Colocalization of neuropeptide Y (NPY)-like and FMRFamide-like immunoreactivities in the brain of the Atlantic salmon (Salmo salar)

Summary The colocalization of the peptides neuropeptide Y (NPY) and Phe-Met-Arg-Phe-NH₂ (FMRFamide) in the brain of the Atlantic salmon was investigated with double immunofluorescence labeling and peroxidase-antiperoxidase immunocytochemical techniques. Colocalization of NPY-like and FMRE amide-like immunoreactivities was observed in neuronal cell bodies and fibers in four brain regions: in the lateral and commissural nuclei of the area ventralis telencephali, in the nucleus ventromedialis thalami, in the laminar nucleus of the mesencephalic tegmentum, and in a group of small neurons situated among the large catecholaminergic neurons in the isthmal region of the brainstem. All cell bodies in these nuclei were immunoreactive to both NPY and FMRF. We consistently observed larger numbers of FMRF-immunoreactive than NPY-immunoreactive fibers. In the nucleus ventromedialis thalami NPY- and FMRFamide-like immunoreactivities were colocalized in cerebrospinal fluid (CSF)-contacting neurons. NPY-immunoreactive, but not FMRF-immunoreactive, neurons were found in the stratum periventriculare of the optic tectum, and at the ventral border of the nucleus habenularis (adjacent to the nucleus dorsolateralis thalami). Neurons belonging to the nucleus of the nervus terminalis were FMRF-immunoreactive but not NPY-immunoreactive. The differential labeling indicates, as do our cross-absorption experiments, that the NPY and FMRFamide antisera recognize different epitopes. Thus, it is probable that NPY-like and FMRFamide-like substances occur in the same neurons in some brain regions.     

Neurotransmitter candidates in the retina of the mudpuppy,Necturus maculosus

Summary Neurons accumulating (³H)-glycine and (³H) GABA were demonstrated with the use of autoradiography. Both were accumulated by different types of amacrine cells, similar those of goldfish. (³H)-GABA was also accumulated by horizontal cells, again similar to the goldfish. These results and physiological studies from other laboratories suggest that GABA and glycine are neurotransmitter candidates in amacrine cells of the mudpuppy.Immunoreactive neuropeptide Y (NPY), glucagon, vasoactive intestinal peptide (VIP), somatostatin, substance P, and neurotensin were found in different types of stratified amacrine cells. Weakly immunoreactive enkephalin and bombesin processes were also seen in the inner plexiform layer. Gastrin-immunoreactive neurons were not detectable.Endogenous 5-hydroxytryptamine was visualized immunohistochemically in a population of diffuse amacrine cells and some cells in the ganglion cell layer. This suggests that 5-hydroxytryptamine may be a neurotransmitter in the retina of the mudpuppy.     

Connectivity of the salamander pretectum: an in-vitro (whole-brain) intracellular tracing study

The amphibian optic tectum and pretectum have been analyzed in detail anatomically and physiologically, and a specific model for tecto-pretectal interaction in the context of the visual guidance of behavior has been proposed. However, anatomical evidence for this model, particularly the precise pattern of pretecto-tectal connectivity, is lacking. Therefore, we stained pretectal neurons intracellularly in an in-vitro preparation of the salamanders Plethodon jordani and Hydromantes genei. Our results demonstrate that the projections of neurons of the nucleus praetectalis profundus are divergent and widespread. Individual neurons may project divergently to telencephalic (ipsilateral amygdala and striatum), diencephalic (ipsi-and contralateral thalamus, contralateral pretectum), and mesencephalic (ipsi- and contralateral tectum and tegmentum) centers, and to the ipsi- and contralateral medulla oblongata and rostral spinal cord. The projection of pretectal cells to the optic tectum is bilateral; axonal structures do not show discernible patterns and are present in all layers of the superficial white matter. A classification of pretectal neurons on the basis of axonal termination pattern or dendritic arborization has not been possible. Our results do not support the hypothesis that a distinct class of pretectal neurons projects to a particular subset of tectal cells. Rather, the pretectum appears to influence the tectum indirectly, acting either on retinal afferents or modulating inhibitory interneurons.     

Cholinergic and catecholaminergic neurons relay striatal information to the optic tectum in amphibians.

In the amphibians Rana perezi and Xenopus laevis, the involvement of cholinergic and catecholaminergic neurons in the relay of basal ganglia inputs to the tectum was investigated. Tract-tracing experiments, in which anterograde tracers were applied to the basal ganglia and retrograde tracers to the optic tectum, were combined with immunohistochemistry for choline acetyltransferase and tyrosine hydroxylase. The results of these experiments suggest that dopaminergic neurons of the suprachiasmatic nucleus and pretectal region, noradrenergic cells of the locus coeruleus and the cholinergic neurons of the pedunculopontine and laterodorsal tegmental nuclei mediate at least part of the basal ganglia input to the tectum in anurans.     

多种神经活性物质在幼年猫和鸡视网膜神经...

An improved post-embedding immunocytochemical technique was used to examine the coexistence of multi-neuroactive substances in neurons of young cat and newly hatched chicken retinas. We found that two classical neurotransmitters-glycine (Gly) and r-aminobutyric acid (GABA) and one neuropeptide, neurotensin (NT) were located in the amacrine cells of cat and chicken retinas. Among them, some cells contain Gly and GABA (Gly/GABA AC), some cells contain Gly and NT (Gly/NT AC) or GABA and NT (GABA/NT AC), and the other cells, a few of them contain Gly, GABA and NT (Gly/GABA/NT AC). In addition, two classical neurotransmitters Gly and GABA were also located or coexisted in the horizontal cells of cat retina. These results indicated that the coexistence of two neurotransmitters or one neurotransmitter and one neuropeptide or two neurotransmitters and one neuropeptide was presented in neurons of developing can and chicken retinas.     

Effect of retinal ablation on the expression of calbindin D28k and GABA in the chick optic tectum

The effect of retinal ablation on qualitative and quantitative changes of calbindin D28k and GABA expression in the contralateral optic tectum was studied in young chicks. Fifteen days old chicks had unilateral retinal ablation and after 7 or 15 days, calbindin expression was analyzed by Western blot and immunocytochemistry. Neuronal degeneration was followed by the amino-cupric silver technique. After 15 days, retinal lesions produced a significant decrease in calbindin immunostaining in the neuropil of layers 5-6 and in the somata of neurons from the layers 8 and 10 of the contralateral tectum, being this effect less marked at 7 days post-lesion. Double staining revealed that 50-60% of cells in the layers 8 and 10 were calbindin and GABA positive, 30-45% were only calbindin positive and 5-10% were only GABAergic neurons. Retinal ablation also produced a decrease in the GABA expression at either 7 or 15 days after surgery. At 7 days, dense silver staining was observed in the layers 5-6 from the optic tectum contralateral to the retinal ablation, which mainly represented neuropil that would come from processes of retinal ganglion cells. Tectal neuronal bodies were not stained with silver, although some neurons were surrounded by coarse granular silver deposits. In conclusion, most of calbindin molecules are present in neurons of the tectal GABAergic inhibitory circuitry, whose functioning apparently depends on the integrity of the visual input. A possible role of calbindin in the control of intracellular Ca2+ in neurons of this circuit when the visual transmission arrives to the optic tectum remains to be studied.     

Expression of Kisspeptins and Kiss Receptors Suggests a Large Range of Functions for Kisspeptin Systems in the Brain of the European Sea Bass

This study, conducted in the brain of a perciform fish, the European sea bass, aimed at raising antibodies against the precursor of the kisspeptins in order to map the kiss systems and to correlate the expression of kisspeptins, kiss1 and kiss2, with that of kisspeptin receptors (kiss-R1 and kiss-R2). Specific antibodies could be raised against the preprokiss2, but not the preoprokiss1. The data indicate that kiss2 neurons are mainly located in the hypothalamus and project widely to the subpallium and pallium, the preoptic region, the thalamus, the pretectal area, the optic tectum, the torus semicircularis, the mediobasal medial and caudal hypothalamus, and the neurohypophysis. These results were compared to the expression of kiss-R1 and kiss-R2 messengers, indicating a very good correlation between the wide distribution of Kiss2-positive fibers and that of kiss-R2 expressing cells. The expression of kiss-R1 messengers was more limited to the habenula, the ventral telencephalon and the proximal pars distalis of the pituitary. Attempts to characterize the phenotype of the numerous cells expressing kiss-R2 showed that neurons expressing tyrosine hydroxylase, neuropeptide Y and neuronal nitric oxide synthase are targets for kisspeptins, while GnRH1 neurons did not appear to express kiss-R1 or kiss-R2 messengers. In addition, a striking result was that all somatostatin-positive neurons expressed-kissR2. These data show that kisspeptins are likely to regulate a wide range of neuronal systems in the brain of teleosts.     

Neurochemical Organization of the Turtle Pretectum: An Immunohistochemical Study. Comparative Analysis

A comparative analysis of neurochemical organization of pretectal nuclei was performed on the basis of results of our studies on immunoreactivity to monoamines (tyrosine hydroxylase—TH and serotonin—5-HT), neuropeptides (substance P—SP, met-enkephalin—mEnk, and neuropeptide Y, NPY), and gamma-aminobutyric acid (GABA) in pretectum of two turtle species, as well as of the corresponding literature data obtained in other reptile species, birds, and mammals. Their presumed homologous pretectal nuclei were shown to have both similar and different immunohistochemical features. A conclusion is made that species of divergent lines of amniotes have not only the evolutionary conservatism of the neurochemical organization of pretectal structures and their projection pathways but also its certain plasticity due to rearrangements in the course of phylogenetic development.     

The distribution of GABA-like-immunoreactive neurons in the brain of the newt,Triturus cristatus carnifex,and the green frog,Rana esculenta

Summary The distribution of -aminobutyric acid (GABA) immunoreactivity was studied in the brain of two amphibian species (Triturus cristatus carnifex, Urodela; Rana esculenta, Anura) by employing a specific GABA antiserum. A noteworthy immunoreactive neuronal system was found in the telencephalic dorsal and medial pallium (primordium pallii dorsalis and primordium hippocampi) and in the olfactory bulbs. In the diencephalic habenular nuclei there was a rich GABAergic innervation, and immunoreactive neurons were observed in the dorsal thalamus. In the hypothalamus the GABA immunoreactivity was found in the preoptic area, the paraventricular organ and in the hypothalamo-hypophysial complex. In the preoptic area of the frog some GABA-immunoreactive CSF-contacting cells were shown. In the optic tectum immunolabeled neurons were present in all the cellular layers. A rich GABAergic innervation characterized both the fibrous layers of the tectum and the neuropil of the tegmentum and interpeduncular nucleus. In the cerebellum, in addition to the Purkinje cells showing a variable immunopositivity, some immunoreactive cell bodies appeared in the central grey. Abundant immunolabeled nerve fibers in the acoustico-lateral area and some immunopositive neurons in the region of the raphe nucleus were observed. In conclusion, the GABAergic central systems, well-developed in the amphibian species studied, were generally characterized by close similarities to the pattern described in mammals.Dedicated to Professor Valdo Mazzi (Dipartimento di Biologia Animale, Università di Torino), in honor of his 70th birthday     

多种神经活性物质在幼年猫和鸡视网膜神经细胞中的共存(简报)

视网膜起源于前脑泡,结构简单层次分明,因此常被视为脑的简化模型。但近期工作证明,视网膜包含的神经活性物质(神经递质、调质和神经肽)种类之多、分布之复杂并不亚于脑。尤其是无长突细胞不仅包含多种递质和肽类,而且还有递质与肽类或肽类与肽类在一个细胞中的共存。对视网膜神经递质、调质和神经肽的研究将是探索脑奥秘的有效途径。     

Neuropeptides and monoamines in the carp (Cyprinus carpio) pretectum: An immunocytochemical study

The distribution of neurotensin, neurokinin A, dynorphin A, galanin, somatostatin-28 (1-12), neuropeptide Y, vasoactive intestinal polypeptide, gastrin-releasing peptide, gamma-melanocyte stimulating hormone, alpha-neo-endorphin, angiotensin H, cholecystokinin-8, serotonin and tyrosine hydroxylase has been studied in the pretectal nuclei of the Cyprinus carpio: nuclei pretectalis superficialis parvicellularis and magnocellularis, pretectalis centralis, pretectalis, and pretectalis periventricularis dorsalis and ventralis using an indirect immunoperoxidase technique. We have found neuropeptide Y and serotonin immunoreactive fibres in all pretectal nuclei, whereas gastrin-releasing peptide immunoreactive fibres were visualized in the nuclei pretectalis superficialis parvicellularis and magnocellularis, pretectalis centralis. pretectalis and pretectalis periventricularis dorsalis; neurokinin A immunoreactive fibres in the nuclei pretectalis superficialis parvicellularis and magnocellularis and pretectalis periventricularis dorsalis; galanin immunoreactive fibres in the nuclei pretectalis superficialis parvicellularis, pretectalis centralis and pretectalis periventricularis dorsalis; and neurotensin immunoreactive fibres in the nucleus pretectalis periventricularis dorsalis. Additionally, immunoreactive cell bodies containing neuropeptide Y were observed in the nuclei pretectalis superficialis parvicellularis and pretectalis periventricularis dorsalis, and serotonin and tyrosine hydroxylase cell bodies were found in the nuclei pretectalis periventricularis dorsalis and ventralis respectively. The presence of the neuroactive substances found in the carp pretectal nuclei suggest that they might be involved in the regulation of certain functions within the visual system.     

Pretectal and Tectal Connections Revealed by Tracing Technique in theTurtle Intergeniculate Leaflet

Using antero- and retrograde tracer techniques, it has been shown that the intergeniculate leaflet nucleus (IGL) in turtles (Testudo horsfieldi, Emys orbicularis) has reciprocal connections with pretectum and tectum. Neurons projecting to this nucleus are located in the retino- and non-retinorecipient nuclei of pretectum and, mostly, in the deep periventricular layer of tectum. Neurons containing neuropeptide Y (NPY) and GABA are a possible source of projections from IGL to the same structures of pretectum and tectum. The data obtained allow concluding the presence of a morphological substrate for interaction of the circadian and non-circadian (visual-motor, perceptive) visual systems in reptiles as well as in other amniotes—birds and mammals.     

Localization and characterization of neuropeptide Y-like peptides in the brain and islet organ of the anglerfish (Lophius americanus)

Summary Results from a previous report demonstrate that more than one molecular form of neuropeptide Y-like peptide may be present in the islet organ of the anglerfish (Lophius americanus). Most of the neuropeptide Y-like immunoreactive material was anglerfish peptide YG, which is expressed in a subset of islet cells, whereas an additional neuropeptide Y-like peptide(s) was localized in islet nerves. To learn more about the neuropeptide Y-like peptides in islet nerves, we have employed immunohistochemical and biochemical methods to compare peptides found in anglerfish islets and brain. Using antisera that selectively react with either mammalian forms of neuropeptide Y or with anglerfish peptide YG, subsets of neurons were found in the brain that labelled with only one or the other of the antisera. In separate sections, other neurons that were labelled with either antiserum exhibited similar morphologies. Peptides from brains and islets were subjected to gel filtration and reverse-phase high performance liquid chromatography. Radioimmunoassays employing either the neuropeptide Y or peptide YG antisera were used to examine chromatographic eluates. Immunoreactive peptides having retention times of human neuropeptide Y and porcine neuropeptide Y were identified in extracts of both brain and islets. This indicates that peptides structurally similar to both of these peptides from the neuropeptide Y-pancreatic polypeptide family are expressed in neurons of anglerfish brain and nerve fibers of anglerfish islets. The predominant form of neuropeptide Y-like peptide in islets was anglerfish peptide YG. Neuropeptide Y-immunoreactive peptides from islet extracts that had chromatographic retention times identical to human neuropeptide Y and porcine neuropeptide Y were present in much smaller quantities. These results are consistent with the hypothesis that peptides having significant sequence homology with human neuropeptide Y and porcine neuropeptide Y are present in the nerve fibers that permeate the islet.     

Colocalization of vasoactive substances in the endothelial cells of human umbilical vessels

Human umbilical vessels are unique in lacking any innervation; thus endothelial cells may play the major role in local control and regulation of the blood flow. In the present study, we examined ultrathin sections of cultured human umbilical vein endothelial cells and tissue preparations of umbilical vein and artery, immunostained by the post-embedding colloidal gold double-labelling technique. We observed colocalization of atrial natriuretic peptide and neuropeptide Y, as well as colocalization of atrial natriuretic peptide and neuropeptide Y with other vasoactive substances, namely, vasoactive intestinal peptide, substance P, calcitonin gene-related peptide and arginine vasopressin. The functional significance of the colocalization of these vasoactive substances in the human umbilical vessel endothelial cells is discussed.     

Intraganglionic laminar endings and their relationships with neuronal and glial structures of myenteric ganglia in the esophagus of rat and mouse

Intraganglionic laminar endings (IGLEs) represent the only vagal mechanosensory terminals in the tunica muscularis of the esophagus and may be involved in local reflex control. We recently detected extensive though not complete colocalization of the vesicular glutamate transporter 2 (VGLUT2) with markers for IGLEs. To elucidate this colocalization mismatch, this study aimed at identifying markers for nitrergic, cholinergic, peptidergic, and adrenergic neurons and glial cells, which may colocalize with VGLUT2 outside of IGLEs. Confocal imaging revealed, besides substantial colocalization of VGLUT2 and substance P (SP), no other significant colocalizations of VGLUT2 and immunoreactivity for any of these markers within the same varicosities. However, we found close contacts of VGLUT2-positive structures to vesicular acetylcholine transporter, choline acetyltransferase, neuronal nitric oxide synthase, galanin, neuropeptide Y, and vasoactive intestinal peptide immunoreactive cell bodies and varicosities, as well as to glial cells. Neuronal perikarya were never positive for VGLUT2. Thus, VGLUT2 was almost exclusively found in IGLEs and may serve as a specific marker for them. In addition, many IGLEs also contained SP. The close contacts established by IGLEs to myenteric cell bodies, dendrites, and varicose fibers suggest that IGLEs modulate various types of enteric neurons and vice versa.     

Intraganglionic laminar endings and their relationships with neuronal and glial structures of myenteric ganglia in the esophagus of rat and mouse

Intraganglionic laminar endings (IGLEs) represent the only vagal mechanosensory terminals in the tunica muscularis of the esophagus and may be involved in local reflex control. We recently detected extensive though not complete colocalization of the vesicular glutamate transporter 2 (VGLUT2) with markers for IGLEs. To elucidate this colocalization mismatch, this study aimed at identifying markers for nitrergic, cholinergic, peptidergic, and adrenergic neurons and glial cells, which may colocalize with VGLUT2 outside of IGLEs. Confocal imaging revealed, besides substantial colocalization of VGLUT2 and substance P (SP), no other significant colocalizations of VGLUT2 and immunoreactivity for any of these markers within the same varicosities. However, we found close contacts of VGLUT2-positive structures to vesicular acetylcholine transporter, choline acetyltransferase, neuronal nitric oxide synthase, galanin, neuropeptide Y, and vasoactive intestinal peptide immunoreactive cell bodies and varicosities, as well as to glial cells. Neuronal perikarya were never positive for VGLUT2. Thus, VGLUT2 was almost exclusively found in IGLEs and may serve as a specific marker for them. In addition, many IGLEs also contained SP. The close contacts established by IGLEs to myenteric cell bodies, dendrites, and varicose fibers suggest that IGLEs modulate various types of enteric neurons and vice versa.     

Localization of neuropeptide Y in human sympathetic ganglia: Correlation with met-enkephalin, tyrosine hydroxylase and acetylcholinesterase

Summary The relationships of immunoreactive neuropeptide Y, enkephalin and tyrosine hydroxylase, on the one hand, and acetylcholinesterase histochemical activity, on the other, were studied in human lumbar sympathetic ganglia. Two thirds of the ganglion cells contained immunoreactive neuropeptide Y. Electron microscopically the immunoreaction was localized in the Golgi apparatus and in large dense-cored vesicles in the nerve endings. Most of the neuropeptide-containing neurons and nerve fibres were also reactive for tyrosine hydroxylase. Nerve fibres reactive for neuropeptide Y were found around ganglion cells regardless of their transmitter contents, whereas enkephalin-reactive nerve terminals surrounded only acetylcholinesterase-containing neurons. The results demonstrate that neuropeptide Y is colocalized with noradrenaline in most of the human sympathetic neurons and that the nerve fibres may innervate selectively the noradrenergic and cholinergic subpopulations of ganglion cells depending on the transmitters of the nerves.     

Colocalization of neuropeptides and NADPH-diaphorase in the intra-adrenal neuronal cell bodies and fibres of the rat

Colocalization of vasoactive intestinal peptide, neuropeptide Y, calcitonin gene-related peptide, substance P, and tyrosine hydroxylase, respectively, with NADPH-diaphorase staining in rat adrenal gland was investigated using the double labelling technique. All vasoactive intestinal peptide- and some neuropeptide Y-immunoreactive intrinsic neuronal cell bodies seen in the gland were double stained with NADPH-diaphorase. Double labelling also occurred in some nerve fibres immunoreactive to vasoactive intestinal peptide and neuropeptide Y in the medulla and cortex. No colocalization of calcitonin gene-related peptide, substance P or tyrosine hydroxylase immunoreactivity with NADPH-diaphorase staining was observed. However, nerve fibres with varicosities immunoreactive for all the neuropeptides examined were closely associated with some of the NADPH-diaphorase-stained neuronal cell bodies. Thus, in rat adrenal gland, nitric oxide is synthesized in all ganglion cells containing vasoactive intestinal peptide and in some containing neuropeptide Y, but not in those containing calcitonin gene-related peptide, substance P or tyrosine hydroxylase.