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Thirty-three million individuals in the United States are projected to suffer from major depressive disorder (MDD) in their lifetime (Kessler et al. 2007). Genetic antecedents and environmental influences are well acknowledged contributors to the precipitation and perpetuation of major depression, and stress is a unifying concept such that the first depressive episode is more likely to be preceded by a severe stressful life event than are recurrent episodes (Foster and MacQueen 2008; Miller et al. 2009; Stroud et al. 2008). Biological markers associated with MDD are largely related to the assessment of HPA axis function (Vreeburg et al. 2009) and inflammatory immune indicators, two interdependent pathways also prominently affected by severe psychological stress (Irwin and Miller 2007; Miller et al. 2009; Miller et al. 2005), as well as glucocorticoid receptor sensitivity (Miller et al. 2005).
A social defeat paradigm, repeated exposures to a dominant resident distributed over 4 weeks, induces depression in the rat in order to causally evaluate the impact of depression on sleep. There is etiological consistency regarding the prominence of extreme stress, particularly of a social nature, as a depression triggering event in humans (Huhman 2006; Stroud et al. 2008). The effects of social defeat that correspond to criteria for MDD in humans including, anhedonia and reduced food consumption and activity, are improved with antidepressant administration (Becker et al. 2008; Rygula et al. 2006a; Rygula et al. 2006b), supporting our contention that chronic social defeat in the rat can be a useful model of first onset depression. To our knowledge, sleep and neuroimmune function have been minimally studied (e.g., Kieran et al. 2010; Razzoli et al. 2007), and immune function has not been studied in this promising preclinical model.
This study is intended to causally evaluate the impact of repeated exposures to social defeat on sleep architecture using electroencephalogram (EEG) / electromyogram (EMG) recordings, as well as neuroendocrine and immune function in Fischer 344 female and male rats. A simple 2´2 experimental design was used: female versus male by repeated exposures to either social defeat or remaining in the home cage. Our goal is to determine the impact of repeated exposures to social defeat on sleep architecture and on neuroimmune function.
Methods:
Mature female and male Fischer 344 and Long Evans rats were maintained on a 12:12 hour dark/light cycle and ambient temperature at 22±1 ºC. Given that the active period for rats is during the dark phase and evidence of greater sleep disruption with light phase manipulations (Chang and Opp 2002), all perturbations were undertaken during the dark phase.
Fischer 344 rats either underwent 12 exposures to social defeat or remained in their home cage. The social defeat paradigm consists of 12 60-minute intrusions over a 4-week period into a resident cage populated by a Long Evans male ex-breeder and a long-term cohabiting ligatured female who continues to cycle, but cannot become pregnant. Female F344 rats are introduced into the resident cage immediately following the removal of the Long Evans male; male F344 rats are introduced into the resident cage with both the female and male Long Evans rats (Becker et al. 2008; Rygula et al. 2005). Immediately following defeat of the intruder, 5 submissive postures or the intruder is pinned for 5 seconds; a protective mesh barrier is placed over the intruder within the resident cage for the remainder of the intrusion period. This barrier allows the stressful nature of the encounter to continue without the threat of physical defeat (Becker et al. 2008; Rygula et al. 2005; Sloman et al. 2003), reflective of entrapment (Gilbert et al. 2002; Sloman et al. 2003). A rotation of residents was used such that intruders were exposed to a minimum of six different residents over the 4 week course.
Telemetric transmitters for EEG/EMG recording were implanted under isoflurane anesthesia and animals were allowed 4 weeks recovery. EEG/EMG recordings were continuous, allowing us to preserve all data, and selectively sample. Arousal state was classified as wakefulness, non-rapid eye movement sleep (NREMS), or REMS as detailed in (Opp 1998). Determination of arousal state was undertaken for a 24-hour period prior to the first social defeat exposure, baseline, and following the final social defeat. Arousal state determination is in progress for the midpoint of the defeat paradigm.
Four biobehavioral outcomes comprise parallels to human MDD criteria. (1) Anhedonia was assessed as sucrose preference, a choice between plain water and a 0.5% sucrose-water solution, reported as % sucrose intake; (2) Changes in body weight reflect decreased food consumption; (3) plasma levels of pro-inflammatory cytokines parallel a pro-inflammatory immune balance evident in humans with MDD; and (4) reduced glucocorticoid receptor sensitivity is reflected by in vitro dexamethasone (DEX) suppression of pro-inflammatory cytokine production. Briefly, diluted whole blood is co-incubated with lipopolysaccharide (LPS) to stimulate pro-inflammatory cytokine production, plus varying concentrations of DEX (0, 1, 10, 50, 100 and 1000 nM) for 48 hours at 37o C. The harvested supernate is then assayed for TNF-alpha levels to determine the 50% inhibition concentration (IC50) of DEX for each individual animal based upon the individual dose response curve generated (Miller et al. 2005).
Results:
Data collection has been completed and analyses are in progress. First, there are changes in light phase sleep architecture from baseline to the final social defeat. Specifically, both %REMS per hour and the total number of REMS bouts in the light phase decreased from baseline to the final social defeat [t(12)=2.350 and 2.522, respectively, p<0.05]. The average duration of wakefulness also decreased from baseline to the final social defeat [t(12)=2.541, p<0.05]. The total light phase NREMS bouts increased from baseline to the final social defeat [t(12)=2.390, p<0.05]. T-tests yielded trends for increasing sleep state transitions, p=0.052. Given that the dark phase is the more active phase of the rat, that we detected no sleep architecture changes from baseline to the final social defeat is not a surprise. Second, over the 4-week paradigm, the social defeat animals exhibited a marked reduction in sucrose intake compared to home animals [F(1,28)=104.783, p<0.001], and among the social defeat animals, females exhibited significantly less sucrose intake compared to the males [F(1,13)=9.807, p<0.01]. Changes in body weight over the 4-week period of social defeat, and terminal plasma pro-inflammatory cytokine levels and DEX suppression are currently under analysis.
Conclusion:
The problems of insufficient sleep and depression are substantial in the U.S. (Kessler et al. 2007; Krueger and Friedman 2009); both are associated with substantial consequences to health (Roth 2009; Rush 2007); and the comorbidity of sleep problems and depression are well known (e.g., Lam 2006; Staner 2010). Sleep disturbance is a common symptom of MDD, affecting 80% or more of individuals with MDD. 15-35% endorse hypersomnia and difficulty with morning arising. Others endorse difficulty falling asleep, staying asleep, and early morning awakenings (Armitage 2007; Germain and Kupfer 2008), consistent with findings of studies using EEG for arousal state determination (Armitage 2007). This study in rats offers a means by which to examine the relevance of this preclinical model with which to study the biobehavioral consequences of an etiologically consistent and profoundly stressful phenomenon, chronic social defeat.
To our knowledge, females have not been included in previous studies of chronic social defeat. (Becker et al. 2008; Razzoli et al. 2007; Rygula et al. 2005; Rygula et al. 2006a; Rygula et al. 2006b; Sgoifo et al. 2002), and although there are a number of reports of sex and estrous phase related sleep differences in rats (e.g., Del Río-Portilla et al. 1997; Fang and Fishbein 1996; Schwierin et al. 1998), the literature focusing on sex differences in stress-induced changes in sleep is scarce; and no reports of sleep in female rats exposed to social defeat are evident.