Environmental and situational factors are important determinants of recreational drug use in humans (for review see Caprioli et al., 2007). It follows that comprehensive laboratory (animal) models of drug-seeking behavior should include these components.
Clinical studies describe a relationship between social isolation and the use of multiple drugs of abuse, as well as more chronic and severe addiction, high rates of dropouts during treatment, and higher rates of relapse after withdrawal attempts (Pelissier and O'Neil, 2000; Dobkin et al., 2002; Compton et al., 2003, 2005; Darke et al., 2005; Westermeyer and Thuras, 2005).
Similarly, preventing rats from normal interaction and communication with other rats has been found to exert major effects on both physiological and behavioral mechanisms (Valzelli and Garattini, 1972; Brain and Benton, 1979). Individual housing increases aggression and interferes with the performance of a cooperation task in male rats (Swanson and Schuster, 1987; Schuster et al., 1993; Wongwitdecha and Marsden, 1996b; Sanchez and Meier, 1997; Vale and Montgomery, 1997; Byrd and Briner, 1999; Miachon et al., 2000). Rats housed individually tend to be more irritable, restless, and hyperactive compared with rats housed in groups (Domeney and Feldon, 1998; Bakshi and Geyer, 1999). They also show patterns of hypersensitivity, anxiety, stress, and depressive-like behavior (Serra et al., 2000; Whittaker-Azmitia et al., 2000; Sudakov et al., 2003; Weiss et al., 2004; Nunes Mamede Rosa et al., 2005; Westenbroek et al., 2005; Grippo et al., 2007; Brenes et al., 2008). Social isolation also affects physiological parameters such as hyperfunction of the hypothalamic-pituitary-adrenal axis, elevated levels of plasma corticosterone, heavier adrenal glands, increased heart rate, hypertension (Nagaraja and Jeganathan, 1999; Serra et al., 2000; Wright and Ingenito, 2003; Weiss et al., 2004; Westenbroek et al., 2005; Grippo et al., 2007), and reduced activity in the serotonergic system (Hall et al., 1998; Whittaker-Azmitia et al., 2000).
Social isolation might interact with the phenomenology of psychoactive drugs and therefore lead to changes in the patterns of drug consumption (for review see Stairs and Bardo, 2009). Results of studies on the role of social isolation on the susceptibility to self administer psychoactive drugs are not consistent, probably because of differences in experimental design, drug delivery system, type of drug, dosage, etc. (Caprioli et al., 2007). However, several studies have reported that rats housed in social isolation tend to consume higher amounts of morphine compared with grouped-housed rats (Alexander et al., 1978, 1981). Morphine has been shown to be effective in reversing isolation effects on various behavioral and physiological parameters (Panksepp et al., 1978, 1980; Jimenez and Fuentes, 1993; Hol et al., 1996; Van den Berg et al., 1999, 2000; Sudakov et al., 2003). The effects of social isolation on physiological or behavioral processes, including drug self-administration, are highly dependent on the age of onset of exposure to the isolation condition. Many studies have focused on the long-term consequences of isolation at infancy or just before weaning (isolation rearing). In some studies, rats were isolated during both weaning and adulthood (isolated housing) confounding whether the resultant behavioral changes could be attributed to isolated rearing, isolated housing, or an interaction between these states. A relatively small number of studies have examined social isolation during adulthood (for review see Hall, 1998; Lu et al., 2003).
In the research presented here, we extend some of the earlier findings on social isolation and morphine intake. We used a relatively short period (21 days) of social restriction in adult rats without limiting auditory, olfactory, or visual contact, and examined the influence of such social isolation in both male and female subjects. Finally, we have studied the reversibility of the effect of social restriction on morphine consumption by (i) switching isolated and social housing in a within-groups design; and, (ii) providing short-term daily social interaction of 60-min per day in otherwise socially isolated rats.
Subjects were adult male (experiments 1 and 3) or female (experiment 2) Wistar rats (Harlan). Their age at the beginning of the experiments was 45–55 days and their weight was 170–220 g. Throughout the study, subjects were maintained in a temperature-controlled room (23±1°C on a reverse 12-h light/dark cycle (lights on 07.00 h) in standard cages with transparent walls and sawdust bedding. The animals had free access to standard dry food. Water and morphine solutions were available through external bottles hanging on the cage. Daily fluid consumption was measured by weighing bottles before presenting them to animals and again after 24 h. All procedures were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the institutional ethics committee. Morphine sulfate was obtained from Rafa Laboratories, Jerusalem.
Upon arrival, animals were housed six per cage and allowed to adapt to the animal facility for 1 week. Rats were then assigned randomly to the different experimental groups. In experiments 1 (males) and 2 (females) there were two groups: isolated housing (n=10; 1 animal per cage measuring 40×25×18 cm) and social housing (n=20; 2 same-sex animals per cage measuring 56×34×19 cm). In experiment 3 there were three groups – isolated housing (n=10), social housing (n=16), and partial isolation (n=10): one rat per cage with access to another male rat (always the same one, in a neutral cage) for 60 min only per day (from 09.00 to 10.00 h). During that time they had access to unlimited physical interaction.
Stage I – adaptation
Animals were maintained under different housing conditions for 21 days with free access to food and water. To make sure there were no differences in initial fluid consumption, baseline water intake was obtained during the last 3 days of this phase (days 19–21).
Stage II – forced consumption (one-bottle test)
Subjects were given access to morphine sulfate solution (0.5 mg/ml) only for 7 days. Morphine solution intake was measured every 24 h. Rats in the social housing group were given one bottle and the total measured intake was divided by two, thus giving an estimate of the consumption of an individual animal.
Stage III – choice (two-bottle test)
Subjects were given access to both water and morphine solution (0.5 mg/ml) for 7 days. Again, intake was estimated by weighing bottles every 24 h. The total measured intake of paired rats was divided by two.
In experiment 1 at the end of stage III (choice test), the housing conditions were reversed/switched (rats previously isolated were housed in pairs and rats previously paired housed in isolation) and the whole procedure was replicated: 21 days in the new housing conditions followed by 4 days of forced morphine consumption (one-bottle test) and then 4 days of choice (two-bottle test).
Within-subjects results (comparison between water and morphine solution intake among isolated and paired rats) were analyzed by paired-samples t-tests.
All of the between-subjects results were analyzed by analysis of variance for repeated measures (mixed design) both for the forced and choice tests, followed by post-hoc Tukey tests.
For pairs of rats, the best estimate of the intake for a single animal is the mean intake of the pair. To reduce statistical bias, we considered each pair as a single animal for analysis. Thus, for example, the intake measures for 10 pairs of rats is calculated on the basis of n=10 rather than n=20.
Numerical results are presented as mean±SEM (in both text and figures) and considered significant for P value less than 0.05.
No differences in body weight were observed among the various housing conditions throughout the four experiments.
Drinking data are presented as mean total volume of morphine solution and water consumed by isolated versus paired subjects. ‘Mean’ represents the average morphine solution or water intake across all days of observation as the most representative estimate of average total consumption.
First (experiment 1), we examined the effect of isolated housing versus social housing (pairs) on morphine and water intake among adult males. Results indicated that isolated housing significantly increased morphine intake both in forced and in choice tests. In the forced test, isolates consumed higher amounts of morphine solution (42.47±2.87 ml) than pairs (29.65±2.01 ml) [F(1,18)=13.37, P<0.002; Fig. 1a]. The days×group interaction was not significant. In the choice test also, isolates consumed more morphine (12.25±1.21 ml), than pairs (6.86±0.85 ml) [F(1,18)=13.26, P<0.002; Fig. 1b]. A significant days×group interaction was found [F(6)=3.57, P<0.002]; there was no significant difference in water intake between the two groups. Isolated as well as paired rats consumed significantly more water (31.21±0.69 and 31.92±1.08 ml; respectively) than morphine solution (12.25±1.24 and 6.86±0.85 ml) [t(9)=10.65, P<0.001 and t(9)=20.22, P<0.001].
These results were replicated when housing conditions were switched, suggesting that the isolation-induced elevation in morphine intake is reversible. In the forced test, isolates (previously paired) consumed more morphine (26.29±2.14 ml) than pairs (previously isolated) (16.12±3.91 ml) [F(1,23)=4.65, P<0.04; Fig. 1c]. The days×group interaction was not significant. In the choice test also, isolates drank more of the morphine solution (7.4±0.2 ml) than pairs (5.52±0.8 ml) [F(1,23)=11.45, P<0.005; Fig. 1d]. The days×group interaction was not significant, and there was no significant difference in water intake between the two groups. Isolated and paired rats consumed significantly more water (28.44±1.31 and 30.24±1.86 ml; respectively) than morphine solution (7.4±0.2 and 5.52±0.8 ml) [t(19)=17.06, P<0.0001 and t(4)=10.04, P<0.001].
Results in females (experiment 2) were similar to those of male subjects. In the forced test, isolated females consumed significantly higher amounts of morphine solution (32.65±2.55 ml) compared with paired females (22.82±1.65 ml) [F(1,18)=10.45, P<0.005; Fig. 2a]. The days×group interaction was not significant. In the choice test also, morphine solution intake of isolated females was higher (15±2.65 ml) than that of socially housed females (3.84±0.48 ml) [F(1,18)=17.19, P<0.001; Fig. 2b]. The days×group interaction was not significant and no difference was found in water intake. Isolated and paired females consumed significantly more water (24.98±2.12 and 26.35±1.24 ml; respectively) than morphine solution (15±2.65 and 3.84±0.48 ml) [t(9)=2.36, P<0.05 and t(9)=19.49, P<0.001].
In the last experiment (experiment 3), we examined the effect of brief physical–social interaction (1 h per day) on morphine intake. One-hour per day of social interaction reversed the increase in morphine intake in isolated rats. In the forced test, the three experimental groups differed significantly from one another [F(2,25)=4.39, P<0.02]. Post-hoc test revealed that isolated rats consumed higher amounts of morphine (45.6±6.61 ml) than rats in the partial isolation (26.76±4.49 ml) (P<0.04) and in the paired group (26.3±3.97 ml) (P<0.04). No significant difference in morphine intake was found between the partial isolation group and the paired group (Fig. 3a). The days×group interaction was not significant. In the choice test, as in the forced test, we found a significant difference in morphine intake between groups [F(2,25)=12.15, P<0.001]. Post-hoc tests showed that isolated rats consumed more morphine (17.18±2.46 ml) than partial isolation group (8.92±0.3 ml) (P<0.002) and than socially housed group (7.16±0.28 ml) (P<0.001). No significant difference was found between partial isolation and paired conditions (Fig. 3b). There were no significant differences in water intake between the various housing conditions, but the days×group interaction was significant [F(2,10)=3.94, P<0.001]. Isolated, partial isolated, and paired subjects consumed more water (36.86±2.82; 33.36±1.43; 32.2±2.09 ml; respectively) than morphine solution (17.18±2.46; 8.92±0.3; 7.16±0.28 ml) [t(9)=5.42, P<0.0001; t(9)=17.8, P<0.0001; t(7)=12.05, P<0.0001].
Experiments 1 and 2 clearly showed that male and female rats in the socially restricted condition consume greater amounts of morphine solution compared with socially housed rats. This pattern was found both in one-bottle and in two-bottle tests. The fact that there were no differences in water intake between the groups suggests that this finding cannot be explained by a general enhancement of fluid intake by isolates, but rather was because of selective enhancement in morphine intake. Moreover, as there were no significant differences in body weight between isolated versus paired subjects during the course of experiments (see also Kretschmer et al., 2005; Thorsell et al., 2005, 2006), these results cannot be explained by differences in body weight that might affect daily intake and/or drug reactivity.
In most studies using social isolation paradigms, subjects are isolated at a young age (usually at infancy or postweaning) for extended periods. The socially housed condition usually consists of three or more animals living together in a group cage often containing additional enrichment items (Heidbreder et al., 2000; Lu et al., 2003; Brenes Saenz et al., 2006).
In contrast, in the present protocol, isolation was initiated in adulthood, the period of isolation was relatively short (21 days), and the animals were housed singly in large adjacent transparent cages, with access to sights, sounds, smells from the colony, but of course without tactile interaction with other animals. The socially housed subjects were housed identically, but with two animals per cage. Thus, our paradigm might be best understood as ‘social restriction in adulthood’. We thus attribute the increase in morphine intake in the socially restricted (isolated) group to the lack of direct tactile contact/social interaction between the animals.
Isolation-induced aggression is a well-documented phenomenon in rats and in other species. Aggressive behaviors of various types are increased as a function of social isolation across a number of isolation paradigms (Valzelli, 1971; Olivier et al., 1987; Byrd and Briner, 1999; Pietropaolo et al., 2004; Ferrari et al., 2005; Miczek and de Boer, 2005). It is generally agreed in these studies that isolation-induced aggression is sex related, the effect being observed in males but not in females (Swanson and Schuster, 1987). A question of interest would be whether the increase in morphine intake by isolated housing reported here is related to isolation-induced aggression. One observation suggesting that different mechanisms may be mediating these two phenomena is that isolation-induced aggression is seen only in males, whereas increase in morphine intake after isolated housing is seen in both males and females.
The impact of socially restricted housing may be temporary and reversible. This is seen in experiment 1 when previously isolated animals were switched to social housing and previously socially housed animals were switched to isolated housing. Under these conditions, morphine intake generally adjusted to the new (switched) housing condition. Animals previously isolated and then socially housed reduced their morphine intake by 62% in one-bottle and 55% in two-bottle tests. We did not observe strictly symmetrical changes when socially housed animals were switched to the isolation condition – their absolute intake of morphine did not change significantly, though the grouped means of the switched groups still differed significantly. This result gives strong support to the assumption that the elevation in morphine intake is indeed caused by the housing manipulation and not by some intrinsic or adventitious variables.
In this study, we show that as little as 60-min per day of social interaction from the onset of housing allocation (partial isolation) completely abolished the isolation-induced increase in morphine intake (experiment 3). To the best of our knowledge, this is the first study to report that brief daily periods of social interaction neutralize the increase in morphine intake in adult isolated rats. In juvenile rats, brief periods of socialization have been shown to ameliorate some of the effects of isolation rearing on later behavior (Einon et al., 1978). In this study, animals in the partial-isolation group consumed significantly less morphine than animals in the complete-isolation group and consumed similar amounts of morphine as those in the socially housed condition. Although 60-min of social interaction per day was sufficient to abolish the effects of 24-h social restriction daily, it is possible that even shorter periods of social interaction would be sufficient.
Indeed, this ‘partial-isolation’ reversal effect has been observed in ‘isolation-induced aggression’ and in an operant cooperation task (Berger and Schuster, 1987; Raz et al., 2005). In these studies, 60-min per day, but not 5-min per day of social contact, were sufficient to reverse the effects of 24-h social restriction daily. In these studies, the short period of social interaction was effective only if the interaction was with a conscious and behaving animal. No reversal of social isolation on aggression was observed when the animal was given the opportunity to ‘interact’ with an anaesthetized rat or with a live rat behind a mesh barrier. Thus, some intermediate period of daily social interaction, between 60 min and 5 min, is likely to be the minimum amount of interaction that is needed to reverse these effects of social isolation. It is assumed that physiological changes occur during these brief daily periods of social interaction in isolated animals that are associated with and mediate the reversal of the effect of isolation on drug seeking and perhaps aggressive behavior. There may be a ‘time window’ of 60 min or less when these changes occur – providing a good opportunity to further investigate these physiological processes, especially using biochemical and electrophysiological methods.
There are a number of possible explanations for the increases reported here in morphine consumption after social isolation. A commonly held view is that, in the rat and in other species, isolated housing and restricted social interaction produce physiological and behavioral changes that may be catalogued under the general category of ‘stress’. Consumption of morphine in isolated rats may be seen as ‘self-medication’, hypothetically bringing relief from the unpleasant state of stress. As socially housed rats are not exposed to the stressful consequences of social isolation, they do not benefit in the same manner from morphine consumption, which might even interfere with the performance of normal social interaction and therefore is consumed at lower doses (Alexander et al., 1978, 1981 Panksepp et al., 1979; McIntosh et al., 1980).
A second and perhaps related explanation is that social isolation may change the sensitivity and reactivity to various stimuli. Therefore, isolated rats may be more or less reactive to the bitter taste of morphine or to the novelty of the taste as opposed to the psychoactive action of the drug. Indeed, in our laboratory we have preliminary data that isolation housing may increase the intake of a bitter solution of quinine. We have also found (Raz and Berger, unpublished) that social isolation produces a startle response, which is a common behavioral test in the literature for assessment of emotional reactivity in rodents, and is often used to assess the effects of antianxiety drugs (Rodgers, 1997; Grillon, 2002; Bourin et al., 2007; Armario et al., 2008; Grillon, 2008).
Similarly, it might be possible to consider the results presented here as reflecting degrees of drug avoidance rather than a higher tendency of isolates to consume the drug for its beneficial psychoactive effects. Morphine solution consumption in our studies and in other studies is always less than water consumption indicating an avoidance of morphine consumption. Housing conditions may modify the degree of morphine avoidance, with single housing resulting in less avoidance than paired housing. Within the limits of this study, we cannot unequivocally state whether the increase in morphine intake after social restriction is because of an increased preference (reinforcing?) action of the drug or to a decreased aversion. Of course, there are other possible interpretations as to why rats consume less morphine solution than water solution, including the obvious pharmacological limitations imposed by the morphine intake. Indeed, in our studies (based on consumption rather than blood levels) we estimate that, in isolated housing, the mean daily intakes of morphine range between 25 mg/kg/24-h (two-bottle test) and 67 mg/kg/24-h (one-bottle test). Doses of 15, 24, 32, or 50 mg/kg/day of morphine in drinking water have been shown to exert a significant analgesic action in the rat (Badawy et al., 1982; Mao et al., 1996; Megens et al., 1998), supporting our assumption of a pharmacological action of the drug as the most appropriate explanation of the pattern of results in our studies. In addition, we have found in preliminary experiments that the opiate antagonist naltrexone reversed the increase in morphine consumption following isolated housing. Isolated rats treated daily with naltrexone (5 mg/kg) consume significantly lower amounts of morphine solution than isolated rats treated with saline and similar amounts to their socially housed counterparts. As naltrexone is not known to affect taste reactivity (Arbisi et al., 1999; Scinska et al., 2000; Goodwin et al., 2001), it is perhaps more likely that it reduces drug intake of isolates because it attenuates the psychopharmacological action of morphine.
A third explanation might be that social isolation somehow affects the rewarding consequences of morphine. However, it is difficult from our study alone to determine whether increased consumption after isolated housing is because of increased sensitivity, making the drug more rewarding, or whether it is an attempt to compensate for a possible decreased effectiveness of the drug (Hall, 1998). A limited number of studies have examined the effect of social and environmental manipulations on sensitivity to μ-opioids. Indeed these studies have shown that group-housed rats are more sensitive than isolated rats to the antinociceptive effects of morphine in the tail-shock and tail-compression tests (Czlonkowski and Kostowski, 1977; Kostowski et al., 1977; Panksepp, 1980; Smith et al., 2005) and are more sensitive to the rewarding effects of morphine and heroin in the place-conditioning procedure (Schenk et al., 1983; Wongwitdecha and Marsden, 1996a).
Social isolation and pathologies of social interaction are associated with a greater use of individual and multiple drugs of abuse, initiating drug use at younger ages, more chronic and more severe levels of addiction, and higher rates of dropout and relapse after withdrawal attempts (Higgins et al., 1994; Holdcraft et al., 1998; Pelissier and O'Neil, 2000; McMahon, 2001; Sayre et al., 2001; Dobkin et al., 2002; Compton et al., 2003, 2005; Darke et al., 2005; Westermeyer and Thuras, 2005). The studies in laboratory animals presented here may have relevance to the study of drug-seeking behavior in humans, and may have clinical implications especially in emphasizing the social context, social interaction, and support in the prevention and effective treatment of substance abuse.
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