Harvey J. Whitfield

Long-term antidepressant administration alters corticotropin-releasing hormone, tyrosine hydroxylase, and mineralocorticoid receptor gene expression in rat brain. Therapeutic implications.

Imipramine is the prototypic tricyclic antidepressant utilized in the treatment of major depression and exerts its therapeutic efficacy only after prolonged administration. We report a study of the effects of short-term (2 wk) and long-term (8 wk) administration of imipramine on the expression of central nervous system genes among those thought to be dysregulated in imipramine-responsive major depression. As assessed by in situ hybridization, 8 wk of daily imipramine treatment (5 mg/kg, i.p.) in rats decreased corticotropin-releasing hormone (CRH) mRNA levels by 37% in the paraventricular nucleus (PVN) of the hypothalamus and decreased tyrosine hydroxylase (TH) mRNA levels by 40% in the locus coeruleus (LC). These changes were associated with a 70% increase in mRNA levels of the hippocampal mineralocorticoid receptor (MR, type I) that is thought to play an important role in mediating the negative feedback effects of low levels of steroids on the hypothalamic-pituitary-adrenal (HPA) axis. Imipramine also decreased proopiomelanocortin (POMC) mRNA levels by 38% and glucocorticoid receptor (GR, type II) mRNA levels by 51% in the anterior pituitary. With the exception of a 20% decrease in TH mRNA in the LC after 2 wk of imipramine administration, none of these changes in gene expression were evident as a consequence of short-term administration of the drug. In the light of data that major depression is associated with an activation of brain CRH and LC-NE systems, the time-dependent effect of long-term imipramine administration on decreasing the gene expression of CRH in the hypothalamus and TH in the LC may be relevant to the therapeutic efficacy of this agent in depression.

Optimization of cRNA probe in situ hybridization methodology for localization of glucocorticoid receptor mRNA in rat brain: a detailed protocol

Summary1.We have described a general ribonucleotide probein situ hybridization methodology for localization of mRNA in frozen, unfixed tissue sections of brain.2.The most important steps in obtaining consistent and reproducible autoradiographs with ribonucleotide probes were tissue acetylation and application of the radiolabeled probe to tissue sections under unsealed, glass coverslips.3.Variability of the hybridization signal in tissue sections has been minimized to achieve a high degree of reproducibility within a given experiment as determined by densitometric analysis of rat glucocorticoid and mineralocorticoid receptor mRNA hybridization autoradiographs.4.Tissue quality has been optimized for high-resolution anatomical localization of mRNA species by nuclear track emulsion.5.The protocol is amenable to rapid, batchwise processing of tissue samples.

Effects of the glucocorticoid antagonist RU 486 on pituitary-adrenal function in patients with anorexia nervosa and healthy volunteers : enhancement of plasma ACTH and cortisol secretion in underweight patients

To further explore whether the hypercortisolism of anorexia nervosa reflects an alteration in the set point for corticotropin-releasing hormone (CRH) secretion or is a manifestation of glucocorticoid resistance, we examined plasma ACTH and cortisol responses to the competitive glucocorticoid antagonist RU 486 (10 mg/kg, p.o. at 8.00 h) versus placebo (PBO) in 7 healthy female volunteers and 8 patients with DSM-III-R anorexia nervosa, all of whom were studied while underweight [64.3 +/- 2.1% average body weight (ABW), mean +/- SE] and 5 of whom were restudied longitudinally following refeeding (> or = 85% ABW, mean 87.4 +/- 0.4% ABW). Blood samples were obtained from 16.00 to 16.30 h and from 4.00 to 8.00 h following dosing. Underweight anorexics were significantly hypercortisolemic by 24 h urinary free cortisol excretion compared with controls (239 +/- 37 vs. 119 +/- 12 nmol/day, p < 0.01). Both controls and underweight anorexics had robust early morning (4.00-8.00 h) plasma cortisol responses to RU 486 (465 +/- 61 and 719 +/- 49 nmol/l) compared with PBO (370 +/- 52 and 451 +/- 31 nmol/l; p < 0.02 and p < 0.01, respectively). The underweight anorexics showed a significant mean early morning plasma ACTH response to RU compared with placebo (3.28 +/- 0.63 vs. 2.01 +/- 0.24 pmol/l, p < 0.05), while the controls showed a trend toward an increase in mean plasma ACTH after RU (3.11 +/- 0.36 pmol/l) compared with PBO (2.31 +/- 0.41 pmol/l, p < 0.13); plasma ACTH means were greater on the RU day than the placebo day at 20 of 25 sampling points (p < 0.001). However, the increment in ACTH on the RU day compared to the placebo day was greater in the underweight anorexics at the first 20 of 25 consecutive time points of the early morning sampling period (p < 0.001). Moreover, underweight anorexics showed a significant plasma ACTH and cortisol response to RU 486 at 16.00-16.30 h (8-8.5 h following administration), while the controls showed no significant response of plasma ACTH or cortisol at this time. When restudied following weight recovery, anorexic patients showed reductions in 24-hour urinary free cortisol excretion (to 191 +/- 40 nmol/day) which were no longer significantly elevated compared with control values.(ABSTRACT TRUNCATED AT 400 WORDS)

Corticotropin-Releasing Hormone: From Endocrinology to Psychobiology

Corticotropin-releasing hormone (CRH) is a brain neuropeptide which coordinates the endocrine, autonomic and behavioral responses to stress. We review the abnormal response to exogenous CRH in various psychiatric syndromes, including major depression and anorexia nervosa. We also contrast pituitary responses to CRH in patients with depression versus Cushings disease. We hypothesize that CRH may play a role in the pathogenesis of various psychiatric syndromes which are characterized during their course by the symptom of depression.

Intrahippocampal Colchicine Alters Hypothalamic Corticotropin-Releasing Hormone and Hippocampal Steroid Receptor mRNA in Rat Brain

The hippocampus appears to be an important modulator of the negative feedback effects of glucocorticoids on the hypothalamic-pituitary-adrenal axis. It is not known if hippocampal subfields CA1-4 or the dentate gyrus differentially alter gene expression of corticotropin-releasing hormone (CRH) in the paraventricular nucleus (PVN) of the hypothalamus. We, therefore, examined the effects of selective destruction of dentate gyrus granule cells, which send excitatory glutaminergic inputs to subfields CA4, CA3 and CA2, on CRH expression in the PVN. To determine the possible involvement of steroid receptors in the regulation of CRH expression, we examined the effects of intrahippocampal colchicine on gene expression of the mineralocorticoid (MR; type I) and glucocorticoid (GR; type II) receptors in hippocampal CA fields and dentate gyrus. Colchicine produced a selective loss of dentate gyrus granule cells without affecting pyramidal cells in CA1-4 as early as 1 day after injection; granule cells were completely destroyed after 3 days. CRH mRNA levels were reduced by 38-48% in the PVN 2-14 days after colchicine. MR mRNA levels were decreased in dorsal and ventral CA fields 1-7 days after colchicine. GR mRNA levels were relatively unchanged, showing a slight decrease only in dorsal CA fields on days 2-7. Unexpectedly, CRH was transiently expressed in dorsal and ventral CA fields 1-3 days after colchicine. In the same time period, mRNA levels of inositol 1,4,5-trisphosphate kinase were decreased, suggesting that increases in neural metabolic activity, indicated by this marker, are not responsible for the transient CRH effect. The results suggest that the dentate gyrus is important for maintenance of steroid hormone receptor mRNA levels in the hippocampus and CRH expression in the hypothalamic PVN, and that CRH gene expression is differentially regulated in the hypothalamus and hippocampus.

The Clinical Implications of Corticotropin-Releasing Hormone

We now appreciate that the brain is the most prolific of all endocrine organs producing scores of neurohormones within and beyond the boundaries of the endocrine hypothalamus. The idea that the brain functions as a gland, however, is not new. Indeed, the evolution of thought leading to the identification of corticotropin releasing hormone (CRH) began around 400 B.C. (1,2). At this time, Hippocrates, in his work entitled De Glandulis, states explicitly, “The flesh of the glands is different from the rest of the body, being spongy and full of veins; they are found in the moist part of the body where they receive humidity... and the brain is a gland as well as the mammae.”

Clinical Studies with Corticotropin Releasing Hormone: Implications for Hypothalamic-Pituitary-Adrenal Dysfunction in Depression and Related Disorders

We now appreciate that the brain is the most prolific of all endocrine organs, producing scores of neurohormones within and beyond the boundaries of the endocrine hypothalamus. The idea that the brain functions as a gland, however, is not new. Indeed, the evolution of thought leading to the identification of corticotropin releasing hormone (CRH) began around 400 B.C. (Zuingerus 1669; Iason 1946). At that time, Hippocrates, in his work entitled De Glandulus, stated explicitly “The flesh of the glands is different from the rest of the body, being spongy and full of veins; they are found in the moist part of the body where they receive humidity ′ and the brain is a gland as well as the mammae.”

Biosynthesis of Rat Insulinlike Growth Factor II in Intact Cells and Cell-Free Translation

Dulak and Temin(1) first reported that the BRL-3A cell line established from normal rat liver secreted a family of polypeptides, which they termed MSA, that had multiplication-stimulating activity for chick embryo fibroblasts. MSA was purified by Moses and colleagues from conditioned media using Dowex chromatography, Sephadex G-75 gel filtration in 1 M acetic acid, and preparative gel electrophoresis, and shown to appear in multiple forms of Mr 16.3K, 8.7K, and 7.1K.(2) Marquardt et al. (3) purified MSA by a different purification scheme (i.e., Bio-Gel P-10 in 1 M acetic acid and high-performance liquid chromatography), and determined the amino acid sequence of a Mr 7484-dalton form. Mr 7484 MSA appears to correspond to our Mr 7.1K species. It is identical to human IGF-II at 62 of 67 amino acid loci, establishing that BRL-MSA represents the rat homologue of IGF-II.(3)