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Dependency of nociception facilitation or inhibition after periaqueductal gray matter stimulation on the context
Behavioural Brain Research 214 (2010) 260–267
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Dependency of nociception facilitation or inhibition after periaqueductal gray matter stimulation on the context M.A. Martins, L. De Castro Bastos, N.E.B. Melo, C.R. Tonussi ∗ Department of Pharmacology, Federal University of Santa Catarina, Florianópolis, SC, 88040-900, Brazil
a r t i c l e
i n f o
Article history: Received 16 October 2009 Received in revised form 17 May 2010 Accepted 23 May 2010 Available online 1 June 2010 Keywords: Pain modulation PAG Stress Hyperalgesia Attention Anxiety
a b s t r a c t Anxiety and/or fear can alter the nociceptive response in humans and animals. Slight stimulation of the dorsal periaqueductal gray matter (DPAG) produced anxiety/fear-related behaviour and hyponociception in escapable, non-anxiogenic nociceptive models. Our aim was to investigate the role of the DPAG in models of persistent, anxiogenic nociception. GLY (1, 10, 20, and 80 nmol/0.3 l/60 s) was injected into the DPAG of rats, 5 min before formalin (2%/50 l) injection either into the knee-joint or hind paw. In the knee-joint incapacitation test, GLY caused hypernociception at lower doses and hyponociception at higher doses. In the paw shacking test, GLY produced only hypernociception with the higher dose. Coinjecting GLY with 7-chlorokynurenic acid (7-CLK) or (+/−)-3-amino-1-hydroxy-2-pyrrolidone (HA-966) completely prevented the GLY effects in incapacitation and paw shacking tests, respectively. GLY injections outside the periaqueductal gray matter (PAG) did not change the nociception. Behavioural analysis indicated that formalin paw injection produced higher stress signals than knee-joint injection, as diminished exploratory behaviour, and stereotypy. The results suggest that activation of the DPAG through the GLYB /NMDA receptor is able to produce either facilitation or inhibition of nociception depending on the nociceptive context. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Anxiety, like pain and fear, plays an adaptive role as an alarm that responds to danger or harm, as in a defensive system. Since these alarms serve similar protective functions it is predictable that they might interact and also elicit collective effects. Indeed, chronic pain states have been found to predict more severe anxiety symptomatology [51], and vice versa, i.e. more anxious patients reported more pain [10,29]. Basic studies have confirmed the relationships between anxiety and nociception, although they have produced contradictory results. For example, a variety of stressful environmental events can produce hyponociception [6,15,23,34,46], but anxiogenic stress models (acute, subchronic and chronic) can also produce hypernociception [1,12,13,20,42,52]. The periaqueductal gray matter (PAG) is a midbrain structure involved in pain, fear, and anxiety modulation [5]. The dorsal columns (DPAG) are proposed to integrate behavioural and autonomic expression of defensive reactions, and nociception similar to that seen in stressful situations (see [3]). DPAG-elicited hyponociception, however, may not be proportional to the aversion-like reactions elicited by its activation since the subtle aversive effect
∗ Corresponding author. Tel.: +55 48 3721 9491x218; fax: +55 48 3337 5479. E-mail address: [email protected] (C.R. Tonussi). 0166-4328/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2010.05.035
produced by the activation of the glycine site at the NMDA type receptor (GLYB /NMDA) in the DPAG [8] has been observed to produce an intense and long-lasting hyponociception in the tailflick test [25]. On the other hand, prostaglandin and capsacin DPAG injections have even been shown to facilitate nociception [30,35]. Slight stimulation of the DPAG is thought to enhance the aversive value of the potential sources of threat to an animal [8], and consequently the animal attention should be directed toward that which seems more threatening at a given time. Different animal models of nociception may exert different aversive effects on the subject [22], i.e. escapable nociceptive tests, such as the heatinduced tail-flick, may have low potential to produce fear/anxiety since the animal has control over the nociception intensity to which it is submitted. On the other hand, inescapable (foot-shock) and/or persistent (formalin) nociceptive models may proportionally be more aversive to the animals. The same reasoning may be followed whether the nociception source is external (from the environment) or internal (from the body). In this case, the possibility exists that the nociception modulation due to the same kind of DPAG stimulation may be a result of a decision between which threat to the animal is more severe (or aversive) in a given situation. The aim of this study was to verify the effect of DPAG stimulation on two models of persistent, inescapable nociception, which differ mainly with respect to the nociceptive source, i.e. subcutaneous (external) or intra-articularly (internal) injected formalin in rats.
M.A. Martins et al. / Behavioural Brain Research 214 (2010) 260–267 2. Methods 2.1. Animals Male Wistar rats (250–350 g) were housed at 21 ± 1 ◦ C under a regular 12/12 h light/dark cycle (lights on 7:00 a.m.). Food and water were available ad libitum. All behavioural tests were performed between 7:00 a.m. and 2:00 p.m. Animal care and handling procedures were in accordance with the ethical guidelines of the International Association for the Study of Pain [16], and also approved by the Ethics Committee for Animal Use of the Federal University of Santa Catarina. All efforts were made to reduce both the animal number and suffering during the experiments. 2.2. Surgical procedure A stainless still guide cannula (length = 13 mm, o.d. = 0.7 mm) was stereotaxically implanted (Paxinos and Watson Atlas [37] coordinates: AP = − 7.6 mm and ML = +1.9 mm from bregma; DV = −2.0 mm from skull and at an angle of 22◦ from the sagittal plane) with its tip intended to be above the dorsal periaqueductal gray (DPAG) area. The cannula was maintained in this position embedded in an acrylic cement cap anchored by two mini-screws fixed to the skull. The surgery was carried out 7 days before the experiment, and the animals were anaesthetised with 1.5 ml/kg of a xylazine (20 mg/ml; Rompun® , Bayer, Brazil)/ketamine (100 mg/ml; Dopalem® , Vetbrands, Brazil) 1:1 mixture. After surgery the rats received an intramuscular injection of Pentabiotic® (0.2 ml/rat; Fort Dodge, Brazil), and were allowed to recover from the anaesthesia in a silent and warm room. In addition, 2% lidocaine with vasoconstrictor was infused (0.2 ml) under the skin before its excision for the skull exposition. Intra-periaqueductal gray microinjections were administered through a thin dental needle (0.3 mm o.d.) which extends 3.25 mm beyond the guide cannula tip. A volume of 0.3 l was injected over a period of 60 s using a hand-driven 5.0 l Hamilton microsyringe. The movement of a small air bubble inside the PE-10 polyethylene tubing, interposed between the upper end of the dental needle and the microsyringe, confirmed successful drug injection. The animals randomly received a single DPAG microinjection of glycine (GLY, 10, 20, 80 and 100 nmol), phosphate buffered saline (PBS) or dimethylsulfoxide (DMSO in PBS, 5%, v/v). The GLYB receptor antagonist, 7chlorokynurenic acid (7CLK; 10 nmol) or (+/−)-3-amino-1-hydroxy-2-pyrrolidone (HA966; 10 nmol) was applied alone or combined with glycine in the same injection, 5 min before formalin injections. At the end of the experiments, the animals were deeply anaesthetised with 2% xylazine and 15% chloral hydrate (0.2 and 1.0 ml/rat) and perfused intracardially with physiological saline (0.9% NaCl) followed by formalin solution (10%). In order to mark the injection site 0.3 l of Evans Blue (0.5%) was then applied through the guide cannula. The brains were removed and stored in 10% formalin solution for posterior histological analysis. Frozen sections (50 m) were obtained using a cryostat, mounted on glass slides and stained using the Giemsa (Sigma–Aldrich, USA) method for microscopic identification of the injection site. Only the rats for which the microinjection site was located within the DPAG were considered. 2.3. Incapacitation test The formalin-induced knee-joint incapacitation model is presented elsewhere [26], as a modification of that described in [47]. In this test, up to three rats are placed simultaneously on a revolving cylinder (30 cm diameter; 3 rpm), and a computerassisted device registers the total time that the ipsilateral hind paw to the stimulated knee-joint fails to make contact with the cylinder surface (i.e. paw elevation time or PET) during a 60-s period of enforced walking. Therefore, PET is an objective measure of the guarding behaviour elicited by articular nociception. The injection site was first shaved and treated with an iodine alcohol antiseptic solution. The animals were gently restrained in a supine position by the hands of the experimenter, and intra-articular injections of formalin (2%, 50 l) were quickly performed with a 30-gauge needle. The concentration of formalin was carefully considered to give a submaximal behavioural response in order to allow that increases or decreases in such a response could be detected. The PET was recorded just after formalin injection and every 5 min thereafter, for a 60-min test period. Between each 60-s recording period, the animals remained on the stopped cylinder, generally exhibiting grooming behaviour or apparently sleeping. There was low exploratory interest probably because the animals were trained one day before on the revolving cylinder in order to spontaneously walk to the top each time the movement started. 2.4. Paw formalin test The other formalin test used was the classical test described by Dubuisson and Dennis [9] with modifications in the registering paradigms. Firstly, one day before the experimental session, the animals were allowed a 20-min habituation period in the observation box. The observation box was a transparent plexiglass cubic chamber (edges = 29 cm) with mirrors angled on the lateral and back walls, adjusted accordingly to allow unhindered observation of the animal’s paws. Immediately after a 2% formalin (50 l) subcutaneous plantar injection in the right hind paw, the animals were observed for the nocifensive response. The numbers of shakes, lifting, and licking/biting of the injected paw were counted and summed as the number of
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nocifensive events (NNE) over 60 min in 5-min cluster periods. Lifting and licking behaviours were sometimes exhibited in long bouts, i.e. more than 3 s, before the return to the resting position. In these cases, the first 3 s of the event was counted as one event, plus one event when the paw returned to the resting position. 2.5. Behavioural evaluation of the animals undergoing paw or knee-joint formalin-induced nociception Apart from the experiments using PAG injections, another group of animals were used for the evaluation of the behavioural signs of stress induced by each kind of nociceptive stimulus. The animals were divided in two main groups, those receiving intraplantar injections and those receiving knee-joint injections. For each injection site, the same number of animals were allocated to receive 50 l of physiological saline or 2% formalin. The experiments were designed such that we had always one paw injected and one knee-joint injected animal being recorded at the same time, irrespective they had received saline or formalin. Immediately after the injection, the animals were put in individual transparent plexiglass boxes (like above) placed side-by-side, in a quiet room and with a light source just above equally illuminating the boxes. The experimenters did not remain in the room, and the sessions were video-recorded for later behavioural analysis. Behaviours chosen for analysis were the time spent in exploratory activity (EA), and the number of occurrences of the tail biting, licking of the floor, and faecal pellet manipulation events. For the adequate pairing of these behaviours with the primary nociceptive behaviour, the NNE were taken for both groups. However, in this case, since the animals were observed in the plexiglass box, the knee-joint injected animals cannot be evaluated in the automated incapacitation test. The same procedure for the evaluation of the NNE used in the traditional paw shaking test above, was also used in the knee-joint test. 2.6. Drugs and vehicles Glycine (Sigma–Aldrich, Brazil) was dissolved in PBS solution. HA966 ((+/−)3-amino-1-hydroxy-2-pyrrolidone) and 7-chlorokynurenic acid (Tocris, USA) were dissolved in PBS and PBS with DMSO (5%, v/v), respectively. Formalin (formaldehyde 37% – Merck AG, Germany) was diluted to 2% in sterile physiological saline, considering the initial concentration as 100%. 2.7. Statistical analysis All statistical analyses were carried out using Graphpad Prism version 5 software. Results are expressed as mean ± S.E.M. (standard error of mean). Multiple comparisons among means in the first phase of formalin response (one single time point) were made using simple ANOVA, but in the second phase (time-course curves) were made using ANOVA for repeated measures. Both were followed by the Dunnett or Tukey post-hoc test. The time-course period analysed were indicated in each case. The minimum significance level considered was P < 0.05.
3. Results Glycine (GLY) microinjection in caudal sites of the dorsal PAG (Fig. 1A and B), at the doses of 10 and 20 nmol, induced a significant increase of the paw elevation time (PET) suggesting hypernociception during the second phase (t = 5–30 min) of the formalin-induced incapacitation (ANOVA; F(3,20) = 6.15). On the other hand, the higher doses of GLY (80 and 100 nmol) inhibited incapacitation in the first (t = 0 min; ANOVA; F(2,22) = 6.04) and second phases (t = 5–30 min; ANOVA; F(2,18) = 29.07), suggesting a hyponociceptive effect. GLY at the dose of 1 nmol did not affect the PET of animals (Fig. 2A and B). Similarly, GLY injections (10, 20 and 80 nmol) that reached immediately dorsal to the DPAG, in the superior colliculus, did not change the formalin response (Fig. 3). Due to the intriguing opposite effect observed with GLY in low and high doses, we decided to compare the effect of DPAG glycine injection in the traditional 2% formalin-induced paw nociception. In this test, the higher dose (80 nmol) induced significant increase of the NNE in the second phase (t = 10–60 min; ANOVA; F(2,30) = 16.95). Interestingly, in the lower dose (20 nmol), glycine did not modify the NNE induced by formalin (Fig. 4). The hypernociceptive effect induced by 80 nmol of GLY into DPAG was blocked by the co-injection of HA966 (10 nmol), an antagonist of the glycine site at the NMDA receptor (GLY-B). HA966 alone did not change the formalin response per se (ANOVA; F(3,29) = 8.76) (Fig. 5). In the knee-joint test, both effects produced by glycine (10 or 80 nmol) were prevented by 7-chlorokynurenic acid (7CLK; 10 nmol), another GLY-B antagonist. The co-administration of
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Fig. 1. (A) Photomicrograph of a slide of the midbrain stained by Giemsa (Sigma–Aldrich, USA) from a representative subject showing an injection site (arrowheads) in the DPAG column section. Section corresponds to −7.30 to −8.00 mm from the bregma in the Paxinos and Watson atlas [37]. (B) Diagrams of rat coronal sections, redrawn from the Paxinos and Watson atlas. The microinjection sites (black dots) as labelled by Evans Blue (0.5%) injection in the dorsal parts of the PAG. The number of points in the figures is less than the total number of rats used because of several site overlaps.
Fig. 2. Hypernociceptive and hyponociceptive effects induced by glycine microinjections into the DPAG in knee-joint incapacitation test induced by formalin. Glycine (GLY) at doses of 1, 10, 20, 50, 80 and 100 nmol/0.3 l/60 s/site was applied to the DPAG 5 min before formalin injection (2% i.a.). The paw elevation time (PET) was evaluated for 60 s every 5 min over a period of 60 min. Data are presented as mean ± S.E.M. Control animals received only PBS (0.3 l/60 s/site). P1 and P2: phases 1 (0–5 min) and 2 (5–30 min) respectively. *P < 0.05; ***P < 0.001 (P1: one-way ANOVA; P2: one-way ANOVA for repeated measures, both followed by the Tukey test).
Fig. 3. Glycine outside the DPAG did not change the articular nociception. Glycine at doses of 10, 20 and 80 nmol/0.3 l/site (GLY10, GLY20, GLY80) was applied to the deep layer of the Superior Colliculus (SC) 5 min before the incapacitation test induced by formalin (2% i.a.). The paw elevation time (PET) was evaluated for 60 s every 5 min over a period of 60 min. Data are presented as mean ± S.E.M. ANOVA for repeated measures followed by Tukey’s test did not reveal significant differences for GLY groups compared with the control group (control, 0.3 l/site). P1 and P2: phases 1 (0–5 min) and 2 (5–60 min), respectively. B: baseline values; n: animals for each group.
Fig. 4. Hypernociceptive effect induced by glycine microinjections into the DPAG in the formalin hind paw test. Glycine (GLY) at doses of 20 and 80 nmol/0.3 l/60 s/site was applied to the DPAG 5 min before formalin injection (2% s.c.). The total number of nocifensive events (NNE) was recorded in 5-min cluster periods over 60 min. Data are presented as mean ± S.E.M. Control animals received only PBS (0.3 l/60 s/site). P1 and P2: phases 1 (0–5 min) and 2 (10–60 min), respectively. ***P < 0.001 (one-way ANOVA for repeated measures followed by the Dunnett test).
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was considered (t = 0). The paw formalin groups (Fig. 7, left column) have shown decreased EA time (Fig. 7C and D: ANOVA for repeated measures, F(3,44) = 7.27. P < 0.001, between formalin and saline paw injected groups, and P < 0.05, between formalin and saline kneejoint injected groups, with Tukey post-hoc test), but increased NLF events (Fig. 7E and F: ANOVA for repeated measures, F(3,44) = 24.39. P < 0.001, between formalin and saline paw injection groups, with Tukey post-hoc test) and NTB (Fig. 7I and J: ANOVA for repeated measures, F(3,44) = 8.51. P < 0.05, between formalin and saline paw injection groups, with Tukey post-hoc test) when compared to the knee-joint group. The faecal pellet manipulation behaviour was also higher in the paw injected group immediately after the formalin injection (Fig. 7G and H: t = 0 min, ANOVA, F(3,28) = 12.24. P < 0.01, between formalin and saline paw injection groups, with Tukey post-hoc test). Fig. 5. Glycine-induced hypernociceptive effect was prevented by HA966. The number of nocifensive events (NNE) of rats which received glycine alone (GLY; 80 nmol/0.3 l/site), vehicle solution (PBS, 0.3 l/site), HA966 (HA; 10 nmol/0.3 l site) alone or mixed with glycine (GLY80 + HA, 0.3 l/site) were recorded 5 min after DPAG injection, and every 5 min thereafter for a period of 60 min. Data are presented as mean ± S.E.M. P1 and P2: phases 1 (0–5 min) and 2 (10–60 min), respectively. ***P < 0.001 (one-way ANOVA for repeated measures followed by the Dunnett test).
7CLK blocked, significantly, the hypernociception in the second phase (ANOVA; F(3,40) = 13.81) and the hyponociception in the first (ANOVA; F(3,23) = 8.29) and second phases (ANOVA; F(3,40) = 14.66) (Fig. 6A and B). Although HA966 was likewise observed to antagonize glycine effects, in this test, it also presented a hypernociceptive effect per se (data not shown). This effect may be due to a reported partial agonistic effect of the drug, and in this case was consistent with the hypernociceptive effect found for the lower glycine dose. Because DPAG has been reported to be involved in the activation of defensive behaviours related to aversive situations, the next set of experiments were designed to behaviourally evaluate the relative aversiveness between formalin injected in the paw and in the knee-joint. By observing the animals, as explained above, the number of the tail biting (NTB), licking of the floor (LF), and faecal pellets manipulation (FPM) behaviours, appeared significantly higher to the animals undergoing paw nociception. On the other hand, the time spent in the exploratory behaviour (EA) appeared significantly lowered in the paw formalin-injected group than in the knee-joint injected animals. In Fig. 7, the behaviours elicited after paw injection were separated from those elicited after kneejoint injection, for the better visualisation. However, statistical analyses were performed taking together all the whole curves of each behaviour. Exception made to the FPM behaviour, for which was found statistical difference only when the first five minutes
4. Discussion The main findings of this study were that glycine microinjection into the dorsal PAG produced opposite effects, i.e. hyper and hyponociception, in two models of persistent nociception. Both hypo and hypernociceptive effects were blocked by 7chlorokynurenic acid or HA966, consistent with the proposal that the GLYB site at the NMDA receptor plays a role in their mediation. The inhibitory role of the PAG in nociception and on pain has been established, as the focal electrical stimulation in this particular structure triggers hyponociception in animals [28,43] and powerful analgesia in humans [27,55]. In addition, our present and previous [25] findings that glycine-induced hyponociception was reversed by GLYB site antagonists is in agreement with data available in the literature implicating glutamate and its NMDA-type receptor as important mediators of the hyponociception elicited by this structure [17,19,33]. However, the same glycine concentration that decreased PET in the present study, which is interpreted as a hyponociceptive effect, increased the nocifensive behaviour elicited by formalin in the hind paw, which is interpreted as a hypernociceptive effect. On the other hand, lower concentrations of glycine, which produced no effect in formalin-induced paw flinches and the tail-flick test, increased formalin-induced articular incapacitation. Recent studies have shown that brainstem modulatory systems can, indeed, exert bidirectional control and that nociception facilitation may even be its most important function. However, determining how this system is recruited to either inhibit or facilitate nociception under different conditions is an important challenge in terms of understanding descending modulation, and
Fig. 6. Glycine-induced hyponociceptive and hypernociceptive effects were prevented by 7-chlorokynurenic acid. The paw elevation time (PET) for rats which received glycine alone (GLY; 10 or 80 nmol/0.3 l/site), vehicle solution (PBS, 0.3 l/site), 7-chlorokynurenic acid (7CLK; 10 nmol/0.3 l site) alone or mixed with glycine (GLY10 + 7CLK or GLY80 + 7CLK, 0.3 l/site) were recorded 5 min after DPAG injection, and every 5 min thereafter over a period of 60 min. Data are presented as mean ± S.E.M. P1 and P2: phases 1 (0–5 min) and 2 (5–30 min), respectively, **P < 0.01; ***P < 0.001 (P1: one-way ANOVA; P2: one-way ANOVA for repeated measures, both followed by the Dunnett test).
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Fig. 7. Behavioural evaluation of the animals undergoing paw or knee-joint formalin-induced nociception. EA: time spent in the exploratory activity; NTB: number of tail biting events; NLF: the number of licking of the floor events; FPM: number of faecal pellet manipulation events; NNE: number of nociceptive behaviour events. Each behaviour was analysed after 2% formalin injection either into the paw (left column) or into the knee-joint (right column), and clustered in 5-min intervals. Control groups received physiological saline instead of formalin. **P < 0.01, ***P < 0.001 (FPM: one-way ANOVA; EA, NTB, NLF: one-way ANOVA for repeated measures, both followed by the Tukey test).
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proposing therapies [see [50] for review). For example, the rostral ventromedial medulla (RVM) was found to be involved in hyperalgesia associated with acute naloxone-precipitated withdrawal, prolonged opioid administration, prolonged heating of the tail and illness induced by endotoxin administration [21,32,49,54]. The RVM also contributes to hyperalgesia and allodynia in inflammatory and neuropathic models, including the formalin test, mustard oil secondary hyperalgesia, and spinal nerve ligation [14,40]. Importantly, this enhancement of nociceptive transmission is selective for certain kinds of inputs, because the RVM seems not to be required for primary or secondary hyperalgesia surrounding a surgical incision site [39] or after long-lasting focal inflammation produced by complete Freund’s adjuvant. Indeed, descending inhibition from the RVM is enhanced in complete Freund’s adjuvant-treated animals [44]. Notwithstanding, there are several differences among these studies which hinders the proposition of a reconciling hypothesis to explain these results, and additional attentional interferences due to brainstem manipulation cannot be discarded. In contrast to the descending inhibition, the involvement of PAG in the nociceptive facilitation circuitry, although suggested, is less clear. Differing from earlier observations after PAG electrical and chemical stimulation, which induced hyponociception often associated with intense aversion-like behaviours, recent investigations have shown that capsaicin and prostaglandin injections into the dorsal PAG can produce hypernociception in formalin and in a fastheating tail-flick models, respectively [30,35]. Similarly to RVM, dorsal PAG projections can also support facilitatory and inhibitory effects on nociception since they indirectly target RVM via a pontine catecholaminergic nucleus, such as the locus coeruleus [4]. Furthermore, locus coeruleus direct catecholaminergic projections to the spinal cord may also produce nociception facilitation or inhibition [18,36]. In an attempt to explain this dual descending circuitries effect, it has been proposed that the nociceptive information is differentially facilitated or inhibited, respectively, depending on whether it is conveyed by A- or C-fibres [24,53]. This conception may fit well to behavioural models based on fast (A-fibres) or slow heating (C-fibres). However, it is difficult to reconcile this idea with the formalin-based models which are mediated by a combination of A- and C-fibre-conveyed action potentials [41], and, indeed, as we have shown here, the same PAG stimulation produced opposite effects in two formalin-based models. Another possible explanation for our results may be related to the intensity of the stimulation. Low intensity electrical stimulation, or low concentrations of chemical substances, in the RVM was seen to cause nociceptive facilitation. On the other hand, high intensity electrical stimulation, or higher concentrations of glutamate or neurotensin, inhibited nociception [48,56]. Similarly, low concentrations of glycine in the PAG facilitated formalin incapacitation, while higher concentrations inhibited this response. However, in the tail-flick test glycine produced only hyponociception even at lower doses [25] than that which reversed incapacitation here, but the higher glycine concentration facilitated the formalin response in the hind paw. Thus, the idea that facilitation and inhibition could be due to the intensity of the PAG activation does not seem to offer a satisfactory explanation. DPAG seems to be pivotal for processing of fear and anxiety [5]. Glycine injection into the DPAG has been shown to produce a progressive attention (or expectancy) to the unknown surrounding environment that may appear potentially more threatening, although it did not produce overt defensive behaviours related to highly stressful conditions, such as exophthalmia, micturition, running, jumping, etc. For example, such procedure has been described to enhance the anxiety-like behaviour observed in specific elevated maze models [8,45]. Different types of anxiogenic stress models
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have been shown to produce hypernociception in persistent nociceptive models, but also to increase the threshold response in phasic nociceptive tests [1,12,13,20,42,52]. One important aspect usually ignored in most studies on nociception and stress is the relative aversion produced by the nociceptive test and the stress model. Furthermore, procedures that will enhance the animal’s anxiety may produce different effects on the aversive component of nociception and other components of aversiveness produced by the experimental manipulation, but not considered in the study. At any given time, the animals should decide among what source of stress may be more threatening, including the nociceptive source. The tail-flick model with a slow heating rate, as an escapable nociceptive model, is expected to be less stressful than formalin-induced inescapable persistent nociception. In addition, formalin injected into the paw seemed to be even more stressful than when injected into the knee-joint, since after injection into the paw the animals exhibited decreased overall exploratory activity, and episodes of stereotypy as the compulsive licking of the floor and manipulation of the faecal pellets. Such behaviours were not observed in the knee-joint injected animals, and may be important indicators of the emotional aversive component elicited by the nociception arising from the paw tissue. One possibility to explain these differences could be the overall nociception perceived by the animals, however it is not easy to compare the relative nociception induced by these two models. The incapacitation registering on the revolving cylinder suggested that nociception elicited by formalin in the knee-joint could have shorter time-course than that elicited by the paw formalin model. However, in the paw formalin model, the animals were not obliged to execute a task, being observed freely in the box. The behavioural evaluation made in the plexiglass box, indicated that both kinds of nociceptive inputs have similar timecourse. Thus, we cannot affirm that paw formalin model hurts longer than knee-joint model. But in spite of a similar time-course, paw formalin-injected animals have shown a high frequency of the biting of the tail behaviour, which was not shown by the knee-joint injected animals. This behaviour suggests nociceptive sensation from the tail, which is consistent with secondary hypernociception. Secondary hypernociception is clearly a function of the intensity of nociceptive input from the primary site of nociception [50]. The presence of such kind of nociceptive behaviour in the paw injected animals, but not in the knee-joint injected ones, could suggest that the intensity of nociception is higher in the paw model. Again, however, this cannot be conclusive because the area of the secondary hypernociception elicited by knee-joint noxious input may be different from that elicited by the paw noxious inputs. Finally, formalin-elicited nociception in the footpad may be more aversive than that induced in the knee-joint, because it could be suggesting to the animal an external attack, against which it must pay all attention. Whatever the explanation, they are all consistent to the idea that paw formalin injection will attract more the animal attention, causing more aversion. Our working hypothesis to explain our present and previous findings is that the increased anxiety/fear produced by DPAG stimulation will enhance nociception when nociception is the major source of anxiety/fear, i.e. in persistent models, but will inhibit it when the major source of anxiety/fear is not the nociceptive model, as in phasic, escapable, nociceptive tests [2,7,31,38]. This may explain why the higher dose of glycine caused hypernociception in the paw formalin model, in contrast to the previously observed hyponociception in the heat-induced tail-flick test [25]. In this case, glycine injected into the DPAG enhanced the attention to factors which were more aversive to the animal, i.e. the persistent nociception, thus enhancing nociception. Supporting this point, an anti-aversive dose of a cannabinoid agonist was shown to reduce the nocifensive response induced by the paw formalin injection [11]. On the other hand, since in phasic, escapable noci-
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ceptive tests, such as the tail-flick test, nociception is gradual, and not persistent, the main focus of the animal’s attention is not likely to be directed toward the tail, but toward some other potentially threatening object – e.g. the experimenter’s hands, surrounding environment, etc. Glycine, in this case, would be diverting the attentional focus away from the tail, thus causing hyponociception. The incapacitation induced by formalin, differently from the other models, presented both glycine effects, i.e. hyper and hyponociception. Interestingly, the hyponociception was observed with the higher glycine dose that caused hypernociception in the paw formalin model, thus excluding the possibility that hyponociception was due to motor impairment. As shown above, formalin injection into the knee-joint did not produce the overt signs of stress observed after injection into the paw, suggesting that the articular model demands less attention toward the nociceptive site than the paw model. In addition, in the knee-joint model the animals are also focused on keeping themselves on the cylinder. Thus, it is conceivable that the animals will be alternating their attention between the walking on the cylinder and the guarding of the painful joint, and this context could be responsible for the observation of the two opposing effects on nociception, depending on the glycine dose injected. In this new, distinct, context the facilitation of DPAG glutamate transmission by glycine seems to be showing that, at least this PAG structure may be functionally plastic in the way it integrates the several stress sources for the nociception modulation, probably determining how the animal will cope with nociception. In conclusion, the results here presented raised the hypothesis that, at least in the dorsal PAG, facilitation or inhibition may be a result of the emotional context in which the nociception occurred, rather than a stereotyped response. This hypothesis needs to be further supported by experiments designed under these predictions, and indeed will be very interesting to extend this idea to other structures involved in nociception modulation, trying to determine whether this functional plasticity is level dependent, and to what extent. For the basic research on nociception in a broader sense, the present results also suggest that the conclusions on the efficacy of analgesic candidates should take into account the contextual aspects in which the nociceptive reflex was observed. Acknowledgements This study was supported by the Brazilian funding agencies CAPES, CNPq and FAPESC (Pronex). MAM and LCB were recipients of doctoral fellowships from CNPq. References [1] Andre J, Zeau B, Pohl M, Cesselin F, Benoliel JJ, Becker C. Involvement of cholecystokininergic systems in anxiety induced hyperalgesia in male rats: behavioral and biochemical studies. J Neurosci 2005;25:7896–904. [2] Arntz A, Dreessen L, De Jong P. The influence of anxiety on pain: attentional and attributional mediators. Pain 1994;56:307–14. [3] Bandler R, Shipley MT. Columnar organization in the midbrain periaqueductal gray: modules for emotional expression? Trends Neurosci 1994;17:379–89. [4] Beitz AJ. The nuclei of origin of brainstem serotonergic projections to the rodent spinal trigeminal nucleus. Neurosci Lett 1982;3:223–8. [5] Behbehani MM. Functional characteristics of the midbrain periaqueductal gray. Prog Neurobiol 1995;46:575–605. [6] Bellgowan PS, Helmstetter FJ. Neural systems for the expression of hypoalgesia during non associative fear. Behav Neurosci 1996;110(4):727–36. [7] Bushnell MC, Duncan GH, Dubner R, Jones RL, Maixner W. Attentional influences on noxious and innocuous cutaneous heat detection in humans and monkeys. J Neurosci 1985;5(5):1103–10. [8] Carobrez AP, Teixeira KV, Graeff FG. Modulation of defensive behavior by periaqueductal gray NMDA/glycine-B receptor. Neurosci Biobehav Rev 2001;25:697–9. [9] Dubuisson D, Dennis SG. The formalin test: a quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats. Pain 1977;4(2):161–74. [10] Ferguson R, Ahles TA. Private body consciousness, anxiety and pain symptom reports of chronic pain patients. Behav Brain Res 1998;36(5):527–35.
[11] Finn DP, Jhaveri MD, Beckett SRG, Roe CH, Kendall DA, Marsden CA, et al. Effects of direct periaqueductal grey administration of a cannabinoid receptor agonist on nociceptive and aversive responses in rats. Neuropharmacology 2003;45:594–604. [12] Gameiro GH, Gameiro PH, Andrade A, da S, Pereira LF, Arthuri MT, et al. Nociception- and anxiety-like behavior in rats submitted to different periods of restraint stress. Physiol Behav 2006;87(4):643–9. [13] Geerse GJ, Van Gurp LCA, Wiegan TVM, Stam R. Individual reactivity to the open-field predicts the expression of stress-induced behavioural and somatic pain sensitization. Behav Brain Res 2006;174:112–8. [14] Heinricher MM, Pertovaara A, Ossipov MH. Descending modulation after injury. In: Dostrovsky JO, Carr DB, Koltzenburg M, editors. Progress in pain research and management. Seattle: IASP Press; 2003. p. 251–60. [15] Helmstetter. FJ Stress-induced hypoalgesia and defensive freezing are attenuated by application of diazepam to the amygdala. Pharmacol Biochem Behav 1993;44(2):433–8. [16] International Association for Study of Pain (IASP). Ethical guidelines for investigation of experimental pain in conscious animals. Pain 1983;16:109– 10. [17] Jacquet YF. The NMDA receptor: central role in pain inhibition in rat periaqueductal gray. Eur J Pharmacol 1988;154(3):271–6. [18] Jasmin L, Boudah A, Ohara PT. Long-term effects of decreased noradrenergic central nervous system innervation on pain behavior and opioid antinociception. J Comp Neurol 2003;460:38–55. [19] Jensen TS, Yaksh TL. The antinociceptive activity of excitatory amino acids in the rat brainstem: an anatomical and pharmacological analysis. Brain Res 1992;569(2):255–67. [20] Jorum E. Analgesia or hyperalgesia following stress correlates with emotional behavior in rats. Pain 1988;32:341–8. [21] Kaplan H, Fields HL. Hyperalgesia during acute opioid abstinence: evidence for a nociceptive facilitating function of the rostral ventromedial medulla. J Neurosci 1991;11(5):1433–9. [22] Le Bars D, Gozariu M, Cadden SW. Animal models of nociception. Pharmacol Rev 2001;53(4):597–652. [23] Lee C, Rodgers RJ. Antinociceptive effects of elevated plus-maze exposure: influence of opiate receptor manipulations. Psychopharmacology 1992;102:507–13. [24] McMullan S, Lumb BM. Midbrain control of spinal nociception discriminates between responses evoked by myelinated and unmyelinated heat nociceptors in the rat. Pain 2006;124(1–2):59–68. [25] Martins MA, Carobrez AP, Tonussi CR. Activation of dorsal periaqueductal gray by glycine produces long lasting hyponociception in rats without overt defensive behaviors. Life Sci 2008;83(3–4):118–21. [26] Martins MA, De Castro LC, Tonussi CR. Formalin Injection into knee joints of rats: pharmacologic characterization of a deep somatic nociceptive model. J Pain 2006;7:100–7. [27] Mayer DJ. Analgesia produced by electrical stimulation of the brain. Prog Neuropsychopharmacol Biol Psychiatry 1984;8(4–6):557–64. [28] Mayer DJ, Wolfle TL, Akil H, Carder B, Liebeskind JC. Analgesia from electrical stimulation in the brainstem of the rat. Science 1971;174:1351–4. [29] McCracken LM, Gross RT, Sorg PJ, Edmands TA. Prediction of pain in patients with chronic low back pain: effects of inaccurate prediction and pain-related anxiety. Behav Res Ther 1993;31(7):647–52. [30] McGaraughty S, Chu KL, Bitner RS, Martino B, El Kouhen R, Han P, et al. Capsaicin infused into the PAG affects rat tail flick responses to noxious heat and alters neuronal firing in the RVM. J Neurophysiol 2003;90(4):2702–10. [31] Miron D, Duncan GH, Bushnell MC. Effects of attention on the intensity and unpleasantness of thermal pain. Pain 1989;39:345–52. [32] Morgan MM, Fields HL. Pronounced changes in the activity of nociceptive modulatory neurons in the rostral ventromedial medulla in response to prolonged thermal noxious stimuli. J Neurophysiol 1994;72(3):1161–70. [33] Morgan MJ, Franklin KB. Stimulation-produced analgesia (SPA) from brainstem and diencephalic sites in the rat: relationships between analgesia, aversion, seizures and catalepsy. Pain 1988;33(1):109–21. [34] Nunes-de-Souza RL, Canto-de-Souza A, da-Costa M, Fornari RV, Graeff FG, Pelá IR. Anxiety-induced antinociception in mice: effects of systemic and intraamygdala administration of 8-OH-DPAT and midazolam. Psychopharmacology 2000;150(3):300–10. [35] Oliva P, Berrino L, De Novellis V, Palazzo E, Marabese I, Siniscalco D, et al. Role of periaqueductal grey prostaglandin receptors in formalin-induced hyperalgesia. Eur J Pharmacol 2006;530(1–2):40–7. [36] Pertovaara A. Noradrenergic pain modulation. Prog Neurobiol 2006;80:53–83. [37] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. San Diego: Academic Press; 1998. [38] Ploghaus A, Becerra L, Borras C, Borsook D. Neural circuitry underlying pain modulation: expectation, hypnosis, placebo. Trends Cogn Sci 2003;7:197–200. [39] Pogatzki EM, Urban MO, Brennan TJ, Gebhart GF. Role of the rostral medial medulla in the development of primary and secondary hyperalgesia after incision in the rat. Anesthesiology 2002;96(5):1153–60. [40] Porreca F, Ossipov MH, Gebhart GF. Chronic pain and medullary descending facilitation. Trends Neurosci 2002;25:319–25. [41] Puig S, Sorkin LS. Formalin-evoked activity in identified primary afferent fibers: systemic lidocaine suppresses phase-2 activity. Pain 1996;64(2):345–55. [42] Quintero L, Moreno M, Avila C, Arcaya J, Maixner W, Suarez-Roca H. Long lasting delayed hyperalgesia after subchronic swim stress. Pharmacol Biochem Behav 2000;67(3):449–58.
M.A. Martins et al. / Behavioural Brain Research 214 (2010) 260–267 [43] Reynolds DV. Surgery in the rat during electrical analgesia by focal brain stimulation. Science 1969;164:444–5. [44] Ren K, Dubner R. Descending modulation in persistent pain: an update. Pain 2002;100:1–6. [45] Santos P, Bittencourt AS, Schenberg LC, Carobrez AP. Elevated T-maze evaluation of anxiety and memory effects of NMDA/glycine-B site ligands injected into the dorsal periaqueductal gray matter and superior colliculus of rats. Neuropharmacology 2006;51:203–12. [46] Teskey GC, Kavaliers M, Hirst M. Social conflict activates opioid analgesic and ingestive behaviors in male mice. Life Sci 1984;35:303–15. [47] Tonussi CR, Ferreira SH. Rat knee-joint carrageenin incapacitation test: an objective screen for central and peripheral analgesics. Pain 1992;48:421–7. [48] Urban MO, Gebhart GF. Characterization of biphasic modulation of spinal nociceptive transmission by neurotensin in the rat rostral ventromedial medulla. J Neurophysiol 1997;78:1550–62. [49] Vanderah TW, Suenaga NM, Ossipov MH, Malan Jr TP, Lai J, Porreca FJ. Tonic descending facilitation from the rostral ventromedial medulla mediates opioid-induced abnormal pain and antinociceptive tolerance. J Neurosci 2001;21(1):279–86.
267
[50] Vanegas H, Schaible HG. Descending control of persistent pain: inhibitory or facilitatory? Brain Res Rev 2004;46:295–309. [51] Varni JW, Rapoff MA, Waldron SA, Gragg RA, Berbestein BH, Lindsley CB. Chronic pain and emotional distress in children and adolescents. J Dev Behav Pediatr 1996;17(3):154–61. [52] Vidal C, Jacob JJ. Stress hyperalgesia in rats: an experimental animal model of anxiogenic hyperalgesia in human. Life Sci 1982;31:1241–4. [53] Waters AJ, Lumb BM. Descending control of spinal nociception from the periaqueductal grey distinguishes between neurons with and without C-fiber inputs. Pain 2008;134(1–2):32–40. [54] Wiertelak EP, Roemer B, Maier SF, Watkins LR. Comparison of the effects of nucleus tractus solitarius and ventral medial medulla lesions on illness-induced and subcutaneous formalin-induced hyperalgesias. Brain Res 1997;748(1–2):143–50. [55] Young RF, Brechner T. Electrical stimulation of the brain for relief of intractable pain due to cancer. Cancer 1986;57(6):1266–72. [56] Zhuo M, Gebhart GF. Biphasic modulation of spinal nociceptive transmission from the medullary raphe nuclei in the rat. J Neurophysiol 1997;78: 746–58.
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Dependency of nociception facilitation or inhibition after periaqueductal gray matter stimulation on the context M.A. Martins, L. De Castro Bastos, N.E.B. Melo, C.R. Tonussi ∗ Department of Pharmacology, Federal University of Santa Catarina, Florianópolis, SC, 88040-900, Brazil
a r t i c l e
i n f o
Article history: Received 16 October 2009 Received in revised form 17 May 2010 Accepted 23 May 2010 Available online 1 June 2010 Keywords: Pain modulation PAG Stress Hyperalgesia Attention Anxiety
a b s t r a c t Anxiety and/or fear can alter the nociceptive response in humans and animals. Slight stimulation of the dorsal periaqueductal gray matter (DPAG) produced anxiety/fear-related behaviour and hyponociception in escapable, non-anxiogenic nociceptive models. Our aim was to investigate the role of the DPAG in models of persistent, anxiogenic nociception. GLY (1, 10, 20, and 80 nmol/0.3 l/60 s) was injected into the DPAG of rats, 5 min before formalin (2%/50 l) injection either into the knee-joint or hind paw. In the knee-joint incapacitation test, GLY caused hypernociception at lower doses and hyponociception at higher doses. In the paw shacking test, GLY produced only hypernociception with the higher dose. Coinjecting GLY with 7-chlorokynurenic acid (7-CLK) or (+/−)-3-amino-1-hydroxy-2-pyrrolidone (HA-966) completely prevented the GLY effects in incapacitation and paw shacking tests, respectively. GLY injections outside the periaqueductal gray matter (PAG) did not change the nociception. Behavioural analysis indicated that formalin paw injection produced higher stress signals than knee-joint injection, as diminished exploratory behaviour, and stereotypy. The results suggest that activation of the DPAG through the GLYB /NMDA receptor is able to produce either facilitation or inhibition of nociception depending on the nociceptive context. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Anxiety, like pain and fear, plays an adaptive role as an alarm that responds to danger or harm, as in a defensive system. Since these alarms serve similar protective functions it is predictable that they might interact and also elicit collective effects. Indeed, chronic pain states have been found to predict more severe anxiety symptomatology [51], and vice versa, i.e. more anxious patients reported more pain [10,29]. Basic studies have confirmed the relationships between anxiety and nociception, although they have produced contradictory results. For example, a variety of stressful environmental events can produce hyponociception [6,15,23,34,46], but anxiogenic stress models (acute, subchronic and chronic) can also produce hypernociception [1,12,13,20,42,52]. The periaqueductal gray matter (PAG) is a midbrain structure involved in pain, fear, and anxiety modulation [5]. The dorsal columns (DPAG) are proposed to integrate behavioural and autonomic expression of defensive reactions, and nociception similar to that seen in stressful situations (see [3]). DPAG-elicited hyponociception, however, may not be proportional to the aversion-like reactions elicited by its activation since the subtle aversive effect
∗ Corresponding author. Tel.: +55 48 3721 9491x218; fax: +55 48 3337 5479. E-mail address: [email protected] (C.R. Tonussi). 0166-4328/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2010.05.035
produced by the activation of the glycine site at the NMDA type receptor (GLYB /NMDA) in the DPAG [8] has been observed to produce an intense and long-lasting hyponociception in the tailflick test [25]. On the other hand, prostaglandin and capsacin DPAG injections have even been shown to facilitate nociception [30,35]. Slight stimulation of the DPAG is thought to enhance the aversive value of the potential sources of threat to an animal [8], and consequently the animal attention should be directed toward that which seems more threatening at a given time. Different animal models of nociception may exert different aversive effects on the subject [22], i.e. escapable nociceptive tests, such as the heatinduced tail-flick, may have low potential to produce fear/anxiety since the animal has control over the nociception intensity to which it is submitted. On the other hand, inescapable (foot-shock) and/or persistent (formalin) nociceptive models may proportionally be more aversive to the animals. The same reasoning may be followed whether the nociception source is external (from the environment) or internal (from the body). In this case, the possibility exists that the nociception modulation due to the same kind of DPAG stimulation may be a result of a decision between which threat to the animal is more severe (or aversive) in a given situation. The aim of this study was to verify the effect of DPAG stimulation on two models of persistent, inescapable nociception, which differ mainly with respect to the nociceptive source, i.e. subcutaneous (external) or intra-articularly (internal) injected formalin in rats.
M.A. Martins et al. / Behavioural Brain Research 214 (2010) 260–267 2. Methods 2.1. Animals Male Wistar rats (250–350 g) were housed at 21 ± 1 ◦ C under a regular 12/12 h light/dark cycle (lights on 7:00 a.m.). Food and water were available ad libitum. All behavioural tests were performed between 7:00 a.m. and 2:00 p.m. Animal care and handling procedures were in accordance with the ethical guidelines of the International Association for the Study of Pain [16], and also approved by the Ethics Committee for Animal Use of the Federal University of Santa Catarina. All efforts were made to reduce both the animal number and suffering during the experiments. 2.2. Surgical procedure A stainless still guide cannula (length = 13 mm, o.d. = 0.7 mm) was stereotaxically implanted (Paxinos and Watson Atlas [37] coordinates: AP = − 7.6 mm and ML = +1.9 mm from bregma; DV = −2.0 mm from skull and at an angle of 22◦ from the sagittal plane) with its tip intended to be above the dorsal periaqueductal gray (DPAG) area. The cannula was maintained in this position embedded in an acrylic cement cap anchored by two mini-screws fixed to the skull. The surgery was carried out 7 days before the experiment, and the animals were anaesthetised with 1.5 ml/kg of a xylazine (20 mg/ml; Rompun® , Bayer, Brazil)/ketamine (100 mg/ml; Dopalem® , Vetbrands, Brazil) 1:1 mixture. After surgery the rats received an intramuscular injection of Pentabiotic® (0.2 ml/rat; Fort Dodge, Brazil), and were allowed to recover from the anaesthesia in a silent and warm room. In addition, 2% lidocaine with vasoconstrictor was infused (0.2 ml) under the skin before its excision for the skull exposition. Intra-periaqueductal gray microinjections were administered through a thin dental needle (0.3 mm o.d.) which extends 3.25 mm beyond the guide cannula tip. A volume of 0.3 l was injected over a period of 60 s using a hand-driven 5.0 l Hamilton microsyringe. The movement of a small air bubble inside the PE-10 polyethylene tubing, interposed between the upper end of the dental needle and the microsyringe, confirmed successful drug injection. The animals randomly received a single DPAG microinjection of glycine (GLY, 10, 20, 80 and 100 nmol), phosphate buffered saline (PBS) or dimethylsulfoxide (DMSO in PBS, 5%, v/v). The GLYB receptor antagonist, 7chlorokynurenic acid (7CLK; 10 nmol) or (+/−)-3-amino-1-hydroxy-2-pyrrolidone (HA966; 10 nmol) was applied alone or combined with glycine in the same injection, 5 min before formalin injections. At the end of the experiments, the animals were deeply anaesthetised with 2% xylazine and 15% chloral hydrate (0.2 and 1.0 ml/rat) and perfused intracardially with physiological saline (0.9% NaCl) followed by formalin solution (10%). In order to mark the injection site 0.3 l of Evans Blue (0.5%) was then applied through the guide cannula. The brains were removed and stored in 10% formalin solution for posterior histological analysis. Frozen sections (50 m) were obtained using a cryostat, mounted on glass slides and stained using the Giemsa (Sigma–Aldrich, USA) method for microscopic identification of the injection site. Only the rats for which the microinjection site was located within the DPAG were considered. 2.3. Incapacitation test The formalin-induced knee-joint incapacitation model is presented elsewhere [26], as a modification of that described in [47]. In this test, up to three rats are placed simultaneously on a revolving cylinder (30 cm diameter; 3 rpm), and a computerassisted device registers the total time that the ipsilateral hind paw to the stimulated knee-joint fails to make contact with the cylinder surface (i.e. paw elevation time or PET) during a 60-s period of enforced walking. Therefore, PET is an objective measure of the guarding behaviour elicited by articular nociception. The injection site was first shaved and treated with an iodine alcohol antiseptic solution. The animals were gently restrained in a supine position by the hands of the experimenter, and intra-articular injections of formalin (2%, 50 l) were quickly performed with a 30-gauge needle. The concentration of formalin was carefully considered to give a submaximal behavioural response in order to allow that increases or decreases in such a response could be detected. The PET was recorded just after formalin injection and every 5 min thereafter, for a 60-min test period. Between each 60-s recording period, the animals remained on the stopped cylinder, generally exhibiting grooming behaviour or apparently sleeping. There was low exploratory interest probably because the animals were trained one day before on the revolving cylinder in order to spontaneously walk to the top each time the movement started. 2.4. Paw formalin test The other formalin test used was the classical test described by Dubuisson and Dennis [9] with modifications in the registering paradigms. Firstly, one day before the experimental session, the animals were allowed a 20-min habituation period in the observation box. The observation box was a transparent plexiglass cubic chamber (edges = 29 cm) with mirrors angled on the lateral and back walls, adjusted accordingly to allow unhindered observation of the animal’s paws. Immediately after a 2% formalin (50 l) subcutaneous plantar injection in the right hind paw, the animals were observed for the nocifensive response. The numbers of shakes, lifting, and licking/biting of the injected paw were counted and summed as the number of
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nocifensive events (NNE) over 60 min in 5-min cluster periods. Lifting and licking behaviours were sometimes exhibited in long bouts, i.e. more than 3 s, before the return to the resting position. In these cases, the first 3 s of the event was counted as one event, plus one event when the paw returned to the resting position. 2.5. Behavioural evaluation of the animals undergoing paw or knee-joint formalin-induced nociception Apart from the experiments using PAG injections, another group of animals were used for the evaluation of the behavioural signs of stress induced by each kind of nociceptive stimulus. The animals were divided in two main groups, those receiving intraplantar injections and those receiving knee-joint injections. For each injection site, the same number of animals were allocated to receive 50 l of physiological saline or 2% formalin. The experiments were designed such that we had always one paw injected and one knee-joint injected animal being recorded at the same time, irrespective they had received saline or formalin. Immediately after the injection, the animals were put in individual transparent plexiglass boxes (like above) placed side-by-side, in a quiet room and with a light source just above equally illuminating the boxes. The experimenters did not remain in the room, and the sessions were video-recorded for later behavioural analysis. Behaviours chosen for analysis were the time spent in exploratory activity (EA), and the number of occurrences of the tail biting, licking of the floor, and faecal pellet manipulation events. For the adequate pairing of these behaviours with the primary nociceptive behaviour, the NNE were taken for both groups. However, in this case, since the animals were observed in the plexiglass box, the knee-joint injected animals cannot be evaluated in the automated incapacitation test. The same procedure for the evaluation of the NNE used in the traditional paw shaking test above, was also used in the knee-joint test. 2.6. Drugs and vehicles Glycine (Sigma–Aldrich, Brazil) was dissolved in PBS solution. HA966 ((+/−)3-amino-1-hydroxy-2-pyrrolidone) and 7-chlorokynurenic acid (Tocris, USA) were dissolved in PBS and PBS with DMSO (5%, v/v), respectively. Formalin (formaldehyde 37% – Merck AG, Germany) was diluted to 2% in sterile physiological saline, considering the initial concentration as 100%. 2.7. Statistical analysis All statistical analyses were carried out using Graphpad Prism version 5 software. Results are expressed as mean ± S.E.M. (standard error of mean). Multiple comparisons among means in the first phase of formalin response (one single time point) were made using simple ANOVA, but in the second phase (time-course curves) were made using ANOVA for repeated measures. Both were followed by the Dunnett or Tukey post-hoc test. The time-course period analysed were indicated in each case. The minimum significance level considered was P < 0.05.
3. Results Glycine (GLY) microinjection in caudal sites of the dorsal PAG (Fig. 1A and B), at the doses of 10 and 20 nmol, induced a significant increase of the paw elevation time (PET) suggesting hypernociception during the second phase (t = 5–30 min) of the formalin-induced incapacitation (ANOVA; F(3,20) = 6.15). On the other hand, the higher doses of GLY (80 and 100 nmol) inhibited incapacitation in the first (t = 0 min; ANOVA; F(2,22) = 6.04) and second phases (t = 5–30 min; ANOVA; F(2,18) = 29.07), suggesting a hyponociceptive effect. GLY at the dose of 1 nmol did not affect the PET of animals (Fig. 2A and B). Similarly, GLY injections (10, 20 and 80 nmol) that reached immediately dorsal to the DPAG, in the superior colliculus, did not change the formalin response (Fig. 3). Due to the intriguing opposite effect observed with GLY in low and high doses, we decided to compare the effect of DPAG glycine injection in the traditional 2% formalin-induced paw nociception. In this test, the higher dose (80 nmol) induced significant increase of the NNE in the second phase (t = 10–60 min; ANOVA; F(2,30) = 16.95). Interestingly, in the lower dose (20 nmol), glycine did not modify the NNE induced by formalin (Fig. 4). The hypernociceptive effect induced by 80 nmol of GLY into DPAG was blocked by the co-injection of HA966 (10 nmol), an antagonist of the glycine site at the NMDA receptor (GLY-B). HA966 alone did not change the formalin response per se (ANOVA; F(3,29) = 8.76) (Fig. 5). In the knee-joint test, both effects produced by glycine (10 or 80 nmol) were prevented by 7-chlorokynurenic acid (7CLK; 10 nmol), another GLY-B antagonist. The co-administration of
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Fig. 1. (A) Photomicrograph of a slide of the midbrain stained by Giemsa (Sigma–Aldrich, USA) from a representative subject showing an injection site (arrowheads) in the DPAG column section. Section corresponds to −7.30 to −8.00 mm from the bregma in the Paxinos and Watson atlas [37]. (B) Diagrams of rat coronal sections, redrawn from the Paxinos and Watson atlas. The microinjection sites (black dots) as labelled by Evans Blue (0.5%) injection in the dorsal parts of the PAG. The number of points in the figures is less than the total number of rats used because of several site overlaps.
Fig. 2. Hypernociceptive and hyponociceptive effects induced by glycine microinjections into the DPAG in knee-joint incapacitation test induced by formalin. Glycine (GLY) at doses of 1, 10, 20, 50, 80 and 100 nmol/0.3 l/60 s/site was applied to the DPAG 5 min before formalin injection (2% i.a.). The paw elevation time (PET) was evaluated for 60 s every 5 min over a period of 60 min. Data are presented as mean ± S.E.M. Control animals received only PBS (0.3 l/60 s/site). P1 and P2: phases 1 (0–5 min) and 2 (5–30 min) respectively. *P < 0.05; ***P < 0.001 (P1: one-way ANOVA; P2: one-way ANOVA for repeated measures, both followed by the Tukey test).
Fig. 3. Glycine outside the DPAG did not change the articular nociception. Glycine at doses of 10, 20 and 80 nmol/0.3 l/site (GLY10, GLY20, GLY80) was applied to the deep layer of the Superior Colliculus (SC) 5 min before the incapacitation test induced by formalin (2% i.a.). The paw elevation time (PET) was evaluated for 60 s every 5 min over a period of 60 min. Data are presented as mean ± S.E.M. ANOVA for repeated measures followed by Tukey’s test did not reveal significant differences for GLY groups compared with the control group (control, 0.3 l/site). P1 and P2: phases 1 (0–5 min) and 2 (5–60 min), respectively. B: baseline values; n: animals for each group.
Fig. 4. Hypernociceptive effect induced by glycine microinjections into the DPAG in the formalin hind paw test. Glycine (GLY) at doses of 20 and 80 nmol/0.3 l/60 s/site was applied to the DPAG 5 min before formalin injection (2% s.c.). The total number of nocifensive events (NNE) was recorded in 5-min cluster periods over 60 min. Data are presented as mean ± S.E.M. Control animals received only PBS (0.3 l/60 s/site). P1 and P2: phases 1 (0–5 min) and 2 (10–60 min), respectively. ***P < 0.001 (one-way ANOVA for repeated measures followed by the Dunnett test).
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was considered (t = 0). The paw formalin groups (Fig. 7, left column) have shown decreased EA time (Fig. 7C and D: ANOVA for repeated measures, F(3,44) = 7.27. P < 0.001, between formalin and saline paw injected groups, and P < 0.05, between formalin and saline kneejoint injected groups, with Tukey post-hoc test), but increased NLF events (Fig. 7E and F: ANOVA for repeated measures, F(3,44) = 24.39. P < 0.001, between formalin and saline paw injection groups, with Tukey post-hoc test) and NTB (Fig. 7I and J: ANOVA for repeated measures, F(3,44) = 8.51. P < 0.05, between formalin and saline paw injection groups, with Tukey post-hoc test) when compared to the knee-joint group. The faecal pellet manipulation behaviour was also higher in the paw injected group immediately after the formalin injection (Fig. 7G and H: t = 0 min, ANOVA, F(3,28) = 12.24. P < 0.01, between formalin and saline paw injection groups, with Tukey post-hoc test). Fig. 5. Glycine-induced hypernociceptive effect was prevented by HA966. The number of nocifensive events (NNE) of rats which received glycine alone (GLY; 80 nmol/0.3 l/site), vehicle solution (PBS, 0.3 l/site), HA966 (HA; 10 nmol/0.3 l site) alone or mixed with glycine (GLY80 + HA, 0.3 l/site) were recorded 5 min after DPAG injection, and every 5 min thereafter for a period of 60 min. Data are presented as mean ± S.E.M. P1 and P2: phases 1 (0–5 min) and 2 (10–60 min), respectively. ***P < 0.001 (one-way ANOVA for repeated measures followed by the Dunnett test).
7CLK blocked, significantly, the hypernociception in the second phase (ANOVA; F(3,40) = 13.81) and the hyponociception in the first (ANOVA; F(3,23) = 8.29) and second phases (ANOVA; F(3,40) = 14.66) (Fig. 6A and B). Although HA966 was likewise observed to antagonize glycine effects, in this test, it also presented a hypernociceptive effect per se (data not shown). This effect may be due to a reported partial agonistic effect of the drug, and in this case was consistent with the hypernociceptive effect found for the lower glycine dose. Because DPAG has been reported to be involved in the activation of defensive behaviours related to aversive situations, the next set of experiments were designed to behaviourally evaluate the relative aversiveness between formalin injected in the paw and in the knee-joint. By observing the animals, as explained above, the number of the tail biting (NTB), licking of the floor (LF), and faecal pellets manipulation (FPM) behaviours, appeared significantly higher to the animals undergoing paw nociception. On the other hand, the time spent in the exploratory behaviour (EA) appeared significantly lowered in the paw formalin-injected group than in the knee-joint injected animals. In Fig. 7, the behaviours elicited after paw injection were separated from those elicited after kneejoint injection, for the better visualisation. However, statistical analyses were performed taking together all the whole curves of each behaviour. Exception made to the FPM behaviour, for which was found statistical difference only when the first five minutes
4. Discussion The main findings of this study were that glycine microinjection into the dorsal PAG produced opposite effects, i.e. hyper and hyponociception, in two models of persistent nociception. Both hypo and hypernociceptive effects were blocked by 7chlorokynurenic acid or HA966, consistent with the proposal that the GLYB site at the NMDA receptor plays a role in their mediation. The inhibitory role of the PAG in nociception and on pain has been established, as the focal electrical stimulation in this particular structure triggers hyponociception in animals [28,43] and powerful analgesia in humans [27,55]. In addition, our present and previous [25] findings that glycine-induced hyponociception was reversed by GLYB site antagonists is in agreement with data available in the literature implicating glutamate and its NMDA-type receptor as important mediators of the hyponociception elicited by this structure [17,19,33]. However, the same glycine concentration that decreased PET in the present study, which is interpreted as a hyponociceptive effect, increased the nocifensive behaviour elicited by formalin in the hind paw, which is interpreted as a hypernociceptive effect. On the other hand, lower concentrations of glycine, which produced no effect in formalin-induced paw flinches and the tail-flick test, increased formalin-induced articular incapacitation. Recent studies have shown that brainstem modulatory systems can, indeed, exert bidirectional control and that nociception facilitation may even be its most important function. However, determining how this system is recruited to either inhibit or facilitate nociception under different conditions is an important challenge in terms of understanding descending modulation, and
Fig. 6. Glycine-induced hyponociceptive and hypernociceptive effects were prevented by 7-chlorokynurenic acid. The paw elevation time (PET) for rats which received glycine alone (GLY; 10 or 80 nmol/0.3 l/site), vehicle solution (PBS, 0.3 l/site), 7-chlorokynurenic acid (7CLK; 10 nmol/0.3 l site) alone or mixed with glycine (GLY10 + 7CLK or GLY80 + 7CLK, 0.3 l/site) were recorded 5 min after DPAG injection, and every 5 min thereafter over a period of 60 min. Data are presented as mean ± S.E.M. P1 and P2: phases 1 (0–5 min) and 2 (5–30 min), respectively, **P < 0.01; ***P < 0.001 (P1: one-way ANOVA; P2: one-way ANOVA for repeated measures, both followed by the Dunnett test).
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Fig. 7. Behavioural evaluation of the animals undergoing paw or knee-joint formalin-induced nociception. EA: time spent in the exploratory activity; NTB: number of tail biting events; NLF: the number of licking of the floor events; FPM: number of faecal pellet manipulation events; NNE: number of nociceptive behaviour events. Each behaviour was analysed after 2% formalin injection either into the paw (left column) or into the knee-joint (right column), and clustered in 5-min intervals. Control groups received physiological saline instead of formalin. **P < 0.01, ***P < 0.001 (FPM: one-way ANOVA; EA, NTB, NLF: one-way ANOVA for repeated measures, both followed by the Tukey test).
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proposing therapies [see [50] for review). For example, the rostral ventromedial medulla (RVM) was found to be involved in hyperalgesia associated with acute naloxone-precipitated withdrawal, prolonged opioid administration, prolonged heating of the tail and illness induced by endotoxin administration [21,32,49,54]. The RVM also contributes to hyperalgesia and allodynia in inflammatory and neuropathic models, including the formalin test, mustard oil secondary hyperalgesia, and spinal nerve ligation [14,40]. Importantly, this enhancement of nociceptive transmission is selective for certain kinds of inputs, because the RVM seems not to be required for primary or secondary hyperalgesia surrounding a surgical incision site [39] or after long-lasting focal inflammation produced by complete Freund’s adjuvant. Indeed, descending inhibition from the RVM is enhanced in complete Freund’s adjuvant-treated animals [44]. Notwithstanding, there are several differences among these studies which hinders the proposition of a reconciling hypothesis to explain these results, and additional attentional interferences due to brainstem manipulation cannot be discarded. In contrast to the descending inhibition, the involvement of PAG in the nociceptive facilitation circuitry, although suggested, is less clear. Differing from earlier observations after PAG electrical and chemical stimulation, which induced hyponociception often associated with intense aversion-like behaviours, recent investigations have shown that capsaicin and prostaglandin injections into the dorsal PAG can produce hypernociception in formalin and in a fastheating tail-flick models, respectively [30,35]. Similarly to RVM, dorsal PAG projections can also support facilitatory and inhibitory effects on nociception since they indirectly target RVM via a pontine catecholaminergic nucleus, such as the locus coeruleus [4]. Furthermore, locus coeruleus direct catecholaminergic projections to the spinal cord may also produce nociception facilitation or inhibition [18,36]. In an attempt to explain this dual descending circuitries effect, it has been proposed that the nociceptive information is differentially facilitated or inhibited, respectively, depending on whether it is conveyed by A- or C-fibres [24,53]. This conception may fit well to behavioural models based on fast (A-fibres) or slow heating (C-fibres). However, it is difficult to reconcile this idea with the formalin-based models which are mediated by a combination of A- and C-fibre-conveyed action potentials [41], and, indeed, as we have shown here, the same PAG stimulation produced opposite effects in two formalin-based models. Another possible explanation for our results may be related to the intensity of the stimulation. Low intensity electrical stimulation, or low concentrations of chemical substances, in the RVM was seen to cause nociceptive facilitation. On the other hand, high intensity electrical stimulation, or higher concentrations of glutamate or neurotensin, inhibited nociception [48,56]. Similarly, low concentrations of glycine in the PAG facilitated formalin incapacitation, while higher concentrations inhibited this response. However, in the tail-flick test glycine produced only hyponociception even at lower doses [25] than that which reversed incapacitation here, but the higher glycine concentration facilitated the formalin response in the hind paw. Thus, the idea that facilitation and inhibition could be due to the intensity of the PAG activation does not seem to offer a satisfactory explanation. DPAG seems to be pivotal for processing of fear and anxiety [5]. Glycine injection into the DPAG has been shown to produce a progressive attention (or expectancy) to the unknown surrounding environment that may appear potentially more threatening, although it did not produce overt defensive behaviours related to highly stressful conditions, such as exophthalmia, micturition, running, jumping, etc. For example, such procedure has been described to enhance the anxiety-like behaviour observed in specific elevated maze models [8,45]. Different types of anxiogenic stress models
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have been shown to produce hypernociception in persistent nociceptive models, but also to increase the threshold response in phasic nociceptive tests [1,12,13,20,42,52]. One important aspect usually ignored in most studies on nociception and stress is the relative aversion produced by the nociceptive test and the stress model. Furthermore, procedures that will enhance the animal’s anxiety may produce different effects on the aversive component of nociception and other components of aversiveness produced by the experimental manipulation, but not considered in the study. At any given time, the animals should decide among what source of stress may be more threatening, including the nociceptive source. The tail-flick model with a slow heating rate, as an escapable nociceptive model, is expected to be less stressful than formalin-induced inescapable persistent nociception. In addition, formalin injected into the paw seemed to be even more stressful than when injected into the knee-joint, since after injection into the paw the animals exhibited decreased overall exploratory activity, and episodes of stereotypy as the compulsive licking of the floor and manipulation of the faecal pellets. Such behaviours were not observed in the knee-joint injected animals, and may be important indicators of the emotional aversive component elicited by the nociception arising from the paw tissue. One possibility to explain these differences could be the overall nociception perceived by the animals, however it is not easy to compare the relative nociception induced by these two models. The incapacitation registering on the revolving cylinder suggested that nociception elicited by formalin in the knee-joint could have shorter time-course than that elicited by the paw formalin model. However, in the paw formalin model, the animals were not obliged to execute a task, being observed freely in the box. The behavioural evaluation made in the plexiglass box, indicated that both kinds of nociceptive inputs have similar timecourse. Thus, we cannot affirm that paw formalin model hurts longer than knee-joint model. But in spite of a similar time-course, paw formalin-injected animals have shown a high frequency of the biting of the tail behaviour, which was not shown by the knee-joint injected animals. This behaviour suggests nociceptive sensation from the tail, which is consistent with secondary hypernociception. Secondary hypernociception is clearly a function of the intensity of nociceptive input from the primary site of nociception [50]. The presence of such kind of nociceptive behaviour in the paw injected animals, but not in the knee-joint injected ones, could suggest that the intensity of nociception is higher in the paw model. Again, however, this cannot be conclusive because the area of the secondary hypernociception elicited by knee-joint noxious input may be different from that elicited by the paw noxious inputs. Finally, formalin-elicited nociception in the footpad may be more aversive than that induced in the knee-joint, because it could be suggesting to the animal an external attack, against which it must pay all attention. Whatever the explanation, they are all consistent to the idea that paw formalin injection will attract more the animal attention, causing more aversion. Our working hypothesis to explain our present and previous findings is that the increased anxiety/fear produced by DPAG stimulation will enhance nociception when nociception is the major source of anxiety/fear, i.e. in persistent models, but will inhibit it when the major source of anxiety/fear is not the nociceptive model, as in phasic, escapable, nociceptive tests [2,7,31,38]. This may explain why the higher dose of glycine caused hypernociception in the paw formalin model, in contrast to the previously observed hyponociception in the heat-induced tail-flick test [25]. In this case, glycine injected into the DPAG enhanced the attention to factors which were more aversive to the animal, i.e. the persistent nociception, thus enhancing nociception. Supporting this point, an anti-aversive dose of a cannabinoid agonist was shown to reduce the nocifensive response induced by the paw formalin injection [11]. On the other hand, since in phasic, escapable noci-
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ceptive tests, such as the tail-flick test, nociception is gradual, and not persistent, the main focus of the animal’s attention is not likely to be directed toward the tail, but toward some other potentially threatening object – e.g. the experimenter’s hands, surrounding environment, etc. Glycine, in this case, would be diverting the attentional focus away from the tail, thus causing hyponociception. The incapacitation induced by formalin, differently from the other models, presented both glycine effects, i.e. hyper and hyponociception. Interestingly, the hyponociception was observed with the higher glycine dose that caused hypernociception in the paw formalin model, thus excluding the possibility that hyponociception was due to motor impairment. As shown above, formalin injection into the knee-joint did not produce the overt signs of stress observed after injection into the paw, suggesting that the articular model demands less attention toward the nociceptive site than the paw model. In addition, in the knee-joint model the animals are also focused on keeping themselves on the cylinder. Thus, it is conceivable that the animals will be alternating their attention between the walking on the cylinder and the guarding of the painful joint, and this context could be responsible for the observation of the two opposing effects on nociception, depending on the glycine dose injected. In this new, distinct, context the facilitation of DPAG glutamate transmission by glycine seems to be showing that, at least this PAG structure may be functionally plastic in the way it integrates the several stress sources for the nociception modulation, probably determining how the animal will cope with nociception. In conclusion, the results here presented raised the hypothesis that, at least in the dorsal PAG, facilitation or inhibition may be a result of the emotional context in which the nociception occurred, rather than a stereotyped response. This hypothesis needs to be further supported by experiments designed under these predictions, and indeed will be very interesting to extend this idea to other structures involved in nociception modulation, trying to determine whether this functional plasticity is level dependent, and to what extent. For the basic research on nociception in a broader sense, the present results also suggest that the conclusions on the efficacy of analgesic candidates should take into account the contextual aspects in which the nociceptive reflex was observed. Acknowledgements This study was supported by the Brazilian funding agencies CAPES, CNPq and FAPESC (Pronex). MAM and LCB were recipients of doctoral fellowships from CNPq. References [1] Andre J, Zeau B, Pohl M, Cesselin F, Benoliel JJ, Becker C. Involvement of cholecystokininergic systems in anxiety induced hyperalgesia in male rats: behavioral and biochemical studies. J Neurosci 2005;25:7896–904. [2] Arntz A, Dreessen L, De Jong P. The influence of anxiety on pain: attentional and attributional mediators. Pain 1994;56:307–14. [3] Bandler R, Shipley MT. Columnar organization in the midbrain periaqueductal gray: modules for emotional expression? Trends Neurosci 1994;17:379–89. [4] Beitz AJ. The nuclei of origin of brainstem serotonergic projections to the rodent spinal trigeminal nucleus. Neurosci Lett 1982;3:223–8. [5] Behbehani MM. Functional characteristics of the midbrain periaqueductal gray. Prog Neurobiol 1995;46:575–605. [6] Bellgowan PS, Helmstetter FJ. Neural systems for the expression of hypoalgesia during non associative fear. Behav Neurosci 1996;110(4):727–36. [7] Bushnell MC, Duncan GH, Dubner R, Jones RL, Maixner W. Attentional influences on noxious and innocuous cutaneous heat detection in humans and monkeys. J Neurosci 1985;5(5):1103–10. [8] Carobrez AP, Teixeira KV, Graeff FG. Modulation of defensive behavior by periaqueductal gray NMDA/glycine-B receptor. Neurosci Biobehav Rev 2001;25:697–9. [9] Dubuisson D, Dennis SG. The formalin test: a quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats. Pain 1977;4(2):161–74. [10] Ferguson R, Ahles TA. Private body consciousness, anxiety and pain symptom reports of chronic pain patients. Behav Brain Res 1998;36(5):527–35.
[11] Finn DP, Jhaveri MD, Beckett SRG, Roe CH, Kendall DA, Marsden CA, et al. Effects of direct periaqueductal grey administration of a cannabinoid receptor agonist on nociceptive and aversive responses in rats. Neuropharmacology 2003;45:594–604. [12] Gameiro GH, Gameiro PH, Andrade A, da S, Pereira LF, Arthuri MT, et al. Nociception- and anxiety-like behavior in rats submitted to different periods of restraint stress. Physiol Behav 2006;87(4):643–9. [13] Geerse GJ, Van Gurp LCA, Wiegan TVM, Stam R. Individual reactivity to the open-field predicts the expression of stress-induced behavioural and somatic pain sensitization. Behav Brain Res 2006;174:112–8. [14] Heinricher MM, Pertovaara A, Ossipov MH. Descending modulation after injury. In: Dostrovsky JO, Carr DB, Koltzenburg M, editors. Progress in pain research and management. Seattle: IASP Press; 2003. p. 251–60. [15] Helmstetter. FJ Stress-induced hypoalgesia and defensive freezing are attenuated by application of diazepam to the amygdala. Pharmacol Biochem Behav 1993;44(2):433–8. [16] International Association for Study of Pain (IASP). Ethical guidelines for investigation of experimental pain in conscious animals. Pain 1983;16:109– 10. [17] Jacquet YF. The NMDA receptor: central role in pain inhibition in rat periaqueductal gray. Eur J Pharmacol 1988;154(3):271–6. [18] Jasmin L, Boudah A, Ohara PT. Long-term effects of decreased noradrenergic central nervous system innervation on pain behavior and opioid antinociception. J Comp Neurol 2003;460:38–55. [19] Jensen TS, Yaksh TL. The antinociceptive activity of excitatory amino acids in the rat brainstem: an anatomical and pharmacological analysis. Brain Res 1992;569(2):255–67. [20] Jorum E. Analgesia or hyperalgesia following stress correlates with emotional behavior in rats. Pain 1988;32:341–8. [21] Kaplan H, Fields HL. Hyperalgesia during acute opioid abstinence: evidence for a nociceptive facilitating function of the rostral ventromedial medulla. J Neurosci 1991;11(5):1433–9. [22] Le Bars D, Gozariu M, Cadden SW. Animal models of nociception. Pharmacol Rev 2001;53(4):597–652. [23] Lee C, Rodgers RJ. Antinociceptive effects of elevated plus-maze exposure: influence of opiate receptor manipulations. Psychopharmacology 1992;102:507–13. [24] McMullan S, Lumb BM. Midbrain control of spinal nociception discriminates between responses evoked by myelinated and unmyelinated heat nociceptors in the rat. Pain 2006;124(1–2):59–68. [25] Martins MA, Carobrez AP, Tonussi CR. Activation of dorsal periaqueductal gray by glycine produces long lasting hyponociception in rats without overt defensive behaviors. Life Sci 2008;83(3–4):118–21. [26] Martins MA, De Castro LC, Tonussi CR. Formalin Injection into knee joints of rats: pharmacologic characterization of a deep somatic nociceptive model. J Pain 2006;7:100–7. [27] Mayer DJ. Analgesia produced by electrical stimulation of the brain. Prog Neuropsychopharmacol Biol Psychiatry 1984;8(4–6):557–64. [28] Mayer DJ, Wolfle TL, Akil H, Carder B, Liebeskind JC. Analgesia from electrical stimulation in the brainstem of the rat. Science 1971;174:1351–4. [29] McCracken LM, Gross RT, Sorg PJ, Edmands TA. Prediction of pain in patients with chronic low back pain: effects of inaccurate prediction and pain-related anxiety. Behav Res Ther 1993;31(7):647–52. [30] McGaraughty S, Chu KL, Bitner RS, Martino B, El Kouhen R, Han P, et al. Capsaicin infused into the PAG affects rat tail flick responses to noxious heat and alters neuronal firing in the RVM. J Neurophysiol 2003;90(4):2702–10. [31] Miron D, Duncan GH, Bushnell MC. Effects of attention on the intensity and unpleasantness of thermal pain. Pain 1989;39:345–52. [32] Morgan MM, Fields HL. Pronounced changes in the activity of nociceptive modulatory neurons in the rostral ventromedial medulla in response to prolonged thermal noxious stimuli. J Neurophysiol 1994;72(3):1161–70. [33] Morgan MJ, Franklin KB. Stimulation-produced analgesia (SPA) from brainstem and diencephalic sites in the rat: relationships between analgesia, aversion, seizures and catalepsy. Pain 1988;33(1):109–21. [34] Nunes-de-Souza RL, Canto-de-Souza A, da-Costa M, Fornari RV, Graeff FG, Pelá IR. Anxiety-induced antinociception in mice: effects of systemic and intraamygdala administration of 8-OH-DPAT and midazolam. Psychopharmacology 2000;150(3):300–10. [35] Oliva P, Berrino L, De Novellis V, Palazzo E, Marabese I, Siniscalco D, et al. Role of periaqueductal grey prostaglandin receptors in formalin-induced hyperalgesia. Eur J Pharmacol 2006;530(1–2):40–7. [36] Pertovaara A. Noradrenergic pain modulation. Prog Neurobiol 2006;80:53–83. [37] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. San Diego: Academic Press; 1998. [38] Ploghaus A, Becerra L, Borras C, Borsook D. Neural circuitry underlying pain modulation: expectation, hypnosis, placebo. Trends Cogn Sci 2003;7:197–200. [39] Pogatzki EM, Urban MO, Brennan TJ, Gebhart GF. Role of the rostral medial medulla in the development of primary and secondary hyperalgesia after incision in the rat. Anesthesiology 2002;96(5):1153–60. [40] Porreca F, Ossipov MH, Gebhart GF. Chronic pain and medullary descending facilitation. Trends Neurosci 2002;25:319–25. [41] Puig S, Sorkin LS. Formalin-evoked activity in identified primary afferent fibers: systemic lidocaine suppresses phase-2 activity. Pain 1996;64(2):345–55. [42] Quintero L, Moreno M, Avila C, Arcaya J, Maixner W, Suarez-Roca H. Long lasting delayed hyperalgesia after subchronic swim stress. Pharmacol Biochem Behav 2000;67(3):449–58.
M.A. Martins et al. / Behavioural Brain Research 214 (2010) 260–267 [43] Reynolds DV. Surgery in the rat during electrical analgesia by focal brain stimulation. Science 1969;164:444–5. [44] Ren K, Dubner R. Descending modulation in persistent pain: an update. Pain 2002;100:1–6. [45] Santos P, Bittencourt AS, Schenberg LC, Carobrez AP. Elevated T-maze evaluation of anxiety and memory effects of NMDA/glycine-B site ligands injected into the dorsal periaqueductal gray matter and superior colliculus of rats. Neuropharmacology 2006;51:203–12. [46] Teskey GC, Kavaliers M, Hirst M. Social conflict activates opioid analgesic and ingestive behaviors in male mice. Life Sci 1984;35:303–15. [47] Tonussi CR, Ferreira SH. Rat knee-joint carrageenin incapacitation test: an objective screen for central and peripheral analgesics. Pain 1992;48:421–7. [48] Urban MO, Gebhart GF. Characterization of biphasic modulation of spinal nociceptive transmission by neurotensin in the rat rostral ventromedial medulla. J Neurophysiol 1997;78:1550–62. [49] Vanderah TW, Suenaga NM, Ossipov MH, Malan Jr TP, Lai J, Porreca FJ. Tonic descending facilitation from the rostral ventromedial medulla mediates opioid-induced abnormal pain and antinociceptive tolerance. J Neurosci 2001;21(1):279–86.
267
[50] Vanegas H, Schaible HG. Descending control of persistent pain: inhibitory or facilitatory? Brain Res Rev 2004;46:295–309. [51] Varni JW, Rapoff MA, Waldron SA, Gragg RA, Berbestein BH, Lindsley CB. Chronic pain and emotional distress in children and adolescents. J Dev Behav Pediatr 1996;17(3):154–61. [52] Vidal C, Jacob JJ. Stress hyperalgesia in rats: an experimental animal model of anxiogenic hyperalgesia in human. Life Sci 1982;31:1241–4. [53] Waters AJ, Lumb BM. Descending control of spinal nociception from the periaqueductal grey distinguishes between neurons with and without C-fiber inputs. Pain 2008;134(1–2):32–40. [54] Wiertelak EP, Roemer B, Maier SF, Watkins LR. Comparison of the effects of nucleus tractus solitarius and ventral medial medulla lesions on illness-induced and subcutaneous formalin-induced hyperalgesias. Brain Res 1997;748(1–2):143–50. [55] Young RF, Brechner T. Electrical stimulation of the brain for relief of intractable pain due to cancer. Cancer 1986;57(6):1266–72. [56] Zhuo M, Gebhart GF. Biphasic modulation of spinal nociceptive transmission from the medullary raphe nuclei in the rat. J Neurophysiol 1997;78: 746–58.