Behavioural Brain Research 271 (2014) 249–257

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Dorsomedial hypothalamus CRF type 1 receptors selectively modulate inhibitory avoidance responses in the elevated T-maze Mariana S.C.F. Silva a , Bruno A. Pereira a , Isabel C. Céspedes a , Juliana O.G. Nascimento b , Jackson C. Bittencourt c , Milena B. Viana a,∗ a

Departamento de Biociências, Universidade Federal de São Paulo, 11060-001 Santos, SP Brazil Departamento de Psiquiatria e Psicologia Médica, Universidade Federal de São Paulo, 04038-020 São Paulo, SP, Brazil c Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo 05508-000, SP, Brazil b

h i g h l i g h t s • • • •

CRF intra-dorsomedial hypothalamus is anxiogenic. Antalarmin intra-dorsomedial hypothalamus is anxiolytic. Antalarmin couteracts the anxiogenic effects of CRF. Panic-related responses and locomotor activity are not altered.

a r t i c l e

i n f o

Article history: Received 8 April 2014 Received in revised form 4 June 2014 Accepted 8 June 2014 Available online 14 June 2014 Keywords: Corticotropin-releasing factor CRF Dorsomedial hypothalamus Elevated T-maze Anxiety Panic

a b s t r a c t Corticotropin-releasing factor (CRF) plays a critical role in the mediation of physiological and behavioral responses to stressors. In the present study, we investigated the role played by the CRF system within the dorsomedial hypothalamus (DMH) in the modulation of anxiety- and panic-related responses. Male Wistar rats were administered into the DMH with CRF (125 and 250 ng/0.2 ␮l, experiment 1) or with the CRFR1 antagonist antalarmin (25 ng/0.2 ␮l, experiment 2) and 10 min later tested in the elevated T-maze (ETM) for inhibitory avoidance and escape measurements. In clinical terms, these responses have been respectively related to generalized anxiety and panic disorder. To further verify if the anxiogenic effects of CRF were mediated by CRFR1 activation, we also investigated the effects of the combined treatment with CRF (250 ng/0.2 ␮l) and antalarmin (25 ng/0.2 ␮l) (experiment 3). All animals were tested in an open field, immediately after the ETM, for locomotor activity assessment. Results showed that 250 ng/0.2 ␮l of CRF facilitated ETM avoidance, an anxiogenic response. Antalarmin significantly decreased avoidance latencies, an anxiolytic effect, and was able to counteract the anxiogenic effects of CRF. None of the compounds administered altered escape responses or locomotor activity measurements. These results suggest that CRF in the DMH exerts anxiogenic effects by activating type 1 receptors, which might be of relevance to the physiopathology of generalized anxiety disorder. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Stress is a complex phenomenon that refers to the observation that any environmental change can disrupt the maintenance of homeostasis, causing a series of physiological and behavioral modifications that together encompass the so-called “stress response” [1]. One of the main physiological alterations that accompany

∗ Corresponding author. Tel.: +55 13 33851535/+55 11 38683203; fax: +55 13 33851535/+55 11 38683203. E-mail addresses: [email protected], [email protected] (M.B. Viana). http://dx.doi.org/10.1016/j.bbr.2014.06.018 0166-4328/© 2014 Elsevier B.V. All rights reserved.

the stress reaction is the activation of the hypothalamic– pituitary–adrenocortical (HPA) axis by corticotropin-releasing factor (CRF) release from the paraventricular nucleus of the hypothalamus. CRF activates the HPA axis by stimulating adrenocorticotropic hormone (ACTH) release from the anterior pituitary. ACTH, in turn, triggers the release of glucocorticoids from the adrenal cortex. Since it regulates the HPA axis activity, CRF is a critical element for the maintenance of an organism’s homeostasis. Nevertheless, nowadays CRF has also been considered for its role within the central nervous system (CNS) [2,3], outside the HPA axis [4]. In fact, CRF neurons have been identified in several different brain regions, i.e.,

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the neocortex, amygdala, medial septal nucleus, thalamus, other hypothalamic nuclei, cerebellum, and in autonomic midbrain and hindbrain nuclei [5,6]. CRF produces its biological effects binding to two major proteincoupled receptors, CRF type 1 (CRFR1) and CRF type 2 (CRFR2). The activation of CRFR1 has been associated with an increased reaction to stress and anxiety-like behavior [7–10]. On the other hand, the involvement of CRFR2 with stress and anxiety-related responses is still a matter of debate [7–10]. The medial hypothalamus has been implicated in a series of different behavioral and physiological functions, including feeding and metabolism, reproduction and stress/anxiety [11,12]. The region is composed of a number of well-circumscribed neuronal groups [12] – i.e., the dorsomedial hypothalamus (DMH), the anterior hypothalamus, the ventromedial hypothalamus and the premamillary nucleus – that are highly interconnected. The medial hypothalamus seems to be particularly involved with the integration of innate stress/anxiety-related responses to environmental threats [12–17]. In fact, the electrical stimulation of the DMH induces escape behavior and autonomic arousal that resemble the ones presented by animals when facing natural threats [18–20]. Administration of glutamate agonists [13,21] and of GABA antagonists into the structure [22,23] also evokes a similar defense pattern. CRF positive neurons have been shown to be present in large numbers in the DMH [24]. Furthermore, the region possesses a substantial concentration in particular of CRF type 1 receptors [25]. It has been previously demonstrated that intracerebroventricular administration of CRF and corticosterone induces neurochemical changes in the DMH, i.e., increases in dopamine and serotonin, that parallel changes observed in the region following exposure to a variety of physical and psychological stress-related stimuli [26]. Also, intraperitoneal injection of JNJ19567470/CRA5626, a CRFR1 antagonist, prevents the sodium lactate-induced panic-like behavioral and cardiovascular responses observed in adult male rats with chronic reduction of GABA levels in the region [27]. Nevertheless, apart from this indirect evidence implicating the DMH CRF system with stress/anxiety, to our knowledge no previous study has investigated the effects of the direct infusion of CRF or CRF-related compounds into the region in the modulation of stress/anxiety-related responses. The present study addresses this question by investigating the effects of intra-DMH CRF and antalarmin – a CRFR1 antagonist [28] – in an animal model of anxiety, the elevated T-maze (ETM). The model, composed of one arm enclosed by walls disposed perpendicularly to two opposed open arms, allows the measurement of an anxiety and a panic-related response. Although the distinction between anxiety and panic is often unclear, the ethopharmacological analysis of the rodent defensive repertoire has provided a sound theoretical framework [29]. According to this view, anxiety is an emotion related to behavioral inhibition and risk assessment, reactions performed in situations of potential danger, either because the context is new or because the aversive stimulus was once present in the past. These responses also seem to be performed when an animal is faced with a threat, which involves a conflict between approach and avoidance (e.g. seeking for food in an area where a predator was once present) [30,31]. On the other hand, panic corresponds to vigorous escape or flight reactions evoked by proximal danger [29]. In terms of psychopathology, it has been proposed that the neurobiological substrates that regulate anxiety are disrupted in generalized anxiety, while dysfunction of the brain circuitry controlling proximal defense reactions has been related to panic disorder [32]. The ETM was developed on the basis of the ideas presented above [33]. The model is composed of one arm surrounded by 40cm high walls, disposed perpendicularly to two opposed open arms.

ETM inhibitory avoidance is an anxiety-related response, measured by placing the animal for three consecutive times at the distal end of the closed arm of the maze and registering the latency to leave this arm with the four paws. Escape, on the other hand, is a panicrelated response, measured by placing the animals directly in one of the open arms of the maze and measuring the latency to leave this arm with the four paws. The pharmacological validation of the ETM has shown that compounds representative of three classes of anxiolytics – namely the agonist of benzodiazepine receptors diazepam, the serotonin 1A agonist buspirone, and the nonselective serotonin type 2 antagonist ritanserin – selectively impair inhibitory avoidance while leaving one-way escape unchanged [34–36]. These results are compatible with the view that inhibitory avoidance relates to generalized anxiety. In contrast the escape task is impaired by chronic, but not acute administration of imipramine [37], clomipramine and fluoxetine [38], drugs that are used to treat panic. As a result, ETM escape has been used as an animal model of panic disorder. To further verify if the anxiogenic effects of CRF were in fact mediated by CRFR1 activation, in the present study we also investigated the effects of the combined treatment with CRF and antalarmin. 2. Materials and methods 2.1. Subjects Male Wistar rats (CEDEME, Universidade Federal de São Paulo, Campus Santos, Brazil), weighing 280–320 g at the beginning of the experiment, were housed in groups of 5–6 per cage. After surgery, animals were housed in pairs in Plexiglas-walled cages until testing. Room temperature was controlled (22 ± 1 ◦ C) and a light–dark cycle was maintained on a 12-h on–off cycle (0700–1900 h lights on). Food and water were available all throughout the experiments. The study was approved by the Ethical Committee for Animal Research of the Federal University of São Paulo and was performed in compliance with the recommendations of the Brazilian Society of Neuroscience and Behavior, which are based on the conditions stated by the “Guide for the Care and Use of Laboratory Animals” (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, 1996). 2.2. Apparatus The elevated T-maze was made of wood and had 3 arms of equal dimensions (50 × 12 cm). One of the arms was enclosed by 40 cm high walls and was oriented perpendicularly to two opposed open arms. The whole apparatus was elevated 50 cm above the floor. To avoid falls, a 1 cm high Plexiglas rim surrounded the open arms. An open field, composed of a round arena (60 × 60 cm), with the floor divided into 12 parts, and walls 50 cm high, was used to evaluate locomotor activity. Luminosity at the level of the T-maze arms or at the open field center was 60 lx. After the experimental sessions, each experimental apparatus was cleaned with a 10% ethanol solution. 2.3. Compounds Rat/human CRF (r/h CRF no. 102-282-15 – PBL, The Salk Institute for Biological Studies, USA) was initially dissolved in a solution of 0.04% acetic acid and 0.02 M KPBS and diluted 1:1 in sterile saline (0.9%). Control animals were administered with a solution of 0.04% acetic acid and 0.02 M KPBS in sterile saline (0.9%) (1:1). The CRF type 1 antagonist antalarmin (no. A8727, Sigma, USA) was dissolved in sterile saline (0.9%) with 2% Tween 80. Control animals were injected with a solution of sterile saline (0.9%) with 2% Tween 80.

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Compounds and vehicles were administered in a volume of 0.2 ␮l 10 min prior to the test sessions.

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Three days after their arrival to the laboratory, rats were anaesthetized with an IP injection of ketamine hydrochloride (80 mg/kg; Agribrands, Brazil) and xylazine (10 mg/kg; Agribrands, Brazil) and fixed to a stereotaxic frame (David Kopf, USA). Local anaesthesia was also performed (2% lidocaine with a vasoconstrictor; Harvey, Brazil) before the implant of stainless steel guide cannulae into the DMH. Guide cannulae (0.6 mm outer diameter and 0.4 mm inner diameter) were inserted into the brain through a hole drilled in the skull above the DMH, following the coordinates from the atlas of Paxinos and Watson [39]: AP = −3.0 mm from bregma; ML = −0.6 mm and DV = −7.2 mm from skull. Cannulae were attached to the skull by means of acrylic resin and two stainless steel screws. Stylets with the same length of the guide cannulae were introduced inside them to prevent obstruction. To prevent infections, at the end of the surgery, all animals were injected (IM) with a 0.2 ml of pentabiotic preparation (Pentabiotico Veterinário Pequeno Porte; Forte Dodge, Brazil). In addition, flunixin meglumine (Schering–Plough, Brazil; 3 mg/kg), a drug with analgesic, antipyretic and anti-inflammatory properties, was administered subcutaneously for post-surgery analgesia. The animals were left undisturbed in their home cages for 6 days after the surgery, except for normal handling for cage cleaning, and monitoring of signs of postoperative pain or behavioral alterations.

saline + Tween 80)/CRF, and antalarmin/CRF. Ten minutes after the last microinjection, the animals were submitted to the behavioral tests as described below. The dose of 250 ng of CRF was chosen for this experiment since it altered both avoidance 1 and 2 latencies in experiment 1. For inhibitory avoidance measurement, each animal was placed at the distal end of the enclosed arm of the elevated T-maze facing the intersection of the arms. The time taken by the rat to leave this arm with the four paws, for the first time, was recorded (baseline latency) and used as a measurement of locomotor activity [34–36]. After baseline latency measurements, the time taken by the rat to leave the enclosed arm was again recorded in two subsequent trials (avoidance 1 and 2) at 30 s inter-trial intervals, during which animals were placed in a Plexiglas cage to which they had been previously habituated. Since being in the open arms seems to be an aversive experience, in these subsequent trials, the animals usually take a longer time to withdraw from the enclosed arm toward the open space, thus showing avoidance learning. Following avoidance measurements (30 s), rats were placed at the end of one of the open arms and the latency to leave this arm with the four paws was recorded for 3 consecutive times (escape 1–3), with 30 s inter-trial intervals. The latencies were always evaluated in the same previously experienced open arm. A cutoff time of 300 s was established for the avoidance and escape latencies. Immediately after being tested in the ETM, each animal was placed for 5 min in the center of the open field and the total number of lines crossed were recorded. ETM and open field measurements were registered by an observer, through the use of a monitor placed in an adjacent room.

2.5. Microinjections

2.7. Histology

For microinjections, needles (0.3 mm outer diameter) were introduced through the guide cannulae until their tip were 1 mm below the cannulae end. Compounds and vehicles were injected over a period of 120 s using 5 ␮l microsyringes (Hamilton 701RN, USA) attached to a microinfusion pump (Insight, Brazil). The displacement of an air bubble inside the polyethylene catheter connecting the syringe needle to the intra-cerebral needle was used to monitor the microinjection. The intra-cerebral needles were removed 60 s after the end of injection.

After the experiments, animals were sacrificed under deep anesthesia with urethane. Their brains were perfused through the heart with saline solution followed by 10% formalin solution, before being removed and fixed in 10% formalin. Frozen sections of 55 ␮m were cut using a microtome in order to localize the site of injections, according to the Paxinos and Watson’s atlas [39]. Only data from rats with injection sites into the DMH were included in the statistical analysis (see Section 2.8).

2.6. Procedure

2.8. Statistical analysis

On the fifth and sixth days after surgery, the experimenter gently handled animals for 5 min. On the sixth day, immediately after handling, rats were exposed to one of the open arms of the Tmaze for 30 min. A wood barrier mounted on the border of the central area of the maze and the open arm’s proximal end isolated this arm from the rest of the maze. It has been shown that this pre-exposure to the open arm renders the escape task more sensitive to the effects of antipanic compounds, because it shortens the latencies of withdrawal from the open arm during the test [40]. On the seventh day after surgery, animals were injected (0.2 ␮l) with CRF (125 or 250 ng; N = 8–11) (experiment 1) or antalarmin (25 ng; N = 7–9) (experiment 2) into the DMH, as described above, and 10 min later tested in the ETM for inhibitory avoidance and escape measurements. The doses of the compounds were chosen on the basis of previously published studies [41,42]. In order to investigate whether antalarmin was able to block the effects of CRF (experiment 3), animals received intra-DMH microinjection of antalarmin (25 ng) or vehicle (KPBS + sterile saline), 10 min before the microinjection of CRF (250 ng) or vehicle (sterile saline + Tween 80). Thus, the following groups (N = 6–9) were formed: vehicle (sterile saline + Tween 80)/vehicle (KPBS + sterile saline), antalarmin/vehicle (KPBS + sterile saline), vehicle (sterile

For experiments 1 and 2, two-way analysis of variance (ANOVA) with repeated measures was used to analyze avoidance and escape data from the ETM, with treatment as the independent factor and trials (baseline, avoidance 1 and 2, or escape 1 to 3) as the dependent factors. A three-factor design was used to analyze ETM results from experiment 3, with the two treatments (treatment 1: vehicle or antalarmin; treatment 2: vehicle or CRF) as the independent factors and trials as the dependent factor. Significant effects of the independent factors or of the interaction between the independent and dependent factors were analyzed by one-way ANOVA followed by the Tukey post-hoc test (experiments 1 and 3) or by the Student’s t-test (experiment 2). Locomotor activity in the open field was analyzed by one or two-way ANOVA followed by the Tukey post-hoc test (experiments 1 and 3, respectively) or by the Student’s t-test (experiment 2). A value of P ≤ 0.05 was considered significant.

2.4. Surgery

3. Results Fig. 1 illustrates the sites of injections (panel A) and a representative photomicrograph of a microinjection tip placement within the DMH (panel B). Only animals with microinjection sites in the DMH were included in Section 2.8.

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Fig. 1. (Panel A) Diagrammatic representation of microinjection sites inside (circles) and outside (triangles) the DMH [39]. Due to overlaps, the number of points represented is fewer than the number of rats actually injected. (Panel B) Representative photomicrograph of a microinjection tip placement within the DMH (circled with a white line, bregma −3.30 mm).

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Fig. 2. Effect (mean ± S.E.M) of the intra-DMH injection of CRF (125 or 250 ng/0.2 ␮l) or vehicle on inhibitory avoidance (panel A) and escape (panel B) latencies of rats in the elevated T-maze. N = 11 (control), 8 (CRF 125 ng) and 8 (CRF 250 ng). * P < 0.05, compared with the control group in the same trial (ANOVA, followed by the Tukey test).

3.1. Experiment 1: Effects of CRF Fig. 2 (upper panel) shows the effects of CRF administered intra-DMH on ETM inhibitory avoidance measurements. Repeated measures ANOVA showed a significant effect of trials (F(2,48) = 13.46; P < 0.001) and treatment (F(2,24) = 7.53; P < 0.003), but not a significant trials by treatment interaction (F(4,48) = 1.27; P = 0.296). The Tukey test showed that in baseline and avoidance 1 animals treated with 250 ng of CRF took a significant longer time to leave the enclosed arm when compared to control animals (P < 0.05). Fig. 2 (lower panel) shows the effects of intra-DMH CRF on ETM escape measurements. Repeated measures ANOVA did not show a significant effect of trials (F(2,48) = 1.83; P = 0.176), treatment (F(2,24) = 1.38; P = 0.270), or trials by treatment interaction (F(4,48) = 1.35; P = 0.267). Table 1 shows the effects of intra-DMH treatment with CRF on the number of crossings in an open field. One-way ANOVA did not show any significant differences between groups of treatment (F(2,26) = 0.152; P = 0.860). 3.2. Experiment 2: Effects of antalarmin Fig. 3 (upper panel) illustrates the effects of intra-DMH administration of antalarmin on ETM inhibitory avoidance. Repeated measures ANOVA showed a significant effect of trials (F(2,28) = 6.77; P = 0.004) and treatment (F(1,14) = 5.24; P = 0.038), but not of the interaction treatment by trials (F(2,28) = 1.50; P = 0.241). Two-tailed unpaired Student t-test showed that

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Fig. 3. Effect (mean ± SEM) of the intra-DMH injection of antalarmin (25 ng/0.2 ␮l) or vehicle on inhibitory avoidance (panel A) and escape (panel B) latencies of rats in the elevated T-maze. N = 7 (control) and 9 (antalarmin). * P < 0.05, compared with the control group in the same trial (unpaired Student t-test).

avoidance 1 latencies were significantly smaller in animals treated with antalarmin when compared to control animals (t(14) = 2.34; P = 0.05). Fig. 3 (lower panel) shows the effects of intra-DMH antalarmin on ETM escape. Repeated measures ANOVA did not show a significant effect of trials (F(2,28) = 0.68; P = 0.514), treatment (F(1,14) = 2.48; P = 0.138), or trials by treatment interaction (F(2,28) = 0.55; P = 0.580). Table 1 shows the effects of intra-DMH antalarmin on the number of crossings in an open field. Two-tailed unpaired Student t-test showed no significant differences between groups of treatment (t(14) = 0.00; P = 1.00).

Table 1 Number of crossings (mean ± SEM) in the open field for each treatment group. Treatment

Number of crossings

Vehicle CRF 125 ng CRF 250 ng Vehicle Antalarmin Vehicle/vehicle CRF/vehicle Antalarmin/vehicle Antalarmin/CRF Vehicle/vehicle (outside DMH) CRF/vehicle (outside DMH) Antalarmin/vehicle (outside DMH) Antalarmin/CRF (outside DMH)

53.36 52.88 42.25 45.71 59.11 52.00 56.75 63.66 74.42 69.54 62.60 56.50 67.50

± ± ± ± ± ± ± ± ± ± ± ± ±

3.69 4.62 5.66 5.61 5.16 5.35 7.00 5.41 10.20 6.30 4.45 5.04 8.17

CRF: corticotropin releasing factor; DMH: dorsomedial hypothalamus.

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Fig. 4. Effects (mean ± SEM) of the combined intra-DMH treatment with CRF (250 ng/0.2 ␮l) and antalarmin (25 ng/0.2 ␮l) on inhibitory avoidance (panel A) and escape (panel B) latencies of rats in the elevated T-maze. N = 7 (vehicle/vehicle), 7 (vehicle/CRF), 6 (antalarmin/vehicle), 6 (antalarmin/CRF). * P < 0.05, compared with all the other groups in the same trial; + P < 0.05, compared to the antalarmin/vehicle and to the antalarmin/CRF groups (ANOVA, followed by the Tukey test).

3.3. Experiment 3: Effects of the combined treatment with CRF and antalarmin Fig. 4 (upper panel) shows the effects of the combined treatment with CRF and antalarmin intra-DMH on ETM inhibitory avoidance measurements. Three-way ANOVA showed a significant effect of trials (F(2,44) = 15.09; P < 0.001), antalarmin by trials (F(2,44) = 12.84; P < 0.001), antalarmin (F(1,22) = 32.03; P < 0.001), CRF (F(1,22) = 5.32; P = 0.031), and of the interaction antalarmin by CRF (F(1,22) = 6.05; P = 0.022). No significant effects of the interaction trials by CRF (F(2,44) = 2.37; P = 0.105) or trials by antalarmin by CRF (F(2,44) = 2.46; P = 0.10) were found. The Tukey post-hoc test showed that in avoidance 1 the CRF-treated group spent a significantly longer time in the closed arm of the ETM when compared to the other three groups (P < 0.001). Also in avoidance 2, the vehicle/vehicle (P = 0.03, when compared to the antalarmin groups) and the CRF/vehicle groups (P = 0.003, when compared to the antalarmin groups) spent a significantly longer time in the closed arm of the maze. Fig. 4 (lower panel) shows the effects of the combined treatment with CRF and antalarmin intra-DMH on ETM escape measurements. Three-way ANOVA showed a significant effect of trials (F(2,44) = 4.64; P = 0.015). No significant effects of trials by antalarmin (F(2,44) = 0.93; P = 0.402), trials by CRF (F(2,44) = 1.02; P = 0.371), trials by antalarmin by CRF (F(2,44) = 0.609; P = 0.548), antalarmin (F(1,22) = 0.09; P = 0.772), CRF (F(1,22) = 0.462; P = 0.504), or antalarmin by CRF (F(1,22) = 1,27; P = 0.273) were found.

Fig. 5. Effects (mean ± SEM) of the combined intra-DMH treatment with CRF (250 ng/0.2 ␮l) and antalarmin (25 ng/0.2 ␮l), for animals with cannulae placements outside the DMH, on inhibitory avoidance (panel A) and escape (panel B) latencies of rats in the elevated T-maze. N = 13 (vehicle/vehicle), 15 (vehicle/CRF), 6 (antalarmin/vehicle), 12 (antalarmin/CRF).

For this experiment, there were enough animals with cannulae placements outside the DMH, allowing the inclusion of a negative control group, to better verify our results. Fig. 5 (upper panel) shows the effects of the combined treatment with CRF and antalarmin intra-DMH on ETM inhibitory avoidance measurements, for animals with cannulae placements outside the DMH. Threeway ANOVA showed a significant effect of trials (F(2,80) = 17.53; P < 0.001), but no significant effect of trial by antalarmin (F(2,80) = 0.30; P = 0.738), trials by CRF (F(2,80) = 2,39; P = 0.098), trials by antalarmin by CRF (F(2,80) = 0.529; P = 0.591), antalarmin (F(1,40) = 0.60; P = 0.443), CRF (F(1,40) = 0.205; P = 0.653) or antalarmin by CRF interaction (F(1,40) = 0.017; P = 0.898). The lower panel of Fig. 5 shows the effects of the combined treatment with CRF and antalarmin intra-DMH on ETM escape measurements, for animals with cannulae placements outside the DMH. Three-way ANOVA showed no significant effects of trials (F(2,80) = 1.72; P = 0.187), antalarmin (F(2,80) = 0.04; P = 0.953), CRF (F(2,80) = 0.46; P = 0.636), trials by antalarmin by CRF (F(2,80) = 1.72; P = 0.842), antalarmin (F(1,40) = 0.19; P = 0.669), CRF (F(1,40) = 0.65; P = 0.427), or antalarmin by CRF interaction (F(1,40) = 0.07; P = 0.789). Table 1 shows the effects of the combined treatment with CRF and antalarmin intra-DMH on the number of lines crossed in an open field. Two-way ANOVA did not show a significant effect of antalarmin (F(1,22) = 0.75; P = 0.395) or CRF (F(1,22) = 0.002; P = 0.964), but showed a significant effect of the interaction between the two treatments (F(1,22) = 5.16; P = 0.033). The Tukey post-hoc test did not show any significant differences between the four treatments (P > 0.05).

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For animals with cannulae placements outside the DMH, two-way ANOVA showed no significant effects of antalarmin (F(1,40) = 0.17; P = 0.682), CRF (F(1,40) = 0.28; P = 0.597) or antalarmin by CRF interaction (F(1,40) = 0.39; P = 0.538) (Table 1).

4. Discussion The results from the present study showed that intra-DMH injection of rat/human CRF, in the higher dose administered (250 ng/0.2 ␮l), facilitated ETM avoidance, an anxiogenic effect (experiment 1). Intra-DMH administration of the CRFR1 antagonist antalarmin significantly decreased avoidance latencies (experiment 2), an anxiolytic effect. To confirm the anxiogenic action of CRF and to verify if antalarmin was able to counteract this effect, experiment 3 was performed. The results of this last experiment confirmed the anxiogenic profile of intra-DMH CRF. Antalarmin administration was able to prevent the drug’s anxiogenic effect. The anxiogenic effect of CRF and the anxiolytic profile of antalarmin were not observed when animals with cannulae placements outside the DMH were analyzed. Our data also showed that none of the compounds administered altered escape responses or locomotor activity measurements. These last results are important since in experiment 1 CRF significantly increased baseline latencies [34–36], which have been traditionally used as a measurement of locomotor activity and not of anxiety-related responses. The observation that the drug did not alter the number of lines crossed in the open field or ETM escape latencies indicates that the results observed in avoidance measurements are not related to a decrease in motor activity, an observation which was also confirmed by experiment 3. Therefore, one possibility is that the significant increase in baseline latencies observed in experiment 1 might reflect an anxious reaction to novelty expressed by some of the animals in this group. Interestingly, activation of the HPA axis seems to follow both reactions to novelty and to potential threat stimuli [43]. Also, previous evidence obtained by our research group indicates that chronic treatment with corticosterone induces a similar response pattern in the ETM, that is, significant increases in baseline latencies, unrelated to locomotor activity alterations [44]. Since CRF possesses a greater affinity for CRFR1 than CRFR2 [3], the anxiogenic effects observed after intra-DMH administration of this peptide suggest a role for the former subtype of receptors in the regulation of stress/anxiety-related responses [3,45]. Previous indirect evidences obtained with the DMH also point to an anxiogenic effect of CRF. As mentioned, it was demonstrated that intracerebroventricular administration of CRF and corticosterone induces neurochemical changes in the DMH, i.e., increases in dopamine and serotonin, that parallel changes observed in the region following exposure to a variety of physical and psychological stress-related stimuli [26]. Also, intraperitoneal injection of JNJ19567470/CRA5626, a CRFR1 antagonist, was shown to prevent the sodium lactateinduced panic-like behavioral and cardiovascular responses observed in adult male rats with chronic reduction of GABA levels in the DMH [27]. On the basis of these results the authors suggest that DMH CRFR1 modulate a panic-related response. Contrarily to the above results [27], in our study, ETM escape responses were unchanged by CRF administration. In fact, the results obtained in the present study suggest that DMH CRF type 1 receptors modulate an anxiety-related, but not a panic-related, response. These observations were also confirmed by the results observed with the administration of intra-DMH antalarmin, a CRFR1 antagonist. Antalarmin impaired ETM avoidance responses, thus showing an anxiolytic (and not a panicolytic) effect. Also, the drug was capable of counteracting the anxiogenic effects of

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intra-DMH CRF, confirming that these last effects were in fact due to the activation of this subtype of CRF receptors. The selectivity of these effects for the DMH were further verified when animals with cannulae placements outside the region were analyzed. For these animals, no significant effects of CRF or antalarmin were observed. The absence of involvement of the DMH with ETM escape responses also contrasts with a previous study performed by our research group [23]. In this particular study, it was shown that intra-DMH injection of the GABAA agonist muscimol significantly impaired ETM escape, not altering, however, ETM avoidance. On the other hand our present data go in the same direction as the ones previously observed for the medial amygdala [46]. In this particular study, intra-medial amygdala administration of CRF presented an anxiogenic effect, facilitating ETM inhibitory avoidance responses. This effect was also counteracted by antalarmin. No changes were observed regarding escape responses or locomotor activity measurements. It is interesting to mention that some of the most important regions to which the medial amygdala projects are the hypothalamic nuclei involved with defense, including the DMH. In fact, using anterograde neuronal tracing with biotinylated and tetrametylrhodamine-conjugated dextran amines to analyze efferent projections from the anterior, posterodorsal and posteroventral subdivisions of the medial amygdala, it was shown that the densest outputs of the region targeted the bed nucleus of the stria terminalis and the hypothalamus [47]. Through its projections to the hypothalamus the medial amygdala regulates the activation of the HPA axis in response to emotional stressors [48]. Concerning the relationship of the medial amygdala with ETM avoidance, previous evidence by our research group [49] has shown that avoidance performance significantly increased FOS immunoreactivity in the region and that the pharmacological inhibition of the medial amygdala by midazolam administration impairs avoidance responses [50], an anxiolytic effect. Therefore, it is not a surprise that the results obtained with the DMH in the present study are similar to the ones previously observed [47] for this amygdaloid nucleus, that is, anxiety-related. The absence of effects of CRF administration in escape responses, in clinical terms related to panic disorder, also seems to corroborate clinical data. In fact, HPA axis abnormalities have been used as an indirect marker of CRF dysregulation and clinical evidence indicates that although increases in HPA function accompany generalized anxiety, panic disorder patients show significantly lower ACTH and cortisol responses to CRF administration [51]. In this same direction, different panicogenic stimuli (for instance, CO2 or sodium lactate) can trigger a panic attack without significantly increasing cortisol release in panic disorder patients [52,53]. Together, these data suggest, as proposed by some authors [54–56], that generalized anxiety and panic disorder differently affect stress hormones. While generalized anxiety disorder activates both the HPA and the sympathoadrenal axes, a panic attack would be an emergency reaction, which caused major sympathetic activation, but little effect on the HPA axis. Interestingly, the neuroendocrine differences between generalized anxiety and panic disorder pointed above were also previously confirmed using the ETM [57]. Corroborating the above clinical findings, plasma levels of corticosterone were shown to be significantly higher in animals that acquired inhibitory avoidance, when compared to control animals and to animals submitted to the escape task. Once again, these results indicate the differential activation of the HPA axis on generalized anxiety and panic-associated behaviors. Taking the above proposition into account, it is indeed possible to conceive that the CRF system of the DMH is preferentially involved with the neurobiology of avoidance responses (in terms of psychopathology related to generalized anxiety disorder), as

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presently suggested. In this regard, it is also important to point out that the previous observations [27] performed with the administration of a CRFR1 antagonist into the DMH, and that indicated a panicogenic role for these receptors, used an animal model of anxiety (the social interaction test) that does not specifically model generalized anxiety or panic disorder, but rather social anxiety [36]. In conclusion, the results reported herein provide direct evidence for the involvement of CRFR1 receptors of the DMH in the modulation of a defensive response that has been associated with generalized anxiety disorder and might be of relevance to the better understanding of the neural mechanisms underlying this pathological condition.

Acknowledgments This study was financed by Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo, FAPESP, Brazil (Grants number: 2011/174710 and 2013/17389-8). Mariana Santos Carvalho de Faria Silva was the recipient of a fellowship grant from Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior, CAPES, Brazil. Bruno Aranha Pereira was the recipient of a fellowship grant from Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo, FAPESP, Brazil. The authors thank José Simões de Andrade for helpful technical support.

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