Seymour Levine

Neonatal maternally deprived rats have as adults elevated basal pituitary-adrenal activity and enhanced susceptibility to apomorphine.

Maternal deprivation of neonatal rats for 24 h enhances the adrenocortical response to stress and/or adrenocorticotropin hormone (ACTH) stimulation during the stress hyporesponsive period (SHRP). The present study tests the hypothesis that such maternally deprived neonatal male rats show altered hypothalamic‐pituitary‐adrenal (HPA) regulation not only immediately after deprivation but also in later life. In addition, we found previously that neonatal changes in HPA activity preceded modulation of nigrostriatal dopamine function. Therefore, we also measured dopamine responsiveness in adult rats which were deprived of their mother during infancy.

Early vs. late maternal deprivation differentially alters the endocrine and hypothalamic responses to stress.

Twenty-four hours of maternal deprivation results in persistent changes in the ACTH response to mild stress. These effects are dependent on the age of the neonate at the time of deprivation. Pups that were separated from the dam at postnatal days (pnd) 3-4 showed an enhanced stress-induced ACTH response at age 20, while pups deprived at pnd 11-12 displayed an attenuated ACTH response to stress at that time. The present study was designed to test the hypothesis that the immediate effects of deprivation at pnd 3 vs. pnd 11 would provide an explanation for these paradoxical effects observed at day 20. For this purpose, we measured the basal and the stress-induced ACTH and corticosterone (CORT) response at days 4 and 12, following 24 h of maternal deprivation. Furthermore, we examined whether similar differences in c-fos and CRH mRNA expression in the paraventricular nucleus (PVN) accompanied the differences in response characteristics of ACTH at pnd 20. The results indicate that changes in the ACTH and CORT responses were minimal after 24 h of maternal deprivation at day 4, whereas these hormones were markedly elevated following deprivation at day 12. The persistent effects also showed age-dependency: pups deprived early showed at pnd 20 an exaggerated ACTH response. Late deprived juveniles exhibited an attenuated ACTH response to stress at pnd 20 while in both conditions the CORT response was not different from the non-deprived litter mates. These persistent endocrine changes were accompanied by the changes in the neural stress markers. The expressions of c-fos and CRH mRNA in the PVN were at pnd 20 significantly greater in their controls in early-deprived animals. The late deprived juveniles showed a reduced response in these neural markers. In conclusion, the persistent alterations in ACTH are reflected by changes in c-fos and CRH mRNA, but these changes in endocrine and in neural stress markers do not appear to be related to any of the hormonal changes that occur at the time of maternal deprivation.

Long-term effects of neonatal maternal deprivation and ACTH on hippocampal mineralocorticoid and glucocorticoid receptors

In the brain, corticosteroids bind to two types of receptors, the classical glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). The effects of different manipulations taking place during early ontogeny on GR and MR binding properties were examined in the adult hippocampus. Infant rats at postnatal day (pnd) 3 were deprived of maternal contact for 24 h and injected with saline or ACTH1-24 at the end of the deprivation period. They were then returned to their dams and weaned on pnd 21. At pnd 48, they were sacrificed (24 h post adrenalectomy) and the hippocampal MR and GR measured using an in vitro cytosol binding assay. In the male rats, deprivation and deprivation + ACTH resulted in a reduction of GRs. MRs were also significantly down regulated in the deprived males. In the female rats, saline injections in deprived female rats resulted in increased GR capacity and ACTH injections led to a further up-regulation of the GRs. None of the early manipulations influenced the regulation of the MRs in females. The binding affinity for corticosteroid receptors was also altered by some of the early manipulations. These results in adult (7-week-old) rats indicate that the receptor systems for corticosteroids in the brain are sensitive to brief manipulations occurring early in development. These changes in receptor capacity and/or affinity may affect corticosteroid-mediated processes in the adult rat.

Maternal deprivation regulates serotonin 1A and 2A receptors in the infant rat

UNLABELLED Several studies have demonstrated that 5-HT1A and 5-HT2A receptors are altered in rat brain following chronic stress. While this is true in the adult animal, this may be different in the developing animal, which has a limited corticosterone response to acute challenges between days 3 and 14 of life. METHODS We investigated the effect of maternal deprivation on 5-HT2A and 5-HT1A receptor mRNA levels in the developing brain. In situ hybridization was used to quantify gene expression in rat pups at three ages: 6, 9, and 12 days old. In each age group, half were maternally deprived for 24 h and half were kept with their mothers. Maternally deprived animals showed elevated ACTH and corticosterone plasma levels when compared to NDEP animals, significantly elevated 5-HT1A mRNA levels in the CA1 hippocampal region and, significantly elevated 5-HT2A mRNA levels in the parietal cortex. No changes were observed in 5-HT1A or 5-HT-transporter mRNA levels in the dorsal raphe. Our results indicate that post-synaptic 5-HT receptors in the developing hippocampus and cortex are sensitive to maternal deprivation. Because hippocampal 5-HT1A gene expressions are known to decrease in the adult animal after chronic glucocorticoid elevation, this data also suggests that other mechanisms, perhaps central, predominate during development.

Stress-Induced Alterations in Corticotropin-Releasing Hormone and Vasopressin Gene Expression in the Paraventricular Nucleus during Ontogeny

From postnatal day (PND) 4 to 14, neonates display a minimal pituitary-adrenal response to mild stress, the so-called ‘stress hyporesponsive period’ (SHRP). During the SHRP, maternal deprivation (MD) alters the pituitary-adrenal system, enabling neonates to become endocrine responsive to specific stimuli. We have previously reported that during the SHRP, mild stress enhances corticotropin-releasing hormone (CRH) messenger RNA (mRNA) expression in the paraventricular nucleus (PVN). Insofar as elevated CRH mRNA was observed both in the presence and absence of adrenocorticotropin (ACTH) release, we hypothesized that other ACTH secretagogues may participate in the pituitary stress response. During the SHRP, does arginine vasopressin (AVP) complement the actions of CRH which might be reflected centrally by the enhanced biosynthesis of both neuropeptides? To test this hypothesis we examined the time course of stress-induced CRH and AVP mRNA in the PVN at PND 6, 12, and 18. As an index of neural activity, c-fos mRNA in the PVN was also examined. Restraint was used as the stressor and MD was employed to enable an endocrine response during the SHRP. Despite the absence of stress-induced ACTH, in nondeprived pups during the SHRP, CRH mRNA was rapidly enhanced. In their maternally deprived (DEP) counterparts, ACTH levels were increased, and a significant induction of CRH mRNA was only observed at day 12. AVP mRNA levels were elevated in DEP 12-day-old pups at 15, 30 and 60 min. In rats beyond the SHRP, plasma ACTH levels, CRH and AVP mRNA were all enhanced following restraint. At PND 18, elevated CRH mRNA was not observed until 4 h after stimulus. Following restraint, c-fos mRNA was increased at all three ages, although the magnitude of c-fos response was less during the SHRP. These results demonstrate that when restraint elicits prototypical ACTH release, the neonatal central response is to enhance the biosynthesis of both AVP and CRH. If nucleic acid changes correlate with release, the increased synthesis of both neuropeptides may indicate the potential for AVP to synergize with CRH during the neonatal stress response.

Effects of a Non-peptide CRF Antagonist (DMP696) on the Behavioral and Endocrine Sequelae of Maternal Separation

We examined whether blockade of corticotropin-releasing factor (CRF) receptors by a non-peptide CRF antagonist (DMP696) would attenuate the stress hyper-responsiveness that occurs in response to maternal separation. In a social interaction test as well as the elevated plus maze, adult male rats, which had been maternally separated as infants, displayed more anxiety-like behavior compared with handled rats. DMP696 increased social interaction in both groups. In the elevated plus maze however, DMP696 significantly increased open arm time in the maternally separated rats but not in the handled group whereas chlordiazepoxide increased open arm time in both groups. DMP696 also appeared to block stress-induced ACTH secretion more readily in the maternally separated group compared with the handled rats. These observations suggest that CRF antagonists are particularly effective in animals that are hyper-responsive to stress and may therefore have utility in the treatment of anxiety and affective disorders where CRF has been implicated.

Separation induced changes in squirrel monkey hypothalamic-pituitary-adrenal physiology resemble aspects of hypercortisolism in humans

When separated from groups, squirrel monkeys respond with significant increases in plasma cortisol and adrenocorticotropic hormone (ACTH). While cortisol remains elevated above pre-separation levels, significant reductions occur in ACTH. Monkeys that respond with greater increases in cortisol subsequently exhibit greater reductions in ACTH, which suggests that reductions in ACTH are mediated by corticosteroid feedback. Monkeys that respond with greater increases in cortisol also tend to exhibit greater cerebrospinal fluid levels of the dopamine metabolite HVA, but not the norepinephrine metabolite MHPG, or corticotropin-releasing factor (CRF). Attenuation of corticosteroid feedback with metyrapone results in significant increases in circulating ACTH, and in older monkeys increases plasma HVA. Similar findings in humans have been reported in clinical studies of hypercortisolism and major depression.

THE ONTOGENY OF THE NEUROENDOCRINE RESPONSE TO ENDOTOXIN

During development, the hypothalamic-pituitary-adrenal (HPA) axis is normally hyporesponsive between postnatal days (pnd) 4 and 14. This interval has been designated as the stress-hyporesponsive period (SHRP). Recent evidence indicates that the neonate can respond to selective stimuli, i.e., exposure to immune signals. The purpose of this study was to investigate the neural correlates of the neonatal stress axis in response to a stimulus that activates the pituitary-adrenal hormones. Thus, lipopolysaccharide (LPS) was administered to neonates at three ages (pnd 6, 12, and 18) during or after the SHRP. In an effort to understand the neonatal hypothalamic paraventricular nucleus (PVN) response to an endotoxin, we measured c-fos immunoreactivity and corticotrophin-releasing hormone (CRH) gene expression. At all ages tested, there was an increase in ACTH and corticosterone (CORT) following LPS compared to controls. During the SHRP, LPS treatment resulted in a marked increase in Fos-positive cells in the PVN, whereas a saline injection had no effect. However, at pnd 18, both LPS and a saline injection elicited equivalent PVN Fos expression. In contrast to the effect on Fos, LPS and a saline injection decreased CRH mRNA at pnd 6 and 12. Outside the SHRP, LPS resulted in an increase in CRH gene expression relative to saline-injected controls. Thus, while the LPS-induced activation of Fos protein and plasma hormones were concordant, CRH mRNA did not positively correlate with the peripheral response. This suggests that the SHRP is not absolute, and the brain is responsive to some stimuli during this period.

Psychobiological Consequences of Social Relationshipsa

Social separations can induce long-lasting increases in cortisol, whereas companionship can result in social buffering. Preliminary evidence from studies of squirrel monkeys suggests that social separation-induced hypersecretion of cortisol is initially driven by hypersecretion of ACTH. From 1-21 days postseparation, however, cortisol remains elevated above pre-separation controls, while ACTH levels are consistently reduced. Hypercortisolism is maintained despite reductions in ACTH, because adrenal responsiveness to ACTH is enhanced. Low circulating ACTH, in turn, is maintained by robust feedback mechanisms that apparently inhibit biosynthesis or release of pituitary ACTH. These findings are consistent with neuroendocrine interactions known or hypothesized to occur during major depressive disorders in humans and raise unique possibilities for comparative research in human and nonhuman primates.

Stress-induced alterations in locus coeruleus gene expression during ontogeny.

Brainstem noradrenergic neurons, particularly the locus-coeruleus (LC), play a pivotal role in modulating the central stress response and have been implicated in regulating the hypothalamic-pituitary-adrenal (HPA) axis. In adult rats, acute stress causes an increase in LC firing and tyrosine hydroxylase (TH) gene expression. While the role of the LC-norepinephrine (LC-NE) system in the adult stress response has been well characterized, there is limited evidence for its participation during development. Previous studies described the neonatal HPA axis as hyporeactive because of stimulus-selective pituitary activation. However, maternal deprivation does reinstate stress-induced endocrine activity and can amplify the neural stress response. Considering that LC neurons can modulate neuroendocrine activity, we hypothesized that the LC-NE system would be stress-responsive during development. Because maternal deprivation (DEP) can alter the central stress response, we examined the LC-NE stress response in both DEP and non-deprived (NDEP) pups. Following an isotonic saline injection (stressor) the time course of TH, c-fos and glucocorticoid receptor (GR) mRNA was examined. Stress-induced TH mRNA was increased in DEP pups at postnatal day (pnd) 12 and in both NDEP and DEP pups at pnd 18. At 15, 30 and 240 min c-fos mRNA was markedly increased in all groups examined. GR mRNA was not altered at pnd 12; however, at pnd 18 NDEP pups showed reduced GR mRNA expression. These data indicate that during ontogeny the LC-NE system is stress-responsive to an acute mild challenge. Activation of LC-NE neurons suggests that this system may participate in modulating the neuroendocrine stress response during development.