Quantitative autoradiographic localization of neuropeptide Y (NPY) binding sites in rat posterior pituitary lobe

1. We have localized and quantified neuropeptide Y (NPY) binding sites in the rat pituitary gland after incubation of tissue sections in the presence of 125I-Bolton-Hunter NPY followed by autoradiography, computerized microdensitometry, and comparison to 125I-standards. 2. In the rat, NPY binding sites are localized exclusively to the part of the posterior pituitary lobe closer to the pituitary stalk. No NPY binding sites could be found in the intermediate or the anterior pituitary lobes. 3. Our results suggest a role for NPY in the regulation of pituitary function and, in particular, that of the neural lobe.     

Corticotropin-releasing factor (CRF)-immunoreactive neurons in the mammillary body of the rat

Summary The presence and distribution of CRF-immunoreactive cells and nerve fibers were studied in the mammillary body of the rat, 12 days after placing various types of lesions within the hypothalamus. Anterior and anteriolateral cuts, placed in the midhypothalamus immediately behind the paraventricular nuclei resulted in an almost complete disappearance of CRF-immunoreactive fibers from the median eminence and simultaneous appearance of CRF-containing neurons in the mammillary body. Posterior or postero-lateral hypothalamic cuts carried out in front of the mammillary body caused the accumulation of CRF-immunoreactive material in neurons and neural processes located behind the cut-line. This type of intervention had no effect on the quantity of CRF fibers in the median eminence. A cut running through the central part of the mammillary body in the frontal plane resulted in appearance of CRF neurons only in the posterior half of the mammillary region. Placing a cut behind and over the mammillary body, CRF-immunoreactive neurons became detectable below the superior cut-line. No immunoreactive neurons were observed in the mammillary body when the frontal cut reached the base of the brain at the posterior border of the nucleus, leaving intact its anterior and superior connections. In all these cases when the mammillo-thalamic tract was transected, CRF neurons became detectable in the mammillary body.     

Corticotropin-releasing factor requires CRF binding protein to potentiate NMDA receptors via CRF receptor 2 in dopamine neurons

Stress increases addictive behaviors and is a common cause of relapse. Corticotropin-releasing factor (CRF) plays a key role in the modulation of drug taking by stress. However, the mechanism by which CRF modulates neuronal activity in circuits involved in drug addiction is poorly understood. Here we show that CRF induces a potentiation of NMDAR (N-methyl-D-aspartate receptor)-mediated synaptic transmission in dopamine neurons of the ventral tegmental area (VTA). This effect involves CRF receptor 2 (CRF-R2) and activation of the phospholipase C (PLC)-protein kinase C (PKC) pathway. We also find that this potentiation requires CRF binding protein (CRF-BP). Accordingly, CRF-like peptides, which do not bind the CRF-BP with high affinity, do not potentiate NMDARs. These results provide evidence of the first specific roles for CRF-R2 and CRF-BP in the modulation of neuronal activity and suggest that NMDARs in the VTA may be a target for both drugs of abuse and stress.     

Corticotropin-releasing factor (CRF) expression in postnatal and adult rat sacral parasympathetic nucleus (SPN)

The neural control of micturition undergoes marked changes during the early postnatal development. During the first few postnatal weeks, the spinal micturition reflex is gradually replaced by a spinobulbospinal reflex pathway that is responsible for micturition in adult animals. Upregulation of brainstem regulation of spinal micturition pathways may contribute to development of mature voiding patterns. We examined the expression of corticotropin-releasing factor (CRF), present in descending projections from Barrington's nucleus to the sacral parasympathetic nucleus (SPN), in postnatal (P0–P36) and adult Wistar rats (P60–90). CRF-immunoreactivity (IR) was present predominantly in the SPN region, although some staining was also observed in the dorsal horn and dorsal commissure in L5–S1 spinal segments. CRF-IR in spinal cord regions was age dependent (R ²=0.87–0.98). The majority of the CRF-IR in the lumbosacral spinal cord was eliminated by complete spinalization (2–3 weeks). Double-label immunohistochemistry was combined with quantitative confocal laser scanning microscopy to quantify the number and percentage of colocalization between CRF-immunoreactive varicosities and preganglionic somas or proximal neurites in the SPN in postnatal and adult rats. Results demonstrate an age-dependent upregulation of CRF-IR in the SPN region and specifically in association with preganglionic parasympathetic neurons identified with neuronal nitric oxide synthase (nNOS)-IR. CRF-immunoreactive varicosities on or within a 1 μm perimeter of nNOS-immunoreactive somas or proximal neurites also increased with postnatal age. The upregulation of CRF-IR in bulbospinal projections to the SPN may contribute to mature voiding reflexes. This work was supported in part through NIH grants DK051369, DK060481, DK065989, NS040796.     

Corticotropin-releasing factor (CRF) antagonist suppresses stress-induced locomotor activity in an amphibian.

Intracerebroventricular (icv) injections of corticotropin-releasing factor (CRF; 25 ng) given to male rough-skinned newts (Taricha granulosa) stimulated locomotor activity tested in a circular arena starting 35 min after the injection. The CRF receptor antagonist, alpha-helical CRF9-41 (ahCRF; 250 or 500 ng), injected icv concurrently with CRF blocked CRF-induced locomotor activity. In contrast, icv injection of ahCRF had no effect on spontaneous locomotor activity. Other studies examined the effect of ahCRF on the elevated locomotor activity that was observed when the animals were stressed (handled or placed in warm water). The CRF antagonist dose dependently attenuated the response to either handling or warm stress tested 2 hr after drug treatment. We also examined the effect of the alpha 2-adrenergic agonist, clonidine, on spontaneous and CRF-induced locomotor activity. Clonidine injected icv dose dependently suppressed spontaneous locomotor activity but not CRF-induced locomotor activity. These studies support the hypothesis that endogenous CRF is involved in mediating stress-induced locomotor activity and indicate that the effects of CRF on locomotor activity are independent of activation of the alpha 2-adrenergic system.     

Corticotropin-releasing factor in humans. I. CRF stimulation in normals and CRF radioimmunoassay

The biological activity of ovine (o) and human (h) corticotropin-releasing factor (CRF) in normal volunteers was investigated, using bolus injections with different CRF dosages. There was a significant increase of ACTH, beta-endorphin and cortisol after the injection of all dosages. Repetitive stimulation and continuous infusion of hCRF lead to repetitive release of identical amounts of ACTH or constant elevation of ACTH levels. oCRF and hCRF serum immunoreactivity was measured with specific radioimmunoassays after bolus injection, pulsatile administration and infusion of CRF. The half-time of serum disappearance after acute injection studies was calculated as 9 min for hCRF dand 18 min for oCRF. The 'metabolic clearance' of hCRF calculated using the infusion study was 2.72 ml/min X kg. Endogenous CRF immunoreactivity was detectable in 14 patients during insulin hypoglycemia and in 86 out of 97 pregnant females. Furthermore, CRF could be extracted from human placenta. The chromatographic pattern of extracted placenta CRF, pregnancy serum CRF and CRF standard preparation was identical. Furthermore, CRF immunoreactivity was detectable in some patients with different causes of ACTH hypersecretion.     

Immunocytochemical localization of corticotropin-releasing factor (CRF) in the rat brain

The immunocytochemical localization of neurons containing the 41 amino acid peptide corticotropin-releasing factor (CRF) in the rat brain is described. The detection of CRF-like immunoreactivity in neurons was facilitated by colchicine pretreatment of the rats and by silver intensification of the diaminobenzidine end-product. The presence of immunoreactive CRF in perikarya, neuronal processes, and terminals in all major subdivisions of the rat brain is demonstrated. Aggregates of CRF-immunoreactive perikarya are found in the paraventricular, supraoptic, medial and periventricular preoptic, and premammillary nuclei of the hypothalamus, the bed nuclei of the stria terminalis and of the anterior commissure, the medial septal nucleus, the nucleus accumbens, the central amygdaloid nucleus, the olfactory bulb, the locus ceruleus, the parabrachial nucleus, the superior and inferior colliculus, and the medial vestibular nucleus. A few scattered perikarya with CRF-like immunoreactivity are present along the paraventriculo-infundibular pathway, in the anterior hypothalamus, the cerebral cortex, the hippocampus, and the periaqueductal gray of the mesencephalon and pons. Processes with CRF-like immunoreactivity are present in all of the above areas as well as in the cerebellum. The densest accumulation of CRF-immunoreactive terminals is seen in the external zone of the median eminence, with some immunoreactive CRF also present in the internal zone. The widespread but selective distribution of neurons containing CRF-like immunoreactivity supports the neuroendocrine role of this peptide and suggests that CRF, similarly to other neuropeptides, may also function as a neuromodulator throughout the brain.     

Corticotropin-releasing factor (CRF) rapidly suppresses apoptosis by acting upstream of the activation of caspases

The physiological role of the corticotropin-releasing factor (CRF) family of peptides has recently been extended by emerging evidence of their cytoprotective effects. To determine whether CRF-mediated cytoprotection is linked to caspase-dependent apoptosis, the effect of CRF on the activation of caspases was investigated in detail in Y79 human retinoblastoma cells. The results presented here demonstrate that the cytoprotective effect of CRF against the actions of camptothecin (CT) was mediated by CRF receptor subtype 1, but not subtype 2. The observed CRF-mediated cytoprotection involved rapid and pronounced suppression of proteolytic processing and activation of procaspase-3, exerted even when CRF was added hours after the application of the cytotoxic agent. Surprisingly, activation of procaspase-3 preceded activation of the initiator procaspases 2, 8, 9 and 10 during CT-induced apoptosis of Y79 cells. The mechanism of the effect of CRF was examined using inhibitors of signalling pathways such as Wortmannin (Akt), cyclic AMP-dependent protein kinase (PKA), extracellular signal-regulated kinase (ERK), protein kinase c (PKC), p38 mitogen-activated protein kinase (p38 MAPK), phospholipase c (PLC), nuclear factor-kappaB (NF-kappaBeta) and c-jun N-terminal kinase (JNK). The involvement of PKA in the mediation of the anti-apoptotic effect of CRF has been established. Taken together, these results demonstrate for the first time that the cytoprotective effect of CRF involved suppression of pro-apoptotic pathways at a site upstream of activation of procaspase-3.     

Corticotropin-releasing factor (CRF) receptor subtypes in mediating neuronal activation of brain areas involved in responses to intracerebroventricular CRF and stress in rats

Corticotropin-releasing factor (CRF) plays an important role in stress responses through activation of its receptor subtypes, CRF1 receptor (CRF₁) and CRF2 receptor (CRF₂). The parvocellular paraventricular nucleus of the hypothalamus (PVNp), the central nucleus of the amygdala (CeA), and the oval nucleus of the bed nucleus of the stria terminalis (BNSTov), which are rich in CRF neurons with equivocal expression of CRF₁ and CRF₂, are involved in stress-related responses. In these areas, Fos expression is induced by various stimuli, although the functions of CRF receptor subtypes in stimuli-induced Fos expression are unknown. To elucidate this issue and to examine whether Fos is expressed in CRF or non-CRF neurons in these areas, the effects of antalarmin and antisauvagine-30 (AS-30), CRF₁- and CRF₂-specific antagonists, respectively, on intracerebroventricular (ICV) CRF- or 60 min-restraint-induced Fos expression were examined in rats. ICV CRF increased the number of Fos-positive CRF and non-CRF neurons in the PVNp, with the increases being inhibited by antalarmin in CRF and non-CRF neurons and by AS-30 in CRF neurons. Restraint also increased Fos-positive CRF and non-CRF neurons in the PVNp, with the increases being inhibited by antalarmin in the CRF neurons. ICV CRF also increased Fos-positive non-CRF neurons in the CeA and the BNSTov, which was inhibited by AS-30 in both areas, and inhibited by antalarmin in the BNSTov only. Restraint increased Fos-positive non-CRF neurons in the CeA and BNSTov, with the increases being almost completely inhibited by either antagonist. These results indicate that both ICV CRF and restraint activate both CRF and non-CRF neurons in the PVNp and non-CRF neurons in the CeA and BNSTov, and that the activation is mediated by CRF₁ and/or CRF₂. However, the manner of involvement for CRF₁ and CRF₂ in ICV CRF- and restraint-induced activation of neurons differs with respect to the stimuli and brain areas; being roughly equivalent in the CeA and BNSTov, but different in the PVNp. Furthermore, the non-CRF_1&2-mediated signals seem to primarily play a role in restraint-induced activation of non-CRF neurons in the PVNp since the activation was not inhibited by CRF receptor antagonists.     

Corticotropin-releasing factor (CRF) is involved in the acute anorexic effect of alpha-melanocyte-stimulating hormone: a study using CRF-deficient mice

Alpha-melanocyte-stimulating hormone (α-MSH) and its receptors are critical and indispensable for maintaining appropriate feeding behavior and energy homeostasis in both mice and humans. Corticotropin-releasing factor (CRF) is a candidate for mediating the anorexic effect of α-MSH. In the present study, we examined whether CRF and its receptors are involved in the anorexic effect of α-MSH, using CRF-deficient (CRFKO) mice and a CRF receptor antagonist. Intracerebroventricular administration of NDP-MSH, a synthetic α-MSH analogue, suppressed food intake in wild-type (WT) mice. This effect was abolished by pretreatment with a non-selective CRF receptor antagonist, astressin, suggesting that the effect of α-MSH-induced anorexia was mediated by a CRF receptor. In CRFKO mice, administration with NDP-MSH did not affect food intake at an early phase (0–4 h). In addition, CRF mRNA levels in the hypothalamus were significantly increased in NDP-MSH-treated mice. Therefore, our findings, using CRFKO, strongly support evidence that CRF is involved in the acute anorexic effect of α-MSH. On the other hand, NDP-MSH administered to CRFKO mice led to suppressed food intake at the late phase (4–12 h), similar to the effect in WT mice. Further, NDP-MSH similarly reduced food intake during the late phase in all types of mice, including WT, CRFKO, and CRFKO with corticosterone replacement. The results would suggest that α-MSH-induced suppression of food intake at late phase was independent of glucocorticoids and CRF.     

Studies on hypothalamo-pituitary corticoliberin system. II. Corticotropin-releasing factor (CRF) and neurophysin (NP) immunoreactive neurocytes in the hamster

The aim of the study was to investigate CRF- and neurophysin-immunoreactive neurocytes in hypothalamo-pituitary system of the hamster. CRF-immunoreactive nerve fibers were observed mainly in the outer layer of the median eminence and pituitary stalk and also in the neurohypophysis. On the contrary, neither intermediate lobe nor anterior pituitary contained CRF-immunoassayable substance. The pattern of distribution of neurophysin-immunoreactive fibres was different from CRF-immunoreactive fibres as far as a median eminence, pituitary stalk and neurohypophysis are concerned. Between the tannocytes of the III ventricle and nervous fibres forming the internal layer of the median eminence a CRF- and neurophysin-immunoreactive perikaryons of neurocytes were found. Results of the study suggest regulatory function of CRF-immunoreactive neurons of the hamster hypothalamo-pituitary system in controlling of ACTH secretion. Moreover, the distribution of CRF-immunoreactive substances in hamster hypothalamo-pituitary system shows some peculiarities if compared with other rodents.     

Neuropeptide Y in the intermediate lobe of the frog pituitary acts as an alpha-MSH-release inhibiting factor

The presence of neuropeptide tyrosine (NPY) in the intermediate lobe of the frog pituitary was demonstrated using indirect immunofluorescence, the immunogold technique and a specific radioimmunoassay combined with high pressure liquid chromatography (HPLC). A high density of NPY-containing fibers, was found among the parenchymal cells of the intermediate lobe. These fibers originated from the ventral infundibular nucleus, travelled via the median eminence to the pars intermedia. At the electron microscopic level, NPY-like material was found exclusively in nerve fibers where the product of the immunoreaction was associated to dense-core vesicles. High concentrations of NPY-like peptide were found in neurointermediate lobe extracts. After Sephadex G-50 gel filtration the major peak of immunoreactive material appeared to co-elute with synthetic porcine NPY. Conversely, HPLC analysis revealed that the NPY-like peptide of the frog pituitary had a retention time shorter than the porcine NPY. The localization of NPY-like material in the pars intermedia suggested a possible role of NPY in the regulation of melanotropic cell secretion. In fact, graded concentrations of synthetic NPY induced a dose-dependent inhibition of alpha-melanotropin (alpha-MSH) release in vitro. The lack of effect of a dopaminergic antagonist on NPY-induced alpha-MSH release inhibition demonstrated that the local dopaminergic system could not account for the NPY action. These results indicate that NPY located in the hypothalamo-hypophyseal system of the frog may act as a melanotropin-release inhibiting factor.     

Thymosin alpha 1-like peptides: localization and biochemical characterization in the rat brain and pituitary gland

Using a radioimmunoassay for thymosin alpha 1, endogenous thymosin-like peptides were characterized in the rat brain and pituitary gland. Thymosin alpha 1-like peptides were present in high concentrations in hypothalamus and pituitary extracts. These peptides were characterized using gel filtration techniques and the main peak of immunoreactive thymosin had a molecular weight similar to that of thymosin alpha 1 (3108 daltons). Using HPLC techniques, one main peak of immunoreactivity was present in brain extracts, whereas two peaks were present in pituitary extracts, one of which coeluted with thymosin alpha 1. The discrete regional distribution of thymosin alpha 1-like peptides was investigated and the highest densities of immunoreactive thymosin were present in the median eminence and arcuate nucleus of the hypothalamus, as well as the neurointermediate lobe of the pituitary. Due to the anatomical proximity of immunoreactive thymosin to loci containing known releasing factors and hormones, thymosin alpha 1-like peptides may function as neuroendocrine regulatory agents.     

Corticotropin-releasing factor (CRF) induced anorexia is not influenced by a melanocortin 4 receptor blockage.

CRF and melanocortin (MSH/ACTH) peptides share a number of central effects including anorexia and grooming. The effects of CRF may be secondary, due to CRF's effects on melanocortin peptide release. We investigated if the newly discovered selective melanocortin 4 receptor antagonist HS014 could influence CRF induced anorexia and grooming. The data show that ICV administration of CRF (3 mg/rat), significantly reduced food intake, feeding time and feeding episodes whereas it increased grooming time and grooming episodes. HS014 (5 mg/rat), that previously has been shown to antagonize the anorectic effect and the excessive grooming induced by alpha-MSH, did however not influence any of the behavioral effects induced by CRF when the peptides were administered together. The data indicate that the anorectic and grooming effects of CRF are independent of pathways involving the MC4 receptors. These data suggest that the anorectic and grooming effect of CRF are not due to a secondary effect caused by increase in release of melanocortins acting on the central MC receptors.     

Corticotrophin-related peptides in the intermediate lobe of the rodent pituitary gland: characterization by high performance liquid chromatography and radioimmunoassay

High performance liquid chromatography (HPLC) and radioimmunoassay have been used to characterize corticotrophin-related peptides in extracts of the intermediate lobe of the rat and mouse pituitary gland. Multiple peaks have been observed, which resemble corticotrophin-like intermediate lobe peptide (CLIP) in that they cross-react with antisera raised against the COOH-terminal region of corticotrophin (ACTH) but not against NH2-terminal directed antisera. These CLIP-like peptides were released from the incubated neurointermediate lobe and their secretion was inhibited in the presence of dopamine. Heterogeneity of peptide species was also observed with antisera raised against alpha-MSH. Multiple peaks of CLIP and alpha-MSH-like material were identified in pituitary extracts from the mouse and levels were elevated in the genetically obese (ob/ob) animal. The nature and possible functions of multiple forms of intermediate lobe peptides are discussed.