Enhanced dorsolateral periaqueductal gray activity counteracts the anxiolytic response to midazolam on the elevated plus-maze Trial 2 in rats

Behavioural Brain Research 162 (2005) 99–107

Research report

Enhanced dorsolateral periaqueductal gray activity counteracts the anxiolytic response to midazolam on the elevated plus-maze Trial 2 in rats Leandro Jos´e Bertoglio a , Cl´audia Anzini b , Cilene Lino-de-Oliveira b , Antˆonio P´adua Carobrez b,∗ a

Departamento de Farmacologia, FMRP-USP, Av. Bandeirantes, 3900, Ribeir˜ao Preto, SP 14049-900, Brazil b Departamento de Farmacologia, CCB, UFSC, Florian´ opolis, SC 88049-900, Brazil Received 25 February 2005; received in revised form 4 March 2005; accepted 7 March 2005 Available online 18 April 2005

Abstract Rodents previously (Trial 1) experienced in the elevated plus-maze (EPM) apparatus no longer respond to anxiolytic-like drugs during retesting (Trial 2). In view of the fact that the dorsolateral periaqueductal gray (dlPAG) modulates fear/anxiety-like behavior, the present study sought to determine its role in this phenomenon. In order to address this issue, EPM-experienced rats that had received lidocaine, a drug which produces a reversible functional deactivation, intra-dlPAG pre-Trial 1, post-Trial 1 or pre-Trial 2, were systemically injected with the benzodiazepine midazolam and submitted to the EPM apparatus. According to the results, 0.25 mg/kg midazolam increased open arms exploration and reduced risk assessment behavior, suggesting an anxiolytic-like effect in EPM-naive rats, regardless of the intra-dlPAG treatment. EPM-experienced rats administered with midazolam only displayed a similar pattern of behavior when lidocaine was administered intra-dlPAG pre-Trial 2, but not pre- or post-Trial 1. These effects were observed in the absence of changes in enclosed arms entries, an EPM general exploratory activity index. The present results suggest that an increased activity of the dlPAG during Trial 2 would explain the lack of anxiolytic-like effect of drugs elicited by prior EPM test experience. © 2005 Elsevier B.V. All rights reserved. Keywords: Anxiety; Fear; Periaqueductal gray; Elevated plus-maze; One-trial tolerance; Benzodiazepine

1. Introduction Rodents tested in the elevated plus-maze (EPM) apparatus display a characteristic increase in open arms exploration after the administration of anxiolytic-like drugs. However, if the subjects have already performed a 5 min EPM task (Trial 1), they no longer respond to anxiolytics in a subsequent test exposure (Trial 2). This phenomenon was initially observed for the benzodiazepine chlordiazepoxide [27] and referred to as “one-trial tolerance” (OTT; [16]). Indeed, it was subsequently demonstrated that drugs binding to the GABAA receptor complex, such as benzodiazepines [5,18,37], ethanol ∗

Corresponding author. Tel.: +55 48 3319491; fax: +55 16 6332301. E-mail address: [email protected] (A.P. Carobrez).

0166-4328/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2005.03.010

[3] and barbiturates [3,14], or to the NMDA/Glycine-Breceptor complex, such as MK-801, HA-966 and memantine [6], fail to produce the expected anxiolytic-like effect in such EPM-experienced rats or mice. Considerable attention has been directed at attempts to either explain or prevent the phenomenon of OTT. Regarding the former issue, several hypotheses have been proposed, including a locomotor habituation [13], an altered state of the binding-sites and/or the receptor complexes involved [3,5,6,21], a fear sensitization [2,4,43] and/or a qualitative shift in the nature of the aversive response elicited between trials [24], against which the anxiolytic-like drugs are ineffective [6,15]. In relation to prevention of the OTT phenomenon, it was demonstrated that rodents continue to display an anxiolytic-like profile in response to drugs on the

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EPM Trial 2 when: (1) a new motivational conflict situation was introduced in the task [36], (2) the EPM trials last 10 min instead of the usual 5 min [17,25], as well as when the time length on Trial 1 was limited to 1 min [12] and (3) amnesic doses of chlordiazepoxide [16] or scopolamine [7] were administered prior to Trial 1. In addition to these aspects, it has been shown that brain areas related to fear/defense systems, such as the amygdala and the medial hypothalamus may modulate this loss of anxiolytic effect of drugs on the EPM Trial 2. For instance, File et al. [19] have reported that a reversible deactivation of the basolateral nucleus of the amygdala with lidocaine [29], immediately after Trial 1, restores the anxiolytic-like effect of a benzodiazepine on Trial 2. A similar pattern of results was observed with the deactivation of the dorsomedial hypothalamus just before Trial 2 [20], suggesting that the OTT phenomenon is controlled by different sets of brain structures, chiefly those involved in the expression of fear/defensive behavior depending on the stage of the phenomenon’s development. In this context, together with the amygdaloid complex and the hypothalamus, the periaqueductal gray matter (PAG), especially its dorsolateral portion (dlPAG), has also been implicated in behavioral expression of fear and possibly anxiety [1,10,22,28,32]. Nevertheless, its role in the present issue, the OTT phenomenon, remains to be determined. In fact, it is noteworthy that a recent work has suggested that the anxiolytic-like effects of WAY-100635, a 5-HT1A receptor antagonist, in the median raphe nucleus (MRN) may be explained by projections from the MRN to the PAG [9]. Interestingly, the anxiolytic response to MRN infusions of WAY100635 is also lost in EPM-experienced animals [9]. Based on this fact, the present study examined the role of the dlPAG in the lack of anxiolytic-like effect of drugs observed in rats previously experienced in the EPM test. In order to address this issue, Experiment 1 examined the effects of the benzodiazepine midazolam in EPM-naive rats that received lidocaine into the dlPAG just before the EPM Trial 1, Experiments 2 and 3 assessed the effects of midazolam on the EPM Trial 2 in rats that received lidocaine intra-dlPAG before or immediately after Trial 1, respectively, while Experiment 4 evaluated the effects of midazolam and lidocaine on the EPM Trial 2 performance.

2. Materials and methods 2.1. Subjects The subjects were 115 male Wistar rats (Universidade Federal de Santa Catarina, SC, Brazil) weighing 300–350 g, aged 13–15 weeks at the time of testing, housed in groups of 2–3 per cage (50 cm × 30 cm × 15 cm), under a standard light cycle (12 h light/dark phase; lights on at 6:00 h) in a temperature-controlled environment (23 ± 2 ◦ C) and with free access to food and water. All procedures were conducted in conformity with the Brazilian Society of Neuroscience and Behavior Guidelines for care and use of laboratory animals, which comply with international

laws and policies. All efforts were made to minimize animal suffering. 2.2. Drugs Midazolam (Dormonid® , Roche, Brazil), initially at a concentration of 5.0 mg/ml, was diluted in 0.9% saline to a concentration of 0.25 mg/ml, which alone served as a vehicle control for the systemic injection. All of which were given in a standard volume of 1.0 ml/kg. Lidocaine (2-diethyl-N-[2,6-diethyphenyl]-acetamide HCl; Sigma, USA) was dissolved to a concentration of 4% (40 mg/ml) in artificial cerebrospinal fluid (CSF) of the following composition (mM): NaCl (126.6), NaHCO3 (27.4), KCl (2.4), KH2 PO4 (0.5), CaCl2 (0.89), MgCl2 (0.8), Na2 HPO4 (0.48) and glucose (7.1), pH 7.4. The doses of midazolam and lidocaine were chosen based on previous dose–response studies [5,19,20,26]. A 0.3 ␮l volume of lidocaine was chosen for these experiments so as to ensure maximum effective diffusion based on the estimate formula outlined in Tehovnik and Somner [41]. According to this estimate of effective radial spread, this volume lidocaine infusion should exert Na+ channel block effects within a ∼0.40 mm radial distance from the injector tip. Furthermore, lidocaine injection should exert effective Na+ channel block within a time-course of ∼10 min, particularly at concentration of 4%. 2.3. Apparatus The elevated plus-maze (EPM) was made of wood and consisted of two opposite open arms, 50 cm × 10 cm (surrounded by a 1 cm high Plexiglas ledge), and two enclosed arms, 50 cm × 10 cm × 40 cm, set up 50 cm above the floor. The junction area of the four arms (central platform) measured 10 cm × 10 cm. In order to avoid urine impregnation, the floor of the apparatus was painted with impermeable dark epoxy resin. 2.4. Surgery Animals were anesthetized using 1.5 ml/kg of a solution containing xylazine (10 mg/ml; Rompun® , Bayer, Brazil) plus ketamine (58 mg/ml; Dopalen® , Agribrands, Brazil) and positioned in a stereotaxic frame. Keeping the skull horizontal between bregma and lambda, a stainless-steel guide cannula (o.d. = 0.6 mm, length = 13 mm), made locally using needles (BD Precision Glide® , Brazil), was lowered at an angle of 22◦ to the sagittal plane aiming at the dlPAG. The following coordinates were used: midline = 1.9 mm; dorsoventral = −2.0 mm from the skull surface; and anterior-posterior = −7.6 mm. The cannula was anchored to the bone with stainless-steel screws and acrylic cement. After this, a stylet was introduced inside the guide cannula to reduce the incidence of occlusion, and an antibiotic association (Pentabi´otico® , Fort Dodge, Brazil; 1.0 ml/kg) was administered subcutaneously. 2.5. Procedures Five days after surgery, after removing the stylet, each rat received a unilateral injection with a thin dental needle (0.3 mm o.d.) introduced through the guide cannula until its tip was 1.0 mm below the cannula end. A volume of 0.3 ␮l of either CSF or lidocaine 4% was injected in 30 s using a microsyringe (Hamilton® , USA) connected to an infusion pump (KD Scientific, KDS100, EUA). A polyethylene catheter was interposed between the upper end of the

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dental needle and the microsyringe. The intracerebral needle was removed 1 min after the end of the injection. The experiments were carried out in a low illumination (44 lx) condition room during the diurnal phase (between 12:00 and 16:00 h). Behavior was recorded by video camera while a monitor and a video-recording system were installed in an adjacent room. After each trial, the EPM apparatus was cleaned with wet and dry towels. A trained observer scored the behavioral parameters from the videotape twice, with an agreement between repeated analyses (intra-observer reliability) of the same test of ≥95%. Behavioral measures were the number of open and enclosed arms entries (EAE) and the amount of time spent on the central platform, open and enclosed arms. These data were used to calculate the percentage of open arms entries {%OAE; [open entries/(open + enclosed entries)] × 100}, the percentage of time spent in open [%OAT; (open time/300) × 100] and enclosed [%EAT; (enclosed time/300) × 100] arms, as well as on the central platform [%CT; (central platform time/300) × 100]. The number of tries (NT; exploratory posture in which the rat stretches forward and then retracts to its original position) to reach the open arms, performed by rats from the central platform or enclosed arms, was also recorded. 2.5.1. Experiment 1: effects of midazolam and lidocaine on the EPM Trial 1 performance Twenty-nine EPM-naive rats were randomly allocated to four groups (n = 6–9) according to either the systemic (saline (SAL) or

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0.25 mg/kg midazolam (MDZ)) or the intra-dlPAG (CSF or 4% lidocaine (LIDO)) treatment given 30 and 5 min, respectively, before the first EPM (Trial 1) exposure, comprising the following groups: SAL-CSF, MDZ-CSF, SAL-LIDO, MDZ-LIDO. All groups were resubmitted undrugged to the EPM 48 h later. 2.5.2. Experiment 2: effects of midazolam on Trial 2 performance of rats that received lidocaine prior to the EPM Trial 1 Seventeen EPM-experienced rats were randomly allocated to two groups (n = 7–10) according to the intra-dlPAG treatment given 5 min prior to Trial 1: Group I, CSF; Group II, 4% lidocaine. Forty-eight hours later, each group was systemically administered with 0.25 mg/kg MDZ and 30 min after submitted to the EPM Trial 2. 2.5.3. Experiment 3: effects of midazolam on Trial 2 performance of rats that received lidocaine immediately after the EPM Trial 1 Thirty-three EPM-experienced rats were randomly allocated to two groups (n = 16–17) according to the intra-dlPAG treatment given immediately after the EPM Trial 1 exposure: Group I, CSF; Group II, 4% LIDO. Each group was further divided in two subgroups (n = 7–10) based on the treatment (saline or 0.25 mg/kg MDZ) given systemically 30 min prior to Trial 2.

Fig. 1. Schematic drawings, based on the atlas of Paxinos and Watson [35], of coronal sections from 7.04 to 7.80 mm posterior to bregma of the rat brain showing the microinjections sites (open circles) into the dorsolateral periaqueductal gray matter (dlPAG). Due to overlapping, the number of points represented is fewer than the number of rats actually injected.

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2.5.4. Experiment 4: effects of midazolam and lidocaine on the EPM Trial 2 performance Thirty-six EPM-experienced rats were randomly allocated to four groups (n = 7–10) according to both the systemic (saline or 0.25 mg/kg MDZ) and the intra-dlPAG (CSF or LIDO) treatment given 30 and 5 min, respectively, before the second EPM (Trial 2) exposure, comprising the following groups: SAL-CSF, MDZ-CSF, SAL-LIDO, MDZ-LIDO. 2.6. Histology At the end of behavioral testing, the rats were anesthetized (as already described in Section 2.4) and perfused with 0.9% NaCl followed by a 10% formaldehyde solution. A 0.3 ␮l injection of Evans Blue was then applied through the guide cannula to mark the exact location of the previous microinjection. The brains were removed and stored in 10% formaldehyde solution for at least 5 days. Frozen brains were sectioned on a microtome and 40 ␮m sections were taken until the site of the needle tip could be seen. Injections into the dlPAG were placed between −7.04 and −7.80 mm (around 50% at −7.64 mm) posterior to bregma. Data from rats with placements falling outside the dlPAG were not included in the statistical analysis. Fig. 1 shows the injection sites in coronal midbrain sections illustrated in diagrams according to the atlas of Paxinos and Watson [35]. 2.7. Statistics Data obtained from experiments were analyzed by one (Experiment 2; “intra-dlPAG drug treatment”) or two-factor (Experi-

ments 1, 3 and 4; “systemic drug treatment” versus “intra-dlPAG drug treatment”) analysis of variance (ANOVA), and followed by Newman–Keuls tests (p < 0.05) using the Statistica® software (StatSoft Inc., Tulsa, OK, USA).

3. Results 3.1. Experiment 1: effects of midazolam and lidocaine on the EPM Trial 1 performance ANOVA overall comparison showed a significant systemic drug treatment effect for %OAT (F (1, 25) = 24.73, p < 0.00001), %OAE (F (1, 25) = 13.36, p < 0.001), NT (F (1, 25) = 21.26, p < 0.00001) and %CT (F (1, 25) = 14.82, p < 0.001). No statistically significant effects were observed regarding the intra-dlPAG drug treatment or the interaction between systemic and central treatments. Post hoc pairwise comparisons showed an increase in both %OAT and %OAE, as well as a reduction in NT, of rats treated with 0.25 mg/kg MDZ relative to saline-treated rats, regardless of the intra-dlPAG treatment (Fig. 2A–C). A reduction in %CT following the 0.25 mg/kg MDZ administration also was observed. However, no effects were observed for EAE in MDZ-treated groups as compared to respective controls (Fig. 2D). In addition, regardless of the pre-Trial 1 drug conditions, no significant changes were observed in any

Fig. 2. Effects on anxiety-like behavior (A and B), risk assessment behavior (C), as well as on general exploratory activity (D) of saline or midazolam (MDZ; 0.25 mg/kg) given systemically and of vehicle (CSF) or lidocaine 4% administered directly into the dlPAG of rats submitted to the elevated plus-maze Trial 1 (n = 6–9). Data are presented as mean ± S.E.M; * p < 0.05 vs. respective control group.

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Table 1 Experiment 1 results: effects on plus-maze Trial 2 performance of systemic (SAL or MDZ) and intra-dlPAG (CSF or LIDO) treatments given pre-Trial 1 (n = 6–9) SAL/CSF Open arms time (%) Open arms entries (%) Number of tries Enclosed arms entries Central platform time (%) Enclosed arms time (%)

6.9 21.2 6.3 6.7 26.8 66.2

± ± ± ± ± ±

2.9 9.3 1.0 0.4 5.8 5.9

MDZ/CSF ± ± ± ± ± ±

7.8 16.3 6.5 6.8 28.0 64.2

3.2 5.7 0.8 1.4 4.8 6.8

SAL/LIDO 3.4 15.2 6.8 8.1 30.8 65.6

± ± ± ± ± ±

1.4 1.5 0.7 1.2 2.6 2.8

MDZ/LIDO 11.0 25.6 7.1 7.8 28.7 60.3

± ± ± ± ± ±

3.8 6.9 1.0 0.6 2.5 5.3

Data are presented as mean ± S.E.M. Table 2 Experiment 2 results: two groups of rats received vehicle (CSF) or lidocaine (LIDO) intra-dlPAG pre-Trial 1; 48 h later, each group was retested (Trial 2) after the midazolam administration (MDZ; 0.25 mg/kg; n = 7–10) Trial 1

Trial 2

CSF Open arms time (%) Open arms entries (%) Number of tries Enclosed arms entries Central platform time (%) Enclosed arms time (%)

12.1 25.6 8.3 9.1 41.1 46.8

LIDO ± ± ± ± ± ±

3.5 4.8 0.8 0.8 4.6 3.4

15.7 33.4 5.5 7.6 31.5 52.8

CSF/MDZ

± ± ± ± ± ±

4.1 5.6 1.1 0.5 2.3 5.7

4.5 31.5 4.1 3.3 32.1 63.4

± ± ± ± ± ±

1.3 7.4 0.9 0.6 11.5 12.6

LIDO/MDZ 7.3 32.5 4.6 5.6 29.9 62.7

± ± ± ± ± ±

1.4 5.6 0.9 1.0 5.4 5.6

Data are presented as mean ± S.E.M.

EPM parameter during the undrugged EPM Trial 2 exposure (Table 1). 3.2. Experiment 2: effects of midazolam on Trial 2 of rats that received lidocaine pre-Trial 1 Table 2 shows the consequences of the pre-Trial 1 microinjection of lidocaine intra-dlPAG on the effects of MDZ on Trial 2. The overall ANOVA comparisons failed to show any statistically significant effect of either systemic or central treatments, as well as the interaction between these factors in all the behavioral measures scored on the EPM Trial 2 (Table 2). 3.3. Experiment 3: effects of midazolam on Trial 2 of rats administered with lidocaine post-Trial 1 Table 3 shows the consequences of the post-Trial 1 microinjection of lidocaine into the dlPAG on Trial 2 MDZ behavioral effects. The overall ANOVA comparisons failed

to show any statistically significant effect of either systemic or central treatments, as well as the interaction between these factors in all the behavioral measures scored on the EPM Trial 2 (Table 3). 3.4. Experiment 4: effects of midazolam and lidocaine on the EPM Trial 2 performance ANOVA overall comparison showed a significant systemic drug treatment versus intra-dlPAG drug treatment interaction for %OAT (F (1, 31) = 4.15, p < 0.05) and a significant systemic drug treatment effect for %OAE (F (1, 31) = 5.03, p < 0.04) and NT (F (1, 31) = 8.56, p < 0.01). Post hoc pairwise comparisons showed an increase in both %OAT and %OAE in the MDZ-LIDO group relative to either the SALLIDO or the MDZ-CSF groups (Fig. 3A and B). In addition, decreased NT was observed in the MDZ-LIDO when compared to the SAL-LIDO group (Fig. 3C). However, no effects were observed for EAE in MDZ-treated groups as compared to respective controls (Fig. 3D).

Table 3 Experiment 3 results: effects on Trial 2 of saline (SAL) or midazolam (MDZ) in rats administered with vehicle (CSF) or lidocaine (LIDO) intra-dlPAG post-Trial 1 (n = 7–10) SAL/CSF Open arms time (%) Open arms entries (%) Number of tries Enclosed arms entries Central platform time (%) Enclosed arms time (%) Data are presented as mean ± S.E.M.

7.0 20.3 8.1 8.4 36.7 56.3

± ± ± ± ± ±

2.2 4.5 0.8 0.5 4.4 5.4

MDZ/CSF 8.5 28.3 7.4 8.2 31.3 60.3

± ± ± ± ± ±

1.5 3.0 0.9 1.1 4.2 4.3

SAL/LIDO 8.7 24.8 9.1 7.4 35.4 55.9

± ± ± ± ± ±

3.2 8.1 1.4 0.9 3.5 4.2

MDZ/LIDO 11.1 21.4 6.9 9.8 28.1 60.8

± ± ± ± ± ±

4.0 5.6 1.1 1.9 3.5 5.8

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Fig. 3. Effects on anxiety-like behavior (A and B), risk assessment behavior (C), as well as on general exploratory activity (D) of saline or midazolam (MDZ; 0.25 mg/kg) given systemically and of vehicle (CSF) or lidocaine 4% administered directly into the dlPAG of rats submitted to the elevated plus-maze Trial 2 (n = 7–10). Data are presented as mean ± S.E.M; * p < 0.05 vs. saline-lidocaine group; # p < 0.05 vs. MDZ-CSF group.

4. Discussion The main experimental findings of the present study are that (1) whatever the intra-dlPAG treatment, midazolam (0.25 mg/kg) produced an anxiolytic-like effect in EPMnaive rats submitted to Trial 1; (2) the anxiolytic-like activity of midazolam was no longer observed on Trial 2 performance of EPM-experienced rats, regardless of the treatment given into the dlPAG prior to or immediately after Trial 1; (3) EPM-experienced rats administered with lidocaine, but not with CSF, into the dlPAG just before Trial 2 responded with an anxiolytic-like profile after the midazolam systemic injection. Results from Experiment 1 showed increased open arms exploration (%OAT and %OAE) and reduced risk assessment behavior, represented by the number of tries in EPM-naive rats after the systemic injection of 0.25 mg/kg midazolam, regardless of the intra-dlPAG treatment, CSF or lidocaine, given just prior to Trial 1. These effects were observed in the absence of significant change in general exploratory activity, represented by enclosed arms entries, and agree with previous studies showing an anxiolytic-like activity of midazolam after either systemic or intra-dlPAG injection in EPM-naive rats [5,11,12,38]. Furthermore, no carryover effects of either the systemic or the intra-dlPAG treatments given to EPM-naive

rats prior to Trial 1 were observed in any behavioral parameter scored on Trial 2 (Table 1). Taken together, these data suggest that the functional integrity of the dlPAG does not seem to be required to express the anxiolytic-like effect of systemically administered midazolam in the EPM Trial 1. In relation to the results of Experiments 2 and 3, despite the direct dlPAG administration of lidocaine pre- or post-Trial 1, respectively, the groups of EPM-experienced rats that received midazolam showed the typical pattern of behaviors on Trial 2, that is, they did not respond to it either by increasing the open arms exploration or reducing behaviors related to risk assessment. These results suggest that the dlPAG does not seem to play a key role in the acquisition/consolidation of an aversive learning response, which is acquired during the initial overall EPM exploration and, in turn, leads to subsequent insensitivity to anxiolytics [6,7]. Indeed, a previous report suggested that the basolateral nucleus of the amygdala seems to be one of the main brain sites recruited at the stage of consolidation for this learning process, since its post-Trial 1 functional deactivation with lidocaine prevented the lack of anxiolytic-like activity of chlordiazepoxide on the EPM Trial 2 [19]. With regard to the acquisition stage of this anxiolyticinsensitive behavioral strategy, further experiments are certainly necessary in order to determine which brain structures are involved.

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According to the Experiment 4 results, EPM-experienced rats administered with 0.25 mg/kg of midazolam and with CSF into the dlPAG (MDZ-CSF group) prior to Trial 2 also failed to demonstrate an anxiolytic-like profile. However, those receiving a lidocaine microinjection responded with an anxiolytic-like profile to midazolam, characterized by increased open arms exploration and reduced risk assessment behavior. Whereas the former result confirmed the usual lack of response to midazolam in EPM-experienced subjects [5,11,12,21], the latter suggests that the functional integrity of the dlPAG is crucial to expression of the OTT phenomenon on Trial 2. A similar finding was observed after the reversible functional deactivation of the dorsomedial hypothalamus [20]. In this context, it is interesting to point out that the dlPAG sends descending projections to the medulla and ascending projections to the dorsal hypothalamus [8]. Thus, the dlPAG would be counteracting the anxiolytic-like effect of midazolam on Trial 2 by influencing caudal motor areas in the medulla directly and/or indirectly via the hypothalamus–limbic system. Despite the consistency of the present findings, no further increase in open arms avoidance was statistically detected on Trial 2. Although this result diverges from previous data from our research group and others (e.g. [6,24,43]), it is probably due to high basal anxiety levels in EPM-naive rats. Besides, as the current experimental design incorporated saline injection prior to EPM testing, an injection effect per se may have concealed an expected experience-induced shift in EPM behavioral baseline. Similar unexpected results have been reported elsewhere [25,37]. Electrical or chemical stimulation of the dlPAG elicits a pattern of responses similar to those naturally adopted by animals when exposed to controllable (or escapable) or uncontrollable threats [10,39]. Of particular relevance to the present work are those data showing increased avoidance behavior in rats submitted to the first experience in the EPM test [10,34,42]. In view of this fact and considering the present results, it is proposed that enhanced dlPAG excitability counteracts the facilitation of approach drive produced by midazolam administration on Trial 2, leading to the occurrence of the phenomenon of OTT. In other words, the increased open arms exploration and reduced risk assessment behavior displayed by the MDZ/LIDO group on Trial 2 reflect a synergic effect between systemic midazolam and intra-dlPAG lidocaine treatments. Interestingly, this synergic effect was not observed on Trial 1 (Experiment 1), indicating that somehow prior test experience lead to a tune up in the importance of the dlPAG during the EPM Trial 2 performance. In salinetreated rats, however, lidocaine per se did not increase open arms exploration on Trial 2. The fact that the lesion itself had no anxiolytic-like effect, but reinstated the anxiolytic response to midazolam, indicates that it is the responsiveness of benzodiazepine receptors outside this brain region that was changed. In this context, Gonzalez and File [21] have shown that in the dorsal raphe nucleus a change in benzodiazepine receptors to an inverse agonist state might underlie the Trial 2 loss of response to benzodiazepines. Although the precise

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nature of this role needs further investigation, another explanation could be an increase in excitatory activity of the glutamatergic system of the dlPAG (for a review see [10]). In a recent update of the theory of Gray and McNaughton [23], McNaughton and Corr [30] argued that brain structures controlling fear and anxiety (as categorically distinct entities) form parallel streams and are represented at all levels of these systems. Although anxiety has a greater neural representation in rostral, cortical brain structures (e.g. prefrontal cortex), the PAG and hypothalamus are the lower level components of this system. Therefore, besides fundamentally controlling fear-like responses (e.g. escape; for a review see [31]), these regions may also control those responses related to anxiety, such as avoidance and risk assessment [10,42]. In general, the present results agree with these findings since the dlPAG exerts, together with the dorsomedial hypothalamus [20], a central role on Trial 2. In consonance with this idea are the majority of the hypotheses proposed to elucidate this lack of anxiolytic-like activity of drugs observed in EPM-experienced rodents, such as the acquisition/sensitization of a phobic-like response to the open arms during Trial 1 [2,15,43]. The EPM test exploration induces the early immediate gene c-fos in the PAG [40], and rats with low open arms exploration have increased expression of the gene encoding limbic system-associated membrane protein in this brain area [33]. In view of these findings, the dlPAG role may no be discarded on the EPM Trial 1. Indeed, it is hypothesized that during Trial 1 a top-down organization would be required to deal with this task and its environment. The dlPAG would participate as a final common pathway for the expression of the defensive responses determined by higher order cortical and subcortical structures as well (e.g. basolateral amygdala, prefrontal cortex and hippocampus). On Trial 2, after an initial contextualization of the task, regions localized in the medial hypothalamic zone and/or in the periaqueductal gray would determine the optimal behavioral response, giving rise to a bottom-up fashion activation of the defensive system. If this hypothesis is correct, while the sensitivity to systemic administration of benzodiazepines during Trial 1 would represent its action in reducing the activity of the higher order cortical and sub-cortical structures that control the behavioral responses expressed during this stage, the insensitivity to these drugs on the EPM Trial 2 would be the result of an increased activity of brain regions, such as the dorsomedial hypothalamus and the dlPAG. Further, these effects could be the result of a reduced GABAergic activity and/or an increased glutamatergic activity within these aforementioned brain areas.

Acknowledgments This work was supported by FAPESP (02/13197-2; 03/13032-6), CAPES and CNPq (521864/96-8), from which A.P. Carobrez receives a research fellowship; L.J. Bertoglio, C. Lino-de-Oliveira and C. Anzini received postgraduate

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scholarships from FAPESP or CNPq. The authors wish to thank Gareth Cuttle for English corrections on the manuscript.

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