Juvenile stress induces a predisposition to either anxiety or depressive-like symptoms following stress in adulthood

European Neuropsychopharmacology (2007) 17, 245 — 256

www.elsevier.com/locate/euroneuro

Juvenile stress induces a predisposition to either anxiety or depressive-like symptoms following stress in adulthood Michael Tsoory a, Hagit Cohen b, Gal Richter-Levin a,* a

Department of Psychology and The Brain and Behavior Research Center, University of Haifa, Mount Carmel, 31905 Haifa, Israel b The Anxiety and Stress Research Unit, Ministry of Health Mental Health Center, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel Received 9 January 2006; received in revised form 30 May 2006; accepted 20 June 2006

KEYWORDS Early-life stress; Animal model; Juvenile; Anxiety; Depression

Abstract Epidemiological studies indicate that childhood trauma is predominantly associated with later emergence of several stress-related psychopathologies. While most dearly-stressT animal models focus on pre-weaning exposure, we examined the consequences of exposure to stress during the early pre-pubertal period, bjuvenile stressQ, on adulthood stress responses. Following two different juvenile stress protocols, predator scent or short-term variable stress, we examined adulthood stress responses using the elevated plus-maze and startle response or exploration and avoidance learning. Employing Cut-off Behavioral Criteria analyses of clustering symptoms on the rats’ altered stress responses discriminated between different patterns of maladaptive behaviors. Exposure to either juvenile stress protocols resulted in lasting alteration of stress responses with the majority of rats exhibiting anxiety-like behaviors, while the remaining third displayed depressive-like behaviors. The results suggest that the presented bJuvenile stressQ model may be relevant to the reported predisposition to develop both anxiety and depression following childhood trauma.

D 2006 Elsevier B.V. and ECNP. All rights reserved.

1. Introduction

* Corresponding author. Tel.: +972 4 824 0962; fax: +972 4 828 8578. E-mail address: [email protected] (G. Richter-Levin).

Traumatic events often trigger the emergence of a variety of stress-related psychopathologies, including not only acute stress disorder (ASD) and Post Traumatic Stress Disorder (PTSD), but also depressive and anxiety symptoms (Moreau and Zisook, 2002). The psychological and behavioral sequelae

0924-977X/$ - see front matter D 2006 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2006.06.007

246 of trauma are associated with multiple endocrine, and anatomical changes in neuronal circuits that are critically involved in modulating stress responses, emotional processing, and learning (Arborelius et al., 1999). Childhood emotional trauma contributes significantly to the emergence of these psychopathologies. Epidemiological studies indicate that early-life stress is predominantly associated with higher prevalence of a range of psychopathologies, particularly depression and PTSD (Agid et al., 1999; Arborelius et al., 1999; Briere and Elliott, 1994; Draijer and Langeland, 1999; Furukawa et al., 1999; Heim and Nemeroff, 2001; Heim et al., 2004; Maughan and McCarthy, 1997; Weiss et al., 1999). Recent years have witnessed growing interest in effectively modeling in animals the long-term effects of childhood emotional trauma on stress responses in adulthood. Most studies concerned with the impact of early-life stress on subsequent stress responses in adulthood in rodents have focused on the post-natal pre-weaning period, i.e. 3—14 days, and involve some form of maternal deprivation or maternal separation producing acute and long-term effects that vary with the pups’ age at exposure to stress (Andersen and Teicher, 2004; Kehoe et al., 1998; Levine, 1994; Levine et al., 1991; Ogawa et al., 1994; Plotsky et al., 1993; Rosenfeld et al., 1992; van Oers et al., 1998; Vazquez et al., 1996; von Hoersten et al., 1993). However, marked differences exist between neonate rats and infants’ stress responses mechanisms (Vazquez, 1998). For example, rat pup’s HPA axis is characterized by a silent hypo-responsive period (Vazquez et al., 1996), while in humans there is no conclusive evidence of a hypo-responsive period in the HPA axis course of development (Gunnar and Donzella, 2002). Indeed, it has been suggested that the ages of 3 to 14 days in the rat roughly corresponds to the 23rd week of gestation in humans (Fitzgerald and Anand, 1993). Furthermore, psychiatric studies often refer to human childhood rather than infancy when investigating the traumatic history of stress-related psychopathologies patients (De Bellis et al., 2002; Nemeroff, 2004; Nemeroff et al., 2003; Penza et al., 2003; Pfefferbaum, 2005; Street et al., 2005; Tupler and De Bellis, 2006; Yang et al., 2004). Thus, we have recently started to examine the consequences of stress exposure at a later early-life period—the juvenile and/or the early adolescent period (Avital et al., 2006; Avital and Richter-Levin, 2005; Tsoory and RichterLevin, 2005). Across species, from rodents to primates, the juvenile brain is a bbrain in transitionQ, which differs markedly both anatomically and neurochemically from that of newborns, weanlings or adults. During this period substantial remodeling occurs in brain areas involved in emotional and learning processes such as the prefrontal cortex (PFC), the hippocampus, and the amygdala, (for review see Spear, 2000). These areas are frequently indicated as being especially sensitive to the effects of stress/trauma. Preteens have enhanced stress perception and responses (Allen and Matthews, 1997; Spear, 2000). Stressful events during this period were associated with later socioemotional maladaptive behaviors (Spear, 2000) and represent a significant risk factor for the later development of stress-related psychopathologies (Heim and Nemeroff, 2001; Heim et al., 2004).

M. Tsoory et al. In order to model in the rat the detrimental effects of childhood and early adolescent emotional trauma on adulthood stress responses observed in humans, we focused on the juvenile period in the rat ontogeny, i.e. at about the age of 4 weeks. Juvenile rats resemble children and preteens in several behavioral features, like reduced dependence on maternal care, increased independence, and increased play-behaviors with peers that diminish with the beginning of puberty (Spear, 2000). Moreover, as in human neural development, in the juvenile rat the HPA axis is fully developed, while other key brain areas involved in both emotional and learning processes, such as the PFC, hippocampus, and amygdala-based neurocircuits, are still undergoing significant maturation processes (Spear, 2000). For example, Andersen and Teicher (2004) demonstrated the overproduction and pruning trajectory that occurs in the rat’s limbic elements, hippocampus (CA1, CA3), amygdala and PFC between pre-puberty and adulthood. Pre-pubertal exposure to stress resulted in more pronounced short-term effects than exposure at other ages. The HPA axis reaches its developmental asymptote around the juvenile period (Vazquez, 1998; Walker et al., 1986). Juvenile rats’ stress response lasts twice longer than adults, indicating slower shutoff of the HPA axis and suggesting less centrally mediated feedback from various forebrain limbic areas at this age (Romeo et al., 2004). In comparison with earlier or later ages, pre-pubertal social isolation produced prominent effects on object exploration (Einon and Morgan, 1977) and fluid intake (McGivern et al., 1996). Juvenile rats differ from adult rats also in the altered behavioral profile following pre-exposure to stress. Juvenile rats (22—24 days) previously isolated during the third week of life were impaired in the Morris water maze task in comparison with control juvenile rats, whereas adult rats (92—94 days) previously isolated during the third week of life demonstrated more rapid learning of the task than controls; the authors suggested that those differences may derive from differential effects of corticosterone elevation on the developing hippocampus (Frisone et al., 2002). Accumulating evidence suggests that stress during juvenility may markedly affect developing brain areas, leading to enduring effects on faculties related to stress coping in adulthood. Social play-behaviors deprivation during juvenility disrupted adulthood social and non-social behaviors and was associated with disregulation of the endogenous opioid systems during juvenility (Van den Berg et al., 1999a,b,c, 2000). Adult rats exposed to chronic variable stress throughout juvenility (21—32 days) exhibited an enhanced acoustic startle response, similar to patients with PTSD (Maslova et al., 2002), suggesting hippocampal and limbic system dysfunction. Avital and Richter-Levin (2005) extended these findings, demonstrating that the combination of juvenile and adulthood exposures to stress increased anxiety levels, not only in comparison with control unstressed rats but also with rats exposed to stress twice in adulthood. Recently, Tsoory and Richter-Levin (2005) showed that exposure to stress during juvenility (27—29 days) has a stronger long-term deleterious effect on learning under stressful conditions in adulthood than exposure to the same stressor during dadolescenceT (33—35 days).

Juvenile stress induces a predisposition to either anxiety or depressive-like symptoms following stress in adulthood Modeling the emergence of stress-related psychopathologies should include not only simulating the etiology, but also emulate the identification of altered behaviors. Clinically diagnosing a person as suffering from a certain stressrelated psychopathology requires identifying a group of symptoms from well-defined symptom-clusters. However, animal stress-exposures studies have routinely compared the responses of the entire stress exposed population with that of bothers/unexposedQ, although in practice the stressed animals displayed a diverse range of responses. In order to proximate animal behavior models and contemporary clinical standards Cohen et al. (2004) have suggested applying ’human diagnostic strategies’ of clustering symptoms on rats’ stress responses using the Cutoff Behavioral Criteria (CBC) based data analysis. The CBC procedure discriminates between different levels and patterns of maladaptive stress responses and thus facilitates segregating animals according to the pattern by which their stress responses were altered or disrupted. Since childhood trauma is associated with later emergence of a range of stress-related psychopathologies, particularly depression and PTSD (Heim and Nemeroff, 2001; Heim et al., 2004; Maercker et al., 2004; Nemeroff, 2004; Pynoos et al., 1999) the current study examined the consequences of juvenile stress on adulthood impaired coping with stress behaviors as indicators of anxiety and or depressive-like behaviors. The study utilized two different stress paradigms, developed individually in the two laboratories which took part in the study. We tested whether the CBC based analysis will disclose similar patterns of long-term effects of exposure to juvenile stress despite the inherent differences in the stress procedures applied at juvenility and the stress responses evaluations in adulthood. To this end two sets of experiments were conducted; juvenile rats were exposed to one of two very different brief stress exposure paradigms, predator scent or a short-term variable stress. In adulthood their coping with stress was assessed using the elevated plus maze and startle response or exploration and avoidance learning. Their responses were analyzed using the CBC procedure (Cohen et al., 2003, 2005, 2004) to distinguish between different patterns of altered stress responses that resemble anxiety and or depressive symptoms.

2. Experimental procedures 2.1. Animals Sixty-three male Sprague Dawley rats, thirty-two in experiment 1 and thirty-one in experiment 2, 24 days old on delivery weighing 35— 49 gr. supplied by Harlan Laboratories Jerusalem were maintained for the entire duration of the experiment on a 12-hr light—dark cycle with lights on at 7 a.m., room temperature 22 F 2 8C., housed four rats per cage (35X60X18 cm.) on sawdust bedding and provided with water and Solid food pellets (Teklad Global Diet 2018S, Harlan Teklad Ltd., Wisconsin, USA) ad libitum. Following the habituation to the vivarium period and the stress exposure, rats were handled periodically. All stress procedures and tests were performed during the light phase under dim illumination. Experiment 1. The effects of juvenile predator scent stress on adulthood stress responses.

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2.2. Study design Rats were randomly assigned into four stress exposure groups: 1) Juvenile Stress (JS): Exposure to predator scent (cat urine), detailed below, at juvenility (28 days); 2) Adulthood Stress (AS): Exposure to predator scent in adulthood (9 weeks); 3) Juvenile and Adulthood Stress (JS + AS): Exposure to predator scent at juvenility (28 days) and in adulthood (9 weeks); 4). Unexposed (Control): these rats were not exposed to any adverse conditions at any time. Rats’ behavior was assessed in adulthood at the age of 9 weeks using two behavioral models: the elevated plus maze (EPM) and the acoustic startle response (ASR) tests.

2.3. Stress procedure Exposing rats to a natural predator without allowing actual contact between them effectively affected stress responses mechanisms (Diamond et al., 1999; Sandi et al., 2005; Woodson et al., 2003). We utilized the predator scent to evoke activation of stress responses mechanisms. Predator scent — 10 min exposure to well-soiled cat litter (in use by the cat for 2 days) sifted for stools (adapted from, Cohen et al., 2000). Unexposed-Control animals were exposed to fresh, unused litter. Each rat was exposed individually to the stressor. Protocols were applied simultaneously to all rats in a cage, so as to not isolate any rat in its home cage. Following completion of the juvenile predator scent exposure, rats were returned to their home cage and were not handled until adulthood re-exposure and/or behavioral assessments, except for weekly cage maintenance.

2.4. Assessment of stress responses in adulthood 2.4.1. The elevated plus maze This maze consists of a plus-shaped platform with two open arms and two closed arms — surrounded by 14-cm high opaque walls on three sides, with arms of the same type located opposite each other (File, 1993). Each rat was placed on the central-platform facing an open arm and was allowed to explore the maze for 5 min. Each test was videotaped and scored by an independent observer. Arm entry was defined as entering an arm with all four paws. The following terms were used: durations in open and closed arms, and on the central-platform; open and closed arm entries; and total entries into all arms. 2.4.2. Acoustic startle response Startle response was measured using two ventilated startle chambers (SR-LAB system, San Diego Instruments, San Diego, CA). Each chamber consisted of a Plexiglas cylinder resting on a platform inside a sound-attenuated, ventilated chamber. A highfrequency loudspeaker inside the chamber produced both a continuous broadband background noise of 68 dB and the various acoustic stimuli. Movement inside the tube was detected by a piezoelectric accelerometer below the frame. Sound levels within each test chamber were measured routinely using a sound level meter (Radio Shack) to ensure consistent presentation. The SR-LAB calibration unit was used routinely to ensure consistent stabilimeter sensitivity between test chambers and over time. The animals were placed inside the tube, the startle session started with a 5-min acclimatization period, with a background noise level of 68 dB(A), which was maintained throughout the session. 30 startle stimuli (110 dB, 15 ms. duration including 0.4 ms. risedecay times); ITI: (randomly varying) 25—35 s. The entire test session was completed in 30 min. Two measures of behavior were assessed: (1) mean whole body startle response amplitude; (2) percent startle responses habituated over repeated presentation of the acoustic pulse was calculated as the percent of amplitude decrease between the mean of the first

248 five values (block 1) and the mean of the last five values out of the total 30 (block 6) amplitude values. Percent habituation = 100  [(block 1 mean startle response amplitude) (block 6 mean startle response amplitude / (block 1 mean startle response amplitude)]. Experiment 2. The effects of a juvenile short-term variable stress exposure on adulthood exploration and avoidance learning.

2.5. Study design Rats were randomly assigned into two groups: 1) Juvenile Variable Stress (JVS): juvenile rats underwent a short-term variable stress protocol at the ages of 27—29 days, as detailed below, and in adulthood at the age of 9 weeks they were exposed to the stressful challenge of learning the two-way shuttle avoidance task; 2) Avoidance Control (AVOI-C): Rats were not exposed to any adverse conditions during juvenility, and were only exposed to the stressful challenge of learning the two-way shuttle avoidance task in adulthood at the age of 9 weeks. Rats’ stress responses were assed in adulthood at the age of 9 weeks using two behavioral models: exploratory behavior in a novel setting (prior to the adulthood stressful challenge) and learning under stressful conditions in the two-way shuttle avoidance task.

2.6. Stress procedures 2.6.1. Juvenile variable stress Since variable stress protocols elicit stronger stress responses than acute or repeated stress protocols in adult rats (Garcia-Vallejo et al., 1998; Prieto et al., 2003; Simpkiss and Devine, 2003), and pre-pubertal rats exhibit an enhanced stress response compared to adults (Romeo et al., 2004), we employed at juvenility (27—29 days) a stress protocol comprised of 3 days of in tandem exposures to different stressors, all chosen for their well documented effects on adult rats (adapted from Tsoory and Richter-Levin, 2005).

! Day 1. (27 d) Forced swim: 10 min forced swim in an opaque circular water tank (diameter 0.5 m; height 0.5 m; water depth 0.4 m), water temperature 22 F 2 8C (adapted from Avital et al., 2001; Hall et al., 2001). Similar forced swim stress procedures affected adult rats’ stress responses mechanisms (Hall et al., 2001). ! Day 2. (28 d) Elevated platform: three 30 min trials; ITI: 60-min in the home cage. Elevated platform: (12  12 cm) 70 cm above floor level, located in the middle of a small closet-like room (adapted from Maroun and Richter-Levin, 2003). Similar elevated platform stress procedures affected adult rats’ stress responses mechanisms (Degroot et al., 2004; Ebner et al., 2004). ! Day 3. (29 d) Foot shock: A 3 min session of six unconditioned shock trials; US: electric foot shock (1 s, 0.8 mA); ITI: 29 s. Apparatus: a small cube-like chamber (31  31  31 cm.) with a metal grid floor connected to a computer-controlled electrical shocker device (Solid State Shocker/Scrambler, Model no. 113—33, Lehigh Valley Electronics Ltd. Lehigh Valley, PA, USA). Similar foot shock stress procedures affected adult rats’ stress responses mechanisms of (Li and Sawchenko, 1998; Passerin et al., 2000; Pezzone et al., 1993, 1992; Rassnick et al., 1998). Each rat was exposed individually to each of the stressors. Protocols were applied simultaneously to all rats in a cage, so as to not isolate any rat in its home cage. At completion of days 1 and 2 stress procedures, rats were returned to their home cage and were not handled until the next

M. Tsoory et al. day. Following completion of day 3 stress procedure, rats were returned to their home cage and were not handled until adulthood behavioral assessments, except for weekly cage maintenance. 2.6.2. Adulthood stressful challenge The two-way shuttle avoidance task, detailed below.

2.7. Assessment of stress responses in adulthood 2.7.1. Exploratory behavior in a novel setting In adulthood, at the age of 9 weeks, exploratory behavior in a novel setting was assessed in the two-way shuttle avoidance apparatus, whilst it was in an inoperative state. Rats were first placed in the apparatus, described below, before it was turned on, and were allowed to explore both compartments for 10 min. If a rat did not shuttle over to the adjacent compartment after 5 min it was manually gently directed through the passage door. The same was performed if the rat failed to shuttle back 5 min later. An observing experimenter recorded the rats’ back and forth exploratory shuttles between compartments. Only voluntary exploratory shuttles were counted. 2.7.2. Learning under stressful conditions: the two-way shuttle avoidance task Immediately following the exploratory behavior assessment, rats were trained in the two-way shuttle avoidance task in a single 100 trials session. 2.7.3. Apparatus The two-way shuttle avoidance box, placed in a dimly-lit, ventilated, sound-attenuated cupboard, is a rectangular chamber (60  26  28 cm) divided by an opaque partition with a small (10  8 cm) passage connecting two equal sized side by side cubeshaped compartments. Both compartments’ metal grid floors are weight sensitive, micro-switches transmit information about the rat’s location to a computer data collection control and program managing both CS presentations (a tone produced by speakers located on the compartments’ distal walls) and electric shocks deliveries (electrical shocker device: Solid State Shocker/Distributor, Coulborn Instruments Inc. Lehigh Valley, PA. U.S.A). 2.7.4. Procedure One session comprised of 100 btrace conditioningQ trials. CS: 10 s tone presentation; US: immediately following the termination of the CS an electric shock (0.5 mA) was delivered for a maximum of 10 s; I.T.I.: (randomly varying) 60 F 12 s. Rats could produce one of three responses: (1) Avoidance — shuttling to the adjacent compartment upon hearing the CS-tone; the tone stopped and an I.T.I. started; the rat avoided the electric shock. (2) Escape — shuttling to the adjacent compartment after the US-shock started; the shock stopped and an I.T.I. started. (3) Escape failure — failing to move to the adjacent compartment; the I.T.I. commenced at the end of the 10 s foot shock. Thus the rat was subjected to the full duration of the electric shock.

2.8. Cut-off Behavioral Criteria (CBC) data analyses In both experiments we have analyses the stress responses in adulthood in a manner that allowed the identification of distinct altered stress response patterns. This was carried in a two step procedure, detailed below (adapted from—Cohen et al., 2003, 2004). 2.8.1. Step I Prior to attempting to distinguish the differentially daffectedT subgroups, we perform a preliminary assessment of the overall response of the exposed population intended to ascertain the

Juvenile stress induces a predisposition to either anxiety or depressive-like symptoms following stress in adulthood accuracy of our zero-hypothesis, i.e. to demonstrate that exposure to the stressor did in fact have significant overall behavioral effects on the exposed rats as a group compared to controls, in each experiment. Altered behaviors following exposure to stress including extremely compromised exploratory behavior in a novel setting or on the elevated plus maze, markedly increased startle reaction that does not undergo any adaptation and impaired learning reflect anxiety-like behaviors, i.e. fearfulness and hyper-vigilance. These observed behaviors are considered to reflect relatively long-term and persistent changes in stress responses mechanisms (for review, Cohen et al., 2004). 2.8.2. Step II The bcut-off behavioral criteriaQ applied to stress exposed rats: Having established that the stressor had an effect on the rats that varied in its intensity, we have further analyzed the patterns of altered responses to form classification criteria based on integrating different levels of the stress responses measurements. Sets of classification criteria representing inclusion and exclusion diagnosis criteria produced distinct patterns of altered stress responses representing psychopathological symptom clusters. The cut off values (detailed in result section) were determined in each of the experiments for each of the parameters (detailed below) by dichotomizing the range of the stress exposed rats, thus distinguishing between rats appearing dmost affectedT or dhardly affectedT by the stress exposure (Cohen et al., 2003). The CBCs analyses allowed for discrimination between clusters of behaviors analogous to anxious or depressive states. Anxiety was indicated by increased harm avoidance behaviors like refraining from exploring unfamiliar and or open arenas, hyper vigilance to stimuli and impaired operant conditioning (for review, Holmes, 2003). Depressive-like state was indicated by immobility or reduced activity and hypo or non responsiveness to stimuli (for a review, Pryce et al., 2005). For experiment 1, the CBCs were applied on the rats’ behaviors in the EPM (parameters: time spent in open or closed arms and total exploration activity) and responses in the ASR (parameters: Mean amplitude of responses and habituation to the stimuli). The CBCs for experiment 2 were applied on rats’ exploratory shuttles in the novel setting (parameter: exploratory shuttles in the avoidance box) and responses while learning the two-way shuttle avoidance task (parameters: total avoidance responses and total escape failures). In order to maximize the accuracy of animals’ classifications and to minimize the chance of including bfalse positivesQ, each animal had to conform to both sets of criteria, inclusion and exclusion, consecutively, to be defined as daffectedT of any pattern. Conversely, in order to be considered to have responded hardly at all, animals had to conform to criteria for bunaffectedQ behaviors. The validity of the criteria were re-affirmed in each experiment by ascertaining that the vast majority of unexposed control animals conform to the dunaffectedT category and none, or almost none, to the daffectedT (Cohen et al., 2003, 2004).

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Differences between groups in the prevalence of animals classified as exhibiting anxious, depressive or unaffected behaviors were determined using v 2 Likelihood Ratio tests.

2.10. Ethical approval All procedures and tests were approved by the Institutional Animal Care Committee and adhered to the guidelines of the NIH Guide for the Care and Use of Laboratory Animals.

3. Results 3.1. Experiment 1 3.1.1. Juvenile predator scent stress alters adulthood behavioral stress response patterns Step I: ascertaining our zero-hypothesis, i.e. demonstrating that exposure to the predator scent stress has significant overall behavioral effects on the exposed rats as a group compared to unexposed rats. Fig. 1 depicts the effects of exposures to predator scent, in juvenility (JS; n = 8), adulthood (AS; n = 8) or their combination (JS + AS; n = 8), enhancing significantly anxiety as indicated by A. avoidance of open spaces and B. hyper vigilance to stimuli. Time spent in the EPM open arms was significantly decreased following exposures to cat urine scent compared to unexposed-control conditions (n = 8), [F(3,27) = 13.74; p b 0.000]. No differences were observed between any of the exposure to stress groups (Fig. 1A). Mean startle amplitude of rats in response to the 30 startle pulses was significantly increased following exposures to cat urine scent compared to control conditions (non-exposed), [F(3,28) = 8.339; p b 0.000]. No differences were observed between any of the exposure to stress groups (Fig. 1B). Percent startle responses habituated over the repeated presentation of the acoustic pulse was significantly decreased following exposures to cat urine scent compared to control conditions (non-exposed), [F(3,28) = 13.8; p b 0.000]. No differences were observed between any of the groups exposed to stress (Fig. 1C).

3.2. Step II: re-analysis of data and applying the cutoff behavioral criteria 3.2.1. Applying the CBCs Using the following criteria we were able to distinguish between rats exhibiting one of three qualitatively distinct stress responses clusters:

2.9. Statistical analysis Acoustic startle responses and the elevated plus maze behaviors, were analyzed using one-way analysis of variance (ANOVA). Where significant group effects were detected, a Bonferroni test indicated significant post-hoc differences between groups. Novel setting exploration data was analyzed using student’s ttest. Differences between groups’ responses in the two-way shuttle avoidance task were determined using a two-way ANOVA for blocks of 10 trials (repeated measure analysis), for groups and for their interaction (blocks groups) which were followed by subsequent student’s t-tests per block.

! Anxiety-like behaviors: (1) time spent in closed arms of EPM = 5 min and open-arms entries = 0; (2) exploration activity in the EPM N 8 total arms entries; (3) Mean amplitude of the startle response N800 and habituation of the ASR b 50%. ! Depressive-like behaviors: (1) t spent in closed arms of EPM = 5 min and open-arms entries = 0; (2) exploration activity in the EPM = 1 total arms entries; (3) Mean amplitude of the startle response b 600 and habituation of the ASR N 60%.

250 ! bUnaffectedQ behaviors: not conforming to the two categories above.

3.3. Juvenile predator scent stress exposure affects the prevalence rates of affected rats displaying both anxiety and depressive-like behaviors in adulthood Juvenile exposure to the predator scent significantly affected the response to it in adulthood. 87.5% of the rats exposed to predator scent both in juvenility and then reexposed to it in adulthood (JS + AS), demonstrated extreme behavioral responses, compared with 50% of those exposed to it only once either in juvenility (JS) or in adulthood (AS)

M. Tsoory et al. and none in the control-unexposed group [v 2 Likelihood Ratio (3) = 16.027, p b .001; u = .623, p b .006; r c = .623, p b .006]. Additional Pearson v 2 Likelihood Ratio test revealed that JS + AS rats differed significantly from both JS and AS rats as well as from Unexposed-Control rats also in the proportions of rats displaying either anxiety or depressive-like behaviors within each group [v 2 Likelihood Ratio (6) = 16.104, p b .013; u = .626, p b .051; r c = .443, p b .051] (Fig. 2). Both juvenile stress (JS) and adulthood stress (AS) resulted in similar prevalence rates of 50% dunaffectedT, 25% displaying anxiety-like behaviors and 25% exhibiting depressive-like behaviors. However, among rats exposed to the stressor both in juvenility and adulthood (JS + AS) significantly higher prevalence were detected. 50% displayed anxiety-like behaviors and 37.5% exhibited depressive-like behaviors, while only 12.5% were categorized as dunaffectedT. Within the Unexposed-Control group all rats were categorized as unaffected, no rat displayed anxiety-like or depressive-like behaviors.

3.4. Experiment 2 3.4.1. Juvenile stress affects adulthood exploration and avoidance learning Step I: ascertaining our zero-hypothesis, i.e. demonstrating that juvenile exposure to the short-term variable stress procedure (JVS) did in fact have significant overall behavioral effect compared with unexposed-control conditions (AVOI-C). 3.4.2. Exploration of a novel setting Juvenile exposure to stress has significantly reduced adulthood exploratory behavior prior to the stressful challenge. Compared with AVOI-C (13.50 F 1.07; n = 8), adult juvenile stressed rats (JVS) exhibited significantly less exploratory shuttles in a novel setting (0 F 0; n = 12), namely the shuttle box prior to avoidance learning [t(7) = 12.63; p b .000] (Fig. 3A).

Figure 1 The effects of predator scent exposures on elevated plus maze exploration and startle responses: Predator scent exposures in juvenility (JS; n = 8), adulthood (AS; n = 8) or their combination (JS + AS; n = 8) significantly affected stress responses. In comparison with unexposed-controls (n = 8), predator scent exposures significantly decreased time spent in the EPM open arms (A) [F(3,27) = 13.74; p b 0.000], significantly increased mean startle response amplitude (B) [F(3,28) = 8.339; p b 0.000], and significantly decreased the habituation to the repeated presentation of the acoustic pulse (C) [F(3,28) = 13.8; p b 0.000]. Bonferroni post-hoc test indicated that UnexposedControl rats spent significantly more time in the EPM open arms ( p b 0.00; **), had a significant lower mean startle response amplitude ( p b 0.00; **) and exhibited a greater significant habituation to the repeated acoustic pulse from all stress exposed rats. No differences were observed between any of the stress exposure groups in time spent in the EPM open arms (A), mean startle response amplitude (B) or habituation to the startling acoustic pulse (C).

Juvenile stress induces a predisposition to either anxiety or depressive-like symptoms following stress in adulthood

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(17.00 F 4.55) performed significantly less avoidance shuttles than AVOI-C rats (57.93 F 4.79) [t(32) = 6.141; p b .000] (Fig. 3B). !Escape failures JVS rats failed to escape the US in significantly more trials than AVOI-C while learning the two-way shuttle avoidance task. Two-way ANOVA with repeated measure

Figure 2 The effects of predator scent exposures on prevalence rates of rats exhibiting altered stress responses profiles in adulthood: Juvenile predator scent exposure significantly enhanced the response to it in adulthood as indicated by the proportion of stress affected animals [v 2 Likelihood Ratio (3) = 16.027, p b .001]. 87.5% of JS + AS rats demonstrated extreme behavioral responses, compared with 50% in the JS or AS groups and none in the Unexposed-Control group. Juvenile predator scent exposure significantly increased the prevalence rates of rats exhibiting an enhanced stress response of either the anxious or depressive-like behavioral profiles [v 2 Likelihood Ratio (6) = 16.104, p b .013]. Among JS + AS rats 50% displayed anxiety-like behaviors and 37.5% exhibited depressive-like behaviors, while only 12.5% were dunaffectedT. Whereas among both JS and AS rats, 25% displayed anxiety-like behaviors, 25% exhibited depressive-like behaviors and 50% were dunaffectedT. Within the Unexposed-Control group no rat displayed anxietylike or depressive-like behaviors.

3.4.3. Two-way shuttle avoidance learning in adulthood Repeated measure analyses of the avoidance, escape and escape failure responses while learning the two-way shuttle avoidance task indicated that exposure to juvenile stress has significantly impaired adulthood coping with learning under stressful conditions, i.e. the two-way shuttle avoidance task. As detailed below, compared to Avoi-C (n = 15) rats, JVS rats (n = 18) performed significantly less avoidance shuttles and presented significantly higher rates of escape failures while learning the two-way shuttle avoidance task. No differences were evident between JVS and AVOI-C rats in escape responses. !Avoidance shuttles JVS rats performed significantly less avoidance shuttles than AVOI-C rats while learning the two-way shuttle avoidance task. Two-way ANOVA with repeated measure analysis for avoidance shuttles per block of 10 trials revealed significant main effects for blocks [F(4,116) = 35.14; p b .000], for groups [F(1,31) = 47.22; p b .000] and for the interaction groups  blocks [F(4,116) = 10.19; p b .000]. Subsequent Student t-test per block showed that JVS rats performed significantly fewer avoidance shuttles than the Avoi-C rats in blocks 1 and 3—10. Similarly, the groups differed significantly in the total number of avoidance shuttles, Student t- test revealed that JVS rats

Figure 3 The effects of a juvenile short-term variable stress exposure on adulthood novel setting exploration and two-way shuttle avoidance learning: Juvenile exposure to the short-term variable stress procedure (JVS) significantly impaired stress coping in adulthood. In comparison with unexposed control rats (Avoi-C; n = 15), JVS rats (n = 18) exhibited significantly less exploratory shuttles in a novel setting prior to avoidance learning (A) [t(7) = 12.63; p b .000; **]; performed significantly fewer avoidance shuttles (B) [t(32) = 6.141; p b .000; **] and failed to escape the US significantly more (C) [t(32) = 4.367; p b .000; **].

252 analysis for escape failures per block of 10 trials found no effects for blocks [F(4,115) = 1.05; n.s.] or for the interaction groups  blocks [F(4,115) = 0.92; n.s.], however did reveal a significant main effect for groups [F(1,31) = 15.96; p b .000]. Student t-tests per block showed that JVS rats failed to escape the US significantly more than AVOI-C rats in all blocks (1—10). Similarly, the groups differed significantly in the total number of escape failures, Student t-test revealed that JVS rats (39.42 F 8.98) failed to escape the US significantly more than AVOI-C rats (.20 F .11), [t(32) = 4.367; p b .000]. (Fig. 3C). !Escape shuttles NO significant differences were observed between the groups in escape shuttles while learning the two-way shuttle avoidance task. Two-way ANOVA with Repeated measure analysis for escape shuttles per block of 10 trials revealed a significant main effect for blocks [F(5,147) = 23.53; p b .000] but not for groups [F(1,31) = .069; n.s.]. The significant interaction between groups and blocks [F(5,147) = 8.94; p b .000] indicates that though in both AVOI-C and JVS groups a decline in the number of escape responses was evident while learning the two-way shuttle avoidance task in the AVOI-C group this decline was more pronounced than in the JVS group. These groups however did not differ significantly in the total number of escape shuttles: AVOI-C (41.93 F 4.72); JVS (43.68 F 6.73); [t(31) = .213; n.s.].

M. Tsoory et al.

3.5. Step II: re-analysis of data and applying the cutoff behavioral criteria 3.5.1. Applying the CBCs Using the following criteria we were able to distinguish between rats exhibiting one of three qualitatively distinct stress responses clusters: ! Anxiety-like behaviors: (1) Not exploring the novel setting; (2) total avoidance shuttles b 41%; (3) total escape failures b 60%. ! Depressive-like behaviors: (1) Not exploring the novel setting; (2) total avoidance shuttles b 41%; (3) total escape failures N 60%. ! bUnaffectedQ behaviors: not conforming to the two categories above.

3.6. Juvenile variable stress affects the prevalence rates of affected rats displaying both anxiety and depressive-like behaviors in adulthood Pearson v 2 Likelihood Ratio test revealed that JVS exposure significantly increased the proportion of bAffectedQ rats [v 2 Likelihood Ratio (1) = 10.221, p b .001; u = .533, p b .002; r c = .533, p b .002]. 74% of the JVS rats displayed significantly altered stress responses in adulthood compared to only 20% among the AVOI-C group. Additional Pearson v 2 Likelihood Ratio test revealed that JVS and AVOI-C rats differed significantly in the proportion of rats displaying bUnaffectedQ, anxiety-like or depressive-like behaviors within each group [v 2 Likelihood Ratio (2) = 13.848, p b .001; u = .573, p b .004; r c = .573, p b .004] (Fig. 4). Within the AVOI-C group 80% were categorized as bUnaffectedQ and the remaining 20% anxious. None of the AVOI-C rats was categorized as depressed. However, within the JVS group only 26% were categorized as bUnaffectedQ. The remaining 74% ’affected’ split evenly between 37% displaying anxiety-like behaviors and 37% displaying depressive-like behaviors.

4. Discussion

Figure 4 The effects of a juvenile short-term variable stress exposure on prevalence rates of rats exhibiting altered stress responses profiles in adulthood: Juvenile exposure to the shortterm variable stress procedure (JVS) significantly increased the proportion of bAffectedQ rats [v 2 Likelihood Ratio (1) = 10.221, p b .001]. 74% of the JVS rats displayed significantly altered stress responses in adulthood compared to only 20% in the AVOIC group. Juvenile exposure to the short-term variable stress procedure (JVS) significantly increased the prevalence rate of rats exhibiting an enhanced stress response of either the anxious or depressive-like behavioral profiles [v 2 Likelihood Ratio(2) = 13.848, p b .001]. Within the JVS group, 37% displayed anxiety-like behaviors; 37% displayed depressive-like behaviors and only 26% were bUnaffectedQ. Whereas within the AVOI-C group, only 20% displayed anxiety-like behaviors, no rat displayed depressive-like behaviors and 80% were dUnaffectedT.

In humans, significant early-life stress and childhood emotional trauma represent major predisposing factors for the emergence of a wide range of stress-related psychopathologies, particularly PTSD and depression (Heim et al., 2003, 2004), both of which have been associated with altered neuronal activity in neurocircuits critically involved in modulating stress responses, emotional processing, and learning (Arborelius et al., 1999). Our results indicate that exposure to stress during juvenility, around the age of 28 days in the rat, induces similar long-term altered stress responses. It is thus a feasible animal model for the induction of a similar predisposition for stress-related psychopathologies. Furthermore, when the stress responses were analyzed using individual cut-off behavioral criteria, two subpopulations could be identified among adult juvenile stressed rats in both experiments. The majority of the rats exposed to stress as juveniles were more prone to respond excessively to adulthood stress. Most commonly they exhibited a behavioral pattern reflecting

Juvenile stress induces a predisposition to either anxiety or depressive-like symptoms following stress in adulthood characteristics of anxiety-like disorder, while about a third displayed a behavioral pattern that corresponded with depressive symptoms. This variance in altered stress-response found in both experiments among adult juvenile stressed rats corresponds with the spectrum of stress-related psychopathologies found in humans. Clinical studies indicate that following exposure to traumatic events, several types of psychopathological responses are observed including acute stress disorder (ASD) and PTSD, depression, a variety of anxiety disorders, somatization symptoms and syndromes, eating disorders, pain syndromes and chronic fatigue syndrome (Cohen et al., 2003, 2004; Moreau and Zisook, 2002). The fact that in both experiments a substantial proportion of the adult juvenile stressed rats exhibited behaviors suggestive of anxiety or depression following short-term and rather mild juvenile stress protocols implies that, as in humans, the developmental stage between weaning and puberty, which we termed the bjuvenileQ age, indeed represents a stress-sensitive period, during which significant adversities may permanently alter stress response mechanisms. In support of this notion a previous study demonstrated that a similar juvenile and adulthood stress exposure was indeed more effective than exposure to the same stressors twice in adulthood (Avital and Richter-Levin, 2005). Childhood, represented here by pre-pubescent weaned rats, is a significant developmental stage in rodents, monkeys and humans (Spear, 2000). During this period maturational changes occur in several key brain areas such as the prefrontal cortex, hippocampus and amygdala neurocircuits that mediate age-specific neurobehavioral and physiological characteristics (Spear, 2000, 2004). In humans, the juvenile and early adolescent periods are normatively recognized as stressful and challenging stages, in which the overwhelming maturational changes may lead to significant levels of stress (Petersen et al., 1996). The ontogenetic alterations in brain function occurring during early youth have been suggested to play an important role in the increasing prevalence of stressrelated psychopathologies during the adolescent period (Bogerts, 1989; Compas et al., 1993; Lipska and Weinberger, 1993; Petersen et al., 1993; Spear, 2000). The juvenile rat brain produce longer lasting stress responses (Romeo et al., 2004) and also undergoes substantial changes in neural mechanisms regulating emotional and learning processing within the prefrontal cortex (Genazzani et al., 1997; Insel et al., 1990; Jernigan et al., 1991; Kalsbeek et al., 1988; Sowell et al., 1999; Van Eden et al., 1990), the hippocampal formation (Insel et al., 1990; Michelson and Lothman, 1989; Nurse and Lacaille, 1999) and the amygdala (Kellogg, 1998; Terasawa and Timiras, 1968). These changes have been suggested to contribute significantly to the susceptibility of the juvenile rat to short-term stress-induced impairments (Einon and Morgan, 1977; McGivern et al., 1996; Spear, 2000, 2004) and therefore these brain structures should be considered as plausible candidates for the long-term deleterious effects of juvenile exposure to stress. Our results correspond with previous findings indicating long-term effects of exposing rats to stress during

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juvenility (Avital et al., 2006; Avital and Richter-Levin, 2005; Maslova et al., 2002; Tsoory and Richter-Levin, 2005), suggesting that, as in humans, the juvenile age in the rat is a stress sensitive period in which even rather short-term exposures to stress produce lasting behavioral disruptions reminiscing both anxiety and depressive symptoms. One must take care not to be too literal in interpreting animal models. They are not to be taken to accurately reflect the human disorder, but merely to approximate certain aspects of it. PTSD and depression involve much more than a physiological imbalance or changes in behavior, aspects which this animal model is unable to approximate. It would be presumptuous to assume that the bcriteriaQ applied in this study in fact reflect behavioral— physiological parameters in the life of the rat which are commensurate with the criteria for PTSD or depression in humans. They do, however, enable to be focused on the distinction between brespondersQ and bnon-respondersQ. Despite the limitations, this study suggests that the presented juvenile stress model combined with the CBCbased analyses of the augmented stress responses in adulthood may be used to model in rats certain facets of the stress-related disorders that emerge in humans who have suffered early-life trauma. It may thus facilitate the investigation of the relevant underlying neural mechanisms associated with both factors related to vulnerability and to resilience in each of the different psychopathologies.

Acknowledgements This project was supported by a 2002 NARSAD Independent Investigator award to G. R-L.

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