Download NK1 receptor-expressing spinoparabrachial neurons trigger diffuse

PAIN 142 (2009) 245–254
PAIN 142 (2009) 245–254
www.elsevier.com/locate/pain
NK1 receptor-expressing spinoparabrachial neurons trigger diffuse noxious
inhibitory controls through lateral parabrachial activation in the male rat
Olivier Lapirot a,b, Raja Chebbi a,d, Lénaic Monconduit a,b, Alain Artola a,b,
Radhouane Dallel a,b,c,*, Philippe Luccarini a,b
a
Inserm U929, Neurobiologie de la douleur trigéminale, Faculté de Chirurgie Dentaire, 11 boulevard Charles de Gaulle, F-63000 Clermont-Ferrand, France
Univ Clermont1, Clermont-Ferrand F-63000, France
c
CHU Clermont-Ferrand, Clermont-Ferrand F-63000, France
d
Faculté médecine dentaire, Monastir, Tunisia
b
a r t i c l e
i n f o
Article history:
Received 13 October 2008
Received in revised form 8 January 2009
Accepted 13 January 2009
Keywords:
Nociception
Pain
Trigeminal
Orofacial
Substance P
NK1 receptor
Parabrachial
Descending inhibitory control
a b s t r a c t
Diffuse noxious inhibitory controls (DNIC) are very powerful long-lasting descending inhibitory controls,
which are pivotal in modulating the activity of spinal and trigeminal nociceptive neurons. The principal
feature of DNIC is that they are subserved by a loop that involves supraspinal structures that have not yet
been identified. Using behavioral, in vivo extracellular electrophysiological and anatomical approaches,
we studied the neuronal network underlying DNIC. Using a new behavioral model of DNIC, in which facial
grooming produced by formalin injection into the vibrissa pad is inhibited by a conditioning noxious
stimulation, formalin injection into the hindpaw, we show that blockade of NK1 receptors in the lumbar
spinal cord – by intrathecal administration of the NK1 receptor antagonist, RP67580 – largely attenuates
DNIC-induced facial analgesia. In a second series of experiments, WDR neurons were recorded from the
trigeminal subnucleus oralis and inhibited their C-fiber-evoked responses by the conditioning noxious
heat stimulation of the hindpaw. We show that inactivating the lateral parabrachial area – by microinjecting the GABAA agonist, muscimol – strongly attenuates DNIC-induced inhibition of C-fiber-evoked
responses. Finally, our neuroanatomical tracing study demonstrates that the descending pathway for
DNIC does not involve direct descending projections from the PB area. We conclude that (1) lamina
I/III spinoparabrachial neurons that express the NK1 receptor and (2) parabrachial neurons are involved
in the ascending part of the loop underlying DNIC and that the descending pathway for DNIC might
include indirect projections to the spinal or medullary dorsal horn.
Ó 2009 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
1. Introduction
Pain is a complex experience that involves sensory-discriminative, cognitive-evaluative, and affective-emotional components.
Conversely, central nervous system networks may modulate the
transmission of nociceptive messages according to the nature of
the painful stimulus and behavioral state of the individual
[24,47]. For instance, descending pathways from the brainstem
can either inhibit or facilitate transmission of nociceptive information at the level of the spinal and medullary dorsal horn (MDH).
One of these naturally activated, descending controls is the diffuse noxious inhibitory controls (DNIC). DNIC are very powerful,
long-lasting inhibitory controls, which have been shown to be pivotal in modulating spinal [37] and trigeminal nociceptive neurons
* Corresponding author. Address: Inserm U929, Neurobiologie de la douleur
trigéminale, Faculté de Chirurgie Dentaire, 11 boulevard Charles de Gaulle, 63000
Clermont-Ferrand, France. Tel.: +33 4 73 17 73 13; fax: +33 4 73 17 73 06.
E-mail address: [email protected] (R. Dallel).
[17,20]. The afferent and efferent pathways of DNIC include the
ventrolateral quadrant and the dorsolateral funiculus of the spinal
cord, respectively [37]. But the fact that sectioning the spinal cord
suppresses DNIC suggests that these controls involve supraspinal
areas [37]. Surprisingly, DNIC are not modified by lesions of area
that are known to modulate pain such as the periaqueductal grey,
parabrachial (PB) area, locus coeruleus/subcoeruleus or rostroventral medulla [37]. On the other hand, lesions of subnucleus reticularis dorsalis (SRD) in the caudal medulla reduced DNIC [8]. But,
importantly, this depression of DNIC is only partial (40%), suggesting that other brain areas are involved in these controls.
More recently, several reports have pointed out the role of neurokinin-1 (NK1) receptor-expressing lamina I/III neurons in not
only transmitting pain to higher brain centers but also setting
inhibitory and excitatory levels in spinal nociceptive neuronal networks [34,35,52]. For example, it was shown, using c-Fos histochemistry, that DNIC are suppressed either following selective
ablation of NK1 receptor-expressing lamina I/III neurons in the
spinal cord, using a substance P and saporin conjugate [52], or in
0304-3959/$36.00 Ó 2009 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.pain.2009.01.015
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O. Lapirot et al. / PAIN 142 (2009) 245–254
NK1/ mice or in wild type mice treated with the NK1 receptor
antagonist, RP67580 [5]. However, these are only indirect evidence
relaying on c-Fos immunohistochemistry. Moreover, supraspinal
areas involved in DNIC are not identified. One possibility is the lateral PB area. Indeed, in the spinal cord, the highest concentration of
NK1 receptor, on which substance P acts, is in the lamina I/III of the
superficial dorsal horn [9]. And the great majority (80%) of projecting lamina I/III neurons expresses the NK1 receptors
[21,43,55] and terminates extensively within the PB area of the
brain [55]. In turn, PB neurons project to the areas in the brain,
such as the amygdala and hypothalamus [2] that are known to
modulate the descending pathways from the brainstem and to regulate nociceptive processing within the spinal DH and MDH [47].
We thus hypothesize that the development of DNIC depends, at
least in part, on the modulation of descending pathways by
ascending NK1R-expressing spinoparabrachial neurons.
Here, we (i) reassessed the role of the spinal neurons that express NK1 receptor in DNIC, using a behavioral model of DNIC,
and (ii) tested the hypothesis that PB neurons are involved in DNIC,
using both electrophysiological and anatomical approaches. These
questions were investigated in the rat trigeminal system.
elicited. The selective neurokinin 1 (NK1) receptor antagonist
RP67580 or its inactive enantiomer (RP67581) (gift from Sanofi Research, Montpellier, France) was injected in a volume of 10 ll/rat.
The dose of RP67580 (20 lg) was selected based on the published
reports [11,15,16,32,48].
4. Electrophysiology
4.1. Trigeminal system
Adult male Sprague–Dawley rats (280–300 g; Charles River Laboratories, L’Arbresle, France) were housed in a temperature-controlled room (22 °C; lights on at 07:00 h and off at 21:00 h). Food
and water were available ad libitum. All efforts were made to minimize as much as possible the number of animals used. All procedures adhered to the ethical guidelines of the International
Association for the Study of Pain and the European Community
Council directive of 24 November 1986 (86/609/EEC).
In the trigeminal system, the medullary dorsal horn (MDH) is
generally considered as the essential brainstem relay of orofacial
nociceptive information [22]. However, findings have established
the presence of nociceptive neurons in the spinal trigeminal nucleus
oralis (Sp5O), the majority of which are WDR neurons [12,13,17].
The properties and plasticity of Sp5O WDR neurons clearly match
those of WDR neurons in lamina V of the spinal DH and MDH
[17,29,30,50]. As for the neurons of the deep laminae of the spinal
DH, Sp5O neurons are indirectly activated by cutaneous C-fiber input [18,58]. Accordingly, there is evidence for ipsilateral connections between the MDH and the Sp5O, emanating from laminae
III–V of the MDH and to a lesser degree from lamina I and external
II [14,57,58]. Although interneurons located in the superficial laminae are likely to relay nociceptive C-fiber-mediated information
from the MDH to downstream WDR Sp5O neurons [14,18,58], interneurons in deep MDH laminae may also be involved since they may
receive C-fiber inputs indirectly, via lamina I–II neurons sending axons into deeper layers [38–40]. Interestingly, unlike the DH where
the laminae are very close, the Sp5O is 3 mm away from the MDH.
This allows various pharmacological or electrophysiological manipulations to be made either in the MDH or in the Sp5O without directly affecting the other structures [12,14,18,58].
3. Behavioral
4.2. Animal preparation
3.1. Facial formalin test
For surgery, the animals were anaesthetized with 2% halothane
in a nitrous oxide/oxygen mixture (2/3–1/3). After intraperitoneal
injection of 100 lg atropine sulphate a tracheal cannula was inserted, the jugular vein was cannulated, the animals were paralyzed by an intravenous perfusion of pancuronium bromide
(0.5 mg/h) and artificially ventilated with a volume-controlled
pump (54–55 strokes/min). The levels of halothane, O2, N2O and
the end-tidal CO2 (3.5–4.5%) were monitored by an anesthetic
gas monitor (Artema MM 200, Sundbyberg, Sweden) during the entire experimental period. These parameters were measured by infra-red absorption, digitally displayed and under the control of
alarms. A sufficient depth of anesthesia was judged from the absence of gross fluctuations of heart rate. The vascularization of
the cutaneous tissues was periodically checked by observing the
color of the paw extremities and the rapidity by which they regained normal color after the application of pressure to the paw.
The core temperature was maintained at 38 ± 0.5 °C with a homeothermic blanket system.
The animals were placed in a stereotaxic frame with the head
fixed in a ventroflexed position (incisor bar dropped 5 mm under
the standard position) by means of an adapted metallic bar. A craniotomy was performed on the right side at the level of the occipitoparietalis suture, and the dura mater was removed. After
surgery, the level of halothane was reduced to 0.6–0.7% and maintained at this level during the recording period.
2. Methods
Rats received 50 ll formalin solution (1.5%), into the right vibrissa pad. Formalin was injected subcutaneously through a 27-gauge
needle into the center of the right vibrissa pad as quickly as possible,
with only minimal animal restraint. Following injection, the animals
were immediately placed back in the test box for a 45-min observation period. The recording time was divided into 15 blocks of 3 min,
and a nociceptive score was determined for each block by measuring
the number of seconds that the animals spent grooming the injected
area with the forepaw. Movements of the ipsilateral forepaw were
accompanied by the movements of the contralateral forepaw. A video camera was used to record the grooming response. Analysis of
the behavior was made by an investigator, who was blinded to the
animal’s group assignment [51].
3.2. Heterotopic conditioning stimulus
Formalin was used as the heterotopic conditioning noxious
stimulus. Rats received a 50 ll subcutaneous injection of diluted
formalin (10%) or saline (0.9%) into the right plantar hindpaw
immediately before formalin injection into the vibrissa pad.
3.3. Direct transcutaneous intrathecal injection
Intrathecal injections were performed as previously described
[46] 5 min before formalin injection. Briefly, the rat was held in
one hand by the pelvic girdle and a 25-gauge needle connected
to a 20 ll Hamilton syringe was inserted into the subarachnoidal
space between lumbar vertebrae L5 and L6, until a tail-flick was
4.3. Microinjections
Drugs were delivered in the left PB area by two-barrel glass
micropipettes (3GC120F-15; Clark Electromedical Instruments,
O. Lapirot et al. / PAIN 142 (2009) 245–254
Pangbourne, UK) fixed on the micromanipulator and connected to
two Hamilton syringes (0.5 ll) by means of polyethylene tubing
[18]. The micropipette was broken back maximally to a diameter
of 70–100 lm. The micropipette was positioned stereotaxically
above the targeted brainstem site 1 h before the injection. The
coordinates used for microinjection sites were 300 lm caudal to
the interaural plane and 2.3 mm lateral to the midline [49]. The
micropipette was placed at this level, because previous electrophysiological, anatomical and immunohistochemical studies have
shown that this PB area contains nociceptive specific neurons
[1,6] and receives a dense projection from the superficial spinal
lamina neurons expressing the NK1 receptor [3,55]. The micropipettes and tubing were filled with GABAA agonist muscimol
(0.25 nmol in 100 nl) or saline with pontamine sky blue, respectively (for location of the injection site). Injections of drugs were
performed with a manual injector over a period of 2 min and monitored by observing the movement of an air bubble in the tubing.
The slow rate of injection was chosen to minimize the chance of
tissue damage. The micropipettes remained in place throughout
the experimental session. The dose of muscimol was selected
based on the published reports [10,28,42,44]. Muscimol (Sigma–
Aldrich, France) was dissolved in 0.9% saline and kept at 4 °C in a
light excluding vial. Fresh muscimol solution was prepared every
testing day.
4.4. Recordings
Unitary extracellular recordings were made from right Sp5O
neurons with glass micropipettes (8–10 M) filled with a mixture
of 5% NaCl and pontamine sky blue. The brainstem was explored
2.4–3.0 mm lateral to the midline and between the frontal planes
AP 1.1 and AP 2.6 mm from the interaural line [49]. Single unit
activities were amplified and displayed on oscilloscopes, and were
also led into a window discriminator connected to a CED 1401plus
interface (Cambridge Electronic Design) and a PC computer (Spike
2.05 software), to allow sampling and analysis of the spontaneous
and evoked neuronal activity.
WDR neurons were recognized based on their responses to
mechanical and percutaneous electrical stimulations of their
receptive field [17]. Specifically, neurons that responded in a
graded manner with increasing firing rates to the stimulus range
from non-noxious to noxious intensity were classified as WDR
cells. Innocuous mechanical stimuli to the skin, mucosa and teeth
included air puff, brushing with a soft brush, gentle stroking and
light pressure with a blunt probe. Noxious mechanical stimuli consisted of heavy pressure, pinprick and pinching with fine forceps
(tip area 1 mm2), which evoked a painful sensation when applied
to the experimenters’ skin. Once a neuron had been identified, its
receptive field was mapped and classified according to its location:
intraoral, perioral or more peripheral regions of the face [17]. Electrical square-wave stimuli (2 ms duration) were applied through a
pair of stainless steel stimulating electrodes subcutaneously placed
into the center of the previously delineated receptive field and
thresholds for eliciting A-fiber and C-fiber responses determined.
In post-stimulus time histograms (PSHs), C- and A-fiber responses
were distinguished according to their latencies. It was previously
shown that burst discharges at latencies >30 ms are elicited by
C-fibers [17,31]. Therefore, all spikes between 30 and 300 ms
post-stimulus were considered as C-fiber-evoked.
4.5. Experimental design
The experimental procedure involved sequences of 105 electrical shocks applied repeatedly (0.66 Hz) to the excitatory RF at
three times the threshold for C-fibers activation every 15 min during 115 min. A 15-min interval was selected because it is the
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shortest one to allow full recovery to baseline response between
sequences. DNIC were triggered by alternatively immersing the
right or the left hindpaw – only one hindpaw being immersed during each sequence – into a 48 °C water bath between the 36th and
60th stimuli (i.e., for 37.5 s). DNIC were thus triggered by noxious
heat stimulation of the left hindpaw, 10, 40, 70 and 100 min and by
noxious heat stimulation of the right hindpaw, 25, 55, 85 and
115 min following muscimol microinjection (Fig. 4).
In each sequence, the PSHs built from 1st–30th responses were
used for studying the unconditioned response, and those built from
the responses to 36th–105th stimuli were used for studying DNIC.
To assess the inhibitory effect of noxious heat stimulation, only the
46th–60th responses were considered, responses to earlier 36th–
45th stimuli during immersion being discarded owing to the latency (5–10 s) of the maximal inhibitory effects [7]. Post-effects
were measured on the 61th–75th and 76th–90th responses, 0–
22 s and 22–44 s after the end of noxious heat stimulation, respectively. In each sequence, PSHs, built from 46th–60th (effect), 61th–
75th (1st post-effect) and 76th–90th (2nd post-effect) responses,
were normalized to that built from the 21th to 35th responses (before noxious heat stimulation; baseline). The inhibitory effect of
DNIC was computed as the percent decrease in the number of
C-fiber-evoked action potentials.
Muscimol (0.25 nmol in 100 nl) was microinjected into the left
PB area after two stable (variation in unconditioned C-fiber response <10%) control sequences had been recorded. There was only
one muscimol injection in each rat.
4.6. Histological analysis
Recording and microinjection sites were visualized by an injection of pontamine sky blue solution at the end of the experiment.
After the animal was killed by an injection of a lethal dose of pentobarbital, the brain was removed and fixed in a 10% formalin solution for 1 week. The tissue was frozen, cut into serial 100-lm-thick
sections, and stained with neutral red. Recording and microinjection sites were determined by microscopic examination, and were
then plotted on camera lucida drawings of serial sections.
5. Neuroanatomy
5.1. Fluorogold injection
Animals (n = 5) were anesthetized with chloral hydrate (7%,
400 mg/kg body weight, i.p.) and placed in a stereotaxic frame. A
2% solution of Fluorogold (hydroxystilbamidine; Molecular Probes,
Eugene, OR, USA), diluted in 0.1 M cacodylic acid, was ejected from
glass micropipettes. These were inserted stereotaxically so that
their tips (30–40 lm diameter) were located within the right
MDH (AP 5.3 mm from the interaural line, ML 2.7 mm, P
1.5 mm) according to Paxinos and Watson [49] at an angle of 80°
to the horizontal plane. Electrophoretic application of Fluorogold
was made by 30 s pulses of positive direct current (3–5 lA) applied
every 60 s for a period of 15–20 min. The microelectrode was left
in situ for a further 5 min before withdrawal from the brain. One
single application was made in the MDH per animal.
One week later, rats were deeply anaesthetized with chloral hydrate and perfused pericardially with warm (37 °C) heparinized
saline (25 IU heparin/mL) followed by cold (10 °C) phosphate-buffered solution (0.1 M, pH 7.6) containing 4% paraformaldehyde and
0.03% picric acid for 15 min. Brainstem and first cervical segment
(C1) were then removed and transferred in the same paraformaldehyde-picric acid solution containing 30% sucrose at 4 °C and left
overnight. Coronal sections were cut on a freezing microtome
(40 lm thick) and collected in a 0.05 M Tris-buffered saline
(TBS). A set of MDH sections was mounted on gelatin-coated slides,
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O. Lapirot et al. / PAIN 142 (2009) 245–254
coverslipped using Vectashield (Vector, Burlingame, CA, USA) and
viewed using a fluorescent microscope to check the location of Fluorogold deposit. Only brainstem sections of animals in which the
injection site was located in the MDH were processed further. A
few selected sections were mounted separately and slightly counterstained with neutral red to help delineating the limits of the
anatomical structures.
5.2. Fluorogold immunocytochemistry
Free-floating brainstem and C1 sections were placed in 1% normal goat serum for 1 h before incubation at 4 °C in a rabbit antibody directed against Fluorogold (1:6000, Chemicon, Temecula,
CA, USA) for 48 h. Sections were then washed in TBS and placed
in biotinylated goat anti-rabbit (1:200, Jackson Immunoresearch,
West Grove, PA, USA) for 2 h at room temperature followed by
incubation in Vector avidin–biotin–peroxidase complex (Vector
ABC, 90 min at room temperature). Immunoreactivity for Fluorogold was visualized in sections using 3,30 -diaminobenzidene tetrahydrochloride (DAB) and ammonium nickel sulphate (kit Vector
Peroxydase substrate DAB). All sections were rinsed in TBS and
transferred to gelatinized slides before being coverslipped using
DPX. All immunolabels were diluted in TBS containing 0.25% bovine serum albumin and 0.3% Triton X-100. Specificity controls
consisted of the omission of the primary antibody and incubation
of sections in inappropriate secondary antibodies. In all these control experiments, no specific staining was evident.
5.3. Data analysis
Computer-assisted bright-field images of injection sites and
representative labeling were obtained using a CCD colour video
camera (Sony DXC-950P) connected to a Nikon Optiphot-2 microscope at 10 magnification. Images were exported to Adobe PhotoShop (v 5.5) to adjust brightness and contrast before adjusting the
image scale. Each injection site was analyzed using coronal sections processed with DAB. The delineation of the MDH was identified using the Paxinos and Watson atlas [49].
Retrogradely labeled cell bodies were counted according to
their location along four different rostrocaudal planes within the
PB area. The delineation of the PB area was based upon the Paxinos
and Watson atlas [49]. Brainstem sections were categorized
according to their approximate rostrocaudal location from the
interaural line. Data are expressed as the sum of the total number
of labeled cells counted in the structure from all four sections
which were analyzed (mean per rat ± SEM).
6. Statistical analysis
Results are expressed as means ± SEM. Statistical analysis was
performed using Student’s t-test, or one-way analysis of variance
(ANOVA) followed by a post hoc Tukey’s test or one-way Repeated
Measures (RM) ANOVA followed by a post hoc Tukey’s test as indicated. The level of significance was set at p < 0.05.
7. Results
7.1. Are the NK1 receptor-expressing neurons involved in DNIC?
To investigate whether neurons in the spinal cord that express
the NK1 receptor are involved in DNIC, we used a behavioral model
of DNIC. In this model, facial grooming behavior produced by formalin injection into the vibrissa pad is inhibited by a conditioning
noxious stimulus, formalin injection into the hindpaw. We thus
examined the effect of a subcutaneous injection of formalin
(50 ll, 1.5%) into the vibrissa pad in four different conditions combining two treatments: subcutaneous injection of either formalin
(50 ll, 10%) or saline into the right hindpaw (heterotopic) together
with intrathecal administration of either RP67580, a NK1 receptor
antagonist, or RP67581.
Typically, subcutaneous injection of formalin into the vibrissa
pad induces a biphasic nociceptive grooming response with an
early, short-lasting phase (starting 20–30 s after formalin injection) and a late, long-lasting one [51]. Such intense facial grooming
was observed in group 1 (control rats receiving heterotopic subcutaneous saline and intrathecal RP67581, n = 8). Durations of the
first (measured during the first 3 min after formalin injection)
and second (measured between blocks 5 and 12, i.e., 15–36 min
after formalin injection) phases were 18 ± 8 s and 251 ± 27 s,
respectively (Fig. 1). In group 2 (rats receiving heterotopic subcutaneous formalin and intrathecal RP67581, n = 8), the grooming response to the formalin injection into the vibrissa pad was
dramatically reduced. Durations of the first and second phases
were only 26 ± 12% (ANOVA followed by Tukey’s test, F3,25 = 3.65,
p = 0.11) and 20 ± 4% (ANOVA followed by Tukey’s test,
F3,25 = 10.861, p < 0.001), respectively, of those in group 1 (Fig. 1).
The NK1 antagonist, RP67580, was present in the last two groups,
3 and 4. Comparison between groups 1 and 3 (rats receiving heterotopic subcutaneous saline and intrathecal RP67580, n = 5)
shows that the administration of RP67580, alone, had no effect
on the duration of facial grooming behavior (Fig. 1). On the other
hand, comparison between groups 3 and 4 (rats receiving heterotopic subcutaneous formalin and intrathecal RP67580, n = 8) shows
that the administration of the NK1 antagonist resulted in a significant reduction in the antinociceptive effect produced by heterotopic formalin. The second phase in group 4 was 57 ± 12% of that
in group 3 (Fig. 1); that is, DNIC were significantly reduced in group
4 compared with those in group 2 (45% reduction; Student’s ttest, p < 0.02).
Thus, the reduction in facial grooming following heterotopic
formalin injection appears to involve the NK1 receptor (comparison between groups 2 and 4) in the lumbar spinal cord (comparison between groups 1 and 3). Indeed, that intrathecal RP67580
alone, i.e. in rats receiving heterotopic subcutaneous saline into
the hindpaw, had no effect on facial grooming which is not consistent with a direct effect of RP67580 on NK1 receptors in spinal trigeminal nucleus.
7.2. Are parabrachial neurons involved in the ascending part of the
loop underlying DNIC?
The PB area is one of the major projection targets of dorsal horn
neurons which express NK1 receptors [55]. This raises the question
as to whether PB area is involved in the neuronal network underlying DNIC. To test this hypothesis, we used an electrophysiological
paradigm of DNIC. In this paradigm, the C-fiber-evoked responses
of Sp5O WDR neurons are inhibited by a conditioning stimulus,
noxious heat of the hindpaw. We examined the effect of pharmacologically inactivating the PB area – by microinjecting the GABAA
agonist, muscimol – on DNIC.
7.2.1. Characteristics of trigeminal WDR neurons
A total of 24 WDR neurons were recorded within the Sp5O.
None of them exhibited spontaneous activity. All had an ipsilateral
receptive field that included the intraoral or perioral region. They
were sensitive to both innocuous and noxious mechanical stimuli
and responded by increasing their firing rate as stimulus intensity
increased into the noxious range. When 2-ms-long percutaneous
electrical stimuli were applied to the center of the receptive field
of the neurons, responses attributable to peripheral activation of
A- and C-fibers could be observed (Fig. 2). The longest latency
O. Lapirot et al. / PAIN 142 (2009) 245–254
Fig. 1. Blockade of spinal NK1 receptors attenuates DNIC. (A) Time course of the
grooming response to subcutaneous formalin injection into the vibrissa pad in rats
receiving [1] heterotopic subcutaneous saline and intrathecal RP67581 (filled circle,
n = 8), [2] heterotopic subcutaneous formalin and intrathecal RP67581 (empty
circle, n = 8), [3] heterotopic subcutaneous saline and intrathecal RP67580 (filled
triangle, n = 5), and [4] heterotopic subcutaneous formalin and intrathecal RP67580
(empty triangle, n = 8). Formalin injections into the vibrissa pad and the hindpaw
were 50 ll, 1.5% and 50 ll, 10%, respectively. Intrathecal administration of RP67580
was 20 lg in 10 ll. (B) Histogram of the duration of the second phase of grooming
behavior in the four groups of rats, as above. Treatments in the different groups of
animals are also indicated below the histograms. Note that formalin injection into
the hindpaw induces a significant inhibition on the facial grooming (group 2) but
not after intrathecal administration of RP67580 (group 4). Statistical analysis was
performed using one-way ANOVA followed by Tukey’s test, F3,25 = 10.861. ns, non
significant; ***p < 0.001.
responses (102 ± 3 ms) were C-fiber-evoked. Indeed, the computed
conduction velocity (0.6 m s1) was in the range of those previously reported for C-fibers [13,14,17,45]. In addition, long latency
responses could only be evoked by high intensity stimulations
(mean threshold: 10.3 ± 1.2 mA) and they exhibited windup
[13,14,17,45]. Fig. 2 shows an example of the progressive increase
of C-fiber-evoked responses to repetitive (0.66 Hz) constant percutaneous suprathreshold electrical stimuli applied to the receptive
field. Finally, it has been already shown that these long latency responses in Sp5O neuron are selectively depressed by intravenous
morphine in a naloxone-dependent way [19] and are reduced by
intracutaneous injection of capsaicin (0.1%), a C-fiber toxin [17].
249
Fig. 2. Effect of muscimol microinjection into the lateral PB area on the C-fiberevoked responses of one Sp5O WDR neuron. (A) Receptive field of the WDR neuron
located ipsilaterally on the muzzle. (B) Semi-schematic drawing of a coronal section
through the Sp5O area illustrating the recording site. (C) Post-stimulus histograms
of C-fiber-evoked responses to 30 successive stimulations (0.66 Hz at three times Cfiber threshold intensity) obtained before (left) and 55 min after (right) muscimol
microinjection (0.25 nmol in 100 nl) into the lateral PB area. (D) Progressive
increase of C-fiber-evoked responses (windup) to 30 successive stimulations
(0.66 Hz at three times C-fiber threshold intensity) before (black) and 55 min after
(grey) muscimol microinjection. Note that the windup is similar before and after
muscimol microinjection.
7.2.2. Effect of muscimol microinjection on the C-fiber-evoked
responses
Anatomical examination of brains revealed that the microinjection site was inside the PB area in 14 experiments, in either the lateral (n = 12) or external medial (n = 2) PB area, whereas it was
‘outside’ in 10 others.
Neither spontaneous activity nor C-fiber-evoked responses were
affected by muscimol microinjection (0.25 nmol in 100 nl) whether
it was within or outside the PB area. Though C-fiber-evoked responses slightly decreased with time, there was no difference
between inside and outside groups (55 min after microinjection:
83 ± 13% and 78 ± 12% of that before microinjection, respectively).
Fig. 2 shows an example of a WDR neuron showing no effect on C-fiber-evoked responses following muscimol microinjection.
7.2.3. Effect of muscimol microinjection on DNIC
All tested units (n = 24) were under the influence of DNIC. Results on DNIC obtained when muscimol microinjection was inside
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the lateral or external medial PB area will be compared with those
when it was ‘outside’.
7.2.3.1. Muscimol microinjection in the lateral PB area. Before muscimol microinjection into the lateral PB area, all tested units (n = 12)
were strongly depressed (85 ± 5%) by the application of noxious
heat (48 °C) to the right or left hindpaw (Fig. 3). The inhibitory of
C-fiber-evoked responses was followed by long-lasting post-effects
of 62 ± 7% and 49 ± 6% during the two consecutive 22-s periods following conditioning, respectively.
Following muscimol microinjection, DNIC triggered by noxious
stimulation of the right hindpaw, i.e. contralateral to the inactivated lateral PB area, were strongly reduced in all recorded neurons (Figs. 3E and 4A). Fig. 3 shows an example in the form of
histograms of C-fiber-evoked responses obtained before and after
muscimol microinjection in the external lateral PB area. The reduction of DNIC was fast: already significant at 25 min, it was maximum at 55 min after muscimol microinjection. And it was longlasting (for at least 115 min after microinjection). Fifty five min
after muscimol microinjection, the mean inhibition (n = 12) of C-fiber-evoked responses was only 51 ± 7%, 36 ± 5% and 30 ± 6% during
noxious heat application and the two consecutive periods following conditioning, respectively. This corresponds to a strong reduction of DNIC: 42 ± 7% (RM ANOVA followed by Tukey’s test,
F4,43 = 16.883, p < 0.001), 47 ± 8% (RM ANOVA followed by Tukey’s
test, F4,43 = 16.883, p < 0.001) and 39 ± 11% (RM ANOVA followed
by Tukey’s test, F4,43 = 16.883, p < 0.013) during noxious heat stimulation and the two consecutive periods following conditioning,
respectively.
On the other hand, muscimol microinjection into the lateral PB
area produced a much smaller effect on DNIC triggered by noxious
stimulation of the left hindpaw, i.e. ipsilateral to the inactivated
lateral PB area (Figs. 3C and 4A). A reduction of DNIC was observed
in only two of the 12 neurons. In average (n = 12), it occurred later
(only 70 min after microinjection) and was much smaller
(Student’s t-test, p < 0.001) than that triggered by the right
hindpaw, the inhibition of C-fiber-evoked responses being
72 ± 5% (n = 12) during noxious heat stimulation, which corresponds to only a 17 ± 3% reduction of DNIC. None of the two
post-effects were affected.
7.2.3.2. Muscimol microinjection in the external medial PB area. DNIC
triggered by noxious stimulation of the right hindpaw were also
reduced after muscimol microinjection into the external medial
PB area (n = 2). The mean inhibition of C-fiber-evoked responses
was, during noxious heat stimulation and the two consecutive
periods following conditioning, 88 ± 3%, 54 ± 10% and 33 ± 2%,
respectively, before muscimol microinjection, and 60 ± 4%,
43 ± 2% and 38 ± 2%, respectively, 55 min after muscimol microinjection. Interestingly, the inhibition of C-fiber-evoked responses
was reduced only during noxious heat stimulation – corresponding
to a 32 ± 2% reduction of DNIC – but not during the two consecutive periods following conditioning. Muscimol microinjection in
the external medial PB area had no effect on DNIC triggered by
noxious stimulation of the left hindpaw.
7.2.3.3. Muscimol microinjection outside the PB area. Before muscimol microinjection into the lateral PB area, all tested units
(n = 10) were strongly depressed (85 ± 5%) by the application of
noxious heat to the right or left hindpaw. The inhibitory of C-fiber-evoked responses was followed by long-lasting post-effects
of 62 ± 7% and 43 ± 7% during the two consecutive periods following conditioning, respectively.
Muscimol microinjection outside the PB area reduced only DNIC
triggered by noxious stimulation of the right hindpaw, i.e. contralateral to the inactivated lateral PB area, and in only two of the 10
recorded neurons (Fig. 4B). The reduction of DNIC was small. In
average (n = 10), 55 min after muscimol microinjection, inhibition
of C-fiber-evoked responses was 71 ± 7% (corresponding to a
18 ± 6% reduction of DNIC). The post-effects were not affected,
and were 59 ± 7% and 47 ± 7% during the two consecutive periods
following conditioning, respectively.
Fig. 3. Muscimol microinjection into the lateral PB area reduces DNIC. (A) Schematic representation of the experimental design. WDR neurons with ipsilateral receptive fields
on the muzzle were recorded in the right Sp5O. DNIC were triggered by immersion of either the left or right hindpaw into 48 °C water bath. Muscimol was microinjected
(0.25 nmol in 100 nl) into the left lateral subnucleus of the PB area. (B–E) Effect of muscimol microinjection into the left external lateral subnucleus of the PB area (B) on DNIC
triggered by the left (C) and right hindpaw (D and E) of one Sp5O WDR neuron. Histogram of C-fiber-evoked responses to 105 successive stimulations (0.66 Hz at three times
the C-fiber threshold intensity) recorded before (D) and 55 min after (C and E) muscimol microinjection. Between the 36th and 60th stimulation was either the left (C) or right
(D and E) hindpaw immersed into 48 °C water bath. Note that after muscimol microinjection into the external lateral subnucleus of PB area, there was a reduction of DNIC
triggered by the stimulation of the right hindpaw (E), whereas those by the stimulation of the left hindpaw (C) were less affected.
O. Lapirot et al. / PAIN 142 (2009) 245–254
251
Fig. 4. Only muscimol microinjection into the PB area reduces DNIC. (A) Time course of the effect of muscimol microinjection into the left lateral PB area (n = 12) on DNIC
triggered by the stimulation of the right (black columns) or left hindpaw (white columns). (B) Time course of the effect of muscimol microinjection outside the left lateral PB
area (n = 10) on DNIC triggered by the stimulation of the right (black columns) and left hindpaw (white columns). (C) Semi-schematic drawing of coronal sections through the
PB area illustrating microinjection sites. Black circles represent microinjection sites within the lateral (n = 12) and external medial (n = 2) PB area, whereas grey circles
represent those outside (n = 10). Abbreviations: bc, brachium conjunctivum; dl, dorsal lateral subnucleus; el, external lateral subnucleus; em, external median subnucleus;
lcr, lateral crescent subnucleus; sl, superior lateral subnucleus; vl, ventral lateral subnucleus. Each section is referred to by its distance (in lm) from the coronal plan, where
the inferior colliculus merges with the pons. Statistical analysis was performed using one-way RM ANOVA followed by Tukey’s test (F4,43 = 16.883). ns, non significant;
*p < 0.05; **p < 0.005; ***p < 0.001.
7.3. Are descending projections from the parabrachial area involved in
the descending part of the loop underlying DNIC?
Thus, inactivating the lateral PB area strongly attenuates DNICinduced inhibition of C-fiber-evoked responses of the Sp5O WDR
neurons. This suggests that, in control conditions, activation of
PB neurons can inhibit the nociceptive responses of Sp5O WDR
neurons. However, are WDR neurons affected by direct descending
fibers originating from PB area or via relays in other brain areas?
Because only one description of the descending projections from
PB area to trigeminal sensory nuclear complex has been made in
rats [59], the present study was designed to provide more detailed
information on these projections.
Retrograde labeling was examined after restricted, small injections of the retrograde tracer fluorogold in the right MDH of five
rats (Fig. 5C). In each case, retrogradely labeled cell bodies were
predominantly or exclusively ipsilateral in the Kölliker-Fuse (KF)
nucleus and to a lesser extent in the external lateral subnucleus
of the PB area, primarily in its caudal third (Fig. 5A and D). Occasional neurons were found scattered among other PB subnuclei.
The contralateral PB (to the injection site) was almost devoid of
labeling. While the absolute numbers of retrogradely labeled neurons varied with the size of the MDH injection, the PB subnuclei in
which these neurons were found were relatively consistent across
cases. These results demonstrate that direct PB projections to the
MDH are mainly ipsilateral, and originate from the caudal KF and
the external lateral subnucleus of the PB area.
8. Discussion
In the behavioral paradigm of DNIC, facial grooming behavior
produced by formalin injection into the vibrissa pad is inhibited
by a conditioning noxious stimulus, formalin injection into the
hindpaw. We show that blockade of NK1 receptors in the lumbar
spinal cord largely attenuates DNIC-induced analgesia.
In the electrophysiological paradigm of DNIC, C-fiber-evoked
responses of Sp5O WDR neurons produced by the electrical stimulation of their receptor field are inhibited by the conditioning stimulus, noxious heat of the hindpaw. We show that inactivating the
PB area, whereas it has no effect on spontaneous activity and
evoked responses of WDR neurons in Sp5O, strongly attenuates
DNIC-induced inhibition of C-fiber-evoked responses.
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O. Lapirot et al. / PAIN 142 (2009) 245–254
Fig. 5. Distribution of PB neurons projecting to the medullary dorsal horn. (A) Numerical view of coronal sections showing retrogradely labeled cells in the contralateral and
ipsilateral PB area following fluorogold injection into the medullary dorsal horn (C). (B) Average number of PB neurons projecting to the medullary dorsal horn. (D) Higher
magnification of retrogradely labeled neurons from the region delineated in (A). Note that retrogradely labeled neurons were predominantly ipsilateral in the Kölliker-Fuse
(KF) nucleus and to a lesser extent in the external lateral subnucleus (el) of the PB area, see Fig. 4 for abbreviations.
Finally, direct projections from the PB area to the MDH are
mainly ipsilateral and originate from the Kölliker-Fuse and to a lesser extent from the external lateral subnucleus of the PB area.
8.1. NK1 receptor-expressing neurons are involved in DNIC
Painful stimuli applied to a body surface can inhibit nociceptive
sensations in other body parts. This phenomenon has been termed
counter-irritation or DNIC [37]. For instance, it has been shown that
capsaicin injection, into either the hindpaw or the back, reduces the
jaw opening reflex [26] and facial grooming behavior [41]. Similarly,
we show here that facial grooming behavior produced by formalin in
the vibrissa pad is inhibited by formalin in the hindpaw.
Inhibition of facial grooming behavior by hindpaw (heterotopic)
noxious stimulation requires the activation of NK1 receptors in the
lumbar spinal cord. Intrathecal administration of the selective NK1
receptor antagonist, RP67580, reduces DNIC. That intrathecal
RP67580 alone, i.e. without the heterotopic conditioning stimulus,
has no effect on facial grooming suggests that the reduction of
DNIC-induced analgesia does not result from a direct inhibition
of NK1 receptors in the spinal trigeminal nucleus. Similarly, in
anesthetized animals, intrathecal administration of the NK1 receptor antagonist, L703606, does not attenuate the jaw opening reflex
[54]. Thus, the activation of NK1 receptors on projection neurons in
the lumbar spinal cord is essential for DNIC-induced analgesia.
The conclusion that activation of NK1 receptor-expressing spinal
projection neurons is essential for triggering DNIC is consistent
with the previous results using c-Fos expression. In this histochemistry paradigm of DNIC, c-Fos expression in the cervical spinal cord
produced by noxious heat of the forepaw is inhibited by a conditioning stimulus, noxious heat of the hindpaw. Such inhibitory effect is suppressed following selective ablation of NK1 receptorexpressing lamina I/III neurons in the lumbar spinal cord, using a
substance P and saporin conjugate [52], in NK1/ mice and in wild
type mice treated with the NK1 receptor antagonist, RP67580 [5].
8.2. Parabrachial neurons are involved in the ascending part of the
loop underlying DNIC
Muscimol microinjection has no effect on the general properties
of recorded neurons. Neither spontaneous activity nor C-fiberevoked responses changed after muscimol microinjection, whether
it was in or outside the PB area. This suggests that the PB area does
not participate directly in the tonic descending inhibitory controls.
This conclusion is consistent with the previous electrophysiological data in the rat [7].
But microinjection of muscimol into the PB area strongly reduces DNIC. This effect is due to the inactivation of the very PB area
rather than that of other neighboring areas – to which muscimol
might have diffused. Indeed, the injected volumes (i.e., diameter
<100 lm) were relatively small compared with the size of the PB
area, and doses were low compared with those used previously
[10,28,42,44]. Moreover, when it was injected outside the PB area,
muscimol did not alter DNIC. Finally, muscimol microinjection
attenuated much more DNIC triggered by the right hindpaw – i.e.
contralateral to the inactivated PB area – than that by the left hindpaw – i.e. ipsilateral to the inactivated PB area. This is consistent
with the previous electrophysiological and anatomical studies.
On the one hand, the surrounding PB area is devoid of nociceptive
neurons and does not receive direct projections from the spinal DH
[3,4,23,55]. On the other hand, direct spinal projections to the PB
area originate predominantly from the contralateral spinal cord
[3,23]. Accordingly, ascending pathways involved in triggering
DNIC are mainly crossed [56].
Although the present experiments cannot indicate which specific subregion of the PB area is involved in DNIC, it seems reasonable to suggest that it is the lateral PB area. Indeed, the most
effective microinjection sites in this study were located in the lateral PB area. This is also consistent with anatomical evidence
showing that the lateral PB area receives direct inputs from spinal
DH lamina I/III neurons [3,23,55]. Either single-unit recordings
[1,4] or immunostaining for c-Fos expression [6,27,36] has demonstrated that spinoparabrachial neurons respond to noxious stimuli.
And lateral PB neurons are nociceptive-specific and able to encode
noxious stimulus intensity [1,6].
However, Bouhassira et al. [7] reported that an ibotenic acid lesion of the PB area does not attenuate DNIC. Interestingly, this
study used a chronic irreversible inactivation of the PB area,
whereas we used an acute pharmacological one. Thus, a parsimonious explanation of these contradictory results is that CNS might
have quickly adapted after PB lesion. Indeed, DNIC likely involve
other neuronal circuits. Inactivation of the PB area strongly
reduced but did not totally suppress DNIC. One candidate is
the SRD. Lesion of the SRD also reduces DNIC (by 40%; 8).
O. Lapirot et al. / PAIN 142 (2009) 245–254
Interestingly, deep dorsal horn laminae V–VI provide the main input to the SRD. One possibility is that there would be at least two
pain pathways involved in the ascending part of the loop underlying DNIC: the spinoparabrachial tract and the spinoreticular tract,
originating from superficial and deep dorsal horn, respectively.
8.3. Projections from the PB area and the descending part of the loop
underlying DNIC
Retrograde tracing reveals that neurons in the PB area of the rat
can project directly to the MDH. Labeled cell bodies are mainly in
the ipsilateral Kölliker-Fuse and to a lesser extent in the external lateral subnucleus of PB area, primarily in the caudal third of PB area.
Importantly, the PB area contralateral to the injection site is almost
devoid of labeling. Similar results have been previously obtained in
the rat [25,59] and cat [53]. It is noteworthy that fluorogold has been
injected in the MDH and not in the Sp5O in which WDR neurons
were recorded. But according to Yoshida et al. [59], PB neurons projecting directly to all subnuclei of spinal trigeminal nucleus – including MDH and Sp5O –have a similar localization.
We show here that inactivation of the left PB area – i.e. contralateral to the recorded site – decreases DNIC on WDR neurons recorded
in the right Sp5O. That there is no direct projection from the contralateral PB area to the spinal trigeminal nucleus [59] indicates that
the left PB area cannot directly affect neurons in the right spinal trigeminal nucleus. Rather, descending control pathways likely involve indirect projections from the PB area to the trigeminal
sensory nuclear complex. Indeed, the lateral PB area sends extensive
projections to multiple areas throughout the CNS that are known to
regulate, directly or indirectly, the nociceptive processing through
various relay nuclei in the brainstem, such as the periaqueductal
gray and the rostroventral medulla [47] These descending projections might include the hypothalamus and amygdala that are believed to have key roles in the modulation of pain [33].
It is important to note that whether the descending part of the
loop underlying DNIC also involves ipsilateral PB-spinal trigeminal
nucleus projections cannot be excluded. However, sites that were
effective in inhibiting DNIC were distributed along the whole
rostrocaudal length of PB area, whereas PB-spinal trigeminal nucleus projecting neurons are almost exclusively concentrated in
the caudal third of PB.
9. Conclusion
In summary, the present results suggest that lamina I/III spinoparabrachial neurons that express the NK1 receptor and PB neurons are involved in the ascending part of the loop underlying
DNIC and that the descending pathway for DNIC might include
indirect projections to the spinal or medullary dorsal horn.
Acknowledgments
We thank A.M. Gaydier for secretarial assistance and Dr. A.
Alloui for technical help during behavioral experiments. This work
was supported by funding from Institut National de la Santé et de
la Recherche Médicale (INSERM), Fondation Benoit, Université
Clermont1 (France) and CHU Clermont-Ferrand (France). None of
the authors have any conflict of interest to declare.
R.C. is supported by a research study grant from Tunisian
Government.
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