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     

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.     

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.     

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.     

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.     

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)     

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)     

A combined light and electron microscopic immunocytochemical method for the simultaneous localization of multiple tissue antigens. Tyrosine hydroxylase immunoreactive innervation of corticotropin releasing factor synthesizing neurons in the paraventricular nucleus of the rat

In order to study the morphological interrelationships between immunocytochemically identified neuronal systems, a double labelling procedure - suitable for correlative light and electron microscopic observations - is introduced. The technique is based on the consecutive use of the silver-gold (SG) intensified and non-intensified forms of the oxidized 3,3'-diaminobenzidine (DAB) chromogen in the framework of the peroxidase-antiperoxidase complex (PAP) indirect immunocytochemical procedure. The first tissue antigen is detected by the SG intensified DAB chromogen, which has a black color and high electron density. The structures containing the second antigen are visualized by the non-intensified DAB-endproduct, which is less electron-dense than the silver-gold amplified form and is brown. The metallic shield that forms around the labeled antibody sequences associated with the first antigen prevents non-specific binding of immunoglobulins used for the detection of the second tissue antigen. The application of this method for the simultaneous detection of tyrosine hydroxylase (TH)- and corticotropin releasing factor (CRF)-immunoreactive structures revealed that black colored TH-immunopositive fibers contacted brown colored CRF-synthesizing neurons in the hypothalamic paraventricular nucleus. The juxtaposition of TH- and CRF-containing elements was apparent in both thick vibratome (40 micron) and semithin (1 micron) sections. At the ultrastructural level, TH-positive terminals - labeled by silver-gold grains - were observed to establish asymmetric synapses with both CRF- and TH-immunoreactive neurons. The former finding indicates a direct, TH-immunopositive, catecholaminergic influence upon the hypothalamic CRF system, while the latter demonstrates the existence of intrinsic connections between TH-positive elements.     

The corticotropin releasing factor (CRF) neurosecretory system in intact, adrenalectomized, and adrenalectomized-dexamethasone treated rats. An immunocytochemical analysis

In general, antisera generated against ovine CFR do not reveal immunopositive neuronal perikarya in the rat. If animals are adrenalectomized significant amounts of immunoreactive CFR are present in the hypothalamus. By using this model, we have visualized the CFR system of the rat. Intact, intact pretreated with dexamethasone, adrenalectomized, and adrenalectomized pretreated with dexamethasone animals were used in the present study. In adrenalectomized and adrenalectomized plus dexamethasone treated animals the CFR-immunopositive neurons were observed in the parvocellular portion of the paraventricular nucleus. Distinct pathways of CRF fibers could be seen emerging from this hypothalamic nucleus. The greatest number of these fibers exited the PVN laterally and crossed either superior to or beneath the fibers of the fornix. The fibers then turned ventrally and cascaded to form a bundle of fibers above the superio-lateral margin of the optic chiasm. They turned caudally and followed the optic tract. As these fibers reached the level of the anterior median eminence, they turned medially to run along the inferior margin of the hypothalamus and enter the median eminence. A few fibers emerged from the PVN along the periventricular margin of the third ventricle, traveled caudally in the periventricular nucleus and entered the median eminence. Adrenalectomized and adrenalectomized-dexamethasone treated rats had very dense accumulations of immunoreactive CRF in the median eminence when compared with controls. Immunoreactive neurons and fibers were also observed in the central nucleus of the amygdala in the adrenalectomized and adrenalectomized-dexamethasone treated animals.     

Evidence for local corticotropin releasing factor (CRF)-immunoreactive neuronal circuits in the paraventricular nucleus of the rat hypothalamus

Summary The interrelationships of corticotropin-releasing factor (CRF) immunoreactive neuronal cell bodies and processes have been examined in the paraventricular nucleus (PVN) of adrenalectomized-dexamethesone 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 feedback mechanism within the PVN for the regulation of CRF production and possibly that of other peptide hormones contained within this complex.Supported by NIH grant NS 19266 to WKP     

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     

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     

Demonstration of distinct corticotropin releasing factor--containing neuron populations in the bed nucleus of the stria terminalis. A light and electron microscopic immunocytochemical study in the rat

Immunocytochemical light and electron microscopic studies revealed two distinct populations of corticotropin releasing factor (CRF) - containing neurons, a dorsolateral and ventrolateral group, located in the bed nucleus of the stria terminalis (BST) of the rat brain. CRF neurons of the dorsolateral group had a smaller diameter and more primary dendrites than those of the ventrolateral group. CRF neurons in the dorsolateral BST had both somatic and dendritic spines, smooth contoured nuclei, and many dense and alveolate vesicles in their cytoplasm. Whereas, CRF neurons in the ventrolateral BST had only dendritic spines, irregularly-shaped indented nuclei and contained only alveolate vesicles in their cytoplasm. The only obvious difference in the type of unidentified afferents that synapsed on the CRF neurons of the BST could be attributed to the presence of the somatic spines on the CRF neurons of the dorsolateral population. Otherwise, the CRF neurons of the BST had a profuse innervation that included axosomatic, axospinous and axodendritic synapses. CRF-containing axons were distributed unevenly throughout the BST. The density of CRF axons was greatest in the lateral subdivisions of the BST, but the ventromedial BST contained many more CRF axons than the dorsomedial BST. The presence of these two CRF neuron populations in the BST suggests functional subdivision beyond previous proposals of a medial and lateral separation of function. Now there is additional morphological evidence to support the proposal of a dorsal and ventral separation of function within the BST.     

Light and electron microscopic immunocytochemical analysis of the dopamine innervation of the rat visual cortex

Brain Cell Biology - The dopaminergic innervation of the rat primary (area 17) and secondary (areas 18 and 18a) visual cortical areas was examined immunocytochemically using an antiserum directed...     

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.     

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.