Effect of protein synthesis inhibition on brain corticotropin-releasing factor and plasma adrenocorticotropin

The effect of inhibiting protein synthesis on concentrations of corticotropin-releasing factor (CRF) in rat brain and plasma adrenocorticotropin (ACTH) was assessed following the administration of the general protein synthesis inhibitor anisomycin. Compared with vehicle-injected controls, protein synthesis inhibition resulted in significantly reduced CRF immunoreactivity (CRF-ir) in median eminence within 1 h (p less than 0.01), remained decreased after 4 h (p less than 0.025), and was nonsignificantly decreased after 24 h. Plasma ACTH levels were greatly increased within 1 h posttreatment (p less than 0.0005), continued elevated after 4 h (p less than 0.01), and returned to normal levels after 24 h. CRF-ir measured in other brain areas 24 h after anisomycin showed decreased levels in medulla-pons (p less than 0.025) and neurointermediate lobe of pituitary (p less than 0.05), with no change noted in frontal cortex, hippocampus, midbrain-thalamus, or cerebellum. Overall these data show that blockade of normal protein synthesis with anisomycin can elicit changes in CRF-ir and ACTH content.     

Sauvagine-like and corticotropin-releasing factor-like immunoreactivity in the brain of the bullfrog (Rana catesbeiana)

Summary Immunocytochemical methods were used to investigate the occurrence and distribution of sauvagine, corticotropin-releasing factor-, or urotensin I-like immunoreactivities (SVG-ir, CRF-ir, UI-ir, respectively) in the bullfrog (Rana catesbeiana) brain, using specific antisera raised against non-conjugated SVG, ovine CRF, rat/human CRF, and UI. In the hypothalamus, SVG-ir was found in the magnocellular perikarya, in the dorsal and ventral regions of the preoptic nucleus, and in the hypothalamo-hypophyseal projections to the external zone as well as the internal zone of the median eminence, to pars nervosa, and in fibres running from the pars nervosa to the pars intermedia of the pituitary. In contrast, CRF-ir was found only in parvocellular perikarya, mainly localized in the rostro-ventral part of the preoptic nucleus, with fine processes protruding through the ependyma of the third ventricle, fibre projections terminating in the anterior preoptic area and in the neuropil of the periventricular gray, and a caudal projection to the external zone of the median eminence. No CRF-ir staining was seen in the pars nervosa and pars intermedia. The use of UI-specific antisera failed to give a positive response in the frog brain. It is concluded that, in the frog brain, two anatomically different CRF-like (or SVG-like) systems co-exist, comparable to the reported co-existence of UI-ir and CRF-ir neuronal systems in fish brain.     

Inhibitory effects of neuropeptide Y (NPY) on CRF and stress-induced cecal motor response in rats

The effects of NPY on CRF and stress-stimulated cecal motility were investigated by electromyography in rats. Intracerebroventricular (ICV) injection of NPY at 300 ng/kg significantly reduced the frequency of spike burst during the first 15 minutes after its administration while no effect was observed at a lower dose (150 ng/kg). Exposure to mental stress (MS) increased significantly (p less than 0.01) during 45 minutes, the frequency of cecal spike bursts. NPY (300 ng/kg) injected ICV, 30 minutes prior to MS periods abolished the excitatory effect induced by stress. The frequency of cecal spike bursts was also increased during the first 15-minutes following ICV injection of CRF (300 ng/kg). Prior (5 min) ICV administration of NPY (150 ng/kg) abolished the stimulatory effect of CRF on cecal motility. It is concluded that central administration of NPY suppresses the stress-induced cecal motor response probably by inhibiting the pathways involved in CRF mediation of these effects.     

Up-regulation of NPY gene expression in hypothalamus of rats with experimental chronic renal failure

Anorexia is possibly one of the most important causes of malnutrition in uremic patients. The cause of this abnormality is still unknown. Considering that: (a) NPY is one of the most important stimulants of food intake; (b) eating is a central nervous system regulated process and (c) NPY is expressed in hypothalamus, we hypothesized that the decrease of NPY gene expression in the hypothalamus could be an important factor contributing to anorexia associated with uremic state. In contrast to the prediction, the results presented in this paper indicate that the NPY gene expression in the hypothalamus of chronic renal failure (CRF) rats was significantly higher than in the hypothalamus of control (pair-fed) rats. Moreover, we found that serum NPY concentration in CRF rats was higher than in control (pair-fed) animals. The increase of plasma NPY concentration in CRF rats may be due to the greater synthesis of the neuropeptide in liver, since higher level of NPY mRNA was found in liver of CRF rats. The results obtained revealed that experimental chronic renal failure is associated with the increase of NPY gene expression in hypothalamus and liver of rats.     

Effect of morphine on hypothalamic corticotropin-releasing factor (CRF) and pituitary-adrenocortical activity

The effects of intraperitoneal and intra-third ventricular administration of morphine on the hypothalamic corticotropin-releasing factor (CRF) and the pituitary-adrenocortical activity were examined in unanesthetized, freely moving rats. Hypothalamic CRF was measured by rat CRF radioimmunoassay. Intraperitoneal or intra-third ventricular administration of morphine increased blood concentrations of ACTH and corticosterone while intraperitoneal administration tended to increase CRF concentration in the whole hypothalamus including the median eminence and intra-third ventricular administration increased CRF concentration in the hypothalamus excluding the median eminence. However, morphine seemed to inhibit the increase in CRF concentration in the hypothalamus induced by the ether-laparotomy stress. The main site of morphine action on the hypothalamo-pituitary-adrenocortical system seemed to be in the hypothalamic area.     

Monoamine oxidase activity in the hypothalamus and various other brain areas and in some endocrine glands of the rat during the estrus cycle

—The activity of monoamine oxidase (MAO, EC 1.4.3.4) was measured in the entire hypothalamus and different hypothalamic regions, in the amygdala, frontal and lateral cerebral cortex, in the pituitary, adrenals and genital organs of male rats and of female rats during the estrus cycle. Activity of MAO changed cyclically in the hypothalamus, amygdala, adrenals and ovaries. The highest levels in the hypothalamus occurred at 10 a.m. on the day of proestrus and during estrus. The lowest levels occurred at 6 p.m. on the day of proestrus, of metestrus and during diestrus. Cyclical variations similar to those found in the whole hypothalamus were also observed in anterior, posterior and lateral portions and the median eminence of the hypothalamus. Activity in the median eminence was greater than that of the whole hypothalamus or its various other portions. The amygdala exhibited less marked cyclical activity which followed the pattern of the hypothalamus by increasing at 10 a.m. and peaking at 3 p.m. on the day of proestrus. At the‘post-critical’period of proestrus, when the activity of MAO in the hypothalamus and amygdala decreased, the activity of MAO in the ovaries and adrenals rose. During the estrus cycle much lower levels of activity of MAO were demonstrated in other regions of the brain (frontal and lateral cerebral cortex), in the pituitary and in the uterus, none of which showed cyclical changes. The changes in activity of MAO in cerebral tissues, endocrine glands and genital organs have been discussed in relation to the probable participation of monoamines in the mechanism(s) of secretion of gonadotrophins by the hypothalamus.     

Hypothalamic corticotropin-releasing factor immunoreactivity is reduced during induction of pituitary tumors by chronic estrogen treatment

The role that estrogen plays in the regulation of corticotropin-releasing factor (CRF) is not known. A radioimmunoassay specific for rat CRF was utilized to measure the CRF-like immunoreactivity (CRF-ir) in the hypothalamus of ovariectomized rats treated with estradiol for periods up to 12 weeks. Compared to ovariectomized controls, estradiol treatment resulted in significantly reduced CRF-ir after 3 and 12 weeks, although no significant change was seen after 8 weeks. Anterior pituitary (AP) weight was greatly increased by estradiol treatment at all time points studied. Bromocriptine treatment for the last 3 weeks of the 12-week period, or removal of estradiol for 3 weeks after 9 weeks of treatment did not reverse the changes in CRF-ir even though significant regression of tumor size was achieved. There was no correlation between AP weight and CRF-ir in individual animals. These data show that chronic treatment with estrogen reduced hypothalamic CRF-ir content. Neither a direct estrogenic effect or an indirect effect mediated through alterations in the adenohypophysis could be ruled out.     

Effect of latent iron deficiency on metal levels of rat brain regions

Seven different metals (iron, copper, zinc, calcium, manganese, lead, and cadmium) were studied in eight different brain regions (cerebral cortex, cerebellum, corpus striatum, hypothalamus, hippocampus, midbrain, medulla oblongata, and pons) of weaned rats (21-d-old) maintained on an iron-deficient (18-20 mg iron/kg) diet for 8 wk. Iron was found to decrease in all the brain regions, except medulla oblongata and pons, in comparison to their respective levels in control rats, receiving an iron-sufficient (390 mg iron/kg) diet. Brain regions showed different susceptibility toward iron deficiency-induced alterations in the levels of various metals, such as zinc, was found to increase in hippocampus (19%, p less than 0.05) and midbrain (16%, p less than 0.05), copper in cerebral cortex (18%, p less than 0.05) and corpus striatum (16% p less than 0.05), calcium in corpus striatum (22%, p less than 0.01) and hypothalamus (17%, p less than 0.02), and manganese in hypothalamus (18%, p less than 0.05) only. Toxic metals lead and cadmium also increased in cerebellum (19%, p less than 0.05) and hippocampus (17%, p less than 0.05) regions, respectively. Apart from these changes, liver (64%, p less than 0.001) and brain (19%, p less than 0.01) nonheme iron contents were found to decrease significantly, but body, liver, and brain weights, packed cell volume, and hemoglobin content remained unaltered in these experimental rats. Rehabilitation of iron-deficient rats with an iron-sufficient diet for 2 wk recovered the values of zinc in both the hippocampus and mid-brain regions and calcium in the hypothalamus region only. Liver nonheme iron improved significantly; however, no remarkable effect was noticed in brain nonheme iron following rehabilitation. It may be concluded that latent iron deficiency produced alterations in various metal levels in different brain regions, and corpus striatum was found to be the most vulnerable region for such changes. It is also evident that brain regions were resistant for any recovery in their altered metallic levels in response to rehabilitation for 2 wk.     

Relationship between neuropeptide Y and luteinizing-hormone-releasing hormone immunoreactivities in the hypothalamus and preoptic region

The purpose of this study was to examine the anatomical relationships of perikarya and fibers containing neuropeptide Y (NPY) and luteinizing-hormone-releasing hormone (LHRH) in the hypothalamus and preoptic region of female rats. In view of our previous report of stimulatory effects of estrogen on LHRH and NPY levels in the median eminence, animals were bilaterally ovariectomized and subsequently implanted subcutaneously with capsules containing estradiol benzoate in oil or vehicle. Following intracerebroventricular injection of colchicine, rats were perfused with fixative and their brains sectioned and processed for immunohistochemical visualization of NPY and LHRH in the same section and in consecutive sections. Estrogen treatment had no discernible effect on the distribution or relationship of these peptides. NPY-immunoreactive fibers were intimately associated with LHRH-labeled primary dendrites and perikarya in the medial preoptic region and horizontal limb of the diagonal band of Broca. Fibers containing NPY or LHRH overlapped extensively in the lateral palisade region of the median eminence and also in the subependymal and internal zones. The external zone of the median eminence displayed relatively less overlap of these peptide systems. LHRH-immunoreactive axons coursed among NPY-labeled perikarya in the arcuate nucleus and appeared to contact these cells. These results suggest that NPY-containing axons may influence LHRH-positive neurons at the cell body and also at the site of axon termination in the median eminence. LHRH-containing axons appear to contact NPY-immunoreactive perikarya in the arcuate nucleus and may interact with terminals in the median eminence. This arrangement may provide a mechanism for communication between NPY and LHRH neurons and for the neuroendocrine coordination of hypothalamic NPY and LHRH secretion before ovulation.     

Immunohistochemical study on the development of CRF-containing neurons in the hypothalamus of the rat

Summary Appearance of immunoreactive corticotropin-releasing factor (CRF)-containing neurons was studied in developing hypothalamus of the rat by use of antisera against rat- and ovine CRF. These neurons were first recognized in the lateral and paraventricular nuclei on days 15.5 and 16.5 of gestation, respectively, when antiserum against rat CRF was employed. Antiserum against ovine CRF revealed the cells two days later exclusively in the latter nucleus. In both nuclei, the neurons increased in number with development. The neurons in the paraventricular nucleus appeared to project their immunoreactive processes to the median eminence via the periventricular and lateral pathways. In the median eminence, the immunoreaction with antiserum to rat CRF was first recognized in its anterior portion in the form of dots on day 16.5 of gestation but as beaded fibers in the external layer on day 17.5; these structures increased in amount with development in rostro-caudal direction. Although antiserum to ovine CRF was less potent in immunostainability than antiserum to rat CRF, it also revealed the beaded fibers in the median eminence on day 17.5 of gestation. Since evidence is available that the paraventricular nucleus is involved in corticotropin release, it is concluded that, in rats, the hypothalamic regulatory mechanism controlling the release of corticotropin initially appears on days 16.5–17.5 of gestation.     

Possible role of CRF peptides in burn-induced hypermetabolism

Hypermetabolism and anorexia are significant problems associated with major burn trauma. Recent studies have shown that hypothalamic corticotropin releasing factor (CRF) elevates metabolic rate, while neuropeptide Y (NPY) reduces it. CRF also elicits anorexia, while NPY stimulates feeding. We hypothesized that elevation of CRF and decrease of NPY may be mediators of these negative effects of burn trauma. Therefore, we assessed concentrations of CRF and NPY in hypothalamus of burned rats one, three, and twenty-one days after a 30% body surface area, full-thickness, open flame burn. In addition we determined whether a biochemical lesion of CRF receptors using 3rd ventricle injections of a saporin-CRF conjugated peptide would decrease resting energy expenditure (REE). We found a three-day period of anorexia, with REE significantly increasing three days after the burn trauma. Concentrations of NPY were increased in the PVN-containing dorsomedial region of the hypothalamus 1 and 3 days after burn trauma, but were increased further in the day 1 pair-fed rats suggesting this change was a consequence of the anorexia. Levels of CRF were decreased in the ventromedial region of the hypothalamus in day 1 and day 3 burned and PF rats. Treatment with the saporin-CRF conjugate normalized REE and reduced CRF receptor-2 density in the hypothalamus of burned rats, and blocked CRF-induced hypermetabolism in sham-burned rats. Although these results suggest a role of CRF receptors in mediating burn-induced hypermetabolism, CRF itself may not be the principle ligand, as suggested by the significant elevation of hypothalamic urocortin 15 days after burn injury.     

Wild ginseng attenuates anxiety- and depression-like behaviors during morphine withdrawal

The purpose of this study was to evaluate whether wild ginseng (WG) administration could attenuate anxiety- and depression-like behaviors and expression of corticotrophin-releasing factor (CRF) and neuropeptide Y (NPY) following withdrawal from repeated morphine administration in rats. Male rats were administered daily doses of WG (50, 100, or 200 mg/kg, i.p.) for 5 days, 30 min before morphine injection (40 mg/kg, s.c). The anxiety- and depression-like behavioral responses were measured 72 h after the last morphine injection using an elevated plus maze (EPM) and forced swimming test (FST), respectively. Changes in hypothalamic CRF and NPY expressions were also examined by analyzing their immunoreactivities in the hypothalamus. Daily administration of WG significantly reduced anxiety-and depression-like behavior, and elicited the suppression of CRF expression and the stimulation of NPY expression in the hypothalamus. Our results demonstrated that WG extract might be effective at inhibiting the anxiety and depression responses due to morphine withdrawal by possibly modulating the hypothalamus CRF and NPY systems. Furthermore, these findings imply that WG extract can be used for developing new medication to cure or alleviate morphine withdrawal symptoms and to prevent relapses of morphine use.     

Increased neuropeptide Y concentrations in the lateral hypothalamic area of the rat after the onset of darkness: possible relevance to the circadian periodicity of feeding behavior

Neuropeptide Y (NPY) is a major hypothalamic peptide which powerfully stimulates feeding when injected into the hypothalamus and is implicated in circadian rhythmicity. To investigate whether NPY is involved in the increased feeding that follows the onset of darkness in rats, NPY levels were measured in discrete hypothalamic areas before and after darkness. Four groups of eight adult female Wistar rats were habituated to a 12:12 hour light:dark cycle, with food presented at the onset of darkness (19.00 hours). One group was sacrificed during the 3 hours before darkness and another in the first 2.5 hours after darkness, with food provided as usual. To distinguish any effects of feeding itself, the study was repeated with two further groups, but food was not provided after darkness. Seven hypothalamic regions were microdissected from slices of fresh brain and acid-extracted for radioimmunoassay of NPY. NPY levels (fmol/microgram protein) were significantly higher (p less than 0.01) in the lateral hypothalamic area (LHA) of the dark-phase group in both studies. In the other six regions, NPY levels did not differ between light and dark phases. The LHA regulates the circadian rhythmicity of feeding and NPY injection here stimulates feeding. Alterations in NPY in the LHA around the onset of darkness may be related to the initiation of dark-phase feeding.     

Immunohistochemical localization of corticotropin-releasing factor (CRF)-containing neurons in the hypothalamus of the Japanese quail,Coturnix coturnix

Immunohistochemical localization of corticotropin-releasing factor (CRF)-like immunoreactivity in the brain of the Japanese quail was studied by means of the peroxidase anti-peroxidase (PAP) method. CRF-immunopositive perikarya of parvocellular neurons were observed mainly in the nucleus praeopticus medialis and nucleus paraventricularis. Additional perikarya were also detected in the nucleus hypothalamicus posterior medialis in the hypothalamus and in the non-hypothalamic nucleus accumbens, nucleus septalis lateralis and nucleus dorsomedialis and dorsolateralis thalami. No CRF immunoreaction was found to coexist with the vasotocin (Vt)-containing system in comparative examination of consecutive sections treated with anti-vasopressin (Vp) serum. The CRF-immunoreactive fibers were detected mainly in the external layer of the anterior median eminence but not in its posterior division. Unilateral adrenalectomy induced the marked reduction in number of the CRF immunopositive fibers in the anterior median eminence.     

Dexfenfluramine treatment and hypothalamic neuropeptides in diet-induced obesity in rats.

Neuropeptide Y (NPY) is a powerful appetite stimulant, and hypothalamic concentrations rise after food deprivation and in experimental diabetes. Serotonergic drugs such as dexfenfluramine are inhibitors of feeding. We measured hyothalamic NPY and NPY mRNA, along with galanin, neurotensin, and somatostatin in chow-fed rats and in rats with dietary obesity, and examined the effect of dexfenfluramine on these peptides in this model. Sixty-five rats were fed a palatable diet (condensed milk, sucrose and chow) for 6 weeks, which produced significant weight gain compared to twenty fed standard chow (145.1 +/- 2.3 g vs. 113.4 +/- 3.2 g, p less than 0.001). Groups of animals were treated for 7 days or 28 days with dexfenfluramine (1.8 mg/kg/day) or saline intraperitoneally via miniosmotic pumps. Hypothalami were dissected into medial and lateral blocks, and NPY, galanin, neurotensin, and somatostatin were measured by radioimmunoassay. Neuropeptide Y mRNA was measured by Northern blotting. Hypothalamic NPY was significantly higher in the palatable diet group compared to chow-fed controls (medial hypothalamus: 86.6 +/- 7.6 vs. 65.7 +/- 4.0 pmol/g tissue, p less than 0.02, lateral hypothalamus 71.2 +/- 6.6 vs. 53.1 +/- 3.6 pmol/g tissue, p less than 0.05), but NPY mRNA was unchanged. Although dexfenfluramine was effective at reducing weight gain in the animals fed the palatable diet, this did not result in any changes in the hypothalamic neuropeptides measured. Neuropeptide Y may be of importance in diet-induced obesity but the weight loss produced by dexfenfluramine in such animals is not mediated by changes in hypothalamic NPY.     

A specific radioimmunoassay for Cortricotropin Releasing Factor (CRF) using synthetic ovine CRF

This newly developed specific radioimmunoassay for corticotropin releasing factor (CRF) had a sensitivity range of 25 pg/tube to 4 ng/tube. Intra and interassay coefficient of variation were 4.6% and 9.8%, respectively. Rat median eminence extracts showed a parallel dose response curve with synthetic ovine CRF and a significant cross reaction was not evident with other tested neuropeptides. The highest mean levels of CRF were found in the median eminence (6.61 ng/mg protein). Considerable amounts of CRF were found in the arcuate nucleus, paraventricular nucleus, dorsomedial nucleus, suprachiasmatic nucleus and ventromedial nucleus. The immunoreactive CRF of the rat medial basal hypothalamus coeluted with bioassayable CRF and with iodinated CRF on Sephadex G-75 chromatography. The results indicate that rat hypothalamus contains a CRF similar to ovine CRF.     

The effect of dexamethasone on neuropeptide Y concentrations in specific hypothalamic regions.

Neuropeptide Y (NPY) is a major hypothalamic peptide which is implicated in the regulation of energy balance and in the activation of the hypothalamo-pituitary adrenal axis. This study aimed primarily to determine the effects on regional hypothalamic NPY levels, of catabolism and weight loss induced in rats by the synthetic glucocorticoid, dexamethasone, injected daily at a dose of 0.4 mg/kg for 7 days. NPY concentrations were significantly raised in the paraventricular nucleus (PVN) of male Wistar rats (45%, p = 0.009; n = 10) compared with saline-injected controls (n = 10). Body weight (p less than 0.001) and food intake (p less than 0.001) were significantly reduced, plasma insulin concentrations were increased (p less than 0.001), but there was no change in glucose concentrations. Chronic dexamethasone treatment did not cause the marked NPY increases in the arcuate nucleus (ARC) and other hypothalamic regions which have been observed in other catabolic states causing weight loss. One possible explanation is the high insulin levels induced by dexamethasone, which may have prevented compensatory hyperphagia by suppressing an increase in hypothalamic NPYergic activity. We also examined the acute effects of a single dexamethasone injection on regional hypothalamic levels, to determine whether the drug had a direct action separate from that due to sustained weight loss. In the acute study, groups of rats (n = 7) were examined at 4 h after a single injection of dexamethasone or saline. NPY concentrations were significantly increased in the lateral hypothalamic area (LHA), (60%, p = 0.008) when compared with saline-injected controls, but there was no change in body weight or glucose or insulin concentrations during the 4h interval. Altered transport or release of NPY in the lateral hypothalamic area may be a result of acute feedback regulation by glucocorticoids on the hypothalamus.     

Corticotropin-releasing factor from the rat brain measured by protein immunoblot

The ability to measure changes in brain levels of corticotropin-releasing factor (CRF) is an important step toward understanding the role of this neuropeptide in mood states. Here, we report for the first time that the protein (Western) immunoblot assay can be used to detect and quantify CRF (4.7 kDa) from the rat brain. Intracerebroventricular (ICV) injections of the neuronal transport-inhibitor colchicine (0, 7.5, 15 and 75 microg) produced a dose-dependent increase in CRF levels within the paraventricular nucleus (PVN) of the hypothalamus with a concomitant and dose-dependent decrease in CRF levels within the median eminence (ME). These data provide a positive validation for the use of the immunoblot assay to detect treatment-induced changes in brain CRF levels.     

Stimulation of Neuropeptide Y Overflow in the Rat Paraventricular Nucleus by Corticotropin-Releasing Factor

Abstract: Neuropeptide Y (NPY) and corticotropin-releasing factor (CRF) are present at high concentrations in the hypothalamus where they mediate important endocrine and autonomic functions. Morphological and physiological studies have suggested an interaction between these peptides, and opposing actions of CRF and NPY have been reported on feeding and other behaviors. This study investigated the effect of CRF on NPY release in vivo, measured by push-pull techniques, in the anesthetized rat. Push-pull probes implanted into the paraventricular nucleus of the hypothalamus (PVN) were perfused with modified Ringer solution containing bovine serum albumin at 15 µl/min, and the perfusate was lyophilized prior to NPY radioimmunoassay. NPY overflow from the rat PVN was increased threefold by perfusion of a depolarizing concentration of potassium (50 mmol/L KCI). When CRF was administered into the PVN via the push-pull cannula at 1 or 5 µg/ml, dose-dependent increases in NPY overflow of two- and fivefold were observed ( p < 0.05). These increases were abolished by prior intracerebroventricular (i.c.v.) administration of the CRF antagonist [ d -Phe¹²,Nle^21,38,C^αMeLeu³²]CRF (12–41) at 1 or 5 µg/µl, respectively. NPY overflow returned promptly to resting levels following CRF administration. In contrast, when CRF was administered by i.c.v. bolus at a similar total dose (2 µg), no significant effect on NPY overflow was observed. These data provide in vivo evidence for an interaction between CRF and NPY at the level of the PVN.