Intrasellar neural-adenohypophyseal choristoma. A morphological and immunocytochemical study.

A patient with mild acromegaly had recurrence of symptoms and signs of a chiasmal-area lesion seventeen years after radiation therapy for a presumed pituitary adenoma. A mass was found anterior to the pituitary gland. Abnormal tissue removed from the sphenoid sinus and sella turcica consisted of a predominantly ganglion-cell lesion. A few ganglion cells were immunoreactive for somatostatin. There were some small cysts lined by cells with immunostaining for glial fibrillary acidic protein, growth hormone or prolactin. Some cells with vacuoles and eosinophilic granules showed immunostaining for growth hormone, prolactin, ACTH, and beta-endorphin and, thus, appeared to be of adenohypophyseal origin. Cases of intrasellar ganglion-cell lesions have been reported, most of them associated with pituitary adenomas and acromegaly. The findings in this case are discussed in relation to the hypothesis that displaced, hypothalamic-type ganglion cells may produce a growth hormone-releasing factor that stimulates the development of a growth hormone-secreting pituitary adenoma. An alternative hypothesis is suggested that includes this concept, but also allows for the influence of non-neuronal cells on neuronal differentiation and for the possible influence of adenohypophyseal hormones on the replication of hypothalamic-type neurons in the lesion.

Osmotic control of vasopressin release by rat hypothalamo-neurohypophyseal explants in organ culture.

The rat hypothalamo-neurohypophyseal system (HNS) in organ culture has been used as an in vitro system for studying the osmotic control of vasopressin (VP) release. The HNS retains osmotically sensitive components as demonstrated by changes in the rate of VP release following alterations in the osmolality of the culture medium. Increasing the osmolality from 295 to 305 mosmol/kg H2O by the addition of NaCl resulted in a 2.5-fold increase in VP release. VP release was significantly decreased subsequent to reducing the osmolality from 295 to 280 mosmol/kg H2O by the addition of distilled water. Also, VP release was stimulated when the osmolality was increased to 300 mosmol/kg H2O by the addition of mannitol, but not by additions of urea or glucose which resulted in comparable increases in the tonicity of the culture medium. These studies demonstrate that the HNS in organ culture responds appropriately to osmotic challenges within the physiological range, and support Verney's concept of an osmoreceptor inasmuch as both NaCl and mannitol were effective osmotic agents.

Evidence for a thyrotropin-releasing hormone inhibitor in the pineal gland.

Extracts of bovine pineal glands were filtered through Sephadex G-25 and tested for their effect on TRH-induced TSH release in cell cultures of dispersed rat pituitary glands. Highly significant anti-TRH activity was found in material which was retarded on G-25. This material was purified sequentially on Sephadex G-10, Sephadex LH-20, and high pressure reverse phase liquid chromatography columns. The ability of partially purified material to inhibit TRH-induced TSH release by dispersed pituitary cells and to displace [3H]TRH from pituitary tumor cell membranes was interpreted as evidence for a TRH inhibitory factor (TRH-IF) in bovine pineal glands. Evidence for binding to TRH receptors included parallel competition displacement curves with synthetic TRH and data showing that excess TRH could overcome both the inhibition of TRH-induced TSH release and the inhibition of [3H]TRH binding by TRH-IF. Copurification of anti-TRH bioactivity (as tested in dispersed pituitary cell cultures) and anti-TRH-binding activity (as tested in membrane preparations) suggests that the inhibitory activity in pineal extracts results from the binding of a TRH antagonist to receptors. Reports by others of PRL release-inhibiting activity in pineal extracts were confirmed. PRL release-inhibiting activity copurified with TRH-IF.

An alternate method utilizing small quantities of ligand for affinity purification of monospecific antibodies.

An alternate method was designed to couple a limited quantity of protein to an affinity support when a conventional technique was unsuccessful. This was achieved through the introduction of a small number of sulfhydryl groups to the ligand by reaction with 2-iminothiolane which resulted in a limited number of reactive sites on the protein. Amino groups on an AH-Sepharose 4B matrix were linked to sulfhydryl groups on the ligand using the heterobifunctional agent m-maleimidobenzoyl sulfosuccinimide ester (sulfo-MBS). This method was employed to prepare an affinity support using a cytosolic protein that activates glyceraldehyde-3-phosphate dehydrogenase as a ligand. Monospecific antibody purified from the affinity column recognized only this protein on a Western blot of a cytosolic extract of kidney epithelial cells.

Immunocytochemical analysis of the rat pineal gland using antisera generated against analogs of luteinizing hormone-releasing hormone (LHRH).

Antisera generated against synthetic luteinizing hormone-releasing hormone (LHRH) or analogs of LHRH, agonists and antagonist, were used to stain the rat pineal gland and the median eminence of the hypothalamus. Of the LHRH analogs used, two revealed immunoreactive material in median eminence and not pineal, two stained pineal only, and three stained median eminence and pineal. Our observations suggest that the immunoreactive material in the pineal gland may be an LHRH-like substance whose affinity characteristics are different from that of hypothalamic LHRH. The affinity properties of hypothalamic LHRH for antibody appear to reside in the C-terminus of the molecule, whereas binding of the antigen in the pineal to antibody appear to be influenced by an N-terminal modification of the LHRH molecule.

Immunocytochemical analysis of the rat pineal gland using antisera generated against luteinizing hormone-releasing hormone (LHRH).

Seven different antisera generated against synthetic luteinizing hormone-releasing hormone (LHRH) were used to stain pineal gland and median eminence of the hypothalamus of normal rats. The LHRH immunogen complexes used to generate these antisera produced either N- or C-terminal directed antibodies or antisera that recognized both portions of the hormone molecule. Of these, four revealed LHRH-like immunoreactive material in the median eminence and pineal gland, two stained median eminence and not pineal, and one was positive in pineal only. These observations suggest that immunoreactive material in pineal gland may be an LHRH-like substance whose affinity characteristics are different from that of hypothalamic LHRH.

Immunocytochemical localization of luteinizing hormone-releasing hormone (LHRH) in Vibratome-sectioned brain.

Immunocytochemical localization of neuropeptides such as luteinizing hormone-releasing hormone (LHRH) is generally performed on Bouin's fixed tissue sections, following tissue dehydration in alcohols and embedment in paraffin. When the final reaction product accurately reflects content and distribution of the neuropeptide has not been examined carefully. Our data indicate a decrease in radioimmunoassayable LHRH content of brain fixed in Bouin's solution and a further significant reduction following dehydration with alcohol. In order to circumvent this loss of hormone, sections of fixed brain were cut on a Vibratome at 30 micron and collected in phosphate-saline buffer. These Vibratome sections revealed a significantly greater amount of overall immunoreactivity and fiber distribution as compared to paraffin-embedded sectioned tissue. These results suggest that certain neuropeptides, soluble in alcohol, can be extracted during dehydration processes necessary for paraffin embedment.

Arginine vasopressin-anti-idiotypic immunostaining of human brain cells.

Abstract Polyclonal anti-idiotypic antibodies, generated against the IgG fraction of antisera to arginine vasopressin (AVP), were shown to recognize two proteins in rat brain and bovine pituitary associated with [(3) H]AVP binding. Immunochemical analyses with these antisera revealed reactivity in paraventricular and supraoptic nucleus neuronal elements and in terminals of the posterior pituitary in the human central nervous system. With the use of a dual immunocytochemical staining technique employing both the anti-idiotype and idiotype for AVP it was possible to demonstrate a pattern of AVP-anti-idiotypic-immunoreactivity on AVP neuronal elements which suggests the existence of autoreceptors.

Pulmonary platelet trapping induced by beta-endorphin injection in the cerebrospinal fluid in dogs.

The effect of beta-endorphin injected into either the lateral ventricle or the cisterna magna on blood pressure, heart rate, peripheral platelet and leukocyte counts, hematocrit levels, catecholamines, and pulmonary platelet trapping was studied. The effect of endotoxin on the endogenous opiocortin system was also investigated. Injection of beta-endorphin caused a significant decrease in blood pressure, bradycardia, and pulmonary platelet trapping. beta-Endorphin had no effect on peripheral platelet and leukocyte counts, catecholamines, or hematocrit levels. Endotoxin shock caused a marked rise in circulating beta-endorphin and a decrease in cerebrospinal fluid beta-endorphin. Our results confirm that endotoxin shock activates the opiocortin system, and we suggest that the endorphins may participate in the evolution of the lung injury seen in septic shock.

Distribution of CRF- and tyrosine hydroxylase-immunoreactive neurons in the brainstem of the domestic fowl (Gallus domesticus).

The distribution of CRF and tyrosine hydroxylase (TH)-immunoreactive neurons was examined in the brainstem of the chicken. Very dense populations of both CRF and TH-immunoreactive (-ir) perikarya are co-extensive in separate neuronal systems throughout a large field of the rostral brainstem, encompassing locus ceruleus, the mesencephalic reticular formation, parabrachial nucleus, and the dorsal and ventral tegmental areas. They are present also in nucleus tractus solitarius, and sparsely in the ventral and lateral areas of the medulla. This co-distribution suggests that the effects of CRF upon central autonomic activity may be mediated via brainstem catecholamine systems. CRF-ir neurons alone are present also in midline nuclei, including n. centralis superior, n.annularis, n.linearis caudalis, and the raphe.

Relationship of alpha MSH-specific neurons to the arcuate opiocortin neuronal system as determined by dual antigen immunocytochemical procedures.

The cross-immunoreactivity, topography, and fiber projections of the alpha MSH-immunoreactive specific neurons in the forebrain of the rat appear to be distinctly different from that of the neurons in the hypothalamic arcuate opiocortin system. The cell bodies, immunoreactive only to alpha-MSH, have a specific pattern of distribution in the dorsal and lateral hypothalamic regions from the level of the retrochiasmatic region to the premammillary area of the posterior hypothalamus. Immunoreactive fibers of these cells appear to extend into regions of the cerebral cortex and hippocampus. An antomical relationship between the immunostained fibers and/or terminals of the arcuate opiocortin pool of neurons and the alpha-MSH-immunoreactive perikarya is described utilizing the ABC (Avidin-Biotin-Peroxidase Complex) and ABC-GO (Glucose Oxidase) or glucose oxidase-antiglucose oxidase complex methods of immunocytochemistry in which two tissue antigens with contrasting colors are demonstrated in the same tissue section.

Methods for the isolation of viable gonadotropin-releasing hormone (LRF) neurons.

Studies were conducted in order to determine if selected neurons could be isolated from the brain using Sepharose-linked recognition complexes directed against or related to the biosynthetic/neurosecretory product of the desired neuronal population. Immunoreactive LRF neurons were precipitated when dispersed cells of adult male rats were incubated successively in media containing free LRF antiserum followed by the exposure of LRF bound to Sepharose-4B. The radioimmunoassayable LRF content of the isolated cells was 88% of that contained in fresh frozen tissue of a contemporary group of rats and trypan blue exclusion indicated that at least 85% of the neurons were viable. Furthermore, based on immunocytochemistry and cresyl violet staining in combination with immunocytochemistry, the isolated cell fraction appeared to be free from other types of cells and also exhibited assayable LRF release when challenged with potassium. These results suggest that the neuroendocrine properties of hypothalamic neurons may be exploited in order to isolate viable cells for acute in vitro experiments.

Melanin-concentrating hormone (MCH) involvement in pentylenetetrazole (PTZ)-induced seizure in rat and guinea pig.

Intraperitoneal injection of 60 mg/kg of pentylenetetrazole (PTZ) induced seizure in rats, but was subthreshold and did not result in seizure in guinea pigs. Three days after intracerebroventricular (i.c.v.) injection of 75 micrograms 6-hydroxydopamine (6-OHDA) in guinea pigs, PTZ induced seizures similar to those seen in rats. In both rats and 6-OHDA-treated guinea pigs, i.c.v. injection of melanin-concentrating hormone (MCH) 15 min before PTZ prevented seizure activity. These results suggest that MCH-containing neurons may participate in the neural circuits involved in expression of PTZ-induced seizure.

Melanotropic peptides in the mammalian brain: the melanin-concentrating hormone.

Melanin-concentrating hormone (MCH) has been identified in neurons of the mammalian brain. This review summarizes some current information regarding the cell biology of this neuropeptide and the topography of MCH-immunoreactive (-IR) neurons in several species including mouse, rat, hamster, guinea pig, rabbit, dog and monkey; and atlas of MCH-IR neurons in the hypothalamus and subthalamus of the brain of guinea pig is presented. Based upon the location of this MCH cell group, it is hypothesized that they may be functionally involved in circuits of extrapyramidal motor systems from striatal centers to the thalamus and cerebral cortex and to the midbrain and spinal cord.

Interaction of putative vasopressin receptors in rat brain and bovine pituitary gland with a vasopressin anti-idiotype antibody as revealed by immunoblotting.

Vasopressin (AVP)-binding proteins were obtained from rat brain and the anterior and posterior lobes of bovine pituitary glands by (a) preparation of crude membranes, (b) solubilization of membrane proteins, (c) passage through an affinity column containing immobilized AVP, and (d) elution from the column with excess AVP. Gel electrophoresis revealed protein bands of 55 and 62 kilodaltons in rat brain, bovine posterior lobe and, to a far lesser extent, in the anterior lobe, which were similar to those previously identified in rat brain to be associated with AVP binding. Immunoblotting demonstrated that the 55 kilodalton bands of rat brain and bovine pituitary gland were selectively immunoreactive with an AVP anti-idiotype antibody. In addition, immunoreactivity occurred with a 62 kilodalton component of rat brain.

Relationship of the central ACTH-immunoreactive opiocortin system to the supraoptic and paraventricular nuclei of the hypothalamus of the rat.

ACTH-immunoreactive (ir) fibers of the central opiocortin system are present in high density in the mid-portion of the paraventricular nucleus (PVN). ACTH-ir fibers, however, do not appear to directly contact oxytocin-ir vasopressin-ir, containing perikarya or axons of vasopressinergic neurons. Magnocellular neurons located more anteriorly and posteriorly in the PVN as well as those located laterally have few ACTH-ir fibers associated with them. No ACTH-ir fibers are present in the supraoptic nucleus.

Topography of the ACTH-immunoreactive neurons in the basal hypothalamus of the rat brain.

Cell bodies of the opiocortin neurons were stained immunocytochemically with ACTH antiserum and their location in the basal hypothalamus of the rat brain was mapped. They are present throughout the entire extent of the hypothalamus, from retrochiasmatic area to mammillary body. In the retrochiasmatic area they are a single, midline group horizontally oriented and lying close to the ventral surface. Throughout the extent of the median eminence, ACTH-ir perikarya are located in the arcuate nucleus, adjacent periventricular stratrum and internuclear space between arcuate and ventromedial nuclei; as a group, they are oriented in a dorso-lateral plane. In the mammillary region, the nucleus assumes a more horizontal orientation again, lying close to the ventral surface. No regional differences were noted in cell density except in the retrochiasmatic area and terminal portion of the mammillary region where they were approximately 30% fewer in number.

Identification of catecholamine and luteinizing hormone-releasing hormone (LHRH)-containing neurons in primary cultures of dispersed cells of the basal hypothalamus.

Primary cultures of dispersed cells were prepared from 3-5 mg pieces of basal hypothalami of 10-12-day-old rats. The tissue included median eminence, arcuate nucleus and variable amounts of adjacent hypothalamus and preoptic area. The dispersion procedure consisted basically of tissue trypsinization and mechanical dissociation of cells. They were cultured in a modified L-15 medium in an air atmosphere. Neurons survived approximately 3 months. On the basis of morphological characteristics, two basic cell types could be distinguished. One was a larger (50 mum diameter) multipolar cell; microspectrofluorometric analysis revealed that a small percentage of these neurons contained a catecholamine. A second type was smaller, fusiform or ovoid and generally bipolar; a significant number of these were immunoreactive for the releasing hormone LHRH.

The effects of nitrous oxide on the central endogenous pro-opiomelanocortin system in the rat.

The hypothesis that nitrous oxide stimulates the central pro-opiomelanocortin system in vivo was explored in this study. A concentration-dependent stimulation of central pro-opiomelanocortin neuropeptides was demonstrated after exposures to variable concentrations of nitrous oxide with oxygen. Rats exposed to 60% and 80% nitrous oxide with oxygen demonstrated an elevation of beta-endorphin concentration along the neuraxis involved with analgesia; no similar effect was observed in alpha-MSH concentration, neither duration of exposure nor acclimation to the enclosed environment altered this stimulation. The discontinuation of nitrous oxide exposure resulted in the diminution of beta-endorphin concentration to pre-exposure levels in 15-30 min. With an ACTH1-39 antisera, a semiquantitative increase in opiocortin immunoreactivity after exposures to nitrous oxide was demonstrated. In conclusion, the increase in beta-endorphin concentration and immunoreactive ACTH1-39 staining in the cells of origin, areas of fiber projection and terminal fields suggest that nitrous oxide stimulates the central pro-opiomelanocortin system in vivo in the rat.

The effects of nitrous oxide on the secretory activity of pro-opiomelanocortin peptides from basal hypothalamic cells attached to cytodex beads in a superfusion in vitro system.

Dispersed cells from adult rat basal hypothalami, attached to Cytodex-3 microcarrier beads, were placed in a column and superfused with aerated high glucose media or media enriched with variable concentrations of nitrous oxide with oxygen. beta-Endorphin and alpha-MSH content was measured in the effluent collected during superfusion and demonstrated a near constant baseline release. Nitrous oxide, 60% (P less than 0.025) and 80% (P less than 0.02), caused significant increases in release of beta-endorphin. Potassium chloride (50 mM) caused a significant increase in release (P less than 0.007) of beta-endorphin whereas saline and 30% nitrous oxide did not. Neither nitrous oxide-enriched media nor potassium chloride had any statistically significant effect on alpha-MSH release. The increase in beta-endorphin secretory activity during exposure to nitrous oxide demonstrates that nitrous oxide may have a stimulatory effect on central pro-opiomelanocortin neurons.