The long-term impact of footshock stress on addiction-related behaviors in rats

Neuropharmacology 60 (2011) 267e273

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The long-term impact of footshock stress on addiction-related behaviors in rats Boglárka Barsy, Éva Mikics, Beáta Barsvári, József Haller* Institute of Experimental Medicine, Department of Behavioural Neurobiology, Hungarian Academy of Sciences, PO Box 67, H-1450 Budapest, Hungary

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 April 2010 Received in revised form 20 August 2010 Accepted 7 September 2010

We investigated the impact of electric shocks e frequently used to model post-traumatic stress disorder in rodents e on behaviors relevant to drug abuse in rats. Rats exposed to 10 shocks of 3 mA over 5 min showed a robust conditioned fear 28 days later, which confirms the traumatic nature of shock exposure. A different set of rats was studied in the conditioned place preference paradigm beginning with the 27th post-shock day. 10 mg/kg morphine induced a marked place preference in both shocked and nonshocked rats. Although the magnitude of place preference was not affected, extinction was markedly delayed in shocked rats. We also investigated tolerance to the hyperthermic effects of morphine. A low dose (5 mg/kg) that was administered 4 weeks after shock exposure robustly increased body temperature in both shocked and non-shocked rats. Repeated injections resulted in a mild tolerance in nonshocked controls; yet, morphine readily increased body temperature in these rats on the 5th day of injections. In contrast, the temperature-heightening effect of morphine was abolished in shocked rats after 2 days. Thus, shock exposure considerably delayed the extinction of place preference induced by, and dramatically accelerated the tolerance to the effects of, morphine. Our study shows that electric shocks durably affect behavior in tests relevant to drug abuse in conjunction with the development of post-traumatic stress disorder-like behavioral dysfunctions. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: PTSD Morphine Conditioned place preference Tolerance Body temperature Rat

1. Introduction Post-traumatic stress disorder (PTSD) is a highly debilitating condition that develops in response to life-threatening stressors and lasts for decades. To get insights into the brain mechanisms of the disorder, a large number of PTSD models were developed. When submitted to traumatic experience, animals develop a series of long-lasting behavioral dysfunctions that are similar to symptoms seen in patients with PTSD, e.g. they avoid trauma-associated contexts, and show contextual fear, altered glucocorticoid production, impaired memory, increased anxiety, decreased social interactions, sleeping problems, and startle deficits (Foa et al., 1992; Garrick et al., 1997; Izquierdo et al., 2004; Louvart et al., 2006; Mikics et al., 2008a; Mikics et al., 2008b; Pawlyk et al., 2005; Rupniak et al., 2003; Servatius et al., 1995; Shimizu et al., 2006). However, one important trauma-related dysfunction received little attention so far in the laboratory, namely the effect of traumatic experience in tests relevant to drug abuse. In humans, PTSD is highly co-morbid with substance abuse (Bronson et al., 2007; De Bellis, 2002; Foa et al., 2006; Keane et al., 1988; Kross et al., 2008; Nadelson, 1989; Owens et al., 2005). Drugs

* Corresponding author. Tel.: þ36 1 2109406; fax: þ36 1 2109951. E-mail address: [email protected] (J. Haller). 0028-3908/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2010.09.009

may be used by PTSD patients as a form of self-medication (Jacobsen et al., 2001; Staiger et al., 2009; Stewart et al., 1998). However, the brain mechanisms underlying the two disorders are highly overlapping; consequently, each may promote the development of the other via the shared mechanisms (Kosten and Krystal, 1988; Tryon, 1998). In line with this assumption, PTSD is a risk factor for substance abuse, and the other way round: substance abuse is a risk factor for PTSD (Dierker and Merikangas, 2001; Dyster-Aas et al., 2008; Gurvits et al., 1997; Roy-Byrne et al., 2004). The aim of this study was to investigate in rats the effects of electric shocks in tests relevant to drug abuse. We studied the impact of traumatic stress on conditioned place preference induced by, and tolerance to the effects of morphine. It was shown earlier that stress exposure has a large impact on behaviors shown in the conditioned place preference and drug self-administration paradigms. Certain stressors (chronic mild stress, social isolation, subordination) hinder, while others (acute restraint, footshocks delivered shortly before behavioral testing, repeated tail-pinch, social stressors) promote opiate place preference and self-administration (Benamar et al., 2003; Coventry et al., 1997; Goeders and Guerin, 1994; Miczek et al., 2008; Papp et al., 1992; Piazza et al., 1990; Shaham and Stewart, 1994; Will et al., 1998). However, the stress-induced changes in such tests have rarely if ever been studied and interpreted in conjunction with trauma-induced behavioral changes. Traumatic

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experience was induced in our subjects by electric shocks, which are frequently used to this end, and which induced long-lasting PTSDlike behavioral alterations in our earlier studies. E.g. we have shown that social behavior was markedly disrupted in both the stressinduced social avoidance and the psychosocial stress paradigms (Mikics et al., 2008b). This behavioral change was associated with a marked over-activation of the amygdala (as shown by increased c-Fos staining) in rats submitted to social interactions. Shocked rats also buried harmless unfamiliar objects (Mikics et al., 2008a). In contrast to mice, rats bury objects in aversive contexts only (e.g. in the shock-prod burying test); therefore, the burying of harmless objects was interpreted as a sign of hyper-vigilance in this study. Both social deficits and object burying were observed for at least 4 weeks after shock exposure. The development of trauma-induced behavioral changes was studied here by means of the conditioned fear paradigm. The conditioned place preference paradigm provides information on the rewarding properties of drugs, which is at the core of substance use disorders (Tzschentke, 2007). Additionally, the development of tolerance to the effects of drugs is one of the main symptoms of drug dependence (American Psychiatric Association, 1994). Morphine was chosen for this study because PTSD patients frequently abuse opiates, while opioid dependence predisposes people to PTSD (Bremner et al., 1996; Clark et al., 2001; Cottler et al., 1992; Saxon et al., 2001). We hypothesized that trauma exposure promotes both morphineinduced conditioned place preference, and tolerance to the hyperthermic effects of morphine. 2. Methods 2.1. Animals Subjects were 2e3 months old male Wistar rats (Charles River Laboratories; Hungary) weighing approximately 250 g at the start of the experiments. Food and water were available ad libitum; temperature and relative humidity were kept at 22  2  C and 60  10%, respectively. Rats were maintained in a reversed light cycle of 12 h with lights off at 10:00 h. Acclimatization to local conditions lasted at least one week. Experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were reviewed and approved by the Animal Welfare Committee of our Institute.

compartments: a black rectangular neutral (‘central’) area (20  20  30 cm) and two test compartments (20  50  30 cm each). The central area was connected to the test compartments by 18  18 cm doors. The test compartments were differently marked: one was striped, the other one was spotted. All experiments were performed in the early hours of the dark phase under dim white light (about 5 lx). The experiment included a habituation phase, a conditioning phase, and a test phase. Two habituation trials were run on the 23rd and on the 24th post-shock day. Rats were placed into the central area, and were allowed to freely explore the apparatus for 10 min. Behavior was recorded in both trials by an experimenter blind to treatment conditions. Data obtained in habituation trial 2 were used as baselines for post-conditioning trials. The conditioning phase started on the 27th post-shocking day, and consisted of 6 trials that were performed on consecutive days; 3 morphine and 3 saline conditioning trials were run on alternate days. Rats were injected intraperitoneally either with saline or with 10 mg/kg morphine. Injections were administered immediately before the introduction of rats into one of the compartments for 20 min. The door of the compartment was closed; thus, the rats were not able to explore the other compartments. Morphine injections were consistently associated with the compartment that was less preferred during the 2nd habituation (baseline) trial. The more preferred compartment was associated with saline. Both arms were associated with saline in saline-conditioned controls. The test phase consisted of six daily test trials that were run on consecutive days, and started on the first post-conditioning day. During test trials, rats were placed in the central area and were allowed to explore the apparatus freely for 10 min. Behavior was recorded by a video camera placed 1.8 m above the conditioning apparatus. Videotapes were analyzed by a trained experimenter blind to the treatments. Time spent in the morphine-associated compartment served as the index of conditioned place preference. The experiment was run in two series that comprised all groups in equal number. 2.2.2.2. Morphine tolerance. Temperature was recorded by the VitalView biotelemetry system (Minimitter, Bend, Oregon). Four groups were studied: saline-treated, not shocked; saline-treated, shocked; morphine-treated, not shocked; and morphinetreated, shocked. The morphine dose was kept constant to promote tolerance. On the first day of the study, rats were implanted with telemetric E-mitters (Minimitter, Bend, Oregon) placed into the abdominal cavity of rats through a midline abdominal incision under ketamineexilazineepromethazine anesthesia (50e10e5 mg/kg intraperitoneally) as described earlier (Haller et al., 2004). After one week recovery, rats were either shocked or exposed to the shocking cage as described above. 27 days later, animals were injected intraperitoneally with saline or morphine (5 mg/kg) on 5 consecutive days. Two injections were administered per day at 0800 h and 1600 h. Temperature was followed for 3 h after the first injection. Values were expressed as changes from body temperatures measured immediately before injections. Sample size was 5e6 per group. Temperature changes did not depend on shock background in saline-treated rats; therefore, these groups were pooled. 2.3. Drugs and doses

2.2. Experimental design 2.2.1. Footshocks Rats were transferred to a separate quiet room, where they received footshocks via the grid floor of a Plexiglas cage (30  30  30 cm) between 1100 and 1300 h. Light intensity in cage was approximately 400 lx. Two shocks trains were administered per minute for 5 min, i.e. each rat received 10 shocks. Each shock train was 1 s-long and consisted of 0.01 s shocks separated by 0.02 s-long breaks. Current potential and intensity were 100 V and 3 mA, respectively. Control rats were placed into a similar box for 5 min, but shocks were not delivered. The box was cleaned after each shocking session with ethanol. 2.2.2. Conditioned fear On the first day of the experiment, rats were either shocked or exposed to the shocking cage as shown above (N ¼ 10 per group). Twenty-eight days after shocks, rats were returned to the shock-box for 5 min between 11.00 and 13.00 h. The experimenter left the room during testing; shocks were not delivered. Behavior was video recorded, and later scored by a trained experimenter blind to treatment conditions. Scoring was made by means of the H77 software developed locally, and frequently used in earlier studies (Haller and Bakos, 2002; Haller et al., 2004; Mikics et al., 2008a,b). The duration of freezing e absence of all observable movements except breathing e was used as an index of conditioned fear and was expressed as percent of total test time. 2.2.2.1. Conditioned place preference. On the first day of the experiment, rats were either shocked or exposed to the shocking cage as shown above. 23 days later, the conditioned place preference paradigm was started. Note that the first treatments were administered on the 27th day. Four groups were studied: non-shocked rats conditioned with saline, shocked rats conditioned with saline; non-shocked rats conditioned with morphine, and shocked rats conditioned with morphine (N ¼ 11e12 per group). Group assignment was random. The conditioned place preference test was performed as described by Barr et al. (1985) with slight modifications as described by Becker et al. (2006) and Vazquez et al. (2006). The apparatus consisted of three

Morphine (morphinium chloratum Ph. Eur. 4) was purchased from ICN Pharmaceuticals Inc. (Budapest, Hungary). The compound was dissolved in saline, and was administered intraperitoneally in a volume of 1 ml/kg. Dose choice was based on detailed doseeresponse curves published earlier. The 10 mg/kg dose reliably induced conditioned place preference in such studies (Moaddab et al., 2009; Rezayof et al., 2007; Wu et al., 2007; Zarrindast et al., 2005), and was used as a single dose in experiments similar to ours (Lu et al., 2005; Wang et al., 2000; Zhai et al., 2008). A lower dose (5 mg/kg) was employed in the morphine tolerance study because this compound affects body temperature in a biphasic manner: low doses increase, while high doses decrease body temperature (Benamar et al., 2003; Fraga et al., 2008; Rawls et al., 2003; Warwick and Schnell, 1976). 2.4. Statistics Conditioned fear responses were analyzed by KruskaleWallis ANOVA followed by ManneWhitney post hoc comparisons. Conditioned place preference was evaluated by two-factor repeated measures ANOVA Factor 1: treatment (repeated measures) Factor 2: time (successive trials). Data were log-transformed to fulfill ANOVA requirements. The effects of morphine on body temperature were analyzed by three factor repeated measures ANOVA. Factor 1 was treatment, Factor 2 was the day of the treatment (levels: day 1 through 5), while Factor 3 (repeated measures factor) was post-injection time (levels: seconds 0e20 through 160e180). NewmaneKeuls post hoc comparisons were also run where appropriate.

3. Results 3.1. Conditioned fear Shocked rats showed a robust freezing behavior when re-exposed to the shock-associated environment 28 days after shocks (Fig. 1).

B. Barsy et al. / Neuropharmacology 60 (2011) 267e273

Freezin g (%time)

100

control

80

Table 1 The effects of saline conditioning in the conditioned place preference test.

shocked

*

*

*

*

60 40 20

*

0 1

2 3 4 minutes of testing

269

5

Fig. 1. Freezing behavior shown in the shock-associated environment, 28 days after shocks. Freezing was not totally absent in controls, but the values are not discernable at the scale appropriate for showing the values seen in shocked rats. *, Significantly different from non-shocked, same time-point (p < 0.0012 at least).

Freezing was significantly increased throughout the whole testing period (H(1,16) values were between 10.50 and 12.88; p < 0.01). 3.2. Conditioned place preference Rats showed a slight preference towards one of the compartments during habituation trials. Compartment preference was not due to the features of the compartments, as the time spent in compartments A and B was similar (41.7  3.1 and 37.4  2.8% of total time, respectively; H(1,48) ¼ 0.61; p > 0.4). During habituation trial 1, compartment preference was stronger in shocked rats, who also showed a decreased number of compartment entries (not shocked: 23.3  1.0; shocked: 16.6  1.8; H(1, 43) ¼ 6.81; p < 0.01). As compartment entries and preference correlated significantly (Spearman R ¼ 0.357; p < 0.02), we attributed these differences to increased fear. The shock-induced change in the magnitude of compartment preference disappeared in the second (baseline) habituation trial, when compartment entries also normalized (22.6  1.3 vs. 19.5  1.5 entries in control and shocked rats, respectively; H(1,43) ¼ 2.19; p < 0.2), and the correlation between compartment preference and locomotion lost significance (R ¼ 0.049; p < 0.8). When baselines and post-conditioning trials were compared, treatment and time (the subsequent trials) showed a significant interaction (Finteraction(18, 234) ¼ 2.30; p < 0.003), demonstrating that place preference depended on the interplay between the two factors. In saline-conditioned rats, the arm preference seen in the baseline trial disappeared after mock conditioning (Table 1). The likely reason was that rats were extensively habituated to the apparatus during conditioning trials. Shocked and non-shocked rats were similar in this respect. As compared to the baseline, not shocked rats trained with morphine spent significantly more time in the morphine-associated compartment during post-conditioning trial 1 (p < 0.015) but not during other trials (p > 0.33) (Fig. 2). In shocked rats, the difference was significant for post-conditioning trials 1e5 (p < 0.01), but not for post-conditioning trial 6 (p > 0.1) (Fig. 2). We note that non-shocked rats spent somewhat more time in the morphine-associated compartment than shocked rats; however, the difference from baseline was statistically similar in the two groups (14.14  2.32 and 12.63  1.75, respectively). Thus, the magnitude of morphine-induced place preference was not affected by shocks, but the extinction of place preference was considerably delayed.

Compartment

Salineassociated

Mock drugassociated

p<

Not shocked. saline-trained Baseline trial Post-conditioning trial 1 Post-conditioning trial 2 Post-conditioning trial 3 Post-conditioning trial 4 Post-conditioning trial 5 Post-conditioning trial 6

41.24 34.08 35.61 39.81 34.09 38.44 39.18

1.73 1.41 2.69 4.19 2.22 3.22 3.49

32.12 32.71 35.02 28.80 33.47 35.07 29.07

1.94 2.65 2.41 2.95 2.98 2.27 3.14

0.01 0.70 0.90 0.20 0.50 0.50 0.20

Shocked. saline-trained Baseline trial Post-conditioning trial Post-conditioning trial Post-conditioning trial Post-conditioning trial Post-conditioning trial Post-conditioning trial

44.94 31.65 32.57 29.70 32.45 33.37 30.73

6.82 2.83 4.03 2.47 2.62 3.16 3.42

24.29 35.40 35.57 38.85 33.45 36.63 35.25

3.41 3.36 3.94 3.75 2.41 3.26 2.73

0.04 0.5 0.6 0.2 0.9 0.6 0.4

1 2 3 4 5 6

Data were expressed as % of total time  standard error of the mean. For data in the morphine-conditioned groups see Fig. 2. Note that 30e35% time was spent in the central (neutral) area. P values refer for post hoc comparisons. For ANOVA see text.

multiple significant interactions (Ftreatmentpost-injection time(16,520) ¼ 22.46; p < 0.0001; Finjection daypost-injection time(32,520) ¼ 2.23; p < 0.002; Ftreatmentinjection daypost-injection time(64,520) ¼ 1.43; p < 0.02). In vehicle-injected controls, the injection per se rapidly and transiently increased body temperature (Table 2). This response was slightly but not significantly reduced over time. Surprisingly, morphine significantly reduced the injection-induced increase in body temperature at 20 min. At later time-points, however, morphine robustly increased body temperature and this effect remained significant throughout in non-shocked rats (Fig. 3). In contrast, shocked rats rapidly habituated to the hyperthermic effect of morphine. Although non-shocked and shocked rats were similar on the first day, hyperthermia rapidly disappeared in the latter. In shocked rats, the increase in body temperature was only transient on the second day, whereas no significant effects were noticed on the following days. Virtually no increase was noticed on the last day of injections, when morphine still increased body temperature in the non-shocked group. Although the hyperthermic effect of morphine was significant on all days, a slight tolerance was noticed in non-shocked rats as well. In these rats, the duration of the increase in body temperature decreased as shown by the area under the curve calculated for those time-points, where the difference from controls was significant (F(4,20) ¼ 2.83; p ¼ 0.05; Fig. 3, bottom left-hand panel). Compared to day 1, the area under the curve decreased marginally significantly on day 4 (p ¼ 0.053 after Bonferroni correction for multiple comparisons), and significant on day 5 (p < 0.03 after correction). In addition, peak temperatures (i.e. those measured at the 5th time-point) also decreased over time (Fig. 3, bottom right-hand panel). The interaction between the day of testing and post-injection time was significant in non-shocked rats (F(32,160) ¼ 4.31, p < 0.0001) and peak temperature on day 5 differed significantly from that measured on day 1 (p < 0.05). 4. Discussion 4.1. Main findings

3.3. Morphine tolerance The temporal evolution of morphine-induced temperature changes depended on treatment, as the three factors showed

Shock exposure induced a lasting conditioned fear, a response frequently used to model PTSD in laboratory rodents. The extinction of conditioned place preference was considerably slower in

B. Barsy et al. / Neuropharmacology 60 (2011) 267e273

Time in drug associated compartment (% time)

270

Morphine conditioned, not shocked 60

4.2. Comparison with earlier findings

* 50

40

30

20

Time in drug associated compartment (% time)

Morphine conditioned, shocked 60

50

* *

40

* * *

30

20 BL 1

shocked as compared to non-shocked rats. In contrast to the nonshocked group, shocked rats rapidly developed tolerance towards the temperature-heightening effects of morphine.

2

3

4

5

6

Post-conditioning trials Fig. 2. Time spent in the morphine-associated compartment in non-shocked and shocked rats during post-conditioning trials. The levels noticed at baselines and their range of variation were outlined by the black horizontal lines and grey horizontal bars, respectively. Note that the ostensible difference in the magnitude of the response is explained by the difference in baselines. For data obtained in saline-conditioned rats see Table 1. *, Significantly different from baseline (p < 0.02 at least after Bonferroni correction). For ANOVA see text.

Although the conditioned fear response is usually studied shortly after shock exposure, it can be detected up to 200 days after shocks (Pamplona et al., 2006; Quinn et al., 2008; Sacchetti et al., 2001; Sutherland et al., 2008). In line with these earlier observations, our rats robustly expressed freezing in the shock-associated environment on the 28th post-shock day. The rapid disappearance of conditioned place preference in our non-shocked rats was somewhat surprising as extinction declined more slowly in a number of reports; moreover, place preference increased over time in some studies (Belda et al., 2008; Lei et al., 2005). There are reports, however, where conditioned place preference disappeared as quickly as in our experiment (i.e. within 2 test trials; Parker and McDonald, 2000; Vazquez et al., 2007). A close look at the literature shows that extinction depends on a multitude of factors. Conditioned place preference lasts longer if the duration of conditioning sessions is large. E.g. place preference was persistent when the duration of conditioning trials was 50 min, but disappeared quickly when conditioning trials lasted just 25 min (Lei et al., 2005; Vazquez et al., 2007). The number of conditioning trials has a similar effect; in the study by Mueller et al. (2002) where 8 conditioning trials of 45 min were run, place preference showed little decline over 12 weeks irrespective to the number of test trials performed (3 or 7 over 12 weeks). However, place preference disappeared rapidly when the number of conditioning trials was just 4, despite the fact that the duration of conditioning trials was only slightly lower (30 min) (Parker and McDonald, 2000). The morphine dose has little impact on the development of place preference but appears to affect extinction (Ribeiro Do Couto et al., 2005). The rapidity of extinction may depend on other conditions as well, e.g. on the number of compartments in the apparatus, and contextual cues (Belda et al., 2008). We conditioned our rats with 10 mg/kg morphine over 6 conditioning trials of 20 min each. Under comparable conditions, the extinction of place preference was similar to that noticed by us (Parker and McDonald, 2000; Ribeiro Do Couto et al., 2005; Vazquez et al., 2007). In rats, morphine may either raise or lower body temperature depending on the dose. In line with our observations, low doses (1e15 mg/kg) dose-dependently increase body temperature by 1e2.5  C for about 2e4 h (Benamar et al., 2003; Fraga et al., 2008; Rawls et al., 2003; Warwick and Schnell, 1976). In some studies, this hyperthermic effect was resistant to tolerance (Cox et al., 1976; Ganesan et al., 1991; Mannisto et al., 1994). In other studies, however, tolerance developed relatively rapidly (Bhalla et al., 2003; Mucha et al., 1987; Sharma et al., 2007; Villar and Bhargava, 1992). These discrepancies are likely explained by variations in either the number of extinction sessions or the time-frame of temperature measurements (Mucha et al., 1987). In our study, the hyperthermic

Table 2 The effects of saline injections on body temperature. Experimental day

1 2 3 4 5

Minutes from injection 20

40

60

80

100

120

140

160

180

1.30  0.06 1.00  0.16 1.01  0.14 1.19  0.13 0.77  0.09

1.45  0.15 0.90  0.16 0.63  0.18 0.55  0.11 0.98  0.32

0.90  0.22 0.41  0.08 0.36  0.14 0.32  0.11 0.86  0.14

0.32  0.13 0.38  0.06 0.23  0.11 0.33  0.14 0.85  0.20

0.13  0.11 0.26  0.06 0.09  0.08 0.06  0.12 0.54  0.15

0.39  0.16 0.28  0.09 0.05  0.13 0.18  0.09 0.26  0.09

0.34  0.27 0.37  0.11 0.38  0.24 0.11  0.17 0.13  0.13

0.13  0.16 0.33  0.09 0.39  0.14 0.13  0.15 0.22  0.13

0.12  0.17 0.56  0.11 0.30  0.20 0.13  0.20 0.41  0.15

Values (mean  SEM) were expressed in  C as differences from baseline. For ANOVA see text.

B. Barsy et al. / Neuropharmacology 60 (2011) 267e273

The effect of morphine on body temperature ( oC, change from control)

3

3

Day 1

* *

* * * * * * * * * *

2 1 0 -1

3

*

Day 2

* * * *

2 1

*

0

*

+

-1

* Day 3

2

3

Day 4

* * *

2

* * * *

1

1

0

0

-1

271

-1

*

* 20

60

100

140

180

Minutes from injection 3

Day 5

2

Morphine training

* * *

*

1

Shock + morphine training range of variation in controls

*

0 -1

significantly different from control (p< 0.05 at least)

* 20

60

100

140

180

Minutes from injection

12

AUC

9

+

6

#

3 0

1

2

3

4

5 Days

Peak temperatures

Changes in non-shocked, morphine trained rats over time 3 2

# 1 0

1

2

1

3

4

5 Days

Fig. 3. Body temperature changes after morphine injections. For clarity, values are shown as differences from control values. Statistics were performed on raw data. The range of variation in controls is indicated by the grey horizontal bars, while the temperatures noticed in controls are shown in Table 2. The bottom panels show that some degree of tolerance occurred in non-shocked rats. Tolerance was obvious in shocked rats; therefore, this was not illustrated by additional panels. AUC, area under the curve for the time-points where significant increases in body temperature were observed; Peak temperatures, average difference from baseline at the 5th time-point; #, significant difference from day 1 (bottom panels); þ, marginal difference from day 1 (p ¼ 0.053 after Bonferroni correction for multiple comparisons).

effect of morphine resulted in mild tolerance in non-shocked rats, as the peak temperatures and the duration of hyperthermia were reduced over time. Yet, non-shocked rats readily responded by hyperthermia to morphine on the fifth day of injections. 4.3. Stressors and conditioned place preference Behavior in the conditioned place preference paradigm provides important information on the motivational effects of drugs, and as

such it is among the most popular models of addiction in experimental animals (Tzschentke, 2007). Earlier studies showed that stressors affect conditioned place preference, but this interaction proved to be complex. Chronic mild stress and subordination reduced morphine place preference, suggesting that stress-induced depression-like states decrease the rewarding potential of opiates, as both paradigms are considered models of depression (Coventry et al., 1997; Fuchs et al., 2004; Lu et al., 2003; Miczek et al., 2008; Papp et al., 1992; Willner et al., 1992). In contrast, acute stressors

272

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(e.g. electric shocks) reliably reinstate opiate place preference, likely via the acute stress-induced activation of opioid neurotransmission (Miczek et al., 2008; Shaham et al., 2000). However, such phenomena cannot be accounted for the delayed extinction of morphine place preference that was observed here. More importantly for the present study, Will et al. (1998) showed that shocks increase the magnitude of morphine place preference. This effect lasted for one week but not for two weeks after shocks. In our study, conditioning started 4 weeks after shocks. At this time-point, the magnitude of the response (the time spent in the drug-associated compartment) was not increased, but the extinction of the response was considerably delayed. Thus, our study shows that the effects of shocks on morphine place preference last considerably longer than one week as suggested earlier (Will et al., 1998). However, in later phases (i.e. 4 weeks after the shocks) not the magnitude of the response but its resistance to extinction is increased. 4.4. Stressors and tolerance to the thermic effects of morphine Tolerance to the effects of drugs is one of the main symptoms of drug dependence (American Psychiatric Association, 1994). As a consequence, tests of tolerance are frequently used to evaluate the abuse potential of compounds (Kota et al., 2008; Naassila et al., 2004; Zachariou et al., 2006). The thermic effects of morphine are readily affected by stressors. However, the effects of these depend largely on experimental conditions. Under the influence of acute stressors, hyperthermic doses of morphine may become hypothermic (Ushijima et al., 1985). In contrast, electric shocks delivered repeatedly over 10 days lead to sensitization, i.e. increase morphine-induced hyperthermia (Benedek and Szikszay, 1985). Sensitization was seen also after repeated acoustic stress, while repeated restraint over 10 days decreased the effects of morphine on body temperature (Benedek and Szikszay, 1985). The effects of morphine on body temperature also depend on the intensity of stressors. Under low levels of stress, sensitization occurred, while a biphasic response was noticed under high stress conditions (Zelman et al., 1985). Here we show that a single exposure to electric shocks markedly accelerates tolerance to the hyperthermic effect of morphine. 5. Conclusions Shock exposure elicited conditioned fear, delayed the extinction of morphine place preference, and accelerated the tolerance to the hyperthermic effects of morphine. Effects were robustly shown one month after shock exposure. These findings show that electric shocks durably affect behavior in tests relevant to drug abuse in conjunction with the development of post-traumatic stress disorder-like behavioral dysfunctions e.g. in conjunction with conditioned fear. One can hypothesize that in an analogous human situation, traumatic experience increases the rewarding effects of morphine, but leads to rapid tolerance, which together promote both drug seeking and persistent drug use. Acknowledgements This study was supported by OTKA Grant No K 72621. References American Psychiatric Association, 1994. Diagnostic and Statistical Manual of Mental Disorders, fourth ed. American Psychiatric Association, Washington, DC. Barr, G.A., Paredes, W., Bridger, W.H., 1985. Place conditioning with morphine and phencyclidine: dose dependent effects. Life Sci. 36, 363e368.

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