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Fear-related behaviors were analyzed in rats exposed to predator-associated cues, a model of psychological trauma, over 10 weeks. Lentiviruses overexpressing CRFR2 were injected into the medial division, posterointermediate part of the BNST of susceptible and resilient rats.
Fear-related behaviors were analyzed in rats exposed to predator-associated cues, a model of psychological trauma, over 10 weeks. Lentiviruses overexpressing CRFR2 were injected into the medial division, posterointermediate part of the BNST of susceptible and resilient rats.
Fear-related behaviors were analyzed in rats exposed to predator-associated cues, a model of psychological trauma, over 10 weeks. Lentiviruses overexpressing CRFR2 were injected into the medial division, posterointermediate part of the BNST of susceptible and resilient rats.
Receptor Type 2 in the Bed Nucleus of Stria Terminalis Improves Posttraumatic Stress Disorder- like Symptoms in a Model of Incubation of Fear Einat Elharrar, Gal Warhaftig, Orna Issler, Yehezkel Sztainberg, Yahav Dikshtein, Roy Zahut, Lior Redlus, Alon Chen, and Gal Yadid Background: Posttraumatic stress disorder (PTSD) is a severe, persistent psychiatric disorder in response to a traumatic event, causing intense anxiety and fear. These responses may increase over time upon conditioning with fear-associated cues, a phenomenon termed fear incubation. Corticotropin-releasing factor receptor type 1 (CRFR1) is involved in activation of the central stress response, while corticotropin-releasing factor receptor type 2 (CRFR2) has been suggested to mediate termination of this response. Corticotropin- releasing factor (CRF) receptors are found in stress-related regions, including the bed nucleus of stria terminalis (BNST), which is implicated in sustained fear. Methods: Fear-related behaviors were analyzed in rats exposed to predator-associated cues, a model of psychological trauma, over 10 weeks. Rats were classied as susceptible (PTSD-like) or resilient. Expression levels of CRF receptors were measured in the amygdala nuclei and BNST of the two groups. In addition, lentiviruses overexpressing CRFR2 were injected into the medial division, posterointermediate part of the BNST (BSTMPI) of susceptible and resilient rats and response to stress cues was measured. Results: We found that exposure to stress and stress-associated cues induced a progressive increase in fear response of susceptible rats. The behavioral manifestations of these rats were correlated both with sustained elevation in CRFR1 expression and long-term downregulation in CRFR2 expression in the BSTMPI. Intra-BSTMPI injection of CRFR2 overexpressing lentiviruses attenuated behavioral impairments of susceptible rats. Conclusions: These results implicate the BNST CRF receptors in the mechanism of coping with stress. Our ndings suggest increase of CRFR2 levels as a new approach for understanding stress-related atypical psychiatric syndromes such as PTSD. Key Words: Anxiety, BNST, CRFR1, CRFR2, fear, PTSD P osttraumatic stress disorder (PTSD) is a chronic and inca- pacitating anxiety disorder that develops in some survivors of a traumatic event and can cause intense fear and a feeling of helplessness (1). Fear and anxiety may increase over time, upon conditioning with fear-associated cues and in the absence of further stress exposure (2). This phenomenon, termed incubation of fear, has been demonstrated in humans (3) and laboratory animals (4). However, the neural pathways involved in generating and regulating incubation of fear and persistent PTSD- like behavior remain unclear (5). Posttraumatic stress disorder and other psychiatric disorders, such as general anxiety disorder and panic disorder, are charac- terized by dysfunctional regulation of the central stress response (6,7). The corticotropin-releasing factor (CRF) family of ligands and receptors are important regulators of the neuroendocrine and behavioral responses to stressful challenges (8). Corticotropin- releasing factor receptor type 1 (CRFR1) and corticotropin- releasing factor receptor type 2 (CRFR2), encoded by different genes and exhibiting unique central and peripheral distribution, display distinct specicities for the CRF/urocortin (Ucn) family of ligands (810). It has been suggested that the two receptor subtypes have opposing roles, wherein CRFR1 participates in initiation of the stress response, inducing fear and anxiety-like responses, while CRFR2 activation re-establishes homeostasis (9). However, results from several studies provide evidence that does not support this notion (11). Corticotropin-releasing factor receptor type 1 is distributed widely in the brain, including regions that play central roles in the neural circuit of fear, such as the central amygdala (CeA) and basolateral amygdala (BLA) (1216). Corticotropin-releasing factor receptor type 2, however, is less prevalent, concentrated in specic stress-related brain areas such as the bed nucleus of the stria terminalis (BNST), dorsal raphe, and lateral septum (17,18). As a subregion of the extended amygdala, the BNST is an integral regulator of the hypothalamic-pituitary-adrenal stress axis. It acts as a critical intermediary, receiving inputs from the corticolimbic system and sending projections to the paraventricular nucleus of hypothalamus, where CRF is released. This induces pituitary activation and initiates the peripheral stress response (19). Initial attempts to link the BNST with conditioned fear responses were mostly unsuccessful; yet, subsequent studies have shown that this region, and in particular the BNST CRF receptors, play a role in some types of anxiety and stress Authors EE and GW have contributed equally to this work. From The Leslie and Susan Gonda (Goldschmied) Multidisciplinary Brain Research Center (EE, GW, RZ, LR, GY), The Mina & Everard Goodman Faculty of Life Sciences (EE, GW, YD, GY), Bar-Ilan University, Ramat- Gan; and Department of Neurobiology (OI, YS, AC), Weizmann Institute of Science, Rehovot, Israel. Address correspondence to Gal Yadid, Ph.D., Bar-Ilan University, Multi- disciplinary Brain Research Center, Geha Street, Ramat-Gan 52900, Israel; E-mail: yadidg@mail.biu.ac.il; goldra@mail.biu.ac.il. Received Nov 15, 2012; revised May 21, 2013; accepted May 24, 2013. 0006-3223/$36.00 BIOL PSYCHIATRY 2013;74:827836 http://dx.doi.org/10.1016/j.biopsych.2013.05.039 & 2013 Society of Biological Psychiatry responses (20). Walker et al. (13) have suggested that the BNST mediates long-duration, but not short-duration, responses, i.e., sustained fear or anxiety, as opposed to phasic fear. More recently, it was suggested that CRF neurons within the lateral BNST modulate conditioned anxiety-like behaviors (21). Chronic stress increases CRF concentrations and CRFR1 immunoreactivity in the BNST (22,23). Corticotropin-releasing factor infusion into the medial and dorsolateral BNST of rats enhances fear- potentiated startle response, an effect blocked by nonselective CRF receptor antagonism (24). Moreover, Arnold et al. (25) showed that intracerebroventricular (ICV) injection of CRF induces c-fos expression in the BNST, in a pattern similar to that found after restraint stress. This effect is blocked by ICV injection of a nonselective CRFR1 and CRFR2 receptor antagonist (25). Bittencourt and Sawchenko (26) found a strong general corre- spondence between the distribution of CRFR1-expressing cells and CRF-stimulated Fos-immunoreactivity, revealing extensive overlap in many brain regions. Although unique sites of CRFR2 expression, including the BNST, were relatively unresponsive to CRF, probably due to the low afnity of CRF to CRFR2 (27,28), these sites were more responsive after ICV administration of Ucn, which interacts preferentially with CRFR2 (26). Taken together, these ndings suggest that stress, anxiety, and fear affect the CRF system in the BNST, and notably the bed nucleus of the stria terminalis, medial division, posterointermediate part (BSTMPI). Indeed, CRFR1 is found in both medial and lateral aspects of the BNST, and CRFR2 is predominantly localized in the BSTMPI and the medial division, posteromedial part (18). In this article, we examined the role of the BSTMPI CRF system in development of fear incubation. We used an established rat model for PTSD, which simulates several types of prevalent PTSD symp- toms, including re-experiencing, avoidance, and hyperarousal (29), after exposure to predator-associated stress. The analysis criteria determine individual PTSD-like behavior, by categorizing the rats into distinct groups according to their magnitude of response to the stressful stimuli (30). Using our model, we found that exposure of rats to stress and stress-associated cues induced a fear response that incubates over time. In addition, the ndings revealed an association between fear incubation and impaired function of the CRF system in the BSTMPI. Susceptible (PTSD-like) rats demonstrated both an inability to suppress CRFR1 messenger RNA (mRNA) levels and a marked decrease in CRFR2 mRNA levels in this region over the long term. Next, we examined a possible interference in susceptible behavior, by application of a viral construct designed to overexpress CRFR2 into the BSTMPI, an approach that to our knowledge has not yet been examined for its benecial effect on fear symptoms or anxiety disorders in general. We found that this treatment signi- cantly attenuated the conditioned fear response in susceptible rats. Methods and Materials Behavioral Procedure Adult male SpragueDawley rats (250300 g; Harlan, Rehovot, Israel) were used. The model described herein is based on Kesner et al. (30). This model consists of several stages (habituation, initial exposure, rst reminder [Re 1 ], second reminder [Re 2 ], and third reminder [Re 3 ]) encompassing 10 weeks, as depicted in Figure 1A and detailed in Supplement 1. Site-Specic Overexpression of CRFR2 Using Lentiviruses After conclusion of the rst testing stage, an additional, separate group of rats were exposed to the same protocol (habituation, initial exposure, Re 1 , Re 2 , and Re 3 ). Rats were divided into susceptible and resilient sets as above, based on their behavioral response. Each subpopulation received a bilateral injection of either CRFR2-overexpressing lentiviruses (treatment) or control lentiviruses into the BSTMPI. Quantitative Real-Time Polymerase Chain Reaction (RT-PCR) Analysis For quantication of receptor levels in the BNST, CeA, and BLA, total RNA was isolated from each region by the single-step method (TriReagent, Sigma, Rehovot, Israel), using the manufac- turers recommended procedure. RT-PCR reactions were carried out using primers for CRFR1 and CRFR2 according to reaction protocols (Supplement 1). Corticosterone Assay After decapitation, trunk blood was collected into chilled tubes containing ethylenediaminetetraacetic acid solution. Blood sam- ples were collected carefully. Samples were centrifuged for 10 minutes at 41C at 2500 rpm, and plasma was stored at 801C until determination. On the day of assay, frozen plasma samples were thawed, and corticosterone levels were measured with a commercial radioimmunoassay kit (Diagnostic Products Corpora- tion, Los Angeles, California). Each group was composed of 13 rats. Design and Construction of Lentiviral Vectors for Site-Specic Overexpression of CRFR2 Vector plasmids were constructed for the production of lentivi- ruses expressing complementary DNA of mouse corticotropin- releasing factor receptor type 2 (CRFR2) using a minimal promoter. The constructed viral plasmid pCSC-minimal promoter tetracycline responsive element-CRFR2-internal ribosome entry site (IRES)-enhanced red uorescent protein was generated by subclon- ing the CRFR2 complementary DNA sequence upstream to an IRES-enhanced red uorescent protein site. The promoter sequence used to drive the expression of CRFR2 in the viruses used in this study was initially designed for conditional overexpression of CRFR2 using the TetOn system. However, due to the leakiness of the target tetracycline responsive element viral construct, we used these viruses (without the effector virus) to obtain constitutive expression of CRFR2 in infected cells. Orientation was conrmed by diagnostic cut and subsequent sequencing. pCSC-cytomegalovirus-IRES-green uorescent protein (GFP) plasmid was used for generating control viruses. Production of Lentiviral Vectors Recombinant lentiviruses are produced by transient trans- fection of HEK293T cells with the plasmid, as described previously (31,32). Infectious lentiviruses are harvested at 48 hours and 72 hours posttransfection, ltered through .45m-pore cellulose acetate lters, and concentrated by ultracentrifugation. In Vitro and In Vivo Validation of CRFR2 Lentiviral Vector Activity To determine the ability of the designed constructs to over- express functional CRFR2, rst an in vitro luciferase reporter assay was performed in HEK293T cells. For this assessment, the CRFR2- expressing plasmid was co-transfected with cyclic adenosine monophosphate responsive element luciferase plasmid. The ability of CRFR2 to activate cyclic adenosine monophosphate signaling in response to different doses of the urocortin-2 (Ucn-2) ligand was measured by luciferase activity, as previously described (33). Next, to determine the ability of the designed construct to overexpress 828 BIOL PSYCHIATRY 2013;74:827836 E. Elharrar et al. www.sobp.org/journal functional CRFR2 in brain tissue, it was microinjected into the BSTMPI. Control animals received intra-BNST injection of the pCSC- cytomegalovirus-IRES-GFP plasmid. Three days later, rats were decapitated, brains were removed and sliced on ice-chilled glass, and the BSTMPI was isolated. mRNA extraction and RT-PCR were performed according to the above protocol, using specic primers for CRFR2 and hypoxanthine-guanine phos- phoribosyltransferase. Intracerebral Injection of Lentiviral Vectors and Analysis of Susceptible Rat Response After Re 2 , susceptible and resilient sets were intraperitoneally anesthetized with ketamine hydrochloride (100 mg/kg) and xylazine (10 mg/kg). Each set was randomly divided into two groups, each receiving a bilateral microinjection (.7 L) of either CRFR2 overexpressing lentivirus or control lentivirus into the BSTMPI. Injection coordinates were as follows: 14 o , anterior .72 mm; lateral 2.9 mm, and ventral 7.0 mm from bregma. Lentiviral injections were conducted using a computer-guided stereotaxic instrument and a motorized nanoinjector (Angle Two Stereotaxic Instrument, St. Louis, Missouri), which is fully inte- grated with the Paxinos Rat Brain Atlas via a control panel. Rats were given a 2-week recovery period, and Re 3 was then performed followed by open eld assessment of performance under the three behavioral conditions. To verify site specicity of this treatment, we examined CRFR2 mRNA levels also in the lateral septum (LS), a stress-related region adjacent to the BNST, in which CRFR2 is also concentrated. Experimental rats in which CRFR2 expression levels in the LS were higher than those in the LS of control rats (due to inaccurate lentiviral injection) were excluded from the analysis. Statistical Analysis See Supplement 1. Figure 1. Behavioral measurements. (A) Schematic description of the experimental design. After 2 weeks of habituation to the open eld (5 min/day), the rats began the experimental procedure. Their baseline response to the three behavioral conditions was monitored on day 1 (1d). Day 7 (7d): stress exposure (initial exposure). Day 14 (14d): rst re-exposure (Re 1 ) to a cue reminder. Day 35 (35e): second re-exposure (Re 2 ). One week later, a group of susceptible and resilient rats received intra-bed nucleus of the stria terminalis, medial division, posterointermediate part lentivirus injection (virus injection). Day 50 (50d): third re-exposure (Re 3 ). (B) Upper gure: The entire distribution of the freezing data in each of the three conditions at Re 2 . The dotted line is the upper level for excluding outliers in each condition. The gray line is the mean of each subpopulation. Lower gure: Correlations between the three conditions for the entire population. There is a strong correlation between exploration versus social interaction, exploration versus hyperarousal, and social interaction versus hyperarousal (R .37, .42, and .59, respectively; p .0001). E. Elharrar et al. BIOL PSYCHIATRY 2013;74:827836 829 www.sobp.org/journal Results Incubation of Fear in Rats Exposed to Stress A total of 180 rats were exposed to the traumatic event (litter with cat odor) and stress-associated cues. Posttraumatic stress disorder-like behavior (measured by duration of freezing response) of each rat was compared both with its own baseline and with the range of the population. This ensured that two unambiguous subpopulations could be signicantly identied, albeit the stressful component of the measurements. The inci- dence of susceptible rats was approximately 20% (36 susceptible rats and 144 resilient). Analysis of all baseline behavioral samples revealed that the upper level for excluding outliers (95% condence) in the exploration and hyperarousal conditions was 1.5 times the interquartile range and for the social interaction condition it was 2 times the interquartile range. Rats expressing behavioral parameters that were two standard deviations from the mean were considered outliers and excluded from the study. The entire distribution of the freezing data is presented for each of the three behavioral conditions at Re 2 (Figure 1B, top). The distribution data show that the majority of rats were concentrated in the resilient subpopulation. Moreover, it reveals a greater increase from baseline in PTSD-like behavior of the susceptible group, for all three behavioral conditions, as com- pared with resilient rats. Pearson product-moment correlations between exploration, social interaction, and hyperarousal were .37, .42, and .59, respectively (Figure 1B, bottom). Thus, while there was a degree of common variance between the three conditions, most of the variance was unique to each condition. Rats were then chosen from the susceptible and resilient sets, based on extreme behavior (n 10 for each subpopulation). A three-way analysis of variance (ANOVA) (with a Greenhouse- Geisser correction) comparing freezing behavior over the four time points (within subjects), the three behavioral conditions (within subjects), and the two groups (susceptible vs. resilient) revealed a statistically signicant three-way interaction (F 3.25,55.16 3.79, p .05). The group time point (F 3,51 7.72, p .001) and the behavioral condition group (F 1.21,20.56 6.21, p .05) interactions, as well as the main effects (which were not a focus of the current investigation), were signicant. The group time point interaction reveals that beyond the particular test, the susceptible group increased its freezing duration over time, signicantly more than the resilient group. The three-way interaction supports this nding, suggesting a different pattern between the different conditions, for each group. The freezing data for the three tests are presented in Figure 2. To clarify the three-way interaction, follow-up one-way ANOVAs compared the different time points in each group. Rats from the susceptible subpopulation showed clear incubation of fear during all three behavioral conditions (Figure 2B). During the exploration conditioning, freezing behavior of susceptible rats was signicantly elevated from baseline after initial exposure, Re 1 , and Re 2 (one-way ANOVA: F 3,8 32.285, p .001; Tukeys post hoc: p .01 [initial exposure and Re 1 ], p .001 [Re 2 ]). For resilient rats, freezing behavior was signicantly elevated after initial exposure and Re 1 and returned to baseline levels at a third reminder (Re 3 ) (F 3,8 5.918, p .05; p .01 [initial exposure], p .05 [Re 1 ]). During social interaction conditioning, freezing behavior of susceptible rats after the Re 1 and the Re 2 were signicantly elevated from baseline (F 3,8 11.659, p .01; p .05 for both time points), while no signicant differences were detected for resilient rats (p .05). During hyperarousal conditioning, freezing behavior of susceptible rats was signicantly elevated after Re 2 , as compared with baseline (F 3,8 2.833, p .05; p .05), while no signicant changes were detected for resilient rats (p .05; Figure 1B). Endocrine data also mimicked clinical observation. We measured corticosterone levels and found the following results for corticosterone: susceptible: 74 5 ng/mL, resilient: 107 14 ng/mL, naive rats: 102 10 ng/mL (one-way ANOVA, p .05; n 13 per group). Low CRFR2 mRNA Expression in the BSTMPI of Susceptible Rats Next, we examined whether the behavioral manifestations of susceptible rats were associated with changes in corticotropin- Figure 2. Behavioral measurements. Freezing behavior during explora- tion, social interaction, and hyperarousal conditions. Freezing time was calibrated to the percentage of total time in the open eld. After exposure to stress and the stress-associated cue, susceptible rats showed an increase from baseline in anxiety-like behavior, i.e., gradual intensication (or incubation) of fear over time for all three behavioral conditions, as opposed to resilient rats (exploration: **p .01, ***p .001, ## p .01, # p .05; social interaction: *p .05; and hyperarousal: *p .05 compared with baseline). Bars represent mean SEM; n 10 per group (subpopulation). Division between susceptible and resilient groups was performed after second reminder (Re 2 ); therefore, gures were constructed based on retrospect data. Re 1 , rst reminder. 830 BIOL PSYCHIATRY 2013;74:827836 E. Elharrar et al. www.sobp.org/journal releasing factor receptor expression in regions linked with anxiety-like behavior, i.e., the CeA, BLA, and BSTMPI. For the CeA and BLA, we found no signicant differences in CRFR1 mRNA levels between control rats versus resilient and susceptible rats after Re 2 (p .05; n 9 for each group; Figure 3B). In the BSTMPI, however, a signicant increase in CRFR1, but not CRFR2, mRNA expression was observed in rats initially exposed to stress (stress group), as compared with control rats (one-way ANOVA: F 2,28 69.16, p .0001; Tukeys post hoc: p .001; n 10 for each group; Figure 3D). Over the longer period, signicantly decreased CRFR1 mRNA levels were found after Re 2 in resilient rats, as compared with susceptible and control rats (one-way ANOVA: F 2,28 69.16, p .0001; Tukeys post hoc: p .01, p .05, respectively; n 10 for each group; Figure 3D). Conversely, a signicant decrease in CRFR2 mRNA expression was demonstrated after Re 2 , in susceptible rats as compared with resilient and control rats (one-way ANOVA: F 2,28 4.004, p .01; Tukeys post hoc: p .05; n 10; Figure 3E). Establishment of a Lentiviral-Based System for Site-Specic Overexpression of CRFR2 Because susceptibility to PTSD-like symptoms coincides with robust changes in BNST CRFR2 levels, we subsequently examined the effect of manipulating levels of this receptor specically within the BNST of susceptible rats. First, we generated a lentiviral system for overexpression of CRFR2 at physiological levels (Figure 3A). We then assessed the ability of this system to express a functional receptor that responds in vitro to its specic ligand, Ucn2 (Figure 3C). Using a cyclic adenosine monophosphate responsive element luciferase reporter assay in HEK293T cells, we found signicantly higher levels of CRFR2 signaling following treatment with 1 or 100 mol/L Ucn2, as compared with 0 and .01 mol/L Ucn2 (one-way ANOVA: F 3,24 17.658, p .0001, Dunnett's post hoc: p .0001 vs. corresponding doses; n 7 for each group). Next, we examined the ability of CRFR2 lentiviruses to overexpress the receptor in vivo in nave rats. Rats that received bilateral, intra-BSTMPI injections of the lenti-CRFR2 showed signicantly higher CRFR2 expression levels in this region than rats that received control virus injection (Student t test, p .05; n 4 per group; Figure 4D). Figure 3. Quantitative real-time polymer- ase chain reaction analysis of corticotro- pin-releasing factor receptor type 1 (CRFR1) and corticotropin-releasing factor receptor type 2 (CRFR2) messenger RNA (mRNA) expression in the central amyg- dala (CeA), basolateral amygdala (BLA), and bed nucleus of the stria terminalis. (A) Rat coronal sections illustrating loca- tion of punches taken from the CeA (upper plate; bregma 2.52) or BLA (lower plate; bregma 2.52). (B) Corticotropin- releasing factor receptor type 1 mRNA levels in the CeA and BLA following the second reminder (Re 2 ) (n 9 for each group) showing no signicant differences between control vs. resilient and suscep- tible rats (p .05). (C) Simplied rat coronal sections illustrating the location of a punch taken from the bed nucleus of the stria terminalis, medial division, pos- terointermediate part (bregma .72). (D) Elevated CRFR1 levels were shown follow- ing initial stressor exposure (stress). Fol- lowing Re 2 , CRFR1 mRNA expression levels were signicantly decreased in resilient as compared with susceptible and control rats (***p .001, **p .01, # p .05, respectively; n 10 for each group). (E) Corticotropin-releasing factor receptor type 2 levels showed no change after initial exposure (p .05; n 10 for each group). After Re 2 , signicantly decreased CRFR2 mRNA levels were found in suscep- tible rats, compared with resilient and control rats (*p .05, # p .05, respec- tively; n 10 for each group). Bars represent mean SEM (A and C reprinted from (77) with permission from Elsevier, copyright 2008). E. Elharrar et al. BIOL PSYCHIATRY 2013;74:827836 831 www.sobp.org/journal Attenuation of Susceptible Behavior by Overexpression of CRFR2 in the BSTMPI Next, to assess the lentiviral effect on susceptible behavior, an additional, separate group of rats were subjected to the behav- ioral protocol. Rats were divided into susceptible and resilient sets as above, and subpopulations were then extracted from these sets (chosen based on extreme response or lack thereof; n 13 for susceptible, n 14 for resilient). Each subpopulation received bilateral, intra-BSTMPI injections of either high-titer CRFR2-express- ing lentivirus (n 7 per group) or control lentivirus (n 6/7). We found that overexpression of CRFR2 in the BSTMPI signicantly attenuated freezing behavior in the exploration, social interaction, and hyperarousal conditions of susceptible rats after Re 3 , as compared with susceptible rats that received the control virus. A three-way ANOVA showed a signicant group treatment time interaction (F 1,20 6.81, p .05) over the three different conditions, i.e., exploration, social interaction, and hyper- arousal. In a four-way ANOVA, the condition group treatment time interaction was not signicant, suggesting that the effect was not signicantly different between tests. As shown in Figure 5, the susceptible overexpression group froze signicantly less than before CRFR2-expressing lentivirus administration (Re 2 ) (paired t test; n 6/7 for each group; p .05, p .01, p .05, p .01 for exploration, social interaction, and hyperarousal, respectively). To verify the site specicity of lentiviral system, mRNA levels in the LS of infected susceptible rats was examined. We found that rats with CRFR2 overexpressing lentiviruses in the BSTMPI expressed higher mRNA levels as compared with susceptible rats that received the control virus or with susceptible rats with no injection (one-way ANOVA: F 3,5 120.3, p .0001; n 5 for each group; Figure 6A). Rats with CRFR2 overexpressing lentiviruses in the LS expressed similar mRNA levels as compared with susceptible rats that received the control virus or with susceptible rats with no injection (one-way ANOVA: F 3,3 .555, p .5; n 5 for each group; Figure 6B). Figure 6C shows GFP staining of the control lentiviruses. Discussion We used a rat model for PTSD to demonstrate a progressive increase in fear response of susceptible (PTSD-like) rats after exposure to stress and stress-associated cues. Moreover, fear incubation in susceptible rats was associated with long-lasting impairment of BNST function, demonstrated as prolonged upre- gulation of CRFR1 expression levels, combined with a prominent decrease in CRFR2 expression levels. Application of a CRFR2- overexpressing lentivirus into the BSTMPI signicantly attenuated the fear response of susceptible rats. Although incubation of fear over a period of 24 hours is well established in rodents (3436), results for longer durations are less conclusive (5). Several reports have shown that conditioned fear responses either remain constant over time or decrease to some extent (3740). Others have shown time-dependent intensication of fear in response to shock-paired contexts over 1 month (41) and 2 months (4) in rats or over 2 weeks in mice (42,43). Yet, this incubation phenomenon was inconsistent across age groups in rats (4), and in mice it diminished after 2 weeks and showed inconsistencies among strains (42,43). Our ndings showed gradual, time-dependent intensication of fear in response to recurring stress cues over a period of 28 days. The maladaptive, PTSD-like symptoms observed in our model correspond with clinical manifestations of PTSD, including anxiety and defensive behavior, social withdrawal and avoidance behaviors, hyperar- ousal, and re-experiencing (5,29,30). Several studies have reported that lower cortisol levels in the acute aftermath of trauma are predictors for subsequent PTSD symptoms (4446). Moreover, lowered circulating cortisol was suggested as a major inuence on the formation and processing of traumatic memories and may be associated with the underlying pathology of PTSD (47,48). In addition, exogenous stressors, such as exposure to a live predator (4951), psychological stress (52), and the predatory cue used herein (5356), correspond well with natural stressors. Therefore, the stressful stimuli used herein may have greater etiological relevance than application of physical stressors, e.g., electric shock (5760), underwater trauma (61), and restraint stress (62,63). Between 5% and 30% of trauma victims develop PTSD, whereas others experiencing the same trauma appear to be resilient (29). Our results, showing that 20% of rats developed maladapted symptoms following exposure to stress and stress cues, correlate well with the above statistics. This also empha- sizes both the importance of individual analysis of behavior and comparison of each exposed animal both with its own baseline and with the entire population. Although this analysis yields a lower percentage of PTSD-like rats, it more accurately simulates Figure 4. Lentiviral system for overexpression of corticotropin-releasing factor receptor type 2 (CRFR2). Schematic illustration of (A) vectors designed to overexpress CRFR2 and (B) its lentivirus control. (C) In vitro biological activity validation of CRFR2-expressing lentiviral construct using a cyclic adenosine monophosphate-dependent luciferase assay. Higher levels of CRFR2 signaling was observed following treatment with 1 or 100 mol/L of urocortin-2 (Ucn-2) (***p .0001 vs. corresponding doses; n 7 per group). (D) In vivo functionality assessment of CRFR2-expressing lentiviral construct. Corticotropin-releasing factor receptor type 2 messenger RNA (mRNA) levels in the bed nucleus of the stria terminalis, medial division, posterointermediate part of rats infected with lenti-CRFR2 were signicantly higher than those of control-infected rats. Mean SEM (*p .05; n 4 per group). CMV, cytomegalovirus; CRFR2, corticotropin-releasing factor receptor type 2; eRFP, enhanced red uorescent protein; GFP, green uorescent protein; IRES, internal ribosome entry site. 832 BIOL PSYCHIATRY 2013;74:827836 E. Elharrar et al. www.sobp.org/journal the heterogeneous pattern of response to stressful events in humans. Thus, the combination of the model components, i.e., exogenous stimuli, simulation of clinical manifestations, and individual categorization, can produce more reliable results for modeling the incubation of fear phenomenon. Recent ndings have demonstrated that the CeA and BNST are functionally complementary, with the CeA mediating short- duration threat responses (i.e., phasic fear) and the BNST mediating long-duration responses (sustained fear or anxiety). Moreover, CRF selectively participates in the BNST-dependent response (13,64). Our results, showing dysfunction of the CRFR2 expressing neurons in BSTMPI, but not CeA or BLA, of susceptible rats provide further support for involvement of this region in sustained fear. Figure 5. Effect of overexpression of corticotropin-releasing factor recep- tor type 2 (CRFR2) in the bed nucleus of the stria terminalis, medial division, posterointermediate part on behavioral manifestation. Suscep- tible and resilient rats received injection of lenti-CRFR2 or control virus into the bed nucleus of the stria terminalis, medial division, poster- ointermediate part. Freezing behavior was assessed in exploration (A), social interaction (B), and hyperarousal (C) conditions 2 weeks (third reminder [Re 3 ]) after lenti-CRFR2 injection. The line represents second reminder (Re 2 ) and the bars represent Re 3 . A signicant reduction in freezing behavior of susceptible rats was detected (Re 3 vs. Re 2 for exploration, social interaction, and hyperarousal respectively; **p .01, *p .05, **p .01; n 6 per group) Mean SEM. Figure 6. Validation of corticotropin-releasing factor receptor type 2 (CRFR2) overexpression in the bed nucleus of the stria terminalis, medial division, posterointermediate part (BSTMPI). Corticotropin-releasing factor receptor type 2 messenger RNA (mRNA) levels were quantied in susceptible rats injected with lenti-CRFR2 (overexpression vector [O.E.]) into (A) the BNSTMPI or (B) lateral septum (LS) in comparison with uninfected or control-infected (control vector [C.V.]) rats (***p .001 and p .05, respectively); mean SEM, n 5 per group. (C) Image of the injection site that conrms bed nucleus of the stria terminalis specic infected neurons via green uorescent protein immunostaining. Dots in a rat coronal illustration indicate site of injection (reprinted from [78] with permission from Elsevier, copyright 2008). E. Elharrar et al. BIOL PSYCHIATRY 2013;74:827836 833 www.sobp.org/journal It has been suggested that CRFR1 activation is critical for initiating stress responses (65), while CRFR2 may be involved in termination of stress responses and restoring homeostasis (66,67). Thus, we postulate that the signicant downregulation of CRFR1 mRNA levels in resilient rats may mediate their behavioral resilience to stress. In contrast, the inability to lower CRFR1 mRNA levels and the dramatic reduction in CRFR2 mRNA levels of susceptible rats may mediate their long-lasting fear response and incompetence. These rats show an exaggerated stress response that remains active over the long term and shutting down of the stress response fails to occur up to 1 month after exposure to stress. In other words, the natural ability to adapt to stress may be damaged in susceptible rats, thereby promoting sustained fear. This hypothesis is further supported by studies showing that CRFR2 knockout mice are hypersensitive to stress and show increased anxiety-related behavior (6870) and that ICV injection of a selective CRFR2 antagonist dose-dependently enhances conditioned fear response (71). Other ndings implicate CRFR2 in mediating stress coping mechanisms (72). Corticotropin- releasing factor receptor type 2, along with its specic ligands, Ucn-2 and urocortin-3 (Ucn-3), are hypothesized to be involved in dampening the stress response (8,28,7375). In addition, Ucn-2 and Ucn-3 are essential for recovery from stressful challenges (75). Thus, our nding showing signicantly low levels of CRFR2 in the BNST of susceptible rats probably affects Ucn-2 and Ucn-3 activity. Thus, in their own right, Ucns may be involved in the pathogenesis and control of various disorders (76). The BNST-CRFR2 system has already been implicated in PTSD-like behavior in a mouse animal model (72). However, this model did not use reminders; rather, mice experienced stress and CRFR2 levels were then examined 2 weeks after rats displayed long-term, steady state PTSD-like behavior. Findings showed that the exposure to stress increased levels of CRFR2 mRNA in the BNST and that knockdown of BNST-CRFR2 reduced the percentage of mice categorized as PTSD-like. In contrast, our model showed a decrease in CRFR2 mRNA levels after incubation of fear and that CRFR2 overexpression modied susceptible behavior. Thus, the decrease in CRFR2 levels following stress -associated cues may mimic physiological re- experiencing of the original stressor after the pathology has been established. Yet, taking together the ndings of both models indicates that balanced levels of CRFR2 are essential for an appropriate response to stressful events. In conclusion, we show a bi-directional association between CRF receptor levels in the BSTMPI and incubation of fear in a rat model for PTSD. The sustained upregulation in CRFR1 expression, combined with reduced CRFR2 expression in the BNST, may have promoted long-term conditioned fear response. Corticotropin-releasing factor receptor type 2 is involved in mechanisms of coping with stressors and challenging situations; thus, we hypothesize that PTSD mainly represents malfunctioning of the ability to contend with stressful stimuli. Even after prolonged dissociation from stress, the susceptible individual may inappropriately react to imminent stress. This emphasizes the need for long-term evaluation of symptoms follow- ing exposure to stress. Moreover, further research focusing on the benecial effect of CRFR2 augmentation in the BNST may provide new insights for the development of PTSD treatments. We thank Dr. Tamar Green-Sadan for critically reviewing the manuscript. 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