Relationship between arginine vasotocin-like and natriuretic peptide-like immunoreactive structures in the brain of the toad Bufo marinus

The distribution of natriuretic peptide-like immunoreactivity was investigated in the brain of Bufo marinus and compared with arginine vasotocin-like immunoreactivity using fluorescence immunohistochemistry. The antisera used were rabbit anti-porcine brain natriuretic peptide, which recognises the three main structural forms of natriuretic peptides, and guinea-pig antivasopressin, which recognises arginine vasotocin. Natriuretic peptide-like immunoreactive fibres were observed in many regions of the brain, being densest in the preoptic/hypothalamic region of the diencephalon and the interpeduncular nucleus of the mesencephalon. Natriuretic peptide-like immunoreactive cell bodies were observed in the dorsal and medial pallium, the medial amygdala, the preoptic nucleus, the ventral hypothalamus, the nucleus posterodorsalis tegmenti mesencephali, and the interpeduncular nucleus. No natriuretic peptide-like immunoreactivity was seen in the pituitary gland. The distribution of arginine vasotocin-like immunoreactivity was similar to that described previously for other amphibian species. Numerous immunoreactive cell bodies were present in the preoptic nucleus whilst immunoreactive fibres were observed in the preoptic/hypothalamic region as well as in extrahypothalamic regions such as the medial amygdala and the medial pallium. Double-labelling immunohistochemistry revealed no colocalisation of arginine vasotocin-like and natriuretic peptide-like immunoreactivities in the same neural elements. The results suggest that natriuretic peptides and arginine vasotocin have distinct distributions in the brain but that natriuretic peptide-like immunoreactive fibres in the hypothalamus could influence the activity of arginine vasotocin-like immunoreactive cell bodies.     

Immunohistochemical characterization of chicken pituitary cells containing the vasotocin VT2 receptor

Research in mammals has demonstrated a variety of regulatory effects of vasopressin and oxytocin on endocrine functions of the anterior pituitary gland. Less evidence is available regarding the hypophysiotropic action of arginine vasotocin (AVT) comprising vasopressic and oxytocic activities in birds. Some hypophysiotropic effects of AVT may result from its interactions with brain circuits controlling pituitary functions, whereas others are caused by its direct affect on pituitary cells. Use of an antiserum to the vasotocin receptor VT2 (VT2R) has revealed numerous immunoreactive cells in the anterior pituitary gland of the chicken. The objective of the present study has been to identify endocrine phenotypes of chicken pituitary cells containing VT2R by means of immunohistochemical labeling. VT2R immunoreactivity has been found in all cells immunoreactive for adrenocorticotropin and alpha-melanotropin. Approximately 10% of labeled lactotropes are also immunoreactive for VT2R and lie around the anatomical boundary dividing the cephalic and caudal lobes. In both corticotropes/melanotropes and lactotropes, immunoreactive VT2R is present in a narrow layer outlining cell bodies. Immunoreactive VT2R is not found in gonadotropes, thyrotropes, or somatotropes. These results provide evidence for the important role of VT2Rs in mediating effects of AVT on endocrine secretion from corticotropes and, partially, from lactotropes.     

Immunocytochemical localization of vasotocin-like immunoreactivity in the brain of the cartilaginous fish,Scyliorhinus caniculus

Summary The distribution of vasotocin-like peptides in the central nervous system of the cartilaginous fish Scyliorhinus canicula was determined by indirect immunofluorescence and peroxidase anti-peroxidase techniques, using a specific antiserum raised in rabbits against synthetic vasotocin. Immunoreactive perikarya were mainly detected in the anterior hypothalamus, within the midcaudal part of the preoptic nucleus. The most rostral positive cell bodies were located in the dorso-lateral parts of the preoptic area, whereas at a more caudal level, they took a ventro-medial position within the deepest layers of the nucleus. Throughout the preoptic region these cells varied in shape according to their location. Occasionally, scattered vasotocin-like immunopositive cells were also identified in the nucleus periventricularis hypothalami. Vasotocin immunoreactivity was detected in numerous varicose nerve fibers of the preopticohypophysial tract. These fibers were seen to course through the medio-basal hypothalamus and caudally, after having passed the hypophysial stem, they reached the neurointermediate lobe of the pituitary. Numerous immunoreactive fibers were also observed within the rostro-medial region of the median eminence. At this level the fibers were in close proximity to the capillary loops. In the preoptic region, some stained cells exibited short processes that appeared to contact non-reactive perikarya. By comparing the distribution of vasotocin- and corticotropin-releasing factor immunoreactivity on adjacent then serial sections, it was revealed that these peptides, in S. canicula, do not coexist in the same perikarya. The present results, are compared with those obtained in other vertebrate groups, and their possible functional implications are discussed.     

An Ultrastructural and Immunocytochemical Study of Gastrodermal Cell Types in Microstomum lineare (Turbellaria,Macrostomida)

The gastrodermal cell types of Microstomum lineare (Turbellaria, Macrostomida) were studied by electron microscopy. Their immunoreactivity (IR) to bovine pancreatic peptide (BPP), FMRF-amide and vasotocin, somatostatin, neurotensin, ACTH, CCK, bombesin, secretin, gastrin/CCK and insulin antisera was tested by light microscopic immunocytochemical methods. In addition to granular club cells and phagocytic cells, neurons and neoblasts occur in the gastroderm of this turbellarian species. This is the first observation of neurons in the gastroderm of a flatworm. Dense-core vesicles (70–100 nm diameter), electron lucent cytoplasm and numerous Golgi complexes characterize the neurons. Unpolarized two-way synapses, neuromuscular junctions and polarized chemical synapses can be observed in the gastroderm. Neoblasts with large nuclei and scanty cytoplasm and differentiating cells containing clusters of basal bodies occur next to the basal lamina of the gastroderm. BPP-like, FMRF-amide-like and vasotocin-like immunoreactivity is demonstrated in the gastroderm. Both BPP and FMRF-amide IR is restricted to the basal cytoplasm of the granular club cells, while a different location for IR to vasotocin antiserum is observed. The status of the neuronal cell in the gastroderm of M. lineare is discussed in relation to endocrine (paracrine) cells and neurons in the gastroderm of invertebrates.     

Immunocytochemical investigation of the hypothalamo-neurohypophysial system in birds

Summary The results of an immunohistochemical investigation of the hypothalamo-neurohypophysial system in several species of birds have shown that: (1) mesotocin and vasotocin are synthesized in separate neurons; (2) in all species investigated the distribution of mesotocinergic and vasotocinergic perikarya follows a common pattern; (3) the external zone of the avian anterior median eminence contains exclusively vasotocinergic nerve fibers, originating in supraoptic and ventral paraventricular regions; (4) the distribution of immunoreactive elements in the neural lobe shows a definite species-dependent pattern.     

The hypothalamo-hypophyseal system of the white seabream Diplodus sargus: immunocytochemical identification of arginine-vasotocin,isotocin, melanin-concentrating hormone and corticotropin-releasing factor

The distribution of the neurosecretory hormones vasotocin, isotocin and melanin-concentrating hormone and the hypophysiotropic hormone corticotropin-releasing factor was studied in the hypothalamo-hypophyseal system of the white seabream (Diplodus sargus) using immunocytochemical techniques. Magnocellular and parvocellular perikarya immunoreactive for arginine-vasotocin and isotocin were present in the nucleus preopticus. Perikarya immunoreactive for arginine-vasotocin extended more caudally with respect to isotocin-immunoreactive perikarya. Parvocellular perikarya were located at rostroventral levels and magnocellular perikarya in the dorsocaudal portion of the nucleus. Arginine-vasotocin and isotocin did not coexist in the same neuron. Fibres immunoreactive for arginine-vasotocin and isotocin innervated all areas of neurohypophysis and terminate close to corticotropic and melanotropic cells. Perikarya immunoreactive for melanin-concentrating hormone and corticotropin-releasing factor were observed in the nucleus lateralis tuberis, with a few neurons in the nucleus periventricularis posterior. In addition, melanin-concentrating hormone immunoreactive perikarya were detected in the nucleus recessus lateralis. The preoptic nucleus did not show immunoreactivity for these antisera. Fibres showing melanin-concentrating hormone and corticotropin-releasing factor immunoreactivity ended close to the melanotropic and somatolactotrophic cells of the pars intermedia, and close to the corticotrophic cells of the rostral pars distalis.     

Estradiol Mediates Effects of Testosterone on Vasotocin Immunoreactivity in the Adult Quail Brain

In adult male quail, the activation of sexual behavior by testosterone (T) is mediated at the cellular level by the interaction of T metabolites with intracellular steroid receptors. In particular, the aromatization of T into an estrogen plays a key limiting role. Nonaromatizable androgens such 5alpha-dihydrotestosterone (DHT) synergize with estradiol (E2) to activate the behavior. Given that the density of vasotocin (VT) immunoreactive structures is increased by T in adult male quail and that VT injections affect male behavior, we wondered whether the expression of VT is also affected by T metabolites such as E2 and DHT. We analyzed here, in castrated male quail, the effects of a treatment with T, E2, DHT, or E2 + DHT on sexual behavior and brain VT immunoreactivity. The restoration by T of the VT immunoreactivity in the medial preoptic nucleus, bed nucleus striae terminalis, and lateral septum of castrated male quail could be fully mimicked by a treatment with E2. The androgen DHT had absolutely no effect on the VT immunoreactivity in these conditions and, at the doses used here, DHT did not synergize with E2 to enhance the density of VT immunoreactive structures. These effects of T metabolites in the brain were not fully correlated with their effects on the activation of male copulatory behavior, suggesting that the increase in VT expression in the brain does not represent a necessary step for the activation of behavior. Although VT expression in the medial preoptic nucleus and bed nucleus striae terminalis is often tightly correlated with the expression of male copulatory behavior, VT presumably does not represent simply one step in the biochemical cascade of events that is induced by T in the brain and leads to the expression of male sexual behavior.     

Non-polar extracts of serum from males contain covert radioimmunoassayable testosterone

Non-polar extracts of sera from human males contain immunoreactive testosterone in a form that is released by mild alkaline hydrolysis. The non-polar derivative shows no immunoreactivity with testosterone antibody prior to hydrolysis. Hydrolyzed non-polar serum extracts from ten adult male volunteers contained 2.0 +/- 0.8 (SD) ng/mL of testosterone. Neither non-polar serum extracts of normal females nor a water blank substituted for non-polar extract of serum yields any immunoreactive testosterone after alkaline hydrolysis. Testosterone palmitate hydrolyzed alone or after addition to non-polar extract of serum yields the expected quantities of radioimmunoassayable testosterone. Previously described conjugates of testosterone are polar and are neither extractable by petroleum ether nor hydrolyzable by alkali. These observations suggest that fatty acid esters of testosterone may be present in serum of human males.     

Estimation of immunoreactive testicular androgen metabolites in the urine of saddle-back tamarins

Hydrolysis, extraction, and radioimmunoassay techniques for the estimation of excreted testosterone metabolites in the urine of saddle-back tamarins have been validated and are described. The steroids measured with the testosterone antiserum used are mostly present as glucuronides and sulfates. Immunoreactivity in high-performance liquid chromatography (HPLC) fractions of urinary extracts and in a standard mixture of cortisol, testosterone, and dihydrotestosterone (DHT) were compared. The fractions with the same retention data as testosterone accounted for the major part of the immunoreactivity. Several other immunoreactive compounds of unknown identity were present in low concentrations. These results suggest that testosterone conjugates are the major steroid metabolites measured with this method in Saguinus fuscicollis. Urinary testosterone levels of castrated males were much lower than those of intact males. Testosterone treatment of castrated males resulted in a temporary superphysiological increase in the levels of urinary testosterone and in an individually variable increase in the levels of the minor immunoreactive compounds. These results suggest that estimation of testosterone metabolite levels in urine is a valid method for the assessment of testicular activity in Saguinus fuscicollis.     

Extrahypothalamic distribution of vasotocin-immunoreactive fibers and perikarya in the avian central nervous system

Immunohistochemical analysis of the extrahypothalamic distribution of vasotocin-like immunoreactive elements within the central nervous system of the domestic fowl and Japanese quail, revealed several mesencephalic, pontine and bulbar target areas topographically identifiable. Extrahypothalamic immunopositive perikarya were observed in diencephalic and mesencephalic locations after glutaraldehyde fixation.     

Comparative study of glial fibrillary acidic protein (GFAP)-like immunoreactivity in the retina of some representative vertebrates.

A polyclonal glial fibrillary acidic protein (GFAP) antiserum was used to study the distribution of GFAP-like immunoreactivity in the retina of adult vertebrates (teleosts, amphibians, reptiles, birds and mammals). GFAP-positive Müller cells were demonstrated in all the species studied, although with different degrees and patterns of immunoreactivity. In nonmammalian vertebrates, Müller cells were the only immunoreactive retinal elements. The staining was located throughout the retina of the species examined, with the exception of the rabbit, which exhibited regional variability in the expression of GFAP. The data indicate that GFAP expression in retinal Müller cells is a common feature of a wide variety of adult vertebrate species.     

Dopaminergic neurones in various retinas and the postnatal development of tyrosine-hydroxylase immunoreactivity in the rabbit retina

The localisation of tyrosine-hydroxylase immunoreactive neurones in retinas of a variety of animals were examined. Immunoreactivity was associated with specific populations of amacrine neurones in all species examined, viz; rabbit, guinea pig, monkey, cow, frog, pigeon and goldfish. Only in the goldfish was immunoreactivity also associated with processes situated in the outer plexiform layer showing that in this species catecholamine interplexiform cells exist. The development of tyrosine-hydroxylase immunoreactive neurones in the rabbit retina was also analysed. The first immunoreactive positive cells were observed by the third postnatal day. The immunoreactive positive neurones at this stage are weak and lack processes. The intensity of the immunoreactivity increases with development, but processes are lacking, until the 10th postnatal day. The immunoreactive neurones only appear fully developed by the 22nd to 28th postnatal day. Autoradiographical analysis of 3H-dopamine uptake strongly suggests that neurones containing tyrosine-hydroxylase immunoreactivity in the different retinas have the capacity to take up exogenous dopamine. It is therefore concluded that localisation of either 3H-dopamine uptake or tyrosine-hydroxylase provides a means of locating catecholamine neurones.     

Dopaminergic neurones in various retinas and the postnatal development of tyrosine-hydroxylase immunoreactivity in the rabbit retina

Summary The localisation of tyrosine-hydroxylase immunoreactive neurones in retinas of a variety of animals were examined. Immunoreactivity was associated with specific populations of amacrine neurones in all species examined, viz; rabbit, guinea pig, monkey, cow, frog, pigeon and goldfish. Only in the goldfish was immunoreactivity also associated with processes situated in the outer plexiform layer showing that in this species catecholamine interplexiform cells exist.The development of tyrosine-hydroxylase immunoreactive neurones in the rabbit retina was also analysed. The first immunoreactive positive cells were observed by the third postnatal day. The immunoreactive positive neurones at this stage are weak and lack processes. The intensity of the immunoreactivity increases with development, but processes are lacking, until the 10th postnatal day. The immunoreactive neurones only appear fully developed by the 22nd to 28th postnatal day.Autoradiographical analysis of ³H-dopamine uptake strongly suggests that neurones containing tyrosine-hydroxylase immunoreactivity in the different retinas have the capacity to take up exogenous dopamine. It is therefore concluded that localisation of either ³H-dopamine uptake or tyrosine-hydroxylase provides a means of locating catecholamine neurones.     

Immunohistochemical investigation of neuropeptides in the central nervous system of the amphioxus,Branchiostoma belcheri

The immunohistochemical localization of nine different neuropeptides was studied in the central nervous system of the amphioxus, Branchiostoma belcheri. In the brain, perikarya immunoreactive for urotensin I and FMRFamide were localized in the vicinity of the central canal. One of the processes of each of these perikarya was found to cross the dorso ventral slit-like lumen of the central canal. Oxytocin-immunoreactive short fibers, but not perikarya, were detected in the ventral part of the brain. Perikarya immunoreactive for arginine vasopressin/vasotocin, oxytocin and FMRFamide were widely distributed in the spinal cord. Arginine vasopressin/vasotocin-immunoreactive fibers often made contacts with Rohde cell axons. Angiotensin II-immunoreactive perikarya were observed in the posterior half of the spinal cord, and urotensin I-immunoreactive perikarya were found in the caudal region of the spinal cord. Cholecystokinin/gastrin-immunoreactive fibers, but not perikarya, were detected in the spinal cord; some extended as far as the ependymal layer of the cerebral ventricle. No colocalization of the peptides examined was observed. No immunoreactivity for atrial and brain natriuretic peptides nor for urotensin II was detected. The present study indicates that there are at least six separate neuronal systems that contain different peptides, respectively, in the central nervous system of the amphioxus. Their functions remain to be determined.Part of this investigation has previously been presented in abstract form (Uemura et al. 1989)     

The vasotocinergic system in the hypothalamus and limbic region of the budgerigar (Melopsittacus undulatus)

We report a morphological and biochemical analysis on the presence, distribution and quantification of vasotocin in the hypothalamus and limbic region of the budgerigar Melopsittacus undulatus, using immunohistochemistry on serial sections and competitive enzyme linked immunoadsorbent assay measurements on tissue extracts. Analysis of the sections showed large vasotocin-immunoreactive neurons in three main regions of the diencephalon, of both male and female specimens. Vasotocinergic cell bodies were located in the ventral and lateral areas of the hypothalamus, dorsal to the lateral thalamus and medial to the nucleus geniculatus lateralis. Immunoreactive neurons were placed also periventricularly, close to the walls of the third ventricle, at the level of the magnocellular paraventricular nucleus. Well evident bundles of immunoreactive fibers were placed ventral to the anterior commissure in the same regions of the hypothalamus and thalamus where vasotocinergic perikarya are localized. Fibers were identified close to the third ventricle, and in the lateral hypothalamic area along the lateral forebrain bundle. In contrast to what reported for other oscine and non-oscine avian species, we were not able to identify immunopositive neurons in any region above the anterior commissure, or detect relevant differences on the distribution of the vasotocin immmunoreactivity between sexes. Competitive enzyme linked immunoadsorption assay and image analysis of the extension of immunoreactivity in the tissue sections were consistent with the qualitative observations and indicated that there is no statistically significant dimorphism in the content of vasotocin or in the location and distribution of vasotocinergic elements in the investigated areas of male and female parrot brains.     

Comparative Physiology of Neurohypophysial Hormone Action on the Vertebrate Oviduct-Uterus

The neurohypophysial hormone, arginine vasotocin, is depletedfrom the hypothalamus, and rises in concentration in the bloodduring oviposition in hens. The contractile responses of isolatedoviducts from birds, reptiles and amphibians are more sensitiveto arginine vasotocin than to oxytocin or mesotocin. This evidenceclearly indicates that arginine vasotocin is involved in parturitionor oviposition in nonmammalian tetrapods. Evidence for a physiologicalrole for specific neurohypophysial hormones in the regulationof oviduct—or in some cases ovarian — contractilityin fishes is unclear and occasionally contradictory. However,it appears unlikely that arginine vasotocin is involved in thefish species that have been investigated. It is evident that,much like the neurohypophysial hormones, the neurohypophysialhormone receptors of the vertebrate myometrium have undergoneevolutionary change.     

Distribution of aromatase-immunoreactive cells in the forebrain of zebra finches (Taeniopygia guttata): Implications for the neural action of steroids and nuclear definition in the avian hypothalamus

Cells immunoreactive for the enzyme aromatase were localized in the forebrain of male zebra finches with the use of an immunocytochemistry procedure. Two polyclonal antibodies, one directed against human placental aromatase and the other directed against quail recombinant aromatase, revealed a heterogeneous distribution of the enzyme in the telencephalon, diencephalon, and mesencephalon. Staining was enhanced in some birds by the administration of the nonsteroidal aromatase inhibitor, R76713 (racemic Vorozole) prior to the perfusion of the birds as previously described in Japanese quail. Large numbers of cells immunoreactive for aromatase were found in nuclei in the preoptic region and in the tuberal hypothalamus. A nucleus was identified in the preoptic region based on the high density of aromatase immunoreactive cells within its boundaries that appears to be homologous to the preoptic medial nucleus (POM) described previously in Japanese quail. In several birds alternate sections were stained for immunoreactive vasotocin, a marker of the paraventricular nucleus (PVN). This information facilitated the clear separation of the POM in zebra finches from nuclei that are adjacent to the POM in the preoptic area-hypothalamus, such as the PVN and the ventromedial nucleus of the hypothalamus. Positively staining cells were also detected widely throughout the telencephalon. Cells were discerned in the medial parts of the ventral hyperstriatum and neostriatum near the lateral ventricle and in dorsal and medial parts of the hippocampus. They were most abundant in the caudal neostriatum where they clustered in the dorsomedial neostriatum, and as a band of cells coursing along the dorsal edge of the lamina archistriatalis dorsalis. They were also present in high numbers in the ventrolateral aspect of the neostriatum and in the nucleus taeniae. None of the telencephalic vocal control nuclei had appreciable numbers of cells immunoreactive for aromatase within their boundaries, with the possible exception of a group of cells that may correspond to the medial part of the magnocellular nucleus of the neostriatum. The distribution of immunoreactive aromatase cells in the zebra finch brain is in excellent agreement with the distribution of cells expressing the mRNA for aromatase recently described in the finch telencephalon. This widespread telencephalic distribution of cells immunoreactive for aromatase has not been described in non-songbird species such as the Japanese quail, the ring dove, and the domestic fowl. © 1996 John Wiley & Sons, Inc.     

GABA neurones in retinas of different species and their postnatal development in situ and in culture in the rabbit retina

Summary The localisation of GABA immunoreactive neurones in retinas of a variety of animals was examined. Immunoreactivity was associated with specific populations of amacrine neurones in all species examined, viz. rat, rabbit, goldfish, frog, pigeon and guinea-pig. All species, with the exception of the frog, possessed immunoreactive perikarya in their retinal ganglion cell layers. These perikarya are probably displaced amacrine cells because GABA immunoreactivity was absent from the optic nerves and destruction of the rat optic nerve did not result in degeneration of these cells. GABA immunoreactivity was also associated with the outer plexiform layers of all the retinas studied; these processes are derived from GABA-positive horizontal cells in rat, rabbit, frog, pigeon and goldfish retinas, from bipolar-like cells in the frog, and probably from interplexiform cells in the guinea-pig retina.The development of GABA-positive neurones in the rabbit retina was also analysed. Immunoreactivity was clearly associated with subpopulations of amacrine and horizontal cells on the second postnatal day. The immunoreactivity at this stage is strong, and fairly well developed processes are apparent. The intensity of the immunoreactivity increases with development in the case of the amacrine cells. The immunoreactive neurones appear fully developed at about the 8th postnatal day, although the immunoreactivity in the inner plexiform layer becomes more dispersed as development proceeds. The immunoreactive horizontal cells become less apparent as development proceeds, but they can still be seen in the adult retina.The GABA immunoreactive cells in rabbit retinas can be maintained in culture. Cultures of retinal cells derived from 2-day-old animals can be maintained for up to 20 days and show the presence of GABA-positive cells at all stages. In one-day-old cultures the GABA immunoreactive cells lacked processes but within three days had clearly defined processes. After maintenance for 10 days a meshwork of GABA-positive fibres could also be seen in the cultures.     

Vasotocinergic innervation of the septal region in the Japanese quail: sexual differences and the influence of testosterone

Summary Vasotocin (VT)-immunoreactive fibres were observed in the nuclei of the quail (Coturnix coturnix japonica) septal region. Their distribution in the nucleus septalis lateralis (SL) and the nucleus striae terminalis (nST) was sexually dimorphic: a dense network of immunoreactive fibres was seen in adult sexually stimulated males but not in females. Experimental manipulation of the hormonal environment influenced this distribution only in males. VT immunoreactivity was absent in SL and nST when male quail were exposed to a shortday photoperiod or castrated. The immunoreactivity was restored to its original level in castrated males by silastic implants of testosterone.