Pharmacology, Biochemistry and Behavior 131 (2015) 112–118
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The antinociceptive effect of stimulating the retrosplenial cortex in the rat tail-flick test but not in the formalin test involves the rostral anterior cingulate cortex Gláucia Melo Reis, Rafael Sobrano Fais, Wiliam A. Prado ⁎ Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, University of Sao Paulo, Ribeirão Preto, SP 14049-900, Brazil
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Article history: Received 13 November 2014 Received in revised form 2 February 2015 Accepted 6 February 2015 Available online 14 February 2015 Keywords: Antinociception Retrosplenial cortex Anterior cingulate cortex Tail-flick test
a b s t r a c t The stimulation of the retrosplenial cortex (RSC) is antinociceptive in the rat tail-flick and formalin tests. The rat RSC is caudal to and send projections to the ipsilateral anterior cingulate cortex (ACC), which is also involved in pain processing. This study demonstrated that pre-treating the rostral (rACC), but not the caudal ACC with CoCl2 (1 mM), or the rACC ablation increased the duration of the antinociceptive effect evoked by a 15-s period of electrical stimulation (AC, 60 Hz, 20 μA) of the RSC in the rat tail-flick. Injecting the GABA-A antagonist bicuculline (50 ng/0.25 μL), but not the GABA-B antagonist phaclofen (300 ng/0.25 μL) into the rACC also increased the duration of the stimulation-induced antinociception from the RSC. In contrast, the effects of rACC stimulation persisted after the injection of CoCl2 (1 mM) into the RSC. The injection of CoCl2 into the rACC did not change the nociceptive behavior of rats during phase 1 of the formalin response but reduced licking response duration during phase 2. This effect was similar in sham or stimulated animals at the RSC. We conclude that the antinociceptive effect of stimulating the RSC in the rat tail-flick test is modulated by the rACC involving GABA-A receptors in this cortex. In contrast, the antinociceptive effect of stimulating the RSC in the formalin test does not involve the rACC. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Retrosplenial cortex (RSC) stimulation produces antinociception in the tail-flick and formalin tests (Reis et al., 2010) and reduces incision pain (Rossaneis et al., 2011) in rats. The RSC is consistently activated during noxious stimulation as revealed by neuroimaging studies in rats (Hess et al., 2007). In addition, regional blood flow is higher in the RSC of fibromyalgic patients compared to control patients (Wik et al., 2003). In contrast, regional blood flow in the RSC of fibromyalgic patients is reduced during externally induced acute pain (Wik et al., 2006). A micro-PET analysis of the rat brain after spinal nerve ligation revealed decreased metabolism in the RSC (Kim et al., 2014). The rat RSC is caudal to and sends projections to the ipsilateral anterior cingulate cortex (ACC) (Vogt and Peters, 1981; Van Groen and Wiss, 2003), which is a brain region involved in pain processing (Quintero, 2013). The stimulation of the rostral ACC (rACC), which is also known as cingulum 1 (Johansen et al., 2001; Paxinos and Watson, 2005), is hyperalgesic (Calejesan et al., 2000; Ohara et al., 2005), and its neural blockade produces analgesia in rats (Vaccarino and Melzack, 1989). In ⁎ Corresponding author at: Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, Av. Bandeirantes 3900, CEP 14049-900 Ribeirão Preto, SP, Brazil. Tel.: +55 16 36023038; fax: +55 16 36332301. E-mail address: [email protected] (W.A. Prado).
http://dx.doi.org/10.1016/j.pbb.2015.02.006 0091-3057/© 2015 Elsevier Inc. All rights reserved.
addition, the rACC mediates the aversive component of pain (Johansen et al., 2001; Kim et al., 2010). An increase in fMRI signaling in the rACC and RSC was discovered in a rat model of pancreatitis-induced abdominal pain (Westlund et al., 2009). The ACC neuronal activity increases during escape from a noxious thermal stimulus (Hutchinson et al., 1999; Koyama et al., 2001; Iwata et al., 2005), and cingulotomy has been proposed for the treatment of several types of chronic pain (Foltz and White, 1962; Fuchs et al., 2014). The rACC activity is necessary for the perception of the aversive component of inflammatory pain in rodents (Johansen et al., 2001). Enhanced transport of Mn2 + observed in fMRI images during noxious stimulation of a rat forepaw were found in several brain regions, including the rACC and RSC; however, only the enhancement in RSC was not attenuated by intraperitoneal morphine (Yang et al., 2011). These findings led to the notion that antinociception evoked by RSC stimulation is not due to spreading to the rACC. However, rACC involvement in the antinociceptive effect of RSC stimulation has yet to be conducted. The present study evaluates whether rACC modulates the stimulationinduced antinociception from the RSC. Changes produced by injecting CoCl2 into or surgical ablation of the rACC against the antinociceptive effect of RSC stimulation was evaluated using the tail-flick and formalin tests in rats. Similar experiments were conducted by injecting GABA-A (bicuculline) or GABA-B (phaclophen) receptor antagonists into the
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rACC because GABA receptors were already demonstrated in this region (Mengod et al., 1990; Bozkurt et al., 2005; Luna-Munguía et al., 2005; Rivera et al., 2008; Palomero-Gallagher et al., 2009). Because a caudal portion of the ACC (cACC) exists between the RSC and the rACC, we also examined whether injecting CoCl2 into the cACC changes the stimulation-induced antinociception from the RSC in the rat tail-flick test. 2. Materials and methods 2.1. Subjects and surgery Male Wistar rats (140–160 g) were housed two to a cage with free access to food and water and maintained at a controlled temperature (23 ± 1 °C) with a 12-h light–dark cycle before and after surgery (light cycle beginning at p.m. 7:00 h). The experiments were approved by the Commission of Ethics in Animal Research, Faculty of Medicine of Ribeirão Preto, University of São Paulo (Protocol number 240/2005). The proposals of the Committee for Research and Ethical Issues of IASP (Zimmermann, 1983) were followed throughout the experiments. Each animal was anesthetized with tribromoethanol (250 mg/kg, i.p.), and a Teflon-insulated monopolar electrode (o.d. = 0.125 mm) was stereotaxically implanted into the skull to lie in the left RSC using the coordinates (in mm) AP = 5.0 (from ear bars); L = 1.0 (from the midline) and H = −1.9 (from the skull surface). A 12-mm length of a 23-gauge stainless steel guide cannula was implanted stereotaxically into the skull until its tip was 0.5 mm above the left rACC using the coordinates (in mm) AP = 9.2; L = 1.0, and H = −2.0. A 12-mm length of a 23-gauge stainless steel guide cannula was implanted stereotaxically into the skull until its tip was 0.5 mm above the left rACC using the coordinates (in mm) AP = 9.2; L = 1.0, and H = − 2.0, or 0.5 mm above the left cACC using the coordinates (in mm) AP = 8.0; L = 1.0, and H = −2.0. The electrode and guide cannula were fixed to the skull with two screws and dental cement. One screw was used as the reference electrode. The guide cannula was kept patent with a sterile obturator until the time of drug administration. Animals were then given penicillin (50 mg/kg, i.m.) and allowed to recover for at least one week before the experiment. Cortical ablations were performed under anesthesia with tribromoethanol (250 mg/kg, i.p.). Animals were placed in a stereotaxic frame and an electrode was implanted into the left RSC. A window including the rACC was then opened in the skull and the rACC was removed by gentle aspiration as previously validated (Lamas et al., 2013). Sham ablated rats also had a window opened in the skull but no aspiration was performed. Rats were given penicillin (50 mg/kg, i.m.) after the ablations and allowed to recover for at least one week before the experiment. 2.2. Tail-flick test Each animal was placed in a ventilated tube with the tail laid across a wire coil maintained at room temperature (23 ± 2 °C). The coil temperature was then increased by the passage of an electric current, and the latency for the tail withdrawal reflex was measured. Heat was applied to a portion of the ventral surface of the tail between 4 and 6 cm from the tip. Tail-flick latency (TFL) was measured in 5-min intervals until a stable baseline was obtained over three or four consecutive trials. The apparatus was fixed to obtain a baseline TFL at approximately 3 (for rACC experiments) or 9 (for cACC experiments) seconds. Only rats showing stable baseline TFL after up to 6 trials were used in each experiment. Each trial was terminated after 6 (for RSC stimulation) or 15 (for ACC stimulation) seconds to minimize the possibility of skin damage. 2.3. Formalin test Rats pre-treated with CoCl2 or saline in the rACC were stimulated at the RSC for 15 s with a current intensity of 20 μA. Soon after this
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procedure, 5% formalin (50 μL) was injected subcutaneously into the dorsal surface of a hind paw with a 25-gauge needle. The hind paw contralateral to the cortical targets was chosen according to Reis et al. (2012). The number of spontaneous injected paw flinches, and the total time in which the animal licked the injected paw were counted during the 5 min of post-injection period (phase 1 of the response to formalin). Separated groups of rats were stimulated at the RSC, 10 min after formalin. The number of spontaneous injected paw flinching, and the total time in which the animal licked the injected paw were counted every 5-min for up to 60 min after formalin (phase 2 of the response to formalin). Flinching was characterized as a rapid and brief withdrawal or flexion of the injected paw. 2.4. Stimulation procedures Twenty minutes after the intracerebral injection, electrical stimulation (AC, 60 Hz) was applied during 15 s to the cortical target at an intensity of 20 μA as suggested elsewhere (Reis et al., 2010). During the stimulation period, rats were gently restrained by hand, and the drop in voltage across a 1-kΩ resistor in series with the electrode was continuously monitored on an oscilloscope. The TFL was recorded 30 s after cortical stimulation and then at a 5-min interval for up to 30 min. No attempt was made to test for the presence of antinociception during stimulation. Sham stimulated rats (control) had identical electrode implant procedures and connections to the stimulator assembly. They also received either saline or a drug into the rACC or cACC but no current was passed through the electrode. 2.5. Intracerebral injection A drug or vehicle was microinjected into the rACC or cACC, or RSC using a glass needle (70–90 μm, o.d.) protected by an assembly of telescoping steel tubes as proposed elsewhere (Azami et al., 1980). The assembly was inserted into the guide cannula and the needle advanced to protrude 1.0 mm beyond the guide cannula tip. The volume of microinjection was 0.25 μL, delivered at a constant rate over a period of 3 min. The needle was removed 20 s after completion of the injection. 2.6. Histology At the end of the experiments, rats were deeply anesthetized with intraperitoneal sodium thiopental (60 mg/kg) and perfused through the heart with 4% paraformaldehyde in 0.1 M phosphate buffered saline. Fast green (0.5 μL) was injected through the guide cannula to label the site of intracerebral injection. The brain was removed and the electrode track or dye spot localized from 50-μm serial coronal sections was stained with neutral red and identified on diagrams from the atlas of Paxinos and Watson (2005). Only animals that had the electrode or dye spot position confirmed by histology were considered for data analysis. 2.7. Experimental design 2.7.1. Effects of neural block of rACC or cACC on the stimulation-produced antinociception from the RSC in tail-flick test Four groups of 8 rats each had an electrode implanted into the rACC and a guide cannula into the RSC. Another four groups of 8 rats each had an electrode implanted into the cACC and a guide cannula into the RSC. Saline (0.25 μL) or 1 mM CoCl2 (0.25 μL) was injected into the rACC or cACC soon after recording of the baseline TFL. Electrical or sham stimulation of the RSC was performed 20 min later, and TFL was recorded within 30 s after the end of the stimulation period and then for up to 30 min at 5-min intervals. Saline/sham stimulated rats were taken as control.
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2.7.2. Effect of ablation of rACC on the stimulation-produced antinociception from the RSC in tail-flick test Cortical ablations were performed in two groups of 8 rats each. One week after real or sham ablation of rACC the animals were taken for recording of the baseline TFL at 5 min intervals. Electrical or sham stimulation of the RSC was then performed and TFL was recorded within 30 s after the end of the stimulation period and then for up to 30 min at 5-min intervals. Sham ablation/sham stimulated rats were taken as control. 2.7.3. Effect of injecting antagonists into the rACC on the stimulationinduced antinociception from the RSC in tail-flick test Saline (0.25 μL), bicuculline (50 ng/0.25 μL) or phaclophen (300 ng/ 0.25 μL) was injected into the rACC soon after recording of the baseline TFL. Electrical or sham stimulation of the RSC was performed 20 min later, and TFL was recorded within 30 s after the end of the stimulation period and then for up to 30 min at 5-min intervals. Saline/sham stimulated rats were taken as control. 2.7.4. Effects of neural block of RSC on the stimulation-produced antinociception from the rACC in tail-flick test Saline (0.25 μL) or 1 mM CoCl2 (0.25 μL) was injected into the RSC or soon after recording of the baseline TFL. Electrical or sham stimulation of the rACC was performed 20 min later, and TFL was recorded within 30 s after the end of the stimulation period and then for up to 30 min at 5-min intervals. Saline/sham stimulated rats were taken as control. 2.7.5. Effect of neural block of the rACC on stimulation-produced antinociception from the RSC in formalin test Formalin (50 μL) was injected subcutaneously in rats pre-treated with CoCl2 or saline in the rACC and the animals were soon submitted to the electrical or sham stimulation of the RSC. The number of spontaneous paw flinches, and the total time in which the animal licked the injected paw were counted during 5 min after formalin (phase 1). In another groups of rats pre-treated with CoCl2 or saline in the rACC, formalin (50 μL) was injected subcutaneously. Ten min later these animals were submitted to electrical or sham stimulation of the RSC and the number of spontaneous injected paw flinching, and the total time in which the animal licked the injected paw were counted every 5-min for up to 60 min after formalin (phase 2). Saline/sham stimulated rats were taken as control in both occasions. 2.8. Drugs Cobalt chloride hexahydrate (CoCl2) was purchased from SigmaAldrich (St. Louis, MO, USA). Bicuculline methiodide and phaclofen were purchased from RBI/Sigma (Natick, MA, USA). Phaclofen was dissolved in 5% dimethyl sulfoxide (DMSO). The other drugs were dissolved in saline. Doses of GABA receptor antagonists were as previously reported (Gilbert and Franklin, 2001; Ciriello and Roder, 1999; Villarreal and Prado, 2007). 2.9. Data analysis Tail-flick latencies (in seconds) are reported as the means ± SD. Comparisons between control and test groups were made by multivariate analysis of variance (MANOVA) with repeated measures to compare the groups over all times. The factors analyzed were treatments, time and treatment × time interaction. In the case of treatment × time interaction, one-way analysis of variance followed by Duncan's test was performed for each time. Data from the formalin tests were compared by ANOVA followed by a Bonferroni post hoc test. The analysis was performed using the statistical software package SPSS/PC+, version 17.0. The level of significance was set at P b 0.05 in all cases.
3. Results 3.1. Effects of neural block or ablation of the rACC or neural block of the cACC on the stimulation-produced antinociception from the RSC in tail-flick test Electrical stimulation of the RSC performed 20 min after injection of saline (0.25 μL) into the rACC (Fig. 1A) or cACC (Fig. 1B), or in rats with sham rACC ablation (Fig. 1C) evoked a prompt increase of the TFL that remained significantly above the control for at least 5 min after stimulation ended. Electrical stimulation of the RSC was performed 20 min after the injection of 1 mM CoCl2 (0.25 μL) into the rACC also had a prompt increase of the TFL, but the effect remained significantly above control for at least 15 min (Fig. 1A). In contrast, RSC stimulation 20 min after the injection of 1 mM CoCl2 (0.25 μL) into the cACC also evoked a prompt increase of the TFL, but the effect did not differ significantly from control regarding the intensity or duration (Fig. 1B). Sham RSC stimulation in rats with sham or real rACC ablation resulted in TFL that did not differ significantly from baseline throughout the observation period. In contrast, RSC stimulation in rats with rACC ablation also induced a prompt increase of the TFL, but the effect remained significantly above control for at least 15 min (Fig. 1C). The curves in Fig. 1A, B and C were significantly different regarding treatment (F3,28 = 11.82; F3,20 = 12.11 and F3,28 = 15.52, respectively), time (F14,392 = 25; F14,280 = 11.56 and F14,392 = 84.31, respectively), and the treatment × time interaction (F42,392 = 13.00; F42,280 = 4.89 and F42,392 = 29.56, respectively), p b 0.0001 in all cases. The locations of the electrode tips in the RSC (Fig. 1A and B) and dye spots in the rACC (Fig. 1A) and cACC (Fig. 1B) are shown in insects. The time during which TFL remained significantly above control after RSC stimulation was significantly longer in rats treated with CoCl2 than in rats treated with saline in the rACC (Fig. 2A). The time in which TFL remained significantly above control after RSC stimulation was significantly longer in rats with rACC ablation than in rats with sham ablation (Fig. 2B). No rat displayed gross disturbance of motor coordination during or after the period of brain stimulation. Animals walked normally and responded to non-noxious stimulation throughout the period of observation. 3.2. Effect of injecting bicuculline or phaclofen into the rACC on the stimulation-induced antinociception from the RSC in tail-flick test Sham RSC stimulation performed 20 min after the injection of saline (0.25 μL) or bicuculline (50 ng/0.25 μL) (Fig. 3A), and 5% DMSO (0.25 μL) or phaclophen (300 ng/0.25 μL) (Fig. 3B) resulted TFL's that were similar to baseline throughout the observation period. RSC stimulation performed 20 min after the saline injection into the rACC evoked a prompt increase of TFL that remained significantly above control for at least 5 min. RSC stimulation performed 20 min after the injection of bicuculline into the ACC also increased the TFL, the effect remaining significantly above control for at least 15 min (Fig. 3A). In contrast, RSC stimulation conducted 20 min after the injection of phaclofen into the rACC evoked an anti-nociceptive effect that did not differ from control in intensity or duration (Fig. 3B). The curves in Fig. 3A and B were significantly different for treatment (F3,28 = 107.64 and 72.29, respectively), time (F13,364 = 88.01 and 120.21, respectively), and the treatment × time interaction (F39,364 = 24.09 and 29.59, respectively), P b 0.0001 in all cases. 3.3. Effect of neural block of the RSC on the reduction of TFL provoked by the stimulation of the ACC Experiments were conducted using a slower increase in coil temperature so that baseline TFL was reached in approximately 9 s. Sham ACC stimulation in rats treated with 1 mM CoCl2 (0.25 μL) or saline (0.25 μL) had TFL that did not differ from baseline throughout the observation
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Fig. 2. The changes in time during which tail-flick latency remained significantly above control after electrical stimulation (ES) of the retrosplenial cortex: (A) in rats injected with saline (0.25 μL) or CoCl2 (1 mM/0.25 μL) in the ipsilateral rostral anterior cingulate cortex; (B) in rats submitted to surgical ablation (abl) of the ipsilateral rostral anterior cingulate cortex. Bars represent the means ± S.D. of 8 animals per group. p b 0.05 compared to sham-stimulated rats (*) or to the remaining groups (#).
3.4. Effect of neural block of the rACC on stimulation-produced antinociception from the RSC in formalin test
Fig. 1. Changes produced by the injection (arrow 1) of saline (sal = 0.25 μL) or CoCl2 (Co = 1 mM/0.25 μL) into the rostral (A) or caudal (B) anterior cingulated cortex on the antinociception induced by the electrical stimulation (ES) (arrow 2) of the ipsilateral retrosplenial cortex of rats in the tail-flick test. (C) Changes produced by the surgical ablation of the rostral anterior cingulated cortex on the antinociception induced by the ES (arrow 2) of the retrosplenial cortex in the rat tail-flick test. The site location of CoCl2 injection into the rostral or caudal anterior cingulated cortex or sites of retrosplenial cortex stimulation is shown on diagrams by Paxinos and Watson (2005). Points represent the means ± S.D. of 8 animals. *p b 0.05 compared to sham-stimulated rats.
period. In contrast, rACC stimulation performed 20 min after the injection of either 1 mM CoCl2 or saline into the RSC evoked a significant reduction in the TFL compared to sham rACC stimulated rats previously treated with saline in the RSC (F3,23 = 72.08; p b 0.0001). However, these effects were not significantly different and lasted less than 5 min (Fig. 4).
Rats injected subcutaneously with saline displayed a small number of spontaneous flinches and had small total time spent licking the injected paw. In addition, these effects were not significantly different in groups of rats submitted to sham or real RSC stimulation (Fig. 5). Phase 1 of the nociceptive responses to the subcutaneous injection of 5% formalin (50 μL) in rats injected with saline (0.25 μL) into the rACC was characterized by a significant increase in the number of spontaneous flinches (Fig. 5A) and in the total time licking the injected paw (Fig. 5B), compared to the response of rats injected with subcutaneous saline. Phase 1 of both nociceptive responses to formalin was significantly inhibited by the RSC stimulation in rats treated with 1 mM CoCl2 (0.25 μL) or saline (0.25 μL) in the rACC. The number of spontaneous flinches during phase 2 of the responses to formalin was not significantly changed by the RSC stimulation in rats treated with saline (0.25 μL) or 1 mM CoCl2 (0.25 μL) in the rACC (Fig. 5C). In contrast, the duration of licking during phase 2 of the formalin response in rats treated with 1 mM CoCl2 (0.25 μL) into the rACC was significantly shorter than in rats treated with saline (0.25 μL). However, this effect was not different in rats subjected to sham or real RSC stimulation (Fig. 5D). The data in Fig. 5A–D were significantly different (F5,47 = 13.39; 165.6; 41.50 and 45.44, respectively; P b 0.0001 in all cases).
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Fig. 4. Changes of tail-flick latency in rats stimulated electrically (ES) in the rostral anterior cingulate cortex after the injection of saline (0.25 μL) or CoCl2 (1 mM/0.25 μL) into the ipsilateral retrosplenial cortex. The site location of CoCl2 injection into the retrosplenial cortex or sites of rostral anterior cingulated cortex stimulation is shown on diagrams by Paxinos and Watson (2005). Bars represent the means ± S.D. of 8 animals. p b 0.05 compared to sham-stimulated rats (*).
Fig. 3. Changes produced by the injection (arrow 1) of saline (sal = 0.25 μL), bicuculline (bic = 50 ng/0.25 μL) (A) or phaclophen (phac = 300 ng/0.25 μL) (B) into the rostral anterior cingulate cortex on the antinociception induced by the electrical stimulation (ES) (arrow 2) of the ipsilateral retrosplenial cortex in the rat tail-flick test. The site location of drug injection into the rostral anterior cingulated cortex or sites of retrosplenial cortex stimulation is shown on diagrams by Paxinos and Watson (2005). Points represent the means ± S.D. of 8 animals. p b 0.05 compared to sham-stimulated rats (*).
4. Discussion These results confirm that electrical stimulation of the RSC is antinociceptive in the rat tail-flick test and inhibits the phase 1, but not the phase 2 responses of rats to formalin as previously demonstrated (Reis et al., 2010). In addition, results demonstrate that the injection of CoCl2 into or ablation of the rACC prolonged the antinociceptive effect of stimulating the RSC. The stimulation-induced antinociception from the RSC was not changed in rats treated with CoCl2 in the cACC. The intracerebral injection of CoCl2 reversibly blocks synaptic transmission but not passage fibers (Kretz, 1984). Therefore, the antinociceptive effect of stimulating the RSC in the tail-flick test is at least partially modulated by the rACC, but does not involve the cACC. The injection of the bicuculline (a GABA-A antagonist), but not phaclofen (a GABA-B antagonist) into the rACC also prolonged the stimulation-induced antinociception from the RSC in the tail-flick test. A dose–response study about the effects of GABA receptor antagonists in the rACC have not been conducted yet. However, the doses of bicuculline and phaclophen used here were similar to those shown earlier to be effective in the anterior pretectal nucleus to change the post-surgical allodynia in rats (Villarreal and Prado, 2007), in the rostral ventromedial medulla to decrease the response of rats to formalin (Gilbert and Franklin, 2001), and in the central nucleus of the amygdala
to inhibit the cardiovascular responses of rats to the injection of glutamate into the same nucleus (Ciriello and Roder, 1999). Consequently, the lack of effect of phaclophen in this study is unlikely the result of an inadequate dose of this antagonist. The presence of GABA receptors, in addition to opioid, serotonergic, dopaminergic and glutamatergic receptors has been demonstrated in the ACC (Mengod et al., 1990; Bozkurt et al., 2005; Luna-Munguía et al., 2005; Rivera et al., 2008; Palomero-Gallagher et al., 2009). Furthermore, inhibitory synaptic transmission within the rACC is mediated by GABA (Descalzi et al., 2009). Therefore, the modulation of the antinociceptive effect of stimulating the RSC in the rat tail-flick test involves GABA-A receptors in the rACC. The presented results also provide additional support for a role of the rACC in supraspinal processing of noxious events and suggest that rACC GABA-A receptors significantly contribute to this processing as proposed elsewhere (LaGraize and Fuchs, 2007). We demonstrated that rACC stimulation reduced the TFL, thus confirming previous demonstration that stimulating the rACC results in hyperalgesia (Calejesan et al., 2000; Tang et al., 2005). In addition, we have shown that the stimulation-induced hyperalgesia from the rACC was not changed in rats treated with CoCl2 in the RSC. Therefore, the RSC is not involved with the pronociceptive effect of the rACC stimulation. The effectiveness of stimulating RSC against phase 1 was the same in rats treated with saline or CoCl2 into the rACC. Injecting CoCl2 into the rACC did not change the rat response during the phase 1, but reduced the licking response duration during phase 2 of the same test as reported elsewhere (Donahue et al., 2001), this effect being similar in sham animals or those stimulated at the RSC. Therefore, the antinociceptive effect of stimulating the RSC in the formalin test does not involve the rACC. By this reason, the participation of the cACC in the modulation of RSC-induced antinociception in the formalin test was not conducted in this study. The stimulation-induced antinociception from the RSC in the rat tailflick test did not occur in animals with dorsolateral funiculus lesions (Reis et al., 2010), which is the main route through which pain inhibition pathways descend to the spinal cord (Millan, 1999). The rACC exerts its hyperalgesic effect by modulating the descending pain inhibition pathways (see Ohara et al., 2005). Our results suggest that rACC exerts an inhibitory role on the stimulation-induced antinociception from the RSC in the tail-flick test, but is not involved in the modulating the effects
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Fig. 5. Changes produced by saline (0.25 μL) or CoCl2 (1 mM/0.25 μL) injection into the rostral anterior cingulate cortex on the effects of electrical stimulation (ES) of the ipsilateral retrosplenial cortex against phases 1 (A and B) and 2 (C and D) of the nociceptive responses of rats to the subcutaneous injection of saline (50 μL) or 5% formalin (50 μL) into the hind paw contralateral to the cortical targets. Stimulation (20 μA) was applied for 15 s immediately before (phase 1) or 10 min after (phase 2) the formalin injection. The number of spontaneous flinching of the injected paw, and the total time during which the animal licked the injected paw, were counted for 5 min after formalin (phase 1) or from 10 to 60 min after formalin (phase 2). The site location of CoCl2 injection into the rostral anterior cingulated cortex or sites of retrosplenial cortex stimulation is shown on diagrams by Paxinos and Watson (2005). Bars represent the means ± S.D. of 8 rats per group. p b 0.05 compared to group sal/sham (*) or formalin/sham (#).
of stimulating the RSC against phase 1 of the animal response to formalin. Therefore, we suggest that the antinociceptive effects of RSC stimulation in the tail-flick and formalin tests might be mediated by different neural systems. The ACC itself send inputs to the periaqueductal gray and the rostral ventral medulla, which are structures involved in the descending endogenous analgesia system (see Millan, 2002). Stimulation of the ACC facilitated spinal tail-flick reflex by acting through brainstem descending pain modulation system probably involving the mesencephalic periaqueductal gray (Fuchs et al., 2014) and the rostral ventral medulla (Calejesan et al., 2000). However, the ACC also send direct descending projections to the spinal dorsal horn, and these projections are activated following peripheral nerve injury (Chen et al., 2014). In summary, the results presented herein show an inhibitory role of the rACC via GABA-A receptors in a pathway activated from the RSC to produce antinociception in the rat tail-flick test. In contrast, the antinociceptive effect of stimulating the RSC in the formalin test does not involve the rACC. Acknowledgments G.M.R. was a recipient of FAPESP fellowship. The authors greatly appreciate the technical assistance of M.A. Carvalho and P.R. Castania. References Azami J, Llewelyn MB, Roberts MHT. An extra-fine assembly for intracerebral microinjection. J Physiol 1980;305:18P–9P.
Bozkurt A, Zilles K, Schleicher A, Kamper L, Arigita ES, Uylings HB, et al. Distributions of transmitter receptors in the macaque cingulate cortex. Neuroimage 2005;25:219–29. Calejesan AA, Kim SJ, Zhuo M. Descending facilitatory modulation of a behavioral nociceptive response by stimulation in the adult rat anterior cingulate cortex. Eur J Pain 2000;4: 83–96. Chen T, Koga K, Descalzi G, Qiu S, Wang J, Zhang LS, et al. Postsynaptic potentiation of corticospinal projecting neurons in the anterior cingulate cortex after nerve injury. Mol Pain 2014;10:33. Ciriello J, Roder S. GABAergic effects on the depressor responses elicited by stimulation of central nucleus of the amygdala. Am J Physiol 1999;276:H242–7. Descalzi G, Kim S, Zhuo M. Presynaptic and postsynaptic cortical mechanisms of chronic pain. Mol Neurobiol 2009;40:253–9. Donahue RR, LaGraize SC, Fuchs PN. Electrolytic lesion of the anterior cingulate cortex decreases inflammatory, but not neuropathic nociceptive behavior in rats. Brain Res 2001;897:131–8. Foltz EL, White Jr LE. Pain “relief” by frontal cingulumotomy. J Neurosurg 1962;19: 89–100. Fuchs PN, Peng YB, Boyette-Davis JA, Uhelski ML. The anterior cingulate cortex and pain processing. Front Integr Neurosci 2014;8:35. Gilbert AK, Franklin KB. GABAergic modulation of descending inhibitory systems from the rostral ventromedial medulla (RVM). Dose–response analysis of nociception and neurological deficits. Pain 2001;90:25–36. Hess A, Sergejeva M, Budinsky L, Zeilhofer HU, Brune K. Imaging of hyperalgesia in rats by functional MRI. Eur J Pain 2007;11:109–19. Hutchinson WD, Davis KD, Lozano AM, Tasker RR, Dostrovsky JO. Pain-related neurons in the human cingulate cortex. Nat Neurosci 1999;2:403–5. Iwata K, Kamo H, Ogawa A, Tsuboi Y, Noma N, Mitsuhashi Y, et al. Anterior cingulate cortical neuronal activity during perception of noxious stimuli in monkeys. J Neurophysiol 2005;94:1980–91. Johansen JP, Fields HL, Manning BH. The affective component of pain in rodents: direct evidence for a contribution of the anterior cingulate cortex. Proc Natl Acad Sci U S A 2001;98:8077–82. Kim SS, Descalzi G, Zhuo M. Investigation of molecular mechanism of chronic pain in the anterior cingulate cortex using genetically engineered mice. Curr Genomics 2010;11: 70–6.
118
G.M. Reis et al. / Pharmacology, Biochemistry and Behavior 131 (2015) 112–118
Kim CE, Kim YK, Chung G, Im HJ, Lee DS, Kim J, et al. Identifying neuropathic pain using (18)F-FDG micro-PET: a multivariate pattern analysis. Neuroimage 2014;86:311–6. Koyama T, Kato K, Tanaka YZ, Mikami A. Anterior cingulate activity during pain-avoidance and reward tasks in monkeys. Neurosci Res 2001;39:421–30. Kretz R. Local cobalt injection: a method to discriminate presynaptic axonal from postsynaptic neuronal activity. J Neurosci Methods 1984;11:129–35. LaGraize SC, Fuchs PN. GABA-A but not GABA-B receptors in the rostral anterior cingulate cortex selectively modulate pain-induced escape/avoidance behavior. Exp Neurol 2007;204:182–94. Lamas V, Alvarado JC, Carro J, Merchán MA. Long-term evolution of brainstem electrical evoked responses to sound after restricted ablation of the auditory cortex. PLoS One 2013;8:e73585. Luna-Munguía H, Manuel-Apolinar L, Rocha L, Meneses A. 5-HT1A receptor expression during memory formation. Psychopharmacology (Berl) 2005;181:309–18. Mengod G, Nguyen H, Le H, Waeber C, Lübbert H, Palacios JM. The distribution and cellular localization of the serotonin 1C receptor mRNA in the rodent brain examined by in situ hybridization histochemistry. Comparison with receptor binding distribution. Neuroscience 1990;35:577–91. Millan MJ. The induction of pain: an integrative review. Prog Neurobiol 1999;57:1–164. Millan MJ. Descending control of pain. Prog Neurobiol 2002;66:355–474. Ohara PT, Vit JP, Jasmin L. Cortical modulation of pain. Cell Mol Life Sci 2005;62:44–52. Palomero-Gallagher N, Vogt BA, Schleicher A, Mayberg HS, Zilles K. Receptor architecture of human cingulate cortex: evaluation of the four-region neurobiological model. Hum Brain Mapp 2009;30:2336–55. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. New York: Academic Press; 2005. Quintero GC. Advances in cortical modulation of pain. J Pain Res 2013;6:713–25. Reis GM, Dias QM, Silveira JW, Del Vecchio F, Garcia-Cairasco N, Prado WA. Antinociceptive effect of stimulating the occipital or retrosplenial cortex in rats. J Pain 2010;11:1015–26. Reis GM, Rossaneis AC, Silveira JW, Prado WA. μ1- and 5-HT1-dependent mechanisms in the anterior pretectal nucleus mediate the antinociceptive effects of retrosplenial cortex stimulation in rats. Life Sci 2012;90:950–5.
Rivera A, Peñafiel A, Megías M, Agnati LF, López-Téllez JF, Gago B, et al. Cellular localization and distribution of dopamine D(4) receptors in the rat cerebral cortex and their relationship with the cortical dopaminergic and noradrenergic nerve terminal networks. Neuroscience 2008;155:997–1010. Rossaneis AC, Reis GM, Prado WA. Stimulation of the occipital or retrosplenial cortex reduces incision pain in rats. Pharmacol Biochem Behav 2011;100:220–7. Tang J, Ko S, Ding HK, Qiu CS, Calejesan AA, Zhuo M. Pavlovian fear memory induced by activation in the anterior cingulate cortex. Mol Pain 2005;1:6. Vaccarino AL, Melzack R. Analgesia produced by injection of lidocaine into the anterior cingulum bundle of the rat. Pain 1989;39:213–9. Van Groen T, Wyss JM. Connections of the retrosplenial granular b cortex in the rat. J Comp Neurol 2003;463:249–63. Villarreal CF, Prado WA. Modulation of persistent nociceptive inputs in the anterior pretectal nucleus of the rat. Pain 2007;132:42–52. Vogt BA, Peters A. Form and distribution of neurons in rat cingulated cortex: areas 32, 24, and 29. J Comp Neurol 1981;195:603–25. Westlund KN, Vera-Portocarrero LP, Zhang L, Wei J, Quast MJ, Cleeland CS. fMRI of supraspinal areas after morphine and one week pancreatic inflammation in rats. Neuroimage 2009;44:23–34. Wik G, Fischer H, Brage'e B, Kristianson M, Fredrikson M. Retrosplenial cortical activation in the fibromyalgia syndrome. Neuroreport 2003;14:619–21. Wik G, Fischer H, Finer B, Bragee B, Kristianson M, Fredrikson M. Retrospenial cortical deactivation during painful stimulation of fibromyalgic patients. Int J Neurosci 2006;116:1–8. Yang PF, Chen DY, Hu JW, Chen JH, Yen CT. Functional tracing of medial nociceptive pathways using activity-dependent manganese-enhanced MRI. Pain 2011;152: 194–203. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983;16:109–10.
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Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
The antinociceptive effect of stimulating the retrosplenial cortex in the rat tail-flick test but not in the formalin test involves the rostral anterior cingulate cortex Gláucia Melo Reis, Rafael Sobrano Fais, Wiliam A. Prado ⁎ Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, University of Sao Paulo, Ribeirão Preto, SP 14049-900, Brazil
a r t i c l e
i n f o
Article history: Received 13 November 2014 Received in revised form 2 February 2015 Accepted 6 February 2015 Available online 14 February 2015 Keywords: Antinociception Retrosplenial cortex Anterior cingulate cortex Tail-flick test
a b s t r a c t The stimulation of the retrosplenial cortex (RSC) is antinociceptive in the rat tail-flick and formalin tests. The rat RSC is caudal to and send projections to the ipsilateral anterior cingulate cortex (ACC), which is also involved in pain processing. This study demonstrated that pre-treating the rostral (rACC), but not the caudal ACC with CoCl2 (1 mM), or the rACC ablation increased the duration of the antinociceptive effect evoked by a 15-s period of electrical stimulation (AC, 60 Hz, 20 μA) of the RSC in the rat tail-flick. Injecting the GABA-A antagonist bicuculline (50 ng/0.25 μL), but not the GABA-B antagonist phaclofen (300 ng/0.25 μL) into the rACC also increased the duration of the stimulation-induced antinociception from the RSC. In contrast, the effects of rACC stimulation persisted after the injection of CoCl2 (1 mM) into the RSC. The injection of CoCl2 into the rACC did not change the nociceptive behavior of rats during phase 1 of the formalin response but reduced licking response duration during phase 2. This effect was similar in sham or stimulated animals at the RSC. We conclude that the antinociceptive effect of stimulating the RSC in the rat tail-flick test is modulated by the rACC involving GABA-A receptors in this cortex. In contrast, the antinociceptive effect of stimulating the RSC in the formalin test does not involve the rACC. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Retrosplenial cortex (RSC) stimulation produces antinociception in the tail-flick and formalin tests (Reis et al., 2010) and reduces incision pain (Rossaneis et al., 2011) in rats. The RSC is consistently activated during noxious stimulation as revealed by neuroimaging studies in rats (Hess et al., 2007). In addition, regional blood flow is higher in the RSC of fibromyalgic patients compared to control patients (Wik et al., 2003). In contrast, regional blood flow in the RSC of fibromyalgic patients is reduced during externally induced acute pain (Wik et al., 2006). A micro-PET analysis of the rat brain after spinal nerve ligation revealed decreased metabolism in the RSC (Kim et al., 2014). The rat RSC is caudal to and sends projections to the ipsilateral anterior cingulate cortex (ACC) (Vogt and Peters, 1981; Van Groen and Wiss, 2003), which is a brain region involved in pain processing (Quintero, 2013). The stimulation of the rostral ACC (rACC), which is also known as cingulum 1 (Johansen et al., 2001; Paxinos and Watson, 2005), is hyperalgesic (Calejesan et al., 2000; Ohara et al., 2005), and its neural blockade produces analgesia in rats (Vaccarino and Melzack, 1989). In ⁎ Corresponding author at: Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, Av. Bandeirantes 3900, CEP 14049-900 Ribeirão Preto, SP, Brazil. Tel.: +55 16 36023038; fax: +55 16 36332301. E-mail address: [email protected] (W.A. Prado).
http://dx.doi.org/10.1016/j.pbb.2015.02.006 0091-3057/© 2015 Elsevier Inc. All rights reserved.
addition, the rACC mediates the aversive component of pain (Johansen et al., 2001; Kim et al., 2010). An increase in fMRI signaling in the rACC and RSC was discovered in a rat model of pancreatitis-induced abdominal pain (Westlund et al., 2009). The ACC neuronal activity increases during escape from a noxious thermal stimulus (Hutchinson et al., 1999; Koyama et al., 2001; Iwata et al., 2005), and cingulotomy has been proposed for the treatment of several types of chronic pain (Foltz and White, 1962; Fuchs et al., 2014). The rACC activity is necessary for the perception of the aversive component of inflammatory pain in rodents (Johansen et al., 2001). Enhanced transport of Mn2 + observed in fMRI images during noxious stimulation of a rat forepaw were found in several brain regions, including the rACC and RSC; however, only the enhancement in RSC was not attenuated by intraperitoneal morphine (Yang et al., 2011). These findings led to the notion that antinociception evoked by RSC stimulation is not due to spreading to the rACC. However, rACC involvement in the antinociceptive effect of RSC stimulation has yet to be conducted. The present study evaluates whether rACC modulates the stimulationinduced antinociception from the RSC. Changes produced by injecting CoCl2 into or surgical ablation of the rACC against the antinociceptive effect of RSC stimulation was evaluated using the tail-flick and formalin tests in rats. Similar experiments were conducted by injecting GABA-A (bicuculline) or GABA-B (phaclophen) receptor antagonists into the
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rACC because GABA receptors were already demonstrated in this region (Mengod et al., 1990; Bozkurt et al., 2005; Luna-Munguía et al., 2005; Rivera et al., 2008; Palomero-Gallagher et al., 2009). Because a caudal portion of the ACC (cACC) exists between the RSC and the rACC, we also examined whether injecting CoCl2 into the cACC changes the stimulation-induced antinociception from the RSC in the rat tail-flick test. 2. Materials and methods 2.1. Subjects and surgery Male Wistar rats (140–160 g) were housed two to a cage with free access to food and water and maintained at a controlled temperature (23 ± 1 °C) with a 12-h light–dark cycle before and after surgery (light cycle beginning at p.m. 7:00 h). The experiments were approved by the Commission of Ethics in Animal Research, Faculty of Medicine of Ribeirão Preto, University of São Paulo (Protocol number 240/2005). The proposals of the Committee for Research and Ethical Issues of IASP (Zimmermann, 1983) were followed throughout the experiments. Each animal was anesthetized with tribromoethanol (250 mg/kg, i.p.), and a Teflon-insulated monopolar electrode (o.d. = 0.125 mm) was stereotaxically implanted into the skull to lie in the left RSC using the coordinates (in mm) AP = 5.0 (from ear bars); L = 1.0 (from the midline) and H = −1.9 (from the skull surface). A 12-mm length of a 23-gauge stainless steel guide cannula was implanted stereotaxically into the skull until its tip was 0.5 mm above the left rACC using the coordinates (in mm) AP = 9.2; L = 1.0, and H = −2.0. A 12-mm length of a 23-gauge stainless steel guide cannula was implanted stereotaxically into the skull until its tip was 0.5 mm above the left rACC using the coordinates (in mm) AP = 9.2; L = 1.0, and H = − 2.0, or 0.5 mm above the left cACC using the coordinates (in mm) AP = 8.0; L = 1.0, and H = −2.0. The electrode and guide cannula were fixed to the skull with two screws and dental cement. One screw was used as the reference electrode. The guide cannula was kept patent with a sterile obturator until the time of drug administration. Animals were then given penicillin (50 mg/kg, i.m.) and allowed to recover for at least one week before the experiment. Cortical ablations were performed under anesthesia with tribromoethanol (250 mg/kg, i.p.). Animals were placed in a stereotaxic frame and an electrode was implanted into the left RSC. A window including the rACC was then opened in the skull and the rACC was removed by gentle aspiration as previously validated (Lamas et al., 2013). Sham ablated rats also had a window opened in the skull but no aspiration was performed. Rats were given penicillin (50 mg/kg, i.m.) after the ablations and allowed to recover for at least one week before the experiment. 2.2. Tail-flick test Each animal was placed in a ventilated tube with the tail laid across a wire coil maintained at room temperature (23 ± 2 °C). The coil temperature was then increased by the passage of an electric current, and the latency for the tail withdrawal reflex was measured. Heat was applied to a portion of the ventral surface of the tail between 4 and 6 cm from the tip. Tail-flick latency (TFL) was measured in 5-min intervals until a stable baseline was obtained over three or four consecutive trials. The apparatus was fixed to obtain a baseline TFL at approximately 3 (for rACC experiments) or 9 (for cACC experiments) seconds. Only rats showing stable baseline TFL after up to 6 trials were used in each experiment. Each trial was terminated after 6 (for RSC stimulation) or 15 (for ACC stimulation) seconds to minimize the possibility of skin damage. 2.3. Formalin test Rats pre-treated with CoCl2 or saline in the rACC were stimulated at the RSC for 15 s with a current intensity of 20 μA. Soon after this
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procedure, 5% formalin (50 μL) was injected subcutaneously into the dorsal surface of a hind paw with a 25-gauge needle. The hind paw contralateral to the cortical targets was chosen according to Reis et al. (2012). The number of spontaneous injected paw flinches, and the total time in which the animal licked the injected paw were counted during the 5 min of post-injection period (phase 1 of the response to formalin). Separated groups of rats were stimulated at the RSC, 10 min after formalin. The number of spontaneous injected paw flinching, and the total time in which the animal licked the injected paw were counted every 5-min for up to 60 min after formalin (phase 2 of the response to formalin). Flinching was characterized as a rapid and brief withdrawal or flexion of the injected paw. 2.4. Stimulation procedures Twenty minutes after the intracerebral injection, electrical stimulation (AC, 60 Hz) was applied during 15 s to the cortical target at an intensity of 20 μA as suggested elsewhere (Reis et al., 2010). During the stimulation period, rats were gently restrained by hand, and the drop in voltage across a 1-kΩ resistor in series with the electrode was continuously monitored on an oscilloscope. The TFL was recorded 30 s after cortical stimulation and then at a 5-min interval for up to 30 min. No attempt was made to test for the presence of antinociception during stimulation. Sham stimulated rats (control) had identical electrode implant procedures and connections to the stimulator assembly. They also received either saline or a drug into the rACC or cACC but no current was passed through the electrode. 2.5. Intracerebral injection A drug or vehicle was microinjected into the rACC or cACC, or RSC using a glass needle (70–90 μm, o.d.) protected by an assembly of telescoping steel tubes as proposed elsewhere (Azami et al., 1980). The assembly was inserted into the guide cannula and the needle advanced to protrude 1.0 mm beyond the guide cannula tip. The volume of microinjection was 0.25 μL, delivered at a constant rate over a period of 3 min. The needle was removed 20 s after completion of the injection. 2.6. Histology At the end of the experiments, rats were deeply anesthetized with intraperitoneal sodium thiopental (60 mg/kg) and perfused through the heart with 4% paraformaldehyde in 0.1 M phosphate buffered saline. Fast green (0.5 μL) was injected through the guide cannula to label the site of intracerebral injection. The brain was removed and the electrode track or dye spot localized from 50-μm serial coronal sections was stained with neutral red and identified on diagrams from the atlas of Paxinos and Watson (2005). Only animals that had the electrode or dye spot position confirmed by histology were considered for data analysis. 2.7. Experimental design 2.7.1. Effects of neural block of rACC or cACC on the stimulation-produced antinociception from the RSC in tail-flick test Four groups of 8 rats each had an electrode implanted into the rACC and a guide cannula into the RSC. Another four groups of 8 rats each had an electrode implanted into the cACC and a guide cannula into the RSC. Saline (0.25 μL) or 1 mM CoCl2 (0.25 μL) was injected into the rACC or cACC soon after recording of the baseline TFL. Electrical or sham stimulation of the RSC was performed 20 min later, and TFL was recorded within 30 s after the end of the stimulation period and then for up to 30 min at 5-min intervals. Saline/sham stimulated rats were taken as control.
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2.7.2. Effect of ablation of rACC on the stimulation-produced antinociception from the RSC in tail-flick test Cortical ablations were performed in two groups of 8 rats each. One week after real or sham ablation of rACC the animals were taken for recording of the baseline TFL at 5 min intervals. Electrical or sham stimulation of the RSC was then performed and TFL was recorded within 30 s after the end of the stimulation period and then for up to 30 min at 5-min intervals. Sham ablation/sham stimulated rats were taken as control. 2.7.3. Effect of injecting antagonists into the rACC on the stimulationinduced antinociception from the RSC in tail-flick test Saline (0.25 μL), bicuculline (50 ng/0.25 μL) or phaclophen (300 ng/ 0.25 μL) was injected into the rACC soon after recording of the baseline TFL. Electrical or sham stimulation of the RSC was performed 20 min later, and TFL was recorded within 30 s after the end of the stimulation period and then for up to 30 min at 5-min intervals. Saline/sham stimulated rats were taken as control. 2.7.4. Effects of neural block of RSC on the stimulation-produced antinociception from the rACC in tail-flick test Saline (0.25 μL) or 1 mM CoCl2 (0.25 μL) was injected into the RSC or soon after recording of the baseline TFL. Electrical or sham stimulation of the rACC was performed 20 min later, and TFL was recorded within 30 s after the end of the stimulation period and then for up to 30 min at 5-min intervals. Saline/sham stimulated rats were taken as control. 2.7.5. Effect of neural block of the rACC on stimulation-produced antinociception from the RSC in formalin test Formalin (50 μL) was injected subcutaneously in rats pre-treated with CoCl2 or saline in the rACC and the animals were soon submitted to the electrical or sham stimulation of the RSC. The number of spontaneous paw flinches, and the total time in which the animal licked the injected paw were counted during 5 min after formalin (phase 1). In another groups of rats pre-treated with CoCl2 or saline in the rACC, formalin (50 μL) was injected subcutaneously. Ten min later these animals were submitted to electrical or sham stimulation of the RSC and the number of spontaneous injected paw flinching, and the total time in which the animal licked the injected paw were counted every 5-min for up to 60 min after formalin (phase 2). Saline/sham stimulated rats were taken as control in both occasions. 2.8. Drugs Cobalt chloride hexahydrate (CoCl2) was purchased from SigmaAldrich (St. Louis, MO, USA). Bicuculline methiodide and phaclofen were purchased from RBI/Sigma (Natick, MA, USA). Phaclofen was dissolved in 5% dimethyl sulfoxide (DMSO). The other drugs were dissolved in saline. Doses of GABA receptor antagonists were as previously reported (Gilbert and Franklin, 2001; Ciriello and Roder, 1999; Villarreal and Prado, 2007). 2.9. Data analysis Tail-flick latencies (in seconds) are reported as the means ± SD. Comparisons between control and test groups were made by multivariate analysis of variance (MANOVA) with repeated measures to compare the groups over all times. The factors analyzed were treatments, time and treatment × time interaction. In the case of treatment × time interaction, one-way analysis of variance followed by Duncan's test was performed for each time. Data from the formalin tests were compared by ANOVA followed by a Bonferroni post hoc test. The analysis was performed using the statistical software package SPSS/PC+, version 17.0. The level of significance was set at P b 0.05 in all cases.
3. Results 3.1. Effects of neural block or ablation of the rACC or neural block of the cACC on the stimulation-produced antinociception from the RSC in tail-flick test Electrical stimulation of the RSC performed 20 min after injection of saline (0.25 μL) into the rACC (Fig. 1A) or cACC (Fig. 1B), or in rats with sham rACC ablation (Fig. 1C) evoked a prompt increase of the TFL that remained significantly above the control for at least 5 min after stimulation ended. Electrical stimulation of the RSC was performed 20 min after the injection of 1 mM CoCl2 (0.25 μL) into the rACC also had a prompt increase of the TFL, but the effect remained significantly above control for at least 15 min (Fig. 1A). In contrast, RSC stimulation 20 min after the injection of 1 mM CoCl2 (0.25 μL) into the cACC also evoked a prompt increase of the TFL, but the effect did not differ significantly from control regarding the intensity or duration (Fig. 1B). Sham RSC stimulation in rats with sham or real rACC ablation resulted in TFL that did not differ significantly from baseline throughout the observation period. In contrast, RSC stimulation in rats with rACC ablation also induced a prompt increase of the TFL, but the effect remained significantly above control for at least 15 min (Fig. 1C). The curves in Fig. 1A, B and C were significantly different regarding treatment (F3,28 = 11.82; F3,20 = 12.11 and F3,28 = 15.52, respectively), time (F14,392 = 25; F14,280 = 11.56 and F14,392 = 84.31, respectively), and the treatment × time interaction (F42,392 = 13.00; F42,280 = 4.89 and F42,392 = 29.56, respectively), p b 0.0001 in all cases. The locations of the electrode tips in the RSC (Fig. 1A and B) and dye spots in the rACC (Fig. 1A) and cACC (Fig. 1B) are shown in insects. The time during which TFL remained significantly above control after RSC stimulation was significantly longer in rats treated with CoCl2 than in rats treated with saline in the rACC (Fig. 2A). The time in which TFL remained significantly above control after RSC stimulation was significantly longer in rats with rACC ablation than in rats with sham ablation (Fig. 2B). No rat displayed gross disturbance of motor coordination during or after the period of brain stimulation. Animals walked normally and responded to non-noxious stimulation throughout the period of observation. 3.2. Effect of injecting bicuculline or phaclofen into the rACC on the stimulation-induced antinociception from the RSC in tail-flick test Sham RSC stimulation performed 20 min after the injection of saline (0.25 μL) or bicuculline (50 ng/0.25 μL) (Fig. 3A), and 5% DMSO (0.25 μL) or phaclophen (300 ng/0.25 μL) (Fig. 3B) resulted TFL's that were similar to baseline throughout the observation period. RSC stimulation performed 20 min after the saline injection into the rACC evoked a prompt increase of TFL that remained significantly above control for at least 5 min. RSC stimulation performed 20 min after the injection of bicuculline into the ACC also increased the TFL, the effect remaining significantly above control for at least 15 min (Fig. 3A). In contrast, RSC stimulation conducted 20 min after the injection of phaclofen into the rACC evoked an anti-nociceptive effect that did not differ from control in intensity or duration (Fig. 3B). The curves in Fig. 3A and B were significantly different for treatment (F3,28 = 107.64 and 72.29, respectively), time (F13,364 = 88.01 and 120.21, respectively), and the treatment × time interaction (F39,364 = 24.09 and 29.59, respectively), P b 0.0001 in all cases. 3.3. Effect of neural block of the RSC on the reduction of TFL provoked by the stimulation of the ACC Experiments were conducted using a slower increase in coil temperature so that baseline TFL was reached in approximately 9 s. Sham ACC stimulation in rats treated with 1 mM CoCl2 (0.25 μL) or saline (0.25 μL) had TFL that did not differ from baseline throughout the observation
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Fig. 2. The changes in time during which tail-flick latency remained significantly above control after electrical stimulation (ES) of the retrosplenial cortex: (A) in rats injected with saline (0.25 μL) or CoCl2 (1 mM/0.25 μL) in the ipsilateral rostral anterior cingulate cortex; (B) in rats submitted to surgical ablation (abl) of the ipsilateral rostral anterior cingulate cortex. Bars represent the means ± S.D. of 8 animals per group. p b 0.05 compared to sham-stimulated rats (*) or to the remaining groups (#).
3.4. Effect of neural block of the rACC on stimulation-produced antinociception from the RSC in formalin test
Fig. 1. Changes produced by the injection (arrow 1) of saline (sal = 0.25 μL) or CoCl2 (Co = 1 mM/0.25 μL) into the rostral (A) or caudal (B) anterior cingulated cortex on the antinociception induced by the electrical stimulation (ES) (arrow 2) of the ipsilateral retrosplenial cortex of rats in the tail-flick test. (C) Changes produced by the surgical ablation of the rostral anterior cingulated cortex on the antinociception induced by the ES (arrow 2) of the retrosplenial cortex in the rat tail-flick test. The site location of CoCl2 injection into the rostral or caudal anterior cingulated cortex or sites of retrosplenial cortex stimulation is shown on diagrams by Paxinos and Watson (2005). Points represent the means ± S.D. of 8 animals. *p b 0.05 compared to sham-stimulated rats.
period. In contrast, rACC stimulation performed 20 min after the injection of either 1 mM CoCl2 or saline into the RSC evoked a significant reduction in the TFL compared to sham rACC stimulated rats previously treated with saline in the RSC (F3,23 = 72.08; p b 0.0001). However, these effects were not significantly different and lasted less than 5 min (Fig. 4).
Rats injected subcutaneously with saline displayed a small number of spontaneous flinches and had small total time spent licking the injected paw. In addition, these effects were not significantly different in groups of rats submitted to sham or real RSC stimulation (Fig. 5). Phase 1 of the nociceptive responses to the subcutaneous injection of 5% formalin (50 μL) in rats injected with saline (0.25 μL) into the rACC was characterized by a significant increase in the number of spontaneous flinches (Fig. 5A) and in the total time licking the injected paw (Fig. 5B), compared to the response of rats injected with subcutaneous saline. Phase 1 of both nociceptive responses to formalin was significantly inhibited by the RSC stimulation in rats treated with 1 mM CoCl2 (0.25 μL) or saline (0.25 μL) in the rACC. The number of spontaneous flinches during phase 2 of the responses to formalin was not significantly changed by the RSC stimulation in rats treated with saline (0.25 μL) or 1 mM CoCl2 (0.25 μL) in the rACC (Fig. 5C). In contrast, the duration of licking during phase 2 of the formalin response in rats treated with 1 mM CoCl2 (0.25 μL) into the rACC was significantly shorter than in rats treated with saline (0.25 μL). However, this effect was not different in rats subjected to sham or real RSC stimulation (Fig. 5D). The data in Fig. 5A–D were significantly different (F5,47 = 13.39; 165.6; 41.50 and 45.44, respectively; P b 0.0001 in all cases).
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Fig. 4. Changes of tail-flick latency in rats stimulated electrically (ES) in the rostral anterior cingulate cortex after the injection of saline (0.25 μL) or CoCl2 (1 mM/0.25 μL) into the ipsilateral retrosplenial cortex. The site location of CoCl2 injection into the retrosplenial cortex or sites of rostral anterior cingulated cortex stimulation is shown on diagrams by Paxinos and Watson (2005). Bars represent the means ± S.D. of 8 animals. p b 0.05 compared to sham-stimulated rats (*).
Fig. 3. Changes produced by the injection (arrow 1) of saline (sal = 0.25 μL), bicuculline (bic = 50 ng/0.25 μL) (A) or phaclophen (phac = 300 ng/0.25 μL) (B) into the rostral anterior cingulate cortex on the antinociception induced by the electrical stimulation (ES) (arrow 2) of the ipsilateral retrosplenial cortex in the rat tail-flick test. The site location of drug injection into the rostral anterior cingulated cortex or sites of retrosplenial cortex stimulation is shown on diagrams by Paxinos and Watson (2005). Points represent the means ± S.D. of 8 animals. p b 0.05 compared to sham-stimulated rats (*).
4. Discussion These results confirm that electrical stimulation of the RSC is antinociceptive in the rat tail-flick test and inhibits the phase 1, but not the phase 2 responses of rats to formalin as previously demonstrated (Reis et al., 2010). In addition, results demonstrate that the injection of CoCl2 into or ablation of the rACC prolonged the antinociceptive effect of stimulating the RSC. The stimulation-induced antinociception from the RSC was not changed in rats treated with CoCl2 in the cACC. The intracerebral injection of CoCl2 reversibly blocks synaptic transmission but not passage fibers (Kretz, 1984). Therefore, the antinociceptive effect of stimulating the RSC in the tail-flick test is at least partially modulated by the rACC, but does not involve the cACC. The injection of the bicuculline (a GABA-A antagonist), but not phaclofen (a GABA-B antagonist) into the rACC also prolonged the stimulation-induced antinociception from the RSC in the tail-flick test. A dose–response study about the effects of GABA receptor antagonists in the rACC have not been conducted yet. However, the doses of bicuculline and phaclophen used here were similar to those shown earlier to be effective in the anterior pretectal nucleus to change the post-surgical allodynia in rats (Villarreal and Prado, 2007), in the rostral ventromedial medulla to decrease the response of rats to formalin (Gilbert and Franklin, 2001), and in the central nucleus of the amygdala
to inhibit the cardiovascular responses of rats to the injection of glutamate into the same nucleus (Ciriello and Roder, 1999). Consequently, the lack of effect of phaclophen in this study is unlikely the result of an inadequate dose of this antagonist. The presence of GABA receptors, in addition to opioid, serotonergic, dopaminergic and glutamatergic receptors has been demonstrated in the ACC (Mengod et al., 1990; Bozkurt et al., 2005; Luna-Munguía et al., 2005; Rivera et al., 2008; Palomero-Gallagher et al., 2009). Furthermore, inhibitory synaptic transmission within the rACC is mediated by GABA (Descalzi et al., 2009). Therefore, the modulation of the antinociceptive effect of stimulating the RSC in the rat tail-flick test involves GABA-A receptors in the rACC. The presented results also provide additional support for a role of the rACC in supraspinal processing of noxious events and suggest that rACC GABA-A receptors significantly contribute to this processing as proposed elsewhere (LaGraize and Fuchs, 2007). We demonstrated that rACC stimulation reduced the TFL, thus confirming previous demonstration that stimulating the rACC results in hyperalgesia (Calejesan et al., 2000; Tang et al., 2005). In addition, we have shown that the stimulation-induced hyperalgesia from the rACC was not changed in rats treated with CoCl2 in the RSC. Therefore, the RSC is not involved with the pronociceptive effect of the rACC stimulation. The effectiveness of stimulating RSC against phase 1 was the same in rats treated with saline or CoCl2 into the rACC. Injecting CoCl2 into the rACC did not change the rat response during the phase 1, but reduced the licking response duration during phase 2 of the same test as reported elsewhere (Donahue et al., 2001), this effect being similar in sham animals or those stimulated at the RSC. Therefore, the antinociceptive effect of stimulating the RSC in the formalin test does not involve the rACC. By this reason, the participation of the cACC in the modulation of RSC-induced antinociception in the formalin test was not conducted in this study. The stimulation-induced antinociception from the RSC in the rat tailflick test did not occur in animals with dorsolateral funiculus lesions (Reis et al., 2010), which is the main route through which pain inhibition pathways descend to the spinal cord (Millan, 1999). The rACC exerts its hyperalgesic effect by modulating the descending pain inhibition pathways (see Ohara et al., 2005). Our results suggest that rACC exerts an inhibitory role on the stimulation-induced antinociception from the RSC in the tail-flick test, but is not involved in the modulating the effects
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Fig. 5. Changes produced by saline (0.25 μL) or CoCl2 (1 mM/0.25 μL) injection into the rostral anterior cingulate cortex on the effects of electrical stimulation (ES) of the ipsilateral retrosplenial cortex against phases 1 (A and B) and 2 (C and D) of the nociceptive responses of rats to the subcutaneous injection of saline (50 μL) or 5% formalin (50 μL) into the hind paw contralateral to the cortical targets. Stimulation (20 μA) was applied for 15 s immediately before (phase 1) or 10 min after (phase 2) the formalin injection. The number of spontaneous flinching of the injected paw, and the total time during which the animal licked the injected paw, were counted for 5 min after formalin (phase 1) or from 10 to 60 min after formalin (phase 2). The site location of CoCl2 injection into the rostral anterior cingulated cortex or sites of retrosplenial cortex stimulation is shown on diagrams by Paxinos and Watson (2005). Bars represent the means ± S.D. of 8 rats per group. p b 0.05 compared to group sal/sham (*) or formalin/sham (#).
of stimulating the RSC against phase 1 of the animal response to formalin. Therefore, we suggest that the antinociceptive effects of RSC stimulation in the tail-flick and formalin tests might be mediated by different neural systems. The ACC itself send inputs to the periaqueductal gray and the rostral ventral medulla, which are structures involved in the descending endogenous analgesia system (see Millan, 2002). Stimulation of the ACC facilitated spinal tail-flick reflex by acting through brainstem descending pain modulation system probably involving the mesencephalic periaqueductal gray (Fuchs et al., 2014) and the rostral ventral medulla (Calejesan et al., 2000). However, the ACC also send direct descending projections to the spinal dorsal horn, and these projections are activated following peripheral nerve injury (Chen et al., 2014). In summary, the results presented herein show an inhibitory role of the rACC via GABA-A receptors in a pathway activated from the RSC to produce antinociception in the rat tail-flick test. In contrast, the antinociceptive effect of stimulating the RSC in the formalin test does not involve the rACC. Acknowledgments G.M.R. was a recipient of FAPESP fellowship. The authors greatly appreciate the technical assistance of M.A. Carvalho and P.R. Castania. References Azami J, Llewelyn MB, Roberts MHT. An extra-fine assembly for intracerebral microinjection. J Physiol 1980;305:18P–9P.
Bozkurt A, Zilles K, Schleicher A, Kamper L, Arigita ES, Uylings HB, et al. Distributions of transmitter receptors in the macaque cingulate cortex. Neuroimage 2005;25:219–29. Calejesan AA, Kim SJ, Zhuo M. Descending facilitatory modulation of a behavioral nociceptive response by stimulation in the adult rat anterior cingulate cortex. Eur J Pain 2000;4: 83–96. Chen T, Koga K, Descalzi G, Qiu S, Wang J, Zhang LS, et al. Postsynaptic potentiation of corticospinal projecting neurons in the anterior cingulate cortex after nerve injury. Mol Pain 2014;10:33. Ciriello J, Roder S. GABAergic effects on the depressor responses elicited by stimulation of central nucleus of the amygdala. Am J Physiol 1999;276:H242–7. Descalzi G, Kim S, Zhuo M. Presynaptic and postsynaptic cortical mechanisms of chronic pain. Mol Neurobiol 2009;40:253–9. Donahue RR, LaGraize SC, Fuchs PN. Electrolytic lesion of the anterior cingulate cortex decreases inflammatory, but not neuropathic nociceptive behavior in rats. Brain Res 2001;897:131–8. Foltz EL, White Jr LE. Pain “relief” by frontal cingulumotomy. J Neurosurg 1962;19: 89–100. Fuchs PN, Peng YB, Boyette-Davis JA, Uhelski ML. The anterior cingulate cortex and pain processing. Front Integr Neurosci 2014;8:35. Gilbert AK, Franklin KB. GABAergic modulation of descending inhibitory systems from the rostral ventromedial medulla (RVM). Dose–response analysis of nociception and neurological deficits. Pain 2001;90:25–36. Hess A, Sergejeva M, Budinsky L, Zeilhofer HU, Brune K. Imaging of hyperalgesia in rats by functional MRI. Eur J Pain 2007;11:109–19. Hutchinson WD, Davis KD, Lozano AM, Tasker RR, Dostrovsky JO. Pain-related neurons in the human cingulate cortex. Nat Neurosci 1999;2:403–5. Iwata K, Kamo H, Ogawa A, Tsuboi Y, Noma N, Mitsuhashi Y, et al. Anterior cingulate cortical neuronal activity during perception of noxious stimuli in monkeys. J Neurophysiol 2005;94:1980–91. Johansen JP, Fields HL, Manning BH. The affective component of pain in rodents: direct evidence for a contribution of the anterior cingulate cortex. Proc Natl Acad Sci U S A 2001;98:8077–82. Kim SS, Descalzi G, Zhuo M. Investigation of molecular mechanism of chronic pain in the anterior cingulate cortex using genetically engineered mice. Curr Genomics 2010;11: 70–6.
118
G.M. Reis et al. / Pharmacology, Biochemistry and Behavior 131 (2015) 112–118
Kim CE, Kim YK, Chung G, Im HJ, Lee DS, Kim J, et al. Identifying neuropathic pain using (18)F-FDG micro-PET: a multivariate pattern analysis. Neuroimage 2014;86:311–6. Koyama T, Kato K, Tanaka YZ, Mikami A. Anterior cingulate activity during pain-avoidance and reward tasks in monkeys. Neurosci Res 2001;39:421–30. Kretz R. Local cobalt injection: a method to discriminate presynaptic axonal from postsynaptic neuronal activity. J Neurosci Methods 1984;11:129–35. LaGraize SC, Fuchs PN. GABA-A but not GABA-B receptors in the rostral anterior cingulate cortex selectively modulate pain-induced escape/avoidance behavior. Exp Neurol 2007;204:182–94. Lamas V, Alvarado JC, Carro J, Merchán MA. Long-term evolution of brainstem electrical evoked responses to sound after restricted ablation of the auditory cortex. PLoS One 2013;8:e73585. Luna-Munguía H, Manuel-Apolinar L, Rocha L, Meneses A. 5-HT1A receptor expression during memory formation. Psychopharmacology (Berl) 2005;181:309–18. Mengod G, Nguyen H, Le H, Waeber C, Lübbert H, Palacios JM. The distribution and cellular localization of the serotonin 1C receptor mRNA in the rodent brain examined by in situ hybridization histochemistry. Comparison with receptor binding distribution. Neuroscience 1990;35:577–91. Millan MJ. The induction of pain: an integrative review. Prog Neurobiol 1999;57:1–164. Millan MJ. Descending control of pain. Prog Neurobiol 2002;66:355–474. Ohara PT, Vit JP, Jasmin L. Cortical modulation of pain. Cell Mol Life Sci 2005;62:44–52. Palomero-Gallagher N, Vogt BA, Schleicher A, Mayberg HS, Zilles K. Receptor architecture of human cingulate cortex: evaluation of the four-region neurobiological model. Hum Brain Mapp 2009;30:2336–55. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. New York: Academic Press; 2005. Quintero GC. Advances in cortical modulation of pain. J Pain Res 2013;6:713–25. Reis GM, Dias QM, Silveira JW, Del Vecchio F, Garcia-Cairasco N, Prado WA. Antinociceptive effect of stimulating the occipital or retrosplenial cortex in rats. J Pain 2010;11:1015–26. Reis GM, Rossaneis AC, Silveira JW, Prado WA. μ1- and 5-HT1-dependent mechanisms in the anterior pretectal nucleus mediate the antinociceptive effects of retrosplenial cortex stimulation in rats. Life Sci 2012;90:950–5.
Rivera A, Peñafiel A, Megías M, Agnati LF, López-Téllez JF, Gago B, et al. Cellular localization and distribution of dopamine D(4) receptors in the rat cerebral cortex and their relationship with the cortical dopaminergic and noradrenergic nerve terminal networks. Neuroscience 2008;155:997–1010. Rossaneis AC, Reis GM, Prado WA. Stimulation of the occipital or retrosplenial cortex reduces incision pain in rats. Pharmacol Biochem Behav 2011;100:220–7. Tang J, Ko S, Ding HK, Qiu CS, Calejesan AA, Zhuo M. Pavlovian fear memory induced by activation in the anterior cingulate cortex. Mol Pain 2005;1:6. Vaccarino AL, Melzack R. Analgesia produced by injection of lidocaine into the anterior cingulum bundle of the rat. Pain 1989;39:213–9. Van Groen T, Wyss JM. Connections of the retrosplenial granular b cortex in the rat. J Comp Neurol 2003;463:249–63. Villarreal CF, Prado WA. Modulation of persistent nociceptive inputs in the anterior pretectal nucleus of the rat. Pain 2007;132:42–52. Vogt BA, Peters A. Form and distribution of neurons in rat cingulated cortex: areas 32, 24, and 29. J Comp Neurol 1981;195:603–25. Westlund KN, Vera-Portocarrero LP, Zhang L, Wei J, Quast MJ, Cleeland CS. fMRI of supraspinal areas after morphine and one week pancreatic inflammation in rats. Neuroimage 2009;44:23–34. Wik G, Fischer H, Brage'e B, Kristianson M, Fredrikson M. Retrosplenial cortical activation in the fibromyalgia syndrome. Neuroreport 2003;14:619–21. Wik G, Fischer H, Finer B, Bragee B, Kristianson M, Fredrikson M. Retrospenial cortical deactivation during painful stimulation of fibromyalgic patients. Int J Neurosci 2006;116:1–8. Yang PF, Chen DY, Hu JW, Chen JH, Yen CT. Functional tracing of medial nociceptive pathways using activity-dependent manganese-enhanced MRI. Pain 2011;152: 194–203. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983;16:109–10.
