The newborn rat's stress system readily habituates to repeated and prolonged maternal separation, while continuing to respond to stressors in context dependent fashion

Abstract

Adrenal corticosterone secretion of newborn mice rapidly desensitizes to repeated maternal absence. The present study investigated the effects of novelty exposure, maternal care and genotype on this phenomenon.

Maternal separation (MS) took place on postnatal days (pnd) 3–5. In Wistar rats, the degree of novelty in the MS-environment was varied by exposing pups to: (i) “home separation”: pups remained in the home cage; (ii) “novel separation”: pups were placed individually in a novel cage. Maternal care was recorded on pnd 1 to 4. To investigate the effect of genotype, we also examined Long Evans in the “home separation” condition. Basal and stress-induced ACTH and corticosterone levels were measured. Adrenal tyrosine hydroxylase (TH) and melanocortin receptor-2 (MCR-2) proteins served as markers for adrenal function.

We show, in both rat strains, that the rise in plasma corticosterone induced by a single 8 h-MS on pnd 5 was abolished, when this separation procedure had also been performed on pnd 3 and 4. Habituation to maternal absence occurred irrespective of housing conditions. However, pups in the “home separation” condition received less maternal care upon reunion than those placed in the “novel separation”. These “home separation” pups appeared more responsive to a subsequent acute novelty-stressor, and their adrenal TH and MCR-2 were higher. Long Evans rats appeared more stress responsive than the Wistars, in the home separation condition.

In conclusion, separation environment, maternal care and genotype do not affect adrenal desensitization to repeated 8 h-MS itself, but may modulate the adrenal stress-responsiveness of separated pups.

Introduction

Aberrant HPA axis activity and corticosterone (CORT) secretion induced by adverse early life experiences is considered a major risk factor for the development of psychiatric disorders in humans (Heim et al., 2008, Lupien et al., 2009). Therefore, rodents deprived as pups from maternal care have been widely used as a laboratory model for early adversity to study the underlying mechanism of CORT-enhanced vulnerabilities (Plotsky et al., 2005, Pryce et al., 2001a). Several adversity paradigms are currently used. One common approach is a single episode of 24 h of maternal absence. Another approach is repeated daily maternal separations (MS), which include repeated periods of 3–8 h absence of the dam during the first two postnatal weeks. It has been proposed that pups experiencing repeated MS display as adult enhanced stress responsiveness, increased anxiety, helplessness and anhedonia, deficits of sensorimotor gating and increased propensity for the intake of addictive drugs (Biagini et al., 1998, Brake et al., 2004, Moffett et al., 2007, Plotsky et al., 2005, Zhang et al., 2005). Interestingly, these rodent behaviors, programmed by early adversity, resemble clinical endophenotypes of depression and schizophrenia, hence providing face and construct validity to these models (Pryce and Seifritz, 2011). However, the outcome of early adversity depends on strain and gender of the animals as well as the frequency, duration, age and time point (within the light cycle) of MS (Claessens et al., 2011, Lehmann and Feldon, 2000). Moreover, the environmental context (housing in groups or in isolation, inside the home cage or in a novel environment) and the ambient temperature have an effect (Lehmann and Feldon, 2000, Ruedi-Bettschen et al., 2004).

Cumulatively, these observations have raised the question to what extent the HPA axis is actually activated during repeated MS. Schmidt and colleagues (Schmidt et al., 2004, Schmidt et al., 2006) found that, in mice, after 8 h of a single MS the basal level of circulating CORT has slowly reached peak levels, while the stress hyporesponsive period (SHRP) has become disrupted resulting in an enhanced responsiveness of the adrenocortical secretion of CORT to mild stressors and exogenous ACTH administration (Levine et al., 1991, Rosenfeld et al., 1992b). Enthoven and colleagues hypothesized that 3 consecutive daily 8 h-MS from postnatal (pnd) 3–5 would amplify neuroendocrine responses to both separation and novelty exposure. Surprisingly, they reported instead that the increase in HPA axis basal activity that is first observed after the initial 8 h-MS was rapidly abolished after repeated 8 h-MS. This rapid desensitization of the neonate's HPA axis to repeated daily separations from the dam was not due to metabolic factors such as ghrelin, and also did not occur because of enhanced glucocorticoid negative feedback (Enthoven et al., 2008). In spite of this rapid desensitization of the HPA axis to the effect of daily separation, the SHRP remained disturbed because a subsequent novelty stressor triggered an enhanced plasma CORT and c-fos mRNA response in the PVN (Enthoven et al., 2008). This finding raised the question whether the environmental context experienced by the pup during MS can influence the outcome (Enthoven et al., 2008).

In the present study we have extended these findings to the rat by examining the immediate effects of 3 daily repeated 8 h-MS from pnd 3–5. The objective of these experiments was to investigate further the apparent “desensitization” of HPA axis activity to repeated MS in different separation contexts in two rat strains. The degree of novelty was varied in the separation environment using: (i) “home separation”: the environment was the home cage and pups remained grouped together; (ii) “novel separation”: the environment did not contain any element of the home cage and pups were additionally isolated from their littermates. The effect of the different MS protocols was investigated on basal and novelty stress-induced ACTH and CORT levels on pnd 5. Since in the study of Enthoven et al.(2008), the apparent adrenal sensitivity to ACTH was altered dramatically during the repeated separations we also measured two biomarkers for adrenal function: tyrosine hydroxylase (TH) levels as index for adrenal medullary function and the level of melanocortin 2 receptor (MCR-2) as an index of adrenal sensitivity to ACTH. Moreover, maternal care was measured for the first 4 postnatal days to explore its possible implication in the outcome of the different separation procedures.

Similar to what we observed in mice, we also found that the MS-induced CORT response is readily abolished in rats if the separations are repeated daily, but that the animals' ability to respond to a novelty stressor depends on the separation context. Genotype and maternal care upon reunion did not affect the desensitization phenomenon, but rather appeared to be associated with stress responsiveness of the pup to an acute novelty stressor.