The effect of different maternal deprivation paradigms on the expression of hippocampal glucocorticoid receptors, calretinin and calbindin-D28k in male and female adolescent rats

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Abstract

Maternal deprivation (MD) is a well-established protocol used to investigate neurobiological changes that are associated with the etiology of and vulnerability to stress-related diseases in animal models. The resulting psychophysiological effects, the timing and duration of these adverse stimuli, and the method by which they exert their effects on the animals remain unclear. This study characterized differences in the hippocampal expression of glucocorticoid receptors (GRs) and the calcium-binding proteins calretinin (CALR) and calbindin-D28k (CALB) in male and female rats that underwent different MD paradigms during the stress hyporesponsive period (SHRP). Both GRs and the two calcium-binding proteins were much more abundant in females than in males. MD paradigms had a significant effect on CALR and CALB expression in both males and females but affected GR levels only in males. Additionally, expression of the two calcium-binding proteins in the hippocampus responded differently to MD-induced stress, especially in females. Taken together, these results indicate that females are able to modulate their response to stress better than males.

Highlights

► We compare three different maternal deprivation (MD) paradigms in this study. ► We examine changes in the hippocampal expression of three proteins in rats on PND21. ► They were glucocorticoid receptors (GRs), calretinin (CALR) and calbindin-D28k (CALB). ► All the three proteins were much more abundantly expressed in females than in males. ► MD paradigms affected CALR and CALB in both genders but affected GRs only in males.

Introduction

Both animal and human behavioral studies have shown that the brain is particularly sensitive to stress during early childhood. Stressful events that occur during this time may induce permanent alterations in neurodevelopment (Plotsky and Meaney, 1993) that may in turn be associated with an increased risk of psychopathology during adulthood (Heim and Nemeroff, 1999, Heim and Nemeroff, 2001). Maternal deprivation (MD) has proven to be an adverse event that can induce significant stress responses in rat pups, even during the stress hyporesponsive period (SHRP) (Lephart and Watson, 1999, Lupien et al., 2009). The maternal deprivation paradigm, which involves the single or repeated separation of newborn pups from their dam for a specific period of time, is a well-established protocol that is used to investigate neurochemical changes associated with the etiology of and vulnerability to psychiatric diseases in animal models (Cirulli, 2003).

Stress triggers the activation of the hypothalamus–pituitary–adrenal (HPA) axis, culminating in the production of glucocorticoids (GCs) by the adrenal glands. An abnormally high level of GCs negatively impacts the functioning of brain regions that regulate GC receptor levels (Csernansky et al., 2006, de et al., 2005, Elgh et al., 2006, Eliner et al., 1995, Lephart and Watson, 1999, Mizoguchi et al., 2008). Because of the high number of glucocorticoid receptors (GRs) located there, one area of the brain that is particularly sensitive to stress and stress-related hormones is the hippocampus, which is involved in learning and memory, behavioral adaptation and regulation of the HPA axis.

An increase in the number of GRs has also been shown to regulate Ca2+ influx into neurons (Karst and Joëls, 2007, Thibault et al., 2007). Calretinin (CALR) and calbindin-D28k (CALB) are two calcium-binding proteins that are implicated in neurogenesis and neuronal survival. They are both widely expressed throughout the nervous system and are considered to be modulators of intracellular calcium concentration (Müller et al., 2005, Schwaller et al., 2002). A recent study demonstrated that that the density of both CALR and CALB increases in the hippocampus of maternally deprived rats when compared to controls (Gruss et al., 2008).

However, the mechanism underlying stress-induced, Ca2+-related neurobiochemical changes in the hippocampus remains unknown. Although the psychophysiological effects of adverse stimuli (such as MD) are well documented in animals, neither the minimum effective intensity of these stimuli nor the molecular basis of their action has been addressed. In this study, we investigated the contribution of the timing and duration of the MD paradigm on stress-induced hippocampal changes in rats. Both GRs and calcium-binding proteins have been reported to play a crucial role in the stress response in rats, and there are no previous data regarding possible alterations in the expression of these proteins when rats of both genders are subjected to different MD paradigms; therefore, we studied the effects of different MD paradigms on the expression of hippocampal GRs, CALR and CALB during the SHRP in male and female adolescent rats.

Section snippets

Animals and maternal deprivation paradigms

Pregnant Sprague–Dawley rats were housed in single cages (40 × 25 × 20 cm) under controlled conditions of temperature (21 ± 1 °C), humidity (55 ± 5%) and light (12-h light/dark cycle beginning at 8:00) and were allowed free access to food and water. They were checked for litters daily. The day of birth was designated as postnatal day zero (PND0). All pups were weaned at PND21 and subsequently housed in sex-matched groups of eight per cage (40 × 25 × 20 cm).

Each litter was randomly assigned as a whole to one

GR expression in the hippocampus

As shown in Fig. 1, GR levels were significantly higher in female vs. male rats [F(1,47) = 63.29, p < 0.001]. Male rats in groups MD and MD9 experienced a significant upregulation of GR when compared to controls (p < 0.05 and p < 0.001, respectively); however, no significant change in GR expression was found among female rats that underwent any of the maternal deprivation paradigms (Fig. 1A and B).

CALR expression in the hippocampus

CALR expression in the hippocampus of male rats was significantly lower than in female rats [F(1,47) = 74.8,

Discussion

The secretion of GC hormones is governed by the activation of the hypothalamic–pituitary–adrenal (HPA) axis during a stressful situation. Our data demonstrated that the abundance of GRs in the hippocampus of female rats was significantly higher than males at PND21, which might be attributed to the greater influence of female-specific gonadal steroids. Females predominantly produce estrogens, whereas androgens are the major sex hormone in males. Female laboratory rodents tend to secrete higher

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