Research report
Hypothalamic and amygdaloid corticotropin-releasing hormone (CRH) and CRH receptor-1 mRNA expression in the stress-hyporesponsive late pregnant and early lactating rat

https://doi.org/10.1016/S0169-328X(01)00137-1Get rights and content

Abstract

This study investigated the expression of corticotropin releasing hormone (CRH) and its receptor CRHR-1, and arginine vasopressin (AVP) mRNAs during the stress hyporesponsive periods of late pregnancy and lactation (day-3) and in virgin stress-responsive females. In situ hybridization histochemistry showed that basal CRH mRNA in the paraventricular nucleus (PVN) decreased in pregnant and increased in lactating rats (compared with virgin controls), whereas it increased after restraint stress only in virgin rats. Basal PVN CRHR-1 mRNA increased markedly in all groups but reached lower levels in pregnant rats. Basal AVP mRNA in the parvocellular PVN was higher in lactating rats, and in contrast to CRH mRNA, it increased after stress in all groups. In medial preoptic area (MPOA) CRH mRNA levels were higher in lactating females compared with virgin and pregnant rats, and unexpectedly they decreased markedly after stress only in virgin rats. CRH mRNA levels in the central and medial nuclei of the amygdala were higher in lactating rats than in virgin or pregnant ones, and stress had no effect in either group. These data suggest that these stress hyporesponsive periods: (1) do not depend on basal CRH mRNA expression in the PVN; (2) appear to have intact stress-activated afferent pathways to the PVN, as shown by preservation of CRHR-1 and AVP responses to stress, but the information may be differently processed; (3) are associated with an alteration in a CRH mediated pathway from the MPOA.

Introduction

Corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP), synthesized by parvocellular neurones of the paraventricular nucleus (PVN), are the principal regulators of pituitary adrenocorticotropic hormone (ACTH) secretion, particularly in response to stress. CRH and AVP also have central effects at hypothalamic and extrahypothalamic sites to integrate autonomic, neuroendocrine, and behavioural responses to stress [4], [16], [21]. In this respect, centrally-administered CRH can partially mimic the responses to stress, including increased arousal, grooming behaviour and locomotor activity, anxiogenesis, and autonomic and neuroendocrine activation [5], [16], [21], [35].

CRH mediates its actions through high-affinity membrane receptors located in the brain and pituitary [1], [13], [37], [41], [57]. CRH binding sites are present in areas of the neocortex, and several nuclei of the brainstem, limbic system and hypothalamus [13]. Most of these areas have direct and indirect connections with the PVN, which is both the main source of CRH peptide and an important relay area for neuroendocrine and autonomic regulation. The expression of CRH receptor type 1 (CRHR-1) mRNA in the PVN is very low or absent in resting conditions [31]. However, mRNA levels increase markedly with stress in a stimulus-specific manner, with an increase in parvocellular neurons following physical/psychological stress, and in the magnocellular PVN and SON following osmotic stimulation [31], [42], [45]. The function of CRH receptors in these nuclei is not clear, but in the PVN they may be part of positive autoregulatory mechanism [26], [31], [32]. This is suggested by the fact that i.c.v. infusion of CRH enhances the expression both of the immediate-early gene c-fos, indicating neuronal activation [24], [39], and of CRH gene [39] in the PVN.

While it is not possible to determine the site(s) of action of i.c.v. administered CRH, it is likely that at least some effects are mediated by receptors in the PVN, since in vitro electrophysiological studies have shown direct excitatory and indirect inhibitory actions of CRH on PVN neurons [59]. On the other hand, some of the behavioural effects appear to be mediated via CRH receptors at other sites, as indicated by the ability of chronic infusion of a CRHR-1 antisense oligodeoxynucleotide into the central nucleus of amygdala (ACe) to reduce anxiety-related behaviour in socially defeated rats [27].

Although it is clear that CRH and AVP mediate both neuroendocrine and behavioural responses to stress, with the latter being more involved in the response to chronic stress, the relative roles of these two peptides during states of stress hyporesponsiveness are not well understood. The periods around birth and lactation in the female are physiological states during which there is a marked alteration of responses to stress [9], [28]. Lactating rats show a reduction in the stress-induced increase in CRH and pro-enkephalin mRNA transcription [29], a markedly reduced stress-induced release of oxytocin [7], [22] and prolactin [23], and severely attenuated hypothalamo-pituitary-adrenal (HPA) responses to a wide range of stressors [9], [53], [54], [56]. Furthermore, late pregnant and lactating females (especially in early lactation) show lower restraint-induced c-fos mRNA expression in the PVN, medial amygdala (AMe) and lateral septum (LS) [9]. The mechanisms by which these areas show less c-fos mRNA expression, and presumably less neuronal activation, during stress in lactating females, are not known.

In addition to displaying attenuated responses to stress, lactating rats lack the increase in c-fos mRNA expression which is observed in the PVN, LS, and AMe of virgin females in response to i.c.v. infusion of CRH [10]. Lactation also abolishes CRH-induced oxytocin secretion [40]. If CRH exerts positive autoregulatory feedback on PVN function, it is possible that an impaired CRH receptor up-regulation in the PVN could contribute to the hyporesponsiveness to stress during lactation. The altered behavioural and neuroendocrine responses to stress during lactation may be due to a decrease in CRH receptor expression and CRH levels in amygdaloid nuclei could account for the disinhibition of the fear responses and increase in aggression in threatening situations [43].

The aims of this study were to assess CRH, CRHR-1 and AVP gene expression in basal and stressed conditions during pregnancy and lactation when the behavioural and neuroendocrine responses to stress are reversibly suppressed.

Section snippets

Animals and restraint procedures

Female virgin (210–250 g), pregnant (17–19 days) and 3-day lactating rats of the Sprague–Dawley strain (Charles River, USA) were housed in a 12-h light, 12-h dark cycle with access to rat chow and water ad libitum. Virgin and pregnant animals were housed in groups of three (cage size: 50×32×18.5 cm) and lactating females were housed with their litters (cage size: 40×24×20 cm). All rats were handled three times a week. For the virgin rats vaginal smears were performed given that the sensitivity

Hypothalamic areas

There was a marked reduction of basal CRH mRNA levels in the PVN of late pregnant females compared with that of virgin control rats (Fig. 2, Fig. 4). In contrast, CRH mRNA was elevated on day 3 of lactation showing levels significantly greater than those of virgin females (P<0.001) (Fig. 2, Fig. 4). Following restraint stress, the expression of CRH mRNA increased in the virgin female group (Fig. 2B) (P<0.05) but not in late pregnant or early lactating females (Fig. 2D,F) (two-way ANOVA; stress

Discussion

These data show marked fluctuations in basal levels of CRH mRNA in the PVN between reproductive states, with a decrease during late pregnancy and an increase during early lactation, changes which do not appear to correlate with the stress hyporesponsiveness characteristic of these states. On the other hand, consistent with the blunted stress responses, CRH mRNA responses to stress were absent during both pregnancy and lactation. In marked contrast, CRHR-1 mRNA and AVP mRNA responses to stress

Acknowledgements

This work was partially supported by a Welcome Trust grant (#053417) to Dr. C. Ingram and Prof. S.L. Lightman and a Short Term Fellowship from the Human Frontier Science Program to Dr. Ana da Costa.

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    Present address: Department of Neuroscience, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3401, USA.

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