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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.2009.01.015 246 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 247 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, 248 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 250 O. Lapirot et al. / PAIN 142 (2009) 245–254 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. 252 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. 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