Corticotropin-releasing factor in the dog adrenal medulla is secreted in response to hemorrhage.

To characterize the nature of CRF-like immunoreactivity (CRF-LI) in the dog adrenal, adrenal medullary, adrenal cortical, or hypothalamic tissue was extracted and subjected to RIA after partial purification on C-18 cartridges or Sephadex G-50. Using N- and C-terminal-directed antisera against rat/human (r/h) CRF, significant levels of CRF-LI were found in the adrenal medulla and hypothalamus, but not in the adrenal cortex. Immunocytochemical analysis revealed that CRF-immunoreactive cells were located in the adrenal medulla, many of them concentrated in the vicinity of blood vessels and at the border between adrenal medulla and cortex. However, the cortex was devoid of any CRF-positive cells. On reverse phase HPLC, CRF-LI in the adrenal medulla coeluted with synthetic r/hCRF. In a bioassay system, using dispersed rat anterior pituitary cells, purified adrenal CRF caused a dose-dependent increase in ACTH secretion parallel to the r/hCRF standard, indicating that dog adrenal medulla contains authentic r/hCRF. Evidence of CRF-LI secretion from the adrenal was supported by its presence in adrenal venous, but not in peripheral arterial, plasma. Adrenal venous plasma CRF-LI coeluted with r/hCRF on reverse phase HPLC after affinity chromatographic purification. The CRF-LI secretory rate in conscious trained dogs was 68 +/- 19 pg/min (concentration, 27 +/- 5 pg/ml). In response to 20% hemorrhage, the CRF-LI secretion rate rose 3-fold within 15 min and was associated with increased catecholamine secretion. The existence of a biologically active CRF-like substance in the dog adrenal medulla and its secretion in conjunction with catecholamines after a hemorrhage suggest a physiological role for this peptide other than pituitary or central nervous system regulation.

Luteinizing hormone-releasing hormone neurons in human preoptic/hypothalamus: differential intraneuronal localization of immunoreactive forms.

Luteinizing hormone-releasing hormone (LRH) may be synthesized as part of a larger prohormone, as are several other neuropeptides. In this study, we sought not only to define the distribution and morphological characteristics of LRH neurons within the human preoptic area and hypothalamus, but also to identify sites of initial synthesis, posttranslational conversion to the decapeptide, and storage of LRH in these neurons. Immunoreactive molecular forms were differentiated using a series of antisera with distinct specificities in the peroxidase-antiperoxidase technique. These antisera were capable of detecting the fully processed hormone as well as extended decapeptide sequences. Immunopositive LRH neurons were more abundant in the infundibular area of the hypothalamus than in the preoptic area. Numbers of immunopositive perikarya and subcellular distribution of reaction product varied with binding requirements of the antisera. After treatment with an antiserum that requires the fully processed decapeptide for binding, the reaction product was associated almost entirely with granules in perikarya and processes, while very little was associated with either rough endoplasmic reticulum (RER) or Golgi apparatus. In contrast, with an antiserum capable of detecting extended forms of the decapeptide, the RER and Golgi were labeled in addition to granules. From these data, we infer that in humans, mature decapeptide is present in granules within LRH neuronal perikarya and processes. Furthermore, the molecular forms associated with RER and Golgi may be precursors in which the decapeptide sequence is extended.

Acrolein: a fixative for immunocytochemical localization of peptides in the central nervous system.

Acrolein was examined as an alternative fixative to formaldehyde for immunocytochemical localization of neuropeptides in the rat brain. A brief (5 min) vascular perfusion with a 5% acrolein solution allowed the identification of thyrotropin-releasing hormone (TRH), vasoactive intestinal peptide (VIP), somatostatin (SRIF), neurotensin (NT), methionine enkephalin (Menk), adrenocorticotropic hormone (ACTH), tyrosine hydroxylase (TH), and luteinizing hormone-releasing hormone (LHRH) in fibers and perikarya within the central nervous system of the rat using the peroxidase-antiperoxidase (PAP) technique. Acrolein appears to be particularly valuable for immunocytochemistry, as it 1) stabilizes heterogeneous peptides and proteins rapidly and effectively, 2) retains antigenicity, and 3) preserves morphological detail.

Vaginocervical stimulation of ferrets induces release of luteinizing hormone-releasing hormone.

Abstract Vaginocervical stimulation of ovariectomized estradiol-primed ferrets (which are reflex ovulators) with a glass rod in the presence of a neck-gripping male induced an increase in plasma luteinizing hormone (LH) from undetectable levels (

Corticotropin-releasing factor in the adrenal medulla.

Immunoreactive and bioactive corticotropin-releasing factor has been identified in the adrenal gland of dogs, rats and humans. Radioimmunoassay and immunohistochemical experiments have clearly demonstrated that localization of the peptide is confined to the adrenal medulla. CRF-containing cells have a characteristic appearance and are often found in close association with blood vessels. Electron microscopic studies suggest that CRF is secreted at blood vessels within the adrenal medullary vasculature. CRF has also been identified in pheochromocytomas. The amount of the peptide made by such tumors is highly variable as the CRF content of pheochromocytomas may be 20 to 100 times higher or lower than that of normal adrenal tissue. The pathophysiological importance of CRF in pheochromocytomas is unknown. Excessive secretion of the peptide into the peripheral circulation may cause prolonged activation of the pituitary adrenal axis. The peptide may also act within the tumor, although its role remains obscure. Studies on chronically cannulated, awake dogs have shown that CRF is secreted into adrenal venous blood. A gradient exists between adrenal venous and peripheral arterial blood, as CRF is undetectable peripherally under resting conditions. Hemorrhage, a hemodynamic stimulus known to activate a sympathetic adrenal response, increases the CRF secretory rate. The time course of CRF secretion in response to this stimulus parallels that of epinephrine secretion. The physiological significance of adrenal medullary CRF remains to be determined. Although CRF has been shown to affect catecholamine secretion, the peptide appears to be only a weak secretagogue for catecholamines. We suggest that CRF may affect local blood flow within the adrenal medulla and may modify catecholamine secretory rates via this mechanism. The localization of CRF cells in close apposition to blood vessels supports this hypothesis.

Biosynthesis of LHRH: inferences from immunocytochemical studies.

Inferences regarding biosynthesis of LHRH in rats are made from immunocytochemical studies using LHRH antisera with varied and specific binding requirements. Immunoreactive perikarya were observed with antisera that could bind putative large molecular weight precursors of LHRH. No cells were detected with an antiserum that requires free decapeptide terminals and could not bind extended precursors. No such differential immunoreactivity was apparent in neuronal processes and neurovascular terminals. Features of intracellular processing of LHRH which can be inferred from these immunocytochemical data are: (1) the decapeptide is initially synthesized within neuronal cell bodies as a larger molecular weight peptide, extended at both the N- and C-terminals; (2) processing occurs as the newly synthesized material is transported along neuronal processes; and (3) intermediate molecular forms are converted to the active decapeptide primarily in distal portions of neuronal fibers, including the neurovascular terminal. Immunocytochemical observations in other mammalian species (humans, monkeys, ferrets and bats) allow us to further suggest that the dynamics of maturation of this hormone may differ among mammals.

LHRH neurons and their projections in humans and other mammals: species comparisons.

Using light microscopic immunocytochemistry, we have identified LHRH neurons and their projections in humans, monkeys, ferrets, bats and rats. In all these species, LHRH neurons project to the vascular contact zone of the ME, but positions of perikarya vary. This cell population, confined largely to rostral forebrain regions in rats, expands into the medial basal hypothalamus in humans, rhesus monkeys, ferrets and bats. Accompanying this expansion is an augmentation of extrahypothalamic LHRH projections. In rats, LHRH projections are primarily confined to the ME and OVLT. In humans, monkeys, ferrets and bats, however, there are also substantial projections to the posterior pituitary, habenular complex and amygdala. Although the significance of these extrahypothalamic projections is unknown, LHRH may function at some of these sites as a neuromodulator. Humans, monkeys, ferrets and bats further differ from rats in the apparent presence of mature decapeptide within perikarya. Whether variations in the dynamics of maturation of LHRH are related to differences in location of these neurons is currently under investigation.

GRF immunoreactive neurons in the paraventricular nucleus of the rat: an immunohistochemical study with monoclonal and polyclonal antibodies.

Our study demonstrates a complex GRF neuronal system within the rat hypothalamus. Using both high affinity polyclonal and high specificity monoclonal antibodies to rat (r) GRF, we have substantiated evidence for immunoreactive GRF (GRF-i) perikarya in the parvocellular portion of the paraventricular nucleus. Other hypothalamic areas containing rGRF-positive perikarya include the lateral arcuate nucleus, lateral hypothalamus, perifornical area and dorsomedial nucleus. GRF-i neuronal terminals were seen in the external zone of the median eminence, more rostrally in the periventricular nucleus, and near the suprachiasmatic nucleus and more caudally in the dorsomedial nucleus and ventral premammillary nucleus.

Characterization of LH-RH immunoreactivity in mammalian pituitary neural lobe by HPLC.

High performance liquid chromatography was used to characterize luteinizing hormone-releasing hormone (LH-RH) immunoreactivity that was previously identified immunocytochemically in the pituitary neural lobes of bats, ferrets and humans. Extracts of bat posterior lobe and hypothalamus, ferret posterior lobe and hypothalamus and human neurohypophysis were partially purified with C-18 Bond-Elut cartridges. Samples were chromatographed using a C-18 reverse phase HPLC column, and LH-RH-immunoreactive moieties were separated by gradient elution (TFA/acetonitrile solvent system). For bats and ferrets, the major peak of neural lobe LH-RH immunoreactivity eluted with a retention time identical to that of hypothalamic LH-RH. Synthetic mammalian standard added to bat and ferret hypothalamic extracts coeluted as a single peak with the predominant form of LH-RH immunoreactivity present in those tissues. In humans, the peak of LH-RH immunoreactivity in neural lobe extracts coeluted with synthetic standard. These results provide strong evidence that the LH-RH-immunoreactive fibers which terminate within the neural lobe contain authentic LH-RH. Additional minor peaks of LH-RH immunoreactivity were observed in posterior lobe and hypothalamic extracts of both bats and ferrets. Comparisons of posterior lobe content of LH-RH immunoreactivity across species verify that the neural lobe projection is a major component of the LH-RH system in bats, whereas it is represented only minimally in the laboratory rat.

Luteinizing hormone-releasing hormone (LH-RH) cells and their projections in the forebrain of the bat Myotis lucifugus lucifugus.

Luteinizing hormone-releasing hormone (LH-RH) neurons and their projections were studied by immunocytochemistry in the brains of little brown bats (Myotis lucifugus lucifugus: Chiroptera: Vespertilionidae ) as a first step in the study of relationships between these neurons and the seasonal reproductive events characteristic of this species. The majority of immunoreactive neurons in adult male, adult female, and fetal bats were ovoid bipolar cells with one thin and one thicker process, both of which gave rise to fine varicose fibers. LH-RH-immunoreactive perikarya were concentrated in the region of the arcuate nuclei in all bats examined. Perikarya were also consistently found dispersed in the mammillary region, anterior hypothalamus, preoptic areas, septum, diagonal band of Broca, and olfactory tracts; they were occasionally observed in the dorsal hypothalamus, organum vasculosum of the lamina terminalis (OVLT), habenula, amygdala, and cingulate gyrus. LH-RH-immunoreactive fibers projected heavily to the median eminence, infundibular stalk, and posterior pituitary. In extrahypothalamic areas, these fibers were especially abundant in the stria medullaris/habenula and stria terminalis/amygdala, but also contributed to the diagonal band of Broca and the olfactory tracts. Immunoreactive fibers that may be components of many different pathways clustered in the rostral septum and permeated the medial hypothalamus. LH-RH-containing fibers frequently entered the subfornical organ, but were observed less often in the OVLT and only occasionally in the pineal. The organization of the LH-RH system in the little brown bat resembles that of primates, but differs considerably from that in the rat. Anatomical characteristics of the LH-RH system in bats thus suggest that this animal may be a particularly suitable species for further study of neuroendocrine control of reproductive function as it may relate to primates, including humans.

Night roosting and the nocturnal time budget of the little brown bat, Myotis lucifugus: Effects of reproductive status, prey density, and environmental conditions.

The insectivorous bat Myotis lucifugus typically apportions the night into two foraging periods separated by an interval of night roosting. During this interval, many bats occupy roosts that are used exclusively at night and are spatially separate from maternity roosts. The proportion of the night which bats spend roosting, and thus the proportion spent foraging, vary both daily and seasonally in relation to the reproductive condition of the bats, prey density, and ambient temperature. A single, continuous night roosting period is observed during pregnancy. During lactation, females return to maternity roosts between foraging bouts, and night roosts are used only briefly and sporadically. Maximum use of night roosts occurs in late summer after young become volant. Superimposed upon these seasonal trends is day-to-day variation in the bats' nightly time budget. Long night roosting periods and short foraging periods are associated with cool nights and low prey density. This behavioral response may minimize energetic losses during periods of food scarcity.

Evidence for diverse pathways of hypophysiotropic hormone transport in mammals.

Comparative studies of mammalian hypothalamic-pituitary relationships have revealed striking variations in hypophysiotropic systems and in portal vascular architecture. Immunocytochemical studies indicate that mammalian GnRH, GHRH and somatostatin systems can project to all portions of the neurohypophysis (median eminence, infundibular stem and pituitary neural lobe). In rats, primary secretion sites are located within the median eminence and upper infundibular stem, whereas in bats, most projections extend into the lower infundibular stem and pituitary neural lobe. In ferrets and monkeys, sites of secretion appear to extend throughout the neurohypophysis, from median eminence to proximal neural lobe. In this review, these interspecific differences are examined in light of observed structural variations in portal vascular systems. Correlations suggest that hypophysiotropic hormones can be delivered to target cells in the pars distalis by diverse routes, with some species relying more heavily on long and others on short portal transport. These patterns may have important functional implications with respect to regulatory mechanisms operating within the hypothalamic-pituitary complex.

Triple-flap medial canthal reconstruction.

A patient with a medial facial defect, following oncological resection involving the medial canthus, nose, upper and lower eyelids, and the cheek, is presented. The defect was reconstructed using a combination of local flaps to provide tissue similar to native tissue, addressing both functional and aesthetic aspects.

Combined light and electron microscope immunocytochemical localization of scattered peptidergic neurons in the central nervous system.

A pre-embedding immunocytochemical technique is described for combined light and electron microscope study of peptidergic neurons in the central nervous system. The protocol is especially designed to overcome the sampling problems inherent in electron microscope study of structures, such as luteinizing hormone-releasing hormone (LHRH) neurons, that are scattered individually across large brain regions. The fixation methods outlined for several mammalian species include immersion and vascular perfusion with acrolein. Fine-structural preservation and LHRH immunoreactivity obtained with this fixative are compared to results with more conventional fixatives. Vibratome sectioning and a "pretreatment" regime, which prepare the tissues for immunocytochemistry, are described. Immunocytochemical labeling is done with free-floating sections and the peroxidase-antiperoxidase unlabeled antibody enzyme technique. Techniques are also described for the subsequent processing of immunoreacted sections for electron microscopy. These methods ensure that the processed sections are readily scanned by light microscopy, so that regions containing immunoreactive structures can be specifically chosen for electron microscope analysis. Sample electron micrographs are shown that illustrate some fine structural features of LHRH neurons in rats, bats, ferrets, and monkeys, as revealed with the techniques described.

Seasonal variations in pituitary LH-gonadotropes of the hibernating bat Myotis lucifugus lucifugus: an immunohistochemical study.

Pituitary gonadotropes were identified throughout the year in the seasonally breeding, hibernating bat Myotis lucifugus lucifugus by means of light microscopic immunohistochemistry. In both male and female bats, these cells were immunoreactive with an antiserum directed to the beta subunit of luteinizing hormone. Some gonadotropes were aggregated near a portion of the infundibular stalk which crosses the anterior lobe, while most were scattered singly in a uniform manner throughout the rest of the pars distalis. This cell population exhibited seasonal variations in both sexes. In males, the proportional volume of the pars distalis occupied by immunoreactive gonadotropes (volume fraction) was significantly reduced in late July, when plasma testosterone levels were approaching their seasonal peak. In females, the volume fraction declined in April, following ovulation, and remained low during pregnancy and lactation. The size and shape of gonadotropes appeared relatively constant throughout the annual reproductive cycle in male bats; the immunoreactive cells were irregular in shape, with cytoplasmic extensions insinuating between and often "cupping" other secretory cell types. In females, the gonadotropes resembled those of males throughout most of the year, except during pregnancy, when these cells became enlarged and ovoid. No evidence of involution was observed in these anterior pituitary cells in either males or females during hibernation.

Seasonal variations in pituitary thyrotropes of the hibernating bat Myotis lucifugus lucifugus: an immunohistochemical study.

Pituitary thyrotropes were identified throughout the year in the hibernating bat Myotis lucifugus lucifugus by means of light microscopic immunohistochemistry. These cells occupied a small proportion of the volume of the pars distalis (mean = 1.36% in males; mean = 1.52% in females) and exhibited a limited distribution pattern that was characteristic of all animals examined. Cells that were immunoreactive with an antiserum directed against the beta subunit of thyroid-stimulating hormone were most numerous in the median rostral and ventral regions; they were scarce or absent in the dorsal portion of the gland and in the extreme lateral wings. No significant seasonal variations were observed in this cell population in females. In males, however, immunoreactive thyrotropes occupied a significantly larger proportion of the pars distalis in June (following arousal from hibernation than at other times of year. No evidence of involution was observed in these anterior pituitary cells in either males or females during hibernation.

A quantitative study of pituitary colloid in the bat Myotis lucifugus lucifugus in relation to age, sex, and season.

Extracellular colloid accumulations were examined in pituitary glands of adult and juvenile, male and female, little brown bats, Myotis lucifugus lucifugus (Chiroptera: Vespertilionidae), collected at various times during the annual reproductive cycle. Round or ovoid periodic acid-Schiff-reactive accumulations of colloid were scattered throughout the pars distalis, pars intermedia, and pars tuberalis. These accumulations were surrounded by processes of agranular "follicular" cells. The proportion of the volume of the pars distalis occupied by colloid (volume fraction) exhibited considerable variation among animals, ranging from 0.00 to 3.18% in the total sample of bats. Pituitaries of juvenile males, yearling males, and nonparous females contained significantly less colloid than those of parous females and adult males. The mean volume fraction for each of these groups suggested that colloid content increased progressively from birth through sexual maturity in this animal. In addition, the frequency distribution of colloid content measurements made in parous females and adult males provided indirect evidence that colloid continues to accumulate throughout life. No sexual dimorphism in colloid content was observed before or after sexual maturity, and colloid content did not fluctuate in relation to the annual reproductive cycle of either males or females.

Immunocytochemical localization of growth hormone and growth hormone-releasing hormone immunoreactivity in the brain and pituitary of the little brown bat.

Anterior pituitary cells exhibiting growth hormone (GH) immunoreactivity and forebrain neurons containing growth hormone-releasing hormone (GHRH) immunoreactivity were identified in little brown bats (Myotis lucifugus) using light microscopic immunocytochemistry. Pituitary somatotropes appeared as ovoid or polyhedral cells that were distributed throughout most of the pars distalis, with the exception of its most rostral region where this cell type was scarce. GH-immunoreactive cells occupied approximately one-third of the total volume of the pars distalis; this proportion did not differ significantly between males and females or in bats collected at different times of year. Neuronal perikarya containing immunoreactive GHRH were observed in the hypothalamic arcuate and suprachiasmatic nuclei, as well as in the cortical and subcortical telencephalon. Fibers were most evident in the median eminence, paraventricular and periventricular nuclei, and molecular layer of the cerebral cortex. Fine fibers were also accumulated in the bed nucleus of the stria terminalis and in the amygdala.