Behavioral Effect of Adolescent Housing Condition
Numerous studies have demonstrated that rats deprived of social interaction during adolescence exhibit a wide range of behavioral alterations when tested in adulthood, many of which recapitulate behaviors seen in children that have been exposed to early life stress. For example, rats raised in single cages during adolescence exhibit increases in anxiety-like behaviors (Hall et al., 1998a; Hellemans et al., 2004; Lim et al., 2011; Wright et al., 1991), hyperactivity in a novel environment (Hall et al., 1998a; Hellemans et al., 2004), deficits in sensory gating (Heidbreder et al., 2000; McCool and Chappell, 2009) and cognitive function (Hedges and Woon, 2011), relative to rats raised in groups and/or in enriched environments. Our data confirm that, with adolescent social interaction as the sole dependent variable, rats deprived of physical contact with their peers during adolescence exhibit significant increases in anxiety-like behavior, assessed on the plus-maze, when compared with rats housed in the same vivarium, but in groups of 4.
We also examined response to novelty by assessing locomotor activity in a novel environment as well as the locomotor response to a novel object. As in other studies (Hall et al., 1997; Hellemans et al., 2004; Lapiz et al., 2000), SI rats exhibited a significant increase in locomotor activity during the first 60 minutes in the novel environment, relative to GH subjects, consistent with an increased behavioral responsivity to an unfamiliar environment. Although this effect was not apparent for the first 10 minutes of the test and the difference in activity dissipated after 60 minutes. Moreover, following the 1 hour acclimation, SI and GH rats showed a similar locomotor response upon the introduction of an immovable novel object. These findings suggest that adolescent social isolation may have a greater effect on response to novelty where there is a significant stress component (i.e., exposure into an inescapable novel environment) (Robinet et al., 1998) than on novelty responding in a context that may be less susceptible to the influence of stress and more related to exploration (animals had already acclimated to the new test cage for 60 minutes prior to the introduction of the novel object and they could choose to explore or ignore the object).
As many rodent EtOH self-administration studies employ single housing conditions, often for extended time periods (Anacker and Ryabinin, 2010; Camarini et al., 2010; Samson and Czachowski, 2003), we sought to examine the behavioral phenotype of singly housed rats purchased to match the age of SI and GH rats, 5 weeks into the juvenile housing manipulation. Surprisingly, STD rats weighed significantly less than the other 2 cohorts upon arrival. Although STD subjects gained weight at a rate comparable with that of SI and GH rats, their weight remained lower than these other 2 cohorts throughout the duration of this study. Moreover, the behavioral profile of STD rats on the plus-maze was very similar to that of SI rats. Therefore, rats obtained as adults from a standard commercial supplier and then housed under conditions commonly used in rodent EtOH-drinking studies display at least some of the behavioral characteristics of animals that have been reared under impoverished conditions. It should be noted that STD rats were only given 1 week of acclimation prior to testing to mimic protocols frequently used in EtOH self-administration studies. Future studies will be needed to determine whether longer acclimation periods can mitigate the anxiogenic behavioral profile observed in STD rats.
Effects of Adolescent Housing Condition on EtOH Self-Administration
To examine the effect of housing condition on EtOH self-administration, subjects were first given 3 days of continuous access to a 10% EtOH solution as their only source of fluid and this was immediately followed by a 5-day 2-bottle choice procedure (10% EtOH/water). The 3-day single-bottle procedure encourages the consumption of pharmacologically relevant amounts of EtOH, as the EtOH solution provides the only source of water to the animals. However, forced EtOH consumption does not always correlate with measures of voluntary EtOH drinking (Chappell and Weiner, 2008). In contrast, 2-bottle choice procedures are frequently used as a simple measure of EtOH's reinforcing effects, as intake in these assays often correlates with EtOH seeking behaviors (Green and Grahame, 2008; although see, Chappell and Weiner, 2008; Samson and Czachowski, 2003). As in our prior study (McCool and Chappell, 2009), no differences in forced EtOH consumption were noted between SI and GH rats, suggesting that adolescent social isolation does not exert nonspecific effects on overall fluid consumption. However, in the 2-bottle choice procedure, SI rats drank significantly more EtOH than GH rats, with a trend toward a significant increase in EtOH preference. These findings are generally consistent with our initial study and further suggest that high levels of antecedent anxiety-like behavior may promote increased EtOH self-administration.
STD rats drank more EtOH than either GH or SI rats in the forced consumption assay and also drank EtOH at levels comparable with SI rats in the 2-bottle choice procedure. If increased levels of anxiety-like behavior promote EtOH drinking, it may not be surprising that STD rats drank as much EtOH as SI subjects as both of these groups exhibited comparable levels of anxiety-like behavior on the plus-maze. However, the observation that SI and STD rats drank similar amounts of EtOH raises the question of whether adolescent social isolation promotes EtOH intake or if group housing lowers EtOH consumption? While this question cannot be conclusively addressed from this study, it should be noted that measures of EtOH intake and preference in SI and STD rats were similar to those observed in a prior study that we conducted with STD Long Evans rats (Chappell and Weiner, 2008) and are also in good agreement with earlier studies conducted with this rodent strain in this same laboratory by Samson and Czachowski (2003).
Although 2-bottle choice procedures are frequently used to assess EtOH self-administration in rats, intake using these models with outbred rats is rather modest and it can be difficult to generate pharmacologically meaningful blood EtOH levels with these protocols (Samson and Czachowski, 2003). Therefore, we also employed an intermittent 2-bottle choice procedure that results in a robust escalation of EtOH intake, even in outbred, commercially procured animals (Simms et al., 2008). Both SI and STD rats escalated their EtOH intake to almost 1 g/kg within the first 30 min of drinking sessions, with some subjects reaching intake levels as high as 8 g/kg per day. In contrast, GH rats did not escalate beyond an average of 0.5 g/kg in the initial 30 minutes of EtOH availability and achieved significantly lower BECs than either SI or STD rats.
These data also support our prior findings, using a limited access operant procedure, in which the adolescent isolation increased consummatory measures of EtOH drinking in Long Evans rats (McCool and Chappell, 2009). However, again, based on the observation that STD rats drank EtOH at levels comparable with SI rats, our findings may be interpreted as evidence that group housing blocks the escalation in EtOH intake that is normally promoted by the intermittent model. One caveat to consider is that cohorts were subjected to 3 distinct EtOH self-administration procedures. It is possible that factors such as stress associated with the 3-day forced EtOH-drinking procedure may have contributed to some of the subsequent differences in voluntary EtOH self-administration. Nevertheless, given that SI and STD rats also displayed comparable levels of anxiety-like behavior on the plus-maze in this study, prior to any EtOH exposure, perhaps the better question may be whether commercially procured animals housed under conditions frequently used in home-cage EtOH-drinking studies can be considered “normal”? Many factors, in addition to the number of cage mates, can impact experimental results. For example, the commercial source of rodent strains can significantly influence the results of diverse studies ranging from an analysis of motor, sensory, and autonomic outcomes associated with a compression model of spinal cord injury (Lonjon et al., 2009) to the induction and long-term consequences of status epilepticus (Langer et al., 2011). Of direct relevance to this study, rats of the same strain, but ordered from different suppliers, have been reported to differ markedly in their initial levels of anxiety-like behaviors (Rex et al., 1996) as well as voluntary EtOH-drinking measures (Palm et al., 2011). Allelic variations that can arise when breeding outbred strains of rats may have contributed to some of the disparate findings noted previously. Such factors are not likely relevant here as all cohorts were obtained from the same supplier. There are, however, many other differences related to housing conditions that may contribute to within-strain differences in experimental results such as those observed in this study (e.g., diet, frequency of cage-cleaning and animal handling, density of animals/cage). Unfortunately, many of these factors are not readily available from suppliers and often go unreported in publications. While we controlled for many of these factors, using a diet, cage-changing schedule, and animal handling frequency similar to those employed by our commercial supplier, there were likely many additional variables that we were either unaware of, or could not readily control for. For example, while GH and STD rats were both group housed during adolescence (4 to 6/cage), there were extreme differences in the density of cages/room. GH subjects were housed in a relatively small vivarium (15 × 30 ft) that contained between 80 to 120 animals. In contrast, while at the commercial supplier, STD rats were housed in large, industrial barrier facilities that contained thousands of animals (Harlan Laboratories, personal communication). Our observations that age-matched STD rats weighed significantly less than GH rats and that large and enduring differences in anxiety-like behavior and EtOH-drinking measures were observed between these groups suggest that early life housing conditions can profoundly influence physiological and behavioral outcomes in adulthood, at least for male, Long Evans rats.
Thus, the question of which cohort should be considered “normal” may really depend on the experimental questions being asked. For studies directed at characterizing the relationship between antecedent anxiety-like behavior and EtOH drinking, our findings suggest that commercially sourced adult rats may not be a suitable model, as the anxiety-like behavior of these animals on the elevated plus-maze was very similar to that of SI rats. In fact, the distribution of open arm times for both STD and SI subjects were clustered within a relatively narrow window, near the upper limit of anxiogenic behavior on the plus-maze. In contrast, animals purchased from a commercial supplier, immediately postweaning, and then group housed during adolescence, exhibited a much broader range of anxiety-like responses on the plus-maze and may, thus, better reflect the spectrum of anxiety-like behaviors expected in “normal” Long Evans rats. In addition, a significant relationship between initial anxiety-like behavior and EtOH drinking, similar to that observed in humans (Chan et al., 2008; Kessler et al., 1997; Kushner et al., 2000), could only be detected in data sets that included GH subjects. Clearly, additional studies will be needed to compare the behavioral profile of STD rats with that of SI and GH animals across a broader range of anxiety-like measures. However, given that seemingly minor environmental factors can significantly influence at least some measures of anxiety-like behavior in rats, and the fact that commercial laboratory animal suppliers do not typically provide detailed information on the rearing conditions of their animals, it would seem that GH subjects may represent a preferable control group in studies examining the complex relationship between anxiety and EtOH drinking.
In summary, our data confirm prior findings that rats reared under conditions in which they are deprived of social contact during adolescence exhibit increased levels of anxiety-like behavior and novelty responding in adulthood, relative to animals raised in small groups during this period. SI rats also consume significantly more EtOH and obtain higher blood EtOH levels in home-cage voluntary drinking procedures. Our data further suggest that rats obtained from a commercial supplier as young adults and then housed under conditions commonly used in home-cage EtOH self-administration studies display an anxiety-like and EtOH-drinking profile similar to that of rats that have been socially isolated during adolescence. Together, these findings provide further evidence that studies with juvenile GH and SI rats may provide a useful model with which to study behavioral and neurobiological substrates linking early life stress and vulnerability to alcoholism. These data also suggest that commercially sourced adult rats may not be suitable for studies examining the relationship between anxiety and EtOH drinking as these animals seem to share an anxiety-like behavioral profile similar to that of rats that have been exposed to chronic early life stress.