Correlative fluorescence-immunocytochemical technique for the localization of monoamines and neurophysin (NP).

A correlative fluorescence-immunocytochemical technique for the localization of monoamines and neurophysin on sections of freeze-dried tissues is described. An extensive network of monoamine-containing perikarya and terminals was found throughout the hypothalamus and median eminence. Immunocytochemical localization of antisera for neurophysin was found in the supraoptic and paraventricular nuclei of the hypothalamus and in both the zona interna and zona externa of the median eminence. This correlative demonstration of both catecholamines and neuropeptides within the same tissue provides a new approach to the study of neurotransmitters and neurohormones and their role in the regulation of the peripheral endocrine system.     

The role of catecholamines and glucagon on serum and liver metallothionein response to restraint stress

The role of glucagon and catecholamines on serum and liver metallothionein (MT) concentrations in basal and stress conditions has been studied. Glucagon showed no effect on serum MT either in basal (unstressed) or stress (20 hours of restraint) conditions. In contrast, glucagon administration increased both unstressed and stressed levels of liver MT. No effect of the alpha + beta-catecholamine blocker labetalol on serum MT levels was observed in unstressed rats. However, the administration of labetalol abolished the increase in serum MT levels caused by stress. These data suggest that catecholamines might be involved in serum MT regulation during stress, while they might not be important in the maintenance of basal serum MT levels. Finally, no significant effect of adrenergic blockade was found on basal and stress levels MT, in agreement with previous data from this laboratory.     

Effects of p-chloroamphetamine, a serotonin-depleting drug, on the median eminence and pituitary pars intermedia

p-Chloroamphetamine (PCA), an agent known to cause depletion of levels of brain serotonin in rodents, was administered to rats in three sequential injections (10mg/kg) to study effects on the hypothalamic median eminence and pituitary gland. One week following the initial sequence of injections of PCA, light and electron micrographs revealed degenerate fibers in the outer zone of the median eminence. Lower drug doses or single 10-mg/kg doses did not lead to morphologic changes. Neuronal processes located in the pituitary intermediate lobe appeared normal although there was a significant increase in the numbers of secretory granules contained within intermediate lobe cells drug-treated rats, as compared to controls. Fluorometric analysis of levels of catecholamine and indoleamine showed a decrease in serotonin in median eminence and pons-medulla, but no change in that of the pituitary. Levels of dopamine and norepinephrine remained unchanged after PCA treatment. The data suggest that fibers affected in the median eminence contain serotonin. Processes in the intermediate lobe may be resistant to the serotonin-lowering effects of PCA observed in brain tissue. In addition, PCA may directly affect granule release from pituitary cells, or may alternatively act on hypothalamic regions which affect the release of intermediate lobe cell hormones.     

Structure of the tail fin in teleosts

The influence of adrenalectomy and administration of hypertonic saline on the amount of vasopressin, oxytocin, and neurophysin contained in the median eminence and the neural lobe of rats was studied by means of the following methods: (i) morphometric and microphotometric analyses of aldehyde fuchsin-stained histological sections of the neurohypophysis; (ii) immunohistochemical demonstration of vasopressin, oxytocin, and neurophysin in the neurohypophysis, and (iii) radioimmunological measurement of vasopressin and oxytocin in extracts of the median eminence and the neural lobe. Adrenalectomy increases the amount of vasopressin and neurophysin in the external layer of the median eminence but does not change the content of oxytocin. It has no influence on the amount of vasopressin, oxytocin, and neurophysin demonstrable in the inner layer of the median eminence and in the neural lobe two weeks after the operation. Hypertonic saline markedly diminishes the vasopressin, oxytocin, and neurophysin content of the inner layer of the median eminence and the neural lobe but reduces only slightly, if at all, the amount of vasopressin and neurophysin in the outer layer of the median eminence. The findings support the concept that osmotic stress reduces only the vasopressin and oxytocin content of the hypothalamus-neural lobe system and has no or only little influence on the vasopressin content of the outer layer of the median eminence.     

Ultrastructural studies on the localization of neurohypophysial hormones and their carrier proteins.

Neurophysin, vasopressin and oxytocin were localized in different portions of the supraopticohypophysial tract (SHT) using the unlabeled antibody enzyme technique at the ultrastructural level. In vasopressin-positive supraoptic perikarya, vasopressin and neurophysin were present in all neurosecretory granules. Within the zona interna of the median eminence, vasopressin and neurophysin were present in two populations of axons, one with granules of 1300-1500 A and one with granules of 900-1300 A. Following exposure of thin sections of median eminence to antiserum to neurophysin, reaction products were present in granules and in the extragranular cytoplasm in the axons with larger granules; in all other cases reaction product was confined to the granules. Vasopressin-positive fibers were also presented in large numbers of the zona externa of the median eminence and many terminated on the pituitary primary portal plexus. A few oxytocin fibers were present on the portal capillaries in the infundibular stalk. In the posterior pituitary all axon profiles were neurophysin positive. Neurophysin was present as both a granular and cytoplasmic pool. Vasopressin-containing axons account for 90% of the neuronal elements in the posterior pituitary and oxytocin for the remaining 10%. Findings on the subcellular distribution of these peptides are related to current theories on transport and release of neurohormones.     

Video-enhanced technique for detecting neurophysin, enkephalin, and somatostatin immunoreactivity in ultrathin sections of cat median eminence

A freeze-drying technique using epoxy-embedded ultrathin serial sections permits critical comparisons of neuropeptides in small fibers and varicosities of the nervous system by video-enhanced, light microscopic immunofluorescence. The desirability of the method was documented by data showing: retention of radioimmunoassayable somatostatin in freeze-substituted blocks of tissue as compared to its loss in tissue dehydrated in an alcohol series; feasibility of OsO4 vapor fixation of freeze-dried tissue and compatibility with neuropeptide immunocytochemistry, and utility of a silicon-intensified-tube video camera for recording low levels of fluorescence from ultrathin sections. Ultrathin serial sections, 150 nm thick, from the inner zone of freeze-dried median eminence of the cat revealed three populations of axons containing various combinations of neurophysin immunoreactivity and enkephalin immunoreactivity. Some elements contained neurophysin immunoreactivity alone, some contained both neurophysin immunoreactivity and enkephalin immunoreactivity, and a few elements contained enkephalin immunoreactivity alone. The adjacent external zone of the median eminence contained immunoreactivity for all three substances, but the structures in this region were too small to permit demonstration of coexistence in 150 nm thick sections.     

Neurosecretion in the external and internal zone of the median eminence of the cat and dog

Summary The immunoglobulin-enzyme bridge technique, in association with rabbit antiporcine neurophysin-II has been applied to the median eminence of the dog and cat in order to study the distribution of neurophysin-like antigens throughout this area of the brain and correlate the findings with the corresponding distribution of neurosecretory material (NSM) as revealed by the crotonaldehyde fuchsin stain.Neurophysin and NSM were both present in the hypothalamo-supraoptico-neurohypophysial system—the pathway taken by oxytocin, vasopressin and neurophysin from the hypothalamus to the posterior pituitary lobe.Whereas the tuberoinfundibular tract of the median eminence was almost devoid of NSM, the presence of neurophysin-like material was clearly evident using immunoperoxidase histochemistry. The significance of a protein in the external median eminence possessing determinants cross-reactive against anti-neurophysin serum is discussed.This work was financed by a grant from the Medical Research Council of New Zealand.     

Effects of stress and beta-funal trexamine pretreatment on morphine analgesia and opioid binding in rats

This study was essentially an in vivo protection experiment designed to test further the hypothesis that stress induces release of endogenous opioids which then act at opioid receptors. Rats that were either subjected to restraint stress for 1 hr or unstressed were injected ICV with either saline or 2.5 micrograms of beta-funaltrexamine (beta-FNA), an irreversible opioid antagonist that alkylates the mu-opioid receptor. Twenty-four hours later, subjects were tested unstressed for morphine analgesia (tail-flick assay) or were sacrificed and opioid binding in brain was determined. [3H]D-Ala2NMePhe4-Gly5(ol)enkephalin (DAGO) served as a specific ligand for mu- opioid receptors, and [3H]-bremazocine as a general ligand for all opioid receptors. Rats injected with saline while stressed were significantly less sensitive to the analgesic action of morphine 24 hr later than were their unstressed counterparts. Beta-FNA pretreatment attenuated morphine analgesia in an insurmountable manner. Animals pretreated with beta-FNA while stressed were significantly more sensitive to the analgesic effect of morphine than were animals that received beta-FNA while unstressed, consistent with the hypothesis that stress induces release of endogenous opioids that would protect opioid receptors from alkylation by beta-FNA. beta-FNA caused small and similar decreases in [3H]-DAGO binding in brain of both stressed and unstressed animals. Stressed rats injected with saline tended to have increased levels of [3H]DAGO and [3H]-bremazocine binding compared to the other groups. This outcome may be relevant to the tolerance to morphine analgesia caused by stress.     

Immunocytochemical identification of neurophysin-secreting neurons in the hypothalamo-neurohypophysial system of some non-mammalian vertebrates

Antiserum raised against a mammalian neurophysin, porcine neurophysin-II, was used in conjugation with the immunoperoxidase histochemical technique to detect neurophysin in the hypothalamus of the chickens, frog and goldfish. In the chickens, the paraventricular and supraoptic nuceli as well as the internal and external zones of the median eminence stained for neurophysin. Material in the perikarya of the frog and goldfish preoptic nucleus also cross-reacted immunologically against anti-porcine neurophysin-II serum. Serial dilutions of the anti-mammalian neurophysins serum were carried out in order to ascertain at which point the 3-layer immunocytochemical reaction ceased to localize neurophysin. In the chicken, frog and goldfish as well as in the rat, neurosecretory structures became difficult to visualize between 12800 and 25400 fold dilution of antiserum. The results demonstrate that the immunological cross-reactivity previously observed between an anti-mammalian neurophysin serum and the neurophysin isolated from mammals of varying phylogeny also extends to certain non-mammalian vertebrates and is suggestive of a structural homology of neurophysin from different species.     

Identification of neurophysin producing cells

Immuno-enzyme histochemical investigations on the bovine hypothalamus showed that the infundibular nucleus contains neurons that produce either neurophysin I or a neurophysin I-like substance. Fine processes of these neurons run in the direction of the median eminence. The possibility that these processes could be the origin of the "neurophysin I-oxytocin" containing nerve fibres of the external region of the median eminence is discussed.     

Naloxone inhibition of stress-induced increase in prolactin secretion

Naloxone, an opiate antagonist that acts by binding to opiate receptors in the brain, was given to rats stressed by immobilization or heat in an attempt to inhibit stress-induced release of prolactin. Both stresses resulted in approximately a 5-fold increase in serum prolactin concentration. Naloxone, at a dose of 0.2 mg/kg b.w. completely or partially inhibited the stress-induced rises in serum prolactin, and reduced serum prolactin concentrations in unstressed rats to below control values. It is concluded that endorphins may be responsible for increased release of PRL during stressful conditions.     

D1 receptor mechanisms in the median eminence and their inhibitory regulation of LHRH release

D1 receptor mechanisms in the median eminence have been studied by means of immunocytochemistry using antisera against dopamine and cyclic AMP-regulated phosphoprotein-32 (DARPP-32) and tyrosine hydroxylase (TH) and by autoradiography using the iodinated analogue of the D1 receptor antagonist SCH-23390. The co-distribution of DARPP-32 and TH immunoreactivity (IR) and of DARPP-32 and luteinizing hormone releasing hormone (LHRH) IR was analysed in the median eminence by means of computer-assisted morphometry and microdensitometry. Functional analysis involved studies on the role of D1 receptors in the regulation of LH serum levels in rats treated with nicotine in the absence and presence of the D1 receptor antagonist. LH serum levels were measured by means of radioimmunoassay procedures.The results on the co-distribution of TH and DARPP-32 IR in the median eminence which were obtained both by analysis of adjacent sections and by two-colour immunocytochemistry on the same section, demonstrated a high degree of overlap of TH and DARPP-32 IR nerve terminals and tanycytes within the medial and lateral palisade zone. Furthermore, studies on LHRH and DARPP-32 IR nerve terminals and tanycytes in the median eminence with the same methodologies demonstrated preferential overlaps within the lateral palisade zone. The overlap area was about 50% of the LHRH or DARPP-32 immunoreactive area in this region. Density maps were also obtained on the distribution of LHRH and DARPP-32 immunoreactive profiles at various rostrocaudal levels. Correlation studies demonstrated a significant and positive co-distribution of LHRH and DARPP-32 immunoreactive terminals and tanycytes within the lateral palisade zone and the subependymal layer (when all DARPP-32 positive squares were considered) of the median eminence. Instead within the medial palisade zone a significant negative correlation coefficient was found, when all the LHRH positive squares were considered.In the receptor autoradiographical analysis a weak-to-moderate labelling was obtained of the part outside the mediobasal hypothalamus using the D1 receptor radioligand [¹²⁵I]SCH-23982, while hardly any labelling was found within the median eminence and the arcuate nucleus.SCH-23390 was found to counteract, in a dose-related way, the inhibitory effects of intermittent nicotine treatment on serum LH levels. The D2 receptor antagonist raclopride in a dose of 1 mg/kg did not counteract the inhibitory effects of nicotine on serum LH levels.The present immunocytochemical, autoradiographic and functional studies suggest the existence of a D1 receptor in the median eminence which can be blocked by the D1 receptor antagonist SCH-23390 in behaviourally relevant doses and which is masked under basal conditions in the male rat. It is proposed that one type of median eminence D1 receptor is located on the axon terminals, not linked to DARPP-32, and which may make possible a rapid regulation of hypothalamic hormone release, e.g. LHRH release from the nerve terminals in the lateral palisade zone as indicated in the present morphological and functional experiments. The other type of median eminence D1 receptor may be located on the tanycytes and linked to DARPP-32. It is suggested that this D1 receptor is responsible for a long-term regulation of hypothalamic hormone release inter alia LHRH release from the terminal and preterminal parts of the LHRH axons in the lateral palisade zone and subependymal layer, respectively.     

Neuropeptides in the median eminence

The presence and possible sources of more than 30 neuropeptides in the median eminence are summarized. The median eminence is the brain area which contains neuropeptides in the highest number and in the highest concentrations in the central nervous system. This area constitutes the final common pathway for signals from the brain to the pituitary. Many peptidergic fibers enter the median eminence and terminate around the pericapillary space and release their neuropeptides into hypophysial portal blood vessels. Other peptidergic fibers traverse the median eminence and terminate in the posterior pituitary. According to their origin, fibers in the median eminence can be classified as intra- or extrahypothalamic fibers. The neuropeptide-containing fibers in the median eminence are mainly intrahypothalamic, they reach the median eminence through either the lateral retrochiasmatic area or the tuberoinfundibular tract. Depending on the site of their action, neuropeptides may be either neurohormones acting on the anterior pituitary cells or neurotransmitters affecting the release of substances from other nerve terminals within the median eminence.     

Effects of Hyperprolactinemia on Plasma Prolactin and Glucose and on Local Cerebral Glucose Utilization

Elevated blood levels of prolactin increase the synthesis, turnover, and release of 3,4-dihydroxyphenylethylamine (dopamine) from the tuberoinfundibular dopaminergic neurons, which project to the median eminence. The present study examined whether hyperprolactinemia also increases local cerebral glucose utilization, as determined by the 2-deoxy-D-[1-14C]glucose method, in the median eminence and other brain structures. Adult male rats were given ovine prolactin (4 mg/kg) subcutaneously every 8 h for 48 h. This treatment exerted an autoregulatory feedback effect on endogenous rat prolactin secretion, as evidenced by decreased circulating levels of rat prolactin. Ovine prolactin treatment also decreased plasma glucose concentrations. However, in both partially immobilized and free-ranging rats, glucose utilization in brain structures containing tuberoinfundibular dopaminergic cell bodies (the arcuate nucleus) and terminals (the median eminence) was not affected by ovine prolactin treatment. Hyperprolactinemia was, however, associated with decreased glucose utilization in the medial forebrain bundle and the CA subfield of the dorsal hippocampus. The lack of a significant effect of prolactin treatment on glucose utilization in the median eminence indicates that the resolution of the deoxyglucose technique, as used here, is not adequate to detect the ovine prolactin-induced increase in tuberoinfundibular dopaminergic neuronal activity, that the median eminence does not utilize glucose as its primary energy substrate, or that ovine prolactin treatment causes a counterbalancing decrease in the activity of other neurons projecting to the median eminence.     

Effects of preconditioning on electrolyte leakasge and lipid composition in black spruce (Picea mariand) stressed with polyethylene giycol

Black spruce ( Picea mariana Mill. B. S. P.) rooted cuttings were grown in solution culture and preconditioned by osmotically stressing plants with polyethylene glycol. After relief from preconditioning stress, water relations, membrane leakiness, and the composition of lipids and fatty acids were compared in preconditioned and control, unconditioned plants. Both groups of plants were subsequently subjected to a severe osmotic stress with polyethylene glycol and examined again. Osmotic stress decreased shoot water potentials and increased the leakage of electrolytes from shoots of stressed, compared with unstressed, plants. However, both unstressed and stressed preconditioned plants leaked less electrolytes compared with unconditioned plants. Changes in sterol, phospholipid and glycolipid composition were observed in preconditioned unstressed and stressed plants. Sterol and phospholipid levels de clined as a result of stress, and preconditioning resulted in a decline in sterol: phospholipid ratios in plants.     

A comparison of neuropeptide immunocytochemistry in fluid-fixed and freeze-dried brains

Summary Immunocytochemical staining of luteinizing hormone-releasing hormone (LHRH), somatostatin, and neurophysin was compared in rat brains fixed with 1) formalin, 2) Bouin's solution, 3) freeze-dried (FD), or 4) freeze-dried + paraformaldehyde vapor perfused (FDV). The distribution of LHRH fibers was similar in all preparations; however, beads of granular reaction product often appeared finer and more numerous in the median eminence of FD- and FDV brains. Positively stained LHRH perikarya were not observed in any of the preparations. In contrast, somatostatin-immunoreactive perikarya were present in the fluid-fixed and FD brains, although few were observed in FDV brains. Somatostatin-immunoreactive fibers were present in all preparations, but appeared most numerous in the median eminence of FD brains. Staining of neurophysin-containing perikarya and fibers was similar in all preparations. These observations suggest that the FD brain can provide a suitable tissue substrate for immunocytochemistry, demonstrating staining comparable to or surpassing that of more conventional preparations. However, staining of antigens in FD brain was not uniformly successful and may depend on stereochemical characteristics of each antigen as well as properties of the primary antisera used in the staining procedure.     

Dual effects of (D-Ala2,Met5)-enkephalinamide on CRF and ACTH secretion

An intra-third ventricular administration of (D-Ala2,Met5)-enkephalinamide (DALA) did not elevate plasma ACTH and corticosterone levels in unanesthetized freely moving rats, but intra-third ventricular administration of DALA and methionine (Met)-enkephalin potentiated a mild stress (hanging for 10 or 30 sec)-induced plasma ACTH and corticosterone elevations in unanesthetized freely moving rats. DALA and Met-enkephalin seemed to stimulate CRF release from the median eminence to increase plasma ACTH, as the CRF concentration in the median eminence area was reduced after injection in these stressed rats. When hypothalamic tissues were perifused in vitro, DALA (1-100 ng/ml) reduced the release of CRF. These results suggest that the opiates seem to have a dual effect on the CRF-ACTH system depending on which action overrides the other.     

Differential regional distribution and release of two forms of gonadotropin-releasing hormone in the chicken brain

The functional significance of two molecular forms of gonadotropin-releasing hormone (GnRH) in the chicken brain was investigated. The differential distribution of [Gln8]GnRH (chicken GnRH-I, cGnRH-I) and [His5,Trp7,Tyr8]GnRH (chicken GnRH-II, cGnRH-II) was determined using high performance liquid chromatography and radioimmunoassay with region-specific antisera. Potassium-stimulated release of immunoreactive cGnRH-I and cGnRH-II from brain regions was assessed in tissue incubations. cGnRH-I and cGnRH-II varied independently in different brain areas. The concentration of cGnRH-I was highest in the median eminence of the hypothalamus, and a small quantity was also detected in the midbrain and cerebrum. cGnRH-II was more widely distributed throughout the brain, with highest concentrations in areas of the hypothalamus outside the median eminence and in the medulla. Potassium stimulated the release of cGnRH-I from the median eminence 4-fold, while cGnRH-II release was not detectable. Neither cGnRH-I nor cGnRH-II was released from the medulla. These data suggest: 1) cGnRH-I is the prime regulator of gonadotropin release from the pituitary, and 2) cGnRH-II may have a neurotransmitter or neuromodulator role in areas of the brain outside the median eminence.     

Memory Enhancing Activity of Naringin in Unstressed and Stressed Mice: Possible Cholinergic and Nitriergic Modulation

The present study was designed to evaluate the effect of Naringin on memory of unstressed and stressed Swiss young albino mice. Naringin (80?mg/kg, i.p.) and donepezil (10?mg/kg) were administered for 21 successive days to separate groups of unstressed and stressed mice. The nootropic activity was evaluated using elevated plus maze and Hebbs Williams Maze. Brain acetylcholinesterase (AChE), brain nitrite and plasma corticosterone levels were also estimated. unpredictable chronic mild stress was produced by using different stressors. Naringin (80?mg/kg) and donepezil significantly showed memory enhancing activity in both unstressed and stressed mice. Naringin significantly reduced brain AChE activity and brain nitrite levels in both unstressed and stressed mice. Naringin (80?mg/kg) significantly reversed scopolamine-induced amnesia in unstressed and stressed mice. 7-Nitroindazole [a neuronal nitric oxide synthase (NOS) inhibitor] and aminoguanidine (an inducible NOS inhibitor) significantly enhanced memory improving activity and brain nitrite decreasing effect of naringin in unstressed and stressed mice respectively. Plasma corticosterone levels were significantly decreased by naringin (80?mg/kg) in stressed mice as compared to its control. Thus, naringin showed memory enhancing activity in unstressed mice probably by decreasing brain AChE activity and by inhibition of neuronal NOS. The memory enhancing activity of naringin in stressed mice might be due to decrease in brain AChE activity, inhibition of inducible NOS and also by decreasing the elevated plasma corticosterone levels.     

Hypothalamo-neurohypophysial system in hagfish: localization of neurophysin-, arg-vasopressin- and oxytocin-like immunoreactivity

Serial paraffin sections of hagfish brain (Eptatretus burgeri) were immunocytochemically examined for porcine neurophysin (NEU), arg-vasopressin (AVP) and oxytocin (OT). A big number of NEU-immunoreactive nerve cells were visible in the frontal part of the ventromedial hypothalamus in close vicinity of the third ventricle. These cells were bipolar in most cases and sometimes additionally stained for AVP but not for OT. NEU-reactive neurons extended axons to the median eminence and the posterior lobe of the pituitary which also contained AVP or OT-like immunoreactivity. Immunostaining outside the hypothalamus could not be observed. It is likely that there exists a mammalian-like oxytocinergic and a vasopressinergic hypothalamo-neurohypophysial system in hagfish, despite the large differences in levels of organization.