Research reportEffect of social isolation on stress-related behavioural and neuroendocrine state in the rat
Introduction
There is now a large body of evidence that stressful early life experiences (maternal deprivation, social isolation) can affect brain development and subsequent adult behaviour [1], [2], [6], [28], [40], [70]. Although the underlying mechanisms are still poorly understood, it seems to be clear that in humans similar environmentally-induced changes could be considered as core factors in the etiology of psychiatric disorders such as schizophrenia, depression or anxiety disorder [33]. In the laboratory rat, early life isolation from social counterparts has been reported to constitute a stressful experience [31], [32], [35] as revealed by its consequences on adult behavioural and hormonal reactions to discrete challenges. The concept of isolation ‘stress’ in rats is derived from early 1960s studies that reported social isolates as abnormally reactive to handling and hyper-emotional [31], [32]. Because social isolation (SI) is conducted during the developmental period from weaning to adulthood, it deprives animals of social contact during a critical phase of their life span characterised by the development of social play [13]. Although, in most studies, isolates maintain olfactory, visual, and auditory contacts by being reared in the same husbandry colony as their socially-reared counterparts (2–4 rats per cage), the lack of physical contact generates a range of behavioural and physiological reactions, which are known to affect considerably the emotional reactivity of adult rats.
An abnormal reactivity of isolated rats to environmental stimuli was described for the first time in the early 1960s by Hatch and co-workers [31], [32]. In these studies, socially isolated rats were described as very reactive to human handling, nervous, and aggressive. In line with this increased emotionality of isolates, one of the most widely reported and reproducible findings following SI is an increased locomotor activity in response to novel situations [10], [14], [22], [54], [63], [68], [71]; however, some strain specificity is apparent, since isolates from the Sprague–Dawley strain do not show this spontaneous hyperactivity [24], [68], [69]. Moreover, isolates need more time to enter a novel environment from a familiar one [8], [12], [13], they show neophobia [29], [30], [44], [45] and increased defecation in the open field [35]. The SI-induced neophobia may be associated with increased anxiety. Indeed, anxiogenic profiles were observed following chronic SI, for example in the elevated plus maze [47], [73], and SI has been proposed as a model for anxiety [47].
The evidence that SI leads to a phenotype consistent with increased emotionality is not a finding shared by all studies. For example, some studies report similar behavioural reactivity to novelty between isolation- and group-reared rats and similar levels of spontaneous activity [18], [20]. In some studies, SI was found to actually reduce the defecation score [21], [23] or not to change it [18]. Locomotion in an open field environment is a complex behaviour, involving neophobia, ‘pure activation’ (increased arousal), and exploration, the balance between them being directly modulated by the emotional state of the animal and its behavioural and physiological reactivity (grooming, defecation, urination). Thus, both increased locomotion (i.e. escape response) or reduced locomotion (i.e. freezing response) may indicate a state of fear or anxiety [3]. Methodological differences, such as SI procedure or test environments [11], could also contribute to the lack of consensus regarding the effects of SI on spontaneous activity. Moreover, abnormalities in the behavioural response of isolates to discrete challenges have not always been associated with alterations of endocrine stress response. The latter has been studied in isolates by measuring the activity of the hypothalamic–pituitary–adrenal (HPA) axis, which is a major system in the adaptive acute response to stress. Changes in basal and stress release of adrenocorticotropic hormone (ACTH) and/or corticosterone (CORT) have not been conclusively demonstrated in isolates. While most studies report no effect of SI on the basal CORT level [21], [34], [35], [43], [46], [66], others demonstrate either increased [18], [19], [32] or reduced basal CORT levels [59] following SI. In response to both ‘physical’ and ‘physiological’ stress treatments, the release of ACTH and CORT were found to be either increased (ACTH injection [32]; open field exposure [35]; footshocks [66]) or unchanged following SI (forced swim [34]). Gender differences have also been reported, with increased CORT production in response to ACTH being greater in females than in males. Finally, both increased [31], [35], [63] as well as unchanged adrenal weight [18], [21], [43], [46], [66] were observed in isolates relative to group-reared controls, a result which depends on the gender, with SI more likely to produce increased adrenal weight in females than in males [5], [32].
After careful scrutiny of the literature, it is therefore not clear whether or not the isolation stress syndrome should be regarded as a developmental manipulation with predictable, robust behavioural and endocrine consequences. The aim of the present study was thus to investigate the behavioural and neuroendocrine changes produced by chronic SI in a large controlled study, where behavioural reactivity in response to graded stress treatments could be assessed within the same animals. We can therefore eliminate the confounding effect of the stress of handling by testing the same animals several times. To evaluate the effect of gender on stress-related behavioural and endocrine state following SI, we tested male and female rats reared in SI or in social groups from weaning until adulthood. The studies were conducted in the Sprague–Dawley strain, since isolates from this strain do not show spontaneous hyperactivity [24], [68], [69]. The latter factor is important to control when evaluating the specific effects of SI on behavioural paradigms sensitive to locomotor activity, such as the elevated plus maze or aversive classical conditioning as measured in the freezing paradigm.
Innate and conditioned aspects of fear and anxiety were evaluated in different paradigms, since it has already been shown in our laboratory that specific brain treatments can affect unconditioned and conditioned fear paradigms differentially [38]. Spontaneous locomotor activity in a novel situation was assessed in the open field environment (mild stressor). Anxiety was measured in the elevated plus maze, a pharmacologically validated anxiety test [49]. Repetitive acoustic stimulations of 120 dB (30 ms duration) were given to the animals to measure their startle reflex. This startle session was further employed as a stressor to elicit the release of stress hormones (ACTH and CORT) [25], [62]. Pavlovian fear-conditioning was studied using the freezing paradigm (strong stressor) [53], [55]. SI neuroendocrine effects were evaluated by measuring plasma ACTH and CORT levels as well as mRNA expression of mineralocorticoid and glucocorticoid receptor in the hippocampus.
Section snippets
Animals
The present studies were conducted on Sprague–Dawley (SD) rats [Zur:SD(Crl:CD®(SD)BR)], bred at the Behavioral Neurobiology Laboratory, Schwerzenbach (CH). Animals were maintained under standard conditions, in temperature (21±1.0 °C) and humidity (55±5%) controlled rooms, on a 12 h–12 h reversed light:dark cycle (lights off at 7 a.m.). During the studies, animals had access to food (Nafag, 9431, Nafag Ecossan, Gossau, Switzerland) and water ad libitum. All experiments were carried out in accordance
Weight of the animals 13 weeks after weaning
Body weight of the animals was significantly different between the genders, F(1,24)=179.1, P<0.001, males (527.0±16.9 g) being heavier than females (283.3±4.6 g). There was no effect of SI on the body weight of the rats.
Locomotion in the arena
All animals showed habituation to the open field environment, as shown by a significant reduction of the total distance moved during the 1-h session (F(5,120)=103.6, P<0.001). Females (1610.1±154.7 cm) were more active than their male counterparts (999.7±109.1 cm; F(1,24)=9.9, P
Discussion
The present study demonstrates that, primarily in males, post-weaning SI produced an emotional state in adult rats associated with an anxiogenic profile and a behavioural and hormonal hyper-reactivity to stressful situations. In contrast, SI did not modify the behavioural reactivity of females in experimental situations assessing fear/anxiety related states. Moreover, the HPA axis activity was not altered by rearing females in SI.
In both genders, SI did not modify spontaneous locomotor
Acknowledgements
This study was supported by the Swiss Federal Institute of Technology Zurich. Isabelle Weiss was further supported by a grant from F. Hoffmann-LaRoche, Ltd. (Basel, Switzerland). We would like to thank the animal facility team of Dr. Isabelle Iselin for the care of the animals, Dr. Annette Domeney and Maud Seguy for helping with blood sampling, Peter Schmid for his precious technical assistance and Jane Fotheringham for her secretarial help.
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- 1
Present address: Institute of Cell Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, HPM D 22, 8093 Zurich, Switzerland.
- 2
Present address: Neuroscience Discovery, ABBOTT GmbH & Co. KG GG RP, 11, 201, Knollstrasse, 67061 Ludwigshafen, Germany. Tel.: +49-621-589-2933; fax: +49-621-589-3232.