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Behavioural Brain Research 246 (2013) 103– 110

Contents lists available at SciVerse ScienceDirect

Behavioural Brain Research

j ourna l h om epage: www.elsev ier .com/ locate /bbr

esearch report

ctivation of 5-HT1A receptors in the rat basolateral amygdalanduces both anxiolytic and antipanic-like effects

hristiana Villela de Andrade Strauss, Maria Adrielle Vicente, Helio Zangrossi Jr. ∗

epartment of Pharmacology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, 14049-900, SP Brazil

i g h l i g h t s

5-HT1A receptor activation in the basolateral amygdala causes anxiolytic effects.These receptors are also involved in the regulation of panic responses.Midazolam in the basolateral amygdala causes anxiolytic effects.

r t i c l e i n f o

rticle history:eceived 12 November 2012eceived in revised form 28 February 2013ccepted 4 March 2013vailable online 13 March 2013

eywords:nxietyanicerotonin and amygdala

a b s t r a c t

The relevance of 5-HT1A and 5-HT2C receptors of the basolateral nucleus of the amygdala (BLA) in themediation of anxiety-related defensive responses has long been acknowledged. Whereas strong evidencesupports that activation of the latter receptors provokes anxiety, conflicting findings have been reportedon the role played by the former binding site. In this study we further investigated the involvement of5-HT1A receptors (5-HT1A-Rs) in the regulation of anxiety- and panic-related defensive behaviors. Theresults showed that intra-BLA injection of the 5-HT1A-R agonist 8-OH-DPAT (0.4–16 nmol) in male Wistarrats impaired the acquisition of inhibitory avoidance in the elevated T-maze, increased the percentageof time spent in the lit compartment of the light–dark transition model and enhanced the number ofpunished drinking events in the Vogel conflict test, all changes compatible with an anxiolytic effect. This

agonist also impaired escape expression in the elevated T-maze, suggestive of a panicolytic-like effect.8-OH-DPAT-induced changes in the elevated T-maze and light–dark tests were blocked by previous localadministration of the 5-HT1A-R antagonist WAY-100635 (0.37 nmol) and were also observed after intra-BLA microinjection of the benzodiazepine receptor agonist midazolam (10–40 nmol). Thus, stimulationof 5-HT1A-Rs in the BLA causes both anxiolytic- and panicolytic-like effects, what may have implicationsfor the pathophysiology and treatment of generalized anxiety and panic disorders.

. Introduction

The amygdala complex has long been implicated in the regu-ation of defensive behaviors and hence in fear and anxiety [1,2].eported evidence indicates that this limbic region, in particular

ts basolateral subdivision (BLA), is critically implicated in theediation of 5-HT-induced behavioral consequences to threat-

ning/aversive stimuli [3–5]. However, the exact role played byhe different 5-HT receptor (5-HT-R) subtypes found in the BLA,articularly 5-HT1A- and 5-HT2C-Rs, in this process is not yet fullynderstood. The importance of these two 5-HT binding sites in the

rocessing of negatively motivated emotion has been supported byvidence indicating that changes in their functioning in differentimbic areas are involved in the mode of action of antidepressants

∗ Corresponding author. Tel.: +55 16 36023353; fax: +55 16 36332301.E-mail address: zangross@fmrp.usp.br (H. Zangrossi Jr.).

166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.bbr.2013.03.005

© 2013 Elsevier B.V. All rights reserved.

[6,7], which are the first choice pharmacological treatment formood and anxiety disorders [8–10].

Several lines of evidence demonstrate that activation of 5-HT2C-Rs within the BLA enhances anxiety [11,12] and this mechanism hasbeen associated with the anxiogenic consequences caused by expo-sure to nociceptive aversive stimuli [13], repeated withdrawalsfrom chronic ethanol diets [14], and short-term treatment withantidepressant drugs [15].

In a recently published paper, Vicente and Zangrossi [15]observed that stimulation of 5-HT2C-Rs in the BLA facilitatesinhibitory avoidance acquisition in the elevated T-maze, indicatingan anxiogenic effect, without interfering with escape expression inthe same test [for details of this experimental model see 16–18].Based on extensive pharmacological validation, these defensive

responses have been related in terms of psychopathology to gen-eralized anxiety (GAD) and panic disorders, respectively [4,19–22].Therefore, facilitation of 5-HT2C-Rs in the BLA seems to primarilyinterfere with behaviors associated with the former condition, in

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ccordance with the proposal on the distinctive role played by 5-HTn fear and anxiety formulated by Deakin and Graeff [4].

Less unequivocal, however, have been the findings on theole played by 5-HT1A-Rs in this amygdala subregion. Thus, localtimulation of these receptors with the 5-HT1A receptor agonist-OH-DPAT has been found to be ineffective [23,24] or to inducenxiogenic [3,24] or anxiolytic [25,26] responses. This discrepancyas been interpreted in terms of procedural variations, such as theoses employed, the use of tests that potentially modulate distin-uished subtypes of anxiety disorders and/or the use of differentpecies or strains of rodents [25,27].

Also, in regard to BLA 5-HT1A-Rs, it is still unclear whetherhey play a distinct role in the regulation of anxiety- and panic-elated defensive behaviors, as recently observed with 5-HT2C-Rs15]. In an earlier study from our group with the elevated T-maze25], it was shown that intra-BLA injection of 8-OH-DPAT impairednhibitory avoidance acquisition, indicating an anxiolytic effect,

ithout affecting escape performance. However, in that study,scape was evaluated in animals without previous experience withne of the open arms of this test apparatus, following the experi-ental protocol originally developed for this model [28,29]. Other

tudies, however, have shown that a short period of exposure tohe open arm increases the pharmacological validity of the escapeask as an index of panic. For instance, chronic administration ofhe antipanic drug imipramine inhibited escape performance, sug-esting a panicolytic-like effect, only in animals submitted to there-exposure procedure [30]. Using this modified protocol, the ele-ated T-maze also revealed the anti-escape effect of other clinicallyffective panicolytic drugs, such as fluoxetine, clomipramine andscitalopram [31,32].

In the present paper, we reassess the role played by BLA 5-T1A-Rs in the regulation of anxiety- and panic-related defensiveehaviors. To this end, the effects in the elevated T-maze of intra-LA microinjection of 8-OH-DPAT or of the 5-HT1A-R antagonistAY-100635, alone or in combination, were compared to that

nduced by the benzodiazepine midazolam. Tests in the elevated-maze were performed using the pre-exposure procedure afore-entioned. For locomotor activity evaluation, animals were also

ested in an open-field immediately after being exposed to thelevated T-maze.

In order to investigate the generality of the results found,he effect of 8-OH-DPAT in the BLA was also investigated in theight–dark transition model and in the Vogel conflict test, whichave been associated with GAD [for further details of these testsee 17, 33].

. Material and methods

.1. Animals

Male Wistar rats weighing 290–310 g were housed in groups of 5 under a 12:12ark/light cycle (lights on at 07:00 h) at 22 ± 1 ◦C and given free access to foodhroughout the experiment, except during testing. Water was also freely available,xcept in experiment 2 where periods of deprivation were followed (see below).ll experiments were carried out in accordance with the Brazilian College of Animalxperimentation and NIH Guides for the care and use of laboratory animals and werepproved by the Experimental Animal Ethical Committee of the School of Medicinef Ribeirao Preto - University of São Paulo. All efforts were made to minimize animaluffering.

.2. Apparatus

The elevated T-maze was made of wood and had three arms of equal dimensions50 cm × 12 cm). One arm, enclosed by 40 cm high walls, was perpendicular to two

pposed open arms. To avoid falls, the open arms were surrounded by a 1 cm highlexiglas rim. The whole apparatus was elevated 50 cm above the floor.

The open-field test was performed in a wooden square arena (60 cm × 60 cm),ith 30 cm high walls. Luminosity at the level of the T-maze arms or open-field was

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Brain Research 246 (2013) 103– 110

The light/dark transition apparatus consisted of a box made of wood with overalldimensions of 48 cm × 24 cm × 27 cm and a grid floor composed of bars spaced 5 mmapart. The box was further divided by a barrier possessing a doorway (10 cm × 10 cm)so that rats could cross into two chambers of equal size (24 cm × 24 cm × 27 cm): onepainted black, not illuminated, and one painted white and illuminated with a 50 lxlight source.

The Vogel conflict test was performed, as described by Pelosi et al. [34], in a Plex-iglas box (length: 42 cm, width: 25 cm, height: 20 cm) with a stainless grid floor. Ametallic spout of a drinking bottle containing water was projected into the box.The contact of the animal with the spout and grid floor closed an electrical cir-cuit controlled by a sensor (Insight Instruments, Brazil), which produced 7 pulses/swhenever the animal was in contact with both components. Each pulse was consid-ered as a lick, and every 20 licks the animal received a 0.5 mA shock for 2 s. The sensorrecorded the total number of licks and shocks delivered during the test period. Theapparatus was located inside a sound attenuated cage.

At the end of each test, the models were cleaned with a 10% ethanol solution.

2.3. Drugs

The following drugs were used: (±)-8-hydroxy-2-(di-n-propyl-amino) tetralinhydrobromide (8-OH-DPAT; Sigma, USA), WAY-100635 (Sigma, USA), and midazo-lam maleate (Roche, Brazil). All drugs were dissolved in sterile saline.

2.4. Surgery

The animals were anaesthetized with 2,2,2-tribromoethanol (250 mg/kg, i.p.)and placed in a stereotaxic frame. In all experiments, two custom-made stainlesssteel guide cannulae (0.6 mm outer diameter, 0.4 mm inner diameter) were bilater-ally implanted in the brain aimed at the BLA. The following coordinates from bregmawere used: BLA: AP = −2.5 mm, lateral = ±5.1 mm, ventral = −6.2 mm [35]. The guidecannulae were fixed to the skull with acrylic resin and two stainless steel screws.Stylets with the same length as the guide cannulae were introduced inside them toprevent obstruction.

At the end of the surgery, all animals were injected intramuscularly with 0.3 mlof antibiotic preparation (benzylpenicillin and streptomycin, Pentabiotico Veter-inário Pequeno Porte; Forte Dodge, Brazil) to prevent possible infections. In addition,flunixin meglumine (Schering–Plough, Brazil; 3 mg/kg), a drug with analgesic,antipyretic and anti-inflammatory properties, was administered subcutaneously forpost-surgery analgesia.

The animals were left undisturbed in their home cages for 6–7 days after thesurgery, except for normal handling for cage cleaning.

2.5. Procedure

2.5.1. Drug microinjectionsFor drug injection, needles (0.3 mm outer diameter, custom-made of com-

mercially available dental needles; BD, Brazil) were introduced through the guidecannulae until their tips were 2 mm below the end of the cannulae. The drugs weremicroinjected into the BLA in a volume of 0.2 �l (for each side) over a period of 120 susing a 10 �l microsyringe (Hamilton 701-RN, USA) attached to a microinfusionpump (KD Scientific, USA). The displacement of an air bubble inside the polyethyl-ene catheters connecting the syringe needles to the intracerebral needles was usedto monitor the microinjection. The intracerebral needles were removed 60 s afterthe end of the injections.

2.5.2. Behavioral testsIn all the experiments described below, independent groups of animals were

used, i.e. each animal received only one injection into the BLA.

2.5.3. Experiments with the elevated T-maze, open-field and light–dark transitiontests

One day before the test, rats were exposed to one of the open arms of the T-mazefor 30 min. A wooden barrier mounted on the border between the maze central areaand the proximal end of the open arm isolated this arm from the rest of the maze.

On the test day, after previous randomization, these animals were intra-BLAinjected either with 8-OH-DPAT (0, 4, 8 or 16 nmol, n = 9–11; experiment 1A), WAY-100635 (0, 0.09, 0.18, 0.37 nmol, n = 5–7; experiment 1B) or midazolam (0, 10, 20or 40 nmol, n = 7–8; experiment 1C). In experiment 1D, we also investigated theeffect of a lower dose of 8-OH-DPAT (0 or 0.4 nmol, n = 8). Ten minutes after themicroinjections, all animals were sequentially tested in the elevated T-maze, open-field and light–dark transition tests, as described below.

In order to investigate whether WAY-100635 was able to block the effectsof 8-OH-DPAT, in experiment 1E the animals received intra-BLA microinjectionof WAY-100635 (0.37 nmol) or saline, 10 min before the microinjection of 8-OH-

DPAT (4 nmol) or saline. Thus, the following groups (n = 6–7) were formed: sal/sal,WAY/sal, sal/DPAT and WAY/DPAT. Ten minutes after the last microinjection, theanimals were submitted to the three behavioral tests mentioned above.

The doses of drugs were chosen on the basis of previously published studieswith the BLA [3,23–25].

oural Brain Research 246 (2013) 103– 110 105

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Fig. 1. Diagrammatic representation of coronal sections of the rat brain showingthe location of injection sites in the BLA (circles). Figures represent coordinates from

C.V. de Andrade Strauss et al. / Behavi

Inhibitory avoidance was the first task measured in the elevated T-maze. Eachnimal was placed at the distal end of the enclosed arm of the elevated T-mazeacing the intersection of the arms. The time taken by the rat to leave this arm withour paws was recorded (baseline latency). The same measurement was repeated inwo subsequent trials (avoidance 1 and 2) at 30-s intervals. When first placed at thend of the enclosed arm, the rat does not see the open arms until it pokes its headeyond the walls of the closed arm. Being on the open arm seems to be an aversivexperience, since rats have an innate fear of height and openness. As such, in the twoucceeding trials the animal gradually leaves the enclosed arm with longer latencies,ndicating the acquisition of inhibitory avoidance [for further details see 16].

Following avoidance training (30 s), rats were placed at the end of the samereviously experienced open arm and the latency to leave this arm with four pawsas recorded 3 consecutive times (escape 1, 2 and 3) with 30-s intertrial intervals.

cutoff time of 300 s was established for the avoidance and escape latencies.In order to assess possible drug effects on locomotor activity, 30 s after testings in

he elevated T-maze, each rat was individually placed at the center of the open-fieldnd the total distance traveled was evaluated by a video tracking system (Ethovision;oldus, Holland) for 5 min.

Immediately after testings in the open-field, all animals were submitted to theight/dark transition test. The rats were individually placed in the middle of the litompartment facing the doorway separating the two compartments. After the firstransition to the dark compartment, the behavior of the animal was recorded forn additional 5 min period, through the use of a video camera connected to a DVDecorder. During this period, the total time spent in the lit compartment and theumber of transitions between the two compartments were measured.

.5.4. Experiment with the Vogel conflict testIn experiment 2, independent groups of naïve animals were water-deprived for

8 h before the test. After the first 24 h of deprivation, they were allowed to drinkreely for 3 min in the test cage in order to find the drinking bottle spout. Somenimals that did not find the spout were not included in the experiment. Twenty-our hours later, animals were injected in the BLA with 8-OH-DPAT (0, 0.4, 2 and0 nmol, n = 7–9) and 10 min later they were again placed in the test cage. The testeriod lasted for 3 min, and the animals received a 0.5-mA shock for 2 s through theottle spout every 20 licks.

.6. Histology

After the experiments, animals were sacrificed under deep urethane anesthe-ia. Their brains were perfused intracardially with saline solution (0.9%) followedy 10% formalin solution before being removed and fixed in 10% formalin. Brainlices of 60 �m were made by means of a microtome in order to localize the sitef drug injection, according to Paxinos and Watson’s atlas [35]. Only animals withnjection sites bilaterally located in the BLA were included in the statistical analysis.

isplacement of guide-cannula was found in 35% of all animals tested.

.7. Statistical analysis

Repeated-measures analysis of variance was used to analyze both avoidancend escape data in the elevated T-maze. In experiment 1A–D, treatment (saline,-OH-DPAT, WAY-100635 or midazolam) was considered the independent factornd trial (baseline, avoidance 1 and 2 or escape 1, 2 and 3 latencies), the depend-nt, repeated measure. In experiment 1E, besides the dependent measure (trial),wo independent factors were considered: pre-treatment with WAY-100635 andreatment with 8-OH-DPAT. Data on locomotion in the open-field, exploration inhe light–dark transition model and number of punished drinking events in theogel test was analyzed by one-way ANOVA (experiments 1A-1D and 2) or two-ay ANOVA (experiment 1E). Multiple comparisons were performed by Duncan’s

ost hoc test.

. Results

Fig. 1 depicts the sites of drug injections into the BLA of animalsested in the current study.

.1. Experiments 1A and 1D: 8-OH-DPAT effects

Fig. 2 (left side, upper panel) shows that 8-OH-DPAT impairednhibitory avoidance acquisition [treatment effect - F(3,37) = 4.58,

< 0.05], suggesting an anxiolytic effect. Repeated measuresNOVA revealed a trial effect [F(2,74) = 6.38, p < 0.01] and a trial

y treatment interaction [F(6,74) = 3.12, p < 0.05]. The post hoc testhowed that 8-OH-DPAT at the three doses tested in experiment 1Aignificantly decreased avoidance 1 and 2 latencies when comparedith the control group.

the Paxinos and Watson [35] rat brain atlas, with respect to bregma. The number ofpoints in the figures is fewer than the total number of rats used because of severaloverlaps.

As can also be seen in Fig. 2 (left side, lower panel), 8-OH-DPATimpaired escape expression, indicating a panicolytic-like effect[treatment effect - F(3,37) = 4.90, p < 0.05]. There were also a trialeffect [F(2,74) = 26.45, p < 0.01] and a marginal trial by treatmentinteraction [F(6,74) = 1.96, p = 0.08]. Duncan’s test revealed that thedrug at a dose of 4 nmol significantly prolonged escape in all trialsmeasured, while at 8 nmol this effect was observed upon escape 1latency.

Fig. 2 (right side) shows that 8-OH-DPAT did not significantlychange the percentage of time spent in the lit compartment or thenumber of transitions between the two chambers of the light–darktransition test.

Table 1 shows that when a lower dose of this agonist was tested(0.4 nmol) no significant changes either in the elevated T-maze orlight–dark transition test were found.

At all doses tested 8-OH-DPAT did not affect locomotion in theopen-field (Table 2).

3.2. Experiment 1B: WAY-100635 effects

Intra-BLA injection of WAY-100635 did not change the behav-ioral responses measured in all tests used in the current study (seeTables 1 and 2).

3.3. Experiment 1C: midazolam effects

As illustrated in Fig. 3 (left side, upper panel) intra-BLA microin-jection of midazolam impaired inhibitory avoidance acquisition

[treatment effect - F(3,25) = 13.47, p < 0.01], suggesting an anx-iolytic effect. Repeated measures ANOVA also revealed a trialeffect [F(2,50) = 64.80, p < 0.01] and a trial by treatment interaction[F(6,50) = 5.09, p < 0.01]. The post hoc test showed that this

106 C.V. de Andrade Strauss et al. / Behavioural Brain Research 246 (2013) 103– 110

Fig. 2. Effects (mean ± SEM) of intra-BLA injection of 8-OH-DPAT (DPAT) on inhibitory avoidance and escape behaviors measured in the elevated T-maze (left side) and on thep compt

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ercentage of time spent in the lit compartment and number of transitions betweeno the saline-injected group.

enzodiazepine at all doses tested significantly decreasedvoidance 1 and 2 latencies when compared to the control group.

Midazolam did not affect escape expression in the elevated T-aze (see Fig. 3, left side, lower panel) or locomotion in the open-

eld (Table 2).In the light–dark transition test (see Fig. 3, right side), the drug

ignificantly increased the time spent by the rats in the lit com-artment [F(3,25) = 3.71, p < 0.05] and the number of transitionsetween the compartments [F(3,25) = 5.46, p < 0.05].

.4. Experiment 1E: previous intra-BLA administration ofAY-100635 and 8-OH-DPAT effects

Fig. 4 (left side, upper panel) shows that intra-BLA injectionf 8-OH-DPAT impaired inhibitory avoidance acquisition and that

his effect was fully counteracted by previous microinjection of

AY-100635. Repeated measures ANOVA showed significantain effects of trial [F(2,42) = 53.81, p < 0.01] and treatmentith the antagonist [F(1,21) = 8.26, p < 0.05]. There were also

able 1ffect (mean ± S.E.M) of intra-BLA injection of 5-HT1A receptor-acting drugs on the beha

Drug (nmol) Baseline (s) Avoidance 1 (s) Avoidance 2 (s) Escape 1 (s)

Experiment 1BSaline 8.8 ± 1.3 57.3 ± 23.8 201.6 ± 61.1 7.2 ± 0.8

WAY 100635 (0.09) 8.0 ± 1.4 15.7 ± 3.4 110.7 ± 43.8 9.0 ± 2.0

WAY 100635 (0.18) 13.2 ± 1.6 25.3 ± 4.5 157.7 ± 53.4 11.3 ± 2.1

WAY 100635 (0.37) 9.1 ± 1.7 77.7 ± 38.1 186.1 ± 53.7 8.7 ± 1.5

Experiment 1DSaline 12.0 ± 1.6 53.0 ± 21.0 232.0 ± 26.0 7.2 ± 0.9

8-OH-DPAT (0.4) 15.0 ± 3.5 75.0 ± 30.5 227.0 ± 31.0 6.9 ± 0.8

artments of the light–dark transition test (right side). n = 9–11. * p < 0.05 compared

significant interactions between trials and treatment with the ago-nist [F(2,42) = 4.15, p < 0.05] and pre-treatment with the antagonist[F(2,42) = 3.60, p < 0.05]. The post hoc test showed that 8-OH-DPATsignificantly decreased avoidance 2 latency when compared to allother groups.

The lower panel of Fig. 4 (left side) shows that 8-OH-DPAT impaired escape expression and this effect was alsofully counteracted by previous microinjection of WAY-100635.Repeated measures ANOVA showed significant main effectsof trial [F(2,42) = 83.64, p < 0.01] and treatment with the ago-nist [F(1,21) = 63.30, p < 0.01] and the antagonist [F(1,21) = 62.16,p < 0.01]. There were also significant interactions between trials andtreatment with the agonist [F(2,42) = 96.06, p < 0.01] and with theantagonist [F(2,42) = 80.18, p < 0.01]. The post hoc test showed that8-OH-DPAT significantly prolonged escape 2 and 3 latencies. Prior

intra-BLA administration of WAY-100635 blocked this panicolytic-like effect.

In the light–dark transition test, 8-OH-PAT significantlyincreased the percentage of time spent in the lit compartment

viors of rats submitted to the ETM and light–dark transition tests.

Escape 2 (s) Escape 3 (s) % Time lit compartment Number transitions

10.2 ± 2.7 5.2 ± 1.0 21.6 ± 6.4 6.0 ± 1.88.8 ± 1.9 7.2 ± 0.9 19.4 ± 2.5 6.6 ± 1.1

10.0 ± 2.1 5.5 ± 1.1 20.2 ± 3.4 5.5 ± 1.27.1 ± 1.1 9.4 ± 1.4 16.4 ± 3.2 5.8 ± 1.0

6.3 ± 0.8 5.3 ± 0.3 13.2 ± 2.6 7.0 ± 1.66.6 ± 0.6 6.8 ± 1.2 10.5 ± 4.0 6.0 ± 1.4

C.V. de Andrade Strauss et al. / Behavioural Brain Research 246 (2013) 103– 110 107

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ig. 3. Effects (mean ± SEM) of intra-BLA injection of midazolam (MDZ) on inhibitorercentage of time spent in the lit compartment and number of transitions betweeo the saline-injected group.

treatment effect - F(1,21) = 5.28, p < 0.05], without affecting the

umber of transitions (see Fig. 4, right side). Previous intra-BLA

njection of WAY-100635 blocked the anxiolytic effect caused byhe agonist [treatment x pre-treatment interaction - [F(1,21) = 7.56,

< 0.05].

able 2ffect (mean ± S.E.M) of intra-BLA injection of 5-HT1A receptor-acting drugs on theistance traveled in the open field test.

Drug (nmol) Distance traveled (m)

Experiment 1ASaline 11.96 ± 1.018-OH-DPAT (4) 15.27 ± 1.708-OH-DPAT (8) 14.22 ± 1.088-OH-DPAT (16) 17.69 ± 3.18

Experiment 1BSaline 17.75 ± 2.12WAY 100635 (0.09) 19.80 ± 0.95WAY 100635 (0.18) 16.69 ± 1.27WAY 100635 (0.37) 18.49 ± 0.75

Experiment 1CSaline 12.34 ± 1.29Midazolam (10) 16.41 ± 2.76Midazolam (20) 13.35 ± 1.36Midazolam (40) 18.03 ± 0.90

Experiment 1DSaline 16.14 ± 2.148-OH-DPAT 13.23 ± 1.41

Experiment 1ESaline/Saline 15.68 ± 0.93WAY/Saline 14.33 ± 1.60Saline/8-OH-DAPT 14.94 ± 1.37WAY/8-OH-DPAT 15.65 ± 2.05

dance and escape behaviors measured in the elevated T-maze (left side) and on thepartments of the light–dark transition test (right side). n = 7–8. * p < 0.05 compared

None of the treatments employed in experiment 1E changedlocomotion in the open-field (Table 2).

3.5. Experiment 2: 8-OH-DPAT effects in the Vogel conflict test

Fig. 5 shows that intra-BLA injection of 8-OH-DPAT significantlyincreased the number of punished drinking events in the Vogelconflict test [F(3,28) = 4.54, p = 0.01]. The number of non-punisheddrinking events during the training section was not different amongthe groups tested (data not shown).

4. Discussion

The results of the present investigation showed that stimu-lation of 5-HT1A-R within the BLA affects defensive behaviorsassociated with both anxiety and panic. Thus, intra-BLA administra-tion of 8-OH-DPAT impaired inhibitory avoidance acquisition andescape expression in the elevated T-maze, suggesting anxiolytic-and panicolytic-like effects, respectively. The anxiolytic effect ofthis agonist was also detected in the light–dark transition modeland Vogel conflict test. It should be highlighted, however, that forthe light–dark transition model, although the anxiolytic effect of8-OH-DPAT was clear in experiment 1E (antagonism with WAY-100635), in experiment 1A (dose-response curve), only an overalltrend toward anxiolysis was detected. However, it is notewor-thy that in experiment 1A when the group tested with a dose of4 nmol was individually compared to the control group, a signifi-

cant increase (p = 0.05) in the time spent by the rats in the aversivecompartment could be observed.

Similarly to 8-OH-DPAT, intra-BLA injection of midazolamalso inhibited avoidance acquisition in the elevated T-maze, but

108 C.V. de Andrade Strauss et al. / Behavioural Brain Research 246 (2013) 103– 110

Fig. 4. Effects (mean ± SEM) of intra-BLA injection of WAY-100635 (WAY; 0.37 nmol) 10 min before the microinjection of 8-OH-DPAT (DPAT; 4 nmol) on inhibitory avoidancea of timo e-inje

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nd escape behaviors measured in the elevated T (left side) and on the percentage

f the light–dark transition test (right side). n = 6–7. * p < 0.05 compared to the salin

n contrast to the 5-HT agonist, this effect was not followed byhanges in escape performance. Midazolam also caused a clearnxiolytic effect in the light–dark transition test.

Local administration of the 5-HT1A-R antagonist WAY-100635ad no effect on the defensive behaviors measured in the elevated-maze and light–dark transition test, but fully blocked the behav-oral effects of 8-OH-DPAT in these two tests. This last findingonfirms the recruitment of 5-HT1A-Rs for the observed effects of

-OH-DPAT.

There is no indication that the effects reported here withhe 5-HT1A-modulating drugs were due to unspecific changes inocomotor activity. Accordingly, none of the compounds tested

ig. 5. Effects (mean + SEM) of intra-BLA injection of 8-OH-DPAT (DPAT) on theumber of punished drinking events measured in the Vogel conflict test. n = 7–9.

p < 0.05 compared to the saline-injected group.

e spent in the lit compartment and number of transitions between compartmentsct group; +p < 0.05 compared to all other groups.

interfered with the total distance traveled by the animals in theopen-field or altered the number of transitions between the twocompartments of the light–dark chamber. The same holds true formidazolam. In this case, no alteration in locomotion in the open-field was observed, although the highest dose increased the numberof transitions in the light–dark model. It is noteworthy, however,that the anxiolytic effect detected with the two other doses testedwas not followed by any significant change in this index. Moreover,besides being an index associated with locomotion, the number oftransitions may also reflect changes in anxiety [17], which in thepresent case reinforces the idea that midazolam was anxiolytic.

The reduction in anxiety induced by 8-OH-DPAT and midazolamin the elevated T-maze confirms earlier findings of our lab usingthe same test, but with animals without previous experience withthe open arm [25]. However, differently from the former study, inwhich 8-OH-DPAT (8 and 16 nmol) failed to alter escape expression,in the present analysis this behavior was significantly inhibited bythe 5-HT agonist. Midazolam was without effect on escape in bothstudies.

Altogether, these findings support the view that pre-exposureto the open arm does not alter the effect of pharmacological manip-ulations on avoidance acquisition, but may have a great impact onescape performance [30,36]. More specifically, it has been shownthat this procedure, by shortening the latencies to withdrawal fromthe open arm during the test, presumably by decreasing reactions tonovelty (e.g. exploration, behavioral inhibition), renders the escapetask more sensitive to the effects of antipanic drugs [30,31] andhence is a better measure of panic attacks [36].

As observed here with midazolam, Bueno et al. [37] reportedthat facilitation of GABAA receptor-mediated neurotransmissionin the BLA by local injection of the agonist muscimol also impairedinhibitory avoidance acquisition, without affecting escape

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xpression in animals pre-exposed to the open arm of thelevated T-maze. Therefore, independently of the test protocol,ctivation of GABAA/benzodiazepine receptors in the BLA onlynterferes with the defensive task that in this test is associated

ith GAD.In line with these findings, administration of benzodiazepines

r GABAA receptor agonists into the BLA have been reported toause anxiolysis in different animal models of anxiety, such as: thelevated plus-maze [23,38,39] and the social interaction test [24].uriously, in two recently published papers neither intra-BLA injec-ion of muscimol [40] nor midazolam [41] changed anxiety in thelevated plus-maze. It is of note that in the work with muscimol,nly one dose was tested and this was 8 times lower than the dosereviously reported to cause anxiolysis in the elevated T-maze [37].esides, in these two studies, the drugs were injected unilaterally,

n the right hemisphere, in contrast to all above cited works wherenjections were made in both hemispheres.

Taking into account the three experimental models of anxietysed here, our results provide compelling evidence that activa-ion of 5-HT1A-Rs in the BLA reduces anxiety. It is important toighlight, however, that two other previous studies [23,24] failedo find significant effects of 8-OH-DPAT in rats tested in the ele-ated plus-maze. In the study of Zangrossi and Graeff [23] whateserves attention is the fact that the agonist was injected withinhe whole basolateral complex, which in addition to the basolat-ral nucleus also comprises the lateral and basomedial subnuclei42]. Notwithstanding this limitation, in that study there was a clearrend toward an anxiolytic effect with a dose of 8 nmol, an effectiveose in present study with the T-maze. In the work of Gonzalez andolleagues [24], a lack of effect was observed within a dose range of.15–0.6 nmol. In none of the tests we performed were doses as lows these effective. It is curious that in the study of Gonzalez, 0.15 and.6 nmol of 8-OH-DPAT increased anxiety in the social interactionest, an effect followed by a decrease in locomotion at the highestose. Within a similar dose range, Hodges et al. [3] also reportednxiogenic effects of this agonist, but in a modified Geller–Seifteraradigm. In this latter study, the drug was microinjected not only

n the basolateral nucleus but also in the lateral subnucleus. There-ore, it is conceivable that methodological differences such as theoses used and the area of injection may underlie these discrepantesults in the literature.

Our present findings are also in agreement with previous evi-ence showing an involvement of the BLA in the modulation ofscape behavior. For instance, lesions of this subnucleus werehown to inhibit flight behavior expressed by wild rats when pur-ued by a human being [43] and to interfere with the escape deficitroduced by uncontrollable stress [44].

One important consideration in the present paper is that intra-LA injection of WAY-100635 did not affect inhibitory avoidancecquisition and escape expression. The lack of effect of this antag-nist suggests that, under physiological conditions, activation of-HT1A-Rs in this amygdala subnucleus is not required for thexpression of these behaviors. This contrasts with the role playedy 5-HT2C-Rs in the same area. In a recent paper, we reported that

ocal administration of the 5-HT2C-R antagonist SB-242084 caused clear anxiolytic effect in the elevated T-maze [15], suggesting that-HT exerts a tonic regulatory role in this area through the activa-ion of these receptors. In that paper we also presented evidencehat activation of BLA 5-HT2C-Rs is implicated in the anxiogenicffect observed after short-term administration of antidepressants.ased on a hypothesis originally formulated by Millan [6], were currently investigating whether the anxiolytic effect caused

y chronic treatment with these drugs involves a decrease inhe functioning of these receptors with a concomitant improve-

ent in the 5-HT1A-R-mediated response in the BLA. In addition,t will be of interest to investigate whether adaptive changes in

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Brain Research 246 (2013) 103– 110 109

BLA 5-HT1A-Rs are also involved in the panicolytic effect caused bylong-term antidepressant administration, as it has been show forthe dorsal periaqueductal gray matter [45], a midbrain area con-sistently associated with the genesis/regulation of panic attacks[46,47].

In conclusion, the current results show that stimulation of 5-HT1A-Rs in the BLA causes both anxiolytic- and panicolytic-likeeffects, what may have implication for the pathophysiology andtreatment of generalized anxiety and panic disorders.

Acknowledgments

The authors thank Afonso Paulo Padovan for helpful techni-cal support. The experiments described in this manuscript werefunded by FAPESP, CAPES and CNPq, Brazil.

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