Long-lasting changes in stress-induced corticosterone response and anxiety-like behaviors as a consequence of neonatal maternal separation in Long–Evans rats

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Abstract

Early neonatal environmental factors appear to have powerful and long-lasting influences on an organism's physiology and behavior. Long–Evans male rats separated from their dam for 3 h daily over the first 2 weeks of life (maternally separated, MS rats) when tested as adults exhibit exaggerated behavioral and neuroendocrine responses to stress compared to 15-min separated (handled, H) animals. The purpose of this study was to compare male and female adult rats that were MS, H or were undisturbed (nonhandled, NH) as neonates in anxiety-like behaviors, in the elevated plus-maze, and in response to startle-inducing auditory stimuli. We confirmed that MS males oversecrete corticosterone (CORT; 2.5–5 times) in response to mild handling stress. MS males and females were less likely to explore open arms of the plus-maze. MS males exhibited 35% higher startle amplitudes compared to controls. Furthermore, MS males were more likely to emit ultrasonic vocalizations in response to startle than were H controls. However, MS and control females did not differ in auditory startle response or in startle-induced ultrasonic vocalizations. Therefore, experiencing maternal separation results in a long-lasting increase in anxiety-like behaviors that occurs in a sex-dependent manner.

Introduction

The last few decades have seen a tremendous increase in interest in the topic of early environmental influences on an organism's physiology and behavior. A large body of evidence indicates that exposure to early adverse life events in the form of childhood neglect and abuse can increase vulnerability to psychopathology in adult life Heim et al., 1997, Heim et al., 2000, Ladd et al., 2000, Caldji et al., 2001.

Periodic neonatal maternal separation in the rat has been used by several investigators as a rodent model of the effects of early adverse life events on adult physiology and behavior. In this procedure, neonatal rats are removed from the mother for several hours daily during the first 2 weeks of life Plotsky and Meaney, 1993, Wigger and Neumann, 1999, Kalinichev et al., 2000, Huot et al., 2001, Boccia and Pedersen, 2001. This procedure is based on the observation by Calhoun (1963) that, in a seminaturalistic environment, subordinate dams are often forced to build their nests at some distance from food and water resources. This results in extension of mother–litter separations from typical 15–30 min to as long as 2–3 h Francis et al., 1999, Liu et al., 2000. When tested as adults, maternally separated (MS) adult offspring exhibit a cluster of behavioral and neuroendocrine signs similar to those observed in patients with depression and anxiety disorders Amsterdam et al., 1987, Heit et al., 1997, Ladd et al., 2000. A primary feature of the MS phenotype has been an enduring dysregulation of hypothalamic–pituitary–adrenal (HPA) axis reactivity to stress Ladd et al., 2000, Caldji et al., 2001. Compared to males that were either separated daily for 15 min (handled, H) or completely undisturbed (nonhandled, NH) as neonates, MS males have elevated basal CRF levels in the median eminence and elevated levels of the CRF mRNA levels in the hypothalamus (Plotsky and Meaney, 1993). In response to stress, such as restraint or air-puff startle, MS males display a potentiation in ACTH and corticosterone (CORT) secretion compared to either H (restraint) or H and animals that received typical animal husbandry (animal-facility reared, AFR; air-puff; Plotsky and Meaney, 1993, Huot et al., 2001). Furthermore, in MS males, there is significant reduction of GABAA receptor levels in the medial prefrontal cortex, as well as in locus coeruleus and nucleus tractus solitarius (Caldji et al., 2000). This decrease in GABA-binding sites might result in potentiated noradrenergic stimulation of CRF synthesis and release (Plotsky et al., 1989) and contribute to increased CORT and ACTH responses to stress observed in MS animals Liu et al., 2000, Huot et al., 2001. In addition, compared to H and NH males, MS males have increased stress-induced norepinephrine responses in the paraventricular nucleus of the hypothalamus, a major source of the CRF that regulates the HPA activity (Liu et al., 2000).

In accord with the neuroendocrine changes, there is some evidence of behavioral alterations in MS animals that resemble those observed in patients with affective disorders. MS rats appear to exhibit increased anxiety-like behavior, anhedonia, increased preference for ethanol and impairment in male sexual behavior Wigger and Neumann, 1999, Caldji et al., 2000, Ladd et al., 2000, Huot et al., 2001, Rhees et al., 2001.

While the majority of investigators examined the effects of maternal separation only in male offspring, those that studied both males and females found sex-dependent effects. For example, while MS and NH males are less likely than H males to explore open arms of the plus-maze and consume a novel food, MS females resemble H females, exhibiting decreased anxiety and neophobia compared to NH females (McIntosh et al., 1999). According to Wigger and Neumann (1999), the impact of maternal separation in decreasing open-arm activity in the elevated plus-maze is more robust in males than in females. Also, exposure to the plus-maze test potentiates the ACTH release in MS males but not in MS females (Wigger and Neumann, 1999). We found that MS males are less sensitive to morphine than are H and NH males in the hot-plate test of antinociception, while this difference is absent among the female groups (Kalinichev et al., 2001).

The increased interest in the paradigm of maternal separation by several laboratories also has resulted in discrepancies among findings, especially concerning behavioral signs of anxiety in MS animals (Lehmann and Feldon, 2000). Some of the differences in outcomes among studies may be, in part, due to the diversity of separation procedures used (e.g., different strains, duration and days of separation, whether the offspring are isolated individually or kept as a litter, ages at testing and behavioral tests applied). For example, Sprague–Dawley males that were separated from their mother for 6 h daily on postnatal days 2–10 when tested as adults did not differ from NH males in the open-field activity (Rhees et al., 2001). In contrast, Sprague–Dawley males separated from the mother for 4.5 h daily during the first 3 weeks of life were less active in the open-field test compared to NH males (Ogawa et al., 1994).

Another source of discrepancy appears to be the control group that is used for a comparison with MS animals. Overall, there is an agreement that MS males of several rat strains exhibit signs of increased anxiety compared to H males in the open-field, plus-maze, auditory startle and novelty-induced suppression of feeding tests McIntosh et al., 1999, Wigger and Neumann, 1999, Caldji et al., 2000, Huot et al., 2001. However, there is lack of agreement on whether MS animals exhibit signs of increased anxiety compared to animals that were completely undisturbed as neonates (NH group). According to some authors, MS males exhibit increased anxiety compared to NH males, as indicated by decreased open-field activity and increased likelihood of approaching food in a novel arena when food deprived Ogawa et al., 1994, Caldji et al., 2000. However, in other measures of novelty-induced suppression of feeding and in the plus-maze test, MS and NH males are similar McIntosh et al., 1999, Caldji et al., 2000. Furthermore, plasma levels of CORT and ACTH following stress are also similar in MS and NH males Plotsky and Meaney, 1993, Liu et al., 2000. According to others, plasma CORT levels during (Ogawa et al., 1994) or following (Pryce et al., 2001) restraint stress are even higher in NH than in MS males.

The primary goal of our study was to shed some light on the MS vs. H vs. NH comparison in anxiety-like responses in both males and females. In particular, elevated plus-maze activity and startle-induced ultrasonic distress vocalizations were studied in Long–Evans rats that experienced 3-h daily separations from the dam during the first 2 weeks of life. Initially, we wanted to confirm that, under our experimental conditions, stress-induced secretion of CORT and ACTH is significantly higher in MS males compared to H males Plotsky and Meaney, 1993, Liu et al., 2000, Huot et al., 2001. We hypothesized that repeated separation from the mother would result in significant alteration in the offspring's reactivity to aversive environmental stimuli in a sex-dependent manner.

Section snippets

Subjects

The subjects were male and female Long–Evans hooded rats (Blue-Spruce, Harlan Sprague–Dawley, Indianapolis, IN) 120 days of age at the time of testing. All the subjects were the offspring of dams that were shipped to our facility on their 12th day of pregnancy. The pups were born and raised in our colony. A total of 108 males and 48 females were used in this study. Food and water were always available ad libitum. The colony room was maintained on a 12:12 light–dark cycle with lights on at 0700

Body weights

Group means (with standard errors) of body weights of MS and control animals on postnatal days 14, 30, 60 and 90 are presented in Table 1. At the age of 14 days, the last day when the mother–litter separation was performed, MS males were significantly (up to 10%) lighter in body weight compared to both H and NH animals [F(2,32)=12.7, P<.0001; Table 1]. At the same age, MS females were significantly (up to 10%) heavier compared to both H and NH females [F(2,29)=6.4, P<.01; Table 1]. Beginning at

Discussion

In Long–Evans rats, repeated 3-h separation from the mother during the first 2 weeks of neonatal life resulted in enhanced secretion of CORT in response to mild stress and in altered anxiety-like behaviors in response to aversive environmental stimuli. Whether a particular environmental challenge (i.e., test for anxiety-like behavior) can reveal behavioral differences between MS, H and NH animals appears to be influenced significantly by the sex of an animal.

Under normal conditions, during the

Acknowledgements

We thank Dr. Michael Davis for his advice on measuring the acoustic startle response. This study was supported by NIH grant DA11384 and Research Scientist Award K05 DA00008 to S.G.H.

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