Hypothalamic Corticotropin-releasing Hormone Research Articles

Reproductive placental corticotropin-releasing hormone and its clinical implications

The hypothalamic-pituitary-adrenal axis and the reproductive system exhibit a complex relationship in both nonpregnant and pregnant women.1,2 The hypothalamic-pituitary-adrenal axis exerts profound, mostly inhibitory, effects on the reproductive axis, with corticotropin-releasing hormone and corticotropin-releasing hormone–induced pro-opiomelanocortin peptides inhibiting hypothalamic gonadotropin-releasing hormone secretion and with glucocorticoids inhibiting pituitary luteinizing hormone and ovarian estrogen and progesterone secretion and rendering estrogen-target tissues, such as the endometrium, resistant to the gonadal steroid. Conversely, estrogen directly stimulates the hypothalamic corticotropin-releasing hormone gene, which may explain the slight hypercortisolism seen in women and the preponderance of depression, anxiety disorders, and anorexia nervosa among women.3 Interestingly, several components of the hypothalamicpituitary-adrenal axis and their receptors are present in reproductive tissues as autacoid regulators of their various functions2, 4-7 (Table I). These include ovarian and endometrial corticotropin-releasing hormone, which may participate in the regulation of steroidogenesis and the inflammatory processes of the ovary (ovulation and luteolysis) and of the endometrium (blastocyst implantation and menstruation), and placental corticotropin-releasing hormone, which is secreted mostly in the latter half of pregnancy and is responsible for the physiologic hypercortisolism seen during this period (Fig 1). Most circulating corticotropin-releasing hormone is neutralized by corticotropin-releasing hormone binding protein produced by the liver. Between weeks 34 and 35 of gestation, however, the concentrations of this protein fall by about 60%, leading to elevations of free corticotropin-releasing hormone. Subsequently plasma free corticotropin-releasing hormone concentrations rise further, to peak at labor and delivery. In addition to the hypercortisolism of pregnancy, placental corticotropin-releasing hormone causes vasodilation of the fetoplacental circulation by activating nitric oxide synthase and participates in labor by stimulating secretion of prostaglandin F2α and prostaglandin E2 by the fetal membranes and by promoting constriction of myometrial cells. Placental corticotropin-releasing hormone secretion is stimulated by glucocorticoids, inflammatory cytokines, and anoxic conditions and therefore by noninflammatory or inflammatory stress, including the stress of preeclampsia or eclampsia.2,6 Premature elevation of placental corticotropin-releasing hormone level has been associated with premature labor and birth. Potent corticotropin-releasing hormone antagonists are in the making and could be tested as labor retardants. The hypercortisolism of pregnancy is followed by a transient suppression of hypothalamic corticotropin-releasing hormone secretion in the postpartum period, which may explain the blues or depression and autoimmune phenomena seen during this period.2,7 Estrogen administration has been shown to ameliorate postpartum depression, probably by returning to normal the secretion of the suppressed corticotropin-releasing hormone neuron. From Section on Pediatric Endocrinology, Developmental Endocrinology Branch, National Institute of Child Health and Development, National Institutes of Health. Reprint requests: G. P. Chrousos, MD, Section on Pediatric Endocrinology, National Institutes of Health, Bethesda, MD 20892-1862. Am J Obstet Gynecol 1999;180:S249-50. 0002-9378/99 $8.00 + 0 6/0/90865 Table I. Reproductive corticotropin-releasing hormone and its potential physiologic functions

Twenty-Four Hour Rhythms of Hypothalamic Corticotropin-Releasing Hormone, Thyrotropin-Releasing Hormone, Growth Hormone-Releasing Hormone and Somatostatin in Rats Injected with Freund’s Adjuvant

The effect of Freund’s adjuvant injection on 24-hour variation of hypothalamic corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH), GH-releasing hormone (GRH) and somatostatin levels was examined in adult rats kept under light between 0800 and 2000 h daily. Groups of rats receiving Freund’s complete adjuvant or its vehicle 3 days before sacrifice were killed at six different time intervals throughout a 24-hour cycle. In the median eminence, adjuvant vehicle-injected rats exhibited significant 24-hour variations for the four hormones examined, with maxima at noon. These 24-hour rhythms were inhibited or suppressed by Freund’s adjuvant injection. In the anterior hypothalamus of adjuvant vehicle-treated rats, CRH content peaked at 1600 h, while two peaks were found for TRH and GRH levels, i.e., at 2400–0400 h and 1600 h. Freund’s adjuvant injection suppressed 24-hour rhythm of anterior hypothalamic CRH, TRH and GRH content and uncovered a peak in anterior hypothalamic somatostatin levels at 0400 h. In the medial hypothalamus of adjuvant vehicle-treated rats, significant 24-hour variations were detectable for TRH (peaks at 1600 and 2400 h) and somatostatin (peak at 2400 h) which disappeared after Freund’s adjuvant injection. In the posterior hypothalamus of adjuvant vehicle-treated rats, two peaks were apparent for CRH, TRH and somatostatin levels, i.e. at 1600 h and 2400–0400 h, this hormonal profile remaining unmodified after Freund’s adjuvant administration. The administration of the immunosuppressant drug cyclosporine (5 mg/kg, 5 days) impaired the depressing effect of Freund’s adjuvant injection on CRH, TRH and somatostatin content in median eminence, but not that on GRH. In the anterior hypothalamus, cyclosporine generally prevented the effect of immunization on hormone levels an revealed a second maximum in TRH at 0400 h. Cyclosporine also restored 24-hour variations in TRH and somatostatin levels of medial hypothalamus of Freund’s adjuvant-injected rats but was unable to modify them in the posterior hypothalamus. The results further support the existence of a significant effect of immune-mediated inflammatory response at an early phase after Freund’s adjuvant injection on hypothalamic levels which was partially sensitive to immunosuppression by cyclosporine.

Neuropeptide Y and Corticotropin-Releasing Hormone Concentrations within Specific Hypothalamic Regions of Lean but Not Ob/ob Mice Respond to Food-Deprivation and Refeeding

Leptin is proposed to control food intake at least in part by regulating hypothalamic neuropeptide Y (NPY), a stimulator of food intake, and corticotropin-releasing hormone (CRH), an inhibitor of food intake. Ob/ob mice are leptin-deficient and would thus be expected to exhibit alterations in hypothalamic NPY and CRH. We therefore measured concentrations of NPY and CRH in discrete regions of the hypothalamus (i.e., ARC, arcuate nucleus; PVN, paraventricular nucleus; VMH, ventromedial nucleus; DMH, dorsomedial nucleus; and SCN, suprachiasmatic nucleus) of 6.5–7-wk-old ob/ob and lean mice with free access to stock diet, 24 h after food deprivation, and 1 h after refeeding. Fed ob/ob mice had 55–75% higher concentrations of NPY in the ARC, VMH and SCN than lean mice. Food deprivation increased NPY concentrations ∼70% in the ARC, PVN and VMH of lean mice, and refeeding lowered NPY concentrations ∼70% in the PVN of these mice. NPY in these hypothalamic regions of ob/ob mice was unresponsive to food deprivation or refeeding. The most pronounced change in CRH concentrations within the regions examined (i.e., ARC, PVN and VMH) occurred in the ARC of lean mice where refeeding lowered CRH concentrations by 75% without influencing ARC CRH concentrations in ob/ob mice. The hypothalamic concentrations of two neuropeptides involved in body weight regulation (i.e., NPY and CRH) in leptin-deficient ob/ob mice respond abnormally to abrupt changes in nutritional status.

Secretory pattern of GH, TSH, thyroid hormones, ACTH, cortisol, FSH, and LH in patients with fibromyalgia syndrome following systemic injection of the relevant hypothalamic-releasing hormones.

To study the hormonal perturbations in FMS patients we injected sixteen FMS patients and seventeen controls a cocktail of the hypothalamic releasing hormones: Corticotropin-releasing hormone (CRH), Thyrotropin-releasing hormone (TRH), Growth hormone-releasing hormone (GHRH), and Luteinizing hormone-releasing hormone (LHRH) and observed the hormonal secretion pattern of the pituitary together with the hormones of the peripheral endocrine glands. We found in FMS patients elevated basal values of ACTH and cortisol, lowered basal values of insulin-like growth factor I (IGF-I) and of triiodothyronine (T3), elevated basal values of follicle-stimulating hormone (FSH) and lowered basal values of estrogen. Following injection of the four releasing-hormones, we found in FMS patients an augmented response of ACTH, a blunted response of TSH, while the prolactin response was exaggerated. The effects of LHRH stimulation were investigated in six FMS patients and six controls and disclosed a significantly blunted response of LH in FMS. We explain the deviations of hormonal secretion in FMS patients as being caused by chronic stress, which, after being perceived and processed by the central nervous system (CNS), activates hypothalamic CRH neurons. CRH, on the one hand, activates the pituitary-adrenal axis, but also stimulates at the hypothalamic level somatostatin secretion which, in turn, causes inhibition of GH and TSH at the pituitary level. The suppression of gonadal function may also be attributed to elevated CRH by its ability to inhibit hypothalamic LHRH release, although it could act also directly on the ovary by inhibiting FSH-stimulated estrogen production. We conclude that the observed pattern of hormonal deviations in FMS patients is a CNS adjustment to chronic pain and stress, constitutes a specific entity of FMS, and is primarily evoked by activated CRH neurons.

Neuropeptide Y (NPY) actions on the corticotroph cell of the anterior pituitary gland are not mediated by a direct effect

Neuropeptide Y has been implicated in the activation of the hypothalamic-pituitary-adrenal (HPA) axis, the regulation of growth and sexual function, and is the most potent stimulant of feeding yet reported. The actions of NPY on the HPA axis are thought to be mediated via an activation of the corticotrophin releasing hormone (CRH) neurones within the paraventricular nucleus of the hypothalamus. The ability of NPY to directly influence the corticotroph cell is currently controversial. These studies investigated whether NPY could have a direct influence on anterior pituitary adrenocorticotrophic hormone (ACTH) release. In dispersed male rat anterior pituitary cells, NPY (1–1000 nM) had no effect on either basal or CRH (1 nM) stimulated ACTH release. Basal release, NPY (1000 nM) 111±6% vs. control 103±5%. CRH stimulated release, CRH (1 nM) with NPY (1000 nM) 226±23% vs. CRH (1 nM) alone 228±20%. In addition, NPY (1000 nM) had no effect on either basal or CRH (1 nM) stimulated ACTH release in the mouse corticotroph cell line, AtT-20. Thus, in two models of the anterior pituitary corticotroph NPY had no effect on ACTH release. NPY induced activation of the HPA axis is likely to be mediated via a modulation of hypothalamic CRH and not via a direct action at the level of the anterior pituitary.

Effect of a high-fat diet on food intake and hypothalamic neuropeptide gene expression in streptozotocin diabetes.

Insulin-deficient diabetic rats are markedly hyperphagic when fed a high-carbohydrate (HC) diet, but normophagic when fed a high-fat (HF) diet. When maintained on a HC diet, diabetic rats also exhibit increased gene expression of the orexigenic peptide neuropeptide Y (NPY) in the hypothalamic arcuate nucleus, and reduced expression of the anorectic peptide corticotropin-releasing hormone (CRH) in the paraventricular nucleus, and these changes are hypothesized to contribute to diabetic hyperphagia. In this experiment we assessed whether the normophagia displayed by HF-fed diabetic rats is associated with the opposite profile of NPY and CRH expression. Our results show that relative to diabetic rats on the HC diet, the diabetic rats on the HF diet exhibited significantly reduced caloric intake (-40%), NPY expression in the arcuate nucleus (-27%), and elevated CRH expression in the paraventricular nucleus (+37%). Insulin and corticosterone, which are known to affect hypothalamic NPY and CRH expression, were not different between these two groups, making it unlikely that they can account for the differences in either feeding behavior or hypothalamic peptide expression. There was a small but significant increase in plasma leptin levels in the diabetic animals maintained on the HF, and large differences in parameters associated with elevated fat oxidation. These observations support the hypothesis that the normalization of food intake observed in diabetic rats consuming a HF diet may in part be mediated by reductions in NPY expression and elevations in CRH expression.

The arginine vasopressin and corticotrophin-releasing hormone gene transcription responses to varied frequencies of repeated stress in rats.

1. Rats habituate to repeated exposure to homotypic stressors. The present studies were designed to define how altered frequency of exposure to a stressor affects the development of habituation and how this habituation is reflected in alterations in basal expression and responsiveness of hypothalamic corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) messenger and heteronuclear RNA (hnRNA). 2. Rats were exposed to a 60 min period of restraint stress every 7th day, every 3rd day, alternate days or daily for 2 weeks and their response to a final episode of stress on day 15 was compared with that of a control group of unstressed rats. 3. The response of plasma corticosterone to the final stressor on day 15 was diminished in animals which had been stressed on only two previous occasions, 7 days apart, and diminished further with increasing frequency of previous stressors until it failed to respond at all in animals stressed daily. 4. The pattern of CRH hnRNA and mRNA responses were similar, decreasing with increasing frequency of exposure to the stressor, while AVP mRNA responses increased in response to repeated stress. 5. The gradual emergence of increased AVP transcription at a time of diminishing CRH response suggests that repeated stress results in a specific facilitation of AVP gene expression, perhaps by impairment of corticosterone feedback.

A corticotropin-releasing hormone type I receptor antagonist delays parturition in sheep.

In sheep, corticotropin-releasing hormone (CRH) can stimulate the fetal release of ACTH to produce a cortisol surge which leads to the onset of parturition. We tested the hypothesis that fetal CRH is a primary factor in the onset of parturition in sheep by using a Type I CRH receptor antagonist, antalarmin, to block the endogenous action of CRH. Pregnant ewes were cannulated at 130-135 days of gestation. Five catheters were placed into the amniotic sac, fetal femoral artery, fetal tarsal vein, maternal jugular vein and carotid artery. After 5 days' recovery, blood samples from maternal and fetal vessels were collected at the following times: a day before the start of infusion, at [-1, 0, 1, 2, 4, 8 and 24]h, on the first day of infusion, and thereafter daily throughout a 10-day infusion. Animals (n=6 per group) received infusions into a fetal vein of either a vehicle comprising 1:1 mixture of ethanol and polyethoxylated castor oil (Cremophor EL) or antalarmin (50 g/L) in the vehicle at a rate of 0.3 mL/h. The plasma samples were assayed for ACTH and cortisol using commercial RIA kits. Fetuses infused with vehicle delivered at a mean gestational age of 141.8 +/- 0.9 days compared with antalarmin-infused sheep at 148.8 +/- 1.6 days (P = 0.0036, unpaired Student's t-test). Fetal ACTH and cortisol did not change in the antalarmin-infused sheep after 3 days' infusion compared to significant increases in vehicle-infused sheep (P=0.004 and P = 0.016 respectively, ANOVA). These data show that CRH receptor antagonism in the fetus can delay the onset of parturition. It supports the hypothesis that hypothalamic CRH drives fetal production of ACTH and is essential for the onset of parturition triggered by a surge in fetal cortisol.

Hypothalamic corticotropin-releasing hormone is a mediator of the anorexigenic effect of leptin.

Leptin, ob gene product, inhibits feeding behavior and stimulates energy expenditure in rodents. Corticotropin-releasing hormone (CRH) and neuropeptide Y (NPY), which act in the hypothalamus to influence energy homeostasis, may mediate the anorexic effect of leptin. The present studies were undertaken to examine the possible involvement of hypothalamic CRH in the anorexigenic action of leptin in male Wistar rats. Recombinant leptin (2 microg/rat), microinjected into the third ventricle, inhibited food intake at 2 h by 33.3% (P < 0.01) in rats that were deprived of food for 18 h. The intracerebroventricular injection of 2 microg leptin also increased hypothalamic CRH content (P < 0.05) at 2 h after its administration. Simultaneous intracerebroventricular administration of 5 microg/rat alpha-helical CRH 9-41 (alpha-hCRH), a CRH antagonist, with 2 microg/rat leptin attenuated the anorexic effect of leptin by 2 h. In contrast, single intracerebroventricular injection of alpha-hCRH did not affect food consumption in food-deprived rats. These results implicate hypothalamic CRH as an important mediator of the anorexic effect of leptin in food-deprived rats.

Reduced Activity of Hypothalamic Corticotropin-Releasing Hormone Neurons in Transgenic Mice with Impaired Glucocorticoid Receptor Function

Loss of central glucocorticoid receptor (GR) function is thought to be involved in the development of neuroendocrine and psychiatric disorders associated with corticotropin-releasing hormone (CRH) hyperactivity. The possible causal relationship between defective GR function and altered activity of CRH neurons was studied in transgenic mice (TG) expressing antisense RNA against GR. Immunocytochemical studies showed significant reductions in CRH immunoreactive neurons in the paraventricular nucleus (PVN) and in CRH and vasopressin (AVP) stores in the external zone of the median eminence. Concomitantly, stimulus-evoked CRH secretion from mediobasal hypothalami of TG mice in vitro was reduced significantly. However, CRH mRNA levels in the PVN of TG mice were marginally lower than those in wild-type (WT) mice. 125I-CRH binding autoradiography revealed no differences between WT and TG animals in any of the brain regions that were studied. Basal plasma corticosterone (cort) levels and 125I-CRH binding, CRH-R1 mRNA, POMC mRNA, and POMC hnRNA levels in the anterior pituitary gland were similar in WT and TG mice. Intraperitoneal injection of interleukin-1beta (IL-1beta) increased plasma cort levels, CRH mRNA in the PVN, and anterior pituitary POMC hnRNA similarly in WT and TG mice. The injection of saline significantly reduced anterior pituitary CRH-R1 mRNA levels in WT mice, but not in TG mice, whereas IL-1beta produced a decrease in these mRNA levels in both strains. The data show that long-term GR dysfunction can be associated with reduced activity of CRH neurons in the PVN and decreased sensitivity of pituitary CRH-R1 mRNA to stimulus-induced downregulation. Moreover, the hypothalamic changes observed in this model suggest that impaired GR function, at least if present since early embryonic life, does not necessarily result in CRH hyperexpression characteristics of disorders such as major depression.

Corticotropin-releasing hormone and inflammation.

Corticotropin-releasing hormone (CRH) is a major regulator of the hypothalamic-pituitary-adrenal axis (HPA) and principal coordinator of the stress response. As in stress, intracerebroventricular administration of CRH suppresses the immune system indirectly, via glucocorticoid and/or sympathetic system-mediated mechanisms. Also, during inflammatory stress, the cytokines TNF alpha, IL-1, and IL-6 stimulate hypothalamic CRH and/or vasopressin secretion as a way of preventing the inflammatory reaction from overreacting. Recently, CRH receptors were described in peripheral sites of the immune system, and CRH was found to promote several immune functions in vitro. We demonstrated a direct role of CRH in the inflammatory immune process in vivo, by first studying the effect of systemic CRH immunoneutralization in an experimental model of carrageenin-induced aseptic inflammation in Spague-Dawley rats. We extended these observations to other forms of experimental inflammation, including streptococcal cell wall polysaccharide- and adjuvant-induced arthritides and peptide R16 (epitope of the interphotoreceptor retinoid-binding protein)-induced uveitis in Lewis rats. We also studied human disease states, including rheumatoid arthritis, Hashimoto thyroiditis, and ulcerative colitis. Inflamed tissues contained large amounts of IR CRH, reaching levels similar to those observed in the hypophyseal portal system. We also demonstrated the presence of CRH mRNA and CRH receptors in inflammatory cells and identified the mast cells as a major immune target for CRH. In addition to production by immune cells, the peripheral nervous system, including the postganglionic sympathetic neurons and the sensory fibers type C, appears to contribute to IR CRH production in inflammatory sites. The production of CRH from the postganglionic sympathetic neurons may be responsible for the stress-induced activation of allergic/autoimmune phenomena, such as asthma and eczema, via mast cell degranulation. Antalarmin, a novel nonpeptide CRH receptor antagonist, displaced 125I-labeled ovine CRH binding in rat pituitary, frontal cortex, and cerebellum, but not heart, consistent with antagonism at the CRHR1 receptor. In vivo antalarmin significantly inhibited CRH-stimulated ACTH release and carrageenin-induced subcutaneous inflammation in rats. Thus, antalarmin and other related compounds that antagonize CRH at the level of its own receptor have therapeutic potential in some forms of inflammation directly mediated by type 1 CRH receptors and promise to enhance our understanding of the many roles of CRH in immune/inflammatory reactions.

Human lymphocytes produce urocortin, but not corticotropin-releasing hormone.

Hypothalamic corticotropin-releasing hormone (CRH) is the principal regulator of the hypothalamus-pituitary-adrenal axis in mammals. In addition, immunoreactive CRH is also present at peripheral sites, where it is thought to act as a proinflammatory peptide. However, the source of peripheral CRH has remained obscure. Human lymphocytes were shown to produce immunoreactive CRH, yet the data on CRH mRNA expression in these cells are equivocal. More recently, Vaughan et al. discovered a new member of the CRH family, termed urocortin. Urocortin was shown to act through the same receptors as CRH. The current study was designed to investigate both mRNA and protein expression of CRH and urocortin in human lymphocytes. Using a commercial CRH(1-41) radioimmunoassay, we demonstrate that normal human lymphocytes and Jurkat T lymphoma cells produce significant amounts of immunoreactive peptide. However, no CRH mRNA was detectable by RT-PCR in these cells. In contrast, a band of the correct size and sequence was amplified with urocortin-specific primers. Immunocytochemical analysis of human lymphocytes using antibodies that could distinguish between CRH and urocortin revealed significant expression of urocortin but not of CRH, consistent with our RT-PCR data. We conclude that human lymphocytes produce urocortin, but not CRH.

Cellular and extracellular regulatory mechanisms of hypothalamic corticotropin-releasing hormone neurons.

CRH is produced in the parvocellular division of the PVN and released into the hypophysial portal circulation to stimulate ACTH synthesis and secretion, thus playing a critical role in the HPA axis regulation. The activity of CRH neurons is regulated by humoral factors such as glucocorticoid and IL-1 as well as numerous kinds of neural inputs within and from outside of the hypothalamus. Circulating glucocorticoids readily have access to the CRH neurons and thereby exert a potent inhibitory action on CRH gene transcription. The molecular mechanisms involved in the glucocorticoid negative feedback remain to be fully understood. IL-1, produced in the periphery during the inflammatory process, is a potent stimulatory factor for CRH neurons. It is not yet clear how a protein of this large molecular weight has access to brain tissues through the blood brain barrier, nor is it clear through which neural pathway the IL-1-mediated signal is conveyed to the hypothalamus.A number of neurotransmitter/modulator candidates have been implicated in the regulation of CRH neurons. Functional roles and anatomical pathways have been explored by pharmacological methods, ablation of specific neural pathway(s), and the neural tracingethod. Despite the substantial knowledge produced by these studies, physiological and/or pathophysiological roles of the respective neural circuitry are still obscure.Two major intracellular signal transduction systems, the CAMP-dependent PKA pathway and diacylglycerol-dependent PKC pathway, are implicated in transcriptional regulation of the CRH gene. The functionality of the CRE, 5'-flanking the CRH gene, has been well established in cell culture system transfected with CRH promoter-reporter constructs, thus the cAMP-dependent PKA pathway may play an essential role in CRH gene expression. The role of the PKC pathway is still controversial and requires further exploration.

Chronic melatonin treatment and the hypothalamo-pituitary-adrenal axis in the rat: Attenuation of the secretory response to stress and effects on hypothalamic neuropeptide content and release

The pituitary-adrenal secretory response to acute and chronic stress, suppressibility of adrenocortical secretions by exogenous glucocorticoids, and hypothalamic content and in vitro release of the two major peptidergic activators of the hypothalamo-pituitary-adrenal (HPA) axis, corticotropin-releasing hormone (CRH) and arginine-vasopressin (AVP), were examined in rats receiving daily melatonin (MEL) injections coincident with the circadian increment of endogenous pineal and adrenocortical secretory activity. After 7 days of MEL administration, the rats displayed a significant attenuation of the adrenocortical secretory response to acute and chronic stress. Chronic MEL treatment also prevented the decline in adrenocorticotropic hormone (ACTH) release resulting from chronic stress exposure. Hypothalamic CRH content was significantly lower in rats receiving MEL treatment, while AVP remained largely unaltered; however, MEL administration counteracted the chronic stress-induced decrease in hypothalamic AVP content and in vitro release. When exposed to dexamethasone in vitro, hypothalamic explants from MEL-treated rats responded with a stronger suppression of CRH and AVP release than those originating from vehicle-injected animals. These observations indicate that MEL attenuates the adrenocortical response to stress and influences the biosynthesis, release and glucocorticoid responsiveness of hypothalamic ACTH secretagogues.

Fetal grafts containing suprachiasmatic nuclei restore the diurnal rhythm of CRH and POMC mRNA in aging rats.

We assessed whether fetal tissue containing the suprachiasmatic nuclei (SCN) can restore age-related changes in the diurnal rhythm of hypothalamic corticotropin-releasing hormone (CRH) and anterior pituitary proopiomelanocortin (POMC) mRNA. Young, middle-aged, and middle-aged SCN-transplanted rats were killed at seven times of day. In young rats, CRH mRNA exhibited a diurnal rhythm in the dorsomedial paraventricular nuclei but not in other subdivisions of the nuclei. No rhythm was detected in aging rats. SCN transplants restored a rhythm in CRH mRNA, but the timing was not precisely the same as in young animals. POMC mRNA exhibited a daily rhythm in young rats. Aging abolished the rhythm and decreased the average mRNA level; fetal transplants restored the rhythm, but the amplitude remained attenuated. These data are the first demonstration that fetal tissue can restore the diurnal rhythm of a neuroendocrine axis that is driven by the SCN. We conclude that the neuroendocrine substrate from the aging host remains capable of responding to diurnal cues to express diurnal rhythmicity in CRH/POMC mRNA when fetal SCN transplants confer the appropriate signals.

Evidence that elevated plasma corticosterone levels are the cause of reduced hypothalamic corticotrophin-releasing hormone gene expression in diabetes

Uncontrolled diabetes mellitus causes both a sustained activation of the hypothalamic–pituitary–adrenal (HPA) axis and reduced expression of corticotrophin-releasing hormone (CRH) mRNA in the hypothalamic paraventricular nucleus (PVN). To investigate the role of glucocorticoids in the regulation of CRH mRNA expression in the PVN of diabetic rats, we studied surgically adrenalectomized (ADX) and sham-operated male Sprague-Dawley rats 4 days after i.v. injection of streptozotocin (STZ; 65 mg/kg i.v.) or vehicle. Among sham-operated animals, AM plasma corticosterone levels were significantly increased in diabetic as compared to nondiabetic animals (1.46±0.54 vs. 0.22±0.05 μg/dl; P<0.05), and were positively correlated to both plasma ACTH levels ( r=0.74; P=0.015) and adrenal gland weight ( r=0.70; P=0.025). In contrast, CRH mRNA levels measured in the PVN by in situ hybridization were inversely related to the plasma corticosterone level ( r=−0.68; P=0.045). In a second experiment, both diabetic and nondiabetic ADX rats received a continuous subcutaneous infusion of either corticosterone at one of two doses or its vehicle for 4 days. Among vehicle-treated ADX animals, STZ diabetes raised hypothalamic CRH mRNA levels, in contrast to the tendency for diabetes to lower CRH mRNA in intact rats in the first experiment. Corticosterone administration lowered CRH mRNA comparably in both diabetic and nondiabetic ADX rats. In contrast, diabetes reduced arginine vasopressin (AVP) mRNA levels in the PVN of ADX rats and blunted the inhibitory effect of glucocorticoids on AVP mRNA levels in this setting. We conclude (1) glucocorticoids are necessary for the effect of diabetes to reduce hypothalamic CRH gene expression, since diabetes causes a paradoxical increase in CRH mRNA levels in adrenalectomized animals; (2) glucocorticoid inhibition of hypothalamic CRH gene expression is intact in diabetic rats; and (3) the activation of the HPA axis by diabetes is associated with a proportionate decrease in PVN CRH gene expression. These findings support a model in which hypothalamic factors additional to CRH activate the HPA axis in uncontrolled diabetes, and inhibit CRH gene expression indirectly by negative glucocorticoid feedback.

Maternal Care, Hippocampal Glucocorticoid Receptors, and Hypothalamic-Pituitary-Adrenal Responses to Stress

Variations in maternal care affect the development of individual differences in neuroendocrine responses to stress in rats. As adults, the offspring of mothers that exhibited more licking and grooming of pups during the first 10 days of life showed reduced plasma adrenocorticotropic hormone and corticosterone responses to acute stress, increased hippocampal glucocorticoid receptor messenger RNA expression, enhanced glucocorticoid feedback sensitivity, and decreased levels of hypothalamic corticotropin-releasing hormone messenger RNA. Each measure was significantly correlated with the frequency of maternal licking and grooming (all r's > -0.6). These findings suggest that maternal behavior serves to "program" hypothalamic-pituitary-adrenal responses to stress in the offspring.

Adrenalectomy Lowers the Body Weight Set-Point in Rats

Gosselin, C. and M. Cabanac. Adrenalectomy lowers the body weight set-point in rats. Physiol Behav 62(3) 519–523, 1997.—In order to evaluate the impact of a complete depletion of glucocorticoids on rats’ body weight set-point, rats were adrenalectomized, and their set-points were estimated before, and after surgery. Body weight set-points were obtained from a quantitative behavioral method based on the rats’ food-hoarding response to weight deficit. In addition, body fat contents were measured in vivo using a total body electrical conductivity analyzer (TOBEC). The hoarding of food showed that adrenalectomized rats had significantly lower body weight set-points than the sham-operated controls (337 ± 11 g vs. 385 ± 8 g) and were also significantly lighter. TOBEC measurements showed that the sham-operated control rats had a higher body fat content than the adrenalectomized rats (16.7 ± 1.1% vs. 10.2 ± 1.2%). The present study demonstrates that adrenalectomy lowers the body weight set-point in rats, suggesting that the concentrations of glucocorticoid hormones, and in turn, the hypothalamic corticotrophin releasing hormone (CRH) participates in the adjustment of the body weight set-point.

Regulation of pituitary corticotropin releasing hormone (CRH) receptor mRNA and CRH binding during adrenalectomy: role of glucocorticoids and hypothalamic factors.

The role of glucocorticoids and hypothalamic factors on CRH receptor expression in the pituitary were studied by analysis of the effects of adrenalectomy and suppression of CRH and VP secretion by hypothalamic lesions in the rat. Consistent with previous in situ hybridization studies, Northern blots showed that pituitary CRH receptor mRNA decreased only transiently after adrenalectomy, falling to 51% of the control levels after 18 h, and returning to control values after 6 days (112%). The early decrease was prevented by dexamethasone injection, 100 micrograms, s.c. The role of increased levels of CRH and VP in the pituitary portal circulation on the transient decrease in CRH receptor mRNA levels after adrenalectomy were studied by in situ hybridization in rats subjected in PVN lesions or median eminence deafferentation by hypothalamic anterolateral cuts (ALC). PVN lesion (12 days) or ALC (8 days) resulted in undetectable irCRH and VP in the external zone of the median eminence and had no effect on basal levels of pituitary POMC mRNA, CRH binding and CRH receptor mRNA. In sham lesioned rats, adrenalectomy for 18 h or 4 days caused the expected increases in pituitary POMC hnRNA and mRNA, and decreases in CRH binding. CRH-R mRNA levels decreased by about 50% after 18 h adrenalectomy but returned to basal by 4 days. PVN lesion or ALC fully prevented the fall in CRH binding after 18 h or 4 days adrenalectomy and the increase in POMC mRNA after 4 days adrenalectomy, whereas only attenuated the decrease in CRH receptor mRNA and increase in POMC mRNA levels after 18 h adrenalectomy. Administration of a CRH antagonist did not affect CRH receptor mRNA and POMC hnRNA and mRNA indicating that residual CRH in the median eminence after hypothalamic surgery is not responsible for the effect of adrenalectomy. These studies confirm previous in situ hybridization studies showing that adrenalectomy causes transient decreases in pituitary CRH receptor mRNA levels. The data indicate that while increases in hypothalamic CRH secretion following glucocorticoid withdrawal mediate pituitary CRH receptor binding loss and the increase in POMC expression after long-term adrenalectomy, CRH only partially accounts for the early changes in CRH receptor mRNA and POMC mRNA.

Intracerebroventricular transplantation of embryonic neuronal tissue from inflammatory resistant into inflammatory susceptible rats suppresses specific components of inflammation.

To more directly define the role of central nervous system factors in susceptibility to peripheral inflammatory disease, we examined the effect of intracerebroventricular transplantation of neuronal tissue from inflammatory resistant into inflammatory susceptible rats on subcutaneous carrageenan-induced inflammation (a measure of innate immunity), and on the relative percentage of naive and memory T helper cells in peripheral blood (a measure of the anamnestic immune response). Female inflammatory disease susceptible Lewis (LEW/N) rats transplanted with hypothalamic tissue from inflammatory resistant Fischer (F344/N) rats exhibited > 85% decrease in carrageenan inflammation compared to naive LEW/N rats, LEW/N rats transplanted with F344/N spinal cord, or sham-operated animals. LEW/N rats transplanted with LEW/N hypothalamic tissue exhibited > 50% decrease in carrageenan inflammation. In contrast, intracerebroventricular transplantation of neuronal tissue did not affect the characteristically twofold higher percentage of naive versus memory T helper cells in LEW/N rats, suggesting that the central nervous system (CNS) and hypothalamus play a greater role in the innate inflammatory response than in the acquired immune processes. Grafted tissue survived well and did not show extensive gliosis or inflammation. Compared to naive LEW/N rats, LEW/N rats transplanted with F344/N or LEW/N hypothalamic tissue expressed significantly greater hypothalamic corticotropin releasing hormone mRNA. LEW/N rats transplanted with F344/N hypothalamic tissue also showed significant increases in plasma corticosterone responses to lipopolysaccharide. These data indicate that intracerebroventricular transplantation of fetal hypothalamic tissue from inflammatory resistant into inflammatory susceptible rats suppresses peripheral inflammation partially through hypothalamic factors. These findings have implications for understanding the contribution of specific neuronal tissue in regulation of components of the immune/inflammatory response and in susceptibility to inflammatory disease. Furthermore, this model could be used in the development of potential new treatments for inflammatory/autoimmune diseases aimed specifically at sites within the CNS.