Morphometrical analysis of the distribution of corticotrophin releasing factor, glucocorticoid receptor and phenylethanolamine-N-methyltransferase immunoreactive structures in the paraventricular hypothalamic nucleus of the rat

By means of the indirect immunoperoxidase technique the corticotrophin releasing factor (CRF) and glucocorticoid receptor (GR) immunoreactive nerve cell bodies and the phenylethanolamine-N-methyltransferase (PNMT) immunoreactive nerve terminals in the paraventricular hypothalamic nucleus of the rat have been mapped out in adjacent vibratome sections (30 micron thick). By means of morphometrical analysis using a semiautomatic image analyser, it was possible to obtain density maps of CRF, GR and PNMT immunoreactive structures within the paraventricular hypothalamic nucleus. The statistical analysis by the use of correlation coefficients gives evidence that the PNMT immunoreactive nerve terminals innervate the majority of the CRF immunoreactive nerve cell bodies and that GR are located in the majority of the CRF immunoreactive neurons.     

Immunohistochemical evidence for the presence of neuropeptides in the hypothalamic suprachiasmatic nucleus of ground squirrels

The cytoarchitecture and immunocytochemical distribution of neuropeptides (corticotropin-releasing factor, CRF; neuropeptide Y, NPY; oxytocin, OXY; vasopressin, VP; and vasoactive intestinal polypeptide, VIP) were studied in the hypothalamic suprachiasmatic nuclei (SCN) in male and female ground squirrels of two species (Spermophilus tridecemlineatus and S. richardsonii). Immunoreactive (IR) perikarya were found in sections incubated with VP or VIP antisera. VP-IR cell bodies were seen in the dorsal and medial parts of the nucleus in colchicine-treated animals. IR fibers were distributed throughout the SCN. In the ventral part of the nucleus, VIP-IR cells were seen in untreated animals and were more pronounced in colchicine-treated animals. VIP-IR fibers and terminals form a dense plexus throughout the nucleus. Furthermore, NPY-IR terminals and fibers with multiple varicosities, but no IR perikarya, were present in the suprachiasmatic nuclei. Within the borders of the SCN, no cell bodies or fibers were stained with CRF or OXY antisera in any animal.     

Quantitative in situ hybridization histochemistry reveals increased levels of corticotropin-releasing factor mRNA after adrenalectomy in rats

A 35S-labeled 48-base synthetic oligonucleotide complementary to a portion of the rat corticotropin-releasing factor (CRF) mRNA was used for in situ hybridization histochemistry. CRF-synthesizing cells were located in the paraventricular nucleus of the hypothalamus. These cells were observed in the medial parvocellular subdivision where there was a 90% increase in the amount of CRF mRNA per unit volume after adrenalectomy.     

Light stimulation of the hypothalamic neuroendocrine system.

This paper demonstrates that, in the mediation of light, the suprachiasmatic nucleus (SCN) functionally associates with the anterior periventricular and parvocellular paraventricular neuron systems in rats. Intact rats (group 1) and rats undergoing a hemicomplete cutting of the SCN (group 2) were housed in a dark room (2-3 weeks) and killed after an exposure to light for 10, 30 or 60 min. Other intact animals (group 3) kept in a dark room (2 weeks) were exposed to light for 10 min, then stored 60 min in the dark room, and killed in darkness. The SCN, anterior periventricular nucleus, and parvocellular paraventricular nucleus were examined immunohistochemically using antisera for vasoactive intestinal polypeptide (VIP), arginine vasopressin, somatostatin, rat corticotropin releasing factor (rCRF), and c-fos protein. In comparison with animals kept in darkness, animals exposed for 10 and 30 min to light indicated a remarkable reduction of VIP immunoreactivity in the SCN and some increase of CRF immunoreactivity in the parvocellular paraventricular nucleus. The diminution of VIP immunoreactivity did not occur in the isolated SCN of group 2 animals. In group 3, a 10 min-light exposure induced a remarkable enhancement of nuclear c-fos immunoreactivity in neurons in the ventrolateral region of the SCN, in the anterior periventricular nucleus, and in the parvocellular paraventricular nucleus, most strongly in the SCN. Double immunolabeling methods have shown that VIP, somatostatin, and CRF neurons in the respective nuclei were c-fos positive.     

Suppression by glucocorticoid of the immunoreactivity of corticotropin-releasing factor and vasopressin in the paraventricular nucleus of rat hypothalamus

The effect of glucocorticoid on the production of corticotropin-releasing factor (CRF) and vasopressin in the paraventricular nucleus of the hypothalamus (PVH) was examined immunocytochemically. Intraperitoneal administration of dexamethasone sulfate in a dose of 0.1 mg/day suppressed the immunoreactivity of CRF and vasopressin in the medial parvocellular divisions of the PVH of the rat subsequent to bilateral adrenalectomy. In the magnocellular divisions, suppression of vasopressin-immunoreactivity was not observed. These results suggest that the vasopressin in the medial parvocellular divisions plays a distinct role from that in the magnocellular divisions, the former having functional significance in the hypothalamo-hypophysio-adrenal axis.     

Immunocytochemical and in situ hybridization detection of hypothalamic neuropeptides from postmortem unfixed rat brains.

The effects of postmortem delay on neuropeptide-containing perikarya was studied in the paraventricular nucleus (PVN) of the rat hypothalamus. Serial sections from brains kept in the skull after death for 6 h and immunocytochemically processed for oxytocin (OT), vasopressin (AVP) and corticotropin releasing factor (CRF) or hybridized in situ for CRF resulted in the well preserved phenotypic expression and stability of mRNA of the aforementioned neuropeptides. Furthermore in most cases, AVP and CRF expression was discernibly enhanced relative to prefixed immunopositive tissue. Results of this study suggest that postmortem variables do not significantly alter the neurochemical coding of magnocellular or parvocellular neurosecretory systems, and support the view that rat and human brain topography can be investigated from tissue left in situ after death for a relatively long period of time.     

Stress induces CRF release in the paraventricular nucleus, and both CRF and GABA release in the amygdala

In the hypothalamus, corticotropin-releasing factor (CRF) initiates the hypothalamic-pituitary-adrenal (HPA) axis response to stress, resulting in the release of glucocorticoids, including cortisol. Extrahypothalamic CRF, particularly in the limbic system, also appears to play a role in the stress response. To further define brain CRF response to stress, immunosensor-based microdialysis probes were used to measure the extracellular levels of CRF in the paraventricular nucleus of the hypothalamus (PVN) and in the amygdala of sheep during a predator (dog) exposure stress. In addition, gamma amino butyric acid (GABA) was measured in the amygdala and cortisol was measured in venous blood. Exposure to the predator stress increased CRF in the PVN and both CRF and GABA in the amygdala. These were followed in time by a rise in venous cortisol. Application of a CRF antagonist to the amygdala, immediately prior to stress, had a small effect on the subsequent observed stress responses. This treatment, however, significantly reduced the responses to a repeat stress administered 2 days later, compared to nontreated animals. Application of a GABA antagonist to the amygdala prior to stress had no effect on the subsequent observed stress response but increased the response to the stress repeated 2 days later. Perfusion with 4-aminopyridine, a neuronal depolarising agent, into the PVN induced a release of CRF accompanied shortly thereafter by a small increase in CRF in the amygdala, and 5-10 min later by an increase in venous cortisol. Perfusion into the amygdala increased the levels of both CRF and GABA but had no effect on either PVN CRF or venous cortisol. These data support roles for both the PVN and amygdala in stress responsiveness. It suggests further that actions at the amygdala can strongly influence subsequent responsiveness to a further stress, mediated in part by both CRF and GABA actions.     

Corticotropin-releasing factor projections from limbic forebrain and paraventricular nucleus of the hypothalamus to the region of the ventral tegmental area

Corticotropin-releasing factor (CRF) is a peptide neurotransmitter with high numbers of cell bodies found in limbic regions of the rat brain including the oval nucleus of the bed nucleus of the stria terminalis (BNSTov) and central nucleus of the amygdala (CeA) as well as in the paraventricular nucleus of the hypothalamus (PVN). CRF systems are activated in response to acute stressors and mediate a wide variety of physiological and behavioral responses to acute stress including aversive responses and responses that support appetitive behaviors. CRF is released in the ventral tegmental area (VTA), the cell body region of the mesocorticolimbic dopaminergic neurons, in response to acute stress and plays a role in stress-activation of appetitive behavior [Wang B, Shaham Y, Zitzman D, Azari S, Wise RA, You ZB (2005) Cocaine experience establishes control of midbrain glutamate and dopamine by corticotropin-releasing factor: a role in stress-induced relapse to drug seeking. J Neurosci 25:5389-5396]. However, although it is known that the VTA region contains significant levels of CRF-immunoreactive fibers [Swanson LW, Sawchenko PE, Rivier J, Vale WW (1983) Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study. Neuroendocrinology 36:165-186], the source of CRF input to the region has not been identified. We used infusions of a fluorescent retrograde tracer, fluorogold, into the VTA region, combined with fluorescent immunocytochemistry for CRF to identify sources of this input. Double-labeled cells were found in BNSTov, CeA and PVN. The percent of fluorogold-labeled cells in each region that were CRF-positive was 30.8, 28.0 and 16.7% respectively. These data point to diffusely distributed sources of CRF-containing fibers in the VTA.     

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

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

Application of avidin-biotinized peroxidase (ABC) method for immunocytochemical localization of corticotropin-releasing factor (CRF) in rat hypothalamus

Studies on immunocytochemical localization of corticotropin-releasing factor (CRF) were performed in the rat hypothalamus using avidin-biotinized peroxidase (ABC) and PAP techniques. In intact and control animals CRF-immunoreactive nerve fibers were observed within outer layer of median eminence. In the adrenalectomized animals, CRF was also demonstrated in perikarya of neurocytes and in their projections in paraventricular nucleus of the hypothalamus. In both immunocytochemical techniques identical localization of CRF was obtained. However, reaction intensity was greater with the ABC technique than with the PAP one. In bilateral adrenalectomized animals, a greater number of CRF-immunoreactive neural fibers were observed in the median eminence than in control rats and rats subjected to sham operation.     

Corticotropin releasing factor and galanin-containing neurons projecting to the median eminence of the rat.

To identify corticotropin releasing factor (CRF)- and galanin (GAL)-containing neurons projecting to the median eminence, a retrograde tracing method with True blue was combined with a double-staining method. It was demonstrated that some True blue-labeled CRF neurons in the paraventricular nucleus were also immunoreactive for GAL. These findings support the hypothesis that GAL and CRF are simultaneously involved in regulation of ACTH secretion from the anterior pituitary.     

Vasopressin excites ventromedial hypothalamic glucose-responsive neurons in vitro

Effects of vasopressin (AVP), oxytocin (OXY), norepinephrine (NE), and glucose on the single-unit activity of hypothalamic ventromedial nucleus (VMN) in tissue slices were studied. While AVP was exclusively excitatory on 58% of the neurons, OXY could be excitatory or inhibitory and affected only 42% of the neurons. There was no correlation between the responses to these two peptides. Each of these two peptides could desensitize neuronal response to itself, but did not cross-desensitize responses to each other. These results indicate that AVP and OXY do not act on the same population of VMN neurons through the same cellular mechanism. Furthermore, only the responses to AVP were correlated to responses to glucose and NE, two agents relevant to central regulation of feeding. This correlation with responses to feeding-relevant agents and the exclusively excitatory action on the VMN, which is involved in the regulation of feeding, suggest that AVP can play a role in the regulation of feeding, particularly the feeding induced by the injection of NE into the paraventricular nucleus, that is known to alter AVP release.     

The distribution of angiotensin II AT1 receptor subtype mRNA in the rat brain.

The present study demonstrates the existence and regional distribution of angiotensin II AT1 receptor subtype mRNA expression in the rat brain by the use of in situ hybridization and RNase protection assay. Substantial expression levels in the brain have only been detected in certain distinct areas, such as the subfornical organ, the parvocellular part of the paraventricular hypothalamic nucleus, and the median preoptic nucleus. The results give further evidence for the involvement of the angiotensin II AT1 receptor subtype in the classical functions of central angiotensin II, like blood pressure control, body fluid homeostasis and in corticotropin-releasing factor (CRF) secretion.     

Analysis of peptide histidine-isoleucine/vasoactive intestinal polypeptide-immunoreactive neurons in the central nervous system with special reference to their relation to corticotropin releasing factor- and enkephalin-like immunoreactivities in the paraventricular hypothalamic nucleus

The distribution of peptide histidine-isoleucine (PHI) and vasoactive intestinal polypeptide (VIP), two peptides derived from the same precursor molecule, was analysed with immunohistochemistry in the central nervous system of the rat, and to a limited extent in some other species including sheep, monkey and man. Special attention was focused on possible cross-reactivity between PHI antisera and corticotropin releasing factor in parvocellular neurons in the hypothalamic paraventricular nucleus projecting to the external layer of the median eminence. (1) Characterization of the PHI and VIP antisera revealed that they recognized different sequences of the peptide molecules. One of the PHI antisera (PHI-N), although mainly N-terminally directed, also probably contained an antibody population directed against the C-terminal amino acid in PHI which is an amidated isoleucine. Rat and human corticotropin releasing factor but not ovine also have an amidated isoleucine in C-terminal position. (2) PHI- and VIP-like immunoreactivity were found with parallel and overlapping distribution in all areas investigated in the rat central nervous system. In many cases coexistence of the two immunoreactivities could be directly demonstrated. PHI neurons were found in some areas so far not know to contain PHI/VIP neurons, including the dorsal septum, the septofimbrial nucleus, the stria terminalis and lamina V of the spinal cord. (3) Using an antiserum directed against the amino acid sequence 111-122 of the VIP/PHI precursor, immunoreactive cell bodies were seen in some areas containing VIP and PHI neurons. PHI- and VIP-like immunoreactivity were expressed in parallel in increasing amounts in the superficial laminae of the dorsal horn after transection of the sciatic nerve [G. P. McGregor et al. (1984) Neuroscience 13, 207-216; S. A. S. Shehab and M. E. Atkinson (1984) J. Anat. 139, 725; S. A. S. Shehab and M. E. Atkinson (1986) Expl Brain Res. 62, 422-430]. (5) The PHI-N antiserum stains large numbers of immunoreactive cells in the parvocellular part of the paraventricular nucleus and these cells are mostly identical with corticotropin releasing factor-positive neurons. Absorption experiments suggested that this PHI-N-like immunoreactivity to a large extent represented cross-reactivity with rat CRF and that earlier demonstration of many PHI-positive neurons in the paraventricular nucleus probably represents an artefact as proposed by F. Berkenbosch et al. (Neuroendocrinology 44, 338-346). However, some cells did, in fact, contain VIP- as well as PHI-like immunoreactivity as was shown with antisera not cross-reacting with corticotropin releasing factor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Corticotropin-releasing factor antagonist reduces activation of noradrenalin and serotonin neurons in the locus coeruleus and dorsal raphe in the arousal response accompanied by yawning behavior in rats

We previously reported that intracerebroventricular (icv) administration of corticotropin-releasing factor (CRF) antagonist attenuates the arousal response during yawning behavior in rats. However, the CRF-related pathway involved in the arousal response during yawning is still unclear. In the present study, we assessed the involvement of the CRF-containing pathway from the hypothalamic paraventricular nucleus (PVN) to the locus coeruleus (LC) and the dorsal raphe nucleus (DRN) in the arousal response during frequent spontaneous yawning, which was induced by several microinjections of l-glutamate into the PVN in anesthetized rats, using c-Fos immunohistochemistry. The PVN stimulation showed significant increases in activation of PVN CRF neurons, LC noradrenalin (NA) neurons and DRN serotonin (5-HT) neurons as well as arousal response during yawning. But icv administration of a CRF receptor antagonist, α-helical CRF (9-41), significantly inhibited the activation of both LC NA neurons and DRN 5-HT neurons except the activation of CRF neurons in the PVN, and significantly suppressed the arousal response during yawning. These results suggest that the CRF-containing pathway from PVN CRF neurons to LC NA neurons and DRN 5-HT neurons can be involved in the arousal response during yawning behavior.     

Distribution of galanin immunoreactivity in the sheep diencephalon

Although the physiological role of galanin has been demonstrated in several endocrine regulations in sheep, the anatomical characteristics of this neuronal system has never been studied. The distribution of galanin-containing neurones was described by immunohistochemistry using galanin antiserum in the diencephalon of adult ewes, both ovariectomized or treated with colchicine. Galanin-immunoreactivity was found throughout the diencephalon. In the ovariectomized ewes, galanin-immunoreactive neurones were mainly observed in the medial preoptic area and the infundibular nucleus. The highest density of immunoreactive fibres was found in the external layer of the median eminence. Numerous galanin-immunoreactive fibres were also observed in the preoptic area, the mediobasal hypothalamus, the periphery of the supraoptic and the paraventricular nuclei. With colchicine treatment, the number of labelled neurones increased, and additional galanin-immunoreactive perikarya were observed in the bed nucleus of the stria terminalis, the lateral septum, the supraoptic, the paraventricular and the periventricular nuclei and the paraventricular nucleus of the thalamus. In the caudal part of the diencephalon, the density of labelled neurones was lower in both groups of animals than in other species studied. Regardless of treatment, labelling was not seen in the suprachiasmatic nucleus and only rarely in the ventromedial nucleus. These results describe, for the first time, the distribution of galanin-immunoreactive neurones in the sheep diencephalon. Compared to other species studied, distribution in the sheep diencephalon has several distinct differences. In ovariectomized animals, the medial preoptic area presents more labelled neurones in sheep than in monkeys, whereas in the supraoptic nucleus the density of labelled neurones is lower in sheep than in humans or opossums. After colchicine treatment only very few differences were observed between sheep and rats, but in contrast to other species, the suprachiasmatic nucleus of the sheep does not contain labelled neurones.     

Paraventricular nucleus region controls pituitary-adrenal function in Brattleboro rats

To study the role of the paraventricular nucleus and of neurohypophysial hormones in the control of ACTH secretion, the paraventricular nuclei (PV) of Brattleboro diabetes insipidus rats (DI) were lesioned (L) with a knife; sham-lesioned DI (S) served as controls. Four days later, the rats were stressed by ether inhalation, and blood samples were taken during and 30-40 min after stress for the determination of corticosterone. The median eminence (ME) and neural lobe (NL) were homogenized in 50 microliters of 0.1 N HCl and frozen pending bioassay of corticotropin-releasing factor (CRF). PV lesion almost abolished the corticosterone secretion to ether and reduced the ME CRF content three- to sevenfold. The NL CRF content in S was about twice that of ME, and oxytocin accounted for more than 60% of NL CRF. However, PV lesion had no effect on NL CRF activity. Low amounts of oxytocin (2 mU/ml) had no significant CRF activity but potentiated the ME CRF effect in L. The results suggest that 1) PV is one of the most important sites for CRF synthesis or CRF fiber transit in DI; 2) corticosterone secretion to ether stress is governed mainly by ME CRF; and 3) a large proportion of CRF fibers to NL probably originates outside PV.     

CRF单克隆抗体和多克隆抗体制备及其在睡眠剥夺模型大鼠脑内的变化

目的:制备并鉴定促肾上腺皮质激素释放因子(CRF)的单克隆抗体(mAb)和多克隆抗体(pAb),并研究其在睡眠剥夺模型大鼠脑内的变化。方法:用CRF分别与牛血清白蛋白(BSA)和牛甲状腺球蛋白(BTG)交联,制成免疫原和检测用的包被抗原。将BSACRF分别免疫新西兰大白兔和雌性BALB/c小鼠,制备其mAb和pAb,经ELISA和免疫细胞化学染色等方法进行鉴定,并用所获抗体观察CRF在正常成年大鼠睡眠剥夺48h后脑内的变化。结果:所获得的CRFpAb和9株mAb具有较高的效价、特异性及亲和力。其中,pAb和2株mAb(1D10,2F4)可用于免疫组织化学染色。睡眠剥夺48h后下丘脑室旁核、杏仁核中央亚核、终纹状核卵圆亚核等核团高表达CRF样阳性产物,而对照组相关核团内则极少或未出现。结论:①成功地获得可用于免疫组化染色的CRFmAb和pAb。②提示CRF在中枢神经系统对睡眠剥夺造成的应激反应的调节过程中具有重要的意义。下丘脑室旁核、杏仁中央核和终纹床核是脑内相关调节环路的一部分。