Evidence for local corticotropin releasing factor (CRF)-immunoreactive neuronal circuits in the paraventricular nucleus of the rat hypothalamus. An electron microscopic immunohistochemical analysis

The interrelationships of corticotropin-releasing factor (CRF) immunoreactive neuronal cell bodies and processes have been examined in the paraventricular nucleus (PVN) of adrenalectomized-dexamethasone treated rats. Antisera generated against ovine CRF (oCRF) were used in the peroxidase-anti-peroxidase-complex (PAP)-immunocytochemical method at both the light and electron microscopic levels. In this experimental model, a great number of CRF-immunoreactive neurons were detected in the parvocellular subdivisions of the PVN and a few scattered labelled parvocellular neurons were also observed within the magnocellular subunits. Characteristic features of immunolabeled perikarya included hypertrophied rough endoplasmic reticulum with dilated endoplasmic cisternae, well developed Golgi complexes and increased numbers of neurosecretory granules. These features are interpreted to indicate accelerated hormone synthesis as a result of adrenalectomy. Afferent fibers communicated with dendrites and somata of CRF-immunoreactive neurons via both symmetrical and asymmetrical synapses. Some neurons exhibited somatic appendages and these structures were also observed to receive synaptic terminals. Within both the PVN and its adjacent neuropil, CRF-immunoreactive axons demonstrated varicosites which contained accumulations of densecore vesicles. CRF-containing axons were observed to branch into axon collaterals. These axons or axon collaterals established axo-somatic synapses on CRF-producing neurons in the parvocellular regions of the PVN, while in the magnocellular area of the nucleus they were found in juxtaposition with unlabeled magnocellular neuronal cell bodies or in synaptic contact with their dendrites. The presence of CRF-immunoreactive material in presynaptic structures suggests that the neurohormone may participate in mechanisms of synaptic transfer. These ultrastructural data indicate that the function of the paraventricular CRF-synthesizing neurons is adrenal steroid hormone dependent. They also provide morphological evidence for the existence of a neuronal ultrashort feed-back mechanism within the PVN for the regulation of CRF production and possibly that of other peptide hormones contained within this complex.     

Evidence for corticotropin releasing factor (CRF) synthesis in the preoptic nucleus of Xenopus laevis tadpoles

Summary For the study of the hypothalamo-hypophysial system of Xenopus laevis tadpoles, hypothalamic lesions were made by means of the electrocoagulation technique. Lesioning of the ventral region of the preoptic nucleus resulted in a decrease of the number of ACTH cells in the pars distalis of the pituitary gland and in a diminution of the PAS-positive reaction of these cells. In addition, regeneration of the neurosecretory cells of the ventral region of the preoptic nucleus observed 6 weeks after lesioning was accompanied by the reappearance of normal PAS-positive ACTH cells in the pars distalis. It is suggested that the neurosecretory cells of the ventral region of the preoptic nucleus of Xenopus laevis tadpoles are related to the ACTH synthesizing cells, probably by producing CRF.Dedicated to Prof. Dr. med. W. Bargmann on the occasion of his 70th birthdayThe authors thank Prof. Dr. J.C. van de Kamer for his interest, Prof. Dr. P.G.W.J. van Oordt for his many helpful comments, and Messrs. H. van Kooten, E. van der Vlist, J.J. van der Vlis and M.C.A. van Pinxteren for preparing the illustrations     

Synaptic interaction of serotonergic axons and corticotropin releasing factor (CRF) synthesizing neurons in the hypothalamic paraventricular nucleus of the rat

Summary The morphological interrelationship between the central serotonergic and hypothalamic corticotropin-releasing factor (CRF) synthesizing systems was studied in the hypothalamic paraventricular nucleus (PVN) of colchicine pretreated male rats. The simultaneous immunocytochemical localization of the transmitter and peptide employed the peroxidase-antiperoxidase complex (PAP) technique using the silver-gold intensified (SGI) and non-intensified forms of the oxidized 3,3-diaminobenzidine (DAB) chromogen.The paraventricular nucleus received a moderate serotonergic innervation as compared with other diencephalic structures. The distribution and arborization of serotonergic axons were more prominent in the parvocellular subnuclei than in the magnocellular units of the nucleus. Serotonin containing axons formed terminal bouton and en passant type synapses with dendrites and somata of parvocellular neurons. The immunocytochemical double labelling technique revealed the overlapping of serotonergic axons and CRF-immunoreactive neurons. Vibratome (40 m) and semithin (1 m) sections indicated that the interneuronal communication may take place on both dendrites and cell bodies of CRF-immunoreactive neurons. Ultrastructural analysis demonstrated that serotonin-containing terminals formed axo-dendritic and axo-somatic synapses with CRF-immunoreactive neurons. These findings indicate that the central serotonergic neuronal system can influence the function of the pituitary-adrenal endocrine axis via a direct action upon the hypophysiotrophic CRF synthesizing neurons.Supported by NIH Grant NS19266     

Neuropeptide-Y and ACTH-immunoreactive innervation of corticotropin releasing factor (CRF)-synthesizing neurons in the hypothalamus of the rat

Summary Corticotropin releasing factor (CRF), synthesized in neurons of the hypothalamic paraventricular nucleus (PVN), is one of the main regulators of the pituitaryadrenal cortex endocrine axis. In order to elucidate the possible involvement of the central neuropeptide-Y (NPY)-and adrenocorticotroph hormone (ACTH)-immunoreactive (IR) systems in the innervation of hypophysiotrophic CRF-synthesizing neurons, immunocytochemical double labelling studies were conducted in the hypothalamus of the rat to localize CRF-synthesizing neurons, as well as neuronal fibers exhibiting NPY and ACTH-immunoreactivity, respectively.The parvocellular subnuclei of the PVN received an intense NPY-and ACTH-IR innervation. At the light microscopic level, these peptidergic axons were associated with the dendrites and perikarya of CRF-IR neurons. Ultrastructural analysis revealed that NPY- and ACTH-IR axons established synaptic specializations with parvocellular neurons expressing CRF-immunoreactivity. These findings indicate that both neuropeptide-Y and adrenocorticotroph hormone containing neuronal systems of the brain are capable of influencing adrenal function via synaptic interactions with hypophysiotrophic CRF-synthesizing neurons. The data also support the concept that NPY and ACTH might be ntilized as neuromodulators within the PVN.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Association of dopaminergic fibers with corticotropin releasing hormone (CRH)-synthesizing neurons in the paraventricular nucleus of the rat hypothalamus

Summary Catecholamines are known to exert a central influence on the hypothalamo-hypophyseal-adrenal neuroendocrine system. The selective dopaminergic innervation of the hypothalamic paraventricular nucleus (PVN) and putative relationships between dopaminergic fibers and corticotropin releasing hormone (CRH)-synthesizing neurons were studied in the male rat by means of immunocytochemistry following the elimination of noradrenergic and adrenergic inputs to the hypothalamus. A 3.0-mm-wide coronal cut was placed unilaterally in the brain at the rostral level of the mesencephalon. All neuronal structures from the cortex to the ventral surface of the brainstem, including the ascending catecholaminergic fiber bundles were transected. This surgical intervention resulted in the accumulation of dopamine--hydroxylase (DBH)-immunoreactivity in axons proximal to the cut, and an almost complete disappearance of DBH activity in those located distal to the lesion. Two weeks following the operation, DBH immunoreactivity was significantly diminished in the PVN located on the side of lesion, while tyrosine hydroxylase (TH)-immunoreactivity was present in a substantial number of fibers in the same nucleus. Both DBH- and TH-immunoreactive axons were preserved in the contralateral PVN. Simultaneous immunocytochemical localization of either DBH- or TH-IR fibers and corticotropin releasing hormone-synthesizing neurons in the hypothalami from brainstem-lesioned, colchicine treated animals revealed that the distribution of catecholaminergic fibers and CRH neurons is homologous within the PVN of the intact side. Only a few scattered DBH-immunoreactive axons were detected among CRH-producing neurons in the PVN on the side of the lesion. In contrast, many tyrosine hydroxylase containing neurons and neuronal processes were observed on the lesioned side and the TH-IR fibers established juxtapositions with CRH-synthesizing neurons.These morphological data demonstrate that following the surgical ablation of noradrenergic and adrenergic afferents to the PVN, a substantial number of tyrosine hydroxylase-IR fibers remained in the nucleus and they were associated with corticotropin releasing hormone synthesizing neurons. Therefore, it is hypothesized that the paraventricular nucleus receives a selective dopaminergic innervation and these dopaminergic axons might influence the function of the pituitary and adrenal glands via the hypothalamic CRH system.Supported by grants from the National Science Foundation (NSF INT 8703030), the Hungarian Academy of Sciences (OTKA 104), the National Institutes of Health (NS 19266) and the National Foundation of Technical Development (OKKFT Tt 286/1986)     

Association of dopaminergic fibers with corticotropin releasing hormone (CRH)-synthesizing neurons in the paraventricular nucleus of the rat hypothalamus

Catecholamines are known to exert a central influence on the hypothalamo-hypophyseal-adrenal neuroendocrine system. The selective dopaminergic innervation of the hypothalamic paraventricular nucleus (PVN) and putative relationships between dopaminergic fibers and corticotropin releasing hormone (CRH)-synthesizing neurons were studied in the male rat by means of immunocytochemistry following the elimination of noradrenergic and adrenergic inputs to the hypothalamus. A 3.0-mm-wide coronal cut was placed unilaterally in the brain at the rostral level of the mesencephalon. All neuronal structures from the cortex to the ventral surface of the brainstem, including the ascending catecholaminergic fiber bundles were transected. This surgical intervention resulted in the accumulation of dopamine-beta-hydroxylase (DBH)-immunoreactivity in axons proximal to the cut, and an almost complete disappearance of DBH activity in those located distal to the lesion. Two weeks following the operation, DBH immunoreactivity was significantly diminished in the PVN located on the side of lesion, while tyrosine hydroxylase (TH)-immunoreactivity was present in a substantial number of fibers in the same nucleus. Both DBH- and TH-immunoreactive axons were preserved in the contralateral PVN. Simultaneous immunocytochemical localization of either DBH- or TH-IR fibers and corticotropin releasing hormone-synthesizing neurons in the hypothalami from brainstem-lesioned, colchicine treated animals revealed that the distribution of catecholaminergic fibers and CRH neurons is homologous within the PVN of the intact side. Only a few scattered DBH-immunoreactive axons were detected among CRH-producing neurons in the PVN on the side of the lesion. In contrast, many tyrosine hydroxylase containing neurons and neuronal processes were observed on the lesioned side and the TH-IR fibers established juxtapositions with CRH-synthesizing neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural localization of glucocorticoid receptor (GR) in hypothalamic paraventricular neurons synthesizing corticotropin releasing factor (CRF)

Summary Corticotropin releasing factor (CRF) synthesizing neurons, located in the hypothalamic paraventricular nucleus (PVN), are the main central regulators of the pituitary-adrenal cortex endocrine axis. The hormone production and release of CRF-synthesizing neurons is regulated by neuronal messages and feedback action(s) of glucocorticoids secreted by the adrenal gland. In order to characterize the latter mechanism, glucocorticoid receptor (GR)-immunoreactive (IR) sites were studied in hypothalamic paraventricular neurons of intact, long-term adrenalectomized, and adrenalectomized plus glucocorticoid treated animals, by means of ultrastructural immunocytochemical labelling. In intact animals, glucocorticoid receptor immunoreactivity was found predominantly in the nuclei of parvocellular neurons. Following adrenalectomy GR-immunoreactivity was localized in the cytoplasm of the cells, and there was a concomitant disappearance of the label from the nuclei. After corticosterone administration to adrenalectomized animals, GR-IR sites were again concentrated within the cell nuclei. Immunocytochemical double labelling studies performed on adrenalectomized plus corticosterone-replaced animals demonstrated glucocorticoid receptor-IR sites in the cell nuclei of parvocellular paraventricular neurons that expressed CRF-immunoreactivity in their cytoplasm.These ultrastructural data indicate that the intracellular location of glucocorticoid receptor is dependent on the availability of glucocorticoids by the neurons. The simultaneous expression of GR- and CRF-immunoreactivity in parvocellular paraventricular neurons supports the concept of a direct feedback action of glucocorticoids upon CRF-synthesizing neurons.Supported by NIH Research Grants NS19266 (W.K.P. and Zs.L.), NS20832 (M.C.B.) and a joint grant (INT-8703030) awarded by the National Science Foundation and the Hungarian Academy of Sciences (Zs.L. and W.K.P.). R.M.U. is a recipient of NIMH Pre-doctoral Fellowship and M.C.B. an NIH Research Carcer Development Award     

Adrenergic innervation of corticotropin releasing factor (CRF)-synthesizing neurons in the hypothalamic paraventricular nucleus of the rat. A combined light and electron microscopic immunocytochemical study

Corticotropin releasing factor (CRF), a neuropeptide synthesized in the parvocellular subnuclei of the hypothalamic paraventricular nucleus (PVN), takes part in the regulation of different stress evoked responses of the organism. In order to elucidate the role of the central adrenergic system in the regulation of these CRF-synthesizing neurons, a novel ultrastructural immunocytochemical dual localization technique was utilized. Phenylethanolamine-N-methyltransferase (PNMT), a specific enzyme marker for the central adrenaline system, and CRF-immunoreactive elements were simultaneously visualized in hypothalamic sections. PNMT-immunoreactive axon terminals established synaptic connections with somata, dendrites and spinous structures of CRF-producing neurons. This morphological finding indicates that the central adrenergic system directly influences CRF-synthesizing neurons in the PVN and provides basis for a more definitive pharmacological manipulation of this system.     

Immunological detection of pro-corticotropin releasing factor (CRF) in rat hypothalamus and pancreatic extracts. Evidence for in vitro conversion into CRF

Extracts of both rat hypothalamus and pancreas were analyzed for their corticotropin releasing factor (CRF)-like immunoreactivity by radioimmunoassay (RIA). In the case of the hypothalamus, besides the rat CRF, further identified by high-pressure liquid chromatography (HPLC), two peptide components, a 20-kDa and a 10-kDa species were detected. The 20-kDa component was stable under acidic pH conditions and was further purified by reverse-phase HPLC. When exposed to proteolytic activities coeluting with 'high-molecular-mass CRF' at pH 6, processing was observed and the CRF generated was identified both by RIA, molecular sieve filtration and HPLC under different experimental conditions. It is concluded that this 20-kDa CRF may represent the CRF precursor and that hypothalamic extracts may contain processing enzymes involved in its selective post-translational cleavage. In the pancreatic extract two immunoreactive forms of CRF were detected, the smaller coeluting with the rat CRF and the other corresponding to the intermediate 10-kDa component detected in the hypothalamus. Pancreatic rat CRF, analyzed using RIA both by molecular sieve filtration and HPLC, was indistinguishable from the hypothalamic rat CRF.     

Synaptic interaction of serotonergic axons and corticotropin releasing factor (CRF) synthesizing neurons in the hypothalamic paraventricular nucleus of the rat. A light and electron microscopic immunocytochemical study

The morphological interrelationship between the central serotonergic and hypothalamic corticotropin-releasing factor (CRF) synthesizing systems was studied in the hypothalamic paraventricular nucleus (PVN) of colchicine pretreated male rats. The simultaneous immunocytochemical localization of the transmitter and peptide employed the peroxidase-antiperoxidase complex (PAP) technique using the silver-gold intensified (SGI) and non-intensified forms of the oxidized 3,3'-diaminobenzidine (DAB) chromogen. The paraventricular nucleus received a moderate serotonergic innervation as compared with other diencephalic structures. The distribution and arborization of serotonergic axons were more prominent in the parvocellular subnuclei than in the magnocellular units of the nucleus. Serotonin containing axons formed terminal bouton and en passant type synapses with dendrites and somata of parvocellular neurons. The immunocytochemical double labelling technique revealed the overlapping of serotonergic axons and CRF-immunoreactive neurons. Vibratome (40 micron) and semithin (1 micron) sections indicated that the interneuronal communication may take place on both dendrites and cell bodies of CRF-immunoreactive neurons. Ultrastructural analysis demonstrated that serotonin-containing terminals formed axo-dendritic and axo-somatic synapses with CRF-immunoreactive neurons. These findings indicate that the central serotonergic neuronal system can influence the function of the pituitary-adrenal endocrine axis via a direct action upon the hypophysiotrophic CRF synthesizing neurons.     

Ultrastructural characteristics of immunolabelled,corticotropin releasing factor (CRF)-synthesizing neurons in the rat brain

Summary The corticotropin releasing factor (CRF)-synthesizing perikarya and neural processes were detected at ultrastructural level in the hypothalamic paraventricular nucleus and in the median eminence of control and colchicine-pretreated rats. The unlabelled antibody peroxidase-antiperoxidase complex (PAP) immunohistochemical method was used in a pre-embedding manner, on thick, non-frozen sections. In CRF-perikarya, neurosecretory granules (80–120 nm in diameter), free ribosomes, and the rough endoplasmic reticulum were labelled. Unlabelled axon terminals formed asymmetric synapses on CRF-containing perikarya and dendrites. Immunolabelled axons terminated in the palisadic zone of the median eminence.     

Focal cerebral ischaemia induces corticotropin releasing factor (CRF) vascular immunoreactivity in rat occluded hemisphere

Corticotropin-releasing factor (CRF) induces the dilatation of cerebral blood vessels and increases cerebral blood flow (CBF). CRF receptor antagonists reduce ischaemic damage in the rat. In the present study, the expression of CRF around cerebral vessels has been investigated in the rat. No CRF immunoreactivity was identified around pial or intracerebral vessels in the absence of cerebral ischaemia. Four hours after middle cerebral artery occlusion (MCAo), intensely CRF-positive blood vessels were evident on the ischaemic cortical surface and in the peri-infarct and infarct zone. Increased CRF immunoreactivity was also detected in swollen axons in subcortical white matter, caudate nucleus and lateral olfactory tract of the ipsilateral hemisphere, consistent with the failure of axonal transport. These data provide morphologic support for a role of CRF in the pathophysiology of cerebral ischaemia.     

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.     

Gastrointestinal secretory, motor and circulatory effects of corticotropin releasing factor (CRF)

This study was designed to determine the effects of CRF on the gastrointestinal functions such as secretion, motility and circulation in dogs. CRF was found to inhibit dose-dependently gastric acid response to pentagastrin but not to histamine. CRF stimulated pancreatic bicarbonate and protein secretion under basal conditions and in response to secretin or cholecystokinin (CCK). This stimulation was accompanied by an increase in plasma levels of pancreatic polypeptide (PP), but not of secretin or gastrin. CRF caused a partial inhibition of the migrating motor complexes in fasted dogs and increased spike activity of the small bowel. These motor effects of CRF probably resulted from the action of the released PP on the intestinal smooth muscle. CRF is also a potent and selective stimulant of the mesenteric blood flow. This effect may be secondary to the stimulation of intestinal motility and metabolism.     

Cognitive disruptions in stress-related psychiatric disorders: A role for corticotropin releasing factor (CRF)

This article is part of a Special Issue “SBN 2014”.Stress is a potential etiology contributor to both post-traumatic stress disorders (PTSD) and major depression. One stress-related neuropeptide that is hypersecreted in these disorders is corticotropin releasing factor (CRF). Dysregulation of CRF has long been linked to the emotion and mood symptoms that characterize PTSD and depression. However, the idea that CRF also mediates the cognitive disruptions observed in patients with these disorders has received less attention. Here we review literature indicating that CRF can alter cognitive functions. Detailed are anatomical studies revealing that CRF is poised to modulate regions required for learning and memory. We also describe preclinical behavioral studies that demonstrate CRF’s ability to alter fear conditioning, impair memory consolidation, and alter a number of executive functions, including attention and cognitive flexibility. The implications of these findings for the etiology and treatment of the cognitive impairments observed in stress-related psychiatric disorders are described.     

Structure-activity studies on 4-substituted-2-anilinopyrimidine corticotropin releasing factor (CRF) antagonists

Structure-activity studies around the 4-position of 2-anilinopyrimidine corticotropin releasing factor (CRF) antagonists suggest that there is a large lipophilic cavity in the rat CRF receptor, which can accommodate a wide variety of substituents at this position in contrast to the steric constraints observed for other positions on the 2-anilinopyrimidine core. The chemical syntheses and biological activities of 2-anilinopyrimidine CRF antagonists with carbon-linked substituents at the 4-position are reported. Significant improvements in rat pharmacokinetic parameters were achieved relative to those for the lead structure. While the lead compound 1 (rCRF Ki = 44 nM) afforded no detectable rat plasma levels after intraperitoneal (i.p.) or oral (p.o.) dosing, compounds 3-3 (rCRF Ki = 16 nM) and 3-4 (rCRF Ki 59 nM) gave high rat plasma levels at 30 mg/kg (i.p., p.o.) (Cmax = 1389 nM and 8581 nM (i.p.) respectively; Cmax = 113 nM and 988 nM (p.o.), respectively). Furthermore 3-3 and 3-4 had superior bioavailabilities at these doses (59 and 46% (i.p.), respectively; 2 and 10%, (p.o.), respectively).     

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.     

Corticotropin releasing factor (CRF) activity in rat plasma.

The ACTH-releasing activity of hypothalamic extract and rat plasma was examined with the dispersed rat pituitary cell technique of Swallow and Sayer (8). Although both plasma and serum caused ACTH release which was dose-related, stress did not enhance the ACTH releasing activity. Furthermore, separation studies of plasma using ultrafiltration and gel separation suggest that the CRF activity in plasma is associated with molecules of a molecular weight greater than 15,000.     

促肾上腺皮质激素释放激素及其受体在痛觉调节中作用的研究进展

最近的大量研究表明,促肾上腺皮质激素释放激素(CRF)和CRF类似肽广泛分布于脑、脊髓和内脏器官。CRF和CRF类似肽均可与CRF受体结合。CRF受体(CRFR)可分为CRFR1、CRFR2和CRFR3三种类型。CRFR2又分为A、B、C三个亚型。各种CRF受体激活时,对细胞产生相应的功能调节。CRF参与机体多层次的痛觉调节,CRF、CRF类似肽及与CRF有关的生物活性物质都有可能成为新一代的镇痛药。