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Addiction Biology
ORIGINAL ARTICLE
doi:10.1111/adb.12184
Social defeat in adolescent mice increases vulnerability to alcohol consumption Marta Rodriguez-Arias1,2, Francisco Navarrete2,3, Maria Carmen Blanco-Gandia1,2, Maria Carmen Arenas1,2, Adrián Bartoll-Andrés3, Maria A. Aguilar1,2, Gabriel Rubio2,4,5, José Miñarro1,2 & Jorge Manzanares2,3 Unidad de Investigación Psicobiología de las Drogodependencias, Departamento de Psicobiología, Facultad de Psicología, Universitat de València, Spain1, Red Temática de Investigación Cooperativa en Salud (RETICS-Trastornos Adictivos), Instituto de Salud Carlos III, MICINN and FEDER, Spain2, Instituto de Neurociencias, Universidad Miguel Hernández-CSIC, Spain3, Unidad de Psiquiatría, Hospital Universitario ‘12 de Octubre’, Spain4 and Instituto de Investigación ‘12 de Octubre’, Spain5
ABSTRACT This study employs an oral operant conditioning paradigm to evaluate the effects of repeated social defeat during adolescence on the reinforcing and motivational actions of ethanol in adult OF1 mice. Social interaction, emotional and cognitive behavioral aspects were also analyzed, and real-time polymerase chain reaction (PCR) experiments were performed to study gene expression changes in the mesocorticolimbic and hypothalamus-hypophysis-adrenal (HHA) axis. Social defeat did not alter anxiety-like behavior in the elevated plus maze or cognitive performance in the passive avoidance and Hebb–Williams tests. A social interaction test revealed depression-like symptoms and social subordination behavior in defeated OF1 mice. Interestingly, social defeat in adolescence significantly increased the number of effective responses, ethanol consumption values and motivation to drink. Finally, real-time PCR analyses revealed that social defeat significantly increased tyrosine hydroxylase and corticotropin-releasing hormone in the ventral tegmental area and paraventricular nucleus, respectively. In contrast, mu-opioid receptor gene expression was decreased in the nucleus accumbens of socially defeated mice. In summary, these findings suggest that exposure to social defeat during adolescence increases vulnerability to the rewarding effects of ethanol without affecting emotional or cognitive performance. The gene expression alterations we have observed in the mesocorticolimbic and HHA axis systems of defeated mice could be related with their increased ethanol consumption. These results endorse future research into pharmacological strategies that modulate these systems for the treatment of social stress-related alcohol consumption problems. Keywords defeat.
Anxiety, ethanol, hypothalamus-hypophysis-adrenal axis, memory, mesocorticolimbic system, social
Correspondence to: Marta Rodríguez-Arias, Unidad de Investigación Psicobiología de las Drogodependencias, Departamento de Psicobiología, Facultad de Psicología, Universitat de València, Avda. Blasco Ibáñez, 21, Valencia 46010, Spain. E-mail: [email protected]
INTRODUCTION Stressful life situations have been widely linked to drug seeking and consumption (Sinha 2001; Stewart 2003). Several studies in humans and animal models have demonstrated that exposure to different kinds of stress increases abuse and relapse to abuse of certain drugs, such as cocaine and ethanol (Derr & Lindblad 1980; Kreibich et al. 2009). Among the different types of stress that individuals may experience, social stress early in life—for example, bullying at school or child abuse—can have critical physiological and behavioral consequences © 2014 Society for the Study of Addiction
in adulthood (de Groot et al. 1999; Lumley et al. 1999). The term bullying is defined as a conscious and willful act of aggression and/or manipulation by one person (bully) against another person (victim) (Sullivan 2000). This behavior produces an emotional state known as social defeat, a phenomenon that can be modeled in animals by means of the resident–intruder paradigm, in which isolated, aggressive mice (residents) are confronted with grouped animals (intruders) through protocols that vary according to the number of social defeat experiences or moment at which the experience is inflicted (Koolhaas et al. 2013). Addiction Biology
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Social defeat represents the main form of social stress that leads to psychopathological changes (Bjorkqvist 2001). In animal models, social defeat stressors have an ecological and ethological validity, as they closely mimic real-life human situations (Shimozuru et al. 2006). Emotional disturbances (e.g. anxiety, depression) have been associated with repeated social defeat experiences and, in turn, with the development of drug addiction (Burke, Burke & Rae 1994). One of the first studies to evaluate the effect of social defeat on the reinforcing effects of cocaine demonstrated that defeated mice acquired cocaine selfadministration behavior in half the time required by controls (Tidey & Miczek 1997). Furthermore, the motor sensitization effects of cocaine have been found to be more pronounced in mice exposed to the resident– intruder paradigm (Miczek et al. 1999), and several social defeat experiences have been shown to significantly increase 24 hours cocaine binge-like consumption (Covington et al. 2005) and cocaine self-administration (Covington & Miczek 2005) in rats. On the other hand, morphine-induced motor sensitization is potentiated by social defeat, although this effect seems to depend on the number of stressful experiences and the interval between stress and locomotor testing (Stohr et al. 1999). Interestingly, our group has observed that social defeat is as effective as physical stress in inducing reinstatement of the morphine-induced conditioned place preference task (Ribeiro Do Couto et al. 2006). On the other hand, there are discrepant data regarding the effect of social defeat on ethanol intake. Acute exposure to social defeat was found to reduce alcohol self-administration in rats (Funk et al. 2005), while rats exposed to five brief episodes of social defeat exhibited increased alcohol drinking on the day of stress-exposure and even higher consumption 1 week later (Caldwell & Riccio 2010). Conversely, a brief social stress experience on five consecutive days was found to significantly decrease alcohol intake and rate of alcohol reinforcement in mice (van Erp & Miczek 2001). Most of the aforementioned studies have been performed in adult animals, while the consequences of social defeat experienced during adolescence have been less studied. This is relevant given that neurobehavioral and hormonal responses to stressors vary between adolescents and adults (Allen & Matthews 1997). Neuroadaptative changes in mesocorticolimbic circuitry in animals exposed to social defeat may explain, at least in part, their higher vulnerability to the reinforcing effects of a drug. Evaluation—using in vivo microdialysis—of dopamine concentrations in the nucleus accumbens (NAcc) of defeated and non-defeated mice has shown that social subordination induces a more pronounced increase in dopamine levels with respect to control mice (Tidey & Miczek 1997). In addition, when Nikulina and colleagues studied the effects of © 2014 Society for the Study of Addiction
social defeat on mu-opioid receptor (MOr) mRNA levels in the ventral tegmental area (VTA) of mice, they observed a clear up-regulatory effect that appeared to be related with enhanced dopaminergic activation in the mesocorticolimbic system (Nikulina et al. 2008). On the other hand, several studies have indicated the crucial role of the hypothalamus-hypophysis-adrenal (HHA) axis, which is activated by social stress experiences and leads to a significant increase in corticotropin-releasing factor in the paraventricular nucleus (PVN) and the hippocampus (HIPP; Keeney et al. 2006). In the present study, we have employed the resident– intruder paradigm to evaluate the effect of social defeat during adolescence on the emotional (anxiety-like behavior) and cognitive (memory) state of OF1 mice during adulthood. An additional goal of our study was to employ an oral self-administration test to analyze if the social defeat protocol applied during adolescence produces any changes in the reinforcing and motivational actions of ethanol in adult mice. Finally, we have performed real-time polymerase chain reaction (PCR) experiments in socially defeated mice and their corresponding controls in order to study gene expression changes in different targets involved in the function of the mesocorticolimbic system and HHA axis.
METHODS Animals A total of 102 male mice of the OF1 strain (Charles River, Barcelona, Spain) were employed in the study: 77 as experimental subjects and 40 as standard opponents and residents. Experimental animals were 21 days old on arrival at the laboratory and were all housed under standard conditions in groups of four (in cages 28 × 28 × 14.5 cm), at a constant temperature (21 ± 2°C), with a reversed light schedule (white lights on 19:30–7:30 hours) and food and water available ad libitum (except during the behavioral test). All studies were conducted in compliance with guidelines of the European Council Directive 2010/63/UE regulating animal research and were approved by the local ethical committees. Repeated social defeat Animals in the experimental group were exposed to four episodes of social defeat lasting 25 minutes each. Each episode consisted of three phases, which began by placing the experimental animal or intruder in the home cage of the aggressive opponent or resident for 10 minutes. During this initial phase, the intruder was protected from attack by a wire mesh wall that permitted social Addiction Biology
Social defeat and ethanol
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Hebb–Williams Social Interaction Passive avoidance Oral ethanol self-administration Gene expression analyses EPM
Fourth social defeat Fourth social defeat Third social defeat Third social defeat Second social defeat Second social defeat First social defeat First social defeat
Third social defeat Second social defeat First social defeat
33 30 27
For the passive avoidance test, a step-through inhibitory avoidance apparatus for mice (Ugo Basile, ComerioVarese, Italy) was employed. This cage is made of Perspex sheets and contains two compartments (15 cm × 9.5 cm × 16.5 cm each). The safe compartment is white
PND
Passive avoidance test
Table 1 Experimental procedure.
EPM tests were carried out essentially following the procedure described by Daza-Losada et al. (2009). In brief, the EPM consisted of two open arms (30 × 5 × 0.25 cm) and two enclosed arms (30 × 5 × 15 cm). The junction of the four arms formed a central platform (5 × 5 cm). The entire apparatus was elevated 45 cm above floor level. In order to facilitate adaptation, mice were transported to the dimly illuminated laboratory 1 hour before testing. The mice’s behavior was videorecorded and later analyzed by a ‘blind’ observer using a computerized method. The experimental room was illuminated with a dim red light (40 lux at 1 m above floor level). The measurements recorded during the test period were frequency of entries and time and percentage of time spent in each section of the apparatus (open arms, closed arms, central platform). An arm was considered to have been visited when the animal placed all four paws on it.
36
Elevated plus maze (EPM)
Fourth social defeat
37–56
57
59–60
61
62–69
70–87
interaction and species-typical threats from the male aggressive resident (Covington & Miczek 2001). In the second phase, the wire mesh was removed from the cage and a 5-minute period of confrontation began. In the third phase, the wire mesh was replaced for a further 10 minutes to allow social threats from the resident. In the third phase, socially defeated animals (n = 15 in the first study, and 16 in the second) were exposed to social defeat on postnatal day (PND) 27, 30, 33 and 36. The exploration group (n = 15 in both experiments) underwent the same protocol, but without the presence of a ‘resident’ mouse in the cage. Following this last phase, animals were kept in the vivarium for 3 weeks, after which the behavioral tests began. The second phase of each social defeat protocol was video-recorded and ethologically analyzed (n = 15). The following behaviors were scored for resident mice: threat and attack; and time needed to perform the first threat and attack (latencies). In the case of intruder mice, the following behaviors were analyzed: avoidance/flee and defensive/submissive behaviors; and time needed to exhibit the first avoidance/flee or defense/submission (latencies). Three different sets of mice were employed in this study. A more detailed description of the experimental procedure is provided in Table 1.
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and illuminated by a light fixture (10 W) fastened to the cage lid, whereas the ‘shock’ compartment is dark and black. The two compartments are divided by an automatically operated sliding door at floor level. The floor is made of 48 stainless steel bars with a diameter of 0.7 mm and situated 8 mm apart. Passive avoidance tests were carried out essentially following the procedure described by Aguilar, Minarro & Felipo (2000). On the day of training, each mouse was placed in the illuminated compartment facing away from the dark compartment. After a 60-second period of habituation, the door leading to the dark compartment was opened. When the animal had placed all four paws in the dark compartment, a footshock (0.5 mA, 3 seconds) was delivered and the animal was immediately removed from the apparatus and returned to its home cage. The time taken to enter the dark compartment (step-through latency) was recorded. Retention was tested 24 hours later by submitting the animal to the same procedure but without applying the shock. The maximum step-through latency was 300 seconds. Social interaction test This test consisted of confronting an experimental animal and a standard opponent in a neutral cage (61 × 30.5 × 36 cm) for 10 minutes following a 1-minute adaptation period prior to the encounter. One day before testing, standard opponents were rendered temporarily anosmic by intranasal lavage with a 4% zinc sulfate solution. This kind of mouse induces an attack reaction in its opponent, but does not outwardly provoke or defend itself since it cannot perceive a pheromone that is present in the urine of the experimental animals and functions as a cue for eliciting aggressive behavior in mice with a normal sense of smell. The videotapes were analyzed using a custom-developed program that estimates the time devoted to different broad functional categories of behavior (non-social exploration, social investigation, threat, attack and avoidance/flee), each of which is characterized by a series of different postures and elements. A more detailed description can be found in Rodriguez-Arias et al. (1998). Hebb–Williams maze The maze (60 cm wide × 60 cm long × 10 cm high) used in our experiment is made of black plastic and contains a start box and a goal box (both 14 cm wide × 9 cm long) positioned at diagonally opposite corners. The maze contains cold water (15°) at a wading depth (3.5 cm), while the goal box is stocked with fresh dry tissue. Several maze arrangements can be made by fixing different barriers to the clear plastic ceiling. This apparatus allows the cognitive process of routed learning and the motivation of water escape to be measured. © 2014 Society for the Study of Addiction
The procedure was based on that employed by Galsworthy et al. (2005), in which mice must navigate the maze and cross from the wet start box to the dry goal box in order to escape the cold water. Animals underwent a 5 minute habituation period (dry sand, no barriers) on day 1 and undertook problem A on day 2 and problem D on day 3 (four trials/day; practice mazes). Mice were subsequently submitted to mazes 1, 5, 3, 4 and 8 on separate days on which eight trials took place. The time limit for a mouse to reach the goal box on its own was 5 minutes, after which the animal was guided to the dry area. The following measurements were recorded: total latency score (sum of latencies of all the problem trials in each maze); and error score, for which a similar total was used (where ‘error’ was considered as entering the error zone, as specified by Galsworthy et al. (2005).
Oral ethanol self-administration Evaluation of ethanol self-administration was carried out in 12 modular operant chambers (Panlab, Barcelona, Spain) in three phases: training, saccharin substitution and 6% ethanol consumption. During the last phase, the number of effective responses and ethanol consumption (μl) were measured under fixed ratio 1 (FR1), fixed ratio 3 (FR3) and progressive ratio (PR) schedules. For more details, see the Supporting Information.
Gene expression analyses—real-time PCR Mice were killed 3 weeks after the last social defeat. Brains were removed from the skull and frozen on dry ice. Coronal brain sections (500 μm) were obtained at plates 19–20 (distance from the bregma: 1.42 and 1.34 mm, respectively) in a cryostat (−10°C) according to Paxinos & Franklin (2001). VTA, PVN and NAcc were microdissected according to the Palkovits method (Palkovits 1983). Total RNA was isolated from brain tissue micropunches using TRI Reagent® (Applied Biosystems, Madrid, Spain) and were subsequently retrotranscribed to cDNA. Quantitative analysis of the relative abundance of tyrosine hydroxylase (TH) (Mm00447546_m1) gene expression in VTA, MOr (Mm01188089_m1) gene expression in NAcc and corticotropin-releasing hormone (CRH; Mm01293920_s1) gene expression in PVN between the social defeat group and control mice was performed using the StepOne Sequence Detector System (Life Technologies, Madrid, Spain). All reagents were obtained from Life Technologies and manufacturers’ protocols were followed. The chosen reference gene was Rn18S (Mm03928990_g1), which was detected using Taqman ribosomal RNA control reagents. All primerprobe combinations were optimized and validated for relative quantification of gene expression. In brief, data for Addiction Biology
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Table 2 Social interaction during the resident–intruder paradigm to induce social defeat (SD). Resident
Threat
Threat latency
Attack
Attack latency
SD1 SD2 SD3 SD4
21.8 ± 2.3 26.2 ± 2.5 21.9 ± 1.6 17 ± 1.8**
3.6 ± 0.8 4.7 ± 2.0 3.2 ± 1.0 3.4 ± 1.3
17.9 ± 2.2 21.2 ± 2.0 24.3 ± 3.4 22 ± 3.6
24.9 ± 12.7 9.2 ± 3.2 6.9 ± 2.5 3.9 ± 1.3
Intruder
Avoidance/flee
Avoidance/flee latency
Defensive/submissive
Defensive/submissive latency
SD1 SD2 SD3 SD4
14.8 ± 2.1 15.8 ± 1.6 16.3 ± 2.5 16.8 ± 2.3
20.2 ± 4.9 6.5 ± 2.0 7.6 ± 2.6 5.3 ± 1.6*
49.9 ± 7.13 75.1 ± 7.16 69.6 ± 3.9 66.8 ± 6.4
33.6 ± 13.4 10.7 ± 2.4 5.5 ± 1.1 9.7 ± 2.6
Data presented as mean values ± SEM. *P < 0.03; differences with respect to the first social defeat (SD1); **P < 0.02; differences with respect to the second social defeat (SD2).
each target gene were normalized to the endogenous reference gene, and the fold change in target gene mRNA abundance was determined using the 2-ΔΔCt method (Livak & Schmittgen 2001) so that the level of each social defeat group was expressed in relation to that of the control group. Statistical analysis The data of the ethological analyses of resident and intruder mice were analyzed by a one-way ANOVA with a within variable—social defeat—with four levels: first, second, third and fourth social defeat. Data relating to social interaction, the EPM and motor activity were analyzed by a one-way ANOVA with a between variable— social defeat—with two levels: exploration or social defeated. The passive avoidance test was analyzed by a two-way ANOVA, with the same between variable— social defeat—and one within variable with two levels: training day or test day. The data of the Hebb–Williams maze were analyzed by a two-way ANOVA with one between subject variable—social defeat—and one within subject variable—number of labyrinths in the maze— with five levels. Mice unable to complete the task within the time limit were attributed maximum latency scores. Statistical analyses for self-administration of ethanol were performed using two-way ANOVA with repeated measures followed by the Student’s-Newman-Keuls test to compare social defeat and control groups at different time points of the oral self-administration paradigm. A Student’s t-test was employed to compare the effects of social defeat on the number of effective responses, breaking point values and ethanol consumption during PR. Student’s t-test was employed to compare the effects of social defeat on TH, MOr and CRH gene expression. Differences were considered significant if the probability of error was less than 5%. SigmaStat v3.11 software © 2014 Society for the Study of Addiction
Table 3 Effects of repeated SD on adolescent mice in the EPM.
Time in open arms % Time in open arms Time in central platform Time in closed arms Entries in open arms % Open entries Entries in closed arms Total entries
Control
Social defeat
39.6 ± 10.4 15.8 ± 4 64.18 ± 10 204.6 ± 12.6 4.2 ± 1.2 17.24 ± 3.9 17 ± 2.1 21.26 ± 2.6
36 ± 7.4 15.44 ± 3.2 69.7 ± 6.5 200 ± 9.7 2.9 ± 0.7 14.54 ± 2.8 15 ± 1.1 18.71 ± 1.3
Data are presented as mean values ± SEM.
(Systat Software Inc., Chicago, IL, USA) was employed for all statistical analyses.
RESULTS Social defeat The ANOVA for resident mice revealed an effect of the time spent engaged in threat [F(3, 42) = 3.288; P < 0.03], showing that mice displayed less threat behavior in the last social defeat than during the second encounter (P < 0.03). In the case of intruder mice, the latency of avoidance/flee showed a significant effect [F(3, 42) = 5.646; P < 0.02]; mice displayed their first avoidance/flee behavior significantly sooner in the fourth social defeat than in the first encounter (P < 0.02; see Table 2). EPM EPM data (see Table 3) revealed no significant differences between the two groups of animals. No significant differences were detected between socially defeated and control Addiction Biology
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Table 4 Means of accumulated times (in seconds) with SEM engaged in different categories of spontaneous behavior in the social interaction test by socially defeated (n = 15) and exploration (n = 15) mice.
Non-social exploration Social investigation Unit of social investigation Threat Attack Avoidance/flee Defensive/submissive
Control
Social defeat
476.52 ± 24.23 56.4 ± 15.69 2.55 ± 0.98 9.68 ± 4.42 1.42 ± 0.86 0±0 0±0
530.42 ± 17.19** 23.57 ± 16.66** 1.73 ± 0.74* 2.37 ± 1.98 0.38 ± 0.28 0.57 ± 0.8* 3.18 ± 4.19**
Differences from their respective control, *P < 0.01; **P < 0.001.
mice with respect to time spent in open arms [F(1, 27) = 0.077; P > 0.05], percentage of time spent in open arms [F(1, 27) = 0.006; P > 0.05] and the number of entries in open arms [F(1, 27) = 0.989). Social interaction test The data for the different types of behavior evaluated in the social interaction test are presented in Table 4. Socially defeated mice spent less time engaged in social investigation than exploration mice [F(1, 28) = 30.866; P < 0.001], although differences between the two groups were not significant (P < 0.001). The mean times spent engaged in each contact (unit of social investigation) were similar [F(1, 28) = 6.614; P < 0.02], being lower among socially defeated animals (P < 0.02), who spent more time engaged in non-social exploration [F(1, 28) = 49.354; P < 0.001] than control animals (P < 0.001). Avoidance/flee [F(1, 28) = 7.545; P < 0.01] and defensive/submissive [F(1, 28) = 8.6; P < 0.01] behaviors were observed only among socially defeated mice (P < 0.01 in all cases). Passive avoidance test and Hebb–Williams maze No significant differences were observed between socially defeated animals and controls in the passive avoidance test or the Hebb–Williams Maze. Additional Supporting Information is provided on the publisher’s website (Supporting Information Figs S2 & S3). The effect of social defeat on self-administration of ethanol Oral ethanol administration was initiated 3 weeks after the last social defeat experience. No differences were detected between control and socially defeated mice during training or substitution phases, which implied that the social defeat procedure did not induce any learning deficit (see Supporting Information Fig. S1). In the last stage of the self-administration protocol (6% ethanol, 0% saccharin), differences were detected between the two groups with respect to both the number of effective © 2014 Society for the Study of Addiction
responses (Fig. 1a; two-way ANOVA with repeated measures followed by the Student’s-Newman-Keuls method: social defeat: F(1, 259) = 27.921, P < 0.001; day: F(9, 259) = 92.450, P < 0.001; social defeat × day interaction: F(9, 259) = 2.882, P = 0.003) and 6% ethanol consumption (Fig. 1b; two-way ANOVA with repeated measures followed by the Student’s-NewmanKeuls method: social defeat: F(1, 259) = 9.624, P = 0.005; day: F(9, 259) = 9.842, P P < 0.001; social defeat × day interaction: F(9, 259) = 1.257, P = 0.262) in the FR1 and FR3 stages. In addition, during PR, breaking point values were significantly higher among socially defeated mice (Fig. 1c, Student’s t-test: t = −2.526, 20 d.f., P = 0.020), although 6% ethanol consumption (Fig. 1d, Student’s t-test: t = −1.073, 28 d.f., P = 0.292) was very similar in both groups. Effect of social defeat on TH, MOr and CRH gene expression Real-time PCR analyses indicated that social defeat affected TH gene expression in the VTA of the experimental mice, which presented significantly higher TH gene expression values (155%) than the control group. (Fig. 2a, n = 8, Student’s t-test, t = −4.263, 14 d.f., P = < 0.001). On the other hand, MOr gene expression was lower in the NAcc of socially defeated mice (−70%) than in that of control animals (Fig. 2b, n = 8, Student’s t-test: t = 3.646, 14 d.f., P = 0.003). Finally, social defeat increased CRH gene expression in the PVN (60%) in relation with levels in control mice (Fig. 2c, n = 7, Student’s t-test: t = 3.633, 12 d.f., P = 0.003). DISCUSSION The results of the present study indicate that repeated experience of social defeat during adolescence reduces social behavior without increasing anxiety, does not produce cognitive disturbances and heightens the reinforcing and motivational effects of ethanol during adulthood in mice. This conclusion is supported by the following facts: (1) socially defeated mice presented Addiction Biology
Social defeat and ethanol
(a)
FR1
80
(b)
FR3
FR1
60
* *
* *
* *
40
20
6% EtOH Consumption (µl)
Effective responses
* *
FR3
* *
1600
CONTROL SOCIAL DEFEAT
* *
* *
* *
1400
7
* *
1200 1000 800
* *
600 400 200
0
0 1
2
3
4
5 6
7
8
9
1
10
2
3
4
(d) 70
350
60
300
6% EtOH Consumption (µl)
Breaking point
(c)
PR
5 6
7
8
9
10
Day
Day
*
50 40 30 20 10 0
250 200 150 100 50 0
Control
Soc. defeat
Control
Soc. defeat
1.6
Ct)
*
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
Ct)
1.8 1.6 1.4 1.2 1.0
*
0.8 0.6 0.4 0.2 0.0
Control
Soc. defeat
Control
Soc. defeat
Corticotropin release hormone (2-
NAcc
VTA 1.8
µ-opioid receptor (2-
Tyrosine hydroxylase (2-
Ct)
Figure 1 Analysis of oral ethanol self-administration in OF1 mice (n = 31).The dots represent means and the vertical lines ± SEM of (a) the number of effective responses and (b) the volume of 6% ethanol consumption during FR1 and FR3.The columns represent means and the vertical lines ± SEM of (c) the breaking point values during PR and (d) the volumes of 6% ethanol consumption. *Represents the values of the socially defeated group that are significantly different (P < 0.05) from those of the control group
PVN 3.0
*
2.5 2.0 1.5 1.0 0.5 0.0
Control
Soc. defeat
Figure 2 (a) Real-time PCR TH relative gene expression evaluation in the VTA brain region of control and socially defeated groups (n = 8). (b) MOr relative gene expression evaluation in the NAcc brain region of these groups (n = 8). (c) CRH relative gene expression evaluation in the PVN brain region of the mice (n = 7).The columns represent means and the vertical lines ± SEM of relative (2-ΔΔCt method) TH gene expression in the VTA. Basal MOr gene expression in the NAcc and the CRH gene expression in the PVN of OF1 mice. *Represents the values of socially defeated mice that are significantly different (P < 0.05) from those of their corresponding control mice
significantly lower values of social investigation and displayed avoidance, flee, defensive and submissive behaviors in the social interaction test; (2) no differences were observed between groups in the time spent in the open arms in the EPM; (3) the latency to enter the dark compartment in the passive avoidance test and the time © 2014 Society for the Study of Addiction
required to reach the goal in each of the Hebb–Williams mazes were similar in defeated and non-defeated mice; (4) the number of effective responses, volume of ethanol consumption and breaking point values were significantly higher in OF1 mice exposed to social defeat during adolescence than in controls; and (5) TH, MOr and CRH gene Addiction Biology
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expression was altered in the VTA, NAcc and PVN, respectively, of socially defeated OF1 mice. Several studies have identified chronic social defeat as a model of depression in adult and adolescent animals for two main reasons: (1) socially defeated animals usually show depression-like behaviors such as social avoidance and behavioral despair; and (2) treatment with antidepressant drugs ameliorates these depression-like symptoms (Becker et al. 2008). Accordingly, we have observed that repeated experience of social defeat during adolescence induces avoidance and flee behaviors in the social interaction test when performed during adulthood. In addition, our socially defeated mice adopted defensive and submissive positions with respect to control animals, thus indicating a significant trend toward social subordination. Several studies have reported an increase in anxiety and despair behaviors in socially defeated adolescent mice (Kinsey et al. 2007; Huang et al. 2013; Iñiguez et al. 2014) and rats (Lio et al. 2011); however, in the present study, we found no differences between the total time spent in the open arms of the EPM by socially defeated versus control mice, as shown in Table 3. This could be due to considerable methodological differences among the studies in question. Firstly, different species and strain of animals have been used. For example, Vidal and colleagues observed that defeated Wistar rats developed social anxiety behavior, whereas defeated WT Groeninger rats did not (Vidal, Buwalda & Koolhaas 2011). Secondly, other studies have inflicted a higher number of social defeat experiences than ours. Moreover, behavioral procedures have been carried out, in most cases, immediately after the stress experience. On the other hand, in line with our results, Watt et al. demonstrated that repeated experience of social defeat during mid-adolescence did not induce anxiety-like behaviors in the EPM 3 weeks later (Watt et al. 2009). Equally, Bourke & Neigh (2011) reported that a chronic mixed modality stressor during adolescence induced sustained changes in depressive-like behavior during both adolescence and adulthood in female but not in male rats. We evaluated the effect of social defeat on aversive memory in the passive avoidance task, finding no differences between control and defeated mice in the time taken to enter the dark compartment. Again, when the animals performed the Hebb–Williams test in order to assess working memory, no significant differences were observed between the two groups. Previous studies have associated exposure to social stress with cognitive impairment; however, in the studies in question cognitive evaluation was carried out immediately or shortly after the end of a chronic stress period or an acute stressor (Park, Campbell & Diamond 2001). However, Buwalda and colleagues found no differences between controls and adult rats that had experienced social defeat three weeks previ© 2014 Society for the Study of Addiction
ously when they evaluated spatial learning and memory in the Morris water maze (Buwalda et al. 2005). Therefore, the cognitive impairment induced by social stress would seem to become apparent in the early stages following a stressful experience, and may be attributable to the evident disturbances reported in the HHA axis. In the literature, there is clear evidence of the relationship between exposure to social stress events and the development of drug addiction. Many studies have shown that social defeat is closely associated with an increase in the locomotor, reinforcing and motivational actions of psychostimulants (Covington & Miczek 2005). However, much less is known about the effect of social defeat on the rewarding effects of ethanol. A recent systematic quantitative study aimed to identify the main factors involved in stress-induced alcohol consumption, showed that increases in alcohol intake are best observed in adult male rats exposed to force swim or foot shock within the context of free-choice home-cage drinking paradigm (Noori, Helinski & Spanagel 2014). In the present study, we evaluated the effects of intermittent social defeat in adolescence on oral self-administration of ethanol during adulthood in OF1 mice. Socially defeated mice displayed increased effective responses to the active lever and higher ethanol consumption values during FR1 and FR3 stages. In the PR, defeated mice had higher breaking point values with respect to their controls, although no differences were detected in ethanol consumption. These results are in accordance with those of previous studies evaluating the effect of social defeat on the reinforcing actions of ethanol. Croft et al. reported that social stress experienced in adulthood increased ethanol consumption and preference in a two-bottle system paradigm for 3 weeks after the last social defeat. Interestingly, they compared a single experience of social defeat with five consecutive daily defeat experiences and a weekly experience over 4 weeks, and concluded that only the repetitive protocol of five consecutive sessions increased vulnerability toward the rewarding effects of ethanol in C57BL10 mice (Croft et al. 2005). On the other hand, in another study employing a similar protocol to induce social defeat in adult rats (five consecutive sessions), ethanol self-administration was significantly higher in non-preferring defeated rats only 2 hours after the last social defeat experience (Caldwell & Riccio 2010). Finally, Funk and colleagues studied the effect of social defeat on ethanol self-administration during extinction and reinstatement stages in adult rats. They concluded that social defeat immediately before self-administration sessions decreased ethanol consumption, did not affect extinction and did not induce a reinstatement effect (Funk et al. 2005). Overall, these previous results provide valuable information about the importance of temporary factors since significant effects only seem to appear when Addiction Biology
Social defeat and ethanol
there is a lapse of time between social defeat and evaluation of ethanol consumption. Therefore, the novelty of our study lies in the following aspects: (1) application of the social defeat protocol during adolescence; and (2) evaluation of the long-lasting effects of social defeat on the reinforcing and motivational actions of ethanol during adulthood (3 weeks later). The reinforcing effects of ethanol have been widely related with activation of the dopaminergic mesolimbic system through different mechanisms, of which the firing rate of dopaminergic neurons in the VTA is 1 (Brodie, Shefner & Dunwiddie 1990). Furthermore, an up-regulation of the TH in VTA under acute (Oliva et al. 2008) or chronic (Lee et al. 2005) ethanol administration has been described. In the present study, social defeat significantly increased TH gene expression, suggesting that enhanced dopamine synthesis is related with increased ethanol self-administration. Accordingly, several studies have found that social defeat increases dopamine signaling in the mesocorticolimbic pathway even 3 weeks later (Razzoli et al. 2011). Another mechanism responsible for the rewarding actions of ethanol is the increase in dopamine release produced through activation of MOr in the VTA and NAcc (Cowen & Lawrence 1999). In addition, several studies have demonstrated the important role played by MOr in the regulation of psychosocial stress effects (Wang et al. 2002; Komatsu et al. 2011). In the present study, real-time PCR gene expression analyses have revealed lower MOr gene expression levels in the NAcc of socially defeated mice. On the contrary, other studies have demonstrated a significant and long-lasting increase in MOr mRNA levels in the VTA (Nikulina et al. 2008). This discrepancy could be due to the different regions studied (NAcc versus VTA) or to the animal species employed (mouse versus rat). Finally, when evaluated to find a possible association between high ethanol self-administration and HHA axis impairments, CRH gene expression in the PVN of socially defeated mice was significantly higher than in control mice, as previously described in another regions, such as HIPP (Keeney et al. 2006). This is relevant, as alterations in the HHA axis have been related with the development of drug addiction (Zorrilla, Logrip & Koob 2014). Among other molecular events, stress-induced elevation in glucocorticoid levels could influence mesolimbic dopaminergic activity via glucocorticoid receptors in D1 receptor-expressing medium spiny neurons of the NAcc regulating the firing rate of mesolimbic dopamine neurons (Spanagel, Noori & Heilig 2014). The rationale behind the real-time PCR analyses performed in the present study is as follows: (1) TH gene expression was measured in the dopaminergic cell bodies of the mesocorticolimbic system located in the VTA, where dopamine synthesis takes place; (2) MOr was analyzed in © 2014 Society for the Study of Addiction
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the NAcc, as several studies have demonstrated that the modulation of dopaminergic neurotransmission by MOr in this specific region is critically involved in the reinforcing effects of ethanol; and (3) CRH was evaluated in the PVN because the parvocellular neurons of this brain region are responsible for the synthesis of this HHA axis regulatory hormone. In conclusion, the results presented in this study show that social defeat experiences during adolescence increase the reinforcing and motivational actions of ethanol during adulthood without impairing cognitive performance or anxiety-like behavior. Interestingly, alterations in gene expression in the mesocorticolimbic system and HHA axis of socially defeated mice could lie behind higher ethanol consumption vulnerability. Further studies are necessary to explore if pharmacological manipulation of these systems might be useful in managing ethanol consumption problems related with early social defeat experiences. Acknowledgements This work was supported by the following research grants: Ministerio de Ciencia e Innovación, (SAF201123420) to Jorge Manzanares; Ministerio de Economía y Competitividad, Dirección General de Investigación (PSI2011-24762) to Jose Miñarro; Generalidad Valenciana, Consejería de Educación (PROMETEO/2009/ 072) to Jose Miñarro; and Instituto de Salud ‘Carlos III’ (FIS), Redes Telemáticas de Investigación Cooperativa en Salud (RETICS), Red de Trastornos Adictivos (RTA), fondos FEDER (RD06/0001/1004, RD12/0028/0019 to Jorge Manzanares; and RD06/001/0016, RD12/0028/ 005 to Jose Miñarro). Authors Contribution All named authors have made an active contribution to the conception, design, analysis and drafting of the article. All authors have contributed equally to the overall coordination of the whole study. MR, MCA, MCBG and MAA performed the social defeat protocol and evaluated social, emotional and cognitive behavioral parameters. FN and AB were responsible for the supervision of oral ethanol self-administration experiments and realtime PCR analysis. All authors critically reviewed the content and approved the final version for publication. References Aguilar MA, Minarro J, Felipo V (2000) Chronic moderate hyperammonemia impairs active and passive avoidance behavior and conditional discrimination learning in rats. Exp Neurol 161:704–713. Allen MT, Matthews KA (1997) Hemodynamic responses to laboratory stressors in children and adolescents: the influences of age, race, and gender. Psychophysiology 34:329–339. Addiction Biology
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Becker C, Zeau B, Rivat C, Blugeot A, Hamon M, Benoliel JJ (2008) Repeated social defeat-induced depression-like behavioral and biological alterations in rats: involvement of cholecystokinin. Mol Psychiatry 13:1079–1092. Bjorkqvist K (2001) Social defeat as a stressor in humans. Physiol Behav 73:435–442. Bourke CH, Neigh GN (2011) Behavioral effects of chronic adolescent stress are sustained and sexually dimorphic. Horm Behav 60:112–120. Brodie MS, Shefner SA, Dunwiddie TV (1990) Ethanol increases the firing rate of dopamine neurons of the rat ventral tegmental area in vitro. Brain Res 508:65–69. Burke JD Jr., Burke KC, Rae DS (1994) Increased rates of drug abuse and dependence after onset of mood or anxiety disorders in adolescence. Hosp Community Psychiatry 45:451– 455. Buwalda B, Kole MH, Veenema AH, Huininga M, de Boer SF, Korte SM, Koolhaas JM (2005) Long-term effects of social stress on brain and behavior: a focus on hippocampal functioning. Neurosci Biobehav Rev 29:83–97. Caldwell EE, Riccio DC (2010) Alcohol self-administration in rats: modulation by temporal parameters related to repeated mild social defeat stress. Alcohol 44:265–274. Covington HE 3rd, Miczek KA (2001) Repeated social-defeat stress, cocaine or morphine. Effects on behavioral sensitization and intravenous cocaine self-administration ‘binges’. Psychopharmacology (Berl) 158:388–398. Covington HE 3rd, Miczek KA (2005) Intense cocaine selfadministration after episodic social defeat stress, but not after aggressive behavior: dissociation from corticosterone activation. Psychopharmacology (Berl) 183:331–340. Covington HE 3rd, Kikusui T, Goodhue J, Nikulina EM, Hammer RP Jr., Miczek KA (2005) Brief social defeat stress: long lasting effects on cocaine taking during a binge and zif268 mRNA expression in the amygdala and prefrontal cortex. Neuropsychopharmacology 30:310–321. Cowen MS, Lawrence AJ (1999) The role of opioid-dopamine interactions in the induction and maintenance of ethanol consumption. Prog Neuropsychopharmacol Biol Psychiatry 23:1171–1212. Croft AP, Brooks SP, Cole J, Little HJ (2005) Social defeat increases alcohol preference of C57BL/10 strain mice: effect prevented by a CCKB antagonist. Psychopharmacology (Berl) 183:163–170. de Groot J, van Milligen FJ, Moonen-Leusen BW, Thomas G, Koolhaas JM (1999) A single social defeat transiently suppresses the anti-viral immune response in mice. J Neuroimmunol 95:143–151. Daza-Losada M, Rodriguez-Arias M, Maldonado C, Aguilar MA, Guerri C, Minarro J (2009) Acute behavioural and neurotoxic effects of MDMA plus cocaine in adolescent mice. Neurotoxicol Teratol 31:49–59. Derr R, Lindblad S (1980) Stress-induced consumption of ethanol by rats. Life Sci 27:2183–2186. Funk D, Harding S, Juzytsch W, Le AD (2005) Effects of unconditioned and conditioned social defeat on alcohol selfadministration and reinstatement of alcohol seeking in rats. Psychopharmacology (Berl) 183:341–349. Galsworthy MJ, Paya-Cano JL, Liu L, Monleon S, Gregoryan G, Fernandes C, Schalkwyk LC, Plomin R (2005) Assessing reliability, heritability and general cognitive ability in a battery of cognitive tasks for laboratory mice. Behav Genet 35:675–692. Huang GB, Zhao T, Muna SS, Bagalkot TR, Jin HM, Chae HJ, Chung YC (2013) Effects of chronic social defeat stress on © 2014 Society for the Study of Addiction
behaviour, endoplasmic reticulum proteins and choline acetyltransferase in adolescent mice. Int J Neuropsychopharmacol 16:1635–1647. Iñiguez SD, Riggs LM, Nieto SJ, Dayrit G, Zamora NN, Shawhan KL, Cruz B, Warren BL (2014) Social defeat stress induces a depression-like phenotype in adolescent male c57BL/6 mice. Stress 17:247–255. Keeney A, Jessop DS, Harbuz MS, Marsden CA, Hogg S, Blackburn-Munro RE (2006) Differential effects of acute and chronic social defeat stress on hypothalamic-pituitaryadrenal axis function and hippocampal serotonin release in mice. J Neuroendocrinol 18:330–338. Kinsey SG, Bailey MT, Sheridan JF, Padgett DA, Avitsur R (2007) Repeated social defeat causes increased anxiety-like behavior and alters splenocyte function in C57BL/6 and CD-1 mice. Brain Behav Immun 21:458–466. Komatsu H, Ohara A, Sasaki K, Abe H, Hattori H, Hall FS, Uhl GR, Sora I (2011) Decreased response to social defeat stress in mu-opioid-receptor knockout mice. Pharmacol Biochem Behav 99:676–682. Koolhaas JM, Coppens CM, de Boer SF, Buwalda B, Meerlo P, Timmermans PJ (2013) The resident-intruder paradigm: a standardized test for aggression, violence and social stress. J Vis Exp 77:e4367. Kreibich AS, Briand L, Cleck JN, Ecke L, Rice KC, Blendy JA (2009) Stress-induced potentiation of cocaine reward: a role for CRF R1 and CREB. Neuropsychopharmacology 34:2609– 2617. Lee YK, Park SW, Kim YK, Kim DJ, Jeong J, Myrick H, Kim YH (2005) Effects of naltrexone on the ethanol-induced changes in the rat central dopaminergic system. Alcohol Alcohol 40:297–301. Lio W, Matsukawa N, Tsukahara T, Kohari D, Toyoda A (2011) Effects of chronic social defeat stress on MAP kinase cascade. Neurosci Lett 504:281–284. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408. Lumley LA, Sipos ML, Charles RC, Charles RF, Meyerhoff JL (1999) Social stress effects on territorial marking and ultrasonic vocalizations in mice. Physiol Behav 67:769–775. Miczek KA, Nikulina E, Kream RM, Carter G, Espejo EF (1999) Behavioral sensitization to cocaine after a brief social defeat stress: c-fos expression in the PAG. Psychopharmacology (Berl) 141:225–234. Nikulina EM, Arrillaga-Romany I, Miczek KA, Hammer RP Jr. (2008) Long-lasting alteration in mesocorticolimbic structures after repeated social defeat stress in rats: time course of mu-opioid receptor mRNA and FosB/DeltaFosB immunoreactivity. Eur J Neurosci 27:2272–2284. Noori HR, Helinski S, Spanagel R (2014) Cluster and metaanalyses on factors influencing stress-induced alcohol drinking and relapse in rodents. Addict Biol 19:225–232. Oliva JM, Ortiz S, Perez-Rial S, Manzanares J (2008) Time dependent alterations on tyrosine hydroxylase, opioid and cannabinoid CB1 receptor gene expressions after acute ethanol administration in the rat brain. Eur Neuropsychopharmacol 18:373–382. Palkovits M (1983) Punch sampling biopsy technique. Methods Enzymol 103:368–376. Park CR, Campbell AM, Diamond DM (2001) Chronic psychosocial stress impairs learning and memory and increases sensitivity to yohimbine in adult rats. Biol Psychiatry 50:994– 1004. Addiction Biology
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Paxinos G, Franklin KBJ (2001) The Mouse Brain in Stereotaxic Coordinates. New York, NY: Academic Press, Harcourt Science and Technology Company. Razzoli M, Andreoli M, Michielin F, Quarta D, Sokal DM (2011) Increased phasic activity of VTA dopamine neurons in mice 3 weeks after repeated social defeat. Behav Brain Res 218:253– 257. Ribeiro Do Couto B, Aguilar MA, Manzanedo C, Rodriguez-Arias M, Armario A, Minarro J (2006) Social stress is as effective as physical stress in reinstating morphine-induced place preference in mice. Psychopharmacology (Berl) 185:459–470. Rodriguez-Arias M, Minarro J, Aguilar MA, Pinazo J, Simon VM (1998) Effects of risperidone and SCH 23390 on isolationinduced aggression in male mice. Eur Neuropsychopharmacol 8:95–103. Shimozuru M, Kikusui T, Takeuchi Y, Mori Y (2006) Socialdefeat stress suppresses scent-marking and social-approach behaviors in male Mongolian gerbils (Meriones unguiculatus). Physiol Behav 88:620–627. Sinha R (2001) How does stress increase risk of drug abuse and relapse? Psychopharmacology (Berl) 158:343–359. Spanagel R, Noori HR, Heilig M (2014) Stress and alcohol interactions: animal studies and clinical significance. Trends Neurosci 37:219–227. Stewart J (2003) Stress and relapse to drug seeking: studies in laboratory animals shed light on mechanisms and sources of long-term vulnerability. Am J Addict 12:1–17. Stohr T, Almeida OF, Landgraf R, Shippenberg TS, Holsboer F, Spanagel R (1999) Stress- and corticosteroid-induced modulation of the locomotor response to morphine in rats. Behav Brain Res 103:85–93. Sullivan K (2000) The Anti-Bullying Handbook. Auckland: Oxford University Press. Tidey JW, Miczek KA (1997) Acquisition of cocaine selfadministration after social stress: role of accumbens dopamine. Psychopharmacology (Berl) 130:203–212. van Erp AM, Miczek KA (2001) Persistent suppression of ethanol self-administration by brief social stress in rats and increased startle response as index of withdrawal. Physiol Behav 73:301–311. Vidal J, Buwalda B, Koolhaas JM (2011) Male Wistar rats are more susceptible to lasting social anxiety than wild-type
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Groningen rats following social defeat stress during adolescence. Behav Processes 88:76–80. Wang J, Charboneau R, Barke RA, Loh HH, Roy S (2002) Mu-opioid receptor mediates chronic restraint stress-induced lymphocyte apoptosis. J Immunol 169:3630–3636. Watt MJ, Burke AR, Renner KJ, Forster GL (2009) Adolescent male rats exposed to social defeat exhibit altered anxiety behavior and limbic monoamines as adults. Behav Neurosci 123:564–576. Zorrilla EP, Logrip ML, Koob GF (2014) Corticotropin releasing factor: a key role in the neurobiology of addiction. Front Neuroendocrinol 35:234–244.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1 Analysis of the evolution of control and socially defeated mice during training (a) and substitution (b) phases of the oral ethanol self-administration task. The dots represent means and the vertical lines ± SEM of the number of effective responses Figure S2 Effects of repeated social defeat on the time taken by adult mice to enter the dark compartment in the training and test sessions (24 h after training) of the passive avoidance test. Data presented as mean values ± SEM ***P < 0.001. Differences with respect to the training day Figure S3 (a) Effects of repeated social defeat during adolescence on the mean latency score in the Hebb–Williams maze. (b) Effects of repeated social defeat during adolescence on the total number of errors in the Hebb–Williams maze. The mazes were classified as easy (1, 3 and 4) or difficult (5 and 8). Data are presented as mean values ± SEM Appendix S1 Materials and methods Appendix S2 Results
Addiction Biology
Addiction Biology
ORIGINAL ARTICLE
doi:10.1111/adb.12184
Social defeat in adolescent mice increases vulnerability to alcohol consumption Marta Rodriguez-Arias1,2, Francisco Navarrete2,3, Maria Carmen Blanco-Gandia1,2, Maria Carmen Arenas1,2, Adrián Bartoll-Andrés3, Maria A. Aguilar1,2, Gabriel Rubio2,4,5, José Miñarro1,2 & Jorge Manzanares2,3 Unidad de Investigación Psicobiología de las Drogodependencias, Departamento de Psicobiología, Facultad de Psicología, Universitat de València, Spain1, Red Temática de Investigación Cooperativa en Salud (RETICS-Trastornos Adictivos), Instituto de Salud Carlos III, MICINN and FEDER, Spain2, Instituto de Neurociencias, Universidad Miguel Hernández-CSIC, Spain3, Unidad de Psiquiatría, Hospital Universitario ‘12 de Octubre’, Spain4 and Instituto de Investigación ‘12 de Octubre’, Spain5
ABSTRACT This study employs an oral operant conditioning paradigm to evaluate the effects of repeated social defeat during adolescence on the reinforcing and motivational actions of ethanol in adult OF1 mice. Social interaction, emotional and cognitive behavioral aspects were also analyzed, and real-time polymerase chain reaction (PCR) experiments were performed to study gene expression changes in the mesocorticolimbic and hypothalamus-hypophysis-adrenal (HHA) axis. Social defeat did not alter anxiety-like behavior in the elevated plus maze or cognitive performance in the passive avoidance and Hebb–Williams tests. A social interaction test revealed depression-like symptoms and social subordination behavior in defeated OF1 mice. Interestingly, social defeat in adolescence significantly increased the number of effective responses, ethanol consumption values and motivation to drink. Finally, real-time PCR analyses revealed that social defeat significantly increased tyrosine hydroxylase and corticotropin-releasing hormone in the ventral tegmental area and paraventricular nucleus, respectively. In contrast, mu-opioid receptor gene expression was decreased in the nucleus accumbens of socially defeated mice. In summary, these findings suggest that exposure to social defeat during adolescence increases vulnerability to the rewarding effects of ethanol without affecting emotional or cognitive performance. The gene expression alterations we have observed in the mesocorticolimbic and HHA axis systems of defeated mice could be related with their increased ethanol consumption. These results endorse future research into pharmacological strategies that modulate these systems for the treatment of social stress-related alcohol consumption problems. Keywords defeat.
Anxiety, ethanol, hypothalamus-hypophysis-adrenal axis, memory, mesocorticolimbic system, social
Correspondence to: Marta Rodríguez-Arias, Unidad de Investigación Psicobiología de las Drogodependencias, Departamento de Psicobiología, Facultad de Psicología, Universitat de València, Avda. Blasco Ibáñez, 21, Valencia 46010, Spain. E-mail: [email protected]
INTRODUCTION Stressful life situations have been widely linked to drug seeking and consumption (Sinha 2001; Stewart 2003). Several studies in humans and animal models have demonstrated that exposure to different kinds of stress increases abuse and relapse to abuse of certain drugs, such as cocaine and ethanol (Derr & Lindblad 1980; Kreibich et al. 2009). Among the different types of stress that individuals may experience, social stress early in life—for example, bullying at school or child abuse—can have critical physiological and behavioral consequences © 2014 Society for the Study of Addiction
in adulthood (de Groot et al. 1999; Lumley et al. 1999). The term bullying is defined as a conscious and willful act of aggression and/or manipulation by one person (bully) against another person (victim) (Sullivan 2000). This behavior produces an emotional state known as social defeat, a phenomenon that can be modeled in animals by means of the resident–intruder paradigm, in which isolated, aggressive mice (residents) are confronted with grouped animals (intruders) through protocols that vary according to the number of social defeat experiences or moment at which the experience is inflicted (Koolhaas et al. 2013). Addiction Biology
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Social defeat represents the main form of social stress that leads to psychopathological changes (Bjorkqvist 2001). In animal models, social defeat stressors have an ecological and ethological validity, as they closely mimic real-life human situations (Shimozuru et al. 2006). Emotional disturbances (e.g. anxiety, depression) have been associated with repeated social defeat experiences and, in turn, with the development of drug addiction (Burke, Burke & Rae 1994). One of the first studies to evaluate the effect of social defeat on the reinforcing effects of cocaine demonstrated that defeated mice acquired cocaine selfadministration behavior in half the time required by controls (Tidey & Miczek 1997). Furthermore, the motor sensitization effects of cocaine have been found to be more pronounced in mice exposed to the resident– intruder paradigm (Miczek et al. 1999), and several social defeat experiences have been shown to significantly increase 24 hours cocaine binge-like consumption (Covington et al. 2005) and cocaine self-administration (Covington & Miczek 2005) in rats. On the other hand, morphine-induced motor sensitization is potentiated by social defeat, although this effect seems to depend on the number of stressful experiences and the interval between stress and locomotor testing (Stohr et al. 1999). Interestingly, our group has observed that social defeat is as effective as physical stress in inducing reinstatement of the morphine-induced conditioned place preference task (Ribeiro Do Couto et al. 2006). On the other hand, there are discrepant data regarding the effect of social defeat on ethanol intake. Acute exposure to social defeat was found to reduce alcohol self-administration in rats (Funk et al. 2005), while rats exposed to five brief episodes of social defeat exhibited increased alcohol drinking on the day of stress-exposure and even higher consumption 1 week later (Caldwell & Riccio 2010). Conversely, a brief social stress experience on five consecutive days was found to significantly decrease alcohol intake and rate of alcohol reinforcement in mice (van Erp & Miczek 2001). Most of the aforementioned studies have been performed in adult animals, while the consequences of social defeat experienced during adolescence have been less studied. This is relevant given that neurobehavioral and hormonal responses to stressors vary between adolescents and adults (Allen & Matthews 1997). Neuroadaptative changes in mesocorticolimbic circuitry in animals exposed to social defeat may explain, at least in part, their higher vulnerability to the reinforcing effects of a drug. Evaluation—using in vivo microdialysis—of dopamine concentrations in the nucleus accumbens (NAcc) of defeated and non-defeated mice has shown that social subordination induces a more pronounced increase in dopamine levels with respect to control mice (Tidey & Miczek 1997). In addition, when Nikulina and colleagues studied the effects of © 2014 Society for the Study of Addiction
social defeat on mu-opioid receptor (MOr) mRNA levels in the ventral tegmental area (VTA) of mice, they observed a clear up-regulatory effect that appeared to be related with enhanced dopaminergic activation in the mesocorticolimbic system (Nikulina et al. 2008). On the other hand, several studies have indicated the crucial role of the hypothalamus-hypophysis-adrenal (HHA) axis, which is activated by social stress experiences and leads to a significant increase in corticotropin-releasing factor in the paraventricular nucleus (PVN) and the hippocampus (HIPP; Keeney et al. 2006). In the present study, we have employed the resident– intruder paradigm to evaluate the effect of social defeat during adolescence on the emotional (anxiety-like behavior) and cognitive (memory) state of OF1 mice during adulthood. An additional goal of our study was to employ an oral self-administration test to analyze if the social defeat protocol applied during adolescence produces any changes in the reinforcing and motivational actions of ethanol in adult mice. Finally, we have performed real-time polymerase chain reaction (PCR) experiments in socially defeated mice and their corresponding controls in order to study gene expression changes in different targets involved in the function of the mesocorticolimbic system and HHA axis.
METHODS Animals A total of 102 male mice of the OF1 strain (Charles River, Barcelona, Spain) were employed in the study: 77 as experimental subjects and 40 as standard opponents and residents. Experimental animals were 21 days old on arrival at the laboratory and were all housed under standard conditions in groups of four (in cages 28 × 28 × 14.5 cm), at a constant temperature (21 ± 2°C), with a reversed light schedule (white lights on 19:30–7:30 hours) and food and water available ad libitum (except during the behavioral test). All studies were conducted in compliance with guidelines of the European Council Directive 2010/63/UE regulating animal research and were approved by the local ethical committees. Repeated social defeat Animals in the experimental group were exposed to four episodes of social defeat lasting 25 minutes each. Each episode consisted of three phases, which began by placing the experimental animal or intruder in the home cage of the aggressive opponent or resident for 10 minutes. During this initial phase, the intruder was protected from attack by a wire mesh wall that permitted social Addiction Biology
Social defeat and ethanol
© 2014 Society for the Study of Addiction
Hebb–Williams Social Interaction Passive avoidance Oral ethanol self-administration Gene expression analyses EPM
Fourth social defeat Fourth social defeat Third social defeat Third social defeat Second social defeat Second social defeat First social defeat First social defeat
Third social defeat Second social defeat First social defeat
33 30 27
For the passive avoidance test, a step-through inhibitory avoidance apparatus for mice (Ugo Basile, ComerioVarese, Italy) was employed. This cage is made of Perspex sheets and contains two compartments (15 cm × 9.5 cm × 16.5 cm each). The safe compartment is white
PND
Passive avoidance test
Table 1 Experimental procedure.
EPM tests were carried out essentially following the procedure described by Daza-Losada et al. (2009). In brief, the EPM consisted of two open arms (30 × 5 × 0.25 cm) and two enclosed arms (30 × 5 × 15 cm). The junction of the four arms formed a central platform (5 × 5 cm). The entire apparatus was elevated 45 cm above floor level. In order to facilitate adaptation, mice were transported to the dimly illuminated laboratory 1 hour before testing. The mice’s behavior was videorecorded and later analyzed by a ‘blind’ observer using a computerized method. The experimental room was illuminated with a dim red light (40 lux at 1 m above floor level). The measurements recorded during the test period were frequency of entries and time and percentage of time spent in each section of the apparatus (open arms, closed arms, central platform). An arm was considered to have been visited when the animal placed all four paws on it.
36
Elevated plus maze (EPM)
Fourth social defeat
37–56
57
59–60
61
62–69
70–87
interaction and species-typical threats from the male aggressive resident (Covington & Miczek 2001). In the second phase, the wire mesh was removed from the cage and a 5-minute period of confrontation began. In the third phase, the wire mesh was replaced for a further 10 minutes to allow social threats from the resident. In the third phase, socially defeated animals (n = 15 in the first study, and 16 in the second) were exposed to social defeat on postnatal day (PND) 27, 30, 33 and 36. The exploration group (n = 15 in both experiments) underwent the same protocol, but without the presence of a ‘resident’ mouse in the cage. Following this last phase, animals were kept in the vivarium for 3 weeks, after which the behavioral tests began. The second phase of each social defeat protocol was video-recorded and ethologically analyzed (n = 15). The following behaviors were scored for resident mice: threat and attack; and time needed to perform the first threat and attack (latencies). In the case of intruder mice, the following behaviors were analyzed: avoidance/flee and defensive/submissive behaviors; and time needed to exhibit the first avoidance/flee or defense/submission (latencies). Three different sets of mice were employed in this study. A more detailed description of the experimental procedure is provided in Table 1.
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and illuminated by a light fixture (10 W) fastened to the cage lid, whereas the ‘shock’ compartment is dark and black. The two compartments are divided by an automatically operated sliding door at floor level. The floor is made of 48 stainless steel bars with a diameter of 0.7 mm and situated 8 mm apart. Passive avoidance tests were carried out essentially following the procedure described by Aguilar, Minarro & Felipo (2000). On the day of training, each mouse was placed in the illuminated compartment facing away from the dark compartment. After a 60-second period of habituation, the door leading to the dark compartment was opened. When the animal had placed all four paws in the dark compartment, a footshock (0.5 mA, 3 seconds) was delivered and the animal was immediately removed from the apparatus and returned to its home cage. The time taken to enter the dark compartment (step-through latency) was recorded. Retention was tested 24 hours later by submitting the animal to the same procedure but without applying the shock. The maximum step-through latency was 300 seconds. Social interaction test This test consisted of confronting an experimental animal and a standard opponent in a neutral cage (61 × 30.5 × 36 cm) for 10 minutes following a 1-minute adaptation period prior to the encounter. One day before testing, standard opponents were rendered temporarily anosmic by intranasal lavage with a 4% zinc sulfate solution. This kind of mouse induces an attack reaction in its opponent, but does not outwardly provoke or defend itself since it cannot perceive a pheromone that is present in the urine of the experimental animals and functions as a cue for eliciting aggressive behavior in mice with a normal sense of smell. The videotapes were analyzed using a custom-developed program that estimates the time devoted to different broad functional categories of behavior (non-social exploration, social investigation, threat, attack and avoidance/flee), each of which is characterized by a series of different postures and elements. A more detailed description can be found in Rodriguez-Arias et al. (1998). Hebb–Williams maze The maze (60 cm wide × 60 cm long × 10 cm high) used in our experiment is made of black plastic and contains a start box and a goal box (both 14 cm wide × 9 cm long) positioned at diagonally opposite corners. The maze contains cold water (15°) at a wading depth (3.5 cm), while the goal box is stocked with fresh dry tissue. Several maze arrangements can be made by fixing different barriers to the clear plastic ceiling. This apparatus allows the cognitive process of routed learning and the motivation of water escape to be measured. © 2014 Society for the Study of Addiction
The procedure was based on that employed by Galsworthy et al. (2005), in which mice must navigate the maze and cross from the wet start box to the dry goal box in order to escape the cold water. Animals underwent a 5 minute habituation period (dry sand, no barriers) on day 1 and undertook problem A on day 2 and problem D on day 3 (four trials/day; practice mazes). Mice were subsequently submitted to mazes 1, 5, 3, 4 and 8 on separate days on which eight trials took place. The time limit for a mouse to reach the goal box on its own was 5 minutes, after which the animal was guided to the dry area. The following measurements were recorded: total latency score (sum of latencies of all the problem trials in each maze); and error score, for which a similar total was used (where ‘error’ was considered as entering the error zone, as specified by Galsworthy et al. (2005).
Oral ethanol self-administration Evaluation of ethanol self-administration was carried out in 12 modular operant chambers (Panlab, Barcelona, Spain) in three phases: training, saccharin substitution and 6% ethanol consumption. During the last phase, the number of effective responses and ethanol consumption (μl) were measured under fixed ratio 1 (FR1), fixed ratio 3 (FR3) and progressive ratio (PR) schedules. For more details, see the Supporting Information.
Gene expression analyses—real-time PCR Mice were killed 3 weeks after the last social defeat. Brains were removed from the skull and frozen on dry ice. Coronal brain sections (500 μm) were obtained at plates 19–20 (distance from the bregma: 1.42 and 1.34 mm, respectively) in a cryostat (−10°C) according to Paxinos & Franklin (2001). VTA, PVN and NAcc were microdissected according to the Palkovits method (Palkovits 1983). Total RNA was isolated from brain tissue micropunches using TRI Reagent® (Applied Biosystems, Madrid, Spain) and were subsequently retrotranscribed to cDNA. Quantitative analysis of the relative abundance of tyrosine hydroxylase (TH) (Mm00447546_m1) gene expression in VTA, MOr (Mm01188089_m1) gene expression in NAcc and corticotropin-releasing hormone (CRH; Mm01293920_s1) gene expression in PVN between the social defeat group and control mice was performed using the StepOne Sequence Detector System (Life Technologies, Madrid, Spain). All reagents were obtained from Life Technologies and manufacturers’ protocols were followed. The chosen reference gene was Rn18S (Mm03928990_g1), which was detected using Taqman ribosomal RNA control reagents. All primerprobe combinations were optimized and validated for relative quantification of gene expression. In brief, data for Addiction Biology
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Table 2 Social interaction during the resident–intruder paradigm to induce social defeat (SD). Resident
Threat
Threat latency
Attack
Attack latency
SD1 SD2 SD3 SD4
21.8 ± 2.3 26.2 ± 2.5 21.9 ± 1.6 17 ± 1.8**
3.6 ± 0.8 4.7 ± 2.0 3.2 ± 1.0 3.4 ± 1.3
17.9 ± 2.2 21.2 ± 2.0 24.3 ± 3.4 22 ± 3.6
24.9 ± 12.7 9.2 ± 3.2 6.9 ± 2.5 3.9 ± 1.3
Intruder
Avoidance/flee
Avoidance/flee latency
Defensive/submissive
Defensive/submissive latency
SD1 SD2 SD3 SD4
14.8 ± 2.1 15.8 ± 1.6 16.3 ± 2.5 16.8 ± 2.3
20.2 ± 4.9 6.5 ± 2.0 7.6 ± 2.6 5.3 ± 1.6*
49.9 ± 7.13 75.1 ± 7.16 69.6 ± 3.9 66.8 ± 6.4
33.6 ± 13.4 10.7 ± 2.4 5.5 ± 1.1 9.7 ± 2.6
Data presented as mean values ± SEM. *P < 0.03; differences with respect to the first social defeat (SD1); **P < 0.02; differences with respect to the second social defeat (SD2).
each target gene were normalized to the endogenous reference gene, and the fold change in target gene mRNA abundance was determined using the 2-ΔΔCt method (Livak & Schmittgen 2001) so that the level of each social defeat group was expressed in relation to that of the control group. Statistical analysis The data of the ethological analyses of resident and intruder mice were analyzed by a one-way ANOVA with a within variable—social defeat—with four levels: first, second, third and fourth social defeat. Data relating to social interaction, the EPM and motor activity were analyzed by a one-way ANOVA with a between variable— social defeat—with two levels: exploration or social defeated. The passive avoidance test was analyzed by a two-way ANOVA, with the same between variable— social defeat—and one within variable with two levels: training day or test day. The data of the Hebb–Williams maze were analyzed by a two-way ANOVA with one between subject variable—social defeat—and one within subject variable—number of labyrinths in the maze— with five levels. Mice unable to complete the task within the time limit were attributed maximum latency scores. Statistical analyses for self-administration of ethanol were performed using two-way ANOVA with repeated measures followed by the Student’s-Newman-Keuls test to compare social defeat and control groups at different time points of the oral self-administration paradigm. A Student’s t-test was employed to compare the effects of social defeat on the number of effective responses, breaking point values and ethanol consumption during PR. Student’s t-test was employed to compare the effects of social defeat on TH, MOr and CRH gene expression. Differences were considered significant if the probability of error was less than 5%. SigmaStat v3.11 software © 2014 Society for the Study of Addiction
Table 3 Effects of repeated SD on adolescent mice in the EPM.
Time in open arms % Time in open arms Time in central platform Time in closed arms Entries in open arms % Open entries Entries in closed arms Total entries
Control
Social defeat
39.6 ± 10.4 15.8 ± 4 64.18 ± 10 204.6 ± 12.6 4.2 ± 1.2 17.24 ± 3.9 17 ± 2.1 21.26 ± 2.6
36 ± 7.4 15.44 ± 3.2 69.7 ± 6.5 200 ± 9.7 2.9 ± 0.7 14.54 ± 2.8 15 ± 1.1 18.71 ± 1.3
Data are presented as mean values ± SEM.
(Systat Software Inc., Chicago, IL, USA) was employed for all statistical analyses.
RESULTS Social defeat The ANOVA for resident mice revealed an effect of the time spent engaged in threat [F(3, 42) = 3.288; P < 0.03], showing that mice displayed less threat behavior in the last social defeat than during the second encounter (P < 0.03). In the case of intruder mice, the latency of avoidance/flee showed a significant effect [F(3, 42) = 5.646; P < 0.02]; mice displayed their first avoidance/flee behavior significantly sooner in the fourth social defeat than in the first encounter (P < 0.02; see Table 2). EPM EPM data (see Table 3) revealed no significant differences between the two groups of animals. No significant differences were detected between socially defeated and control Addiction Biology
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Table 4 Means of accumulated times (in seconds) with SEM engaged in different categories of spontaneous behavior in the social interaction test by socially defeated (n = 15) and exploration (n = 15) mice.
Non-social exploration Social investigation Unit of social investigation Threat Attack Avoidance/flee Defensive/submissive
Control
Social defeat
476.52 ± 24.23 56.4 ± 15.69 2.55 ± 0.98 9.68 ± 4.42 1.42 ± 0.86 0±0 0±0
530.42 ± 17.19** 23.57 ± 16.66** 1.73 ± 0.74* 2.37 ± 1.98 0.38 ± 0.28 0.57 ± 0.8* 3.18 ± 4.19**
Differences from their respective control, *P < 0.01; **P < 0.001.
mice with respect to time spent in open arms [F(1, 27) = 0.077; P > 0.05], percentage of time spent in open arms [F(1, 27) = 0.006; P > 0.05] and the number of entries in open arms [F(1, 27) = 0.989). Social interaction test The data for the different types of behavior evaluated in the social interaction test are presented in Table 4. Socially defeated mice spent less time engaged in social investigation than exploration mice [F(1, 28) = 30.866; P < 0.001], although differences between the two groups were not significant (P < 0.001). The mean times spent engaged in each contact (unit of social investigation) were similar [F(1, 28) = 6.614; P < 0.02], being lower among socially defeated animals (P < 0.02), who spent more time engaged in non-social exploration [F(1, 28) = 49.354; P < 0.001] than control animals (P < 0.001). Avoidance/flee [F(1, 28) = 7.545; P < 0.01] and defensive/submissive [F(1, 28) = 8.6; P < 0.01] behaviors were observed only among socially defeated mice (P < 0.01 in all cases). Passive avoidance test and Hebb–Williams maze No significant differences were observed between socially defeated animals and controls in the passive avoidance test or the Hebb–Williams Maze. Additional Supporting Information is provided on the publisher’s website (Supporting Information Figs S2 & S3). The effect of social defeat on self-administration of ethanol Oral ethanol administration was initiated 3 weeks after the last social defeat experience. No differences were detected between control and socially defeated mice during training or substitution phases, which implied that the social defeat procedure did not induce any learning deficit (see Supporting Information Fig. S1). In the last stage of the self-administration protocol (6% ethanol, 0% saccharin), differences were detected between the two groups with respect to both the number of effective © 2014 Society for the Study of Addiction
responses (Fig. 1a; two-way ANOVA with repeated measures followed by the Student’s-Newman-Keuls method: social defeat: F(1, 259) = 27.921, P < 0.001; day: F(9, 259) = 92.450, P < 0.001; social defeat × day interaction: F(9, 259) = 2.882, P = 0.003) and 6% ethanol consumption (Fig. 1b; two-way ANOVA with repeated measures followed by the Student’s-NewmanKeuls method: social defeat: F(1, 259) = 9.624, P = 0.005; day: F(9, 259) = 9.842, P P < 0.001; social defeat × day interaction: F(9, 259) = 1.257, P = 0.262) in the FR1 and FR3 stages. In addition, during PR, breaking point values were significantly higher among socially defeated mice (Fig. 1c, Student’s t-test: t = −2.526, 20 d.f., P = 0.020), although 6% ethanol consumption (Fig. 1d, Student’s t-test: t = −1.073, 28 d.f., P = 0.292) was very similar in both groups. Effect of social defeat on TH, MOr and CRH gene expression Real-time PCR analyses indicated that social defeat affected TH gene expression in the VTA of the experimental mice, which presented significantly higher TH gene expression values (155%) than the control group. (Fig. 2a, n = 8, Student’s t-test, t = −4.263, 14 d.f., P = < 0.001). On the other hand, MOr gene expression was lower in the NAcc of socially defeated mice (−70%) than in that of control animals (Fig. 2b, n = 8, Student’s t-test: t = 3.646, 14 d.f., P = 0.003). Finally, social defeat increased CRH gene expression in the PVN (60%) in relation with levels in control mice (Fig. 2c, n = 7, Student’s t-test: t = 3.633, 12 d.f., P = 0.003). DISCUSSION The results of the present study indicate that repeated experience of social defeat during adolescence reduces social behavior without increasing anxiety, does not produce cognitive disturbances and heightens the reinforcing and motivational effects of ethanol during adulthood in mice. This conclusion is supported by the following facts: (1) socially defeated mice presented Addiction Biology
Social defeat and ethanol
(a)
FR1
80
(b)
FR3
FR1
60
* *
* *
* *
40
20
6% EtOH Consumption (µl)
Effective responses
* *
FR3
* *
1600
CONTROL SOCIAL DEFEAT
* *
* *
* *
1400
7
* *
1200 1000 800
* *
600 400 200
0
0 1
2
3
4
5 6
7
8
9
1
10
2
3
4
(d) 70
350
60
300
6% EtOH Consumption (µl)
Breaking point
(c)
PR
5 6
7
8
9
10
Day
Day
*
50 40 30 20 10 0
250 200 150 100 50 0
Control
Soc. defeat
Control
Soc. defeat
1.6
Ct)
*
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
Ct)
1.8 1.6 1.4 1.2 1.0
*
0.8 0.6 0.4 0.2 0.0
Control
Soc. defeat
Control
Soc. defeat
Corticotropin release hormone (2-
NAcc
VTA 1.8
µ-opioid receptor (2-
Tyrosine hydroxylase (2-
Ct)
Figure 1 Analysis of oral ethanol self-administration in OF1 mice (n = 31).The dots represent means and the vertical lines ± SEM of (a) the number of effective responses and (b) the volume of 6% ethanol consumption during FR1 and FR3.The columns represent means and the vertical lines ± SEM of (c) the breaking point values during PR and (d) the volumes of 6% ethanol consumption. *Represents the values of the socially defeated group that are significantly different (P < 0.05) from those of the control group
PVN 3.0
*
2.5 2.0 1.5 1.0 0.5 0.0
Control
Soc. defeat
Figure 2 (a) Real-time PCR TH relative gene expression evaluation in the VTA brain region of control and socially defeated groups (n = 8). (b) MOr relative gene expression evaluation in the NAcc brain region of these groups (n = 8). (c) CRH relative gene expression evaluation in the PVN brain region of the mice (n = 7).The columns represent means and the vertical lines ± SEM of relative (2-ΔΔCt method) TH gene expression in the VTA. Basal MOr gene expression in the NAcc and the CRH gene expression in the PVN of OF1 mice. *Represents the values of socially defeated mice that are significantly different (P < 0.05) from those of their corresponding control mice
significantly lower values of social investigation and displayed avoidance, flee, defensive and submissive behaviors in the social interaction test; (2) no differences were observed between groups in the time spent in the open arms in the EPM; (3) the latency to enter the dark compartment in the passive avoidance test and the time © 2014 Society for the Study of Addiction
required to reach the goal in each of the Hebb–Williams mazes were similar in defeated and non-defeated mice; (4) the number of effective responses, volume of ethanol consumption and breaking point values were significantly higher in OF1 mice exposed to social defeat during adolescence than in controls; and (5) TH, MOr and CRH gene Addiction Biology
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expression was altered in the VTA, NAcc and PVN, respectively, of socially defeated OF1 mice. Several studies have identified chronic social defeat as a model of depression in adult and adolescent animals for two main reasons: (1) socially defeated animals usually show depression-like behaviors such as social avoidance and behavioral despair; and (2) treatment with antidepressant drugs ameliorates these depression-like symptoms (Becker et al. 2008). Accordingly, we have observed that repeated experience of social defeat during adolescence induces avoidance and flee behaviors in the social interaction test when performed during adulthood. In addition, our socially defeated mice adopted defensive and submissive positions with respect to control animals, thus indicating a significant trend toward social subordination. Several studies have reported an increase in anxiety and despair behaviors in socially defeated adolescent mice (Kinsey et al. 2007; Huang et al. 2013; Iñiguez et al. 2014) and rats (Lio et al. 2011); however, in the present study, we found no differences between the total time spent in the open arms of the EPM by socially defeated versus control mice, as shown in Table 3. This could be due to considerable methodological differences among the studies in question. Firstly, different species and strain of animals have been used. For example, Vidal and colleagues observed that defeated Wistar rats developed social anxiety behavior, whereas defeated WT Groeninger rats did not (Vidal, Buwalda & Koolhaas 2011). Secondly, other studies have inflicted a higher number of social defeat experiences than ours. Moreover, behavioral procedures have been carried out, in most cases, immediately after the stress experience. On the other hand, in line with our results, Watt et al. demonstrated that repeated experience of social defeat during mid-adolescence did not induce anxiety-like behaviors in the EPM 3 weeks later (Watt et al. 2009). Equally, Bourke & Neigh (2011) reported that a chronic mixed modality stressor during adolescence induced sustained changes in depressive-like behavior during both adolescence and adulthood in female but not in male rats. We evaluated the effect of social defeat on aversive memory in the passive avoidance task, finding no differences between control and defeated mice in the time taken to enter the dark compartment. Again, when the animals performed the Hebb–Williams test in order to assess working memory, no significant differences were observed between the two groups. Previous studies have associated exposure to social stress with cognitive impairment; however, in the studies in question cognitive evaluation was carried out immediately or shortly after the end of a chronic stress period or an acute stressor (Park, Campbell & Diamond 2001). However, Buwalda and colleagues found no differences between controls and adult rats that had experienced social defeat three weeks previ© 2014 Society for the Study of Addiction
ously when they evaluated spatial learning and memory in the Morris water maze (Buwalda et al. 2005). Therefore, the cognitive impairment induced by social stress would seem to become apparent in the early stages following a stressful experience, and may be attributable to the evident disturbances reported in the HHA axis. In the literature, there is clear evidence of the relationship between exposure to social stress events and the development of drug addiction. Many studies have shown that social defeat is closely associated with an increase in the locomotor, reinforcing and motivational actions of psychostimulants (Covington & Miczek 2005). However, much less is known about the effect of social defeat on the rewarding effects of ethanol. A recent systematic quantitative study aimed to identify the main factors involved in stress-induced alcohol consumption, showed that increases in alcohol intake are best observed in adult male rats exposed to force swim or foot shock within the context of free-choice home-cage drinking paradigm (Noori, Helinski & Spanagel 2014). In the present study, we evaluated the effects of intermittent social defeat in adolescence on oral self-administration of ethanol during adulthood in OF1 mice. Socially defeated mice displayed increased effective responses to the active lever and higher ethanol consumption values during FR1 and FR3 stages. In the PR, defeated mice had higher breaking point values with respect to their controls, although no differences were detected in ethanol consumption. These results are in accordance with those of previous studies evaluating the effect of social defeat on the reinforcing actions of ethanol. Croft et al. reported that social stress experienced in adulthood increased ethanol consumption and preference in a two-bottle system paradigm for 3 weeks after the last social defeat. Interestingly, they compared a single experience of social defeat with five consecutive daily defeat experiences and a weekly experience over 4 weeks, and concluded that only the repetitive protocol of five consecutive sessions increased vulnerability toward the rewarding effects of ethanol in C57BL10 mice (Croft et al. 2005). On the other hand, in another study employing a similar protocol to induce social defeat in adult rats (five consecutive sessions), ethanol self-administration was significantly higher in non-preferring defeated rats only 2 hours after the last social defeat experience (Caldwell & Riccio 2010). Finally, Funk and colleagues studied the effect of social defeat on ethanol self-administration during extinction and reinstatement stages in adult rats. They concluded that social defeat immediately before self-administration sessions decreased ethanol consumption, did not affect extinction and did not induce a reinstatement effect (Funk et al. 2005). Overall, these previous results provide valuable information about the importance of temporary factors since significant effects only seem to appear when Addiction Biology
Social defeat and ethanol
there is a lapse of time between social defeat and evaluation of ethanol consumption. Therefore, the novelty of our study lies in the following aspects: (1) application of the social defeat protocol during adolescence; and (2) evaluation of the long-lasting effects of social defeat on the reinforcing and motivational actions of ethanol during adulthood (3 weeks later). The reinforcing effects of ethanol have been widely related with activation of the dopaminergic mesolimbic system through different mechanisms, of which the firing rate of dopaminergic neurons in the VTA is 1 (Brodie, Shefner & Dunwiddie 1990). Furthermore, an up-regulation of the TH in VTA under acute (Oliva et al. 2008) or chronic (Lee et al. 2005) ethanol administration has been described. In the present study, social defeat significantly increased TH gene expression, suggesting that enhanced dopamine synthesis is related with increased ethanol self-administration. Accordingly, several studies have found that social defeat increases dopamine signaling in the mesocorticolimbic pathway even 3 weeks later (Razzoli et al. 2011). Another mechanism responsible for the rewarding actions of ethanol is the increase in dopamine release produced through activation of MOr in the VTA and NAcc (Cowen & Lawrence 1999). In addition, several studies have demonstrated the important role played by MOr in the regulation of psychosocial stress effects (Wang et al. 2002; Komatsu et al. 2011). In the present study, real-time PCR gene expression analyses have revealed lower MOr gene expression levels in the NAcc of socially defeated mice. On the contrary, other studies have demonstrated a significant and long-lasting increase in MOr mRNA levels in the VTA (Nikulina et al. 2008). This discrepancy could be due to the different regions studied (NAcc versus VTA) or to the animal species employed (mouse versus rat). Finally, when evaluated to find a possible association between high ethanol self-administration and HHA axis impairments, CRH gene expression in the PVN of socially defeated mice was significantly higher than in control mice, as previously described in another regions, such as HIPP (Keeney et al. 2006). This is relevant, as alterations in the HHA axis have been related with the development of drug addiction (Zorrilla, Logrip & Koob 2014). Among other molecular events, stress-induced elevation in glucocorticoid levels could influence mesolimbic dopaminergic activity via glucocorticoid receptors in D1 receptor-expressing medium spiny neurons of the NAcc regulating the firing rate of mesolimbic dopamine neurons (Spanagel, Noori & Heilig 2014). The rationale behind the real-time PCR analyses performed in the present study is as follows: (1) TH gene expression was measured in the dopaminergic cell bodies of the mesocorticolimbic system located in the VTA, where dopamine synthesis takes place; (2) MOr was analyzed in © 2014 Society for the Study of Addiction
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the NAcc, as several studies have demonstrated that the modulation of dopaminergic neurotransmission by MOr in this specific region is critically involved in the reinforcing effects of ethanol; and (3) CRH was evaluated in the PVN because the parvocellular neurons of this brain region are responsible for the synthesis of this HHA axis regulatory hormone. In conclusion, the results presented in this study show that social defeat experiences during adolescence increase the reinforcing and motivational actions of ethanol during adulthood without impairing cognitive performance or anxiety-like behavior. Interestingly, alterations in gene expression in the mesocorticolimbic system and HHA axis of socially defeated mice could lie behind higher ethanol consumption vulnerability. Further studies are necessary to explore if pharmacological manipulation of these systems might be useful in managing ethanol consumption problems related with early social defeat experiences. Acknowledgements This work was supported by the following research grants: Ministerio de Ciencia e Innovación, (SAF201123420) to Jorge Manzanares; Ministerio de Economía y Competitividad, Dirección General de Investigación (PSI2011-24762) to Jose Miñarro; Generalidad Valenciana, Consejería de Educación (PROMETEO/2009/ 072) to Jose Miñarro; and Instituto de Salud ‘Carlos III’ (FIS), Redes Telemáticas de Investigación Cooperativa en Salud (RETICS), Red de Trastornos Adictivos (RTA), fondos FEDER (RD06/0001/1004, RD12/0028/0019 to Jorge Manzanares; and RD06/001/0016, RD12/0028/ 005 to Jose Miñarro). Authors Contribution All named authors have made an active contribution to the conception, design, analysis and drafting of the article. All authors have contributed equally to the overall coordination of the whole study. MR, MCA, MCBG and MAA performed the social defeat protocol and evaluated social, emotional and cognitive behavioral parameters. FN and AB were responsible for the supervision of oral ethanol self-administration experiments and realtime PCR analysis. All authors critically reviewed the content and approved the final version for publication. References Aguilar MA, Minarro J, Felipo V (2000) Chronic moderate hyperammonemia impairs active and passive avoidance behavior and conditional discrimination learning in rats. Exp Neurol 161:704–713. Allen MT, Matthews KA (1997) Hemodynamic responses to laboratory stressors in children and adolescents: the influences of age, race, and gender. Psychophysiology 34:329–339. Addiction Biology
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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1 Analysis of the evolution of control and socially defeated mice during training (a) and substitution (b) phases of the oral ethanol self-administration task. The dots represent means and the vertical lines ± SEM of the number of effective responses Figure S2 Effects of repeated social defeat on the time taken by adult mice to enter the dark compartment in the training and test sessions (24 h after training) of the passive avoidance test. Data presented as mean values ± SEM ***P < 0.001. Differences with respect to the training day Figure S3 (a) Effects of repeated social defeat during adolescence on the mean latency score in the Hebb–Williams maze. (b) Effects of repeated social defeat during adolescence on the total number of errors in the Hebb–Williams maze. The mazes were classified as easy (1, 3 and 4) or difficult (5 and 8). Data are presented as mean values ± SEM Appendix S1 Materials and methods Appendix S2 Results
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