Traumatic stress reactivity promotes excessive alcohol drinking and alters the balance of prefrontal cortex-amygdala activity

Abstract

Post-traumatic stress disorder (PTSD) and alcoholism are highly comorbid in humans and have partially overlapping symptomatic profiles. The aim of these studies was to examine the effects of traumatic stress (and stress reactivity) on alcohol-related behaviors and neuronal activation patterns. Male Wistar rats were trained to respond for alcohol, were exposed to predator odor (bobcat urine) paired with context and were tested for short- and long-term avoidance of the predator odor-paired context, alcohol self-administration and compulsivity of alcohol responding. Rats were re-exposed to the odor-paired context for western blot analysis of ERK phosphorylation in subregions of the medial prefrontal cortex (mPFC) and the amygdala. Rats that avoided the predator-paired chamber (Avoiders) exhibited persistent avoidance up to 6 weeks post conditioning. Avoiders exhibited increases in operant alcohol responding over weeks, as well as more compulsive-like responding for alcohol adulterated with quinine. Following re-exposure to the predator odor-paired context, Avoiders and Non-Avoiders exhibited unique patterns of neuronal activation in subregions of the mPFC and the amygdala, which were correlated with changes in avoidance and alcohol drinking. Furthermore, activity of upstream regions was differentially predictive of downstream regional activity in the Avoiders versus Non-Avoiders. An animal model for assessing the effect of traumatic stress on alcohol drinking reveals individual differences in neuronal activation patterns associated with re-exposure to traumatic stress-related stimuli, and may provide insight into the neural mechanisms underlying excessive alcohol consumption in humans with PTSD.

OPEN

Citation: Transl Psychiatry (2013) 3, e296; doi:10.1038/tp.2013.70 & 2013 Macmillan Publishers Limited All rights reserved 2158-3188/13

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ORIGINAL ARTICLE

Traumatic stress reactivity promotes excessive alcohol drinking and alters the balance of prefrontal cortex-amygdala activity

S Edwards1, BB Baynes2, CY Carmichael3, ER Zamora-Martinez1, M Barrus4, GF Koob1 and NW Gilpin2,5

Post-traumatic stress disorder (PTSD) and alcoholism are highly comorbid in humans and have partially overlapping symptomatic proles. The aim of these studies was to examine the effects of traumatic stress (and stress reactivity) on alcohol-related behaviors and neuronal activation patterns. Male Wistar rats were trained to respond for alcohol, were exposed to predator odor (bobcat urine) paired with context and were tested for short- and long-term avoidance of the predator odor-paired context, alcohol self-administration and compulsivity of alcohol responding. Rats were re-exposed to the odor-paired context for western blot analysis of ERK phosphorylation in subregions of the medial prefrontal cortex (mPFC) and the amygdala. Rats that avoided the predator-paired chamber (Avoiders) exhibited persistent avoidance up to 6 weeks post conditioning. Avoiders exhibited increases in operant alcohol responding over weeks, as well as more compulsive-like responding for alcohol adulterated with quinine. Following re-exposure to the predator odor-paired context, Avoiders and Non-Avoiders exhibited unique patterns of neuronal activation in subregions of the mPFC and the amygdala, which were correlated with changes in avoidance and alcohol drinking. Furthermore, activity of upstream regions was differentially predictive of downstream regional activity in the Avoiders versus Non-Avoiders. An animal model for assessing the effect of traumatic stress on alcohol drinking reveals individual differences in neuronal activation patterns associated with re-exposure to traumatic stress-related stimuli, and may provide insight into the neural mechanisms underlying excessive alcohol consumption in humans with PTSD.

Translational Psychiatry (2013) 3, e296; doi:http://dx.doi.org/10.1038/tp.2013.70

Web End =10.1038/tp.2013.70 ; published online 27 August 2013

Keywords: alcohol abuse; avoidance; escalation; individual differences; PTSD

INTRODUCTIONPost-traumatic stress disorder (PTSD) is a debilitating disorder dened by the DSM-IV in terms of three major symptom clusters: persistent avoidance of trauma-related stimuli, hyperarousal and intrusive re-experiencing of the traumatic event.1 Notably, PTSD occurs only in a subset of humans exposed to a traumatic stressor, estimated to be between 10 and 25% of individuals, depending on the stress stimulus and the criteria used to assess PTSD (for example, Breslaun et al.2). Important for the present report, PTSD and alcohol-use disorders are highly comorbid in humans and have partially overlapping symptomatic proles (for example, Engdahl et al.3).

PTSD has been described in terms of co-occurring maladaptive fear conditioning processes (that is, excessive fear learning or decient fear extinction) and sensitized stress responses,4 which are mediated by changes in partially overlapping neural circuits (for example, Pape and Pare,5 and Shin and Liberzoni6). Studies attempt to model these pathological neural cascades by exposing animals to a stimulus that mimics traumatic stress exposure in humans (for reviews, see Cohen et al.7 and Logrip et al.8), although different stressors (for example, social defeat, footshock and predator odor) elicit different sets of behavioral and biological responses in rats.9 Animals can be divided into subgroups based on behavioral responses to the stressor, as long as the behavioral response is not maximal or uniform across rats. Predator odor stress allows for the division of animals into subgroups that exhibit

either multiple co-occurring signs of behavioral dysregulation (for example, hyperarousal and high anxiety) or collective absence of these signs.10 This clustering of behavioral changes suggests a common underlying etiology for multiple behavioral counter-adaptations in animals that do not cope well with a traumatic stressor and in humans with PTSD.11

Humans with pre-existing PTSD are more likely to develop alcohol dependence than non-PTSD controls.12 PTSD and alcohol dependence are both dened by negative affective states (for example, hyperarousal and dysphoria) that result from maladaptive physiological responses to environmental stimuli.13,14 Further

more, an accumulation of evidence points to dysregulation of overlapping neural circuits following exposure to traumatic stress5 and the transition to alcohol dependence.15,16 The high rate of

comorbidity and similar symptomatology of these two disorders in humans, as well as the recruitment of brain stress systems in both disorders, support a self-medication hypothesis in which affected individuals may consume excessive amounts of alcohol to ameliorate PTSD symptoms. Importantly, past studies have not explicitly assessed alcohol- and drug-taking behaviors in rodents that do and do not exhibit a PTSD-like state following exposure to a traumatic stressor.8

Animal models of PTSD-like behavioral symptoms can be used to dissect neural perturbations produced by maladaptive responses to traumatic events.17 In this study, we assessed neuronal signaling patterns by measuring ERK phosphorylation

1Committee on the Neurobiology of Addictive Disorders, The Scripps Research Institute, La Jolla, CA, USA; 2Department of Physiology, Louisiana State University Health Sciences Center, New Orleans, LA, USA; 3Department of Pharmacology, Boston University, Boston, MA, USA; 4Department of Psychology, University of British Columbia, Vancouver, BC, Canada and 5Department of Neuroscience, Louisiana State University Health Sciences Center, New Orleans, LA, USA. Correspondence: Dr NW Gilpin, Department of Physiology Louisiana State University Health Sciences Center, 1901 Perdido Street, New Orleans, LA 70112, USA.

E-mail: mailto:[email protected]

Web End [email protected] Received 24 April 2013; revised 5 June 2013; accepted 15 July 2013

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following exposure to a context previously paired with traumatic stress, mimicking symptom provocation studies in human PTSD patients. The ERK pathway is an intracellular signal transduction cascade that regulates synaptic plasticity in several limbic brain regions via its ability to relay extracellular signals into nuclear responses,18 and ERK phosphorylation has been widely used as a measure of stimulus-driven neuronal activation.19,20

Here, we demonstrate that rats exhibiting persistent high avoidance of trauma-related stimuli also exhibit long-lasting post-stress increases in alcohol drinking, that ERK phosphorylation patterns are predictive of behavioral dysregulation in individual rats, and that high-avoidance, high-drinking rats exhibit altered neuronal activation patterns in the prefrontal cortex and amygdala, as well as a potential modication of connectivity between these regions.

MATERIALS AND METHODSEthics statementAll experiments were conducted in accordance with the institutional (LSUHSC, IACUC 2908; TSRI, IACUC 08-0015) and NIH guidelines.

General methodsSubjects. Adult male Wistar rats were used in all experiments. All experiments were performed during the dark period (08002000 h) of a 12 h:12 h cycle. In all cases, rats were experimentally naive at the start of experiments, and were habituated to housing conditions and experimenter handling for X1 week before the start of experimental manipulations.

Behavioral proceduresAlcohol self-administration (Experiments 1 and 2): Rats were trained to respond for alcohol (10% w/v) and water in a concurrent, two-lever, free-choice operant situation. Volumes of 0.1 ml each of alcohol and water were delivered on a xed ratio-1 schedule. Before the start of operant training, rats were given access to 10% w/v alcohol and water in the home cage for a single 24-h period. Intakes were not recorded and the purpose of this access was to avoid neophobic response to alcohol in operant chambers. On the rst day of operant training, rats were given 12 h access to a single lever (right lever) that produced 0.1 ml deliveries of water on an xed ratio-1 schedule with ad libitum food available on the oor of the operant chamber. On the second day of operant training, rats were given 2 h access to two levers that produced 0.1 ml deliveries of alcohol versus water on xed ratio-1 schedules. During the second session and all subsequent sessions going forward, alcohol was paired with the right lever and water was paired with the left lever. On the third day of operant training, rats were given 1 h access to the two-lever contingency, and starting on the fourth day of training and for all subsequent sessions going forward rats were given 30 min access to the two-lever alcohol versus water contingency. This procedure allows rats to learn to press for alcohol in an operant situation without the use of sweeteners or uid deprivation. Rats were allowed B15 sessions (see design of each experiment below) of operant responding for alcohol versus water until operant responding was stable and reliable for these rats, at which point they were divided into two groups matched for intakes across the nal 5 days of this baseline period: rats to be exposed to traumatic (predator odor) stress and rats not to be exposed.

Aversive contextual conditioning (Experiments 13): Rats were exposed to predator odor versus no odor (or no odor vs no odor for controls) in two separate contexts that differed on both visual (wall patterns) and tactile (oor grids) cues following stabilization of operant alcohol responding in Experiments 1 and 2. On the rst day of this procedure, rats were allowed 5 min of free exploration of the conditioning apparatus, which consisted of two conditioning chambers (36 cm length 30 cm width 34 cm height)

connected by a small triangular compartment. The time that rats spent in the two conditioning chambers was recorded as the pre-conditioning baseline. The details of pre-conditioning baseline compartment times and assignment of unconditioned stimuli to compartments is detailed below in the Experiment-Specic Procedures section for Experiments 13.

On the day following the pre-conditioning preference test, rats were placed in one of the two contexts for 15 min in the absence of odor, then

they were returned to the home cage. On the third day of the procedure, rats were placed in the other context for 15 min and a sponge soaked in bobcat urine (or no odor for controls) was placed in the room. On the fourth and nal day of this procedure (24 h post-odor exposure), rats were again allowed 5 min of free exploration of the conditioning apparatus. For all experiments, rats in the control group were never exposed to bobcat urine for the duration of the experiment.

Avoidance of predator-paired context was quantied as post-conditioning time in predator context minus pre-conditioning time in predator context. Rats that exhibited 410 s decrease in time spent in predator-paired chamber following conditioning (relative to baseline) were termed highly reactive and will be called Avoiders from here on in this study, whereas rats that exhibited o10 s decrease in time spent in predator-paired chamber following conditioning (relative to baseline) will be called Non-Avoiders. This group division criterion (4oro10-s decrease) was chosen, because in our experiments not many animals exhibited avoidance scores approximately equal to 10 s (providing an unambig

uous distinction between groups). In addition, we dene avoidance as the presence of a behavior and non-avoidance as the absence of that behavior (rather than the presence of another behavior); therefore, we decided not to classify animals that exhibited very small reductions (o10 s) in time spent in predator odor-paired chamber as Avoiders.

Finally, we intentionally chose a criterion for groups that represented a data point that would remain constant across experiments (that is, time spent in predator odor-paired context) rather than a cohort-specic criterion (for example, median split) that was likely to change across experiments. This division criterion is at least partly validated by its predictive value for subsequent changes in brain and behavior, as described in the Results section. The percentages of animals designated as Avoiders versus Non-Avoiders are detailed below in the Experiment-Specic Procedures section for Experiments 13.

We modeled traumatic stress in humans by exposing rats to predator odor for the following reasons: (1) the effects of other more commonly used stressors (for example, restraint and footshock) on alcohol drinking are transient, variable and inconsistent across studies and time,8,21

(2) to avoid using a stressor that administers physical pain to the rat and(3) the perceived presence of a predator has powerful long-lasting behavioral and biological effects across species.22 Predator stress models also lend themselves particularly well to the detection of individual differences,7 a critical component of animal models of the PTSD symptomatology.

Appetitive contextual conditioning (Experiment 3): In this procedure, rats were allowed to consume a sweet solution (supersac, 3% glucose, 0.125% saccharin;23) or water on alternating days in two separate contexts that differed on both visual (wall patterns) and tactile (oor grids) cues. On the rst day of this procedure, rats were allowed 5 min of free exploration of the conditioning apparatus, which consisted of two conditioning chambers connected by a small triangular compartment. The time that rats spent in the two compartments was recorded as the pre-conditioning baseline, and rats were assigned supersac solution in one compartment and water solution in the other compartment. On the day following the preconditioning preference test, rats were placed in one of the two contexts for 15 min and were allowed to drink water ad libitum from a graduated bottle, then they were returned to the home cage. On the third day of the procedure, rats were placed in the other context for 15 min and were allowed to drink supersac ad libitum from a graduated bottle, then they were returned to the home cage. The procedure for days 2 and 3 was repeated twice more (days 47), such that rats received water-context pairings for 3 total days and supersac context pairings for 3 total days. On day 8, rats were allowed 5 min of free exploration of the conditioning apparatus. Preference for supersac-paired context was quantied as postconditioning time in supersac context minus pre-conditioning time in supersac context.

Compulsivity of alcohol self-administration (Experiment 1): Rats that had previously been trained to self-administer alcohol were allowed to respond for 10% w/v alcohol adulterated with gradually increasing concentrations of quinine, as previously described.24 Briey, rats were allowed to respond for ethanol/quinine solutions versus water during a 30-min operant self-administration over 7 consecutive days. On the rst day of this procedure, 0% quinine was added to the ethanol solution, and quinine concentrations on following days were 0.0005, 0.001, 0.0025, 0.005, 0.01 and 0.025% quinine. Operant response data are presented as percent change from baseline (10% w/v ethanol 0% quinine) for each rat.

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Elevated plus-maze (Experiment 3): The elevated plus-maze (EPM) test was used to test anxiety-like behavior in conditions previously described.25

Briey, the maze was made of black Plexiglas and consisted of four arms (50 cm long 10 cm wide); two closed arms had 40-cm high, dark walls

and two open arms had no walls. The height of the apparatus was 50 cm from the oor. During testing, open arms received 22350 lux and closed arms received o1 lux of illumination. These lighting conditions produce %

open arm times of 1520% in our lab, consistent with the literature for EPM results observed in genetically heterogeneous rats (in results reported below, mean % open arm time is approximately equal to 14%). Individual rats were placed in the center of the maze facing a closed arm and were removed after 5 min. Behavior was recorded by a digital camera and was scored by an experimenter blind to treatment conditions. The apparatus was cleaned between subjects. Primary measures were percent open arm time (open/open closed) and number of closed-arm entries (dened as

all four paws entering).

Western blot analysis. Individual ERK phosphorylation levels in the brain regional homogenates were determined as described in Edwards et al.20

Immediately upon completion of the nal 15-min context exposure (see below), animals were killed under light isourane anesthesia by decapitation. Brains were rapidly dissected and snap frozen in isopentane. Regional tissue samples were obtained using 1216 gauge punches from frozen coronal brain slices (0.5 mm thick) obtained by the use of a cryostat. Tissue samples were homogenized by sonication in lysis buffer (320 mM sucrose, 5 mM

HEPES, 1 mM EGTA, 1 mM EDTA and 1% SDS, with protease inhibitor cocktail and phosphatase inhibitor cocktails II and III diluted 1:100; Sigma, St Louis, MO, USA), heated at 100 1C for 5 min and were stored at 80 1C until the

determination of protein concentration by a detergent-compatible Lowry method (Bio-Rad, Hercules, CA, USA). Samples of protein (15 mg) were subjected to SDS-polyacrylamide gel electrophoresis on 10% acrylamide gels by using a Tris/Glycine/SDS buffer system (Bio-Rad), followed by electro-phoretic transfer to polyvinylidene diuoride membranes (GE Healthcare, Piscataway, NJ, USA). Membranes were blocked overnight in 5% non-fat milk at 4 1C and were then incubated in primary antibody recognizing the dual phosphorylated form of ERK (1:1000, 5% non-fat milk; Cell Signaling, Danvers, MA, USA). Membranes were washed and labeled with species-specic peroxidase-conjugated secondary antibody (1:10 K; Bio-Rad) for 1 h at room temperature. Following chemiluminescence detection (SuperSignal West Pico; Thermo Scientic, Rockford, IL, USA), blots were stripped for 20 min at room temperature (Restore; Thermo Scientic) and were reprobed for total protein levels of ERK (1:10 K; Cell Signaling). Immunoreactivity was quantied by densitometry (ImageJ 1.45S; NIH) under linear exposure conditions. Separate gel sets composed of control and experimental (Avoider and Non-Avoider) animals were processed in parallel for each region. Densitized values were expressed as a percentage of the mean of control values for each gel to normalize data across blots. Individual phosphoprotein levels were normalized to individual total protein levels to generate ERK phosphorylation (pERK)/ERK ratio values for statistical comparison.

Experiment-specic proceduresExperiment 1. The purpose of Experiment 1 was three-fold: (1) to assess alcohol drinking and compulsivity of alcohol drinking in rats exposed to predator odor stress, (2) to assess the persistence of avoidance behavior and the predictive value of avoidance for subsequent changes in alcohol drinking, and (3) to generate tissue from these groups of rats to assess neural correlates of behavioral changes.

Rats (n 48) were trained to self-administer 10% w/v ethanol versus

water in a two-lever operant situation during 30-min self-administration sessions, as described above, for a period of 18 days. Rats then underwent the 4-day aversive contextual conditioning procedure described above. For rats in the experimental group, predator odor exposure was assigned to compartments in a biased design (baseline time for predator odor-paired chamber 145 s and baseline time for neutral chamber 94 s). Following

the 4-day aversive conditioning procedure, rats were once again allowed to self-administer 10% w/v ethanol versus water on days 2, 5, 8 and 11 following exposure to predator odor (or no odor in controls). Six weeks following exposure to predator odor, rats were again tested for avoidance of the predator-paired chamber. Finally, rats were tested for compulsivity of alcohol responding using the quinine adulteration procedure described above. At the end of behavioral experiments, all rats were left in the home cage undisturbed for a period of 1 week, at which point they were given one nal 15-min exposure to the predator-paired context (neutral context

in unstressed controls) and were killed, and the brains were removed for western blot analysis.

Experiment 2. The purpose of Experiment 2 was to conrm Experiment 1 ndings of post-stress escalation of alcohol drinking in Avoider rats, and also to test animals for a longer period of time (19 days instead of 11 days) to assess the persistence of this effect. Rats (n 29) were trained

to self-administer 10% w/v ethanol versus water in a two-lever operant situation during 30-min self-administration sessions, as described above, for a period of 18 days. Rats then underwent the 4-day aversive contextual conditioning procedure described above. To conrm that the Experiment 1 results were not due to the biased conditioning design, rats in the experimental group were assigned predator odor exposure in an unbiased design such that pre-conditioning preference for predator and neutral contexts was counterbalanced (baseline times 133 vs 136 s in the two

contexts subsequently paired with predator odor or no odor, respectively). Following the 4-day aversive conditioning procedure, rats were once again allowed to self-administer 10% w/v ethanol versus water on days 2, 8, 12, 15 and 19 following exposure to predator odor (or no odor in controls).

Experiment 3. The purpose of Experiment 3 was to determine whether innate anxiety-like behavior or the ability to classically condition an appetitive stimulus (to assess learning ability of subsequent Avoider and Non-Avoider groups) was predictive of subsequent avoidance behavior. One week following vivarium arrival, rats (n 20) were tested for innate

anxiety-like behavior on the EPM, as described above. Four days following the EPM test, rats were allowed to explore an apparatus with three chambers that differ in the visual (wall pattern) and tactile (oor composition) cues. For each rat, the three compartments were assigned one of the three unconditioned stimuli: supersac solution, predator odor and water solution/no odor (that is, neutral context). Using two of these three compartments, rats underwent the 8-day appetitive contextual conditioning procedure as described above. Then, 3 days following the post-conditioning preference test for the supersac-paired context, rats underwent the 4-day aversive contextual conditioning procedure described above (using the same neutral context and the third previously unused compartment for predator odor exposure). Rats in this experiment were not divided into Avoider and Non-Avoider groups; instead, the variability of conditioned preference and aversion times were analyzed to assess whether predator odor-paired context aversion might be attributable to the rats ability to classically condition contextual pairings with unconditioned stimuli. To facilitate conditioning and the variability of conditioning, a biased design was used for this experiment (in a three-chamber pre-test, baseline times 122 s for predator odor-paired cham

ber, 95 s for neutral chamber and 62 s for supersac-paired chamber).

Statistical analysis. Data are reported as means.e.m. Data were analyzed with one-way, two-way and three-way analysis of variance (ANOVA) as described in text, and signicant group differences were probed with a StudentNewmanKeuls or Bonferroni post-hoc analysis. Pearsons correlations were used to determine relationships between behavioral and molecular outcomes. In all cases, statistical signicance was set at the Po0.05 level. Where appropriate, data in gures are presented as difference scores relative to baseline or percent change from baseline.

RESULTSTraumatic stress produces persistent avoidance in some but not all rats

Figure 1a shows that Avoiders exhibited signicantly greater avoidance of the predator-paired chamber at 24 h and 6 weeks post-predator odor exposure, F(1,35) 29.49, Po0.001, relative to

Non-Avoiders. Avoidance of the predator odor-paired chamber at 24 h was positively and signicantly correlated with the avoidance of the same chamber 6 weeks later, r(35) 0.37, P 0.025. In

animals trained to self-administer alcohol and then exposed to the 4-day aversive conditioning procedure, 59% of rats (61% in Experiment 1; 55% in Experiment 2) were designated as Avoiders according to the criteria outlined above. It is worth noting that the proportion of animals that met Avoider criteria was similar in the two experiments even though Experiment 1 utilized a biased conditioning design and Experiment 2 utilized an unbiased conditioning design.

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Avoiders Non-Avoiders

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Figure 1. (a) Mean (s.e.m.) change in time (s) spent in predator-paired context from pre-conditioning test to post-conditioning test, quantied as post-test score minus pre-test score. Rats (Experiment 1) were divided into Avoiders (n 23) and Non-Avoiders (n 14) based on avoidance of

predator-paired chamber at 24 h post conditioning, and then were tested again for avoidance 6 weeks later. Rats that exhibited high avoidance of the predator-paired chamber at 24 h post-conditioning also exhibited avoidance 6 weeks later. (b) Scatter plot for individual rat (Experiment 3) shows change in preference for a context repeatedly paired with an appetitive stimulus (saccharin glucose solution) versus change in preference

for a context repeatedly paired with predator odor. There was no predictive value by the degree to which a rat conditioned the appetitive stimulus for the degree to which a rat conditioned the aversive stimulus. This also suggests that differences in avoidance are not attributable to differences in ability of rats to learn. (c) Scatter plot for individual rat (Experiment 3) shows percent time spent in open arms of EPM (5-min test) in experimentally naive rats (index of innate anxiety-like behavior) versus change in preference for a context repeatedly paired with predator odor. There was no predictive value by innate anxiety-like behavior for the degree to which a rat conditioned the aversive stimulus. (d) Mean operant alcohol responses (s.e.m.) per 30 min session for Avoider, Non-Avoider and non-stressed control groups (Experiments 1 and 2 combined) across the last 3 days of the pre-conditioning baseline self-administration period. Groups did not differ in baseline alcohol self-administration. These data also provide further support for the notion that differences in avoidance are not attributable to differences in ability of rats to learn. (e) Mean (s.e.m.) operant presses for alcohol per 30-min self-administration session by rats in Experiment 2 (results similar in Experiment 1, see text) during intermittent testing across the 19 days following exposure to predator odor (Avoider and Non-Avoider groups) or no odor (control group). Avoider rats exhibited persistent increases in operant alcohol responding relative to the two other groups and their own baseline across the 19 days following exposure to odor. *Po0.05 signicant main effect of group. Avoiders exhibited marginally non-signicant increases in alcohol responding relative to Non-Avoiders (P 0.06) and unstressed Controls (P 0.055). (f) Mean (s.e.m.) self-administration of alcohol solution adulterated with progressively increasing

quantities of quinine, quantied as percent change from baseline (10% w/v ethanol plus 0% quinine) for each rat (Experiment 1). A 10 higher

concentration (0.025% vs 0.0025%) of quinine was required to reduce operant alcohol self-administration in Avoider rats vs Non-Avoider and Control rats, indicative of more compulsive-like alcohol drinking in Avoider rats. **Po0.001 Avoiders responded signicantly more for alcohol 0.0025% quinine relative to unstressed Controls. There was a non-signicant tendency of Avoiders to press more for this solution than

Non-Avoiders (P 0.10). (g) Scatter plot for individual rat (Experiment 2) shows change in preference for predator-paired context versus percent

change in operant alcohol responding at day 19 post-odor exposure relative to baseline. Rats that exhibited high avoidance of the predator-paired context 24 h post-odor exposure self-administered more alcohol at 19 days post-odor exposure.

Figures 1bd show the results of several behavioral measures that failed to predict avoidance of predator-paired chamber in individual rats. The conditioned place preference exhibited by rats for a supersac-paired context did not predict avoidance of a predator odor-paired context, r 0.03, P40.05 (Figure 1b). Time

spent in the open arms of the EPM also did not predict the avoidance of a predator odor-paired context, r 0.11, P40.05

(Figure 1c). Baseline alcohol drinking (pooled across rats in Experiments 1 and 2) did not differ between control rats and rats that exhibited high or low avoidance of predator odor-paired

context, F(2,75) 0.23, P40.05, suggesting that limited-access

operant alcohol self-administration training before odor exposure does not make rats more or less likely to exhibit high reactivity to a traumatic stress.

Avoiders exhibit excessive alcohol self-administrationIn Experiments 1 and 2, rats that exhibited high avoidance of the predator odor-paired chamber 24 h following exposure also exhibited lasting increases in operant alcohol self-administration

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Figure 2. (a) ERK phosphorylation (pERK) in the medial prefrontal cortex (PFC) after re-exposure (15 min) to the predator odor-paired context. Relative to neutral-paired animals (controls), re-exposure to the context produced a bidirectional regulation of pERK levels in the vmPFC in the odor-paired groups, with Avoiders displaying relatively higher ERK phosphorylation compared with that of Non-Avoiders. This relationship was not observed in the dorsomedial PFC (dmPFC). **Po0.01 higher ERK phosphorylation levels in Avoiders versus Non-Avoiders or *Po0.05 higher ERK phosphorylation levels in vmPFC versus dmPFC in Avoiders. (b) ERK phosphorylation in the amygdala subregions after re-exposure to the odor-paired context. As a percentage of neutral-paired control animals, Avoider and Non-Avoider stress-paired groups displayed more ERK phosphorylation in the central nucleus of the amygdala (CeA) versus basolateral amygdala (BLA). **Po0.01 higher ERK phosphorylation levels in the CeA versus BLA of pooled stress-paired groups by analysis of variance (main effect of group). Data reect the means.e.m. of pERK/ERK ratios and are expressed as a percentage of neutral-paired controls. Inset: representative western blot images for pERK and ERK levels in vmPFC, CeA and BLA in Control (C), Avoider (A) and Non-Avoider (NA) groups.

relative to Non-Avoiders and unstressed controls. A two-way (group days) repeated measures ANOVA of alcohol lever presses

from the 11 days following stress exposure in Experiment 1 (data not shown) yielded a signicant main effect of the group, F(2,45) 4.55, P 0.016, and post-hoc analyses revealed that

Avoiders exhibited signicant post-stress increases in operant alcohol responding relative to both Non-Avoiders (P 0.015) and

unstressed controls (P 0.05). A two-way (group days) repeated

measures ANOVA of alcohol lever presses from the 19 days following stress exposure in Experiment 2 (Figure 1e) yielded a signicant main effect of the group, F(2,26) 3.47, P 0.046, and

post-hoc analyses revealed that Avoiders exhibited marginally non-signicant increases in alcohol responding relative to Non-Avoiders (P 0.06) and unstressed controls (P 0.055). Increases

in alcohol drinking appeared to incubate with time in Avoiders, as evidenced by the fact that Avoider rats exhibited peak alcohol responding 19 days post stress. Importantly, high avoidance of the predator odor-paired context by exposed rats was predictive of increased alcohol drinking 19 days post stress, r 0.47, P 0.03, suggesting that high stress reactivity

24 h post stress (as measured by conditioned place aversion) produces long-lasting neural changes that promote excessive alcohol drinking (Figure 1g).

To test compulsivity (that is, persistence) of alcohol responding when alcohol is paired with an aversive stimulus, rats were tested on consecutive days for operant responding for alcohol adulterated with progressively increasing quantities of quinine. As illustrated in Figure 1f, a 10 higher concentration of quinine

(0.025% vs 0.0025%) was required to reduce operant alcohol responding in Avoider rats relative to Non-Avoider rats and unstressed controls. A two-way (group concentration) repeated

measures ANOVA of lever presses for alcohol adulterated with0.0025% versus 0.025% quinine revealed signicant main effects of the group, F(2,43) 5.10, P 0.01, and the concentration,

F(1,43) 33.37, Po0.001, as well as a signicant group

concentration interaction effect, F(2,43) 3.32, Po0.05. Post-hoc

analysis of alcohol 0.0025% (moderate concentration) quinine

data revealed that Avoiders responded signicantly more for this solution relative to unstressed controls (Po0.001); there was a non-signicant tendency of Avoiders to press more for this solution than Non-Avoiders (P 0.10). In contrast, none of the

groups differed in their pressing for the alcohol solution adulterated with the highest quinine concentration (0.025%).

Traumatic stress drives neuronal activation according to avoidance behavior

At the end of behavioral testing, animals were exposed to a nal 15-min exposure to the predator-paired context (neutral context in unstressed controls) and were killed, and the brains were removed for western blot analysis to measure context-associated ERK phosphorylation. As illustrated in Figure 2a, a two-way (group medial prefrontal cortex (mPFC) region) ANOVA revealed

a signicant interaction effect on pERK levels, F(2,98) 3.25,

Po0.05, and post-hoc analyses indicated that pERK levels in ventromedial PFC (vmPFC) were signicantly higher in Avoiders relative to Non-Avoiders (Po0.01). A separate two-way (group

mPFC region) ANOVA of data only from the stressed groups (data normalized to controls) revealed a signicant interaction effect on pERK, F(1,64) 5.55, Po0.05), and post-hoc analyses indicated that

normalized pERK levels were signicantly higher in vmPFC versus dorsomedial PFC (dmPFC) of Avoiders (Po0.05). As illustrated in

Figure 2b, a two-way (group amygdala region) ANOVA of data

only from the stressed groups (data normalized to controls) revealed a signicant main effect of region on pERK, F(1,63) 8.15,

Po0.01), which indicated that Avoider and Non-Avoider groups exhibited collectively higher pERK expression in the central nucleus of the amygdala (CeA) versus basolateral amygdala (BLA). In accordance with subgroup data, avoidance of the odor-paired environment was signicantly correlated with vmPFC pERK levels (r2 0.17, Po0.05). We next determined within-subject,

inter-regional correlations of ERK phosphorylation upon re-exposure to the predator odor-paired context (Figure 3). Signicant correlations were found between BLA and CeA in both Avoiders (r2 0.46, Po0.01) and Non-Avoiders (r2 0.42, Po0.05),

but not in stress-naive controls (r2 0.19, P 0.21). Furthermore,

in Avoiders, but not in Non-Avoiders or Controls, there was a signicant correlation between dmPFC and BLA (r2 0.68,

Po0.01) and a marginally non-signicant correlation between vmPFC and CeA (r2 0.20, P 0.06). In most cases, inter-regional

correlations were bidirectional, that is, correlative data comprised both increases and decreases in ERK phosphorylation.

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Avoiders Non-Avoiders

BLA pERK

150

150

-100 -50 0 50 100

(% of Controls)

-150

BLA pERK

(% of Controls)

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-50

-100 -50 0 50 100 dmPFC pERK (% of Controls)

-150

r2=0.68, p<0.01 r2=0.10

dmPFC pERK (% of Controls)

Avoiders Non-Avoiders Controls

vmPFC-CeA r

2=0.19, p=0.06 r2=0.10, n.s. r2=0.12, n.s.

(+) (+) (-)

BLA-CeA r

2=0.46, p<0.01 r2=0.42, p<0.05 r2=0.19, n.s.

(+) (+) (+)

Figure 3. Within-subject between-region correlations of ERK phosphorylation upon re-exposure to the predator odor-paired context. (a) In Avoiders (left panel), but not in Non-Avoiders (right panel) or Controls (data not shown), there was a signicant correlation between dorsomedial prefrontal cortex (dmPFC) and basolateral amygdala (BLA) ERK phosphorylation (pERK) expression. Panel b reports the strength and direction of correlations between pERK expression in ventromedial PFC (vmPFC) and central nucleus of the amygdala (CeA; top row), and also between BLA and CeA (bottom row) in Avoiders, Non-Avoiders and Controls upon re-exposure to predator odor-paired context.

Avoiders Non-Avoiders

100

-100 -50 0 50 100

100

-100 0 100

r2=0.25, p<0.05

r2=0.24, p<0.05

r2=0.13

r2=0.15

50

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Post-Conditioning Alcohol Responding

(Sum of from Baseline over 5 days)

0

dmPFC

BLA

0

-50

-50

-100

100

-100 -50 0 50 100 150

-100

100

-100 -50 0 50 100 150

50

50

0

0

-50

-50

-100

-100

Predator Context-Induced pERK (% Controls)

Figure 4. Relationship between alterations in individual levels of alcohol drinking (19 days post conditioning) and odor-paired-context-induced ERK phosphorylation in the dorsomedial prefrontal cortex (dmPFC) and basolateral amygdala (BLA). Elevations in drinking after odor exposure signicantly correlated with context-induced reductions in ERK phosphorylation (pERK) levels in both the dmPFC (a, r2 0.25,

Po0.05) and BLA (b, r2 0.24, Po0.05) of Avoiders, but not of Non-Avoiders or Controls.

Elevated drinking levels correspond to traumatic context-associated ERK phosphorylation

Finally, we examined individual relationships between alterations in ethanol drinking behavior and biochemical reactivity to the stress-paired context. Elevations in ethanol self-administration after predator odor exposure were signicantly and negatively correlated with context-induced pERK levels in both the dmPFC (r2 0.25, Po0.05) and BLA (r2 0.24, Po0.05) in Avoiders

(Figure 4). In contrast, weak positive correlations (r2 0.130.15)

were observed between post-odor drinking and ERK phosphorylation in these two regions in Non-Avoiders.

DISCUSSIONPTSD is a complex psychiatric disorder that is highly comorbid with alcohol-use disorders, although the underlying neural and

molecular perturbations have not been identied. In the present study, animals that persistently avoided a traumatic context exhibited persistent increases in alcohol drinking over weeks. This result supports the notion that traumatic stress reactivity is associated with increased risk for alcohol-use disorders. It is important to emphasize that drinking tests in these experiments occurred in a separate and distinct environment from the traumatic stress experience, consistent with the human PTSD situation. Previous studies have used avoidance as an index for dividing subpopulations of stressed rodents (for example, see Russo et al.26), and in our study the animals that displayed avoidance of the odor-paired context (Avoiders) did so persistently, consistent with previous ndings from other labs.27

Further, a single odor exposure was sufcient to elevate drinking in Avoiders for at least 19 days, and avoidance behavior across individual animals was predictive of alcohol drinking 19 days later.

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Finally, Avoiders exhibited compulsive-like alcohol drinking relative to Non-Avoiders and unstressed controls, as evidenced by the attenuated ability of quinine adulteration to reduce alcohol drinking in Avoiders.

Re-exposure to the odor-paired environment produced a bidirectional modulation of pERK levels in the vmPFC in stressed groups, with Avoiders displaying higher ERK phosphorylation compared with that of Non-Avoiders. Although neither stress-paired group differed from unstressed (and unselected) controls at the group level, avoidance of the stress-paired environment was predictive of context-driven vmPFC ERK phosphorylation levels (Figure 2c). These data appear to reect the critical nature of individual sensitivity to traumatic stress-associated contextual challenges. Hyperactivation of the vmPFC is associated with a negative effect28,29 and major depressive disorder,30,31 including antidepressant responsiveness.32 War veterans with vmPFC damage are less likely to develop PTSD;31 some humans with PTSD exhibit heightened vmPFC activity;33,34 and in healthy

humans, increased blood ow in the vmPFC is predictive of self-reported anxiety during an anticipatory anxiety task.35

Nevertheless, there is also a large human imaging and animal literature that links PTSD-like symptom provocation with vmPFC hypoactivity.36 Differences across human imaging studies may be explained by functionally distinct subdivisions of the human vmPFC and/or the use of symptom provocation in some studies (for review, see Myers-Schulz and Koenigs37). It is important to note that in our study the vmPFC was dened as the infralimbic cortex plus the ventral portion of the prelimbic cortex, whereas the dmPFC was dened as the dorsal portion of the prelimbic cortex plus the anterior cingulate. A limitation of the current analysis is that the neuronal (and even cellular) phenotypes associated with observed changes in ERK phosphorylation are unknown. This limits functional interpretations of ERK phosphorylation changes in the mPFC, which comprises both excitatory and inhibitory neuronal elements. Nevertheless, the between-region correlations presented here likely reect a second-order connectivity between subregions of the mPFC and amygdala (see Figure 3).

Animal studies report elevated pERK in vmPFC (but not dmPFC) dendrites after repeated, but not acute, footshock stress in rats,38

suggesting that multiple exposures to stress and/or stress-paired stimuli may differentially alter the balance of vmPFC plasticity in stress-resilient versus susceptible populations. A critical question is whether this plasticity contributes to the persistent behavioral dysregulation seen in individuals with high stress reactivity. In support of this hypothesis, re-exposure to a context previously paired with footshock triggers vmPFC activation (as measured by c-Fos levels) that is partly predictive of a freezing behavior in that context 28 days (but not 1 day) following the initial stress.39 The

ERK/mitogen-activated protein kinase signaling cascade activates numerous transcription factors, including c-Fos40 and cAMP

response element-binding protein,41 and is thereby believed to promote long-term memory maintenance.42 In our study, high avoidance and excessive alcohol drinking were observed in the same rats over weeks, and context-induced ERK phosphorylation in the vmPFC was also observed weeks after conditioning and was predictive of context avoidance, which suggests that ERK interacts with downstream transcriptional mechanisms to mediate persistent behavioral effects of traumatic stress. Similar to the results reported here, vmPFC ERK phosphorylation is associated with enhanced cocaine-seeking following protracted withdrawal periods.43

PTSD patients exhibit a strong synchronization of the amygdala and the dmPFC/anterior cingulate cortex upon symptom provocation.44 Here we show much stronger pERK correlations between dmPFC and BLA in Avoider rats relative to Non-Avoiders and controls (Figure 3). In rodents, intra-BLA infusions of the b-adrenoceptor agonist clenbuterol enhance avoidance memory

and increase plasticity-related proteins (that is, Arc and CaMKIIa) in the dmPFC, whereas BLA inactivation reduces levels of these signaling proteins in the dmPFC.45 In accordance with our own ndings, this avoidance memory-enhancing intra-BLA infusion of clenbuterol reduces BLA ERK phosphorylation.46 We also found

that the odor context-associated changes in ERK phosphorylation in the BLA and CeA were positively and strongly correlated in both groups of stressed rats, but not in stress-naive controls, consistent with the role of this circuit in anxiety.47 These data also provide validation for our measure of stimulus-driven neuronal activity intended to model symptom provocation studies in PTSD subjects. Increased activation of the CeA, the major output region of the amygdala, in stressed rats likely drives downstream effector regions (for example, the hypothalamus and periaqueductal gray) responsible for mediating stress- and alcohol-related behavioral and physiological processes.48

Within-subject biochemical measurements have been used to assess regional activation patterns associated with avoidance memory (for example, Holloway-Erickson et al.45 and McIntyre et al.49). Here we show that avoidance of a stress-paired environment associates with a modication of the mPFC amygdala signaling as assayed by context-induced ERK phosphor-ylation. Interestingly, most of our correlative data comprise both increases and decreases in pERK, suggesting elaborate changes in connectivity between the mPFC and amygdala. The vmPFC projects to the CeA50 and the CeA drives both the peripheral and central stress systems,51 highlighting the importance of this circuitry in stress-related disorders. Both top-down models, in which the vmPFC hypoactivity disinhibits stress-associated limbic circuitry (for example, the amygdala28,37), and bottom-up models,

in which the amygdala hyperactivity drives PTSD symptomatology (for example, Rauch et al.52), have been proposed. Numerous other PTSD symptom provocation studies suggest the importance of altered amygdalamPFC connectivity44,53,54 in accordance with the

design and results of the current study. It should be noted that the vmPFC also regulates behavioral and physiological responses to stress by activating the hypothalamicpituitaryadrenal axis,55,56

and circulating glucocorticoids sensitize CeA activity,5759 resulting

in a functionally positive CeAhypothalamicpituitaryadrenal feedback loop that promotes long-term stress-related pathology.60

As a functional correlate linking stress-induced plasticity and alcohol drinking, context-induced reductions in both the dmPFC and BLA ERK phosphorylation were strongly predictive of postconditioning increases in alcohol drinking in Avoiders (Figure 4). This relationship was dampened and inverted in Non-Avoiders, implying a distinct relationship between stress reactivity and drinking between these two populations. As mentioned above, the b-adrenoceptor activation may mediate reductions in BLA ERK activity and drive stress-induced escalation of drinking in the Avoider population, whereas the Non-Avoider population may be protected from this mechanism. Support for this hypothesis is provided by evidence that propranolol reduces excessive, but not moderate, alcohol drinking in rats.61 The ERK/mitogen-activated protein kinase signaling cascade is also linked with other stress-related receptors in the limbic system, particularly a-1 adrenergic receptors62 and CRF1Rs.63 Antagonism of both a-1 adrenergic receptors and CRF1Rs reduces excessive alcohol drinking and other dependence-related behaviors in animals.6467 Indeed,

the brain norepinephrine systems are being investigated as a therapeutic target for PTSD.6870

Collectively, our data suggest unique alterations of the mPFC amygdala signaling in Avoider rats (Figure 5) that drive persistent, excessive and compulsive-like alcohol drinking. Going forward, this model will be useful for elucidating the neurobiology of enhanced vulnerability for alcohol-use disorders in humans with PTSD, and may provide valuable targets for behavioral and pharmacological intervention into these oft co-occurring disorders.

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7 Cohen H, Zohar J. An animal model of posttraumatic stress disorder: the use of cut-off behavioral criteria. Ann NY Acad Sci 2004; 1032: 167178.

8 Logrip ML, Zorrilla EP, Koob GF. Stress modulation of drug self-administration: implications for addiction comorbidity with post-traumatic stress disorder. Neuropharmacology 2012; 62: 552564.

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11 Yehuda R. Advances in understanding neuroendocrine alterations in PTSD and their therapeutic implications. Ann NY Acad Sci 2006; 1071: 137166.

12 Jacobsen LK, Southwick SM, Kosten TR. Substance use disorders in patients with posttraumatic stress disorder: a review of the literature. Am J Psychiatry 2001; 158: 11841190.

13 Bonne O, Charney DS. Neuropathology of stress: prospects and caveats. Psychiatry

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14 Koob GF. Alcoholism: allostasis and beyond. Alcohol Clin Exp Res 2003; 27: 232243.15 Koob GF. A role for brain stress systems in addiction. Neuron 2008; 59: 1134.16 Edwards S, Koob GF. Neurobiology of dysregulated motivational systems in drug addiction. Future Neurol 2010; 5: 393401.17 Miller MM, McEwen BS. Establishing an agenda for translational research on PTSD. Ann NY Acad Sci 2006; 1071: 294312.18 Sweatt JD. Mitogen-activated protein kinases in synaptic plasticity and memory. Curr Opin Neurobiol 2004; 14: 311317.19 Girault JA, Valjent E, Caboche J, Herve D. ERK2: a logical AND gate critical for drug-induced plasticity? Curr Opin Pharmacol 2007; 7: 7785.20 Edwards S, Bachtell RK, Guzman D, Whisler KN, Self DW. Emergence of context-associated GluR(1) and ERK phosphorylation in the nucleus accumbens core during withdrawal from cocaine self-administration. Addict Biol 2011; 16: 450457.21 Becker HC, Lopez MF, Doremus-Fitzwater TL. Effects of stress on alcohol drinking: a review of animal studies. Psychopharmacology (Berl) 2011; 218: 131156.22 Zanette LY, White AF, Allen MC, Clinchy M. Perceived predation risk reduces the number of offspring songbirds produce per year. Science 2011; 334: 13981401.23 Valenstein ES, Kakolewski JW, Cox VC. Sex differences in taste preference for glucose and saccharin solutions. Science 1967; 156: 942943.24 Vendruscolo LF, Barbier E, Schlosburg JE, Misra KK, Whiteld Jr. TW, Logrip ML et al. Corticosteroid-dependent plasticity mediates compulsive alcohol drinking in rats. J Neurosci 2012; 32: 75637571.25 Rassnick S, Heinrichs SC, Britton KT, Koob GF. Microinjection of a corticotropin-releasing factor antagonist into the central nucleus of the amygdala reverses anxiogenic-like effects of ethanol withdrawal. Brain Res 1993; 605: 2532.26 Russo SJ, Murrough JW, Han MH, Charney DS, Nestler EJ. Neurobiology of resilience. Nat Neurosci 2012; 15: 14751484.27 Blanchard RJ, Yang M, Li CI, Gervacio A, Blanchard DC. Cue and context conditioning of defensive behaviors to cat odor stimuli. Neurosci Biobehav Rev 2001; 25: 587595.28 Koenigs M, Grafman J. Posttraumatic stress disorder: the role of medial prefrontal cortex and amygdala. Neuroscientist 2009; 15: 540548.29 Zald DH, Mattson DL, Pardo JV. Brain activity in ventromedial prefrontal cortex correlates with individual differences in negative affect. Proc Natl Acad Sci USA 2002; 99: 24502454.30 Greicius MD, Flores BH, Menon V, Glover GH, Solvason HB, Kenna H et al. Resting-state functional connectivity in major depression: abnormally increased contributions from subgenual cingulate cortex and thalamus. Biol Psychiatry 2007; 62: 429437.31 Koenigs M, Huey ED, Calamia M, Raymont V, Tranel D, Grafman J. Distinct regions of prefrontal cortex mediate resistance and vulnerability to depression. J Neurosci 2008; 28: 1234112348.32 Mayberg HS, Brannan SK, Tekell JL, Silva JA, Mahurin RK, McGinnis S et al. Regional metabolic effects of uoxetine in major depression: serial changes and relationship to clinical response. Biol Psychiatry 2000; 48: 830843.33 Morey RA, Petty CM, Cooper DA, Labar KS, McCarthy G. Neural systems for executive and emotional processing are modulated by symptoms of posttraumatic stress disorder in Iraq War veterans. Psychiatry Res 2008; 162: 5972.

dmPFC

Hypo-reactivity to Reminder Cue Predicts High Drinking in Avoiders

Stronger Connectivity in Avoiders

vmPFC

Hyper-reactivity to Reminder Cue Predicts High Avoidance

Stress - & Alcohol-Related Behavior and Physiology

BLA

Hypo-reactivity to Reminder Cue Predicts High Drinking in Avoiders

CeA

Figure 5. Proposed model of corticolimbic neurocircuitry underlying traumatic stress-induced avoidance and increases in excessive alcohol drinking. Traumatic stress reminder cue-induced neuronal activation (that is, ERK phosphorylation (pERK) expression) in the ventromedial prefrontal cortex (vmPFC) predicts high avoidance behavior. Also depicted here is that the hyporeactivity of the dorsomedial PFC (dmPFC) and basolateral amygdala (BLA) to a traumatic stress reminder is highly predictive of each other and is also highly predictive of escalated post-stress alcohol drinking only in Avoider rats, suggesting an increased dmPFCBLA connectivity in Avoider rats. Two potentially important aspects of this circuitry are not depicted here: (1) the population of intercalated g-aminobutyric acid (GABA) cells that act as a relay for some but not all central nucleus of the amygdala (CeA) afferents, and (2) the neuroendocrine (hypothalamicpituitaryadrenal) stress axis that is driven by vmPFC activity and inhibited by dmPFC activity,55,56 and which itself may

interact with the CeA in a reciprocally excitatory loop.60

CONFLICT OF INTEREST

The authors declare no conict of interests.

ACKNOWLEDGEMENTS

This work was supported by NIH grants AA018400 (NWG), AA020839 (SE), AA006420, AA008459, AA012602 and AA020608 (GFK), and by The Pearson Center for Alcoholism and Addiction Research at TSRI. This is publication number 22051 from The Scripps Research Institute.

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