Corticotropin-releasing Factor Binding Sites Research Articles

Functional characterization of corticotropin-releasing factor type 1 receptor endogenously expressed in human embryonic kidney 293 cells

The endogenous expression in human embryonic kidney 293 (HEK293) cells of corticotropin-releasing factor (CRF) receptors was detected. High-affinity binding sites for human CRF ( K i=3.6 nM), ovine CRF ( K i=4.6 nM), rat urocortin ( K i=2.2 nM), sauvagine ( K i=2.4 nM) and astressin ( K i=4.3 nM) with the pharmacological characteristics for CRF type 1 (CRF 1) receptors and B max values of ∼30 fmol/mg protein were determined. The four CRF receptor agonists nonselectively stimulated cAMP production in HEK293 cells at low agonist concentrations, whereas the antagonist astressin shifted the dose–response curve for ovine CRF significantly rightward. Transfection of the pcDNA3 vector into HEK293 cells strongly reduced the expression of the endogenous CRF receptor. Northern blot analysis revealed the expression of a CRF 1 transcript in human neuronal tissues, HEK293, human NTera-2 (NT2) carcinoma, Y-79 retinoblastoma and African green monkey kidney (COS-7) cells. Neither by Northern blot analysis nor by reverse transcriptase PCR (RT-PCR), the expression of CRF 2 could be detected. In cAMP stimulation experiments, functional CRF receptors were detected in these cell lines. These data show that HEK293 and other cell lines endogenously express CRF 1 receptors.

Anxiety-like behavior in mice lacking the angiotensin II type-2 receptor

The main biological role of angiotensin II type 2 receptor (AT 2) has not been established. We made use of targeted disruption of the mouse AT 2 gene to examine the role of the AT 2 receptor in the central nervous system (CNS). AT 2-deficient mice displayed anxiety-like behavior compared with wild-type mice. However, AT 2-deficient mice showed no depressant-like activity and no change in hexobarbital-induced sleeping time as compared with findings in wild-type mice. Both noradrenergic and corticotropin-releasing factor (CRF) neuronal systems appear to be involved in this anxiety-like behavior. Diazepam, captopril (angiotensin I converting enzyme inhibitor), prazosin (α 1 antagonist) reversed the anxiety-like behavior in these AT 2-deficient mice, whereas yohimbine (α 2 antagonist), phenylephrine (α 1 agonist), clonidine (α 2 agonist), isoproterenol (β 1/β 2 agonist), propranolol (β 1/β 2 antagonist) and α-helical CRF 9-41 (CRF receptor antagonist) has no apparent effects on anxiety-like behavior in AT 2-deficient mice. In addition, concentrations of plasma adrenocorticotropic hormone (ACTH) and corticosterone in AT 2-deficient mice did not differ from these in wild-type mice, hence, there are probably no endocrine abnormalities involving the hypothalamic-pituitary-adrenal axis (HPA). The amygdala appears to play an important role in many of the responses to fear and anxiety. The number of [ 3 H ]prazosin but not [ 125 I ]CRF binding sites in the amygdala was significantly reduced in AT 2-deficient mice. These findings indicate that the noradrenergic system is involved in mediating the anxiety-like behavior in AT 2-deficient mice.

Central and peripheral serotonergic correlates in violent suicidal attempters

The endogenous expression in human embryonic kidney 293 (HEK293) cells of corticotropin-releasing factor (CRF) receptors was detected. High-affinity binding sites for human CRF (Ki=3.6 nM), ovine CRF (Ki=4.6 nM), rat urocortin (Ki=2.2 nM), sauvagine (Ki=2.4 nM) and astressin (Ki=4.3 nM) with the pharmacological characteristics for CRF type 1 (CRF1) receptors and Bmax values of ∼30 fmol/mg protein were determined. The four CRF receptor agonists nonselectively stimulated cAMP production in HEK293 cells at low agonist concentrations, whereas the antagonist astressin shifted the dose–response curve for ovine CRF significantly rightward. Transfection of the pcDNA3 vector into HEK293 cells strongly reduced the expression of the endogenous CRF receptor. Northern blot analysis revealed the expression of a CRF1 transcript in human neuronal tissues, HEK293, human NTera-2 (NT2) carcinoma, Y-79 retinoblastoma and African green monkey kidney (COS-7) cells. Neither by Northern blot analysis nor by reverse transcriptase PCR (RT-PCR), the expression of CRF2 could be detected. In cAMP stimulation experiments, functional CRF receptors were detected in these cell lines. These data show that HEK293 and other cell lines endogenously express CRF1 receptors.

Corticotropin releasing factor modulates interleukin-1-induced prostaglandin synthesis in fibroblasts: receptor binding and effects of antagonists

Corticotropin releasing factor (CRF) is a predominant regulator of the neuroendocrine, autonomic and behavioral responses to stress. In addition, numerous studies support autocrine/paracrine roles for this peptide at peripheral sites. CRF and CRF binding sites have been identified in different regions of the central nervous system as well as in the heart, spleen, adrenal and testis, and high levels of CRF were detected in inflamed fibroblasts. However, the precise physiological or pathophysiological role of peripheral CRF cannot yet be discerned. Here we show that CRF, through interaction with specific membrane receptors, blocks the interleukin-1α (IL-1α)-stimulated prostaglandin (PG) synthesis in fibroblasts. Binding of [ 125I]-labeled CRF in fibroblasts was saturable and fitted a two sites model. K D for the higher-affinity class of receptors was 20±2.2 pM, and B max 1.95±0.22 fmol/mg protein. For the lower-affinity class of receptors K D was 160±17 nM, and B max 2.38±0.27 fmol/mg protein. CRF blocked the effect of IL-1α on PGE 2 synthesis, and this was antagonised by d-PheCRF 12–41. In addition, the CRF receptor antagonists α helical CRF 9–41 and d-PheCRF 12–41 at high concentrations inhibited the IL-1α-induced PG synthesis similarly to CRF, suggesting partial agonistic action. Taken together, these results suggest a modulatory role of CRF in inflammation.

Structure and function of the ovine type 1 corticotropin releasing factor receptor (CRF 1) and a carboxyl-terminal variant

Corticotropin releasing factor (CRF) is the major neuropeptide regulating the hypothalamo–pituitary–adrenocortical axis in most species. A pituitary receptor for CRF (designated CRF 1) belonging to the seven-transmembrane helix, G-protein-coupled receptor superfamily has been cloned for human, rat, mouse and xenopus. Since ovine CRF shares only 84% identity to human/rat CRF (h/rCRF) we postulated that the sheep pituitary CRF 1 receptor may have similarly diverged from the rodent and human CRF 1. We report the molecular cloning of an ovine pituitary cDNA containing a 1245 bp open reading frame encoding a 415 amino acid sheep CRF 1 receptor 78, 86, 94, and 95% homologous to xenopus, chicken, rat, mouse, and human CRF 1, respectively. The divergence in primary structure between the sheep CRF 1 and the other mammalian CRF 1s is primarily localized to the extracellular amino terminal domain of the receptor (18 of 22 divergent residues, ovine vs human CRF 1). A variant of the oCRF 1 was also isolated (oCRF 1var) with 133 bp deleted from nucleotide (nt) 1080 to nt 1213 of the open reading frame (ORF) resulting in a new ORF of 1176 nt predicting a 392 residue CRF 1 variant receptor. The 133 bp deletion would cause a frame-shift at residue 358 within the carboxyl-third of the seventh transmembrane domain (TM7) resulting in a shortened cytoplasmic tail with a new amino acid sequence from residue 358 to 392. Scatchard analysis of saturation curves using membrane prepared from Cos 7 cells transfected with oCRF 1 or oCRF 1var indicated that both wild-type and variant receptors were expressed similarly (number of CRF binding sites) and both bound oCRF with high affinity [oCRF 1 ( K d): 2.5+1.6 nM; oCRF 1var: 5.1+2.3 nM]. The non-hydrolyzable GTP analogue (GTP γS) lowered the affinity of both wild-type and variant oCRF 1 receptors to a similar extent (oCRF 1: 18.2 nM; oCRF 1var: 22.4 nM). Both wild-type and variant oCRF 1 receptors exhibited ≈10-fold greater selectivity for oCRF and sauvagine compared to h/rCRF or α-helical [9–41]oCRF. CRF effectively stimulated the accumulation of cAMP (EC 50=51 pM) in Cos 7 cells transiently transfected with wild-type but not variant oCRF 1 receptor. In Cos 7 cells transfected with oCRF 1var, cAMP accumulation was only observed at the highest concentration of oCRF utilized (100 nM). Basal (unstimulated) levels of cAMP in Cos 7 cells transfected with oCRF 1var (in the presence of 2 mM IBMX) were ≈50% lower than for the wild-type oCRF 1. Differences in cAMP accumulation could not be attributed to differences in receptor number since total binding sites in the transfected cells were not different between wild-type or variant oCRF 1 receptors. Agonist-induced receptor internalization, determined as the percent of total [ 125I] Tyr 0-oCRF bound located in the acid-resistant fraction of transfected Cos 7 cells, increased with time (0–60 min at 37°C) for both wild-type and variant oCRF 1. Wild-type CRF 1 internalized ≈2-fold greater percent of total [ 125I] Tyr 0-oCRF bound compared to the variant receptor. In summary, an ovine CRF 1 and a CRF 1 cytoplasmic tail receptor variant displaying high affinity binding to oCRF as well as selectivity for oCRF vs h/rCRF, were cloned from an adult sheep pituitary cDNA library. GTP γS studies indicate that both variant and wild-type receptors couple efficiently to G αs however, only the wild-type oCRF 1 is capable of stimulating cAMP production at physiological levels of CRF. Agonist-induced internalization of the ovine CRF 1var is also reduced compared to the wild-type CRF 1 receptor. We suggest that the oCRF 1var interacts efficiently with G αs but is unable (post-hormonal binding) to effectively stimulate G-protein activation of adenylate cyclase, indicating that the cytoplasmic tail of the CRF 1 can modulate receptor function related to signal transduction. The cytoplasmic carboxyl terminus of the CRF 1 also appears to play a role in agonist-induced internalization of this member of the class II subfamily of G-protein coupled receptors (GPCRs).

Regional binding to corticotropin releasing factor receptors in brain of rats exposed to chronic cocaine and cocaine withdrawal.

Cocaine, as does exposure to other physiological stressors, releases brain corticotropin releasing factor (CRF), and this release habituates during the course of repeated cocaine administration in animals. Due to the many signs of anxiety and responses to stress that are produced by cocaine withdrawal in humans, the present study was designed to assess the effects of chronic cocaine and its withdrawal on regional 125I-Tyr-oCRF binding to the CRF1 receptor in brains of male Lewis rats. Cocaine or saline was intravenously infused for 10 days in a regimen that resembled a self-administration paradigm (1 mg/kg every 12 min for 2 h each day). Tissues were harvested either 15 min after or 10 days after the last cocaine infusion, and the brains were sectioned and prepared for CRF1 receptor autoradiography. Compared with findings in saline controls, there was a 31% lower level of CRF binding sites in the basolateral nucleus of the amygdala immediately after the last cocaine infusion, but not 10 days later. Neuroendocrine and non-neuroendocrine mechanisms associated with CRF1 receptors do not appear to contribute to long-term withdrawal effects.

Chapter 4 The distribution of corticotropin-releasing factor (CRF), CRF binding sites and CRF1 receptor mRNA in the mouse cerebellum

The purpose of the present study is to determine the distribution of CRF containing afferents, and correlate these findings with the distribution of CRF binding sites and the neuronal localization of mRNA for the CRF1 receptor in the cerebellum of a single species, the mouse. Corticotropin releasing factor (CRF) has been localized within climbing fibers and mossy fibers throughout the cerebellar cortex of the mouse using immunohistochemistry. CRF immunoreactive, axonal varicosities also are present within all four of the cerebellar nuclei. 125I-labeled CRF binding sites are evident throughout all three layers of the cerebellar cortex (molecular, Purkinje and granule cell layers), but are not seen within the cerebellar nuclei. In situ hybridization histochemistry was employed using an antisense riboprobe corresponding to the full length sequence of the rat mRNA for the CRF1 receptor. Positive signal is present throughout the cerebellum in Purkinje cells and the granule cell layer. CRF1 receptor mRNA also is expressed within all four of the cerebellar nuclei. Further experiments are required to reconcile the lack of CRF binding sites in the cerebellar nuclei with the positive mRNA receptor expression and the presence of immunoreactive axonal varicosities. In previous physiological experiments, iontophoretic application of CRF enhances spontaneous as well as quisqualate-induced activity of Purkinje cells in slice preparations of the mouse cerebellum. When the results of the anatomical techniques are compared to the physiological data, there is convergent evidence to suggest that CRF influences the firing rate or responsiveness of Purkinje cells directly via release of the peptide from the climbing fiber system and indirectly via the mossy fiber-granule cell-parallel fiber circuit. Taken together, these anatomical and physiological data provide strong evidence to suggest that, in the adult cerebellum, CRF functions as a neuromodulator.

The temporal and spatial development of corticotropin-releasing factor (CRF) binding sites and CRF afferents in the opossum cerebellum

The intent of this study is to determine the developmental timecourse of the appearance and distribution of corticotropin releasing factor (CRF) binding sites within the developing opossum cerebellum, and to correlate this with the temporal and spatial distribution of CRF-labeled axons. 125I-labeled ovine CRF was used to identify the distribution and temporal expression of CRF binding sites in the opossum cerebellum. By PD8, binding sites are evident over cells in the external granular layer, as well as the subjacent immature Purkinje cell layer, but not over the ventricular layer or the intermediate zone. At PD 8, the intermediate zone, located between the ventricular and immature Purkinje cell layers, contains migrating nuclear, Golgi and Purkinje cells. By PD12, binding sites are present over all layers of the immature cerebellum (the external granular layer, the multitiered Purkinje cell layer, and the intermediate zone of migrating cells), except the ventricular layer. The adult distribution of CRF binding sites is evident by PD30-38 which includes the molecular, Purkinje and internal granule cell layers. The present results provide the first account of the ontogeny of CRF binding sites in the developing cerebellum. The early expression and distribution of CRF receptors, when correlated with the temporal expression and distribution of the peptide, provide additional evidence to support our working hypothesis that CRF functions as a regulator of developmental events which is distinct from its proposed function as a neuromodulator in the mature cerebellum.

Diurnal variation of corticotropin-releasing factor binding sites in the rat brain and pituitary

1. Corticotropin-releasing factor (CRF) is thought to be involved in the regulation of the diurnal activity of the hypothalamus-pituitary-adrenal (HPA) axis and to act as a neurotransmitter in the brain. To date it is unknown whether the binding sites of the central CRF system are subject to diurnal variations. 2. We measured the number of CRF binding sites over the course of a complete 24-hr light-dark cycle in the pituitary, amygdala, bed nucleus of the stria terminalis (BNST), cingulate cortex, visceral cortex, paraventricular nucleus of the hypothalamus, hippocampus, and locus ceruleus of rats by in vitro receptor autoradiography with iodinated ovine CRF. A 24-hr time course was also established for plasma CRF and corticosterone. 3. The diurnal pattern of plasma CRF does not correlate with the pattern of plasma corticosterone. Within the brain, CRF binding in the basolateral nucleus of the amygdala showed a U-shaped curve with maximum levels in the morning and a wide hallow between 1500 and 0100. A biphasic profile with a small depression in the afternoon and a more pronounced depression in the second half of the activity period is characteristic for the other brain areas and the pituitary. The profile for the pituitary correlates with those for the BNST and the area of the locus ceruleus. Furthermore, the diurnal pattern of CRF binding sites in the BNST correlates with that of the hippocampus, and the daytime pattern of the visceral cortex is similar to that of both the hippocampus and the BNST. 4. Since the CRF-binding profiles in the brain and the pituitary clearly differ from the profiles of both plasma CRF and corticosterone, one may assume that the diurnal pattern of central CRF binding sites is not directly coupled to the activity of the HPA axis.

Distribution of corticotropin-releasing factor (CRF) binding sites in the opossum cerebellum

Corticotropin-releasing factor (CRF) has been demonstrated in the hypothalamic-pituitary-adrenal axis and studied in extrahypothalamic sites throughout the brain. Localization of CRF immunoreactivity, CRF mRNA, and CRF receptors within specific brain areas supports an extrahypothalamic function for CRF. Previous reports have revealed the localization of several peptides, including CRF, in the cerebellar cortex and nuclei of the North American opossum ( Didelphis marsupialis virginiana); climbing fibers, mossy fibers, and a beaded plexus of axons in the Purkinje cell layer demonstrate CRF immunoreactivity. CRF also is localized within neurons in the inferior olivary complex and other brainstem nuclei which are known to project to the cerebellar cortex. Physiological recordings indicate CRF potentiates the excitatory effects of both aspartate and glutamate, the putative transmitters of the major afferent inputs to the cerebellum. The present study reports that specific CRF binding sites are present in all lobules of the opossum cerebellar cortex, with the greatest density in vermal lobules V through X, the flocculus, and the paraflocculus. The cerebellar nuclei do not appear to be labeled. CRF binding sites are present over all neuronal layers of the cerebellar cortex. The presence of CRF immunoreactivity in climbing fibers, mossy fibers, and a beaded axonal plexus, and CRF binding sites within the cerebellar cortex, as well as the fact that CRF potentiates the excitatory effects of both aspartate and glutamate, indicate that this peptide may function as a neuromodulator in the cerebellum of the North American opossum. An attempt is made to correlate the distribution of CRF-IR neurons and fibers, CRF mRNA, and CRF receptors to the extrahypothalamic function of CRF as it relates to the olivocerebellar pathway.

Early development of cerebellar afferent systems that contain corticotropin-releasing factor.

Corticotropin-releasing factor (CRF) and CRF binding sites have been described in the cerebellum of several species, including the North American opossum (Didelphis marsupialis virginiana), the species used in the present study. Inotophoretic application of this peptide in the adult cerebellum enhances the spontaneous and amino acid-induced firing rate of Purkinje cells and overcomes the GABA-induced suppression of Purkinje cell activity. The present account provides immunohistochemical evidence for the localization of CRF in the North American opossum within developing axons and their growth cones prior to the formation of the Purkinje cell and granule cell layers. CRF mRNA is present on postnatal day (PD) 1 within the internal migratory stream of the ventral lateral medulla, which contains migrating olivary neurons, and within the ventral medulla in the region, where inferior olivary neurons first aggregate to form the inferior olivary complex. The olivary complex can first be identified on PD2 and is well defined by PD3. CRF-immunoreactive axons are evident within the cerebellar primordium on PD4 and penetrate the nascent Purkinje cell layer between PD14 and PD26. By PD26, CRF-immunoreactive puncta are organized within the Purkinje cell layer as parasagittal bands. Thus, olivary neurons express CRF mRNA prior to the time that the first CRF-labeled axons are present in the cerebellar anlage (PD4), suggesting that olivary axons are among the first to reach the developing cerebellum. Coincident (PD1-3) with the early transcription of CRF mRNA in the inferior olive, cells in the medullary reticular formation (PD1) and locus coeruleus (PD2) also transcribe CRF mRNA. These brainstem sites also could provide CRF-immunoreactive axons to the developing cerebellum; however, based on the results of this study and correlative data reported in the literature, we propose that the primary source of early-arriving CRF fibers is the inferior olivary complex. The early arrival of CRF-containing axons in the cerebellum prior to synaptogenesis and migration of both granule cells and Purkinje cells suggests a role for this peptide in target recognition and synaptic organization.

Corticotropin releasing factor receptors in inflamed tissue: Autoradiographic identification

We have shown that injecting corticotropin releasing factor (CRF) into inflamed rat paws causes the local release of endogenous opioids to inhibit pain. The purpose of the current study was to visualize CRF receptors by means of in vitro receptor autoradiography. We used 125I-CRF as a ligand on sections of popliteal lymph nodes, subcutaneous tissue of hind paws and spleen of rats. The density of 125I-CRF lebelled receptors was higher in inflamed lymph nodes than in noninflamed lymph nodes ( P=0.0431 , Wilcoxon test). Similarly, the density of CRF receptors was significantly higher in inflamed than in noninflamed paws ( P=0.0431 , Wilcoxon test). This increase in CRF receptors indicates an upregulation of CRF binding sites in inflamed tissue. These findings are consistent with our in vivo studies which have shown potent antinoceciptive effects of locally applied CRF in inflamed but not in noninflamed tissue.

Corticotropin‐Releasing Factor Binding Sites Undergo Axonal Transport in the Rat Vagus Nerve

Corticotropin-releasing factor (CRF) binding sites were found to be present in the rat vagus nerve and underwent axonal transport. Binding sites accumulated on both sides of ligatures placed on the nerve and at similar rates following ligation of right or left cervical vagal trunks of either male or female rats. CRF binding sites also accumulated proximal and distal to ligatures on subdiaphragmatic vagal trunks. Binding was specific, reversible and inhibited by the CRF receptor antagonist α-helical-CRF(9-41). [(125) l]Tyr(0) -ovine-CRF binding to rat vagus nerve was not guanine nucleotide-sensitive. CRF and cholecystokinin binding sites were transported at a similar rate in the cervical vagus, although turnover of CRF binding sites was more rapid. No differences in CRF binding site transport were observed between Zucker rats of lean or obese genotype.

Corticotropin-releasing factor receptors in the rabbit brain visualized by in vitro autoradiography

Corticotropin-releasing factor (CRF) binding sites were visualized in the rabbit brain by in vitro autoradiography using the radioligand 125I-[Tyr 0]ovine CRF. The radioligand binding to sections of rabbit cingulate cortex were competed for by ovine and rat CRF with inhibitory constants (K i) of 26 and 37 nM, respectively, whereas sauvagine and alpha-helical CRF 9–41 were approximately 10-fold less potent. In the rabbit brain, the highest densities of binding sites for CRF are found in the pineal gland and the choroid plexus. The cerebral cortex is labelled throughout, with the highest concentration of binding sites in the piriform and primary olfactory divisions. In the cerebellar cortex, the granular layer is more intensely labelled than the molecular layer. The distribution of CRF binding sites in the hippocampus follows a laminar pattern; the molecular layer of the dentate gyrus is intensely labelled, the oriens, radiatum and lacunosum moleculare layers of Ammon's horn contain moderate densities of binding and no binding is observed in the granular layer of the dentate gyrus and the pyramidal cell layer. The ventral subnucleus of the lateral septum, the zonal and superficial layers of the superior colliculus contain high densities of receptors. A moderate concentration of binding sites is observed in the caudate nucleus, putamen, bed nucleus of the stria terminalis, paraventricular, anterodorsal and anteroventral thalamic nuclei and the medial nucleus of the mammillary body. The fundus striati, the dorsal subnucleus of the lateral septum, the lateral dorsal thalamic, lateral and medial geniculate nuclei, the lateral and anterior cortical amygdaloid nuclei, central and posterior interpeduncular nuclei all contain low but significant levels of CRF binding sites. No detectable binding is observed in the pons, medulla oblongata or fibre tracts. The high densities of CRF binding sites found in the cortex, hippocampus and structures associated with the limbic system supports a role for CRF in the modulation of behavioural and physiological responses associated with stress. However, the extremely high density of CRF binding sites found in the pineal gland and choroid plexus suggest a more extensive role for CRF in the rabbit central nervous system.

Functional corticotropin-releasing factor (CRF) receptors in mouse spleen: Evidence from adenylate cyclase studies

Radioligand binding studies have previously identified a high affinity, magnesium-dependent, guanine nucleotide-sensitive binding site for corticotropin-releasing factor (CRF) in mouse spleen. In order to determine the functional nature of these CRF binding sites, we examined the effects of CRF on adenylate cyclase activity in mouse spleen homogenates. The stimulation of adenylate cyclase activity was dependent on time, tissue protein concentration, and guanine nucleotides. CRF-stimulated adenylate cyclase activity was evident in the presence of guanosine-5′-triphosphate (GTP) and its precursor guanosine-5′-diphosphate (GDP) but was not detected in the presence of the hydrolysis-resistant GTP analogs, guanyl-5′-imidodiphosphate [Gpp(NH)p] and guanosine-5′-gamma-thiotriphosphate (GTP-γ-S). The rank order of potency for CRF analogs and fragments in stimulating adenylate cyclase activity was comparable to their affinities for CRF binding sites in mouse spleen homogenates. The putative receptor antagonist, alpha helical ovine CRF(9–41), did not stimulate adenylate cyclase activity but did attenuate the stimulation by various concentrations of rat/human CRF. In summary, these data demonstrate the functional nature of CRF receptors in mouse spleen as evidenced by CRF stimulation of cAMP production and suggest that this peptide may play a physiological role in regulating immune function.

Distribution of corticotropin-releasing factor in the cerebellum and precerebellar nuclei of the opossum: a study utilizing immunohistochemistry, in situ hybridization histochemistry, and receptor autoradiography.

This study reports 1) a nonhomogeneous distribution of three morphologically distinct, corticotropin-releasing factor (CRF)-immunoreactive axonal phenotypes within the cerebellum of the opossum: climbing fibers, mossy fibers, and beaded fibers within the ganglionic plexus; 2) the existence of CRF binding sites within the cerebellar cortex; and 3) the distribution of CRF-containing neurons in brainstem precerebellar nuclei identified with immunohistochemistry and in situ hybridization histochemistry. CRF-immunoreactive climbing and/or mossy fibers were identified within all cerebellar lobules. The density of CRF-immunoreactive fibers was greatest in the vermis, where longitudinal bands of intensely immunoreactive climbing and mossy fibers were interspersed with regions containing fibers demonstrating lower levels of immunolabeling. CRF-immunoreactive fibers were present within all deep cerebellar nuclei. The topography of CRF-containing cerebellar fibers is discussed with respect to possible sites of origin within the brainstem. CRF-immunoreactive neurons were identified in all nuclei of the inferior olivary complex, although the number and intensity of immunostaining of CRF-containing cells varied within and among individual nuclei. CRF-immunoreactive somata were also present in brainstem nuclei known to give rise to cerebellar mossy fibers. In situ hybridization histochemistry utilizing an 35S-labeled synthetic 48-base oligodeoxynucleotide complementary to amino acids 22-37 of rat CRF proper revealed that CRF mRNA is transcribed in precerebellar nuclei. Variation in the level of CRF mRNA was detected among inferior olivary nuclei, in correspondence with variations detected in the levels of immunostaining. Data from this study suggest that variation in the level of CRF immunoreactivity detected within cerebellar afferent fibers may correlate with the level of CRF mRNA within cell bodies of origin of the projections. In vitro receptor autoradiography, utilizing 125I-Tyro-ovine CRF, revealed correspondence between CRF binding sites and CRF-immunoreactive fibers in the cerebellar cortex. Results of this study support suggestions for CRF-mediated circuitry in the cerebellum.

The ontogeny of brain receptors for corticotropin-releasing factor and the development of their functional association with adenylate cyclase

This study reports the ontogeny of corticotropin-releasing factor (CRF) receptor binding sites in rat brain, using both membrane binding assays and in vitro receptor autoradiography. CRF binding sites are evident by prenatal day 17, increase to 312% of their adult density by postnatal day 8, then decrease to reach adult values by day 21. Not only the density, but the distribution of CRF binding undergoes major modifications in development. CRF binding sites are most numerous in striatum prenatally, but postnatally, binding is more dense in the cortex, reaching the adult laminar distribution by postnatal day 14. Brain CRF receptors are linked to adenylate cyclase early in postnatal life. This contrasts with the later appearance of most of the guanine nucleotide stimulatory protein and catalytic subunit sites in the rat brain and suggests that CRF receptors may become functional earlier than several other brain receptors that are linked to adenylate cyclase.

Intrahypothalamic microinfusion of corticotropin-releasing factor elevates blood glucose and free fatty acids in rats

Three experiments examined whether intrahypothalamic microinfusions of corticotropin releasing factor (CRF) can affect circulating levels of the metabolic fuels, glucose and free fatty acids. Infusions of CRF into the paraventricular nucleus dose-dependently increased serum glucose levels; greater increases were seen in acute than in chronic preparations. The greater effectiveness could not be accounted for by anesthetization per se. CRF infusion into the ventromedial nucleus did not affect serum glucose. Infusions into both sites, however, significantly increased serum free fatty acids. Neither glucose nor free fatty acids were altered by infusions into the lateral hypothalamus or the caudate-putamen. These data suggest that the previously identified CRF binding sites and CRF neuronal terminals in the paraventricular and ventromedial nuclei may be involved in the central regulation of metabolic fuel release. Additionally, it appears that the importance of CRF in the paraventricular nucleus in regulating serum glucose may be greater under some conditions than others.

Reduced corticotropin releasing factor binding sites in the frontal cortex of suicide victims.

Previous studies have provided evidence that corticotropin releasing factor (CRF) is hypersecreted in patients with major depression. This CRF hypersecretion is believed to contribute at least in part to hyperactivity of the hypothalamic-pituitary-adrenal axis in depressed patients. If CRF is chronically hypersecreted in depressed patients, then, due to down-regulation, a reduced number of CRF receptor binding sites should be present in patients with profound depressive disorder. To test this hypothesis, we measured the number and affinity of CRF binding sites in the frontal cortex of 26 suicide victims and 29 controls who died of a variety of causes. There was a marked (23%) reduction in the number of CRF binding sites in the frontal cortex of the suicide victims compared with the controls. These data are consistent with the hypothesis that CRF is hypersecreted in depression.

Effect of passive immunization against corticotropin-releasing factor (CRF) on the postadrenalectomy changes of CRF binding sites in the rat anterior pituitary gland.

The concentration of corticotropin-releasing factor (CRF)-binding sites decreases in the rat anterior pituitary after adrenalectomy; this change may be related either to a direct effect of the circulating glucocorticoids at the pituitary level or to a desensitization of CRF receptors through an increased CRG release in hypophysial portal blood. In order to examine the latter possibility we have measured plasma adrenocorticotropin hormone (ACTH) levels and the number of anterior pituitary CRF binding sites in sham-operated and 24-hour adrenalectomized rats after blockade of endogenous CRF by passive immunization with an antiserum anti-rat CRF (CRF-AS), or after injection of normal rabbit serum (NRS). In NRS-injected rats, after sham operation, plasma ACTH concentration increased (227 +/- 34 vs. 118 +/- 19 pg/ml in controls) without change in CRF-binding sites capacity (20.7 +/- 2.6 vs. 24.6 +/- 3.5 fmol/mg protein in controls). Adrenalectomy induced a large rise in plasma ACTH (785 +/- 89 pg/ml) and a decrease in the number of CRF-binding sites (12.2 +/- 1.7 fmol/mg protein). After CRF-AS injection, plasma ACTH was normalized in sham-operated animals (149 +/- 24 pg/ml) and significantly reduced in adrenalectomized rats (472 +/- 76 pg/ml); the adrenalectomy-induced decrease in the number of CRF-binding sites was unaffected by the CRF-AS administration (12.2 +/- 1.7 fmol/mg protein). The administration of dexamethasone to adrenalectomized rats significantly reduced plasma ACTH concentrations (23.3 +/- 10.6 pg/ml) and prevented the loss in CRF-binding sites capacity (20.7 +/- 1.3 fmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)