1 cell after SP-SAP pretreatment before and at 1 h
after transection. This cell exhibited spontaneous activity before transection of
the DLF that did not change after transection. The maximal extent of the DLF
lesion (left) in this animal and the recording site in the spinal cord for this
neuron (right) are also illustrated.
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SP-SAP-treated groups that exhibited spontaneous activity was
74% (20 neurons) and 72% (18 neurons), respectively, and did
not differ. Similarly, mean spontaneous activity did not differ
between the control (4.2 ⫾ 1.5 imp/s) and SP-SAP (4.3 ⫾ 1.7
imp/s) groups.
Responses evoked by the standard mechanical stimulus, a
von Frey monofilament with a bending force of 178 mN
applied for the duration of 2 s did not differ between the
groups. The mean number of impulses evoked by the mechanical stimulus was 28.5 ⫾ 6.6 and 32.2 ⫾ 7.0 for vehicle- and
SP-SAP-treated groups, respectively.
Responses evoked by heat stimuli also did not differ between neurons recorded from control and SP-SAP-treated animals. Mean response thresholds were 42.2 ⫾ 0.5°C for neurons in vehicle-treated animals and 42.0 ⫾ 0.7°C in animals
that received SP-SAP. As illustrated in Fig. 3, stimulus-responses curves that plot the mean number of impulses evoked
by each stimulus temperature did not differ between the groups
(2-way ANOVA with repeated measures). Also, the mean
cumulative number of impulses evoked by all the heat stimuli
was 1,056.1 ⫾ 208.0 imp for neurons recorded from the
vehicle group and 1,068 ⫾ 297 from those recorded from the
Acute transection of the DLF at the thoracic level produced
an immediate and long-lasting increase in spontaneous activity
of all 27 lumbar nociceptive neurons (21 WDR and 6 HT) in
vehicle-treated animals (Fig. 4A). The peak increase in discharge rate occurred 20 – 45 min after transection. At 1 h after
transection of the DLF, the mean discharge rate for all neurons
increased from 4.2 ⫾ 1.5 imp/s before transection to 13.4 ⫾ 2
imp/s after, an increase of 233% (paired t-test, P ⬍ 0.01; Fig.
5). To avoid the possibility that the increase in spontaneous
activity was due to activation of descending axons resulting
from trauma, 4% lidocaine was apply to the transected area
(n ⫽ 4). Local anesthesia of the area of transection did not alter
the increase in spontaneous activity. For these four neurons (2
WDR and 2 HT), spontaneous activity increased 240% at 1 h
after transection. This suggests that the increase in spontaneous
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KHASABOV, GHILARDI, MANTYH, AND SIMONE
activity after DLF transection results from the interruption of
descending axons that contribute to tonic inhibition.
In contrast, transection of the DLF had no affect on the
spontaneous activity of nociceptive neurons in animals treated
with SP-SAP (Fig. 4B). Only 3 of 24 neurons exhibited a small
mean increase (34%) in the rate of spontaneous discharge after
transection, whereas the rate of spontaneous activity either did
not change or decreased in the remaining 21 neurons. The
mean rate of spontaneous activity for all neurons was 4.3 ⫾ 1.7
imp/s before and 4.4 ⫾ 2.0 imp/s after DLF transection, which
did not differ (t-test; Fig. 5).
FIG. 6. Effect of DLF transection on heat-evoked responses of dorsal horn
neurons in animals pretreated with vehicle. A: representative example of
responses of a single neuron to stimuli of 39, 43, and 51°C before (top) and at
1 h after transection (bottom). Responses to heat clearly increased after
transection. A digitized trace of each temperature and a time scale are
provided. B: the maximal extent of the DLF lesion in the animal from which
this neuron was recorded. C: reconstruction of the recording site in the dorsal
horn for this neuron.
before transection and 42.3 ⫾ 0.8°C after). Furthermore, the
mean cumulative numbers of impulses evoked by all heat
stimuli were 1,068.0 ⫾ 297.6 before transection and 1,095.2 ⫾
361.0 after (Fig. 9C) and did not differ significantly.
DLF transection and responses to mechanical stimuli
DLF transection and responses evoked by heat stimuli
In animals pretreated with vehicle, transection of the DLF
significantly increased responses of nociceptive dorsal horn
neurons to heat stimuli. A representative example of the
increased response to heat for one neuron is provided in Figure.
6. At 1 h after DLF transection, the mean number of impulses
evoked by heat stimuli of 35–51°C for all neurons increased
(ANOVA and Bonferroni t-test; Figure. 7A), and the mean heat
response threshold (Fig. 7B) decreased 3.4°C (from 42.2 ⫾ 0.5
to 38.9 ⫾ 0.5°C; paired t-test, P ⬍ 0.001). In addition, the
mean cumulative number of impulses evoked by all intensities
of heat stimuli was increased by 100% at 1 h after transection
(from 1,056.1 ⫾ 208.0 imp before to 2,115.3 ⫾ 393.3 imp after
transection; paired t-test, P ⬍ 0.01; Fig. 7C). Although responses of WDR and HT neurons to heat before and after
transection did not differ, the effect of DLF transection was
more pronounced in HT neurons. Thus at 1 h after transection
the mean cumulative number of impulses evoked in HT neurons increased 133% (from 1,360.5 ⫾ 961.8 imp before transection to 3,167.6 ⫾ 1,910.2 imp after, paired t-test, P ⬍ 0.05),
whereas responses of WDR neurons increased 91% at this time
(from 1,002.4 ⫾ 197.1 imp to 1,918.9 ⫾ 335.7 imp, paired
t-test, P ⬍ 0.01).
In animals pretreated with SP-SAP, transection of the DLF
did not alter responses of dorsal horn neurons to heat (Fig. 8).
The mean number of impulses evoked by each heat stimulus
was similar before and after transection (Fig. 9A), and mean
response thresholds (Fig. 9B) were not altered (42.0 ⫾ 0.7°C
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As illustrated by the representative example in Fig. 10A,
transection of the DLF in animals pretreated with vehicle
produced an increase in responses of dorsal horn neurons to
mechanical stimuli, and responses were typically increased
throughout the RF. The mean number of impulses evoked by a
single application of the von Frey monofilament for all neurons
FIG. 7. Responses or all neurons to heat before and at 1 h after transection
of the DLF in animals pretreated with vehicle. A: mean ⫾ SE number of
impulses evoked by heat stimuli of 35–51°C before and after the DLF
transection. Responses to stimuli of 41–51°C were significantly elevated
following transection. B: mean ⫾ SE response threshold (°C) for all neurons
before and after the transection. Thresholds decreased significantly after
transection. C: the cumulative number of impulses evoked by all heat stimuli.
The sum of impulses (imps) across all stimuli increased significantly after
transection. Significant differences after transection are indicated by * (P ⬍
0.05) and ** (P ⬍ 0.01).
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FIG. 5. Mean ⫾ SE rate (imp/s) of spontaneous activity for all cells before
and after transection of the DLF in animals pretreated with vehicle (left) and
SP-SAP (right). Spontaneous activity of nociceptive neurons increased significantly after transection in animals pretreated with vehicle but not with
SP-SAP. **, a significant difference in mean discharge rate after transection
(P ⬍ 0.01).
SPINAL NK-1 EXPRESSING NEURONS AND DESCENDING MODULATION
increased 82.6%, from 28.5 ⫾ 6.6 to 52.1 ⫾ 9.8 imp (paired
t-test, P ⬍ 0.01; Fig. 10B). As was found for responses to heat
stimuli, transection of the DLF resulted in a greater increase in
mechanically evoked responses of HT neurons as compared
with WDR neurons. At 1 h after transection, the mean number
of impulses for HT neurons increased 1,026% (from 5.8 ⫾ 1.8
to 65.3 ⫾ 24.1 imp, paired t-test, P ⬍ 0.05), whereas responses
of WDR neurons increased 43% (from 34.6 ⫾ 7.6 to 49.5 ⫾
9.1 imp, paired t-test, P ⬍ 0.01).
Transection of the DLF had no effect on responses to
mechanical stimuli in animals pretreated with SP-SAP (Fig.
11A). The mean numbers of impulses evoked by the von Frey
monofilament for all neurons was 32.2 ⫾ 7.0 imp before and
33.9 ⫾ 6.9 imp after transection (Fig. 11B).
FIG.
9. Responses to heat for all neurons before and at 1 h after transection
of the DLF in animals pretreated with SP-SAP. A: mean ⫾ SE number of
impulses evoked by heat stimuli of 35–51°C before and after the DLF
transection. Responses to heat were not altered after transection. B: mean ⫾ SE
response threshold (°C) for all neurons before and after DLF transection. Mean
response thresholds were not changed after transection. C: the cumulative
number of impulses evoked by all heat stimuli. The sum of impulses (imps)
across all stimuli remained constant after transection.
J Neurophysiol • VOL
FIG. 10. Enhanced responses of dorsal horn neurons to mechanical
stimuli (178 mN bending force) at 1 h after transection of the DLF. A:
representative examples of evoked responses of a single neuron to mechanical stimuli at various locations within the RF before and after transection.
The RF is 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 where pairs of responses (before and after transection) were
obtained. Horizontal bars between pairs of responses represent period of
stimulation (2 s). B: the mean ⫾ SE number of impulses evoked by a single
mechanical stimulus before and after the DLF transection. Responses were
averaged across all test sites. **, a significant difference after transection
(P ⬍ 0.01).
DISCUSSION
In earlier studies, we found that ablation of spinal NK-1expressing neurons in the superficial dorsal horn resulted in
a dramatic decrease in nocifensive behaviors and hyperalgesia produced by intraplantar injection of capsaicin, inflammation, peripheral nerve injury (Mantyh et al. 1997;
Nichols et al. 1999), and spinal cord injury (Yezierski et al.
2004). After SP-SAP, the remaining nociceptive neurons in
the dorsal horn responded weakly to injection of capsaicin
and lost their ability to become sensitized (Khasabov et al.
2002). The lack of central sensitization did not result from
a decrease in the initial excitation evoked by capsaicin
because injection of a high dose of capsaicin, which elicited
discharge similar to that evoked in control animals, also did
not result in sensitization (Khasabov et al. 2002). Consequently, we hypothesized that the development of central
sensitization is dependent, at least in part, on the regulation
of descending modulatory pathways by ascending NK-1positive spinal neurons. This hypothesis is supported by the
present study in which loss of NK-1-positive neurons re-
FIG. 11. The effect of DLF transection on responses of dorsal horn
neurons to mechanical stimuli in animals pretreated with SP-SAP. A:
examples of mechanically evoked responses of a single neuron before and
at 1 h after transection. Format is the same as in Fig. 9. Responses after
transection were similar to those obtained before transection at all test sites
within the RF. B: mean ⫾ SE number of impulses evoked by a single
application of the von Frey monofilament before and after the DLF
transection. Responses were averaged across all test sites and were not
changed after transection.
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FIG. 8. Effect of DLF transection on heat-evoked responses of dorsal horn
neurons in animals pretreated with SP-SAP. A: representative example of
responses of a single neuron to stimuli of 39, 43, and 51°C before (top) and at
1 h after transection (bottom). Responses of this neuron to heat were not altered
after transection. A digitized trace of each temperature and a time scale are
provided. B: the maximal extent of the DLF lesion in the animal from which
this neuron was recorded. C: reconstruction of the recording site in the dorsal
horn for this cell.
1003
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KHASABOV, GHILARDI, MANTYH, AND SIMONE
sulted in a dramatic loss of the facilitation of spinal neurons
that normally follows DLF transection. Consistent with
these results, it has been shown that mice lacking the NK-1
receptor also exhibited a decrease in descending control of
nociceptive transmission (Bester et al. 2001).
Descending modulation via the DLF
J Neurophysiol • VOL
Potential mechanisms by which NK-1-expressing spinal
neurons alter descending modulation
The mechanisms by which NK-1 positive ascending neurons
affect descending modulation from supraspinal structures are
unclear. Although the increased responses of spinal neurons
after DLF transection were abolished by SP-SAP, it is unlikely
that NK-1-positive neurons drive descending inhibition because animals that received intrathecal SP-SAP exhibited normal behavioral responses to noxious stimuli (Mantyh et al.
1997; Nichols et al. 1999) and responses of nociceptive dorsal
horn neurons to acute stimuli were unchanged (Khasabov et al.
2001). Rather, it appears that superficial NK-1-expressing
dorsal horn neurons provide the excitatory drive that engages
descending facilitatory mechanisms. Perhaps transection of the
DLF unmasks descending facilatatory mechanisms that travel
outside the DLF. It has been proposed that facilatatory pathways descend via the VLF (Zhuo and Gebhart 1997, 2002). In
terms of ON and OFF cell activation in the RVM, excitation of
NK-1-expressing neurons may lead to a state in which ON cells
dominate the activity in the RVM and thereby produce facilitation of nociceptive transmission. It remains to be determined
whether this occurs through the inhibition of descending inhibitory pathways, excitation of descending facilitatory pathways, or both.
Conclusions
Results of the present study demonstrate that NK-1-positive
spinal neurons are part of circuitry that includes rostral medullary structures involved in descending modulation of nociceptive transmission in the spinal cord. As more becomes
known about the anatomical organization of NK-1-positive
ascending neurons and brain stem descending modulatory
neurons, it will be important to determine the transmitter
systems and receptors involved at various levels within this
circuitry. These studies will provide new information on the
mechanisms by which descending systems modulate excitability of dorsal horn neurons.
ACKNOWLEDGMENTS
SP-SAP was obtained from Advanced Targeting Systems.
GRANTS
This work was supported by National Institute on Drug Abuse Grant
DA-11986.
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Although there are several descending projections through
the DLF (Watkins et al. 1981), those originating from the RVM
are a main source of the projections through this pathway
(Watkins et al. 1980). Two types of RVM neurons directly
related to nociceptive stimulation have been identified according to their responses evoked by noxious stimuli and the
correlation between their responses and withdrawal reflex activity (Basbaum and Fields 1978; Fields et al. 1991; Hirakawa
et al. 2000). ON cells exhibit a burst-like increase in their
discharge that is correlated with a nociceptive withdrawal
reflex, and prolonged noxious thermal stimulation causes prolonged activation of these neurons (Morgan and Fields 1994).
Ablation of ON cells decreased hyperalgesia (Porreca et al.
2001). OFF cells appear to inhibit nociceptive transmission
because their discharge rate decreases or pauses during noxious
stimulation. The notion that ON cells facilitate and OFF cell
inhibit nociceptive transmission is supported by the activity of
these cells during opioid analgesia. ON cells are inhibited and
OFF cells are excited by opioids. Furthermore, the antinociceptive effects of morphine were blocked with selective blockade
of OFF cell activity (Heinricher et al. 1999). It has been
proposed that either OFF or ON cell activity within descending
systems can be dominant and result in a state of decreased
nociceptive transmission and analgesia (e.g., after opioid administration) or enhanced nociceptive transmission and hyperalgesia (e.g., prolonged noxious stimulation) (Fields 2004).
Indeed persistent nociceptive input appears to be necessary for
the development of neuronal plasticity in descending systems
(Pertovaara 2000; Ren and Dubner 2002).
We found that descending systems that travel via the DLF
are regulated by spinal NK-1-expressing neurons, the majority
of which project to supraspinal structures (Marshall et al. 1996;
Todd et al. 2000, 2002). In SP-SAP-treated animals, transection of the DLF did not increase spontaneous activity of
remaining dorsal horn neurons and did not enhance their
responses to mechanical and heat stimuli. These results demonstrate that descending modulatory mechanisms, perhaps
originating in the RVM, are altered after the ablation of spinal
NK-1-positive neurons. Results of the present study are in
agreement with an earlier study (Suzuki et al. 2002) demonstrating that NK-1 expressing spinal neurons activate descending systems to control neuronal excitability in the spinal cord.
In that study, loss of NK-1-expressing neurons in the superficial dorsal horn using SP-SAP attenuated serotonin-dependent
descending facilitation during persistent inflammatory nociception. Because the majority of NK-1-positive spinal neurons
project to supraspinal structures, it is suggested that spinal
neurons possessing the NK-1 receptor play a pivotal role in
driving descending pathways, including those that travel
through the DLF. Transection of the DLF interrupted descending projections to the spinal cord but not ascending nociceptive
pathways because these ascend primarily via the contralateral
ventrolateral funiculus (Tracey 1995).
Transection of the DLF in naı̈ve animals increased spontaneous activity of nociceptive dorsal horn neurons and enhanced
their responses to heat and mechanical stimuli (Li et al. 1998;
Pubols et al. 1991). It is well established that the DLFdependent descending inhibition of spinal neurons results from
activity in the RVM (Basbaum and Fields 1984; Li et al. 1998;
Pubols et al. 1991). Injection of local anesthetic into the RVM
eliminated descending inhibition of spinal nociceptive neurons
(Li et al. 1998) and inhibited the antinociceptive effect produced by stimulation of the periaqueductal gray (Sandkuhler
and Gebhart 1984). Complete transection of the spinal cord did
not enhance the inhibition of dorsal horn neuronal responses
produced by transection of the DLF alone (Li et al. 1998),
suggesting that the RVM, with its descending axons in the
DLF, is the primary source of descending inhibition.
SPINAL NK-1 EXPRESSING NEURONS AND DESCENDING MODULATION
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