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PubMed1991

Colocalization of insulin-like growth factor-binding protein with insulin-like growth factor I.

We report the localization of insulin-like growth factor I (IGF-I) and a 25-kDa form of insulin-like growth factor-binding protein (IGF-BP-1) in adult rat kidney. The antigens were localized using a rabbit anti-human IGF-I antibody, and a rabbit anti-human IGF-BP-1 antibody raised against human 25-kDa IGF-BP-1 purified from amniotic fluid. Immunohistochemistry by the avidin-biotin peroxidase conjugate technique showed that both peptides are located in the same nephron segments, in the same cell types. The most intense staining was in papillary collecting ducts. There was moderate staining also in cortical collecting ducts and medullary thick ascending limbs of Henle's loop. In collecting ducts the antigens were shown to be present in principal cells but not in intercalated cells. In distal convoluted tubules, cortical thick ascending limbs, and in structures presumptively identified as thin limbs of Henle's loops there was only modest staining. The macula densa, however, lacked immunoreactivity. Colocalization of IGF-I and IGF-BP-1 in the same cells supports the notion, derived from studies on cultured cells, that the actions of IGF-I may be modified by IGF-BPs that are present in the same location.

Animals↗

PubMed1992

Colocalization and release of angiotensin and renin in renal cortical cells.

Angiotensin is generated within the kidney, but the precise loci for the formation of angiotensin I (ANG I) and angiotensin II (ANG II) have not been demonstrated. We performed electron microscopy immunocytochemistry in kidney sections of 10-day-old (newborn) and adult Wistar-Kyoto (WKY) rats using specific antibodies to renin, ANG I, ANG II, and angiotensinogen (AO). Renin, ANG I, ANG II, and AO were present in juxtaglomerular (JG) cells. Renin was largely confined to cytoplasmic granules; ANG I and ANG II were colocalized to these granules but also were present in the cytoplasm; AO was distributed throughout the cytoplasm. AO also was present in a renal cortical distribution in proximal tubular cells. Northern blot analysis demonstrated AO mRNA in total kidney and liver but not in renal microvessels. Using the reverse hemolytic plaque assay, we demonstrated release of ANG I and renin from individual renocortical cells of adult WKY rats. Under control conditions, the number of releasing cells was 11 +/- 1 for ANG I and 10 +/- 1 for renin. Addition of rat renin inhibitor (RI) (1 x 10(-5) M), which inhibited renin activity in the medium from 37 to 9 pg ANG I.ml-1.h-1, did not alter ANG I plaque number. Addition of rat AO increased ANG I plaque number to 17 +/- 2 (P less than 0.05). Incubation with both RI and AO prevented the increase in ANG I plaque number obtained with AO alone. Enalapril treatment (7 days; n = 5) increased the number of plaque-forming cells to 22 +/- 2 for ANG I (P less than 0.0005) and to 39 +/- 7 for renin (P less than 0.001). The results suggest an intracellular location for AO and angiotensin and release of renin and ANG I by renal cortical cells and suggest that released angiotensin is produced intracellularly and that secretion of ANG I is augmented by converting enzyme inhibition.

Angiotensin I↗

PubMed1998

Dynein and dynactin colocalize with AQP2 water channels in intracellular vesicles from kidney collecting duct.

We investigated whether the motor protein cytoplasmic dynein and dynactin, a protein complex thought to link dynein with vesicles, are present in rat renal collecting ducts and associated with aquaporin-2 (AQP2)-bearing vesicles. Immunoblotting demonstrated cytoplasmic dynein heavy and intermediate chains in kidney, with relative expression levels of inner medulla > outer medulla > cortex. In addition to being present in cytoplasmic fractions, dynein was abundant in membrane fractions enriched for intracellular vesicles. Dynactin was also abundant in membrane fractions enriched for intracellular vesicles. Furthermore, both dynactin and dynein were present in vesicles specifically immunoisolated using anti-AQP2 antibodies. Immunocytochemistry revealed labeling for dynein in the collecting duct principal cells with a pattern consistent with labeling of intracellular vesicles. Moreover, quantitative double immunogold labeling confirmed colocalization of AQP2 and dynein in the same vesicles at the electron microscopic level. Thus the microtubule-associated motor protein dynein and the associated dynactin complex are present in rat renal collecting duct principal cells and are associated with intracellular vesicles, including those bearing AQP2, consistent with the view that dynein and dynactin are involved in vasopressin-regulated trafficking of AQP2-bearing vesicles.

Animals↗

PubMed1999

Cellular and subcellular immunolocalization of ClC-5 channel in mouse kidney: colocalization with H+-ATPase.

To determine the immunolocalization of ClC-5 in the mouse kidney, we developed a ClC-5-specific rat monoclonal antibody. Immunoblotting demonstrated an 85-kDa band of ClC-5 in the kidney and ClC-5 transfected cells. Immunocytochemistry revealed significant labeling of ClC-5 in brush-border membrane and subapical intracellular vesicles of the proximal tubule. In addition, apical and cytoplasmic staining was observed in the type A intercalated cells in the cortical collecting duct. In contrast, the staining was minimal in the outer and inner medullary collecting ducts and the thick ascending limb. Western blotting of vesicles immunoisolated by the ClC-5 antibody showed the presence of H+-ATPase, strongly indicating that these two proteins were present in the same membranes. Double labeling with antibodies against ClC-5 and H+-ATPase and analysis by confocal images showed that ClC-5 and H+-ATPase colocalized in these ClC-5-positive cells. These findings suggest that ClC-5 might be involved in the endocytosis and/or the H+ secretion in the proximal tubule cells and the cortical collecting duct type A intercalated cells in mouse kidney.

Amino Acid Sequence↗

PubMed2001

SNAP-25-associated Hrs-2 protein colocalizes with AQP2 in rat kidney collecting duct principal cells.

The vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels in kidney collecting duct is likely mediated by vesicle-targeting proteins (N-ethylmaleimide-sensitive factor attachment protein receptors). Hrs-2 is an ATPase believed to have a modulatory role in regulated exocytosis. To examine whether Hrs-2 is expressed in rat kidney, we carried out RT-PCR combined with DNA sequence analysis and Northern blotting using a digoxigenin-labeled Hrs-2 RNA probe. RT-PCR and Northern blotting revealed that Hrs-2 mRNA is localized in all zones of rat kidney. The presence of Hrs-2 protein in rat kidney was confirmed by immunoblotting, revealing a 115-kDa protein in kidney and brain membrane fractions corresponding to the expected molecular size of Hrs-2. Immunostaining and confocal laser scanning microscopy of LLC-PK(1) cells (a porcine proximal tubule cell line) transfected with Hrs-2 DNA confirmed the specificity of the antibody and revealed that Hrs-2 is mainly localized in intracellular compartments, including cathepsin D-containing lysosomal/endosomal compartments. The cellular and subcellular localization of Hrs-2 in rat kidney was examined by immunocytochemistry and confocal laser scanning microscopy. Hrs-2 immunoreactivity was observed in collecting duct principal cells, and weaker labeling was detected in other nephron segments. The labeling was predominantly present in intracellular vesicles, but labeling was also observed in the apical plasma membrane domains of some cells. Colabeling with AQP2 revealed colocalization in vesicles and apical plasma membrane domains, suggesting a role for Hrs-2 in regulated AQP2 trafficking.

Adenosine Triphosphatases↗

PubMed1990

Origin and colocalization of CGRP- and SP-reactive nerves in cat airway epithelium.

A combination of neuroanatomic techniques was used to examine the origin and neuropeptide content of nerve fibers in the airway epithelium of adult cats. By the use of immunocytochemical methods, the peptides substance P (SP) and calcitonin gene-related peptide (CGRP) were colocalized in airway epithelial nerve fibers. Two days after wheat germ agglutinin (WGA) was injected into the nodose ganglion, fibers containing WGA immunoreactivity (IR) were detected in the airway epithelium. SP-like immunoreactivity (LI) and CGRP-LI were demonstrated separately in the WGA-IR fibers, establishing their origin from nerve cell bodies of nodose ganglion. Vagal transection inferior to the nodose ganglion reduced the number of SP- and CGRP-IR fibers by greater than 90% in ipsilateral airways. In contralateral airways, SP-IR fibers were substantially reduced, whereas the effect on CGRP-IR fibers was not statistically significant. Vagotomy superior to the nodose ganglion did not alter the density of peptide-IR fibers. The results prove that SP- and CGRP-IR nerve fibers of cat airway epithelium originate from nerve cell bodies in the nodose ganglion and that SP- and CGRP-like peptides may be stored together in some nerve fibers of the airway epithelium.

Animals↗

PubMed2001

Distribution and colocalization of neurotransmitters and receptors in the pre-Bötzinger complex of rats.

The pre-Bötzinger complex (PBC), thought to be the center of respiratory rhythm generation, is a cell column ventrolateral to the nucleus ambiguus. The present study analyzed its cellular and neurochemical composition in adult rats. PBC neurons were mainly oval, fusiform, or multipolar in shape and small to medium in size. Neurokinin-1 receptor, a marker of the PBC, was present in the plasma membrane of mostly medium and small neurons and their associated processes and boutons. Among neurons immunoreactive for different neurotransmitter or receptor candidates, various numbers were colocalized with neurokinin-1 receptor. The highest ratio was with nitric oxide synthase (52.72%), and the lowest was with glycine receptors (31.93%). Glutamic acid decarboxylase- and glycine transporter 2-immunoreactive boutons, as well as GABA(A) receptor-immunoreactive plasma membrane processes and boutons, were also identified in the PBC. PBC neurons exhibited different levels of cytochrome oxidase activity, indicating their various energy demands. Our results suggest that synaptic interactions within the PBC of adult rats involve a variety of neurotransmitter and receptor types and that nitric oxide may play an important role in addition to glutamate, GABA, glycine, and neurokinin.

Amino Acid Transport Systems, Neutral↗

PubMed2000

Colocalization of neuronal NO synthase with urotensins I and II in the caudal neurosecretory neurons and the urophysis of the teleost oreochromis niloticus: a gold immunoelectron microscopic study.

The intracellular distribution of neuronal nitric oxide synthase (nNOS) was studied in the caudal neurosecretory system of a teleost, Oreochromis niloticus (Cichlids), by means of post-embedding immunogold labeling with a polyclonal antibody directed against nNOS of human origin. Ultrastructural examination demonstrated that neuronal NOS-like molecules are distributed within the Dahlgren cell perikarya, the neurosecretory axons, and the urophysial axon terminals. In the neurosecretory somata, gold particles for nNOS were mainly cytosolic, whereas in the neurosecretory axons and axon terminals they were associated with the membrane and/or the dense core of neurosecretory granules. Double immunogold labelings for nNOS/urotensin I (UI) and nNOS/urotensin II (UII) demonstrated that nNOS-like molecules are colocalized with UI and/or UII in the neurosecretory granules contained within the urophysial terminals. The present findings suggest that both a soluble cytosolic and a particulate neuronal NOS are expressed in the caudal neurosecretory neurons. They confirm previous biochemical data on the same species.

Animals↗

PubMed2001

NADPH-diaphorase activity and nitric oxide synthase-like immunoreactivity colocalize in the electromotor system of four species of gymnotiform fish.

The electric organ discharge (EOD) of gymnotiform electric fish is controlled by a well-characterized neural circuit in the brainstem and spinal cord. NADPH-diaphorase (NADPH-d) activity was previously found in phase-locking and/or rapidly firing neurons in the electromotor and electrosensory systems of Apteronotus leptorhynchus [Turner and Moroz, 1995]. These findings suggested that nitric oxide synthase (NOS) is expressed in these neurons and may regulate their precise, high frequency firing. We extended these results by examining the distribution of both NADPH-d activity and NOS-like immunoreactivity (NOS-lir) in the electromotor systems of four gymnotiform species that differ in the frequency and modulation of their EODs. NOS-lir colocalized with NADPH-d staining throughout the electromotor system, indicating that NADPH-d is a faithful indicator of NOS in this system. The distribution of NOS-lir and NADPH-d was similar in the electromotor systems of all four species in this study, with one exception: NOS and NADPH-d staining was consistently less intense in pacemaker and relay cells in Sternopygus macrurus, which produces low frequency EODs, than in the three other species that produce higher frequency EODs. This species difference in NOS expression in the pacemaker nucleus may be related to species differences either in EOD frequency or in modulations of the EOD (e.g., the jamming avoidance response). In Apteronotus species, NOS-lir and NADPH-d were concentrated in bands along the axons of their nerve-derived electric organs. These bands corresponded to regions surrounded by little or no staining with a Schwann cell-specific antibody, suggesting that the NOS-positive regions lie near nodes of Ranvier. In Sternopygus and Eigenmannia, the innervated, posterior membranes of muscle-derived electrocytes were more intensely labeled for NADPH-d and NOS than inexcitable portions of the membrane. Thus, in both muscle- and nerve-derived electric organs, NOS is concentrated near excitable membranes. These results indicate that NOS is well-positioned within the electromotor system to regulate the frequency, precision, amplitude, and waveform of EODs.

Animals↗

PubMed1998

Androgen receptors and estrogen receptors are colocalized in male rat hypothalamic and limbic neurons that express Fos immunoreactivity induced by mating.

Conversion of testosterone into estradiol is important for male rat sexual behavior, and both steroids probably contribute to mating. The distributions of neurons containing androgen receptors (AR) and estrogen receptors (ER) overlap, and many AR-immunoreactive (AR-ir) neurons express Fos immunoreactivity (Fos-ir) induced by mating. Because mating-induced Fos-ir in the male rat occurs mainly in AR-ir neurons, and because both steroids are important for mating, we hypothesized that (i) AR-ir and ER-ir are colocalized and that (ii) some of these neurons are activated during mating. We examined, in adjacent sections from the medial preoptic area (MPN) through the central tegmental field (CTF), the expression of ER-ir in: (i) AR-ir-containing neurons, and (ii) Fos-ir-expressive neurons. PG21 anti-AR, OA-11-824 anti-c-fos, H222 or 1D5 anti-ER primary antibodies were visualized, respectively, with cyanine-conjugated, fluorescein- or cyanine-conjugated, and fluorescein-conjugated secondary antibodies in male rats which were killed 1 h after ejaculating with a receptive female. In MPN, bed nucleus of the stria terminalis (BNST), and medial amygdala (MEA), 80-90% of ER-ir labeling occurred in AR-ir-positive neurons but only about 30% of AR-ir neurons were ER-ir-positive. No ER-ir was found in the CTF. This suggests the presence of three types of brain neurons sensitive to gonadal steroid hormones: neurons sensitive to androgens only, neurons sensitive to both androgens and estrogens, and neurons sensitive to estrogens only. About 50% of ER-ir labeling occurred in cells expressing mating-induced Fos-ir but only about 30% of Fos-ir neurons were ER-ir-positive. These findings suggest that, in the MPN, at least two different neuronal populations are activated during mating: the first contains AR-ir only and the second contains AR-ir and ER-ir. In the BNST and MEA, at least three hormonally sensitive populations are activated during mating: the two described above plus a third population which expresses ER-ir only.

Animals↗

PubMed2000

Estrogen and tamoxifen differentially regulate beta-endorphin and cFos expression and neuronal colocalization in the arcuate nucleus of the rat.

Estrogen regulates hypothalamic gene expression, synthesis and release of the endogenous opioid peptide beta-endorphin (betaEND), although a consensus estrogen response element sequence has not been identified in the rat proopiomelanocortin (POMC) gene. POMC gene expression is also regulated by the activation of AP-1 promoter elements, which are known to be estrogen sensitive. The present studies examine whether estrogen modulates the hypothalamic POMC system through a non-classical mechanism involving AP-1 binding proteins such as cFos. Immunohistochemical double-labeling for betaEND and cFos was used and immunoreactive (-ir) populations were quantified in the arcuate nucleus and periarcuate area across time using unbiased stereological methods. Ovariectomized rats were injected with 50 microg estradiol (E2), 500 microg tamoxifen citrate (TAM) or both (E2+TAM) and were perfused 1, 2, 4 or 48 h later. E2 rapidly increased numbers of cFos-ir, betaEND-ir and doubly-labeled cells after 4 h, and the number of betaEND-ir cells remained high 48 h later, suggesting that the stimulatory effects of cFos on POMC in the hypothalamus persist after the cFos signal decays. Treatment with TAM alone did not affect the numbers of immunoreactive cells, although E2+TAM blocked the E2-mediated induction in all immunoreactive populations. Similar effects were seen at the transcriptional level. E2 increased hypothalamic POMC mRNA after 4 h, while TAM treatment or coadministration of E2+TAM did not significantly change the levels of POMC mRNA. Cellular colocalization of betaEND-ir and cFos-ir supports a possible intracellular co-regulation of these peptides by an estrogen-dependent mechanism within a subset of hypothalamic neurons. It does not, however, appear that E2 acts directly through an AP-1 site within the POMC gene.

Animals↗

PubMedDate not supplied

Colocalization of aldehyde dehydrogenases and Fe/NADPH-induced lipid peroxidation in tissue sections of rat retina.

Epidemiological and experimental studies suggest the involvement of lipid peroxidation (LPO) in retinal diseases. Clinicians usually prescribe antioxidants to help in the treatment of proliferative diabetic vitreoretinopathy and age-related macular degeneration. In spite of this, these processes inexorably induce visual impairment and may progress towards blindness. In addition to other pathogenic mechanisms not fully understood, it may be that peroxidic aldehydes from LPO occurring in the eyes, acting as cytotoxic chemicals, mediate in these chronic disorders. To test the mechanisms of removing peroxidic aldehydes from retinal cells and in an attempt to understand long-lasting changes induced by LPO, the distribution and activity of aldehyde dehydrogenases (ALDH) in the rat retina were studied and compared with the LPO sites induced by iron/nicotine adenine dinucleotide phosphate. Histochemical and immunocytochemical assays revealed the colocalization of LPO and ALDH, mainly in the photoreceptors and inner retinal layers. This suggests the involvement of ALDH in detoxifying peroxidic aldehydes from the retina. Any change in ALDH retinal expression and distribution might be of crucial importance in assessing the paths of LPO-mediated vitreoretinopathies. Further research is needed to evaluate these findings and their application to new ophthalmic therapy.

Aldehyde Dehydrogenase↗

PubMedDate not supplied

Calcitonin gene-related Peptide and choline acetyltransferase colocalization in the human vestibular periphery.

Within the vestibular system, calcitonin gene-related peptide (CGRP) has been localized in the efferent terminals and their brainstem neuronal cell bodies in several animal models. Presently, very few studies have verified these findings in the vestibular system in adult primates or humans. CGRP immunoreactivity (CGRPi) and its colocalization with choline acetyltransferase immunoreactivity (ChATi) in human vestibular end organs and Scarpa's ganglion were studied using polyclonal antibodies against CGRP and ChAT, at the light-microscopic level. The CGRPi axons ramified to produce numerous CGRPi terminals throughout the neurosensory epithelium of the maculae and cristae, primarily in the basal and midbasal areas. Numerous CGRPi efferent terminals made contact with both type II vestibular hair cells and the afferent chalices surrounding type I vestibular hair cells. All CGRP immunoreactive fibers also exhibited ChATi. As in the animal models, no CGRPi was found within Scarpa's ganglion. This study provides evidence for CGRPi in the human vestibular periphery and validates the biomedical relevance of the current animal models.

Calcitonin Gene-Related Peptide↗

PubMed2002

Candidate genes colocalized to linkage regions in inflammatory bowel disease.

BACKGROUND/AIMS: The genes encoding for tumor necrosis factor-alpha (TNF-alpha), epidermal growth factor receptor (EGFR) and the vitamin D receptor (VDR) are colocalized to inflammatory bowel disease-associated linkage regions on chromosomes 6, 7 and 12. An association study of these gene polymorphisms with ulcerative colitis or Crohn's disease and a stratification according to disease phenotypes was performed in order to identify genetically homogenous subgroups. PATIENTS AND METHODS: 119 healthy, unrelated controls, 95 patients with Crohn's disease and 93 patients with ulcerative colitis were genotyped for the (G to A) -308 TNF-alpha promoter polymorphism on chromosome 6, the codon 497 EGFR polymorphism on chromosome 7 and the TaqI polymorphism of the VDR gene on chromosome 12. After genotyping, patients were stratified according to the respective disease phenotype. RESULTS: A disequilibrium in the distribution of the VDR genotypes was found in patients with ulcerative colitis compared to controls (p = 0.024). In fistulizing and fibrostenotic Crohn's disease the 'TT' genotype was significantly reduced compared with other phenotypes (p = 0.006), whereas the 'tt' genotype was found more frequently (p = 0.04). The frequency of the WT allele of the EGFR gene was significantly higher in ulcerative colitis (p = 0.04) than in controls. Further significant differences, concerning the associations of the different polymorphisms and disease susceptibility or clinical phenotypes, were not observed. CONCLUSIONS: Regardless of the disease phenotype, the associations between the polymorphisms and inflammatory bowel disease investigated herein are modest, even after stratification for the disease phenotypes. Hence, these polymorphisms are unlikely to confer the reported linkage between inflammatory bowel disease and chromosomes 6, 7 and 12.

Adult↗

PubMed2003

Colocalization of progesterone receptors and thyroid hormone receptors alpha in the ovine diencephalon: no effect of estradiol.

Progesterone and thyroid hormones are key hormones in the control of reproduction in the ewe since both have been shown to inhibit GnRH secretion. GnRH neurons do not express progesterone receptors (PR) but half of them contain thyroid hormone receptors alpha (THRalpha), two nuclear receptors potentially able to act on gene transcription. PR and THRalpha distributions overlap in most regions of the ovine preoptic area (POA) and hypothalamus. To determine whether progesterone and thyroid hormones may also have common neuronal targets for the control of GnRH cell activity, we searched for coexpression of PR with THRalpha within neurons of the POA and hypothalamus of ovariectomized ewes treated with estradiol plus progesterone or with progesterone alone. Double-labeled cells were found throughout the medial POA, the periventricular part of the paraventricular nucleus (PVNpe), the arcuate nucleus (ARC) and the ventrolateral ventromedial nucleus (VMNvl). Colocalization ratios were not statistically different between steroid treatment groups. 84% of PR-immunoreactive cells in VMNvl, 90% in POA and ARC and 95% in PVNpe contain THRalpha. More than two-thirds of THRalpha-immunoreactive cells contain PR in each area examined. This study provides evidence that progesterone and thyroid hormones may act within the same cells to modulate physiological functions such as reproduction.

Animals↗

PubMed1986

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)

Animals↗

PubMed1991

Repeated stress-induced activation of corticotropin-releasing factor neurons enhances vasopressin stores and colocalization with corticotropin-releasing factor in the median eminence of rats.

Stress-induced release of corticotropin-releasing factor (CRF) and vasopressin (AVP) was studied in rats by measuring the decline of CRF and AVP stores in the median eminence after blockade of fast axonal transport with colchicine (5 micrograms per rat intracisternally). Quantitative immunocytochemistry was used to detect changes in CRFi and AVPi in the external zone of the median eminence (ZEME) selectively. Immobilization stress induced a fast ACTH response to 1,000-2,000 pg/ml which was associated with a fall in both CRFi and AVPi of 34% during the first 30 min. This is followed by different time courses of further AVPi and CRFi depletion. In addition, we investigated the effect of repeated daily stress exposure on CRFi and AVPi in the ZEM 1 day after stress exposure. Repeated daily immobilization for 9 or 16 subsequent days did not affect the CRFi stores in the ZEME, but increased the AVPi stores to 161 +/- 13% and 218 +/- 11% respectively. Quantitative analysis of electron microphotographs of repeatedly handled rats showed a mean density of CRF positive profiles in the ZEME of 45.5 +/- 2.5 per 500 microns 2 of which 25% also stained for pro-AVP-derived peptides. After 9 subsequent days of immobilization the total density of CRF-positive profiles remained unchanged, but the fraction of CRF swellings that also stained for pro-AVP-derived peptides increased approximately 2-fold. We conclude that (1) the secretion of AVPi and CRFi from the ZEME are independently controlled, indicating differential activation of AVP containing and AVP deficient CRF neurons during acute immobilization, and (2) repeated stress leads to plastic changes in hypothalamic CRF neurons resulting in increased AVP stores and colocalization in CRF nerve terminals.

Adrenocorticotropic Hormone↗

PubMed1992

Fetal development of delta-sleep-inducing-peptide-like immunoreactivity in hypothalamus of guinea pig with special regard to the prenatal colocalization with gonadotropin-releasing-hormone-like immunoreactivity.

Delta-sleep-inducing peptide (DSIP) colocalizes within gonadotropin-releasing-hormone (GnRH)-containing neurons in adult hypothalamus and could play a role in the regulation of hypothalamic-pituitary axis in adults. To support the possibility that DSIP also participates in fetal neuroendocrine events and to demonstrate the ontogenic evidence of coexisting neuropeptides, we have performed a detailed immunocytochemical study of DSIP- and GnRH-immunoreactivity in fetal hypothalamus of guinea pig. Using indirect immunofluorescent and sequential double-immunolabeling (elution-restaining) techniques, the results indicated that DSIP immunoreactivity was initially detected at the 38th day of gestation. In contrast to the first appearance of GnRH immunoreactivity at day 28, therefore, a 10-day delay was found. Such a delay remains as yet unexplained. From its first occurrence, DSIP immunoreactivity was always labeled with GnRH, whereas some of GnRH-immunoreactive structures did not display a DSIP immunoreactivity. But with the growth of fetus, especially before and after birth, a complete overlap between DSIP and GnRH immunoreactivity was observed throughout various regions of hypothalamus. Attention was also focused on prenatal morphological development of DSIP/GnRH- and GnRH-immunolabeled neurons. Initially, labeled neurons were visualized as uni- or bipolar types. Thereafter, their smooth and irregular subtypes could be distinguished. During later fetal age, relatively mature features were evident such as the increase of multipolar and irregularly labeled neurons. Taken together, these data provide, for the first time, anatomical evidence that DSIP exists in fetal hypothalamus and that, like GnRH, it could regulate the hypothalamic-pituitary axis during ontogenesis.

Animals↗