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available at www.sciencedirect.com

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Research Report

Blockade of fear-induced antinociception with intra-amygdala infusion of midazolam: Influence of prior test experience
Daniela Baptista a,b , Karina Bussadori a , Ricardo Luiz Nunes-de-Souza b,c , Azair Canto-de-Souza a,b,
a

Grupo de Psicobiologia/Depto de Psicologia/CECHUFSCar, Rod. Washington Lus, Km 235, 13565-905 So Carlos, SP, Brazil Programa de Ps-Graduao em Cincias Fisiolgicas-UNESP-UFSCar, Rod. Washington Lus, Km 235, 13565-905 So Carlos, SP, Brazil c Laboratory of Pharmacology, School of Pharmaceutical Sciences, So Paulo State UniversityUNESP, Araraquara, SP, 14801-902, Brazil
b

A R T I C LE I N FO Article history: Accepted 16 July 2009 Available online 25 July 2009 Keywords: Fear Antinociception Amygdala Midazolam Elevated plus-maze Mice

AB S T R A C T Intra-amygdala infusion of midazolam, a benzodiazepine receptor agonist, produces anxiolytic-like effects in mice when first exposed to the elevated plus-maze (EPM) and blocks antinociception induced in mice confined in the open arm of the EPM. However, benzodiazepines fail to alter anxiety in maze-experienced rodents, a phenomenon defined as one-trial tolerance (OTT). The main purpose of the present study was to investigate whether intra-amygdala midazolam attenuates the open arm-induced antinociception (OAA) in maze-experienced mice. Nociception was assessed by the writhing test (intraperitoneal injection of 0.6% acetic acid). In Experiment 1, nociception was recorded in mazeexperienced mice without prior drug treatment. Experiment 2 investigated the effects of systemic midazolam (0.5, 1.0 and 2.0 mg/kg, s.c.), injected before EPM trial 2, on OAA in maze-experienced mice. In Experiment 3, the effects on OAA of intra-amygdala midazolam (30 nmol/0.1 l), injected before trial 1 (maze-naive) or before trial 2 (maze-experienced), were observed. The effects on OAA of intra-amygdala midazolam injected before trial 1 and trial 2 were also investigated (Experiment 4). The results showed that OAA remained unchanged in maze-experienced mice and was insensitive to systemic midazolam. However, intra-amygdala midazolam attenuated OAA in maze-naive mice, but not in maze-experienced mice. Even when given before both trial 1 and trial 2, intra-amygdala midazolam failed to alter OAA in maze-experienced mice. Taken together, these results confirm that the GABAA/benzodiazepine receptor complex located within the amygdala plays a role in OAA in maze-naive mice. The lack of effects following systemic or intraamygdala midazolam on OAA in maze-experienced mice suggests that the OTT is also observed in the modulation of nociception and that the GABAA/benzodiazepine receptor located within this limbic forebrain structure participates in this process. 2009 Elsevier B.V. All rights reserved.

Corresponding author. Depto de Psicologia/CECH UFSCar, Rod. Washington Lus, Km 235, 13565-905 So Carlos, SP, Brazil. Fax: +55 16 3351 8489. E-mail address: souzaalm@ufscar.br (A. Canto-de-Souza). 0006-8993/$ see front matter 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.07.055

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1.

Introduction

The influence of emotional states such as anxiety and fear on the pain reaction has been widely investigated (e.g., Bolles and Fanselow, 1980; Fields et al., 2006; Kelly, 1986; Siegfried et al., 1990). It is thus known that, besides displaying defensive behaviors (e.g., fight, flight, freezing, vocalization), and neurovegetative (e.g., tachycardia, hypertension, defecation) responses characterized as a fear reaction, animals exposed to innate or learned threatening situations usually exhibit antinociception (Kelly, 1986, Rodgers, 1995). Evidence of the involvement of opioid and non-opioid mechanisms in the modulation of environmentally induced antinociception has been raised by many (e.g., Kelly, 1986; Rodgers, 1995; Heinricher et al., 2009). For instance, it has been shown that defeated mice display opioid-like antinociception which was both blocked by the opiate receptor antagonist naloxone (Miczek et al., 1982; Rodgers and Hendrie, 1983) and cross-tolerant to morphine (Miczek et al., 1982). However, nonopioid antinociception has also been reported in defeated mice (Rodgers and Randall, 1986; Rodgers and Shepherd, 1989; Rodgers et al., 1990; Canto-de-Souza, et al., 1997). In this context, there is evidence suggesting that mechanisms underlying defeat-induced non-opioid antinociception involve 5HT1A receptors (Rodgers and Randall, 1986; Rodgers and Shepherd, 1989; Rodgers et al., 1990; Canto-de-Souza, et al., 1997), and 5HT3 receptors (Shepherd and Randall, 1990), as well as benzodiazepine receptors (Rodgers and Randall, 1987). The search for animal models to reveal the influence of emotional state on nociception has resulted in the use of the elevated plus-maze (EPM) test (Lee and Rodgers, 1990), a widely-used animal model of anxiety (e.g., Carobrez and Bertoglio, 2005; Lister, 1987; Pellow et al., 1985). Rodgers et al. were the first to show that mice display not only defensive behavioral reactions but also a non-opioid type of antinociception (as assessed by the tail-flick test) when exposed to the EPM. They later showed that EPM-induced antinociception is attenuated by anxiolytic drugs such as buspirone and diazepam (for review see Rodgers, 1995). In some studies we have tried to identify the central mechanisms underlying the EPM-induced antinociception (Mendes-Gomes and Nunes-de-Souza, 2005, 2009; Nunes-deSouza et al., 2000) and have demonstrated that this type of pain inhibition is attenuated by intra-amygdala (Nunes-deSouza et al., 2000) but not by intra-periaqueductal gray (Mendes-Gomes and Nunes-de-Souza, 2005) microinjections of midazolam, suggesting a site-specific control of this type of pain inhibition by benzodiazepine receptors. In the amygdala, this benzodiazepine receptor full agonist induced an anxiolytic-like effect in a standard EPM test (sEPM: two enclosed arms and two open arms) and completely blocked EPM-open arm confinement-induced antinociception (OAA) (Nunes-deSouza et al., 2000), indicating that anxiety and OAA share similar modulatory mechanisms within this limbic forebrain structure. An intriguing finding observed in many laboratories is a change in the behavioral profile in the second or subsequent exposures of rodents to the EPM. In general, maze-experienced rodents explore the open arms of the EPM less and become

insensitive to the antianxiety effects of benzodiazepines (e.g., Bertoglio and Carobrez, 2002a,b; File et al., 1990; Holmes and Rodgers 1998; Rodgers and Shepherd, 1993) and other anxiolytic drugs (e.g., Bertoglio and Carobrez, 2002a,b; Canto-deSouza et al., 2002; Nunes-de-Souza et al., 2002; Pellow et al., 1985) during a second trial in the maze. This phenomenon, which is defined as one-trial tolerance (OTT) (File et al., 1990), has been explained variously as a manifestation of locomotor habituation (Dawson et al., 1994), an altered state of the binding-site on the receptors involved (Bertoglio and Carobrez, 2002a,b, 2003; Gonzalez and File, 1997), an experimentally induced sensitization of fear/anxiety (Bertoglio and Carobrez, 2003; Treit et al., 1993), a qualitative shift in the emotional state between trials, from unconditioned fear to learned avoidance (Bertoglio and Carobrez, 2003, 2004; Dal-Cl et al., 2003; File and Zangrossi, 1993; Holmes and Rodgers, 1999) and/or activation of cognition-related telencephalic structures involved in the control of learned fear (Albrechet-Souza et al., 2008). In addition, recent results from our laboratory showing that intra-cerebral injection of 5-HT2 receptor agonists could attenuate anxiety at trial 2 only when they were also injected prior to trial 1 (Gomes and Nunes-de-Souza, in press) have raised the possibility of pre-trial 1 treatment preventing the occurrence of OTT in mice. Interestingly, while previous studies have emphasized the involvement of GABAA/benzodiazepine receptors in OTT (e.g., Bertoglio and Carobrez, 2002a,b; File et al., 1990; Holmes and Rodgers 1999; Lister, 1987; Rodgers and Shepherd, 1993), recent results have shown that intra-amygdala infusion of midazolam is capable of attenuating anxiety in maze-experienced mice (Barbalho et al., 2009). Thus, considering (i) the close relationship between anxiety/fear emotional states and antinociception (e.g., Bolles and Fanselow, 1980; Fields et al., 2006; Kelly, 1986; Siegfried et al., 1990), (ii) the fact that EPM-induced antinociception is not attenuated by several exposures to the maze in rats (Cornlio and Nunes-de-Souza, 2009) and (iii) the involvement of GABAA/benzodiazepine receptors located within the amygdala in the modulation of OAA and anxiety in maze-naive (Nunes-de-Souza et al., 2000) and mazeexperienced (Barbalho et al., 2009) mice, respectively, it would be of interest to know whether GABAA/benzodiazepine receptors located within this forebrain structure are also involved in the modulation of OAA in maze-experienced mice. In this context, the present study was designed to reveal whether the OAA and anxiety share underlying mechanisms in maze-experienced mice. Thus, we investigated the effects of intra-amygdala midazolam on OAA in maze-experienced mice. The OAA was also recorded in maze-experienced mice without any prior drug treatment. Given that OAA was insensitive to systemic midazolam in maze-naive mice (Nunes-de-Souza et al., 2000), we also tested the effects of systemic midazolam on OAA in mazeexperienced mice.

2.

Results

The histological analysis confirmed that 129 mice received cannulae in amygdala (Fig. 1). Nineteen subjects were used to investigate the effects of intra-amygdala midazolam (MDZ: 0

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Fig. 1 Drawings of serial sections showing microinfusion sites within the amygdala of the mice. The gray area represents the area reached by the injected drug. The solid black circles show the sites of injection outside the amygdaloid complex. Sections are between 1.82 and 2.18 mm from the bregma in the atlas of Franklin and Paxinos (1997).

or 30 nmol) on OAA in maze-naive mice [MDZ 0: n = 5 for open arm confinement (OAC) and n = 5 for enclosed arm confinement (EAC); MDZ 30: n = 5 for OAC and n = 4 for EAC]. Forty-

eight animals were used to investigate the effects of intraamygdala midazolam (0 or 30 nmol), injected prior to trial 2, on OAA in maze-experienced mice (MDZ 0: n = 12 for OAC and

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n = 12 for EAC; MDZ 30: n = 12 for OAC and n = 12 for EAC). Sixtytwo animals were used to investigate the effects of intraamygdala midazolam (0 or 30 nmol) injected prior to trial 1 and prior to trial 2 on OAA in maze-experienced mice (MDZ 0: n = 16 for OAC and n = 14 for EAC; MDZ 30: n = 15 for OAC and n = 17 for EAC).

2.1. Experiment 1: Evaluation of OAA in maze-naive and maze-experienced mice

The Student t-test for independent samples revealed that OAconfined animals showed a lower number of writhes than EAconfined subjects in both maze-naive (t(23) = 7.09, P < 0.001) and maze-experienced mice (t(24) = 5.94, P < 0.001) (Table 1). Fig. 2 Lack of effect of systemic midazolam (0, 0.5, 1.0 or 2.0 mg/kg, s.c.) on OAA in maze-experienced mice (n = 1112). Data are presented as mean SEM. #P < 0.05 versus EA-confined group.

2.2. Experiment 2: Lack of effect of systemic midazolam on OAA in maze-experienced mice

Two-way ANOVA revealed significant effects for the type of confinement factor (F(1,87) = 27.31, P < 0.05), but no significant effects for the treatment factor (F(3,87) = 1.98, P > 0.05) or the type of confinement treatment interaction (F(3,87) = 0.07, P > 0.05). Duncan's post hoc test showed that the OAconfined animals showed fewer writhes than EA-confined animals (Fig. 2).

hoc comparisons revealed that OA-confined animals have fewer writhes than EA-confined mice (Fig. 3B).

2.4. Experiment 4: Lack of effect of intra-amygdala midazolam, injected prior to trial 1 and prior to trial 2, on OAA in maze-experienced mice
Intra-amygdala midazolam injected before trial 1 and before trial 2 was incapable of changing OAA in maze-experienced mice. Two-way ANOVA revealed significant effects for the type of confinement factor (F(1,58) = 20.39, P < 0.05), but no significant differences for the treatment factor (F(1,58) = 1.85, P > 0.05) or type of confinement treatment interaction (F(1,58) = 0.34, P > 0.05). As shown in Experiments 13, OAconfined animals exhibited fewer writhes than EA-confined animals (Table 2).

2.3. Experiment 3: Effects of intra-amygdala midazolam on OAA in maze-naive and maze-experienced mice
Analyzing the results for maze-naive mice, two-way ANOVA revealed no statistically significant effects for the type of confinement factor (F(1,15) = 0.77, P > 0.05), but showed significant effects for the treatment factor (F(1,15) = 24.38, P < 0.05) and the treatment type of confinement interaction (F(1,15) = 10.91, P < 0.05). Post hoc comparisons showed that midazolam increased the total number of writhes in OA-confined animals. Although ANOVA did not reach significant values for the type of confinement, the post hoc Duncan test indicated that the number of writhes was significantly higher in EA-confined animals than in the OA group, but only for saline-treated animals (Fig. 3A). Regarding the effects of intra-amygdala midazolam on OAA in maze-experienced mice, two-way ANOVA revealed significant effects for the type of confinement factor (F(1,41) = 29.61, P < 0.05), but no significant effects for the treatment factor (F(1,41) = 3.57, P > 0.05) or the type of confinement treatment interaction (F(1,41) = 3.21, P > 0.05). Post

Table 1 Assessment of OAA in maze-naive and mazeexperienced mice (n = 1114). Previous exposure to maze
Naive Experienced

Number of writhes Open arm

4.27 0.81 6.64 1.24#
#

Enclosed arm
17.35 1.50 17.58 1.35

Data are presented as mean SEM. P < 0.05 versus group confined in the enclosed arm (EA).

Fig. 3 Effects of intra-amygdala midazolam (0 or 30 nmol/ 0.1 l) on OAA in (A) maze-naive (n = 45) and (B) maze-experienced mice (n = 12 per group). Data are presented as mean SEM. #P < 0.05 versus EA-confined group. P < 0.05 versus respective control group.

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Table 2 Lack of effect of intra-amygdala midazolam (0 or 30 nmol/0.1 l) injected before trial 1 and before trial 2 on OAA in maze-experienced mice (n = 1417). Treatment Number of writhes Open arm
Saline Midazolam (30 nmol/0.1 l)
#

Enclosed arm
7.21 1.04 9.12 1.20

3.38 0.58 4.13 0.94#

Data are presented as mean SEM. P < 0.05 versus EA-confined group.

3.

Discussion

The present results showed that OAA was unchanged in mazeexperienced mice, indicating that this type of pain inhibition is not altered by previous experience to the aversive environment (i.e. the open arms of the EPM). Systemic treatment with the benzodiazepine receptor agonist midazolam did not alter the antinociceptive response profile in maze-experienced mice. Importantly, when injected intra-amygdala, midazolam completely blocked OAA in maze-naive mice, confirming previous results from our laboratory (Nunes-de-Souza et al., 2000). However, intra-amygdala midazolam was incapable of altering OAA in maze-experienced mice, suggesting that a widely described OTT phenomenon, namely the lack of anxiolytic-like effects of benzodiazepine receptor agonists in maze-experienced rodents (e.g., Bertoglio and Carobrez, 2002a, 2002b; File et al., 1990; Holmes and Rodgers 1999; Rodgers and Shepherd, 1993), may also be observed in another fear-induced response, the OAA. Our results corroborate previous studies demonstrating that EPM exposure induces antinociception in mice (e.g., Conceio et al., 1992; Lee and Rodgers, 1990; Mendes-Gomes and Nunes-de-Souza, 2005; Nunes-de-Souza et al., 2000) and rats (Cornlio and Nunes-de-Souza, 2009), particularly when nociception is assessed by the writhing test (Conceio et al., 1992; Nunes-de-Souza et al., 2000). Given the strength of the antinociceptive response observed in the present and previous studies (Cornlio and Nunes-de-Souza, 2009; MendesGomes and Nunes-de-Souza, 2005; Nunes-de-Souza et al., 2000), it is likely that the type of nociceptive test is a relevant factor in the assessment of antinociception induced by exposure to the EPM. For example, in some previous studies, exposure to the EPM caused weak antinociception of low magnitude in mice (e.g., Lee and Rodgers, 1990) or failed to alter the nociception in rats (Albrechet-Souza et al., 2005) subjected to the tail-flick test. In fact, Albrechet-Souza et al. (2005) also reported that rats did not show antinociception following both trial 1 and trial 2 in the EPM. The present study also showed that systemic administration of midazolam did not alter the nociceptive response in maze-experienced mice. This result does not corroborate our previous finding that systemic administration of midazolam (1.0 mg/kg) intensified the OAA and provoked pain inhibition in maze-naive enclosed arm confined mice (Nunes-de-Souza et al., 2000). Since the present results showed that midazolam did not change the nociceptive response in either open or enclosed arm confined animals, it seems reasonable to

suggest that maze experience somehow changes the antinociceptive effects of midazolam. However, it has also been emphasized that midazolam failed to change the nociceptive response assessed by the tail-flick test in both maze-naive and maze-experienced rats (Albrechet-Souza et al., 2005). Midazolam has also been observed to have no effect on antinociception induced by exposure to an open EPM (oEPM: an EPM with all open arms), following intra-cerebral injection of this benzodiazepine receptor agonist. Mendes-Gomes and Nunes-de-Souza (2005) demonstrated that intra-periaqueductal gray matter injections of midazolam did not change oEPMinduced antinociception in maze-naive mice. Although the Mendes-Gomes and Nunes-de-Souza study involved intracerebral injection (against systemic injection), maze-naive mice (against maze-experienced mice) and a different nociception test (formalin test writhing test), its principal similarity with the present study was the type of aversive situation animals were placed in: exposure to an oEPM confinement in the open arm of the EPM. In contrast, the present study demonstrated that intraamygdala infusion of midazolam attenuated OAA in mazenaive mice. This result corroborates a previous report that midazolam blocked OAA when injected into this limbic forebrain structure (Nunes-de-Souza et al., 2000). Since intraamygdala midazolam also attenuated anxiety in mice exposed to the standard EPM (with two open arms and two closed arms), Nunes-de-Souza et al. (2000) have suggested that the blockade of the antinociceptive response could be a consequence of the antiaversive effect of midazolam. In the present study, the response to intra-amygdala midazolam confirmed the results obtained by Nunes-de-Souza et al. (2000), indicating a modulatory role of the benzodiazepine receptors in the OAA. Previous studies have demonstrated a reduction in the exploration of the open arms (the potentially aversive areas) when animals are re-exposed to the EPM (e.g., Gonzalez and File, 1997; Griebel et al., 1997; Rodgers et al., 1997). This open arm avoidance enhancement is generally accompanied by a loss of the anxiolytic-like effects of benzodiazepines in mazeexperienced animals. The failure of anxiolytic drugs to attenuate anxiety in retest paradigms is known as the onetrial tolerance phenomenon (OTT) (e.g., File et al., 1993). As described in the Introduction section, several hypothesis have been proposed to explain the OTT in maze-experienced animals. Present results indicate that OAA per se is not changed in maze-experienced animals, in that mice do not show an increased number of the writhes induced by i.p. acetic acid injection at trial 2 in the EPM. In fact, OAA remained unchanged in the retest. In this connection, it has recently been demonstrated that repeated exposure (26 exposures) to the open EPM do not alter the antinociceptive profile in rats (Cornlio and Nunes-de-Souza, 2009). In addition, the lack of the anti-antinociceptive effects following intra-amygdala midazolam in maze-experienced mice indicates that the OTT is not a phenomenon unique to emotional states such as anxiety/fear, but also affects nociception. The failure of midazolam to attenuate OAA in maze-experienced mice suggests that GABAA/benzodiazepine receptors located within the amygdaloid complex somehow lose their modulatory function in the pain response.

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Although a lack of anxiolytic-like effects in maze-experienced rodents has been widely described for benzodiazepines, we have recently found that intra-amygdala midazolam does not lose its anxiolytic-like effects in maze-experienced mice (Barbalho et al., 2009), suggesting that the benzodiazepine receptor complex located within this forebrain limbic structure does not play a role in the OTT phenomenon. However, the present study demonstrated that even when given before trial 1, intra-amygdala midazolam loses its effects in antagonizing OAA during trial 2 (Experiment 4). Taken together, these findings suggest that intra-amygdala benzodiazepine receptors have distinct roles in anxiety and in OAA, particularly in mazeexperienced mice. In other words, while amygdala GABAA/ benzodiazepine receptors keep their antianxiety response during trial 2 (Barbalho et al., 2009), they somehow become incapable of altering antinociception induced by open armconfinement in maze-experienced mice. The underlying mechanisms for this differential action of GABAA/benzodiazepine receptor activation (i.e., on anxiety versus on antinociception) remain unclear. One explanation might be the differential involvement of anatomically distinct populations of GABAA/benzodiazepine receptors within different nuclei of the amygdaloid complex. Interestingly, several studies have shown that the anxiolytic-like effects of benzodiazepine agonists depend on the site where the drug is injected (e.g., central nucleus or basolateral nucleus of amygdala) (for a review, see Engin and Treit, 2008). Thus, further research should be done on the part played by the different nuclei of the amygdala in the discrepant effects of midazolam on anxiety and OAA. In conclusion, OAA remained unchanged in maze-experienced mice (Exp. 1) and was insensitive to systemic treatment with midazolam (Experiment 2). When injected into the amygdala complex, midazolam blocked OAA completely, but only in maze-naive mice (Experiment 3). The lack of effect of midazolam on OAA in maze-experienced mice did not depend on whether the drug was administered before or after trial 1, since this benzodiazepine receptor agonist failed to inhibit OAA even when given both pre-trial 1 and pre-trial 2 in the EPM (Experiment 4). Importantly, the absence of effects on OAA in maze-experienced mice, following infusion of midazolam into the amygdala, is probably not due to any failure in its antiaversive properties, since we have recently found that intra-amygdala midazolam attenuated anxiety in maze-experienced mice (Barbalho et al., 2009). Finally, the lack of effects, following systemic or intra-amygdala midazolam injection, on OAA in maze-experienced mice suggests that the OTT phenomenon is also observed in the modulation of nociception and that the GABAA/benzodiazepine receptor complex located within this limbic forebrain structure participates in this process.

cage (cage size: 41 34 16 cm). They were maintained under a normal 12:12 h lightdark (LD) cycle (lights on: 700 a.m.) in a temperature (24 1 C) and humidity (55 5%) controlled environment. Food and drinking water were freely available except during the brief test periods. The experiments were carried out during the light phase of the LD cycle (09:0016:00). Different batches of experimentally-naive mice were used for each experiment.

4.2.

Drugs

The drug used was midazolam maleate (kindly donated by Marcus L. Brando, INeC/USP, Ribeiro Preto, Brazil) dissolved in 0.9% NaCl. The doses of midazolam used were: 0.5, 1.0 or 2.0 mg/kg s.c. (systemic) and 30 nmol/0.1 l (intraamygdala) (MW: 441.8 g). Both systemic and intra-amygdala doses were based on a previous study (Nunes-de-Souza et al., 2000).

4.3.

Surgery and microinjection

Stainless-steel guide cannulae (26-gauge 7 mm; Insight Instruments, Brazil) were bilaterally implanted in mice under ketamine + xylazine anesthesia (100 mg/kg and 10 mg/kg, i.p.) in a stereotaxic frame (Insight Instruments, Brazil). The guide cannulae were fixed to the skull with dental acrylic and jeweler's screws. Stereotaxic coordinates (Franklin and Paxinos, 1997) for the amygdala were anteroposterior (AP) = 0.8; lateral (L) = 2.7 and ventral (V) = 2.0 from the skull surface. During implantation, the guide cannula was aimed to terminate 1.0 mm above the target site. A dummy cannula (33-gauge stainless steel wire; Fishtex, Brazil), inserted into each guide cannula at the time of surgery, served to reduce the incidence of occlusion. Before tests, mice were allowed 4 to 5 days to recover from surgery. Postoperative analgesia was provided for 3 days by adding acetaminophen solution (200 mg/ml) to the drinking water at a dilution of 0.2 ml acetaminophen: 250 ml water (final concentration 0.16 mg/ml). Solutions were injected into the amygdala by a microinjection unit (33-gauge stainless steel cannula, Insight Instruments, Brazil), that extended 1.0 mm beyond the tip of the guide cannula. The microinjection unit was connected to a 10 l Hamilton microsyringe via polyethylene tubing (PE-10) and the rate of flow was controlled by an infusion pump (BI 2000Insight Instruments, Brazil) programmed to deliver 0.1 l of each solution over a period of 60 s. The microinjection procedure consisted of gently restraining the mice, inserting the injection unit, infusing the solution for 60 s and keeping the injection unit in place for 90 s. The movement of a small air bubble in the PE-10 tubing, during and after the microinjection, confirmed the delivery of the solution.

4.
4.1.

Experimental procedures
Subjects

4.4.

Apparatus and general procedure

Subjects were adult male Swiss mice weighing 2530 g (Federal University of So Carlos, SP, Brazil), housed in groups of 10 per

The basic EPM design was very similar to that originally described (Lister, 1987). It comprised two open arms (OA: 30 cm 5 cm 0.25 cm) and two enclosed arms (EA: 30 cm 5 cm 15 cm) that extended in a cross from a

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common central platform (5 cm 5 cm) and the entire apparatus was elevated to a height 38.5 cm above floor level. Confinement to an OA or EA was achieved by placing an easily removable gate at the proximal end of each arm of the EPM. All testing was conducted under moderate illumination (77 lx; measured on the central platform of the EPM) during the light phase of the LD cycle. Nociception was assessed by the writhing test previously describe by Vanderwende and Margolin (1956). In the present study, writhes were induced by i.p. injection of 10 ml/kg b.w. 0.6% acetic acid. Thus, following the appropriate drug injection-test interval (s.c. injection: 20 min; intra-cerebral injection: 5 min), animals received an i.p. injection of 0.6% acetic acid. They were then individually confined to either an OA or an EA of the EPM for 5 min, during which the number of writhes was recorded. Between subjects, the maze was thoroughly cleaned with 20% ethanol and dried with a cloth. All sessions were video-recorded with a camera linked to a monitor and VCR in an adjacent laboratory.

intra-amygdala and five minutes later they received an injection of 0.6% acetic acid i.p. Half of each group was confined to the OA or EA of the EPM to record the number of writhes during a 5-minute period (trial 2: maze-experienced animals).

4.4.4. Experiment 4: Effects of intra-amygdala midazolam, injected prior to trial 1 and prior to trial 2, on OAA in maze-experienced mice
Two groups of mice were injected with saline or midazolam (30 nmol/0.1 l) intra-amygdala and five minutes later, half of animals of each group were individually confined to the OA or EA of the EPM for a 5-minute period (trial 1: maze-naive animals that have not received prior nociceptive stimulus). Twenty-four hours later the mice received a second injection of saline or midazolam (30 nmol/0.1 l) intra-amygdala and five minutes later an injection of 0.6% acetic acid i.p. and were then confined to the OA or EA of the EPM to record the number of writhes during a 5-minute period (trial 2: maze-experienced animals).

4.4.1. OAA

Experiment 1: Effects of prior experience of maze on the

4.5.

Histology

The nociception test was carried out on four independent groups of mice. Two groups received 0.6% acetic acid i.p. and five minutes later they were confined to the OA (n = 11) or EA (n = 14) of the EPM, to record the number of writhes during a 5-minute period (trial 1: maze-naive animals). The other two groups of mice were confined to the OA (n = 14) or EA (n = 12) for 5 min (trial 1: without prior nociception stimulus). Twenty-four hours later, they were injected with 0.6% acetic acid i.p. and then individually confined to the OA or EA to record the nociception response (trial 2: maze-experienced animals).

At the end of testing, all animals received a 0.1 l infusion of 1% methylene blue by the microinjection procedure described above. The animals were then killed by anesthetic overdose and their brains were removed and accommodated in recipients containing 10% formaldehyde solution and later coronally sectioned with a cryostat microtome (ANCAP 300), following which the injection sites were microscopically verified with reference to the Atlas of Franklin and Paxinos (1997). Data from animals with injection sites outside the amygdaloid complex were excluded from the study.

4.4.2. Experiment 2: Effect of systemic treatment with midazolam on OAA in maze-experienced mice
Twenty-four hours after being confined to the OA or EA of the EPM for five minutes, 95 mice were injected with midazolam [MDZ: 0, 0.5, 1.0 or 2.0 mg/kg, s.c.; OA (n = 12 per treatment) or EA (n = 1112)] and 20 min later they were injected i.p. with 0.6% acetic acid. Five minutes later, each mouse was reexposed to the OA or EA to record the number of writhes during a 5-minute period.

4.6.

Statistical analysis

Data were analyzed by the independent Student t-test (Experiment 1) or two-way analyses of variance (ANOVA) (treatment and type of confinement). Significant F values were followed up by Duncan's multiple range tests. A P value of 0.05 or less was required for significance.

4.7.

Ethics

4.4.3. Experiment 3: Effects of intra-amygdala midazolam on OAA in maze-naive and maze-experienced mice
Five to six days after surgery for guide cannula implantation, two groups of mice were injected with saline or midazolam (30 nmol/0.1 l) intra-amygdala and five minutes later were injected with 0.6% acetic acid i.p. Half of the animals in each group were confined to the OA or EA of the EPM, to record the number of writhes during a 5-minute period (trial 1: mazenaive animals). To investigate the effects of intra-amygdala midazolam on OAA in maze-experienced animals, two groups of mice were confined to the OA or EA for 5 min (trial 1: without prior nociception stimulus). Twenty-four hours later the mice were injected with saline or midazolam (30 nmol/0.1 l)

The experiments reported in this study were performed in compliance with the recommendations of the Brazilian Society of Neuroscience and Behavior (SBNeC), based on the US National Institutes of Health Guide for Care and Use of Laboratory Animals. This study was approved by the Ethics Committee on Animal Experiments of the Federal University of So Carlos (Res. 005/2007).

Acknowledgments
D. Baptista received a scholarship from FAPESP (06/06855-4) and R.L. Nunes-de-Souza received a CNPq research fellowship (309407/2006-0). The authors would like to thank Adriana

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Maria Corsi and Jos Carlos Gaban for their technical assistance.
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