Dajan O'Donnell

Molecular basis for the development of individual differences in the hypothalamic-pituitary-adrenal stress response

Summary1.Several years ago, investigators described the effects of infantile handling on the development of hypothalamic-pituitary-adrenal (HPA) responses to stress in the rat. Rat pups exposed to brief periods of innocuous handling early in life showed reduced HPA responses to a wide variety of stressors, and the effect persists throughout the life of the animal. These effects are robust and provide an excellent model for understanding how early environmental stimuli, which are external to the organism, alter neural differentiation and, thus, neuroendocrine responsivity to stress.2.This paper reviews the endocrine mechanisms affected by early handling and our current understanding of the neural transduction of environmental events and their effects at the level of the target neurons (in the hippocampus and frontal cortex).3.In brief, handling serves to increase glucocorticoid receptor gene transcription, increasing sensitivity to glucocorticoid negative feedback regulation and, thus, altering the activity within hypothalamic corticotropin-releasing factor/vasopressin neurons. Together these changes serve to determine neuroendocrine responsivity to stress.

Postnatal handling alters glucocorticoid, but not mineralocorticoid messenger RNA expression in the hippocampus of adult rats

Postnatal handling alters hypothalamic-pituitary-adrenal (HPA) responses to stress in the rat. Handling also increases hippocampal glucocorticoid receptor density, and this effect appears to form, in part at least, the basis for the effect of handling on HPA responsiveness to stress. In the present study we have used in situ hybridization techniques to examine the effect of postnatal handling on the expression of glucocorticoid and mineralocorticoid receptor mRNAs in various cell fields of the dorsal hippocampus in adult rats. Grain counting analysis over individual cells showed that postnatal handling significantly increased (40-50%) glucocorticoid receptor mRNA in all hippocampal cell fields. In contrast, handling had no effect on mineralocorticoid receptor mRNA expression. These findings are consistent with the results of receptor binding studies showing that handling increases hippocampal glucocorticoid receptor, but not mineralocorticoid receptor density. Thus, the increase in glucocorticoid receptor binding in handled animals is likely associated with altered rates of receptor biosynthesis. Moreover, the handling effect is quite specific, altering glucocorticoid receptor, but not mineralocorticoid receptor mRNA expression. The mechanism(s) whereby glucocorticoid receptor gene expression is permanently increased by postnatal handling remains to be determined.

Environmental regulation of the development of glucocorticoid receptor systems in the rat forebrain. The role of serotonin.

Several years ago Levine, Denenberg, Ader, and others’” described the effects of postnatal “handling” on the development of behavioral and endocrine responses to stress. As adults, handled rats exhibited attenuated fearfulness in novel environments and a reduced adrenal glucocorticoid response to a variety of stressors. These findings demonstrated that the development of rudimentary, adaptive responses to stress could be modified by environmental events. In addition the handling paradigm provided a marvelous opportunity to examine how subtle variations in the early environment alter the development of specific neurochemical systems, leading to stable individual differences in biological responses to stimuli that threaten homeostasis. The effects of handling and other environmental manipulations, such as maternal separation or early immune challenge, on the development of HPA responses to stress have been reviewed recently in detail.@ In this paper the focus is on how these early environmental events alter the differentiation of the forebrain neurons which regulate HPA function, and thus we will present only a brief summary of the neuroendocrine differences between adult animals that were handled vs nonhandled early in life. The important point to be gleaned here is that the handling effect on pituitary-adrenal responses to stress is mediated, in part at least, by changes in hippocampal glucocorticoid receptor levels. In these studies rat pups are handled from the day following birth until weaning. The animals are then housed normally in groups and tested as fully mature adults. With the exception of some subtle differences (see Ref. 7) the handling effect on HPA function is the same for both males and females. Briefly, postnatal handling reduces adult HPA responses to stress and this is apparent in plasma levels of either ACI’H or corticosterone.g-12 Interestingly, there is no difference (as best as we can detect) between handled (H) and nonhandled (NH) animals in basal levels of ACTH or corticosterone.lOJ1 At the level of the paraventricular n. of the hypothalamus, H animals how lower levels of the nRNAs for corticotropin releasing-hormone (CRH) and arginine vaso-

Glucocorticoid receptor gene expression is unaltered in hippocampal neurons in Alzheimer's disease

Excessive glucocorticoid levels increase the metabolic vulnerability of hippocampal neurons to a wide variety of insults. Since glucocorticoid hypersecretion occurs in Alzheimers-type dementia it has been proposed that a primary reduction in hippocampal glucocorticoid receptor expression leads to failure of feedback, hypercortisolemia and hence further neuronal loss. However, we have recently found that lesions of the cholinergic innervation of the hippocampus--known to be severely affected in Alzheimers disease--increase corticosteroid receptor gene expression in the rat hippocampus. We have now examined both glucocorticoid (GR) and mineralocorticoid (MR) receptor gene expression in individual neurons in human postmortem hippocampus, using in situ hybridization histochemistry in 5 patients with Alzheimers disease (81 +/- 3 years) and 7 controls (81 +/- 7 years) without neurological disease. The distribution and intensity of MR and GR mRNA expression in the hippocampus of Alzheimers disease were similar to that in control tissue, with high expression in dentate gyrus and CA2-4, but significantly lower expression in CA1. In a separate group of patients with Alzheimers disease we found significantly increased 24 h integrated plasma cortisol levels (59% greater than age-matched controls) and reduced cortisol-binding globulin (21% lower). These data do not suggest a primary deficiency of biosynthesis of hippocampal corticosteroid receptors in Alzheimers disease. The maintenance of hippocampal GR and MR gene expression, in the face of an increased glucocorticoid feedback signal, may reflect loss of the cholinergic innervation.

Effects of adrenalectomy and corticosterone replacement on glucocorticoid receptor levels in rat brain tissue: a comparison between Western blotting and receptor binding assays

A sensitive Western blotting technique, using a commercially available antibody, was developed herein to study glucocorticoid receptor (GR) autoregulation in brain tissue. A prominent immunoreactive band at approximately 94 kDa, representing the GR, was observed in soluble fractions prepared from rat hippocampus whereas two bands (approximately 97 and 94 kDa) were detected in frontal cortex preparations. Four-day adrenalectomy significantly increased immunoreactive GR levels in both brain regions. In contrast, adrenalectomized animals implanted with corticosterone pellets of varying concentrations displayed dose-dependent decreases in immunodetectable GR levels. Radioligand binding assays ([3H]dexamethasone +/- RU 28362), performed on these same tissue preparations, revealed a similar pattern of GR response to that measured by Western blotting. However, changes in GR binding capacity were generally greater in magnitude than corresponding changes in immunoreactive GR levels. This discrepancy was most pronounced in adrenalectomized animals administered a bolus of corticosterone 1 h prior to sacrifice where a 60-70% reduction in receptor binding sites occurred, in sharp contrast to the 25-30% decrease in immunoreactive GR levels. Taken together, our findings suggest that Western blotting can be used to study GR regulation in brain tissue and that changes in steroid-binding capacity may not necessarily reflect changes in receptor protein levels.

Aldosterone modulates glucocorticoid receptor binding in hippocampal cell cultures via the mineralocorticoid receptor.

The regulation of corticosteroid receptor expression in the rat brain by adrenal steroids remains controversial. The results of in vivo studies [Brinton and McEwen, 1988; Luttge et al., 1989] suggest that activation of type I receptors can modulate both mineralocorticoid (MR; type I) and glucocorticoid (GR; type II) receptor binding in selected brain regions. The present study utilized primary hippocampal cell cultures from rats sacrificed at E19-20 days of gestation to examine the effects of RU 28362, corticosterone (CORT) and aldosterone (ALDO) on GR binding ([3H]dexamethasone +/- RU 28362). Four days of exposure to 10 nM RU 28362, a highly selective GR agonist, resulted in a robust (approximately 70%) decrease in GR binding. Similar exposure to 10 nM of either CORT or ALDO also produced a significant (50-55%) decrease in GR binding capacity. Scatchard analyses confirmed that the diminished GR binding capacity in response to ALDO was due to a decrease in total number of binding sites (Bmax for Control = 112 +/- 25 fmol/mg vs. ALDO = 48 +/- 12 fmol/mg) with no significant change in the affinity constant. The calculated EC50 from the ALDO concentration response curve was 3.5 nM. Competition studies demonstrated that such low nM concentrations of ALDO were unable to displace specific [3H]dexamethasone +/- RU 28362 binding. Spironolactone, a highly specific MR antagonist, inhibited the ALDO-induced down-regulation of GR binding. These findings support the hypothesis that MR activation can modulate GR binding in hippocampal cells.

Regulation of glucocorticosteroid receptor expression in rat hippocampal cell cultures by nerve growth factor.

Dispersed hippocampal cells cultured in serum-free conditions were used to study the effects of nerve growth factor (NGF) on the expression of type I (mineralocorticosteroid or MR) and type II (glucocorticosteroid or GR) corticosteroid receptors. Cells, plated at a density of 1.2 x 10(6) cells/ml in 60 mm Petri dishes, were mainly identified as neurons (90-95%) and maintained for at least 2 weeks. A 7-day treatment with 10-50 ng NGF/ml induced a concentration-dependent decrease of GR binding (40% decrease) compared to untreated cells. In contrast, MR density was unaffected by a 7-day treatment with 50 ng NGF/ml. Data are discussed as possible direct and/or indirect effects of NGF at the level of both neuronal and glial cells.

Entorhinal Cortex Lesions Transiently Alter Glucocorticoid but Not Mineralocorticoid Receptor Gene Expression in the Rat Hippocampus

Abstract: Entorhinal cortex lesions destroy an important hippocampal input and lead to axonal sprouting in the dentate gyrus. Glucocorticoids are known to inhibit this reinnervation process. In the present study, we examined changes in hippocampal glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) mRNA expression using in situ hybridization following unilateral entorhinal cortex lesioning (ECL) in the rat. As early as 1 day postlesioning, a 33% bilateral decrease in GR mRNA expression was observed in the dentate gyrus. By contrast, a 36% bilateral increase in GR mRNA expression was detected in the CA1 cell field. GR mRNA levels in both regions returned to those of control animals 2 days postlesioning, indicating that these effects were transient. Adjacent sections hybridized with probes to MR mRNA revealed no changes in hippocampal MR gene expression as a result of ECL. The selective decrease in GR mRNA expression observed in the dentate gyrus following ECL is specific to the hippocampal subregion targeted for reactive synaptogenesis and thus may serve to attenuate the inhibitory actions of circulating glucocorticoids.