Immunoreactive Corticotropin-releasing Hormone Research Articles

Evidence that corticotropin-releasing hormone inhibits cell growth of human breast cancer cells via the activation of CRH-R1 receptor subtype

It has been previously shown that corticotropin-releasing hormone (CRH) exerts antiproliferative activity on an estrogen-dependent tumor cell line, i.e. human endometrial adenocarcinoma Ishikawa (IK) cells. Here we have investigated the effects of CRH on another estrogen-dependent tumor cell line, human breast cancer MCF7 cells. In this paradigm, CRH given at a fixed concentration of 100 nM significantly inhibited cell growth induced by 100 nM estradiol (E2) after 48 and 72 h of incubation. This effect was not associated with the induction of apoptosis. CRH inhibition of cell proliferation was counteracted in a concentration-dependent manner by the non-selective CRH receptor antagonist, astressin, as well as by a CRH-R1 selective receptor antagonist, antalarmin. RNase protection assays carried out on MCF7 under basal conditions showed that these cells express in a constitutive manner the CRH-R1 receptor subtype. We have also investigated the putative source of CRH acting on breast cancer cells; we found that MCF7 cells express CRH mRNA under basal conditions and secrete sizable amounts of immunoreactive CRH, which leads to postulate the existence of paracrine–autocrine inhibitory mechanism operated by CRH in breast cancer cells.

Corticotropin releasing hormone and the immune/inflammatory response

Hypothalamic corticotropin-releasing hormone (CRH) acts as the major physiologic ACTH secretagog. Moreover, CRH is distributed in the brain and spinal cord, adrenal medulla, testes, ovaries, gastrointestinal tract, pancreas, myometrium, endometrium, placenta, and diverse inflammatory sites. Immunoreactive CRH has been found in the cytoplasm of immune accessory cells (macrophages, endothelial cells, and tissue fibroblasts), and in inflammatory sites of both acute and chronic inflammation (synovial lining cell layers and blood vessels from the joints of patients with rheumatoid arthritis and osteoarthritis). Additionally, the local presence of CRH in the uveitic eyes, cytoplasm of inflammatory cells (macrophages, lymphocytes, and polymorphonuclear cells) infiltrating the iris, ciliary body, vitreous, retina, and choroid appears to be of pivotal importance in the process of experimental autoimmune uveoretinitis. Traditionally, hypothalamic CRH has been considered to act indirectly in an anti-inflammatory fashion, since the end product of the hypothalamic–pituitary–adrenal axis is cortisol, a well-known anti-inflammatory compound. However, CRH produced at peripheral inflammatory sites has been shown to participate in an autocrine/paracrine stimulation of inflammation. Thus, CRH may have a peripheral, primarily activating role on the immune system. The mechanisms of the CRH-mediated component of the immune/inflammatory response are still unclear. CRH in inflammatory sites seems to be involved in the activation of the Fas/Fas ligand system. Furthermore, locally produced embryonic and endometrial CRH plays a role in both the aseptic inflammatory process of implantation and the anti-rejection process that protects the fetus from the maternal immune system. There are two types of G-protein-coupled CRH receptors (CRH-R1 and CRH-R2). Pyrrolopyrimidine compounds, such as antalarmin, have been developed as CRH-R1 receptor antagonists. Confirming the peripheral pro-inflammatory actions of CRH, antalarmin has been shown to suppress experimental aseptic inflammation. Thus, antalarmin may represent the first in a new class of anti-inflammatory agents operating through CRH-R1. Studies of CRH genetics have provided new insights on the pathogenesis of rheumatoid arthritis in humans. DNA variation across the CRH gene-containing region has been examined in families with multiple cases of rheumatoid arthritis. Transmission Disequilibrium Test analysis showed significant association at the CRH locus.

Cushing's Syndrome Due to Ectopic CRH Secretion by Adrenal Pheochromocytoma Accompanied by Renal Infarction

Ectopic production of corticotropin-releasing hormone (CRH) by a pheochromocytoma is an infrequent cause of Cushing's syndrome. We report the case of a 43-year-old man with Cushing's syndrome due to a CRH-producing adrenal pheochromocytoma. The patient had clinical and biochemical evidence of hypercortisolism in conjunction with high ACTH levels and non-suppressible serum cortisol levels on low-dose and high-dose dexamethasone suppression testing. In addition to these clinical features of one month's duration, the patient developed symptoms of pheochromocytoma including headache, hypertension that was resistant to conventional therapy and excessive sweating. Biochemical testing confirmed elevated 24-hour urinary catecholamines and metabolites. Abdominal CT revealed a 4.5 x 4 x 3.5 cm mass in the left adrenal gland. He underwent elective left adrenalectomy. Light microscopic and immunochemical studies revealed a pheochromocytoma that contained immunoreactive CRH and was negative for ACTH. Plasma ACTH and dexamethasone supression tests normalized after surgery. This is an unusual case of a CRH-secreting pheochromocytoma. This was complicated by renal infarction, illustrating further the complexity of Cushing's syndrome in a patient with pheochromocytoma caused by CRH hypersecretion.

Acute Stress and Intravesical Corticotropin-Releasing Hormone Induces Mast Cell Dependent Vascular Endothelial Growth Factor Release From Mouse Bladder Explants

Corticotropin-releasing hormone is typically released from the hypothalamus but it has proinflammatory effects outside of the brain, possibly through the activation of mast cells. These cells express corticotropin-releasing hormone receptors with selective secretion of vascular endothelial growth factor, which may be involved in the pathogenesis of painful bladder syndrome/interstitial cystitis. This condition is characterized by bladder inflammation and worsened by stress. We investigated the effect of intravesical corticotropin-releasing hormone and acute restraint stress on vascular endothelial growth factor release from mouse bladder explants and the role of mast cells. The bladder of C57BL/6 mast cell deficient (W/W(v)) and normal congenic (+/+) female mice (Jackson Laboratories, Bar Harbor, Maine) at ages 10 to 12 weeks was catheterized using anesthesia. After emptying urine 1) normal saline or corticotropin-releasing hormone was introduced for 45 minutes, urine was collected and the mice were allowed to recover for 4 hours before sacrifice or 2) the mice were stressed by placing them in a restrainer for 4 hours before sacrifice and urine was collected 2 hours after stress. The bladder was removed 4 hours after stress and processed for corticotropin-releasing hormone immunohistochemical staining. In other experiments the bladder was removed, minced into 1 mm(2) pieces and cultured with or without corticotropin-releasing hormone overnight. Urine and medium were frozen for histamine, interleukin-6, tumor necrosis factor-alpha and vascular endothelial growth factor assay. Corticotropin-releasing hormone (100 nM) or acute restraint stress (4 hours) increased histamine release in urine and vascular endothelial growth factor release in medium without increasing interleukin-6 or tumor necrosis factor-alpha in the bladder explants of C57BL/6 or +/+ but not W/W(v) mice. No vascular endothelial growth factor, interleukin-6 or tumor necrosis factor-alpha was detected in urine before or after stimulation. Corticotropin-releasing hormone immunoreactivity was present in control bladders but it increased dramatically in the bladder of stressed mice. Intravesical corticotropin-releasing hormone and acute restraint stress induced mast cell dependent vascular endothelial growth factor release from bladder explants. These findings suggest that stress, corticotropin-releasing hormone, mast cells and vascular endothelial growth factor might participate in the pathogenesis of painful bladder syndrome/interstitial cystitis, which is worsened by stress, and provide for new therapeutic targets.

Corticotropin-releasing hormone-receptor 1 (CRH-R1) and CRH-binding protein (CRH-BP) are expressed in the gills and skin of common carpCyprinus carpioL. and respond to acute stress and infection

We established that corticotropin-releasing hormone (CRH), CRH-binding protein (CRH-BP) and CRH-receptor 1 (CRH-R1) are expressed in the gills and skin of common carp Cyprinus carpio, an early vertebrate. Immunoreactive CRH was detected in macrophage-like cells in gills and skin, in fibroblasts in the skin and in endothelial cells in the gills. The involvement of the CRH system in gills and skin was investigated in response to infection and in an acute restraint stress paradigm. Carp were infected with the protozoan leech-transmitted blood flagellate Trypanoplasma borreli and subjected to acute restraint stress by netting for 24 h. The expression of CRH-BP and CRH-R1 genes in the gills and in the skin is downregulated after both infection and restraint. Thus the peripheral CRH system reacts to infection and stress. The gills and skin separate the internal from the external environment and are permanently exposed to stress and pathogens. Because of their pivotal role in maintaining the homeostatic equilibrium, these organs must act locally to respond to diverse stresses. Clearly, the CRH system is involved in the response of the integument to diverse stresses at the vulnerable interface of the internal and external milieu.

Aging associated changes in amygdalar corticotropin-releasing hormone (CRH) and CRH-binding protein in Fischer 344 rats

Behavioral adaptation in aging may become impaired from abnormal expression of amygdalar corticotropin-releasing hormone (CRH) and/or CRH-binding protein (CRH-BP). In this study, we serially sectioned the amygdala in 4-, 12-, and 24-month-old Fischer 344 rats following perfusion with 4% paraformaldehyde. We determined the amount of CRH and CRH-BP containing cells as well as the density of fibers expressing CRH or CRH-BP utilizing densitometric methods. Images were digitized using Zeiss Axiovision software and densitometrically analyzed using Scion Image. Both sides were analyzed in sections cut at 30 μm thickness. Cell counts of CRH-BP containing cells in the basolateral and lateral nucleus of the amygdala were lower in 24-month-old rats vs. 4-month-old rats, respectively (mean cells/section ± SE): 31 ± 6 vs. 72 ± 10 ( n = 3; P < 0.05 via ANOVA and Fisher's PLSD). There was a trend for cell counts of CRH containing cells in the central nucleus of the amygdala to be lower in 24-month-old rats vs. 4-month-old rats, respectively 28 ± 7 vs. 47 ± 9 ( n = 3; P = 0.07 via ANOVA). Densitometric analysis of the number of CRH-BP positive fibers revealed no age differences in CeA; however, with regards to CRH-positive fibers, both 4- and 12-month rats had greater CeA CRH immunoreactivity relative to 24-month-old rats ( Ps < 0.05 via ANOVA and Fisher's PLSD). These changes may contribute to impaired adaptations to stress, cognitive decline, and other pathophysiological processes during aging.

Reduced Anorexigenic Efficacy of Leptin, But Not of the Melanocortin Receptor Agonist Melanotan-II, Predicts Diet-Induced Obesity in Rats

Leptin gains access to the central nervous system where it influences activity of neuronal networks involved in ingestive behavior, neuroendocrine activity, and metabolism. In particular, the brain melanocortin (MC) system is important in leptin signaling and maintenance of energy balance. Although leptin or MC receptor insensitivity has been proposed to be associated with obesity, the present study compared central leptin and MC receptor stimulation on some of the above-mentioned parameters and investigated whether these treatments predict proneness to diet-induced obesity (DIO) in outbred Wistar rats. Third-cerebroventricular administration of equi-anorexigenic doses of leptin and of the MC agonist melanotan-II caused comparable increases in plasma ACTH and corticosterone levels and c-Fos-labeling in approximately 70% of paraventricular hypothalamic (PVN) neuronal cell bodies containing CRH. This reinforces involvement of paraventricular CRH neurons in the short-term neuroendocrine and ingestive effects of leptin and melanocortins. In the DIO prediction study, anorexigenic efficacy of melanotan-II was not correlated with any parameter linked to DIO but was highly correlated with MC in situ binding (with labeled [Nle(4),D-Phe(7)]alpha-MSH) as well as CRH immunoreactivity in the PVN of DIO rats. This suggests intricate relationships among MC signaling, the CRH system, and ingestive behavior unrelated to DIO. In the same animals, leptin's anorexigenic efficacy was not correlated with PVN MC in situ binding or CRH immunoreactivity but correlated inversely to post-DIO plasma leptin, liver weight, and abdominal adiposity, the latter being correlated to insulin resistance. Thus, differences in leptin but not MC signaling might underlie DIO, visceral obesity, and insulin resistance.

Co-localization and distribution of corticotrophin-releasing hormone, arginine vasopressin and enkephalin in the paraventricular nucleus of sheep: A sex comparison

The paraventricular nucleus (PVN) is integral to regulation of the hypothalamo-pituitary-adrenal (HPA) axis and contains cells producing corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and enkephalin. We used immunohistochemistry to map these peptides and to resolve the extent of co-localization within PVN cells in intact and gonadectomized male and female sheep. Immunoreactive (ir) CRH, AVP and enkephalin cells were mapped in two rams and two ewes at 180 μm intervals throughout the rostro-caudal extent of the PVN. Similar distributions of AVP-ir cells occurred in both sexes whereas CRH-ir and enkephalin-ir cells extended more rostrally in rams. In groups ( n=4) of intact and gonadectomized sheep of both sexes, co-localization and distribution of neuropeptides was influenced by sex and gonadectomy. Males had more AVP and CRH cells than females. Intact animals had more AVP cells than gonadectomized animals. There were no differences between groups in the number or percentage of cells that stained for both CRH and AVP or in the number of cells that stained for both CRH and enkephalin. Differences were observed in the percentage of enkephalin cells that contained CRH with males having a greater percentage of co-localized cells than did females. Differences were also observed in the number and percentage of cells that stained for both enkephalin and AVP; the number of cells that stained for both neuropeptides was greater in males than in females and greater in intact animals than in gonadectomized animals. Differences were observed in the percentage of AVP cells that contained enkephalin, and in the percentage of enkephalin cells that contained AVP with males having a greater percentage of co-localized cells than did females. We conclude that sex and gonadal status affect peptide distribution in the PVN of the sheep which may provide an anatomical basis for sex differences in HPA axis responses to stress.

Distribution and Acute Stressor‐Induced Activation of Corticotrophin‐Releasing Hormone Neurones in the Central Nervous System of Xenopus laevis

In mammals, corticotrophin-releasing hormone (CRH) and related peptides are known to play essential roles in the regulation of neuroendocrine, autonomic and behavioural responses to physical and emotional stress. In nonmammalian species, CRH-like peptides are hypothesized to play similar neuroendocrine and neurocrine roles. However, there is relatively little detailed information on the distribution of CRH neurones in the central nervous system (CNS) of nonmammalian vertebrates, and there are currently no comparative data on stress-induced changes in CRH neuronal physiology. We used a specific, affinity-purified antibody raised against synthetic Xenopus laevis CRH to map the distribution of CRH in the CNS of juvenile South African clawed frogs. We then analysed stress-induced changes in CRH immunoreactivity (CRH-ir) throughout the CNS. We found that CRH-positive cell bodies and fibres are widely distributed throughout the brain and rostral spinal cord of juvenile X. laevis. Strong CRH-immunoreactivity (ir) was found in cell bodies and fibres in the anterior preoptic area (POA, an area homologous to the mammalian paraventricular nucleus) and the external zone of the median eminence. Specific CRH-ir cell bodies and fibres were also identified in the septum, pallium and striatum in the telencephalon; the amygdala, bed nucleus of the stria terminalis and various hypothalamic and thalamic nuclei in the diencephalon; the tectum, torus semicircularis and tegmental nuclei of the mesencephalon; the cerebellum and locus coeruleus in the rhombencephalon; and the ventral horn of the rostral spinal cord. To determine if exposure to an acute physical stressor alters CRH neuronal physiology, we exposed juvenile frogs to shaking/handling and conducted morphometric analysis. Plasma corticosterone was significantly elevated by 30 min after exposure to the stressor and continued to increase up to 6 h. Morphometric analysis of CRH-ir after 4 h of stress showed a significant increase in CRH-ir in parvocellular neurones of the anterior preoptic area, the medial amygdala and the bed nucleus of the stria terminalis, but not in other brain regions. The stress-induced increase in CRH-ir in the POA was associated with increased Fos-like immunoreactivity (Fos-LI), and confocal microscopy showed that CRH-ir colocalized with Fos-LI in a subset of Fos-LI-positive neurones. Our results support the view that the basic pattern of CNS CRH expression arose early in vertebrate evolution and lend further support to earlier studies suggesting that amphibians may be a transitional species for descending CRH-ergic pathways. Furthermore, CRH neurones in the frog brain exhibit changes in response to a physical stressor that parallel those seen in mammals, and thus are likely to play an active role in mediating neuroendocrine, behavioural and autonomic stress responses.

Mirtazapine acutely inhibits basal and K(+)-stimulated release of corticotropin-releasing hormone from the rat hypothalamus via a non-genomic mechanism.

Originally described as a pivotal mediator of acute neuroendocrine responses to stress, corticotropin-releasing hormone (CRH) is currently envisioned as a peptide neurotransmitter involved in the pathogenesis of anxiety and depressive disorders; it has been postulated that antidepressant drugs are clinically effective insofar as they are able to reduce central CRH production and release. In this study we used a well validated in vitro model, i.e. acute rat hypothalamic explants, to investigate the effects of the antidepressant mirtazapine on the production and release of CRH from the hypothalamus in short-term experiments. CRH release was assessed through the measurement of CRH immunoreactivity in the incubation medium. We found that mirtazapine reduces in a concentration-dependent manner both basal and K(+)-stimulated CRH release in 30-min and 60-min experiments. Mirtazapine had no effect on CRH mRNA expression in 1-h and 3-h experiments; the intra-hypothalamic levels of peptide were not reduced, and even tended to increase, with respect to controls. Mirtazapine reduces CRH release from CRH-containing neurons in the rat hypothalamus through a mechanism independent from the modulation of CRH gene expression and peptide production.

Distribution of corticotropin releasing hormone in the fetus, newborn, juvenile, and adult baboon.

Corticotropin releasing hormone (CRH) has previously been identified in extrahypothalamic tissues and may act in a paracrine fashion within these tissues. To date, CRH production and its role in the fetus and newborn have not been investigated. The aim of this study was to explore the distribution and ontogeny of CRH in extrahypothalamic tissues of the fetus, newborn, juvenile, and adult baboon. Pituitary, adrenal, kidney, liver, and lung tissues from baboons at 125 d gestation, 140 d gestation, 185 d gestation (term), juveniles, and adults were obtained at necropsy. The tissues were quantified for protein and immunoreactive CRH was determined by a RIA. CRH levels were normalized to the protein content of each tissue. CRH was present in all tissues and varied over a 100-fold range according to tissue type. The highest concentration of CRH was found in the pituitary, which did not differ with the gestation and/or age of the animal. In the lung tissues of 125- and 140-d gestation animals, CRH was greater than the term, juvenile, and adult lung (p < 0.02). CRH in the adrenal gland of the 125-d samples was greater than the other four ages tested (p < 0.02). Liver CRH levels were higher in the term animals compared with the juvenile baboons. Our study documents the existence of CRH in extrahypothalamic tissues of the baboon from 125 d of gestation to adulthood. Given its presence and distribution, we speculate that CRH may exert ongoing paracrine and/or autocrine actions in these tissues from the time of intrauterine life throughout adulthood.

Maternal and Fetal Hypothalamic‐Pituitary‐Adrenal Axes During Pregnancy and Postpartum

The principal modulators of the hypothalamic-pituitary-adrenal (HPA) axis are corticotropin-releasing hormone (CRH) and arginine-vasopressin (AVP). Corticotropin-releasing hormone is not exclusively produced in the hypothalamus. Its presence has been demonstrated at peripheral inflammatory sites. Ovulation and luteolysis bear characteristics of an aseptic inflammation. CRH was found in the theca and stromal cells as well as in cells of the corpora lutea of human and rat ovaries. The cytoplasm of the glandular epithelial cells of the endometrium has been shown to contain CRH and the myometrium contains specific CRH receptors. It has been suggested that CRH of fetal and maternal origin regulates FasL production, thus affecting the invasion (implantation) process through a local auto-paracrine regulatory loop involving the cytotrophoblast cells. Thus, the latter may regulate their own apoptosis. During pregnancy, the plasma level of circulating maternal immunoreactive CRH increases exponentially from the first trimester of gestation due to the CRH production in the placenta, decidua, and fetal membranes. The presence in plasma and amniotic fluid of a CRH-binding protein (CRHbp) that reduces the bioactivity of circulating CRH by binding is unique to humans. Maternal pituitary ACTH secretion and plasma ACTH levels rise during pregnancy-though remaining within normal limits-paralleling the rise of plasma cortisol levels. The maternal adrenal glands during pregnancy gradually become hypertrophic. Pregnancy is a transient, but physiologic, period of hypercortisolism. The diurnal variation of plasma cortisol levels is maintained in pregnancy, probably due to the secretion of AVP from the parvicellular paraventricular nuclei. CRH is detected in the fetal hypothalamus as early as the 12th week of gestation. CRH levels in fetal plasma are 50% less than in maternal plasma. The circulating fetal CRH is almost exclusively of placental origin. The placenta secretes CRH at a slower rate in the fetal compartment. AVP is detected in some neurons of the fetal hypothalamus together with CRH. AVP is usually detectable in the human fetal neurohypophysis at 11 to 12 weeks gestation and increases over 1000-fold over the next 12 to 16 weeks. The role of fetal AVP is unclear. Labor appears to be a stimulus for AVP release by the fetus. The processing of POMC differs in the anterior and intermediate lobes of the fetal pituitary gland. Corticotropin (ACTH) is detectable by radioimmunoassay in fetal plasma at 12 weeks gestation. Concentrations are higher before 34 weeks gestation, with a significant fall in late gestation. The human fetal adrenal is enormous relative to that of the adult organ. Adrenal steroid synthesis is increased in the fetus. The major steroid produced by the fetal adrenal zone is sulfoconjugated dehydroepiandrosterone (DHEAS). The majority of cortisol present in the fetal circulation appears to be of maternal origin, at least in the nonhuman primate. The fetal adrenal uses the large amounts of progesterone supplied by the placenta to make cortisol. Another source of cortisol for the fetus is the amniotic fluid where cortisol converted from cortisone by the choriodecidua, is found. In humans, maternal plasma CRH, ACTH, and cortisol levels increase during normal labor and drop at about four days postpartum; however, maternal ACTH and cortisol levels at this stage are not correlated. In sheep, placental CRH stimulates the fetal production of ACTH, which in turn leads to a surge of fetal cortisol secretion that precipitates parturition. The 10-day-long intravenous administration of antalarmin, a CRH receptor antagonist, significantly prolonged gestation compared to the control group of animals. Thus, CRH receptor antagonism in the fetus can also delay parturition. The HPA axis during the postpartum period gradually recovers from its activated state during pregnancy. The adrenals are mildly suppressed in a way analogous to postcure Cushing's syndrome. Provocation testing has shown that hypothalamic CRH secretion is transiently suppriently suppressed at three and six weeks postpartum, normalizing at 12 weeks.

Oxytocin innervation of caudal brainstem nuclei activated by cholecystokinin

The integration of ‘long-term’ adiposity signaling with the ‘short-term’ meal-related signal cholecystokinin (CCK) is proposed to involve descending hypothalamic projections to areas of the caudal brainstem (CBS) that regulate the amount of food consumed during a single meal. One such projection extends from cell bodies in the hypothalamic paraventricular nucleus (PVN) to the nucleus tractus solitarius (NTS), where cells that respond to peripheral CCK are concentrated. Candidate neuronal cell types that may comprise this PVN–NTS projection includes those expressing corticotropin-releasing hormone (CRH) or oxytocin. We therefore sought to determine whether oxytocin or CRH axons are preferentially located in close anatomical proximity to neurons of the NTS that are activated by peripheral administration of CCK, as determined by immunocytochemical staining for Fos protein. Rats received injections of either an anorexic dose of CCK (8 nmol/kg, i.p.) or vehicle and were perfused 2 h later with 4% paraformaldehyde. Immunocytochemistry was performed on cryostat sections (14 μm) of caudal brainstem, using a polyclonal antibody to Fos protein and either a monoclonal antibody to oxytocin or a polyclonal antibody to CRH. As expected, CCK administration significantly increased the numbers of Fos-positive neurons by 489% ( p<0.01) and 400% ( p<0.01), respectively, in the medial and gelatinosus subdivisions of the NTS. These same regions received dense oxytocin axon innervation, whereas CRH immunoreactivity was not as prevalent in these areas. In areas outside the NTS, such as the dorsal motor nucleus of the vagus (DMV), Fos activation was absent despite a dense oxytocin and CRH innervation. To investigate whether CCK-induced reductions of food intake require intact oxytocin signaling, we performed a separate study in which CCK injection was preceded by injection into the fourth ventricle of an oxytocin receptor antagonist [d(CH 2) 5, Tyr (Me) 2, Orn 8]-vasotocin (OVT). This study showed CCK was 23% and 22% less effective at inhibiting food intake at 30 min ( p<0.05) and 1 h ( p<0.05) food intake, respectively, in the presence of OVT. Taken together, the data indicate that oxytocin axons within the descending pathway from the PVN to the NTS are anatomically positioned to interact with NTS neurons that respond to vagally mediated peripheral CCK signals such as those that occur following ingestion of a meal. These findings support the hypothesis that oxytocin exerts a tonic stimulatory effect on the response of key neurons within the NTS to CCK and further reduce meal size.

Sensitization associated with stressors and cytokine treatments

Like stressors, interleukin-1β and tumor necrosis factor-α (TNF-α) increase hypothalamic–pituitary–adrenal (HPA) activity and monoamine turnover at hypothalamic and extrahypothalamic sites. These effects can be re-elicited more readily upon reintroduction of these challenges (sensitization), depending on their time of re-exposure and the particular system being assessed. Following TNF-α administration, the co-expression of corticotropin releasing hormone (CRH) and arginine vasopressin increased within the median eminence, peaking 7–14 days after treatment, and was associated with an early corticosterone sensitization. However, the re-elicitation of sickness symptoms and corticosterone release was most pronounced at lengthy re-exposure intervals (28 days), possibly reflecting histamine release from mast cells. In addition, the cytokine engendered the sensitization of norepinephrine and serotonin utilization, and CRH immunoreactivity at mesocorticolimbic sites, but these effects were most prominent at brief re-exposure intervals (1–7 days). Cytokines may independently prime multiple regulatory systems, and by virtue of the neurochemical changes imparted, have both immediate and proactive influences on the evolution of psychopathology.

Increased corticotropin-releasing hormone immunoreactivity in monoamine-containing pontine nuclei of depressed suicide men.

A number of clinical investigations and postmortem brain studies have provided evidence that excessive corticotropin-releasing hormone (CRH) secretion and neurotransmission is involved in the pathophysiology of depressive illness, and several studies have suggested that the hyperactivity in CRH neurotransmission extends beyond the hypothalamus involving several extra-hypothalamic brain regions. The present study was designed to test the hypothesis that CRH levels are increased in specific brainstem regions of suicide victims with a diagnosis of major depression. Frozen tissue sections of the pons containing the locus coeruleus and caudal raphe nuclei from 11 matched pairs of depressed suicide and control male subjects were processed for radioimmunocytochemistry using a primary antiserum to CRH and a ([125])I-IgG secondary antibody. The optical density corresponding to the level of CRH-immunoreactivity (IR) was quantified in specific pontine regions from the film autoradiographic images. The level of CRH-IR was increased by 30% in the locus coeruleus, 39% in the median raphe and 45% in the caudal dorsal raphe in the depressed suicide subjects compared to controls. No difference in CRH-IR was found in the dorsal tegmentum or medial parabrachial nucleus between the subject groups. These findings reveal that CRH-IR levels are specifically increased in norepinephrine- and serotonin-containing pontine nuclei of depressed suicide men, and thus they are consistent with the hypothesis that CRH neurotransmission is elevated in extra-hypothalamic brain regions of depressed subjects.

Differential release of corticotropin-releasing hormone (CRH) in the amygdala during different types of stressors

The purpose of this study was to determine if differences exist between the effects of acute treadmill running and restraint stress on corticotropin-releasing hormone (CRH) release within the amygdala of rats. Extracellular CRH immunoreactivity (CRH-IR) was measured in microdialysate collected from the central nucleus of the amygdala (CeA) during exposure to an inactivated treadmill (TC), during 1 h treadmill running to exhaustion (RUN), and 1 h restraint (RES). Extracellular CRH-IR increased from control levels during the first 20-min period for TC, RUN, and RES, with the largest increase during RES. During the second 20-min period, only RES maintained levels higher than control values. CRH release was higher than control during the third 20-min period of RES and RUN. A second experiment consisted of four groups of either cage controls (CC), TC, RUN, or RES. Immediately following the 60-min treatment, brains were removed and trunk blood collected for analysis of tissue CRH-IR and plasma corticosterone. While amygdala tissue CRH-IR was not different in the CC, TC and RUN rats, these groups had significantly lower levels than the RES animals. Hypothalamic tissue CRH-IR was not different between the CC and TC rats, but the levels were significantly higher in the RES and RUN rats than in the two control groups. Plasma corticosterone levels were elevated only in RES and RUN rats. Results from tissue analysis indicate that increased tissue CRH-IR in the amygdala and hypothalamus can be elicited by RES, while only the hypothalamus shows an increase following RUN. Further, extracellular CRH release in the CeA is increased throughout the period of RES, when rats are placed on the treadmill, and when the animals are approaching physical exhaustion. No increase is observed during the running period between placement on the treadmill and intense exertion. Overall, the data suggest that amygdala CRH release is regulated differently during treadmill running and restraint.

Strain Differences in the Expression of Corticotropin-Releasing Hormone Immunoreactivity in Nerves That Supply the Spleen and Thymus

The existence of nerve fibers containing corticotropin-releasing hormone (CRH) immunoreactivity in primary and secondary lymphoid organs from three strains of young adult male rats was examined. Spleens and thymuses from Fischer 344 (F344), Sprague-Dawley (SD) and Lewis (LEW) rats were prepared for immunocytochemistry using antisera directed against CRH. In F344 and SD rats, we were unable to demonstrate CRH-immunoreactive nerves in either the thymus or the spleen. Despite the lack of CRH-containing nerves, CRH immunoreactivity was present in pleotropic cells in the septum, cortex and medulla of the thymus, and in the red and white pulp of spleens from F344 and SD rats. In contrast, CRH+ nerves were found in thymuses and spleens from LEW rats. CRH+ nerves coursed in the interlobular septa, capsule, cortex and medulla of the LEW rat thymus. Large CRH-immunoreactive nerve bundles were present in the hilar region of the LEW rat spleen, and individual CRH+ fibers coursed in the capsule, trabeculae, red pulp, venous sinuses and marginal zone of the white pulp of the spleen. These findings indicate strain differences in neurotransmitter-specific nerves that innervate the rat spleen and thymus under basal conditions.

Time-dependent sensitization of corticotropin-releasing hormone, arginine vasopressin and c-fos immunoreactivity within the mouse brain in response to tumor necrosis factor-α

Stressor or cytokine treatments, such as interleukin-1β, promote time-dependent alterations of hypothalamic-pituitary-adrenal functioning, including increased arginine vasopressin stores within corticotropin-releasing hormone (CRH) terminals in the external zone of the median eminence. Likewise, we have previously shown that the proinflammatory cytokine, tumor necrosis factor-α (TNF-α), provoked a time-dependent sensitization of neuroendocrine and brain monoamine activity. To further explore the protracted consequences of TNF-α, the present investigation determined whether the cytokine sensitized activity of neuroendocrine regulatory brain regions, as assessed by c-fos expression, and had protracted consequences on amygdaloid CRH, as well as hypothalamic corticotropin secretagogues. Indeed, immunoreactivity for arginine vasopressin and corticotropin-releasing hormone, and their colocalization within cell terminals of the median eminence, varied over time following an initial 4.0-μg tumor necrosis factor-α treatment, peaking after 7 days and normalizing within 28 days. Within the central amygdala, a sensitization effect was evident as reflected by increased CRH immunoreactivity, but this effect required re-exposure to the cytokine, unlike the median eminence changes that simply evolved with the passage of time. As well, tumor necrosis factor-α provoked a marked sensitization of c-fos staining within the paraventricular nucleus of the hypothalamus, supraoptic nucleus and the central amygdala. From these data we suggest that tumor necrosis factor-α influences responsivity of stressor-reactive brain regions and has protracted effects on central neuropeptide expression within the hypothalamus and central amygdala, although the time course for the effects vary across brain regions. Evidently, exposure to tumor necrosis factor-α may promote neuroplasticity of brain circuits involved in mediating neuroendocrine, sickness or inflammatory responses. It is suggested that such a sensitization may influence the response to immunological and traumatic insults and may thus be relevant to behavioral pathology.

Effects of interleukin-2 on the expression of corticotropin-releasing hormone in nerves and lymphoid cells in secondary lymphoid organs from the Fischer 344 rat

This study examined the influence of interleukin (IL)-2 on corticotropin releasing hormone (CRH) immunoreactivity in the Fischer 344 (F344) rat spleen. Rats were given either vehicle or 1, 10, 25, 50, 100, or 200 ng of human recombinant (hr)IL-2 by intraperitoneal (i.p.) injection, and were sacrificed 0.5, 1, 4, 12, or 24 h after treatment. Spleens and mesenteric lymph nodes were prepared for immunocytochemistry to localize CRH. In spleens from vehicle-treated animals, CRH immunoreactivity was present in several types of cells of the immune system, but CRH + nerves were not observed in either spleens or lymph nodes from vehicle-treated animals. Treatment with IL-2 induced CRH expression in nerves in the spleen in a dose- and time-dependent manner. CRH + nerves were not found in the mesenteric lymph nodes after IL-2 treatment, instead a dramatic time- and dose-dependent accumulation of CRH + cells (resembling small lymphocytes and large granular mononuclear cells) in the cortex and medulla. These findings indicate that IL-2 stimulates the synthesis of CRH in nerves that innervate the F344 rat spleen, and promote the appearance of CRH + immunocytes into draining mesenteric lymph nodes.

Stress-induced sensitization of CRH-ir but not P-CREB-ir responsivity in the rat central nervous system

There is some evidence that a traumatic life event can induce long-term alterations in corticotropin-releasing hormone (CRH) producing neurons in humans, which may play a role in the pathophysiology of anxiety disorders, including post-traumatic stress disorder (PTSD). To study the long-term effects of a traumatic event on brain CRH-immunoreactivity (CRH-ir) and phospho-cAMP response element binding protein-immunoreactivity (P-CREB-ir), rats were exposed to a single session of foot shocks (preshocked) or no shocks (control). Two weeks later half of the control rats and half of the preshocked rats received an electrified prod in the home cage for 15 min and behavior was recorded. Fifteen minutes after the removal of the prod rats were perfused and brain sections were stained for CRH-ir and P-CREB-ir. There was no basal difference between preshocked and control rats in brain CRH-ir and P-CREB-ir. Exposure to the electrified prod induced a significant increase in CRH-ir in the paraventricular nucleus of the hypothalamus, the median eminence and the central amygdala in preshocked rats, but not in control rats. The electrified prod increased the number of P-CREB-ir neurons in the paraventricular nucleus of the hypothalamus and the locus coeruleus, but the preshock experience did not affect this response. In an additional experiment with a similar design plasma hormone levels were measured 14 days after the foot shocks. The preshock experience sensitized the shock prod-induced ACTH and corticosterone response. No behavioral differences between preshocked and control rats were found during the shock prod tests. We suggest that long-term stress-induced changes in neuropeptide dynamics of CRH-ir neurons may play a role in long-term stress-induced neuroendocrine sensitization.