Pharmacology, Biochemistry and Behavior 124 (2014) 188–195

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High-dose corticosterone after fear conditioning selectively suppresses fear renewal by reducing anxiety-like response Hongbo Wang a,b, Xiaoli Xing a,b, Jing Liang a, Yunjing Bai a, Zhengkui Lui a, Xigeng Zheng a,⁎ a b

Key Laboratory of Mental Health, Institute of Psychology, Chinese Academy of Sciences, Beijing 100101, PR China University of Chinese Academy of Sciences, Beijing, PR China

a r t i c l e

i n f o

Article history: Received 11 January 2014 Received in revised form 22 May 2014 Accepted 7 June 2014 Available online 14 June 2014 Keywords: Corticosterone Fear conditioning Fear renewal Anxiety Elevated plus-maze

a b s t r a c t Exposure therapy is widely used to treat anxiety disorders, including posttraumatic stress disorder (PTSD). However, preventing the return of fear is still a major challenge after this behavioral treatment. An increasing number of studies suggest that high-dose glucocorticoid treatment immediately after trauma can alleviate the symptoms of PTSD in humans. Unknown is whether high-dose glucocorticoid treatment following fear conditioning suppresses the return of fear. In the present study, a typical fear renewal paradigm (AAB) was used, in which the fear response to an auditory cue can be restored in a novel context (context B) when both training and extinction occur in the same context (context A). We trained rats for auditory fear conditioning and administered corticosterone (CORT; 5 and 25 mg/kg, i.p.) or vehicle with different delays (1 and 24 h). Forty-eight hours after drug injection, extinction was conducted with no drug in the training context, followed by a test of tone-induced freezing behavior in the same (AAA) or a shifted (AAB) context. Both immediate and delayed administration of high-dose CORT after fear conditioning reduced fear renewal. To examine the anxiolytic effect of CORT, independent rats were trained for cued or contextual fear conditioning, followed by an injection of CORT (5 and 25 mg/kg, i.p.) or vehicle at a 1 or 24 h delay. One week later, anxiety-like behavior was assessed in the elevated plus maze (EPM) before and after fear expression. We found that high-dose CORT decreased anxietylike behavior without changing tone- or context-induced freezing. These findings indicate that a single highdose CORT administration given after fear conditioning may selectively suppress fear renewal by reducing anxiety-like behavior and not by altering the consolidation, retrieval, or extinction of fear memory. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Exposure to a traumatic event may result in the development of anxiety disorders, such as posttraumatic stress disorder (PTSD). Anxiety disorders are characterized by excessive fear and sustained anxiety (Myers and Davis, 2007; Shin and Liberzon, 2010). Fear can be extinguished by repeated exposure to a fear-provoking stimulus in the absence of an aversive consequence, a process termed extinction (Chang et al., 2009; Craske et al., 2008). Extinction only contextdependently suppresses the expression of fear memory, but it does not erase fear memory. An extinguished stimulus can still result in fear renewal when it is presented in a distinct environment from where extinction occurs (Bouton, 2004; Myers and Davis, 2007). The renewal effect appears general and robust. It can occur after extensive extinction training (Bouton, 2002). Fear renewal poses a serious problem for the clinical treatment of anxiety disorders because the fears of patients are often extinguished in a context (e.g., the therapist's office), but the patients are likely to reencounter extinguished fear stimuli in another new environment (Craske et al., 2008). Therefore, attenuating ⁎ Corresponding author. Tel.: +86 10 64877528; fax: +86 10 64872070. E-mail address: [email protected] (X. Zheng).

http://dx.doi.org/10.1016/j.pbb.2014.06.003 0091-3057/© 2014 Elsevier Inc. All rights reserved.

fear renewal has become one of most important problems for improving the long-term effectiveness of exposure-based treatment. Growing evidence suggests that treatment with high-dose glucocorticoids (GCs; corticosterone in animals and cortisol in humans) may be beneficial during the early stages after a traumatic event. In the clinic, high-dose hydrocortisone administration after trauma exposure decreases the incidence of PTSD in long-term survivors and also improves their quality-of-life outcomes (Schelling et al., 2001, 2004, 2006). Early single high-dose hydrocortisone treatment attenuates the anxiety- and fear-related core symptoms of both the acute stress and subsequent PTSD in patients (Zohar et al., 2011). In preclinical studies, immediate post-trauma corticosterone (CORT) treatment can significantly reduce anxiety-like behavior and the conditioned fear response (Cohen et al., 2008). The effects of high-dose CORT treatment on behavior may be associated with its role in modulating neural plasticity, including increases in dendritic growth, spine density, and brain-derived neurotrophic factor levels and decreases in postsynaptic density-95 levels in the dentate gyrus (Zohar et al., 2011). The memory consolidation process occurs immediately after fear acquisition, and GCs can mediate memory consolidation in an inverted U-shaped dose–response relationship. Midrange doses have optimal effects on enhancing memory, whereas high doses are less effective or even impair memory (Roozendaal, 2000,

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2003). Thus, the reduction of conditioned fear appears to be attributable to the ability of high-dose CORT to impair the consolidation of fear memory. Conditioned fear and anxiety-like behavior have many similarities at the neuroanatomical (Davis, 1992) and genetic (Ponder et al., 2007) levels. Additionally, conditioned fear is also attenuated by anxiolytic drugs (Bitencourt et al., 2008; Resstel et al., 2006; Santos et al., 2005). Therefore, an unresolved issue is whether high-dose CORT treatment immediately after fear conditioning can inhibit the return of fear by reducing anxiety levels or blocking fear memory consolidation. In the present study, we used a tone fear conditioning model, which is an amygdala-dependent task (Bergstrom et al., 2013; Johansen et al., 2011), to examine the effect of immediate (1 h) and delayed (24 h) administration of high-dose CORT on fear renewal and retrieval. The effect of high-dose CORT on anxiety was assessed in the elevated plus maze. Corticosterone may selectively modulate the memory consolidation of spatial/contextual tasks, depending on hippocampal function (Atsak et al., 2012; Roozendaal et al., 2004). We further examined freezing levels that are possibly altered by CORT administration 1 and 24 h after fear conditioning based on a contextual fear memory model.

The preparation of the CORT dissolution was described by Hellsten and Wennstrom (Hellsten et al., 2002). Corticosterone (5 and 25 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) was suspended in 100% sesame oil and administered intraperitoneally (i.p.) in a volume of 1.5 ml/kg body weight. The rats were injected with CORT or vehicle (sesame oil) 1 or 24 h after fear conditioning. These doses were chosen according to the behavioral data described in a previous study (Cohen et al., 2008).

2. Materials and methods

2.4. Behavioral experiments

2.1. Animals and housing

2.4.1. Experiment 1: Effect of CORT administered 1 h after conditioning on fear renewal Based on the finding that a single treatment with high-dose CORT immediately after stress exposure might disrupt the consolidation of fearful memories (Cohen et al., 2008), the aim of this experiment was to investigate whether immediate CORT treatment after stress exposure facilitates the ability of extinction to reduce fear return. This experiment comprised three different phases: tone fear conditioning, extinction training, and fear renewal test.

The subjects were adult male Sprague–Dawley rats (240–260 g) obtained from a commercial supplier (Vital River Animal Center, Beijing, China). The rats were housed four per cage in standard steel hanging cages (50 × 29.5 × 21.5 cm) and kept on a 12 h light/12 h dark cycle (lights on at 7:00 AM) with free access to food and water. The experiments were performed during the lights-on phase. For the following experiment, the rats were uniformly handled for 5 days before each experiment began. The experimental protocol and procedures were in compliance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. 2.2. Apparatus 2.2.1. Observation chamber for fear conditioning, extinction and testing Four identical observation chambers (30.5 × 25.4 × 30.5 cm, Coulbourn Instruments, Allentown, PA, USA) were used for fear conditioning, extinction, and testing. The chamber was constructed of aluminum (two side walls and ceiling) and Plexiglas (rear wall and hinged front door) and was situated in a sound-attenuating cabinet. The floor of the chamber consisted of 18 stainless-steel rods (6 mm diameter) spaced 1.5 cm apart (center to center), which were wired to a shock generator and solid-state grid scrambler (Coulbourn, H13-15) for the delivery of footshock (unconditioned stimulus [US]). A speaker was mounted on one side panel of the chamber to deliver the tone (conditioned stimulus [CS]). Both the shock and tone deliveries were controlled by a computerized system. A video camera was mounted on the ceiling of the chamber to videotape behavior. To maximally reduce the influence of context on cued fear conditioning, tactile, visual, and olfactory cues were manipulated to create two distinct contexts (context A and context B). Context A consisted of the original construction described above. A small yellow light bulb (6 watt) was mounted on one side panel to provide illumination inside the chamber. The chambers were scented with 75% alcohol before and after use for each rat. The experimental manipulations in context A were conducted in a room with one fluorescent room light (60 watt). Context B was modified from context A, in which black acrylic boards with nine circular holes (1.5 cm diameter) were fitted to the sidewalls of the chamber. The chamber light was changed to a white light bulb (6 watt). The floor was replaced with a steel sieve-shaped plate (1 × 1 cm). The chambers were scented with diluted perfume (1%). The experimental manipulations in context B were conducted in the same experimental room that was lit with three red lamps (20 watts each).

2.2.2. Elevated plus maze The EPM apparatus was constructed of black Plexiglas and consisted of two opposing open arms (50 × 10 cm, surrounded by a 1-cm-high Plexiglas edge), perpendicular to two opposing closed arms (50 × 10 × 40 cm). Connecting these arms was a center area that measured 10 × 10 cm. The maze was elevated 50 cm above the floor. Video tracking software was used to measure behavior and the time the rats spent in each section (ANY-Maze, Stoelting Co., Wood Dale, IL, USA). 2.3. Corticosterone treatment

2.4.1.1. Tone fear conditioning (day 1). The rats were transported to context A and underwent a 3 min acclimation period in the experimental chamber. This provided a baseline (BL) measure of contextual fear. Following the initial 3 min acclimation period, all of the rats were exposed to five conditioning tones (30 s, 80 dB, 2 kHz) that co-terminated with footshock (1 s, 1.0 mA). The mean intertrial interval (ITI) was 2 min (range, 1-3 min). After the last shock, the rats were kept in the conditioning chamber for an additional 2 min and then returned to their home cages. The rats were assigned to matched experimental groups based on average freezing behavior in response to the last four CS presentations during fear conditioning (the first CS was not included because it preceded the first US). Corticosterone or vehicle was injected 1 h after conditioning. 2.4.1.2. Fear extinction (day 3 and day 5). Forty-eight hours after the injection, the rats were returned to context A for the first extinction training. During extinction training, a 2-min BL was followed by 40 trials with a 60-s ITI. Forty-eight hours later, extinction training was repeated a second time. 2.4.1.3. Renewal test (day 8). Seventy-two hours after the last extinction session, the animals were tested in context A for the retrieval of extinction memory or in a novel context (context B) for fear renewal. Rats in the vehicle- and CORT-treated groups were allocated to one of two subgroups (AAA group and AAB group) that were matched for the levels of freezing on the last extinction day. Thus, the animals were conditioned, extinguished, and tested in context A (i.e., AAA group) or conditioned and extinguished in context A but tested in context B (AAB renewal group). The renewal test consisted of a 2-min BL period followed by a single continuous, 2-min tone presentation. Fear was indexed by defensive freezing behavior, defined as the absence of all visible movement except respiration. The percent time freezing during the acclimation period (baseline pre-CS measure) and

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CS presentations was quantified using automated motion-sensitive software (Coulbourn FreezeView software). 2.4.2. Experiment 2: Effect of CORT administered 24 h after conditioning on fear renewal This experiment examined the effect of delayed high-dose CORT treatment on fear renewal to determine whether the effect results from impairment of the consolidation of fear memory. The procedure in this experiment was identical to Experiment 1, with the exception that CORT or vehicle was administered 24 h after fear conditioning. 2.4.3. Experiment 3: Effect of CORT administered 1 or 24 h after tone fear conditioning on fear-enhanced behavior in the EPM This experiment compared the effects of immediate and delayed high-dose CORT treatment after stress exposure on anxiety-like behavior in the EPM. The retention of tone-induced fear memory was measured prior to anxiety-like behavior in the EPM. This procedure can enhance “state anxiety” and is sensitive to the effects of anxiolytic drugs (Korte, 2001; Korte and De Boer, 2003; Korte et al., 1995). 2.4.3.1. Tone fear conditioning (day 1). Rats were conditioned as described in Experiment 1. After conditioning, the rats were assigned to matched experimental groups with comparable levels of post-shock freezing and then were injected at different delays (1 or 24 h). 2.4.3.2. Fear-potentiated behavior in the EPM (day 8). One week after the injection, the rats were placed in a novel context (context B) to assess the retention of tone fear memory (fear expression). During the retention test, a 2-min BL (pre-CS period) was followed by five CSs with a 60-s ITI. One hour after the test, anxiety-like behavior was assessed in the EPM. As our laboratory reported previously (Li et al., 2010), the rats were individually placed in the center area, facing one of the open arms. At the end of each test session, the maze was carefully cleaned with 10% alcohol before the next animal was tested. We assessed the following behaviors: time spent (duration) in the open and closed arms and on the central platform and number of open and closed arm entries. Anxiolytic-like behavior (i.e., increased open arm time or increased open arm entries) can be determined simultaneously with a measure of spontaneous motor activity (total and/or closed arm entries) (Walf and Frye, 2007). Additionally, Cohen's laboratory proposed an “anxiety index” that integrates behavioral measures in the EPM, which is calculated as the following: "
Anxiety index ¼ 1−



time spent in the open arms total time on the maze

þ



#

number of entries to the open arms total exploration on the maze

2

Anxiety index values range from 0 to 1, in which an increase in the index represents an increase in anxiety-like behavior (Cohen et al., 2008). 2.4.4. Experiment 4: Effect of high-dose CORT administered 1 or 24 h after contextual fear conditioning on anxiety-like behavior and context-induced freezing behavior This experiment compared the effects of immediate and delayed high-dose CORT treatment after stress exposure on context-induced fear memory and anxiety-like behavior in the EPM in a contextual fear conditioning model. Additionally, contextual fear conditioning, as opposed to cued fear conditioning, may best produce sustained anxiety (Grillon, 2002); thus, anxiety-like behaviors were assessed directly. 2.4.4.1. Contextual fear conditioning (day 1). The rats received five footshocks (1.0 mA, 1 s) in context A. After conditioning, the animals were randomly assigned to the three groups and then injected with CORT (5 or 25 mg/kg) or vehicle at a different delay (1 or 24 h).

2.4.4.2. Elevated plus maze test (day 8). One week after injection, the rats were assessed in the EPM. The test procedure was the same as described in Experiment 3.

2.4.4.3. Contextual fear test (day 9). Twenty-four hours after the EPM test, the rats were placed again in context A for 8 min in the absence of shock to assess the retention of contextual fear memory (fear expression).

2.5. Statistical analysis The freezing data were statistically analyzed using two-way repeated-measures (trial) analysis of variance (ANOVA) where appropriate. For the fear renewal and EPM results, the statistical analyses were performed using two-way ANOVA followed by Fisher's Least Significant Difference post hoc test. The statistical analyses were performed using SPSS 16.0 software. The level of significance was set to p b 0.05. The data are expressed as mean ± SEM, and “n” refers to the number of rats used.

3. Results 3.1. Experiment 1: Immediate administration of high-dose CORT attenuated fear renewal in the absence of impairing consolidation of fearful memory Fig. 1B shows the freezing behavior data during fear acquisition (left panel) and fear extinction (middle and right panels). During fear acquisition, the rats displayed a progressive increase in freezing behavior and were equally split into three groups based on the five CS presentations during fear conditioning (repeated-measures ANOVA, F4,180 = 91.78, p b 0.001; Fig. 1B, left panel). Because the level of freezing in the 10th trial was already low and comparable to subsequent extinction trials, we only present the first 10 trials of each extinction session. During the first 2 min of the extinction session, prior to the first tone presentation (i.e., BL), the conditioned animals exhibited high levels of fear before the onset of the extinction trials (i.e., a consequence of fear conditioned to the context). Once extinction training commenced, CS presentations yielded robust freezing behavior in both the vehicle- and CORT-treated groups. The repeated-measures (trial) ANOVA of extinction session data (Fig. 1B, middle and right panels) revealed significant extinction learning across tone trials (session 1, F9,405 = 30.33, p b 0.001; session 2, F9,405 = 14.17, p b 0.001), with no main effect of group (F b 1) and no group × trial interaction (F b 1). In the tone test after extinction, the two-way ANOVA revealed no effects of group or context and no group × context interaction during the baseline period prior to the renewal tone test (F b 1; Fig. 1C). The two-way ANOVA (group × context) of CS freezing in the renewal test revealed main effects of group (F2,42 = 4.21, p b 0.05) and context (F 1,42 = 13.33, p b 0.01) and a group × context interaction (F2,42 = 3.84, p b 0.05; Fig. 1D). The post hoc tests revealed lower freezing in the high-dose CORT group than in the vehicle and low-dose CORT groups. Simple-effects analysis revealed a strong renewal effect in the vehicle group (F1,44 = 10.73, p b 0.01) and 5 mg/kg CORT group (F1,44 = 8.45, p b 0.01), which was not evident in rats that received 25 mg/kg CORT (F b 1). These data demonstrate that systemic high-dose CORT administration immediately following fear conditioning neither disrupted the consolidation of fear memory (see extinction session 1) nor facilitated the consolidation of extinction memory (see extinction session 2). Furthermore, because rats that received high-dose CORT did not display fear renewal, CORT appeared to act through mechanisms other than influencing the consolidation of fear memory.

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Fig. 1. Effects of immediate administration of high-dose corticosterone (CORT) on fear renewal: (A). Behavioral procedure for the experiment. Vertical gray arrow represents intraperitoneal injection (CORT or vehicle). (B). Mean (±SEM) percent baseline (BL) freezing and tone freezing during fear acquisition and extinction. (C and D). Mean (±SEM) percent BL freezing and tone freezing in either the same context as acquisition/extinction (Same) or in a novel context (Shifted) at test. Vehicle and 5 mg/kg CORT-treated rats showed significant renewal (Same vs. Shifted: ⁎⁎p b 0.01), which was blocked by 25 mg/kg CORT treatment (p N 0.05). High-dose CORT-treated rats showed a significant decrease in freezing in the Shifted condition (25 mg/kg vs. vehicle: ^^p b 0.01; 25 mg/kg vs. 5 mg/kg: #p b 0.05). n = 7–9 per group. Cond., conditioning; Ext., extinction; h, hour; Shk, shock.

3.2. Experiment 2: Delayed administration of high-dose CORT attenuated fear renewal Freezing levels in the acquisition and extinction sessions were similar among groups (Fig. 2B). As shown in Fig. 2C, the two-way ANOVA revealed no effects of group or context and no group × context interaction during the baseline period prior to the renewal tone test (F b 1). The two-way ANOVA (group × context) of CS freezing in the renewal test revealed main effect of context (F 1,46 = 12.50, p b 0.01), and group × context interaction (F 2,46 = 3.23, p b 0.05), and that the effect of group approached near significance (F2,46 = 3.12, p = 0.054) (Fig. 2D). Simple effects analysis revealed that a strong renewal effect in vehicle (F1,48 = 9.97, p b 0.01) and 5 mg/kg CORT-treated rats (F1,48 = 8.18, p b 0.01), which was lost in rats with 25 mg/kg CORTtreated (F b 1). These data indicate that the delayed administration of high-dose CORT also reduced fear renewal.

3.3. Experiment 3: Both immediate and delayed administration of high-dose CORT reduced fear-potentiated behavior in the EPM Freezing behavior during tone fear conditioning and testing is shown in Fig. 3B. No significant difference in the levels of freezing was found among groups during the tone fear test (F5,51 = 0.43, p N 0.05). The two-way ANOVA (dose × time) of anxiety-like behavior in the

EPM test revealed a main effect of dose (time spent in the open arms: F2,51 = 18.65, p b 0.0001, Fig. 3C; anxiety index: F2,51 = 9.30, p b 0.0001, Fig. 3D) but no main effect of time (F b 1) and no dose × time interaction (F b1). No difference was observed in closed arm entries among groups (F b 1; Fig. 3E). The post hoc tests revealed that administration of 25.0 mg/kg CORT elicited a significant increase in the time spent in the open arms and decreased the anxiety index compared with the vehicle group (p b 0.01) and 5 mg/kg CORT group (p b 0.001). Administration of 5.0 mg/kg CORT elicited a significant decrease in the time spent in the open arms relative to vehicle (p b 0.05). The data showed that high-dose CORT did not affect the consolidation of tone fear memory. Overall, the results of Experiment 3 demonstrated that post-conditioning high-dose CORT reduced anxiety-like behavior. 3.4. Experiment 4: Both immediate and delayed administration of high-dose CORT reduced EPM behavior without affecting contextual fear response Freezing behavior during the contextual fear test is shown in Fig. 4B. No difference in the levels of freezing was observed among groups during the contextual fear test (F5,44 = 0.44, p N 0.05). The two-way ANOVA (dose × time) of anxiety-like behavior in the EPM test indicated a main effect of dose (time spent in the open arms: F2,44 = 7.11, p b 0.01, Fig. 4C; anxiety index: F2,44 = 7.08, p b 0.01, Fig. 4D) but no main effect of time (F b 1) and no dose × time interaction (F b 1). No difference was observed in closed arm entries among groups (F b 1; Fig. 4E). The

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Fig. 2. Effects of delayed injection of high-dose CORT on fear renewal. (A). Behavioral procedure used for the experiment. Vertical gray arrow represents intraperitoneal injection (CORT or vehicle). (B). Mean (±SEM) percent baseline (BL) freezing and tone freezing during each tone presentation. (C). Mean (±SEM) percent freezing to 120-sec tone presentations in either the Same or Shifted context at test. Vehicle and 5 mg/kg CORT-treated rats showed significant renewal (Same vs. Shifted: ⁎⁎p b 0.01), which was blocked by CORT treatment (p N 0.05). Highdose CORT-treated rats showed a near significant decrease in freezing as compared with the other two groups (p = 0.054). n = 8–10 per group. Abbreviations as in Fig. 1.

post hoc tests revealed that administration of 25.0 mg/kg CORT elicited a significant increase in the overall time spent in the open arms and a significant decrease in the anxiety index compared with the vehicle and 5 mg/kg CORT groups (all p b 0.001). No significant difference in anxiety-like behavior was observed between the vehicle and 5.0 mg/kg CORT groups. 4. Discussion The present study showed that both immediate (1 h) and delayed (24 h) high-dose CORT administration after fear conditioning significantly reduced fear renewal and anxiety-like behavior. However, no difference was observed in the expression of tone- or context-induced fear memory between CORT- and vehicle-treated rats. During extinction training, neither high-dose nor low-dose CORT treatment produced any changes in the level of freezing compared with vehicle treatment. These results indicate that post-stressor intervention with a single high-dose CORT administration suppressed fear renewal in a novel environment and also reduced anxiety-like behavior but did not impair the consolidation or retrieval of fear memory. High-dose CORT may reduce fear renewal (Figs. 1D, 2D) by impairing fear memory consolidation (Cohen et al., 2008), decreasing the retrieval of previous aversive learning episodes (Aerni et al., 2004; Soravia et al., 2006), or enhancing the consolidation of extinction learning (Abrari et al., 2008; Blundell et al., 2011; Cai et al., 2006; Lu et al., 2006; Siegmund et al., 2011). Our finding that early post-conditioning

intervention with high-dose CORT failed to attenuate tone-induced fear expression (Fig. 3B) or context-induced fear expression (Fig. 4B) suggests that single treatment with high-dose CORT immediately after fear conditioning did not impair fear memory consolidation or retrieval, which is inconsistent with a previous report, in which an early post-stressor intervention with high-dose CORT reduced cue-induced freezing behavior (Cohen et al., 2008). This disparity might be attributable to methodological differences between our experiments and the study by Cohen et al., including the animal model, phase of the light/dark cycle during which the experiments were performed, and timing of the test. Cohen et al. used an animal model of predator scent stress and performed the experiments during the dark phase, whereas all of the experiments in the present study were conducted during the light phase in the tone and contextual fear conditioning model. Additionally, these studies had substantially different injection-test intervals. Cohen et al. (2008) conducted the behavioral tests 30 days after CORT injection, whereas we performed these tests 7 days after CORT administration. The levels of freezing in the vehicle- and CORT-treated groups were equivalent during the entire extinction training (Figs. 1B, 2B), indicating that neither impaired retrieval nor enhanced extinction was responsible for the high-dose CORT-induced reduction of fear renewal. The reduction of fear renewal was also unlikely attributable to the protective effects of CORT against the delayed behavioral response triggered by acute stress (Rao et al., 2012) because fear expression and fear renewal were both assessed at the same time point (1 week) after fear conditioning.

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Fig. 3. Effects of immediate and delayed post-stressor administration of high-dose CORT on retention of tone fear memory and fear-potentiated plus-maze behavior: (A) Behavioral procedure used for the experiment. Vertical gray arrow represents intraperitoneal injection (CORT or vehicle). (B) The percent freezing to tone during fear conditioning and test. (C) Time spent in the open arms of the EPM. (D) Anxiety index. (E) Closed arm entries. All data represent group mean ± SEM. (#) p b 0.05, (##) p b 0.01 vs. vehicle, (⁎⁎⁎) p b 0.001 vs. 25 mg/kg, n = 8–10 per group. w, week; EPM, elevated plus-maze.

Our finding that 25 mg/kg CORT administered immediately after fear conditioning resulted in a significant reduction of anxiety-like behavior is consistent with previous reports (Cohen et al., 2008; Zohar et al., 2011). There may be a “window of opportunity,” namely the first 6 h after the trauma exposure, for the beneficial effects of high-dose CORT treatment to become evident (Zohar et al., 2011). Furthermore, the present results suggest another time point when post-stress anxiety can be reduced by high-dose CORT (i.e., 24 h after fear conditioning training). Preclinical data indicate that threat stimuli elicit two classes of defensive behaviors: those that are associated with imminent danger (fear) and those that are associated with less specific and less predictable threats (anxiety) (Grillon, 2008). Conditioned fear and anxietylike behavior may partly share the same neural circuits and protein pathways. For example, both fear and anxiety are modulated by amygdala activity (Davis, 1992). Ponder and Kliethermes (Ponder et al., 2007) selectively bred mice for high and low levels of contextual fear and found that the high fear learning mouse line exhibited greater levels of anxiety-like behavior in the open field and zero maze. Conditioned fear and anxiety-like behavior are also both attenuated by anxiolytic drug (Bitencourt et al., 2008; Santos et al., 2005). Here, the significant reduction of fear renewal might be attributable to the anxiolytic effects of high-dose CORT.

One issue is why high-dose CORT administration did not affect the conditioned fear response through its anxiolytic effect in the absence of extinction. One important consideration is that corticosteroids may produce opposite effects on emotional behavior via different receptors in the brain (Korte, 2001). Therefore, the phase of the stress response and its context need to be considered when interpreting the results of behavioral experiments. Because of the ambiguity of the CS, the moderate level of fear, and involvement of the hippocampus after extinction, the CS-elicited aversive states before extinction and after extinction might be associated with fear and anxiety, respectively. First, although fear and anxiety are obviously overlapping, aversive, activated states that are centered on threat (Shin and Liberzon, 2010), they are in fact two independent entities that merit operational definitions (Öhman, 2008; Walker et al., 2003). Anxiety is characterized as a state of being that arises from general and nonspecific stimuli that are perceived as being potentially threatening in the future. In contrast, fear is stimulated by specific stimuli (Dias et al., 2013). Some authors pointed out that the origins of anxiety are unclear or uncertain [for review, see (Steimer, 2002)]. After fear conditioning, the CS has a predictable and specific “danger” meaning; thus, the CS elicits a “fear” state during the tone or context fear memory retention test (Figs. 3B, 4B), so that the CS-induced freezing response is unaffected by the anxiolytic effect of high-dose CORT. However, after extinction training, the CS

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Fig. 4. Effects of early and delay post-stressor administration of high-dose CORT on anxiety-like behavior and retention of contextual fear memory. (A) The behavioral procedure used for the experiment. Vertical gray arrow represents intraperitoneal injection (CORT or vehicle). (B) The percent freezing to context during each minute. (C) Time spent in the open arms of the EPM. (D) Anxiety index. (E) Closed arm entries. All data represent group mean ± SEM. (⁎⁎) p b 0.01 vs. vehicle, (##) p b 0.01 vs. 25 mg/kg, n = 8–9 per group.

acquires a second “safe” meaning that is available together with the first meaning (Bouton, 2002). Extinction turns a CS into an ambiguous stimulus with two opposing meanings (Vervliet et al., 2013). The current CS is less predictable and less specific when the extinguished CS is presented in a novel context (renewal test), and the animal does not “know” which meaning the CS provides, leading to a state of “anxiety”. Thus, the CS-induced freezing response is inhibited by the anxiolytic effect of high-dose CORT. Second, the level of fear was moderate in the renewal test. A previous study proposed that moderate and intense aversive states might be related to “anxiety” and “phobic” conditions, respectively, and anxiolyticsensitive freezing is triggered by a moderate level of fear (Santos et al., 2005). More recent work from our laboratory showed that high-dose CORT reduced freezing in rats with a low level of fear compared with rats with a high level of fear (divided according to their freezing level during conditioning) (An et al., 2013). In the present study, after two extinction sessions (2 × 40 trials), the level of freezing was very moderate in the renewal test (Figs. 1D, 2D) and was significantly less than the level in the tone or context fear memory retention test (Figs. 3B, 4B). Therefore, high-dose CORT reduced fear renewal. Lastly, after extinction, the hippocampus, which serves as a key structure of glucocorticoid action in the brain, is critical for fear renewal (Corcoran and Maren, 2001, 2004; Ji and Maren, 2005, 2007). The

hippocampus is involved in disambiguating the meaning of the CS using contextual cues (Orsini and Maren, 2012) and novelty detection (Maren, 2013). Glucocorticoids could affect anxiety by acting on ventral hippocampal network activity (Albrecht et al., 2013). Further studies are needed to determine the role of the hippocampus in the ability of post-conditioning high-dose CORT to reduce fear renewal. Collectively, these multiple factors, including the ambiguity of the CS, moderate level of fear, and involvement of the hippocampus, might regulate the effect of post-conditioning high-dose CORT treatment on the renewal of conditional fear. Although the hippocampus also underlies contextual fear expression, the meaning of the CS (training context) is specific, and the level of fear is high. Thus, high-dose CORT treatment had little effect on contextual fear expression (Fig. 4B). If the reduction of fear renewal results from an anxiolytic effect of CORT, then it is not surprising to observe that delayed CORT administration also reduced fear renewal. 5. Conclusions The major finding of the present study was that both immediate and delayed posttrauma intervention with high-dose CORT significantly reduced fear renewal and anxiety-like behavior in the EPM. This reduction of fear renewal appeared to be related to the anxiolytic effect of high-

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dose CORT rather than the effect of CORT on the consolidation of fear memory. The present results extend the findings of previous studies (Cohen et al., 2008; Zohar et al., 2011). Further studies are needed to reveal the explicit underlying mechanisms of high-dose CORT in the reduction of fear renewal. Acknowledgements This study was supported by 1) the Chinese Natural Science Foundation (30971057) to X. G. ZHENG, 2) Key Laboratory of Mental Health, Institute of Psychology, Chinese Academy of Sciences, 3) Knowledge Innovation Program of Chinese Academy of Sciences (KSCX2-EW-J-8; KJZD-EW-L04-2). References Abrari K, Rashidy-Pour A, Semnanian S, Fathollahi Y. Administration of corticosterone after memory reactivation disrupts subsequent retrieval of a contextual conditioned fear memory: dependence upon training intensity. Neurobiol Learn Mem 2008;89(2): 178–84. Aerni A, Traber R, Hock C, Roozendaal B, Schelling G, Papassotiropoulos A, et al. Low-dose cortisol for symptoms of posttraumatic stress disorder. Am J Psychiatry 2004;161(8): 1488–90. Albrecht A, Caliskan G, Oitzl MS, Heinemann U, Stork O. Long-lasting increase of corticosterone after fear memory reactivation: anxiolytic effects and network activity modulation in the ventral hippocampus. Neuropsychopharmacology 2013;38(3):386–94. An Xian-Li, Zheng Xi-Geng, Liang Jing, Bai Yun-Jing. Corticosterone combined with intramedial prefrontal cortex infusion of SCH 23390 impairs the strong fear response in high-fear-reactivity rats. PsyCh J 2013;2:1–10. Atsak P, Hauer D, Campolongo P, Schelling G, McGaugh JL, Roozendaal B. Glucocorticoids interact with the hippocampal endocannabinoid system in impairing retrieval of contextual fear memory. Proc Natl Acad Sci U S A 2012;109(9):3504–9. Bergstrom HC, McDonald CG, Dey S, Tang H, Selwyn RG, Johnson LR. The structure of Pavlovian fear conditioning in the amygdala. Brain Struct Funct 2013;218(6):1569–89. Bitencourt Rafael M, Pamplona Fabrício A, Takahashi Reinaldo N. Facilitation of contextual fear memory extinction and anti-anxiogenic effects of AM404 and cannabidiol in conditioned rats. Eur Neuropsychopharmacol 2008;18(12):849–59. Blundell J, Blaiss CA, Lagace DC, Eisch AJ, Powell CM. Block of glucocorticoid synthesis during re-activation inhibits extinction of an established fear memory. Neurobiol Learn Mem 2011;95(4):453–60. Bouton ME. Context, ambiguity, and unlearning: sources of relapse after behavioral extinction. Biol Psychiatry 2002;52(10):976–86. Bouton ME. Context and behavioral processes in extinction. Learn Mem 2004;11(5): 485–94. Cai WH, Blundell J, Han J, Greene RW, Powell CM. Postreactivation glucocorticoids impair recall of established fear memory. J Neurosci 2006;26(37):9560–6. Chang CH, Knapska E, Orsini CA, Rabinak CA, Zimmerman JM, Maren S. Fear extinction in rodents. Curr Protoc Neurosci 2009. [Chapter 8, Unit8 23]. Cohen H, Matar MA, Buskila D, Kaplan Z, Zohar J. Early post-stressor intervention with high-dose corticosterone attenuates posttraumatic stress response in an animal model of posttraumatic stress disorder. Biol Psychiatry 2008;64(8):708–17. Corcoran KA, Maren S. Hippocampal inactivation disrupts contextual retrieval of fear memory after extinction. J Neurosci 2001;21(5):1720–6. Corcoran KA, Maren S. Factors regulating the effects of hippocampal inactivation on renewal of conditional fear after extinction. Learn Mem 2004;11(5):598–603. Craske MG, Kircanski K, Zelikowsky M, Mystkowski J, Chowdhury N, Baker A. Optimizing inhibitory learning during exposure therapy. Behav Res Ther 2008;46(1):5–27. Davis M. The role of the amygdala in fear and anxiety. Annu Rev Neurosci 1992;15:353–75. Dias BG, Banerjee SB, Goodman JV, Ressler KJ. Towards new approaches to disorders of fear and anxiety. Curr Opin Neurobiol 2013;23(3):346–52. Grillon C. Startle reactivity and anxiety disorders: aversive conditioning, context, and neurobiology. Biol Psychiatry 2002;52(10):958–75. Grillon C. Models and mechanisms of anxiety: evidence from startle studies. Psychopharmacology (Berl) 2008;199(3):421–37. Hellsten J, Wennstrom M, Mohapel P, Ekdahl CT, Bengzon J, Tingstrom A. Electroconvulsive seizures increase hippocampal neurogenesis after chronic corticosterone treatment. Eur J Neurosci 2002;16(2):283–90. Ji J, Maren S. Electrolytic lesions of the dorsal hippocampus disrupt renewal of conditional fear after extinction. Learn Mem 2005;12(3):270–6.

195

Ji J, Maren S. Hippocampal involvement in contextual modulation of fear extinction. Hippocampus 2007;17(9):749–58. Johansen JP, Cain CK, Ostroff LE, LeDoux JE. Molecular mechanisms of fear learning and memory. Cell 2011;147(3):509–24. Korte SM. Corticosteroids in relation to fear, anxiety and psychopathology. Neurosci Biobehav Rev 2001;25(2):117–42. Korte SM, De Boer SF. A robust animal model of state anxiety: fear-potentiated behaviour in the elevated plus-maze. Eur J Pharmacol 2003;463(1–3):163–75. Korte SM, de Boer SF, de Kloet ER, Bohus B. Anxiolytic-like effects of selective mineralocorticoid and glucocorticoid antagonists on fear-enhanced behavior in the elevated plus-maze. Psychoneuroendocrinology 1995;20(4):385–94. Li Y, Zheng X, Liang J, Peng Y. Coexistence of anhedonia and anxiety-independent increased novelty-seeking behavior in the chronic mild stress model of depression. Behav Processes 2010;83(3):331–9. Lu KT, Yang YL, Chao PK. Systemic and intra-amygdala administration of glucocorticoid agonist and antagonist modulate extinction of conditioned fear. Neuropsychopharmacology 2006;31(5):912–24. Maren S. Fear of the unexpected: hippocampus mediates novelty-induced return of extinguished fear in rats. Neurobiol Learn Mem 2013;14:417–28. Myers KM, Davis M. Mechanisms of fear extinction. Mol Psychiatry 2007;12(2):120–50. Öhman A. Fear and anxiety: overlaps and dissociations. In: Lewis M, Haviland-Jones JM, Barrett LF, editors. Handbook of emotions. 3rd ed. New York, NY: Guilford Press; 2008. p. 709–29. Orsini Caitlin A, Maren Stephen. Neural and cellular mechanisms of fear and extinction memory formation. Neurosci Biobehav Rev 2012;36(7):1773–802. Ponder CA, Kliethermes CL, Drew MR, Muller J, Das K, Risbrough VB, et al. Selection for contextual fear conditioning affects anxiety-like behaviors and gene expression. Genes Brain Behav 2007;6(8):736–49. Rao RP, Anilkumar S, McEwen BS, Chattarji S. Glucocorticoids protect against the delayed behavioral and cellular effects of acute stress on the amygdala. Biol Psychiatry 2012; 72(6):466–75. Resstel LBM, Joca SRL, Moreira FA, Correa FMA, Guimaraes FS. Effects of cannabidiol and diazepam on behavioral and cardiovascular responses induced by contextual conditioned fear in rats. Behav Brain Res 2006;172(2):294–8. Roozendaal B. 1999 Curt P. Richter award. Glucocorticoids and the regulation of memory consolidation. Psychoneuroendocrinology 2000;25(3):213–38. Roozendaal B. Systems mediating acute glucocorticoid effects on memory consolidation and retrieval. Prog Neuropsychopharmacol Biol Psychiatry 2003;27(8):1213–23. Roozendaal B, de Quervain DJ, Schelling G, McGaugh JL. A systemically administered betaadrenoceptor antagonist blocks corticosterone-induced impairment of contextual memory retrieval in rats. Neurobiol Learn Mem 2004;81(2):150–4. Santos JM, Gargaro AC, Oliveira AR, Masson S, Brandao ML. Pharmacological dissociation of moderate and high contextual fear as assessed by freezing behavior and fearpotentiated startle. Eur Neuropsychopharmacol 2005;15(2):239–46. Schelling G, Briegel J, Roozendaal B, Stoll C, Rothenhausler HB, Kapfhammer HP. The effect of stress doses of hydrocortisone during septic shock on posttraumatic stress disorder in survivors. Biol Psychiatry 2001;50(12):978–85. Schelling G, Kilger E, Roozendaal B, de Quervain DJ, Briegel J, Dagge A, et al. Stress doses of hydrocortisone, traumatic memories, and symptoms of posttraumatic stress disorder in patients after cardiac surgery: a randomized study. Biol Psychiatry 2004;55(6): 627–33. Schelling G, Roozendaal B, Krauseneck T, Schmoelz M, DE Quervain D, Briegel J. Efficacy of hydrocortisone in preventing posttraumatic stress disorder following critical illness and major surgery. Ann N Y Acad Sci 2006;1071:46–53. Shin LM, Liberzon I. The neurocircuitry of fear, stress, and anxiety disorders. Neuropsychopharmacology 2010;35(1):169–91. Siegmund A, Koster L, Meves AM, Plag J, Stoy M, Strohle A. Stress hormones during flooding therapy and their relationship to therapy outcome in patients with panic disorder and agoraphobia. J Psychiatr Res 2011;45(3):339–46. Soravia LM, Heinrichs M, Aerni A, Maroni C, Schelling G, Ehlert U, et al. Glucocorticoids reduce phobic fear in humans. Proc Natl Acad Sci U S A 2006;103(14):5585–90. Steimer T. The biology of fear- and anxiety-related behaviors. Dialogues Clin Neurosci 2002;4(3):231–49. Vervliet B, Craske MG, Hermans D. Fear extinction and relapse: state of the art. Annu Rev Clin Psychol 2013;9:215–48. Walf Alicia A, Frye Cheryl A. The use of the elevated plus maze as an assay of anxietyrelated behavior in rodents. Nat Protoc 2007;2(2):322–8. Walker DL, Toufexis DJ, Davis M. Role of the bed nucleus of the stria terminalis versus the amygdala in fear, stress, and anxiety. Eur J Pharmacol 2003;463(1–3):199–216. Zohar J, Yahalom H, Kozlovsky N, Cwikel-Hamzany S, Matar MA, Kaplan Z, et al. High dose hydrocortisone immediately after trauma may alter the trajectory of PTSD: interplay between clinical and animal studies. Eur Neuropsychopharmacol 2011;21(11): 796–809.