Adrenalectomy enhances endotoxemia-induced hypophagia: higher activation of corticotrophin-releasing-factor and proopiomelanocortin hypothalamic neurons

Inflammatory and infectious processes evoke neuroendocrine and behavioral changes known as acute-phase response that includes activation of the hypothalamo-pituitary-adrenal (HPA) axis and reduction of food intake. Besides its action as the most important ACTH secretagogue, corticotrophin-releasing factor (CRF), synthesized in the paraventricular nucleus (PVN), is also involved in the control of food intake. Alpha-melanocyte stimulating hormone (α-MSH) in the arcuate nucleus also plays a role in the energy homeostasis, possessing anorexigenic effects. To investigate the participation of neuropeptides involved in the regulation of food intake during endotoxemia, we administrated lipopolysaccharide (LPS) in sham-operated and adrenalectomized (ADX) male Wistar rats to evaluate food intake, hormone responses and Fos-CRF and Fos-α-MSH immunoreactivity in the PVN and arcuate nucleus, as well as CRF and POMC mRNA expression in these hypothalamic nuclei. In sham-operated rats, treatment with LPS (100 µg/kg) showed lower food intake, higher plasma ACTH and corticosterone levels, as well as an increase in Fos-CRF double labeled neurons and CRF mRNA expression in the PVN, with no changes in Fos-α-MSH immunoreactivity and POMC mRNA expression in the arcuate nucleus, compared to saline treated rats. After LPS treatment, ADX rats showed further increase in plasma ACTH levels, marked decrease of food intake, higher Fos-CRF immunoreactive neurons in the PVN and CRF mRNA expression, as well as an increase in Fos-α-MSH immunoreactivity and POMC mRNA expression in the arcuate nucleus, compared to sham-operated rats treated with LPS. In conclusion, the present data indicate that the marked hypophagia during endotoxemia following ADX is associated with an increased activation of CRF and POMC neurons in the hypothalamus and an increased mRNA expression of these neuropeptides.     

A double sequential immunofluorescence method demonstrating the co-localization of urotensins I and II in the caudal neurosecretory system of the teleost,Gillichthys mirabilis

Summary A double immunofluorescence method was devised to localize simultaneously urotensin-I (UI) and -II (UII) immunoreactivities in the caudal neurosecretory system of the goby, Gillichthys mirabilis. In a sequential fashion, sections of the posterior spinal cord and urophysis were treated with antiserum to corticotropin-releasing factor (CRF) that cross-reacts with UI, fluorescein-conjugated sheep anti-rabbit IgG, biotinylated anti-UII and rhodamine-conjugated avidin. UI and UII immunoreactivities appeared to coexist in some neurons and in most fibers and urophysial tissue; the remainder of the fibers and urophysis and the majority of neurons were immunoreactive for CRF/ UI only. No convincing evidence of immunoreactivity for UII only was found. A few nonreactive cells were seen, but these may not be neurosecretory neurons. The two immunoreactive cell types were not segregated topographically, and the intensity of perikaryal immunofluorescence for CRF/UI was variable. To explain these results a hypothesis that all caudal neurosecretory cells may synthesize both UI and UII and that immunoreactive differences may reflect different states of cellular activity, is suggested. This sequential double immunofluorescence method offers several advantages over other techniques and is especially useful for co-localization studies when primary antisera from different species are not available.     

Time course of vasopressin and oxytocin secretion after stress in adrenalectomized rats.

To characterize the participation of vasopressin (AVP) and oxytocin (OT) in hypothalamus-pituitary-adrenal regulation after adrenalectomy (ADX), we evaluated corticosterone, ACTH, AVP and OT plasma concentrations and AVP and OT content of the paraventricular nucleus (PVN) at different periods (3 h, 1, 3, 7 and 14 days) in sham or ADX rats under basal conditions and after immobilization stress. ADX animals showed undetectable corticosterone levels, while sham animals showed a marked increase in corticosterone and ACTH 3 h after surgery, then lowering to basal control levels. ADX rats showed high basal ACTH levels with a triphasic response without changes after immobilization. After three hours, the ADX group showed higher OT levels than the sham group. OT was increased after immobilization stress in sham and ADX groups. AVP plasma levels did not change throughout the basal or stress studies in either group. There was a decrease in hypothalamic AVP content 1 and 3 days after ADX under basal and stress conditions. Plasma osmolality showed a significant decrease in the ADX group at 3, 7, and 14 days. In conclusion, there are different pituitary-adrenal axis set points after removal of the glucocorticoid negative feedback. The role of vasopressinergic and oxytocinergic neurons in the ACTH secretion after ADX or immobilization stress appears to differ. Magnocellular AVP is unlikely to contribute to ACTH secretion in response to ADX or immobilization stress. On the other hand, OT is elicited by immobilization stress and might contribute to the ACTH secretion during short-term ADX.     

Glucocorticoids regulate peptidyl-glycine alpha-amidating monooxygenase gene expression in the rat hypothalamic paraventricular nucleus

Peptidyl-glycine alpha-amidating monooxygenase (PAM) is a posttranslational processing enzyme which catalyzes the formation of biologically active alpha-amidated peptides. The two major neuropeptides involved in the regulation of ACTH secretion [CRF and arginine vasopressin (AVP)], synthesized in the parvocellular part of the hypothalamic paraventricular nucleus (PVN), are amidated, and their synthesis and/or release is negatively regulated by glucocorticoids. In this study, using in situ hybridization, we have shown that PAM mRNA is abundantly expressed in the hypothalamic paraventricular and supraoptic nucleus. Surgical adrenalectomy (ADX) induced increases in PAM, CRF, and AVP mRNA in the parvocellular part of the PVN, while corticosterone treatment normalized these values. PAM and AVP gene expression were not changed in the magnocellular part of the PVN or in the supraoptic nucleus. These observations suggest that in addition to stimulation of CRF and AVP synthesis, ADX induces an increase in PAM synthesis in the PVN and, thus, support the hypothesis of increased secretion of both CRF and AVP after ADX.     

Long-term hypoxia enhances proopiomelanocortin processing in the near-term ovine fetus

Secondary stressors in long-term hypoxic (LTH) fetal sheep lead to altered function of the hypothalamic-pituitary-adrenal axis. Although ACTH is considered the primary mediator of glucocorticoid production in fetal sheep, proopiomelanocortin (POMC) and 22-kDa pro-ACTH (22-kDa ACTH) have been implicated in the regulation of cortisol production in the ovine fetus. This study was designed to determine whether POMC expression and processing are altered after LTH. Pregnant ewes were maintained at high altitude (3,820 m) from day 30 of gestation to near term, when the animals were transported to the laboratory. Reduced Po2 was maintained by nitrogen infusion through a maternal tracheal catheter. On days 139-141, fetal anterior pituitaries were collected from normoxic control and LTH fetuses. We measured POMC and corticotrophin-releasing factor type 1 receptor (CRF1-R) mRNA using quantitative real-time PCR, and we used Western blot analysis for quantitation of ACTH, ACTH precursor, and CRF1-R proteins. We measured plasma ACTH1-39 using a two-site immunoradiometric assay specific for ACTH1-39. Plasma ACTH precursors were measured by ELISA. Anterior pituitary POMC mRNA levels were not different between groups, whereas CRF1-R levels were significantly higher in the LTH anterior pituitaries compared with control (P<0.05). In contrast, protein levels of POMC, CRF1-R, 22-kDa ACTH, and ACTH1-39 were significantly lower in the LTH group. Plasma concentrations of both ACTH precursors and ACTH1-39 were significantly elevated in LTH fetuses, whereas the ratio of plasma precursors to ACTH was significantly lower. We conclude that LTH results in enhanced POMC processing and/or release to ACTH and increased hypothalamic drive.     

Desensitization of corticotropin-releasing factor receptors

Pretreatment of rat anterior pituitary cells with corticotropin releasing factor (CRF) rapidly and markedly reduced the ability of CRF to restimulate cyclic AMP formation and adrenocorticotropic hormone (ACTH) release. The effect was dependent on the length of time of pretreatment as well as the concentration of CRF. Neither basal nor intracellular immunoreactive ACTH levels nor basal cyclic AMP content were affected. CRF's stimulatory action on cyclic AMP formation and ACTH release recovered within one hour following CRF pretreatment. Forskolin, a compound that directly activates adenylate cyclase also releases ACTH from these cells. Pretreatment with CRF did not alter forskolin-stimulated cyclic AMP accumulation or ACTH secretion. Furthermore, CRF pretreatment did not change epinephrine's ability to increase the release of ACTH. These results indicate that CRF can regulate the responsiveness of its own receptor.     

Neuroanatomical connections between corticotropin-releasing factor (CRF) and somatostatin (SRIF) nerve endings and thyrotropin-releasing hormone (TRH) neurons in the paraventricular nucleus of rat hypothalamus.

In order to investigate the possible involvement of corticotropin-releasing factor (CRF) and somatostatin (SRIF) on thyrotropin-releasing hormone (TRH) neuronal cell activity in the rat hypothalamic paraventricular nucleus, we have proceeded to the simultaneous localization of CRF or SRIF and TRH. For this purpose, we used a dual immunostaining procedure that employed antibodies to CRF and SRIF and peroxidase-labeled goat anti-rabbit IgG as a first sequence, and antibodies to a cryptic fragment (Phe178-Glu199) of pro-TRH (to label TRH neurons) and alkaline phosphatase-labeled goat anti-rabbit IgG as the second sequence. A rich innervation of the paraventricular nucleus by immunoreactive CRF and SRIF fibers was observed. A large number of CRF and SRIF nerve endings were seen intimate anatomic proximity and often appeared to surround TRH-containing cell bodies. These results strongly suggest that TRH neurons might be regulated by both CRF and SRIF. These interactions might be the neuroanatomical basis for the already observed inhibitory effects of CRF and SRIF on TRH release.     

Pregnancy alters cortisol feedback inhibition of stimulated ACTH: studies in adrenalectomized ewes

These studies test the hypothesis that pregnancy alters the feedback effects of cortisol on stimulated ACTH secretion. Ewes were sham-operated (Sham), or adrenalectomized (ADX) at approximately 108 days gestation and replaced with aldosterone (3 microg x kg(-1) x day(-1)) and with cortisol at either of two doses (ADX + 0.6 and ADX + 1 mg x kg(-1) x day(-1)); ewes were studied during pregnancy and postpartum. Mean cortisol levels produced in ADX ewes were similar to normal pregnant ewes (ADX+1) or nonpregnant ewes (ADX+0.6), respectively. Plasma ACTH concentrations in response to infusion of nitroprusside were significantly increased in the pregnant ADX+0.6 ewes (1,159 +/- 258 pg/ml) relative to pregnant Sham ewes (461 +/- 117 pg/ml) or the ADX+1 ewes (442 +/- 215 pg/ml) or the same ewes postpartum (151 +/- 69 pg/ml). Plasma ACTH concentrations were not significantly different among the groups postpartum. Increasing plasma cortisol to 20-30 ng/ml for 24 h before hypotension produced similar inhibition of ACTH in all groups. Pregnancy appears to decrease the effectiveness of low concentrations of cortisol to inhibit ACTH responses to hypotension.     

Responses of the rat pituitary-adrenal axis to hypotensive infusions of corticotropin-releasing factor, vasoactive intestinal peptide and other depressor agents.

We have assessed in male rats the response of the hypothalamo-pituitary-adrenal axis to hypotension induced by 30 min i.v. infusions of corticotropin-releasing factor (CRF; 0.1, 0.2 and 0.5 nmol/kg/min), calcitonin gene-related peptide (CGRP; 0.25 nmol/kg/min), vasoactive intestinal peptide (VIP; 0.25 nmol/kg/min) and nitroprusside (NP; 150 micrograms/kg/min). Infusions of CRF produced dose-dependent decreases in mean arterial blood pressure of 10, 35 and 43 mmHg at 30 min, and the other treatment had depressor effects comparable with the higher CRF doses (between -35 and -44 mmHg). Plasma ACTH levels were increased from 383% to 595% by CGRP, NP and the three different CRF infusions (P less than 0.001 vs. controls), whereas they were raised more than 10-fold by VIP administration (P less than 0.001 vs. other treatments), a level 60% higher than the maximum achieved with CRF. Corticosterone levels were increased by 112% to 146% following infusion of the three different CRF doses, CGRP and NP (P less than 0.001 vs. controls), and by 240% after VIP (P less than 0.001 vs. other treatments). Plasma aldosterone values were increased by 112% to 140% after infusion of NP and the two higher CRF doses (P less than 0.01 vs. controls), and by 223% following VIP (P less than 0.05 vs. CRF 0.2 and NP). CGRP infusion, although resulting in similar haemodynamic changes, did not alter circulating aldosterone. The levels measured after CGRP were identical to those observed after the infusion of atrial natriuretic peptide (ANP; 1 nmol/kg/min), a known inhibitor of aldosterone secretion. These results demonstrate that the combination of hypotension and direct pituitary stimulation by CRF does not increase circulating ACTH levels above those obtained with hypotension alone (NP and CGRP), whereas VIP, which has only minimal direct effects on corticotroph function, markedly enhanced the ACTH response, suggesting that it may modulate ACTH release by an indirect mechanism. Evaluation of aldosterone levels after the different infusions indicates that CGRP prevented the rise normally associated with acute hypotension, thus confirming recent observations in other species that stimulated aldosterone secretion can be inhibited by CGRP.     

Proliferation of pituitary corticotrophs following adrenalectomy as revealed by immunohistochemistry combined with bromodeoxyuridine-labeling

Proliferation of corticotrophs following adrenalectomy (ADX) was studied by a combination of bromodeoxyuridine (BrdU)-labeling and immunohistochemistry. Rats were adrenalectomized, allowed to survive for 1, 3, 7, and 14 days and given 100 mg/kg body wt BrdU 3 h before sacrifice. BrdU and adrenocorticotropic hormone (ACTH) were detected in the same sections of the anterior pituitary using double-labeling immunohistochemistry. BrdU-labeled cells in the pituitary showed a tendency to increase until 1 week after ADX and slightly decreased at 2 weeks. Corticotrophs were increased to about 1.5 times of the control level 1–2 weeks after ADX. The number of cells double-labeled with both BrdU and ACTH increased markedly after ADX, suggesting active mitosis of existing corticotrophs. On the other hand, the ratios of these double-stained cells to all BrdU-labeled cells and to all corticotrophs were 5–7% and 0.9–1.3%, respectively, even after ADX, suggesting that the majority of corticotrophs which were increased after ADX were recruited from some other type of immature cells. The extent to which the two mechanisms are involved in hyperplasia of corticotrophs after ADX remains to be elucidated.     

ACTH determination in a petrosal sinus venous specimen after corticotrophin releasing factor provides the best clue on the laterality of microadenoma in Cushing's disease

A 42-year-old woman with Cushing's disease was endocrinologically cured after transsphenoidal selective excision of a left-sided microadenoma. Simultaneous samplings from the bilateral inferior petrosal sinuses with ovine corticotrophin-releasing factor (CRF) stimulation were performed preoperatively to evaluate some relations between the localization of a possible microadenoma and the levels of ACTH in inferior petrosal sinuses. The data for the venous samplings were as follows:-(1) The basal levels of ACTH obtained simultaneously from both inferior petrosal sinuses and peripheral vein were about the same. (2) A significant difference, with a high level of ACTH from the ipsilateral side of the microadenoma, was demonstrated after CRF stimulation. (3) The magnitude of increase in ACTH on the contralateral side after CRF was similar to that of the peripheral response. It is recommended that all patients with ACTH dependent Cushing's syndrome and negative radiological findings should have bilateral simultaneous inferior petrosal venous sampling with CRF stimulation.     

The effect of serotonin agonist 1-(trifluoromethylphenyl)-piperazine on corticotropin releasing factor and arginine vasopressin in rat hypothalamic nuclei

Effects of 1-(m-trifluoromethylphenyl)-piperazine, a serotonin agonist, were examined on rat plasma levels of adrenocorticotropin (ACTH) and arginine vasopressin (AVP), and on hypothalamic contents of corticotropin releasing factor (CRF) and AVP, to investigate the role of brain serotonin in ACTH regulation. Both plasma ACTH and AVP levels increased markedly 30 min after injection of the compound and were still elevated at 80 min. CRF and AVP contents in the median eminence decreased 30 min after injection but returned to the basal levels by 80 min. The AVP content in the supraoptic nucleus was elevated 80 min after injection. The CRF and aVP content did not significantly change in the paraventricular, suprachiasmatic and arcuate nuclei. Serotonin or 1-(m-trifluoromethylphenyl)-piperazine did not stimulate the release of ACTH in pituitary cell cultures. These results suggest that both CRF and AVP were secreted into the portal vessels by 1-(m-trifluoromethylphenyl)-piperazine to release ACTH from the anterior pituitary and that both the ACTH and AVP release were stimulated via the brain serotonergic mechanism.     

Immunocytochemical demonstration of proopiomelanocortin- and other opioid-related substances and a CRF-like peptide in the gut of the american cockroach, Periplaneta americana L

Using the peroxidase-antiperoxidase technique, we showed the presence of peptides which are immunologically resembling mammalian corticotropin releasing hormone (CRF)-, adrenocorticotropic hormone (ACTH)-, beta-endorphin (beta-END)-, alpha-melanocyte stimulating hormone (alpha-MSH)-, methionine-enkephalin (met-ENK)- and leucine enkephalin (leu-ENK)- like immunoreactivity in hundreds to thousands of endocrine cells and nerve fibers in the midgut of the American cockroach Periplaneta americana. In the cockroach hindgut no immunoreactive cell bodies could be observed, although nerve fibers were clearly noticed to be recognized by antisera to CRF, ACTH1-24, ACTH11-24 and beta-END. Nothing is exactly known as to the function(s) of the demonstrated materials, but one can speculate that these numerous immunoreactive cells, might have important paracrine and/or endocrine functions in the insect physiology.     

Corticotrophin-releasing factor mediates hypophagia after adrenalectomy, increasing meal-related satiety responses

Adrenalectomy-induced hypophagia is associated with increased satiety-related responses, which involve neuronal activation of the nucleus of the solitary tract (NTS). Besides its effects on the pituitary–adrenal axis, corticotrophin-releasing factor (CRF) has been shown to play an important role in feeding behaviour, as it possesses anorexigenic effects. We evaluated feeding-induced CRF mRNA expression in the paraventricular nucleus (PVN) and the effects of pretreatment with CRF₂ receptor antagonist (Antisauvagine-30, AS30) on food intake and activation of NTS neurons in response to feeding in adrenalectomised (ADX) rats. Compared to the sham group, ADX increased CRF mRNA levels in the PVN of fasted animals, which was further augmented by refeeding. AS30 treatment did not affect food intake in the sham and ADX + corticosterone (B) groups; however, it reversed hypophagia in the ADX group. In vehicle-pretreated animals, refeeding increased the number of Fos and Fos/TH-immunoreactive neurons in the NTS in the sham, ADX and ADX + B groups, with the highest number of neurons in the ADX animals. Similarly to its effect on food intake, pretreatment with AS30 in the ADX group also reversed the increased activation of NTS neurons induced by refeeding while having no effect in the sham and ADX + B animals. The present results show that adrenalectomy induces an increase in CRF mRNA expression in the PVN potentiated by feeding and that CRF₂ receptor antagonist abolishes the anorexigenic effect and the increased activation of NTS induced by feeding in the ADX animals. These data indicate that increased activity of PVN CRF neurons modulates brainstem satiety-related responses, contributing to hypophagia after adrenalectomy.     

Discordances in the temporal pattern of the ACTH/beta-endorphin releasing effects of synthetic CRFs and partially purified bovine CRF in a superfusion system

Temporal characteristics of ACTH and beta-endorphin secretion induced by bovine hypothalamic CRF-A (void volume) and CRF-B (Kav = 0.583) separated by Sephadex G-100 were compared to those of synthetic ovine or rat CRF, sauvagine and vasopressin. Dispersed cells or minced fragments of rat adenohypophyses perifused in a column were exposed to various secretagogues, and ACTH and/or beta-endorphin concentrations of the effluent were measured by radioimmunoassays. CRF-A or CRF-B induced an immediate brisk rise of ACTH and beta-endorphin within 1 min after initiation of CRF perifusion. The maximum rate of ACTH or beta-endorphin secretion was reached 1-2 min later. Hormone secretion persisted throughout a 10-min exposure to these secretagogues. More than 80% of the total ACTH or beta-endorphin secretion induced by 10-min perifusion with bovine CRF occurred during exposure to CRF. With 10-min perifusion with 300 ng/ml ovine or rat CRF, the onset of the major CRF-stimulated ACTH or beta-endorphin secretion was markedly delayed compared to that following bovine CRF. During perifusion with ovine or rat CRF, a modest slow increase in ACTH or beta-endorphin secretion was observed. More than 60-70% of the total ACTH or beta-endorphin secretion induced by 10-min perifusion with rat or ovine CRF occurred after CRF withdrawal. The ACTH secretory patterns for sauvagine were very similar to those for synthetic rat or ovine CRF. These results suggest some qualitative differences between partially purified bovine CRF and synthetic ovine or rat CRF.     

Corticotropin-releasing factor in humans. II. CRF stimulation in patients with diseases of the hypothalamo-pituitary-adrenal axis

A stimulation test with 100 micrograms ovine or human corticotropin-releasing factor (CRF) is a useful diagnostic tool in diseases of the hypothalamo-pituitary-adrenal axis. No serious side effects were observed during the test procedure. The results showed that the CRF test is useful in making the differential diagnosis of established Cushing's syndrome (n = 42). The CRF test was also repeated after transsphenoidal surgery in 25 patients with Cushing's disease. Successfully operated patients exhibit no, blunted or normal adrenocorticotropic hormone (ACTH) responses to CRF (n = 15), whereas patients who did not show remission remained hyperresponsive (n = 10). In patients with autonomous adrenal cortisol secretion, the ACTH response to CRF was suppressed (n = 10). After surgery the ACTH response to CRF can already be demonstrated when cortisol levels are still undetectable. Pulsatile administration of CRF in one patient after unilateral adrenalectomy and another patient under corticoid therapy revealed that ACTH responses to CRF normalize rapidly but cannot be sustained if CRF administration is withdrawn, suggesting that the cause of adrenal failure after unilateral adrenalectomy for Cushing's syndrome or long-term corticoid therapy is due to hypothalamic CRF deficiency. The decrease of the ACTH responses to CRF in glucocorticoid-treated patients correlated directly to the daily corticoid dosage. Since the ACTH hyperresponse to CRF in 6 patients with Cushing's disease was also suppressed by short-term dexamethasone treatment, the pituitary level as target site for the acute feedback inhibition is also demonstrated. The evaluation of the CRF-induced ACTH response in patients with secondary adrenal failure without detectable pathology in the sella and suprasellar region (n = 6) enables the differentiation between hypothalamic and pituitary adrenal insufficiency. In patients with hypothalamic lesions the ACTH response to CRF was normal whereas insulin hypoglycemia failed to induce an ACTH rise.     

Inhibition of spontaneous ACTH release and improved response to CRF in sheep premedicated with the enkephalin analogue DAMME

The effect of IV injections of an enkephalin analogue (DAMME) in sheep prior to the administration of CRF has been compared with chlorpromazine-morphine (CPZ-M) injections as well as a chlorpromazine-morphine-nembutal (CPZ-M-N) regime. The results showed that the administration of DAMME 60 minutes prior to CRF injections, gave more consistent and more sustained inhibition of spontaneous plasma ACTH and plasma cortisol secretion than either CPZ-M or CPZ-M-N pre-treatment. Changes in plasma ACTH and plasma cortisol levels were more readily detectable in DAMME-treated sheep as compared with saline controls when 50 micrograms injections of CRF were given into the carotid artery. This suggests that DAMME may inhibit endogenous CRF release by an action at hypothalamic level or above.     

Radioimmunocytochemistry using a tritiated goat anti-rabbit second antibody

Affinity-purified goat anti-rabbit immunoglobulin G (GAR) was conjugated with (3H)-propionyl succinimidate and used to localize substance P (SP), enkephalin (ENK), and serotonin immunoreactive sites in the spinal dorsal horn and medulla of the rat and cat. Autoradiographic localization was demonstrated on paraffin, frozen, Vibratome, and 2 micron plastic sections. The latter were obtained from radiolabeled Vibratome sections that were embedded in epoxy resin. The distribution of SP, ENK, and serotonin demonstrated by radioimmunocytochemistry was comparable to that observed on semiadjacent sections using peroxidase-antiperoxidase (PAP) immunocytochemistry. The autoradiograms, however, were generated using primary antibody concentrations up to five times more dilute than concentrations used for the PAP procedure. Indirect radioimmunocytochemistry using a (3H) anti-immunoglobulin G second antibody can be used to localize a variety of monoclonal and polyclonal antisera. It is quantifiable at the light microscopic level and can be potentially used with peroxidase histochemistry to double label immunoreactive structures at the ultrastructural level.     

Localization of corticotropin-releasing factor immunoreactivity in the brain of the teleost Sparus aurata

The distribution of perikarya and fibers containing corticotropin-releasing factor (CRF) was studied in the brain of the teleost Sparus aurata by immunocytochemistry using the peroxidase-antiperoxidase method. Antisera against rat CRF, arginine vasotocin, and human adrenocorticotropin (ACTH) were used. Most CRF-immunoreactive neurons were located in the nucleus lateralis tuberis, but they were absent from the nucleus preopticus, which only contained arginine vasotocin neurons. Few CRF perikarya were identified in the nucleus preopticus periventricularis and in the mesencephalic tegmentum. A conspicuous bundle of immunoreactive fibers ran along the diencephalic floor and pituitary stalk to end near the cells of the hypophysial pars intermedia. No CRF was seen near the adenohypophysial rostral pars distalis. Our results suggest that, in Sparus aurata, CRF is a releasing factor for melanotropic cells. Its role as a releasing factor for ACTH is discussed.