Neuropharmacology 79 (2014) 201e211

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Paradoxical mineralocorticoid receptor-mediated effect in fear memory encoding and expression of rats submitted to an olfactory fear conditioning task Rimenez R. Souza a, Silvia Dal Bó b, E. Ronald de Kloet c, d, Melly S. Oitzl e, Antonio P. Carobrez a, * a Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Trindade, 88040-900 Florianópolis, SC, Brazil b Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil c Department of Medical Pharmacology, LACDR, Leiden University Medical Center, Leiden, The Netherlands d Department of Endocrinology and Metabolism, Leiden University Medical Center, Leiden, The Netherlands e Swammerdam Institute for Life Sciences, SILS-CNS, University of Amsterdam, Amsterdam, The Netherlands

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 June 2013 Received in revised form 30 October 2013 Accepted 21 November 2013

There is general agreement that the substantial modification in memory and motivational states exerted by corticosteroids after a traumatic experience is mediated in complementary manner by the mineralocorticoid (MR) and glucocorticoid (GR) receptors. Here we tested the hypothesis that pharmacological manipulation of MR activity would affect behavioral strategy and information storage in an olfactory fear conditioning (OFC) task. Male Wistar rats were submitted to the OFC with different training intensities. We observed that following high intensity OFC acquisition, a set of defensive coping strategies, which includes avoidance and risk assessment behaviors, was elicited when subjects were exposed to the conditioned stimulus (CS) 48 h later. In addition, following either OFC acquisition or retrieval (CS-I test) a profound corticosterone secretion was also detected. Systemic administration of the MR antagonist spironolactone altered the behavioral coping style irrespective the antagonist was administered 60 min prior to the acquisition or before the retrieval session. Surprisingly, the MR agonist fludrocortisone given 60 min prior to acquisition or retrieval of OFC had similar effects as the antagonist. In addition, posttraining administration of fludrocortisone, following a weak training procedure, facilitated the consolidation of OFC. Fludrocortisone rather than spironolactone reduced serum corticosterone levels, suggesting that, at least in part, the effects of the MR agonist may derive from additional GR-mediated HPAaxis suppression. In conclusion, the present study suggests the involvement of the MR in the fine-tuning of behavioral adaptation necessary for optimal information storage and expression, as revealed by the marked alterations in the risk assessment behavior. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Fear memory encoding Mineralocorticoid receptor Olfactory fear conditioning Stress Coping strategy Risk assessment behavior

1. Introduction Memories for emotional-linked events are highly adaptive, consistently stronger and more resistant to extinction than those for trivial information (Maren, 2011; Pitman et al., 1987; Vermetten et al., 2007; Yehuda, 1998). This emotional memory resilience depends on neural substrates activated by sensory information and neuromodulators that mediate the stress response (Joels et al.,

* Corresponding author. Tel.: þ55 48 3721 4845; fax: þ55 48 3721 9813. E-mail addresses: [email protected], [email protected] P. Carobrez). 0028-3908/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2013.11.017

(A.

2006; Pacak and Palkovits, 2001; Roozendaal et al., 1997). Among the primary mediators of the stress response are the corticosteroids (corticosterone in rodents and cortisol in man), which are secreted by the adrenal cortex shortly after a threat is detected and an emotional state emerges (Cordero et al., 1998; De Kloet, 2004; Palkovits, 1987). These hormones readily cross the bloodebrain barrier binding to the high affinity mineralocorticoid (MR) and the tenfold lower affinity glucocorticoid (GR) receptors, thus exerting a wide range of actions over emotional and cognitive systems, allowing the individual to cope with trauma-linked cues, while promoting information storage (De Kloet, 1991; Joels et al., 2006). Both human and rodent studies have indicated that high corticosteroid levels or severe stress situations may cause a dissociative

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state in which affected individuals respond fearfully to cues normally not predicting threat (Christianson, 1992; Kaouane et al., 2012), a phenomenon that is described to be dependent on genetic background (Brinks et al., 2009). Such impairment in predicting the precise trauma-related cue even in safe contexts may be in the central core of the intrusive recollections observed in posttraumatic stress disorder (PTSD) (American Psychiatric Association, 2000). In this sense, it has been suggested that central MR mediate the effects of corticosterone in the appraisal of stressful information with consequences for behavioral flexibility and the sensitivity of the stress response system (Berger et al., 2006; Gass et al., 2001; Oitzl and de Kloet, 1992; Sandi and Rose, 1994a), while subsequent activation of GR promotes recovery and information storage linked to the event (Oitzl and de Kloet, 1992). However, disruption in cognitive processing may occur during imbalance in these complementary actions mediated by MR and GR. Such an imbalance occurs if the stress system is activated in an inappropriate manner out of the context to be remembered (de Quervain et al., 1998; de Quervain et al., 2000; Okuda et al., 2004; Rimmele et al., 2010; Zhou et al., 2011). According to the hypothesis that MR are involved in the selection of an appropriate coping strategy during challenge (Oitzl and de Kloet, 1992; Oitzl et al., 1994), it seems worthwhile to explore if its manipulation may lead to long-term behavioral and cognitive disturbances in an olfactory fear conditioning (OFC) paradigm, a task that requires an essential sense of the rodent’s interaction with the environment. In humans, the olfactory system has been described as an important substrate in the processing of a conditioned stimulus (CS) operating in the core of memory recall in combat-related PTSD (Vermetten et al., 2007). However, only a limited number of studies use olfactory cues in a rodent fear conditioning paradigm, in spite of the substantial importance attributed to their olfactory system. Using rats, we have recently demonstrated that the OFC task would be a useful tool to investigate trauma-related behavioral coping strategies (Canteras et al., 2008; Kroon and Carobrez, 2009). The OFC task is sensitive to anxio-selective compounds, such as benzodiazepines and beta-adrenoceptor blockers, allowing a longitudinal study of specific components of aversive memory processes such as acquisition, consolidation and retrieval, as well as higherorder conditioning, which are recognized windows for pharmacological intervention in the study of emotional memory (Cavalli et al., 2009; Kroon and Carobrez, 2009; Pavesi et al., 2011). Given the role of MR in the selection of an appropriate coping strategy during challenging situations, we herein investigated whether the blockade or activation of these receptors would modulate fear encoding and expression in the OFC paradigm. Therefore, the study was designed to test possible time-dependent effects of a pharmacological manipulation of MR on OFC memory formation and retrieval. The present results demonstrated paradoxical effects after MR blockade or activation influencing the rat’s risk assessment analysis and consequently the appropriate behavioral expression of the conditioned fear. 2. Material and methods 2.1. Animals Adult male Wistar rats (12e15 weeks; weighing 330e400 g) from the University in-house colony were used in this study. Animals were housed in four to five per cage (50  30  15 cm) under controlled temperature (22  2  C) and light (12:12 lightedark cycle; lights on at 07:00 a.m.), and with free access to food and water. All rats were naïve at the beginning of the experiments. Experiments were always conducted between 09:00 and 16:00 h. Animal care procedures and maintenance were followed in accordance with the Guide for the Care and Use of Laboratory Animals of the NIH (Institute for Laboratory Animal Research, 2011) and had been previously approved by the Ethics Committee on Use of Laboratory Animals from the Federal University of Santa Catarina, Brazil (protocol 23080.008789/2009-46).

3. Drugs Spironolactone (SPI, a rather selective MR antagonist; Tocris BioscienceÒ, USA) at 10 and 20 mg/kg, Fludrocortisone (FLU, a potent MR agonist with residual GR agonist activity; Sigmae AldrichÒ, USA) at 1 and 3 mg/kg and Dexamethasone (DEX, a potent GR agonist with residual MR agonist activity; Tocris BioscienceÒ, USA) at 1 mg/kg, were dissolved in sterile 0.9% NaCl solution (plus 1% tween 80 as solvent), which alone was used as vehicle control (VEH). All drugs and vehicle were administered subcutaneously (s.c.) at a volume of 1.0 ml/kg. Drugs dosage were chosen based on pilot experiments from our laboratory and previous studies from the literature (Hall and Hall, 1961; Ninomiya et al., 2010; Oitzl and de Kloet, 1992; Otte et al., 2003). 3.1. Blood sampling and determination of serum corticosterone by ELISA Blood sampling procedures were carried out by tail incision according to Fluttert et al. (2000) 30 min after each respective test. In brief, immediately after the experiment, animals were placed in a cage with their home bedding where they remained undisturbed until blood sample collection. For blood collection, the animals were individually transported to an adjacent room where blood was collected from the tail ending (minimum of 300 ml/rat). Blood collection lasted no more than 2 min. Blood samples were centrifuged at 3000 rpm during 10 min (4  C) and sera samples were separated immediately afterward and stored at 20  C until the assay. Corticosterone concentration was assessed in duplicate by solid phase enzyme-linked immunosorbent assay kit (Corticosterone ELISA, IBL InternationalÒ, Germany). At the day of the assay, thawed samples were restored by vortex mixing. Twenty microliter of each standard and blood sera samples were used in the assays. Standard curves were constructed using 0, 5, 15, 30, 60, 120 and 240 nmol/L standard samples provided by the kit manufacturer. The optical density (450 nm) for each microliter plate was measured using an InfiniteÒ 200 Pro reader (TECAN Group Ltd., Switzerland) within 10 min after adding stop solution for ending the enzymatic reaction. Determination of the concentration curve was made by an automated method with 4 parameter logistics curve fit using MasterPlexÒ 2010 software (Hitachi Software Engineering Co. Ltd., USA). Calculation of the results was made after constructing a standard curve by plotting the mean absorbance value obtained from each standard against its concentration. Intraand inter-assay variations were 6.7% and 13.6%, respectively. 3.2. Apparatuses and experimental procedures The OFC protocol was conducted using two different apparatuses as previously described by Kroon and Carobrez (2009) (see Fig. 1). Memory acquisition was performed using a conditioning chamber (50  26  35 cm), which was constructed with stainlesssteel walls and a grid floor composed of stainless-steel bars (0.5 cm spaced), and connected to a shock generator (InsightÒ Equipamentos, Brazil). The conditioned fear responses during OFC retrieval were measured in a test chamber, which was made up of black PlexiglasÒ consisting of two zones, an open area (40  26  36 cm) and an enclosed roofed compartment (20  26  36 cm). The two zones were connected by a small open door (7  7 cm) which allowed the animals to traverse both spaces. On the opposite wall, distal to the entrance of the enclosed space, a small pierced acrylic pack (1  5  5 cm) was attached, serving as a dispenser for the olfactory stimulus embedded in a filter paper. Both apparatuses had a frontal wall made up of transparent PlexiglasÒ that allowed the behavioral recording by a DVD

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Fig. 1. Experimental design used to evaluate the Olfactory Fear Conditioning task. CS ¼ amyl acetate odor as conditioned stimulus; US ¼ foot-shock as unconditioned stimulus; CS-I Test ¼ odor exposure test session and CS-II Test ¼ contextual no-odor exposure test.

recording system connected to a video camera. Five percent amyl acetate solution (99%, SigmaeAldrichÒ, USA) diluted in propylene glycol was used as the olfactory stimulus for conditioning (Pavesi et al., 2011). Sessions were carried out in sound-attenuated rooms with an exhaust system. In order to reduce probability of scent information between each individual’s experimental session (from urine/fecal cues), the apparatus was cleaned with 10% alcohole water solution between each rat’s experiment and with 70% alcoholewater solution before and after each experimental session. In the first and the second case, a period of 10 or 30 min, respectively, was observed to guarantee the removal of any alcohol residue by the exhauster system. 3.3. General procedure The experimental paradigm of the OFC consisted of two consecutive phases: the conditioning (OFC acquisition; 2 days) and the retrieval (OFC expression; 3 days), with sessions spaced 24 h apart, as illustrated by Fig. 1. The acquisition of OFC was carried out in the conditioning chamber. On day 1, each subject was placed in the apparatus and allowed to freely explore the chamber during 4 min, in a familiarization session. On the following day (day 2), subjects were placed back into the chamber and received 1, 3 or 5 pairings of electrical foot shock (US; 0.4 mA/2 s; 40 s inter-trial interval) in the presence of 100 ml of amyl acetate (CS) embedded in a filter paper (4  4 cm) placed under the grid floor. The expression of OFC was performed in the test chamber and consisted of familiarization (day 3), CS-I test (day 4) and CS-II test (day 5). In the familiarization session, the subjects were allowed to freely explore the apparatus and baseline behavioral parameters were measured in the presence of a clean filter paper (neutral odor). On CS-I test session, each rat was re-exposed to the apparatus in the presence of the olfactory CS placed inside the dispenser. On the CSII test, the rats were re-exposed to the apparatus without the CS (context only) to test for second order conditioning as the result of CS-context association during the CS-I test. 3.4. Behavioral measurements Based on previous data showing that similar set of behavioral defensive responses is evoked in rats exposed to predator odor or to the OFC (Dielenberg and McGregor, 2001; Do Monte et al., 2008; Kroon and Carobrez, 2009), the following behavioral parameters were scored and therefore interpreted as defensive coping strategies: the time spent in close proximity (within 7 cm) to the odor source (approach time), the time spent in the enclosed compartment (hide time), and the time performing risk assessment behavior by stretching-out from the enclosed compartment towards the open compartment (head-out time).

3.5. Experimental groups 3.5.1. Experiment 1 e behavioral and hormonal response of rats exposed to the OFC Thirty-six subjects were randomly assigned to four groups in the behavioral characterization of the OFC. Group 1 (unpaired CS-5US) was exposed to the conditioning chamber in the presence of CS during the familiarization, and re-exposed twenty-four hours later when then received 5US (day 2), in absence of CS, in order to evaluate possible non-associative memory encoding. Groups 2, 3 and 4 were regularly familiarized without odor on day 1 in the conditioning chamber, and on day 2 they were exposed to pairings of 1, 3 or 5US in the presence of the CS. They were designated as CS1US, CS-3US, and CS-5US, respectively. On day 3, all the groups were familiarized in the test chamber and in the following day (CS-I test; day 4) they were re-exposed to the test chamber in the presence of olfactory CS. On day 5 (CS-II test), subjects were returned to the test chamber in the absence of the olfactory CS, where the second order conditioning was evaluated. Defensive coping strategy was evaluated on days 3, 4, and 5. The hypothalamusepituitaryeadrenal (HPA) axis activation following OFC acquisition and expression was assessed by measuring serum corticosterone concentrations 30 min after the conditioning (day 2) or retrieval (day 4). In the first set, 20 subjects were randomly assigned to 4 independent groups, no CS-US, CS-only (no US), CS1US, and CS-5US. Immediately after conditioning, each subject was placed in a housing cage with their home bedding where they remained undisturbed until the collection of blood sample at 30 min after the conditioning session. In a second experimental setting, 21 subjects were familiarized to the conditioning chamber in the absence of odor (except for the unpaired CS-5US group which was exposed to the odor in this session). On day 2, the subjects were exposed to the conditioning chamber according to the following blueprint: no CS-US, unpaired CS-5US, CS-1US, or CS-5US. Twentyfour hours later, all groups were familiarized (day 3) to the test chamber in the absence of odor stimulus. On day 4, immediately after the CS-I test session, animals were placed in a housing cage with their home bedding where they remained undisturbed until the collection of blood samples at 30 min after the conditioning session. 3.5.2. Experiment 2 e OFC acquisition and expression after MR blockade The role of the MR blockade on OFC acquisition and expression was tested by the administration of the MR antagonist SPI 60 min prior to the conditioning (day 2) or retrieval (day 4), respectively. The MR antagonist SPI (10 or 20 mg/kg) or VEH were given prior to the conditioning with a CS-5US association or prior to the CS-I test in independent groups. Defensive coping strategy was evaluated on days 3, 4, and 5 as previously described.

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3.5.3. Experiment 3 e evaluation of the MR activation in the acquisition and expression of OFC The temporal effects of MR activation on the acquisition of OFC were assessed by the systemic administration of the MR agonist FLU prior to conditioning (day 2) or retrieval (day 4), respectively. Independent groups of animals were injected with FLU (1 or 3 mg/ kg) or VEH prior (60 min) to conditioning with CS-5US or to the CS-I test. An additional group was injected with FLU (1 mg/kg) 20 min before the conditioning or retrieval to test the time-dependency effect. Defensive coping strategy was evaluated on days 3, 4, and 5 as previously described. 3.5.4. Experiment 4 e the effects of the MR activation on OFC consolidation Here we evaluated the effects of the MR agonist on OFC facilitation. On day 1, all animals were familiarized in the conditioning chamber without odor. Evaluation of the OFC consolidation was accessed by s.c. injection of FLU (1 or 3 mg/kg) immediately after a weak OFC training (CS-1US) conditioning session (day 2). To evaluate possible pro-associative effects of FLU per se, an additional group was injected with FLU (3 mg/kg) after a CS-only (no US) conditioning session. Defensive coping strategy was evaluated on days 3, 4, and 5 as previously described. 3.5.5. Experiment 5 e the effects of MR blockade or activation on serum corticosterone levels HPA axis response to OFC stress in the presence of MR antagonist or agonist was tested in two independent groups of animals. In the first set, 24 h after familiarization in the conditioning chamber, independent groups were systemically injected with vehicle or with the MR antagonist SPI (10 mg/kg) 60 min before being submitted to a CS-only (no US) or a CS-5US conditioning regimen. In a second cohort, 24 h after familiarization in the conditioning chamber, independent groups were systemically injected with vehicle, or the GR agonist DEX (1 mg/kg) as a positive control, or with the MR agonist FLU (1 mg/kg) 60 min before the conditioning with CS-5US. In both cases, immediately after the conditioning, the subjects returned to their home cage remaining undisturbed until blood sample collection 30 min later.

limits (within 95%) of collapsed data obtained during the 10 min familiarization session in the test chamber for each group comparisons. 4.1. Experiment 1 e behavioral and hormonal response of rats exposed to the OFC The possibility that different training intensities could affect OFC was evaluated in experiment 1. Data shown in Fig. 2aec represent the defensive coping strategy exhibited during familiarization, CS-I and CS-II sessions by rats previously submitted to various CS-US conditioning schedules. ANOVA carried out with CS-I and CS-II data, showed a significant (p < 0.05) conditioning effect represented by the %approach time [F(3,30) ¼ 10.77; p < 0.0005], %hide time [F(3,30) ¼ 15.10; p < 0.0005], and %head-out time [F(3,30) ¼ 19.59; p < 0.0005]. Post hoc comparisons revealed a significant decrease (p < 0.05) in the %approach time, and increase in the %hide time and %head-out time during the CS-I and CS-II test for the subjects paired with CS-5US compared to the unpaired CS-5US group. No significant statistical differences were detected in the groups paired with CS-1US or CS-3US compared to the unpaired CS5US group. Fig. 2d illustrates the serum corticosterone levels 30 min after the exposure to the conditioning chamber in the following conditions: no CS-US, CS only, CS-1US, and CS-5US. ANOVA revealed a significant (p < 0.05) treatment effect on corticosterone levels [F(3,16) ¼ 40.37; p < 0.0005]. Subsequent, NewmaneKeuls’ test revealed that the CS-5US group presented a marked (p < 0.05) increase in corticosterone levels when compared to all other groups. Fig. 2e illustrates the serum corticosterone levels 30 min after OFC retrieval (CS-I test) on rats previously exposed to the conditioning chamber in the following schedule: no CS-US, unpaired CS-5US, CS-1US, and CS-5US. ANOVA detected a significant (p < 0.05) treatment effect on corticosterone levels after the CS-I test [F(3,17) ¼ 62.82; p < 0.0005]. NewmaneKeuls’ post hoc test revealed a significant (p < 0.05) increase in corticosterone levels of rats exposed to the CS-I test that were previously paired with either CS-1US or with CS-5US compared to no CS-US group. In addition, rats from CS-5US group presented a significantly (p < 0.05) higher corticosterone level from the CS-1US group.

3.6. Statistics The defensive coping strategy represented by the approach, hide, and head-out time during the 10 min sessions were transformed in percentage of the total session time and taken as the dependent variables. Baseline data on familiarization in the test chamber were initially analyzed by one-way ANOVA for possible fear generalization effect. CS-I and CS-II data were analyzed by repeated measures ANOVA (treatment vs CSI/CSII). Data from corticosterone levels were also analyzed by one-way ANOVA (experiment 1 and experiment 5 on SPI treatment) or by two-way ANOVA (experiment 5 FLU/DEX treatment). Post hoc NewmaneKeuls’ tests were performed when appropriate. The minimum statistical level of significance adopted was p < 0.05. Statistical analyses were performed using the StatisticaÒ10 software package (StatSoftÒ, USA) and graphic representation was made using the software GraphPad PrismÒ v.5 (GraphPad Software Inc, San Diego, USA). 4. Results During the familiarization session in the test chamber, one-way ANOVA showed no statistical differences among the groups in the behavioral measurements evaluated. Therefore the hatched horizontal lines in Figs. 2e7 represent the mean and the confidence

4.2. Experiment 2 e systemic administration of MR antagonist spironolactone impairs OFC acquisition and conditioned fear expression Data from experiment 2 are displayed in Fig. 3. Systemic SPI treatment 60 min prior to CS-5US conditioning was able to impair OFC acquisition. ANOVA on CS-I and CS-II data, showed a significant treatment effect in %hide time [F(2,22) ¼ 5.07; p < 0.05] and %headout time [F(2,22) ¼ 11.98; p < 0.0005]. In the CS-I test, post hoc comparisons revealed a significant (p < 0.05) decrease in the %hide and in the %head-out time in the SPI 10 mg/kg treated group and a significant (p < 0.05) decrease in %head-out time in the SPI 20 mg/ kg compared to the vehicle group. During the CS-II test, both doses of SPI reduced (p < 0.05) only the %head-out time as revealed by NewmaneKeuls’ test. Fig. 4 depicts the effects of the MR antagonist administered 60 min prior to the OFC expression (CS-I test). ANOVA revealed a significant treatment effect in the %approach time [F(2,19) ¼ 5.14; p < 0.05],%hide time [F(2,19) ¼ 3.71; p < 0.05] and %head-out time [F(2,19) ¼ 13.73; p < 0.0005]. In the CS-I session, post hoc comparisons revealed a significant (p < 0.05) increase in the %approach time, and a significant decrease in the %hide and %head-out time in SPI 20 mg/kg treated group. A significant (p < 0.05) decrease in % head-out time in the SPI 10 mg/kg compared to vehicle group was

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Fig. 2. Behavioral (left panels) and hormonal (right panels) measurements of the conditioned fear in rats submitted to the OFC protocol. The hatched horizontal lines represent the mean  95% confidence limits for all rats in the familiarization session. Histograms on left panels represent the mean þ S.E.M. of the approach time (%) (a), hide time (%) (b), and head-out time (%) (c). *p < 0.05 compared to the unpaired CS-5US group in the respective session (n ¼ 8e9/group). Histograms on the right panels represent the mean þ S.E.M of serum corticosterone (mg/dL) measured 30 min after the conditioning session (d) and 30 min after the CS-I test (e). *p < 0.05 compared to all other groups and b) þp < 0.05 compared to no CS-US and unpaired CS-5US groups (n ¼ 5e6/group).

also observed. During the CS-II test, post hoc NewmaneKeuls revealed that both doses of SPI reduced (p < 0.05) the %head-out time. 4.3. Experiment 3 esystemic administration of fludrocortisone impairs OFC Fig. 5 illustrates the conditioned fear responses of rats treated with FLU prior to (60 min) the CS-5US conditioning, exhibited during CS-I and CS-II sessions. ANOVA revealed a significant treatment effect in the %hide time [F(3,31) ¼ 6.55; p < 0.005] and % head-out time [F(3,31) ¼ 17.22; p < 0.0005]. During the CS-I, post hoc test revealed a decreased (p < 0.05) %hide time and %head-out time in the FLU 1 mg/kg, and a decreased %head-out time in the FLU 3 mg/kg compared to vehicle treated groups. In the CS-II, post hoc test revealed only a decreased (p < 0.05) %head-out time in the FLU 1 mg/kg. No effects were observed when FLU 1 mg/kg was administered 20 min prior to conditioning. Fig. 6 depicts the effects of FLU treatment 60 min prior CS-I test. ANOVA revealed a significant treatment effect in the %head-out

time [F(3,36) ¼ 6.89; p < 0.005]. Post hoc analysis showed a significant reduction in %head-out time in FLU 1 mg/kg group compared to the vehicle in the CS-I (p < 0.05) and CS-II (p < 0.05) test sessions. In addition, FLU 1 mg/kg had no effect when injected 20 min prior the CS-I. 4.4. Experiment 4 e systemic administration of MR agonist facilitates a weak training OFC Fig. 7 exhibits the data representing the effects of FLU administration immediately after a weak training (CS-1US) conditioning. ANOVA detected a significant treatment effect for the %approach time [F(3,36) ¼ 8.55; p < 0.0005], %hide time [F(3,31) ¼ 9.79; p < 0.0005] and %head-out time [F(3,31) ¼ 23.43; p < 0.0005]. Post hoc analyses revealed an increased (p < 0.05) %hide time in FLU 3 mg/kg, and an increased %head-out time in FLU 1 and 3 mg/kg treated groups compared to vehicle during the CS-I test. Post hoc analyses also revealed a reduced (p < 0.05) %approach time in FLU 1 and 3 mg/kg groups when compared to FLU 3 (w/o US) group. This defensive response pattern remained present in the CS-II test,

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to the groups from the CS-only condition. In addition, a significant reduction in corticosterone release was detected after SPI (10 mg/ kg) treatment when compared to vehicle in the CS-5US group. Fig. 8b illustrates the serum levels of corticosterone 30 min after CS-5US conditioning in rats pre-treated with vehicle, DEX (1 mg/ kg), or FLU (1 mg/kg) 60 min before. ANOVA detected a significant treatment effect [F(2,15) ¼ 34.76; p < 0.0005] on corticosterone levels and post hoc test revealed a significant (p < 0.05) reduction on corticosterone levels of rats previously treated with DEX or FLU compared to vehicle group. 5. Discussion The present study demonstrates that systemic administration of the MR antagonist spironolactone was able to alter the defensive coping strategy adopted towards the olfactory CS, irrespective of the antagonist being administered 60 min prior to acquisition or before the retrieval session. Surprisingly, the MR agonist FLU given 60 min, but not 20 min, prior to acquisition or retrieval of the OFC had similar effects as the antagonist, except when the agonist was

Fig. 3. Effects of spironolactone (SPI) infusion prior to OFC acquisition. The hatched horizontal lines represent the mean and confidence limits (95%) for all rats during the familiarization session. Histograms represent the mean þ S.E.M. of the approach time (%), hide time (%), and head-out time (%). VEH, SPI 10, and SPI 20 mg/kg were administered 60 min prior to the CS-5US conditioning. *p < 0.05 compared to the vehicle group in the respective session (n ¼ 8e9/group).

represented by an increased (p < 0.05) %hide time in FLU 3 mg/kg, and %head-out time in FLU 1 and 3 mg/kg groups. No effects were detected in rats from the group receiving FLU 3 mg/kg treatment without US association during the conditioning session when compared to vehicle group. 4.5. Experiment 5 e systemic administration of MR antagonist increases circulating levels of corticosterone while MR agonist suppresses HPA-axis Fig. 8a presents the serum levels of corticosterone 30 min after the CS only or to CS-5US training in animals previously treated with vehicle or SPI (10 mg/kg). ANOVA revealed a significant condition effect (CS  CS-5US) [F(1, 18) ¼ 199.82; p < 0.0005] and a condition  treatment effect [F(1, 18) ¼ 20.81; p < 0.0005]. Further test showed a significant increase in corticosterone levels in rats from the CS-only group treated with SPI (10 mg/kg) when compared to CS-only vehicle group. Moreover, corticosterone levels were significantly increased in animals exposed to CS-5US, regardless the treatment (vehicle or SPI 10 mg/kg), in comparison

Fig. 4. Effects of spironolactone (SPI) prior to OFC retrieval. The hatched horizontal lines represent the mean and confidence limits (95%) for all rats in the familiarization session. Histograms represent the mean þ S.E.M. of the approach time (%), hide time (%) and head-out time (%). VEH, SPI 10, and SPI 20 mg/kg were administered 60 min prior the CS-I session. *p < 0.05 compared to the vehicle group in the respective session (n ¼ 7e8/group).

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fear conditioning results from the rodent’s association of the context with the predator. In the OFC approach, it is possible to observe that rats first learn that the presence of the odor predicts the possibility of foot-shock (conditioning). The CS exposure induces a defensive response and consequently a contextual association, implying that the context also predicts the CS. Thus, defensive behavior is further observed during the CS-II test, even in the absence of the olfactory CS, suggesting the establishment of a second-order conditioning. This confirms previous data from our group (Canteras et al., 2008; Kroon and Carobrez, 2009) and is in line with findings demonstrating the adaptive importance of the olfactory system in rodents (Brennan and Keverne, 1997; Paschall and Davis, 2002). 5.1. Behavioral and endocrine response to olfactory fear conditioning

Fig. 5. Effects of fludrocortisone (FLU) infusion prior to OFC acquisition. The hatched horizontal lines represent the mean and confidence limits (95%) during the familiarization session. Histograms represent the mean þ S.E.M. of the approach time (%), hide time (%), and head-out time (%). VEH, FLU 1, and FLU 3 were administered 60 min, while FLU 1(20 min) was administered 20 min prior to the acquisition. *p < 0.05 compared to the vehicle group in the respective session (n ¼ 6e11/group).

given immediately after the OFC acquisition. Furthermore, FLU rather than SPI suppressed stress-induced corticosterone secretion to a similar extent as the potent synthetic glucocorticoid DEX. These findings suggest complex interactions linked to MR at different levels of the HPA axis in response to the OFC. To obtain our findings three behavioral parameters were scored as defensive coping strategy elicited by the olfactory CS. These three measures represent at least two different decision making processes: a) the head-out time, which is evidence of risk assessment analysis; and b) approach and hide time, which represents the movement towards or away from the danger source, respectively. In both cases these defensive coping styles can be related to the acquisition of information and the memory encoding, needed either to make the defense pattern effective or to make it unnecessary (Blanchard and Blanchard, 1988). A similar pattern of defensive behavior was observed previously in rats exposed to a cat odor (Dielenberg and McGregor, 2001). This congruence in OFC defensive responses and the overlap of neural sites involved in processing the predator odor and the olfactory fear conditioning suggest the engagement of similar defensive motivational states (Canteras et al., 2008). However, there is a peculiar difference between these two approaches. In the predator odor approach, the

During the familiarization session in the test chamber, the similar behavioral strategy to cope with novelty was found regardless any previous drug treatment or foot-shock schedule, suggesting that no fear generalization was induced by the previous experimental manipulation. This same behavioral strategy, adopted during the familiarization day, was maintained during CS-I and CSII sessions for the subjects belonging to groups unpaired, CS-1US and CS-3US, but not for those under the CS-5US conditioning schedule. In this last group, a remarkable increase in defensive behavior was observed during the CS-I test, indicating a shift in the behavioral strategy used by the subjects to cope with the danger represented by the olfactory CS. Moreover, such defensive behavior profile was kept the same during the CS-II test, when the olfactory stimulus was no longer present, suggesting a second order contextual conditioning, which confirms previous studies using a similar procedure (Canteras et al., 2008; Cavalli et al., 2009; Kroon and Carobrez, 2009; Pavesi et al., 2011). The CS-5US group also showed increased circulating corticosterone levels 30 min after the conditioning session or the CS presentation, which is compatible with the well-described rise in corticosterone secretion shortly after the detection of a stressor, therefore, facilitating the subjects’ adaptation (Joels et al., 2006; Pacak and Palkovits, 2001). These results show a significant rise in corticosterone levels in rats exposed to a CS-5US but not to CS1US schedule and are in accordance with the aforementioned description linking the release of corticosteroids with appropriate memory storage for emotional-related events (Cordero et al., 1998; Cordero and Sandi, 1998; Pacak and Palkovits, 2001). Furthermore, the rise in corticosterone levels in the CS-5US group after the CS exposure (CS-I test) is also consistent with the notion that stress hormones are important for memory retrieval (Dudai, 2004; Gewirtz and Davis, 2000). Although showing no elevation in corticosterone levels after conditioning, the exposure to the CS was able to induce a significant increase in corticosterone levels in the CS-1US group, a fact that was not translated into an observable behavioral defensive response. Therefore, it is possible that, even in the absence of an explicit behavioral manifestation, some degree of aversive emotional information was stored during the weak OFC training that was retrieved and appeared capable to induce corticosterone release when the subjects were presented to the CS. Supporting the idea of specific boundaries for facilitatory effects of corticosteroids over memory formation, Marin et al. (2012) showed in a recent study with humans that after reading negative rather than neutral news, the cortisol levels were significantly increased only after subsequent stressful tasks, where a better memory for the negative news excerpts was observed. Similarly, it was demonstrated in both chicks and rats that the development of long-term memory for

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that corticosterone acting via MR affects OFC encoding not only agree with results on the role of MR in distinct phases of memory (Oitzl and de Kloet, 1992; Oitzl et al., 1994; Sandi and Rose, 1994a), but also with previous reports demonstrating a more prominent effect of MR knockout or blockade over appraisal and behavioral strategy during learning (Berger et al., 2006; Oitzl and de Kloet, 1992; Oitzl et al., 1994; Schwabe et al., 2010; Ter Horst et al., 2012). This notion has been recently reinforced by Harris et al. (2013), showing that the forebrain MR over-expression may lead to retention-enhanced effects and enhanced fixed memories, supporting a previously described positive role for MR in information processing (Ferguson and Sapolsky, 2008; Lai et al., 2007). Furthermore, we tested whether the blockade of MR, prior to the CS exposure, would alter the defensive behavioral strategy in the test chamber. The results showed that spironolactone administered prior to the CS-I test interferes with all three parameters scored, suggesting a reduced defensive behavioral strategy, which was maintained during the CS-II test. It is important to mention that the consistent effect observed for the head-out parameter, on previously treated subjects, could be explained by a direct spironolactone effect on this type of behavioral strategy during retrieval in both sessions.

Fig. 6. Effects of fludrocortisone (FLU) infusion prior to OFC retrieval. The hatched horizontal lines represent the mean and confidence limits (95%) during the familiarization session. Histograms represent the mean þ S.E.M. of the approach time (%), hide time (%), and head-out time (%). VEH, FLU 1, and FLU 3 were administered 60 min, while FLU 1(20 min) was administered 20 min prior to the CS-I test session. *p < 0.05 compared to the vehicle group in the respective session (n ¼ 9e11/group).

aversive events shows a positive correlation between training intensity and corticosterone release (Cordero et al., 1998; Sandi and Rose, 1997). It was also described that in chicks assigned to a weak training procedure, which did not result in memory retention, corticosterone levels during memory retrieval were comparable to those observed in untrained chicks (Sandi and Rose, 1997). Altogether, these findings lead to the conclusion that the strength of fear memory formation depends to a great extent on the concentration of corticosterone. 5.2. MR blockade by spironolactone impairs OFC We observed that the administration of the MR antagonist spironolactone, prior to the conditioning, was able to interfere with the OFC encoding. As a consequence, a reduced behavioral defensive response was detected with a clear effect revealed for the head-out time measurement. It has been shown that the head-out parameter is included in the risk assessment pattern of the rodents’ behavior, which also encompasses stretched attend postures and scanning to non-discrete or potential danger such as a predatory odor or an olfactory CS (Blanchard and Blanchard, 1988; Kroon and Carobrez, 2009; Ribeiro-Barbosa et al., 2005). Thus, our findings

Fig. 7. Effects of fludrocortisone (FLU) administered immediately after a CS-1US conditioning. The hatched horizontal bars represent the mean and confidence limits (95%) during the familiarization session. Histograms represent the mean þ S.E.M. of the approach time (%), hide time (%), and head-out time (%). VEH, FLU 1, and FLU 3 were administered immediately after CS-1US OFC acquisition, while FLU 3(CS-only) was administered following the conditioning chamber exposure without US. *p < 0.05 compared to the vehicle group in the respective session (n ¼ 8e9/group).

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Fig. 8. Serum corticosterone levels in rats submitted to various treatment regimens in an olfactory fear conditioning paradigm. a) serum corticosterone levels 30 min after CS-only or CS-5US conditioning session in rats previously (60 min) treated with vehicle, or spironolactone (SPI 10 mg/kg). *p < 0.05 compared to Vehicle/CS-only, #p < 0.05 compared to Vehicle/CS-5US; b) serum corticosterone levels 30 min after CS-5US conditioning session in rats previously (60 min) injected with vehicle, dexamethasone (DEX 1 mg/kg), or fludrocortisone (FLU 1 mg/kg). *p < 0.05 compared to Vehicle.

5.3. Pre- and post-learning fludrocortisone have opposite effects on OFC Surprisingly, the MR agonist fludrocortisone likewise reduced head-out behavior either when administered 60 min prior to CS5US association or before the CS-I test. However, the impairment was not observed when the administration of fludrocortisone occurred 20 min before either acquisition or retrieval. Altogether, these findings suggest a genomic rather than a membranemediated action, in agreement with the previously described time-dependent effect for the corticosteroid hormones (Joels et al., 2006; Zhou et al., 2010). Although the present results demonstrate FLU impairment of OFC acquisition and expression if the steroid was given prior to the tests, a facilitatory effect was observed when the MR agonist FLU was administered immediately after a weak training schedule (CS1US). This post-learning administration supported the OFC by increasing the defensive behavioral strategy during subsequent CSI and CS-II exposure. FLU injected in the absence of the US was not as successful to induce the association in the OFC task, suggesting that the aversive stimulus provided by the weak training is required for the facilitatory effects. These findings are in agreement with previous evidence showing that during a stressful situation coincident recruitment of limbic and sensory regions and the activation of corticosteroid receptors in such areas must be associated in time with the context for memory facilitation (Joels et al., 2006). Furthermore, although the weak training procedure did not result in observable defensive behavior in the subsequent CS-I test exposure, it increased corticosterone level, suggesting that an emotional memory trace formed during the weak training acquisition was potentiated through further MR, and possibly GR, activation by FLU. Overall, the data are in agreement with several studies which have demonstrated that post-training systemic administration of corticosterone or GR agonists can enhance memory consolidation (Abrari et al., 2009; Flood et al., 1978; Hui et al., 2004; Ninomiya et al., 2010; Roozendaal et al., 1996; Sandi and Rose, 1994b). 5.4. Risk assessment Animal studies often neglect the anticipatory behavioral defensive responses such as the long-term avoidance of trauma-

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related cues and/or risk assessment directed towards the potential sources of danger (Canteras et al., 2008; Dielenberg and McGregor, 2001; Ribeiro-Barbosa et al., 2005). Risk assessment is a scanning strategy involved in the detection and evaluation of threats. It is a highly adaptive process that facilitates the choice of a specific defense strategy and consequently helps rodents to better cope with the aversive situation (Blanchard and Blanchard, 1989). In the present study, the prominent role of MR stands out in modulating head-out risk assessment behavior. Systemic administration of the MR antagonist or the agonist prior to the OFC acquisition or expression, had similar effects on interfering with the risk assessment. On the other hand, MR activation immediately after a weak training procedure increased the head-out behavior in the CS-I and CS-II sessions. Notably, these effects following MR antagonist or agonist prior or immediately after conditioning could be observed from 1 to 48 h later, indicating a possible genomic effect (De Kloet et al., 2008; Prager and Johnson, 2009), although non-genomic effects on behavioral flexibility cannot be discarded, since the MR antagonist impaired OFC expression. The apparent prevalence of MR effects on the choice for appropriate strategy has been demonstrated by studies using forebrain-specific MR knockout mice and central administration of MR antagonist (Berger et al., 2006; Oitzl and de Kloet, 1992; Ter Horst et al., 2012). In this study, the effect of MR modulation was observed consistently for the head-out behavior which is part of a mediation process towards the adoption of the most appropriate defensive repertoire to cope with the aversive situation, reinforcing the previously demonstrated role for MR on appraisal and suggesting that standard fear conditioning tasks that only measure behavioral actions such as freezing, may not be able to detect such subtle responses related to behavioral control systems. 5.5. The fludrocortisone paradox The current study presents an interesting paradox: the MR agonist and the antagonist exert similar effects when given prior to the training and retrieval sessions. This finding foremost is a reminder that, although the main effect of these synthetic steroids is mediated by MR, the compounds are not selective. The pharmacological profile of spironolactone includes actions such as antiandrogenic, progestogenic and estrogenic (Delyani, 2000; Menard, 2004), but it does not act on GR. Additionally, it was demonstrated that this antagonist is quickly metabolized with a half life of 5 min after a single 2 mg intravenous administration in rats, and its actions may also derived from its metabolites, like canrenone and 7athyomethyl-spironolactone (Sadee et al., 1974). On the other hand, although FLU is characterized as a potent MR agonist, it has appreciable affinity for the GR, while the steroid has a biological half life of 3e6 h (Ribot et al., 2013). Hence, this mixed agonist likely will have complementary MR- and GR-mediated effects. With these facts in mind the following two mutually not exclusive scenarios’ to explain the paradox may be testable predictions in future studies. First, do GR-mediated effects of FLU participate? The answer to this question is affirmative. FLU, but not SPI, suppresses stressinduced HPA axis activity at the pituitary level as DEX does (de Kloet et al., 1974; De Kloet et al., 1975; Grossmann et al., 2004), an effect that was also recently observed in humans (Buckley et al., 2007; Karamouzis et al., 2013; Lembke et al., 2013; Otte et al., 2003). This suppression of pituitary ACTH release will deplete circulating corticosterone from blood and brain (Karssen et al., 2005; Meijer et al., 1998). Also, multidrug resistance P-glycoprotein in the bloodebrain barrier recognizes the synthetic glucocorticoid as substrate and will hamper access to the central target sites (Meijer et al., 1998). The net effect of FLU will be, therefore, to replace the corticosterone depleted from the brain in binding to MR

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and GR. To what extent and in what ratio this dual receptor occupancy occurs we do not know, but it is perhaps sufficient to mimic in brain the well-established GR-facilitated consolidation of the fear conditioning response, if administered post-learning. Thus, the impaired retrieval response can also be interpreted as a GRmediated response. Second, does blockade of MR allow prevalence of activated GR? Also this is possible, since it is well established that MR and GR have complementary actions, if expressed in the same cell. Under basal conditions blockade of MR disinhibits the MR-mediated tonic control of the HPA axis. Blockade of MR under high levels of corticosterone will not only minimize a contribution from this receptor, but also permit a relatively larger contribution of GRmediated actions by the increased stress-induced levels of endogenous corticosterone. As a consequence, after SPI, this peculiar pharmacology would swing under stress the pendulum towards GR-mediated actions as a tentative explanation for these effects. Taking these data together, the complementary effects mediated by MR- and GR (de Kloet et al., 2005; De Kloet et al., 2008; De Kloet et al., 1998) may be of relevance to explain the similarity in SPI and FLU effects; FLU will activate both receptors in an as yet to determine stoichiometry, while after SPI during stress the endogenous hormone participates by predominantly activating GR, because MR is blocked. This differential occupancy scenario of both receptors has consequences for the U-shaped relationship established for coordinative MR- and GR mediated regulation of neuronal excitability (Joels and Baram, 2009) and behavioral performance (Oitzl and de Kloet, 1992). It also affects differentially the release and action of additional stress mediators (e.g. CRH, vasopressin, POMC peptides) that act in concert with the steroids on information processing in the brain (Joels and Baram, 2009). A conclusive answer to these considerations requires much more study to differentiate the role of each hormone, peptide and their receptors in OFC. 5.6. Implications of our findings The literature has pointed out many paradoxical effects of stress on memory performance. It is generally accepted that stress-related situations are well remembered, up to the point to become a psychiatric condition, such as the PTSD (Pitman et al., 1987; Yehuda, 1998). On the other hand, it has been described that stress can also impair cognitive processes, since individuals experiencing stressful events often show impaired recall or poor representation of memories that are not related to the trauma (Christianson, 1992). Thus, the results presented here represent an attempt to identify temporal activation properties of MR in a complex and framed fear conditioning task to assess learning and memory encoding/acquisition, consolidation, retrieval and further higher order conditioning, as well as specific components of defensive reaction, related to avoidance and risk assessment. Our results are in agreement with previous human and animal findings demonstrating that complementary MR and GR-mediated actions are indispensable for memory formation and also can interfere with emotional memory processes. We demonstrated a clear and consistent effect of MR modulation on the head-out behavior, a parameter representing risk assessment or cost benefit analysis. This is in accordance with the fact that the MRs are involved in appraisal processes and can influence information processing allowing a better coping style with fearful situations (Berger et al., 2006; Oitzl and de Kloet, 1992; Oitzl et al., 1994; Smythe et al., 1997). Along with the fact that OFC is a highly reproducible test which engages clear and measurable conditioned responses such as defensive avoidance and risk assessment, both observable reactions in PTSD patients (Pitman et al., 1987; Vermetten et al., 2007;

Yehuda, 1998), our results also imply the OFC test as an important tool to investigate the stress responsive cognitive and neuroendocrine systems. However, further biochemical studies are needed to unveil if the prior acquisition/expression impairing effects of MR activation derive from: a) changes in the cellular machinery which becomes occupied and cannot be properly activated when needed; b) MR:GR imbalance; or c) suppression of the HPA-axis. Disclosure The authors RRS, SDB, MSO and APC declare that they do not have any commercial associations that impact on the present work. The founding for this study was provided by Brazilian public agencies CAPES, CNPq and FAPESP, which have no further role on the study design; in the collection, analysis, and interpretation of the data; in the writing of the report; and in the decision to submit the paper for publication. ERdK is scientific advisor to Corcept Therapeutics and Dynacort Therapeutics. Author contributions R.R.S. and A.P.C. designed the experimental research; R.R.S. performed all experiments as part of his PhD thesis; R.R.S. and S.D.B. performed corticosterone assays; R.R.S., E.R.dK., M.S.O. and A.P.C. analyzed data and contributed in writing the manuscript. Acknowledgments This research was supported by CAPES, FAPESP, and CNPq from which RRS received a doctoral fellowship and APC a research fellowship. ERdK was supported by the Royal Netherlands Academy of Arts and Sciences. References Abrari, K., Rashidy-Pour, A., Semnanian, S., Fathollahi, Y., Jadid, M., 2009. Posttraining administration of corticosterone enhances consolidation of contextual fear memory and hippocampal long-term potentiation in rats. Neurobiol. Learn. Mem. 91, 260e265. American Psychiatric Association, 2000. Diagnostic Criteria from DSM-IV-TR. American Psychiatric Association, Washington, D.C. Berger, S., Wolfer, D.P., Selbach, O., Alter, H., Erdmann, G., Reichardt, H.M., Chepkova, A.N., Welzl, H., Haas, H.L., Lipp, H.P., Schutz, G., 2006. Loss of the limbic mineralocorticoid receptor impairs behavioral plasticity. Proc. Natl. Acad. Sci. U. S. A. 103, 195e200. Blanchard, D.C., Blanchard, R.J., 1988. Ethoexperimental approaches to the biology of emotion. Annu. Rev. Psychol. 39, 43e68. Blanchard, R.J., Blanchard, D.C., 1989. Antipredator defensive behaviors in a visible burrow system. J. Comp. Psychol. 103, 70e82. Brennan, P.A., Keverne, E.B., 1997. Neural mechanisms of mammalian olfactory learning. Prog. Neurobiol. 51, 457e481. Brinks, V., de Kloet, E.R., Oitzl, M.S., 2009. Corticosterone facilitates extinction of fear memory in BALB/c mice but strengthens cue related fear in C57BL/6 mice. Exp. Neurol. 216, 375e382. Buckley, T.M., Mullen, B.C., Schatzberg, A.F., 2007. The acute effects of a mineralocorticoid receptor (MR) agonist on nocturnal hypothalamic-adrenal-pituitary (HPA) axis activity in healthy controls. Psychoneuroendocrinology 32, 859e864. Canteras, N.S., Kroon, J.A., Do-Monte, F.H., Pavesi, E., Carobrez, A.P., 2008. Sensing danger through the olfactory system: the role of the hypothalamic dorsal premammillary nucleus. Neurosci. Biobehav. Rev. 32, 1228e1235. Cavalli, J., Bertoglio, L.J., Carobrez, A.P., 2009. Pentylenetetrazole as an unconditioned stimulus for olfactory and contextual fear conditioning in rats. Neurobiol. Learn. Mem. 92, 512e518. Christianson, S.A., 1992. Emotional stress and eyewitness memory: a critical review. Psychol. Bull. 112, 284e309. Cordero, M.I., Merino, J.J., Sandi, C., 1998. Correlational relationship between shock intensity and corticosterone secretion on the establishment and subsequent expression of contextual fear conditioning. Behav. Neurosci. 112, 885e891. Cordero, M.I., Sandi, C., 1998. A role for brain glucocorticoid receptors in contextual fear conditioning: dependence upon training intensity. Brain Res. 786, 11e17. De Kloet, E.R., 1991. Brain corticosteroid receptor balance and homeostatic control. Front. Neuroendocrinol. 12, 95e164. De Kloet, E.R., 2004. Hormones and the stressed brain. Ann. N. Y. Acad. Sci. 1018, 1e15.

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