Corticotropin-releasing Factor Immunostaining Research Articles

Immunolocalization of corticotropin-releasing factor (CRF) and corticotropin-releasing factor receptor 2 (CRF-R2) in the developing gut of the sea bass (Dicentrarchus labrax L.)

Our previous data indicated an important role for adrenocorticotropic (ACTH)-like molecules co-operating with macrophages to control the modifications in body homeostasis during the first period of the life of sea bass (up to 30 days post-hatching) before the lymphoid cells have reached complete maturation. The aim of the study was to determine the immunolocalization of corticotropin-releasing factor (CRF), which is a very important mediator of stress-related responses. Our data showed that immunostaining for CRF is localized already at 8 days after hatching in nerve fibers of the gastrointestinal tract wall from the pharynx to the anterior gut, when the larvae are still feeding on yolk. This pattern of immunolocalization appeared similar to that in 24-day-old larvae, but at this stage there were also large cells immunopositive to CRF located in the wall of the midgut and hindgut. Lipopolysaccharide (LPS) treatment, which is a known stimulator of stress hormone responses, did not modify the CRF immunostaining pattern, though it did affect the immunolocalization of the peripheral CRF receptor, i.e. CRF-R2. Immunolocalization of CRF-R2 appeared in nerve fibers of the gut wall in larvae fixed 1h after the end of lipopolysaccharide (LPS) treatment. The present results suggest that CRF plays important autocrine and/or paracrine roles in the early immune responses at the gut level in the larval stages of sea bass (Dicentrarchus labrax L.) as already proposed for ACTH. Moreover, our studies taken together with other research on fish, in comparison with mammals, suggest a phylogenetically old role of CRF in immune–endocrine interactions.

Spatial distribution of corticotropin-releasing factor immunopositive climbing fibers in the mouse cerebellum: Analysis by whole mount immunohistochemistry

This study examined the spatial organization of corticotropin-releasing factor (CRF) immunopositive climbing fibers in the mouse cerebellum by whole mount immunohistochemistry. A striking pattern of parasagittal stripes of CRF staining was revealed. Cryosections of whole mount CRF stained cerebellum showed that anti-CRF immunostaining is restricted to climbing fibers in the molecular layer and does not penetrate deeper into the granular layer. The array of CRF stripes was reminiscent of zebrin II immunopositive Purkinje cell stripes in the anterior vermis and the hemispherical lobules. However, a direct comparison of the two distributions showed that the CRF-defined parasagittal stripes and transverse zones in the posterior vermis are different from those defined by the expression of zebrin II: in particular, CRF immunostaining revealed a transverse boundary between lobules VIb and VII and the presence of four CRF-immunopositive climbing fiber stripes in lobule VIII. Furthermore, an array of CRF stripes was seen in lobule X, the flocculus and the paraflocculus, despite uniform zebrin II expression in these areas. In these cases some, but not all, CRF-immunopositive stripes shared boundaries with Purkinje cell stripes that were visualized by the expression of heat shock protein 25. The results reveal a reproducible pattern of CRF-immunopositive climbing fiber innervation in the mouse cerebellum that bears a complex relationship to the stripes delineated by Purkinje cell compartmentation antigens.

Chronic stress enhances vasopressin but not corticotropin-releasing factor secretion during hypoglycemia.

Quantitative immunocytochemistry was used to investigate the effect of chronic intermittent stress on the storage and release of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) in the zona externa of the median eminence (ZEME). Wistar rats were subjected to repeated once daily insulin stress and studied 24 h after the last episode of hypoglycemia. Chronic intermittent hypoglycemia differentially affected CRF immuno-staining (CRFi) and AVPi stores in the ZEME, characterized by a transitory fall in CRFi (7 days) and a gradual increase in AVPi (11 days). The acute hypothalamic response to stress was studied by measuring insulin-induced depletion of CRFi and AVPi in the ZEME after blockade of fast axonal transport with colchicine (5 micrograms/rat ic). Three hours after insulin, CRFi was reduced by 24 and 28% in naive and repeatedly (11 times) stressed animals, respectively. In contrast, insulin did not affect CRFi in rats exposed to hypoglycemia for 7 days. In addition, hypoglycemia reduced AVPi in the ZEME by 37, 42, and 54% in naive and repeatedly (7 and 11 times) stressed rats, respectively. We conclude that chronic intermittent hypoglycemia induces a shift in hypothalamic signals for adrenocorticotropic hormone release in favor of AVP. This may result in altered sensitivity of the corticotrophs for hypophysiotropic factors and corticosteroids that are of physiological importance for the pituitary response to subsequent stressors.

Corticotropin-releasing hormone-containing neurons in the hypothalamo-hypophyseal system in rats six weeks after bilateral lesions of the paraventricular nucleus

Corticotropin-releasing factor-like immunoreactive nerve fibers and varicosities are present in the pituitary stalk and median eminence 6 weeks after bilateral lesioning of the hypothalamic paraventricular nucleus. The total immunoreactivity may reach 10% of the control density. The origin of these fibers was investigated 3 days after hypothalamic transections in paraventricular-lesioned (6 weeks postoperatively) rats. Accumulations of corticotropin-releasing factor immunostaining were observed in the proximal portions of the transected axons and in neuronal perikarya. Fibers with retrograde labeling were seen lateral and dorsolateral from the sagittally oriented knife cuts which transected the retrochiasmatic area and cells were found in the supraoptic nucleus and in the perifornical nucleus (dorsal-dorsolateral to the fornix), ipsilateral to the lesion. No corticotropin-releasing factor immunostained cells were seen in other hypothalamic or preoptic nuclei which project to the median eminence or the posterior pituitary. Corticotropin-releasing factor containing cells in the supraoptic and perifornical nuclei may have an importance in stress response in rats with long-term paraventricular lesions.

Effects of Catecholamine-Depleting Medullary Knife Cuts on Corticotropin-Releasing Factor and Vasopressin Immunoreactivity in the Hypothalamus of Normal and Steroid-Manipulated Rats

Single and dual immunohistochemical staining techniques were used to assay the effects of disruption of brain stem catecholaminergic inputs on corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) immunoreactivity in parvocellular neurosecretory neurons of the paraventricular nucleus of the hypothalamus (PVH) in normal and in adrenalectomized (ADX) rats treated with dexamethasone or vehicle. The results may be summarized as follows: (1) In adrenally intact rats, confirmed unilateral transection of ascending catecholaminergic pathways near their origins in the medulla produced a decrement in the number of CRF-immunoreactive cells that could be detected on the side of the brain ipsilateral to the cut. No effect on AVP immunoreactivity in the parvocellular division of the PVH was evident. Staining for both peptides in terminals in the external lamina of the median eminence tended to show modest decreases on the lesioned side of the brain. Compatible results were obtained in comparing the effects of bilateral transections with controls. (2) Unilaterally lesioned ADX rats treated with vehicle showed the expected enhancement in CRF immunostaining in the parvocellular division of the PVH, though the response on the side ipsilateral to the lesion was blunted relative to that seen contralaterally; the effect of ADX on AVP immunoreactivity on the ipsilateral side was more markedly reduced, but still showed evidence of enhancement (i.e., could be colocalized in some CRF immunoreactive perikarya). (3) Unilaterally lesioned ADX rats treated with dexamethasone showed no evidence of enhanced CRF or AVP immunoreactivity in perikarya or terminals on either side of the brain. The generally lower levels of staining for both peptides seen on the lesioned side across conditions suggest that the effect of interruption of ascending catecholaminergic pathways on peptide dynamics in the PVH is dissimilar to that of ADX, is manifested via different mechanisms, and that at least some types of feedback inhibition of corticotropin-releasing peptides by adrenal steroids do not require intact catecholaminergic inputs to be exhibited.

Corticotropin-Releasing Factor-Producing Neurons in the Rat Activated by Interleukin-1

Intraperitoneal administration of human recombinant interleukin-1 (IL-1) to rats can increase blood levels of corticosterone and adrenocorticotropic hormone (ACTH). The route by which IL-1 affects pituitary-adrenal activity is unknown. That the IL-1-induced pituitary-adrenal activation involves an increased secretion of corticotropin-releasing factor (CRF) is indicated by three lines of evidence. First, immunoneutralization of CRF markedly attenuated the IL-1-induced increase of ACTH blood levels. Second, after blockade of fast axonal transport in hypothalamic neurons by colchicine, IL-1 administration decreased the CRF immunostaining in the median eminence, indicating an enhanced release of CRF in response to IL-1. Third, IL-1 did not stimulate ACTH release from primary cultures of anterior pituitary cells. These data further support the notion of the existence of an immunoregulatory feedback circuit between the immune system and the brain.

Adrenalectomy-induced enhancement of CRF and vasopressin immunoreactivity in parvocellular neurosecretory neurons: anatomic, peptide, and steroid specificity

Following adrenalectomy (ADX), corticotropin-releasing factor (CRF) and vasopressin immunoreactivity are jointly expressed by a population of parvocellular neurosecretory neurons in the paraventricular nucleus of the hypothalamus (PVH). Because these cells stain positively for CRF, but not for vasopressin, after pretreatment with colchicine, the results suggest the existence of state-dependent alterations in the expression of peptides by neuroendocrine neurons. The present study sought to determine whether other neuropeptides (e.g., neurotensin, met-enkephalin) that have been colocalized with CRF in the parvocellular division of the PVH are influenced similarly by ADX; whether the enhancement of CRF and/or vasopressin immunoreactivity after ADX is limited to neurons of the PVH; and what factors might be involved in the regulation of the expression of these peptides in the PVH. The results confirmed that CRF and vasopressin immunoreactivity are both enhanced, and may be colocalized in a substantial population of parvocellular neurosecretory neurons after ADX; no comparable enhancement of staining for met-enkephalin or neurotensin was observed. The effect of ADX on CRF immunoreactivity was not limited to cells in the PVH, as neurons in the cerebral cortex, amygdala, and the bed nucleus of the stria terminalis also showed heightened CRF immunostaining after ADX; vasopressin immunoreactivity was never colocalized with CRF in these extrahypothalamic sites. Hypophysectomy produced an enhancement of CRF and vasopressin staining in the PVH that was comparable to that seen after ADX, implicating adrenal steroids as primary regulators of peptide expression in this system. Corticosteroid replacement studies in ADX rats indicated that lower doses of dexamethasone attenuated, and higher doses essentially abolished, the expected enhancement of both CRF and vasopressin immunoreactivity after ADX. The relative potency of steroids in mitigating these effects was dexamethasone greater than corticosterone greater than deoxycorticosterone greater than aldosterone. Collectively, these results indicate that the ADX-induced enhancement of CRF and vasopressin immunoreactivity in parvocellular neurosecretory neurons is at least somewhat specific to these peptides and to this cell type. Both peptides would appear to be regulated similarly by adrenal steroids, with glucocorticoids playing a primary role.

Corticotropin-releasing factor immunostaining in the rat spinal cord and medulla oblongata: An unexpected form of cross-reactivity with substance P

By use of two antisera (α-CRF A, α-CRF B) raised against conjugates of o-CRF bovine thyroglobulin, cryostat sections of formaldehyde-fixed gelatin models containing o-CRF can be stained. The staining intensity was quantitated by use of an automated microfluorimeter and was shown to be dependent on the concentration of o-CRF (1–300 μM) added to the gel. Determination of the CRF staining intensity after incorporation of o-CRF-related peptides and fragments indicated that both antisera reacted with the C-terminal region of o-CRF. They showed poor cross reactivity with r-CRF fixed in the gel. In the same models, r-CRF could be immunostained efficiently by use of an antiserum (α-CRF C) raised to a conjugate of r-CRF and thyroglobulin. This antiserum reacted with the N-terminal and midportion parts but not with the C-terminal fragment of o-CRF fixed in the gels. By use of both o-CRF antisera nerve fibers can be stained in the rat hypothalamus (median eminence) and in the medulla oblongata (spinal trigeminal tract and nucleus) and spinal cord (dorsal horn). Immunoinhibition experiments showed that o-CRF caused a concentration-dependent quenching (0.001–1 μM) of the immunostaining of o-CRF-containing models, rat median eminence and medulla oblongata preparations. α-CRF C also stained CRF immunoreactive (CRFi) fibers in the rat hypothalamus with an equal distribution to that found with the o-CRF antisera. However, no immunostaining was found in the spinal trigeminal nucleus and tract and in the dorsal horn, indicating that these fibers store different CRF-related products from those found in the hypothalamus. The CRFi in the medulla oblongata and spinal cord induced by α-CRF A was completely abolished 1 week after treatment of adult rats with capsaicin, a substance known to deplete Substance P (SP) from those areas. Gels incorporated with SP showed a concentration-dependent increase (range 10–1000 μM) in immunostaining with both o-CRF sera but not with the r-CRF antiserum. In addition, incubation of o-CRF sera with SP caused a concentration-dependent quenching (range 10–100 μM) of immunostaining in SP-containing models. SP at a concentration of 100 μM was also effective in quenching the CRFi in the dorsal horn and spinal trigeminal area. Quenching was also obtained with the C-terminal part of o-CRF (range 0.002–0.1 μM), which indicates that both CRF antisera contain an immunoglobulin which recognizes determinants on CRF as well as on SP. We conclude that CRFi in primary sensory nerves in the medulla oblongata and spinal cord may be due to cross reaction of CRF antisera with SP, located in high concentrations in such areas. In addition, the data show that the non-biological gelatin model is a good tool to investigate unexpected cross reactions in immunocytochemistry.

Colocalization of peptide histidine isoleucine amine and corticotropin-releasing factor immunoreactivity in neurons of the rat hypothalamus: a surprising artefact.

Indirect immunocytochemistry of corticotropin-releasing factor (CRF) and peptide histidine isoleucine amide (PHI) was performed by the use of antibodies raised to CRF and PHI. The staining intensity was quantitated by using an automated microfluorimeter. CRF and PHI immunoreactive fibres showed a similar pattern of distribution in the zona externa of the median eminence of the rat hypothalamus. Administration of colchicine (50 micrograms i.c.v.) resulted in the appearance of PHI and CRF immunoreactive cell bodies in the parvocellular part of the paraventricular nucleus. The PHI immunoreactive cell bodies were of low intensity and less abundant than those stained with the CRF antisera. Microfluorimetric measurements of the immunostaining in the median eminence showed parallel changes of PHI and CRF immunostaining after adrenalectomy, administration of reserpine and/or pargyline. In order to evaluate whether these data demonstrate that PHI and CRF are colocalized in hypothalamic neurons, we studied the specificity of PHI immunostaining by the use of a nonbiological gelatin model. Although CRF and PHI do not show structural homologies, the PHI antisera caused staining of PHI containing gels (range: 0.001-1 microM) but also of rat CRF (rCRF)-containing gels (range: 10-300 microM). In addition, preincubation of one of the PHI antisera with PHI or rCRF both caused a concentration-dependent quenching of the immunostaining in PHI- and CRF-containing gels and in preparations of the median eminence. Again, higher concentrations of rCRF (100 microM) than PHI (0.1 microM) were needed to show immunoinhibition, suggesting that the PHI antiserum has much lower avidity for native and fixed rCRF than for native and fixed PHI.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of urotensin I- and corticotropin-releasing factor-like immunoreactivity in the central nervous system of Catostomus commersoni

The distribution of urotensin I (UI) and corticotropin-releasing factor (CRF) immunoreactive (IR) structures was studied in the central nervous system (CNS) of the white sucker using the peroxidase-antiperoxidase immunocytochemical procedure. The close sequence homology between both peptides resulted in a high degree of crossreactivity. This was resolved by saturating the antisera solutions with heterologous antigens and specificity tests were done by adding excess of homologous peptides. UI immunoreactivity was seen in all of the identifiable caudal spinal cord neurosecretory cells, in their processes projecting to the urophysis, in thin beaded fibres coursing along the spinal cord, in brain stem, hypothalamus, proximal pars distalis and, expecially, in the telencephalon. Some IR-UI specific and IR-CRF specific parvocellular neurons were also identified in the caudo-ventral tuberal region and ventral telencephalon. The IR-CRF was mainly present in parvocellular and magnocellular perikarya of the nucleus preopticus and in the preoptic-neurohypophysial pathway. Dense networks of IR-CRF reacting beaded fibres were also located in the lateral and posterior recessus nuclei. In the pituitary, IR-CRF fibre bundles were seen mainly in the neurointermediate lobe and in the rostral pars distalis. The cells of origin of the extraurophyseal system of IR-UI fibres in the sucker CNS have not been identified. The distribution of CRF immunostaining correlates well with the documented knowledge of CNS structures involved in the control of ACTH secretion in the goldfish. The probability of the occurence of two UI-CRF related molecules, or of two different forms resulting from a common precursor molecule, forming two separate neuronal systems in the sucker CNS seems likely.

Co-expression of corticotropin-releasing factor and vasopressin immunoreactivity in parvocellular neurosecretory neurons of the adrenalectomized rat.

To clarify the anatomical organization that allows for the synergy of vasopressin and oxytocin with corticotropin-releasing factor (CRF) in promoting adrenocorticotropic hormone secretion from the anterior pituitary, immunohistochemical double staining methods were used to compare the distribution of these peptides in the hypothalamic paraventricular nucleus of normal, colchicine-treated, and adrenalectomized male rats. In untreated animals, a few CRF-stained cells were found in the parvocellular division of the paraventricular nucleus, while brightly stained oxytocin- and vasopressin-immunoreactive cells were centered in the magnocellular division. In animals treated with colchicine, and inhibitor of axonal transport, large numbers of CRF-stained cells were found in the parvocellular division of the nucleus, and 1-2% of these also stained with antivasopressin. As reported previously, a substantial number of oxytocin-stained cells, centered in a discrete anterior part of the magnocellular division, also expressed CRF immunoreactivity. In contrast, after adrenalectomy, CRF immunostaining of cells in the parvocellular division was enhanced selectively and greater than 70% of these cells also stained positively for vasopressin. The distribution of oxytocin-stained cells was not influenced by adrenalectomy. The unusual localization of vasopressin immunoreactivity in parvocellular neurosecretory neurons in the adrenalectomized rat suggests that a single population of cells can produce CRF and vasopressin, both of which are potent promoters of adrenocorticotropic hormone secretion. These findings indicate that there is a state-dependent plasticity in the expression of biologically active peptides by individual neuroendocrine neurons.

Corticotropin releasing factor (CRF): origin and course of afferent pathways to the median eminence (ME) of the rat hypothalamus.

8-10 days after making various lesions in the rat hypothalamus, the presence of corticotropin releasing factor (CRF) immunoreactive neural structures was studied in paraffin and vibratome sections with CRF immunocytochemistry. Bilateral anterolateral deafferentation of the medial basal hypothalamus (MBH) caused complete disappearance of CRF immunoreactivity from the median eminence (ME) in brains where the posterior edge of the cut reached the level of the pituitary stalk. A shorter cut resulted in positive immunostaining caudal to the caudal edge of the cut. Unilateral deafferentation of the MBH caused significant decrease in CRF immunostaining in the ipsilateral ME. Unilateral posterolateral deafferentation of the MBH caused no changes in CRF immunostaining in the rostral ME, while fewer CRF-containing processes were observed in the more caudal regions. A horizontal cut ventral to the paraventricular nuclei (PVN) caused a slight decrease in the number of CRF-immunoreactive profiles in the ME. A wider and complete unilateral horizontal cut resulted in a significant decrease in CRF immunoreactivity on the operated side. Following various surgical interventions, hormone accumulation in cell bodies was detected in the paraventricular, periventricular preoptic, dorsomedial, periventricular, lateral and posterior hypothalamic, and premammillary nuclei. Fibers arising from most of these nuclei formed a fan-like projection to the ME. The majority of the CRF-fibers ran through the lateral tract of the fan, and reached the ME by the lateral-basal retrochiasmatic area (LBRCA). Scattered fibers were detected in the lateral-basal hypothalamus as far caudally as the level of the pituitary stalk. Unilateral anterolateral and horizontal cuts did not result in complete disappearance of CRF immunoreactivity from the ipsilateral ME, indicating the existence of CRF-fibers of contralateral origin in the ME.

Immunoreactive corticotropin releasing factor (CRF) and vasopressin are colocalized in a subpopulation of the immunoreactive vasopressin cells in the paraventricular nucleus of the hypothalamus

In the magnocellular cells of the paraventricular nucleus of hypothalamus, corticotropin releasing factor (CRF) immunoreactivity was observed in a subpopulation of vasopressin immunoreactive cells. No CRF immunostaining was observed in the supraoptic nucleus, a region with marked vasopressin staining.