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PubMed2006

Sexually dimorphic immunoreactivity of galanin and colocalization with arginine vasotocin in the chicken brain (Gallus gallus domesticus).

The bed nucleus of the stria terminalis medialis (BSTM) of adult chickens (Gallus gallus domesticus) was previously shown to synthesize arginine vasotocin (AVT) in males only and coincides spatially and temporally with steroid activity regulating male reproductive behavior. Galanin has been shown to be a potent modulator of the behavioral and neuroendocrine responses in the mammalian BSTM and in other sexually dimorphic brain regions. In the present study of adult chickens the morphological relationship of AVT and galanin was examined by immunohistochemical analysis of two limbic structures, the BSTM and the lateral septum (SL). The analysis also included the hypothalamic nuclei supraopticus (SON) and paraventricularis (PVN). In males galanin and AVT were both synthesized in the BSTM, while in females neither galanin nor AVT was present. Furthermore, in the males galanin and AVT were colocalized in the majority of neurons within BSTM and in fibers of the SL. In both sexes galanin neurons in the PVN were scattered between the distinct clusters of AVT neurons and there was no colocalization of galanin and AVT in single PVN neurons. Furthermore, AVT immunoreactivity was significantly higher in the SON than in the PVN in both sexes. In the SON, galanin was colocalized with AVT in significantly more neurons in hens than in males (P </= 0.05%). These results demonstrate that the distributions of galanin and AVT are sexually dimorphic not only in the limbic BSTM but also in the hypothalamic SON. It is tempting to speculate that galanin in the SON is involved in regulation of oviposition as an AVT-dependent female-specific function.

Animals↗
PubMed1989

Immunoreactivity in Limulus: III. Morphological and biochemical studies of FMRFamide-like immunoreactivity and colocalized substance P-like immunoreactivity in the brain and lateral eye.

FMRFamide-like immunoreactivity (FLI) and the colocalization of FMRFamide and substance P-like (SPLI) immunoreactivities were examined in the brain and lateral eye of the horseshoe crab with FITC- and TRITC-labeled secondary antibody techniques. In the brain, fibers with FLI were localized in the neuropils of the lamina, medulla, central body, corpus pedunculatum, optic tract, circumesophageal connective, and central neuropil. An extensive network of reactive fibers innervatives the brain's vascular sheath. Somata with FLI were found in the dorsal medial group, dorsal lateral posterior groups #1 and #2, and ventral posterior lateral groups #1 and #2. Several distinct subgroups of reactive somata were noted in both the medullar and ventral medial groups. The distribution of fibers in the brain with colocalized FLI and SPLI includes those which innervate the vascular sheath and widespread populations of small-diameter beaded fibers in the central neuropil and circumesophageal connective. Somata with colocalized FLI and SPLI constitute minority populations in the medullar and dorsal medial groups but form the majority population of a subgroup in the ventral medial group. Overall localization of SPLI was reevaluated and is reported here according to the nomenclature of the new Chamberlain and Wyse brain atlas. In addition to those previously reported, somata with SPLI were found in the dorsal lateral posterior groups #1 and #2, the ventral lateral posterior groups #1 and #2, and several distinct subgroups of the medial and ventral medial groups. In the retina of the lateral eye, fibers with both FLI and SPLI ramify in the lateral plexus and ultimately innervate the corneal epidermis. Brain homogenates were examined for immunoreactive (ir) FMRFamide and ir-substance P with radioimmunoassay techniques. Ir-FMRFamide and ir-substance P eluted in different fractions from both gel filtration chromatography and HPLC. Furthermore, the binding curves for both substances were similar to those of the corresponding synthetic compounds. Brain homogenates were also bioassayed on the lateral eye. Three gel filtration fractions mimic natural circadian activity by increasing the sensitivity of the lateral eye, but they were not coincident with ir-FMRFamide or ir-substance P. Although it is not completely resolved what the active molecules in these fractions are, it is clear that neither ir-FMRFamide nor ir-substance P is a possible candidate.

Animals↗
PubMed1993

Distribution and colocalization of choline acetyltransferase immunoreactivity and NADPH diaphorase reactivity in neurons within the medial septum and diagonal band of Broca in the rat basal forebrain.

NADPH diaphorase histochemistry and choline acetyltransferase immunocytochemistry were used to assess quantitatively the presence of nitric oxide synthase in the cholinergic neurons of the magnocellular basal forebrain complex. Virtually all (97%) NADPH diaphorase reactive magnocellular neurons in the medial septum and the vertical and horizontal limbs of the diagonal band of Broca were choline acetyltransferase immunoreactive, whereas only a proportion of the choline acetyltransferase immunoreactive neurons were NADPH diaphorase reactive. Thus NADPH diaphorase histochemistry identified a subpopulation of the magnocellular cholinergic neurons. Occasionally, NADPH diaphorase reactive neurons were observed within the medial septum and diagonal band of Broca that were not choline acetyltransferase immunoreactive, and in general were morphologically distinct from the magnocellular neurons; such neurons are probably representatives within the medial septum and diagonal band of more widely distributed phenotypically distinct populations of NADPH diaphorase reactive neurons. The proportions of the neurons in which choline acetyltransferase and NADPH diaphorase colocalized in the medial septum and in the diagonal bands of Broca were similar in any one coronal section, but there was a considerable difference in the proportions throughout the rostrocaudal extent of these nuclei. In the most rostral sections of the medial septum and diagonal band, approximately 70% of the choline acetyltransferase immunoreactive neurons were NADPH diaphorase reactive, whereas the proportion decreased progressively to about 30% at the level of the decussation of the anterior commissure. To examine further the extent of colocalization throughout the magnocellular basal forebrain complex, sections of the magnocellular preoptic nucleus, substantia innominata, and nucleus basalis magnocellularis were examined. While there was little total colocalization of choline acetyltransferase immunoreactivity and NADPH diaphorase reactivity in any particular section (approximately 18%), almost all of the double labelled neurons were in the substantia innominata, with very few in the other nuclei. Thus although there is a caudal to rostral gradient of the proportion of magnocellular cholinergic neurons that are NADPH diaphorase reactive throughout the entire basal forebrain magnocellular complex, subregions, such as the substantia innominata and magnocellular preoptic nucleus, may not follow this trend. The recent demonstration that the NADPH diaphorase histochemical reaction localizes a nitric oxide synthase suggests that attention should be given to the NADPH diaphorase subpopulation in pathological and experimentally induced alterations of the basal forebrain.

Animals↗
PubMed2006

Hexose transporters GLUT1 and GLUT3 are colocalized with hexokinase I in caveolae microdomains of rat spermatogenic cells.

Postmeiotic spermatogenic cells, but not meiotic spermatogenic cells respond differentially with glucose-induced changes in [Ca2+]i indicating a differential transport of glucose via facilitative hexose transporters (GLUTs) specifically distributed in the plasma membrane. Several studies have indicated that plasma membrane in mammalian cells is not homogeneously organized, but contains specific microdomains known as detergent-resistant membrane domains (DRMDs), lipid rafts or caveolae. The association of these domains and GLUTs isoforms has not been characterized in spermatogenic cells. We analyzed the expression and function of GLUT1 and GLUT3 in isolated spermatocytes and spermatids. The results showed that spermatogenic cells express both glucose transporters, with spermatids exhibiting a higher affinity glucose transport system. In addition, spermatogenic cells express caveolin-1, and glucose transporters colocalize with caveolin-1 in caveolin-enriched membrane fractions. Experiments in which the integrity of caveolae was disrupted by pretreatment with methyl-beta-cyclodextrin, indicated that the involvement of cholesterol-enriched plasma membrane microdomains were involved in the localization of GLUTs and uptake of 2-deoxyglucose. We also observed cofractionation of GLUT3 and caveolin-1 in low-buoyant density membranes together with their shift to higher densities after methyl-beta-cyclodextrin treatment. GLUT1 was found in all fractions isolated. Immunofluorescent studies indicated that caveolin-1, GLUT1, and hexokinase I colocalize in spermatocytes while caveolin-1, GLUT3, and hexokinase I colocalize in spermatids. These findings suggest the presence of hexose transporters in DRMDs, and further support a role for intact caveolae or cholesterol-enriched membrane microdomains in relation to glucose uptake and glucose phosphorylation. The results would also explain the different glucose-induced changes in [Ca2+]i in both cells.

3-O-Methylglucose↗
PubMed2006

Choline acetyl transferase and neuropeptide immunoreactivities are colocalized in somata, but preferentially localized in distinct axon fibers and boutons of cat sympathetic preganglionic neurons.

Cholinergic sympathetic preganglionic neurons (SPN) coexpress the biosynthetic enzyme for acetylcholine, choline acetyl-transferase (ChAT), and neuropeptides such as enkephalin (ENK) in their cell bodies. However, it is not clear whether they also coexpress ChAT and neuropeptides in axon fibers and boutons. To explore coexpression of ChAT and neuropeptides in somata and axon processes of SPN, we investigated, using immunohistochemistry, retrograde labeling, confocal analysis, and tridimensional reconstruction, whether ChAT and the peptides neurotensin, methionine-ENK, somatostatin, calcitonin gene-related peptide, and vasoactive intestinal peptide colocalize in somata, axons fibers, and boutons of cat SPN. Practically, complete colocalization for these peptides and ChAT was observed in SPN somata. Conversely, in most instances we observed independent localization of immunoreactivity (IR) for ChAT and the peptides in axon fibers and boutons. The minor colocalization between ChAT- and peptide-IR in preganglionic fibers could correspond to a sequential axonal transport of ChAT and peptides, since we observed coexistence of these transmitters after blocking axonal transport. Contrary to Dale's principle, our results suggest that SPN can synthesize ChAT and peptides in their cell bodies and route them to distinct axon boutons or terminals in sympathetic ganglia. Presence of axon boutons containing either ChAT or neuropeptides lead us to suggest a new neurochemical pattern of cotransmission in sympathetic ganglia based on the concurrent release of transmitters and cotransmitters from distinct presynaptic boutons, rather than in the corelease of these mediators from the same axon process. The possibility that cellular segregation could be transient and depend on functional requirements is considered.

Animals↗
PubMed1988

Immunohistochemical and biochemical analysis of serotonin and substance P colocalization in the nucleus tractus solitarii and associated afferent ganglia of the rat.

In a previous study of afferent projections to the nucleus tractus solitarii (NTS), it was shown that over half of the retrogradely-labelled neurons in the nucleus raphe pallidus contained serotonin-immunoreactivity and over half of these neurons contained substance P-immunoreactivity, suggesting that these two putative neurotransmitters are colocalized in NTS-afferent neurons. The objectives of the present study were to 1) directly determine if varicosities in the NTS, the area postrema (AP), and the dorsal motor nucleus of the vagus nerve (DMN) do contain both transmitters, 2) determine if primary afferent neurons in the nodose and pretrosal ganglia might also colocalize serotonin and substance P, and 3) quantify the amount of substance P that is contained in serotonergic varicosities in the NTS. Distributions and colocalization of substance P and serotonin in the NTS were studied using dual-color immunohistochemistry, while the quantity of substance P in serotonergic varicosities was assessed by radioimmunoassay (RIA) using micropunches from the NTS of 5,7-dihydroxytryptamine-(5,7 DHT-) and vehicle-treated rats. Varicosities that contained both serotonin- and substance P-immunoreactivity were found in the NTS, the DMN, and the AP. Double-labelled varicosities were common in the NTS and DMN (i.e., qualitatively similar to the density seen in the hypoglossal nucleus and in the ventral horn of the cervical spinal cord); however, the vast majority of the varicosities in these autonomic areas only displayed immunoreactivity for one or the other of these transmitters. This paucity of doubly-labelled varicosities, in comparison to the number of singly-labelled varicosities, was reflected in the lack of a significant decrease in substance P levels as determined by RIA of micropunches taken from caudal and intermediate levels of the NTS in 5,7 DHT- and vehicle-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Afferent Pathways↗
PubMed1997

Method to screen for relevant yeast two-hybrid-derived clones by coimmunoprecipitation and colocalization of epitope-tagged fragments--application to Bcl-xL.

The yeast two-hybrid system is a powerful genetic method to screen cDNA libraries to identify protein-protein interactions. A screen with a typical bait may yield many clones, including ones that are not biologically relevant which need to be eliminated by evaluating each clone in an alternative assay to confirm the interaction with the bait protein. We have developed an efficient assay to quickly screen two-hybrid-derived clones for coimmunoprecipitation and colocalization with the bait protein in mammalian cells. Gene fragments derived from a two-hybrid screen are cloned into an epitope tagging vector that can generate high levels of epitope-tagged protein in mammalian cells. The vector expressing an epitope-tagged protein is then cotransfected into mammalian cells with an expression vector for the bait protein. Interaction between the bait protein and epitope-tagged protein is evaluated by coimmunoprecipitation and colocalization. We demonstrate the utility of this approach by applying it to clones isolated in a two-hybrid screen using Bcl-xL as bait, showing that two-hybrid-derived fragments of Bad and Bax, previously known to interact with Bcl-xL, both colocalize and coimmunoprecipitate with Bcl-xL.

Animals↗
PubMed1999

Colocalization within the nucleolus of two highly related IFN-induced human nuclear phosphoproteins with nucleolin.

We have previously reported the identification of two interferon (IFN)-induced cDNAs which code for two proteins, named 41 and 75, which have homology to a number of proteins involved in regulating gene expression. Here we establish that these cDNAs correspond to in vivo synthesized mRNAs. Expression of the 41 and 75 cDNAs, both in vitro and in vivo, generated proteins of 30 and 68 kDa, respectively. In a variety of mammalian cells, 41 and 75 were found to be located in the nucleus, with 41 being localized to the nucleolus, whereas 75, although it is mainly concentrated at the periphery of the nucleolus, is also found throughout the nucleoplasm. Treatment with interferon results in a translocation of 41 to the periphery of the nucleolus and it is in this region that the two proteins colocalize. 41 and 75 were found to colocalize with nucleolin but not with B23 or fibrillarin, three nucleolar proteins involved in ribosome synthesis. This colocalization was not affected by low concentrations of actinomycin D. In view of this and since 41 and 75 have homology to proteins involved in regulating gene expression, we suggest that, in association with nucleolin, they play a role in ribosome biogenesis.

Amino Acid Sequence↗
PubMed2001

MMP-2 colocalizes with caveolae on the surface of endothelial cells.

We examined the spatial distribution of MMP-2 on the surface of human endothelial cells using immunofluorescence and confocal microscopy. Staining endothelial cells with MMP-2-specific antibodies revealed a punctate labeling at the basolateral side of the cell periphery, which colocalized with patches of caveolin-1, a major constituent of the caveolae. This colocalization was confirmed by immunogold electron microscopy. MT1-MMP, TIMP-2, and the alphavbeta3 integrin exhibited a similar pattern of staining, with pericellular patches that colocalized with either MMP-2 or caveolin-1. The presence of MT1-MMP and TIMP-2 in caveolae patches could be seen only after treatment with concanavalin A, which induced MMP-2 activation but had no noticeable effect on the pattern or intensity of MMP-2 immunostaining. In contrast, MMP-9 and TIMP-1 staining showed a pattern completely different from that of MMP-2 and TIMP-2, with positive spots uniformly distributed throughout the cell body. Our data show that MMP-2, its activator the MT1-MMP, and its proposed receptor, the alphavbeta3 integrin, are all targeted to the same membrane microdomains on the endothelial cell, thereby restricting matrix proteolysis to a limited microenvironment at the cell surface.

Adult↗
PubMed2000

Statistical evaluation of colocalization patterns in immunogold labeling experiments.

The ultrastructural localization of various antigens in a cell using antibodies conjugated to gold particles is a powerful instrument in biological research. However, statistical or stereological tools for testing the observed patterns for significant clustering or colocalization are missing. The paper presents a method for the quantitative analysis of single or multiple immunogold labeling patterns using interpoint distances and tests the method using experimental data. The clustering or colocalization of gold particles was detected using various characteristics of the distribution of distances between them. Pair correlation and cross-correlation functions were used for exploratory analysis; second order reduced K (or cross-K) functions were used for testing the statistical significance of observed events. Confidence intervals of function values were estimated by Monte Carlo simulations of the Poisson process for independent particles, and results were visualized in histograms. Furthermore, a suitability of K functions modified by censoring or weighting was tested. The reliability of the method was assessed by evaluating the labeling patterns of nascent DNA and several nuclear proteins with known functions in replication foci of HeLa cells. The results demonstrate that the method is a powerful tool in biological investigations for testing the statistical significance of observed clustering or colocalization patterns in immunogold labeling experiments.

Cell Nucleus↗
PubMed2001

Human-herpesvirus-8-encoded K8 protein colocalizes with the promyelocytic leukemia protein (PML) bodies and recruits p53 to the PML bodies.

Promyelocytic leukemia protein (PML) bodies are nuclear sites for both input viral genome deposition and immediate-early (IE) gene transcription during infection with certain human DNA viruses, such as human cytomegalovirus (HCMV), herpes simplex virus type 1, and adenovirus. In this study, we showed that the K8 (K-bZIP) protein, an early protein encoded by the human herpesvirus 8 (HHV-8), colocalized with the PML bodies in HHV-8-infected primary effusion lymphoma cells. Cotransfection of two plasmids expressing the K8 protein and green-fluorescence protein (GFP)-PML fusion protein into 293T cells revealed that the K8 protein colocalized with PML in cells with high PML expression. Overexpression of the K8 protein in Chinese hamster ovary (CHO) cells with stable GFP-PML expression did not induce the dispersion of the PML bodies, unlike the IE1 protein of HCMV. Transfection of a truncated K8 gene revealed that the leucine zipper domain of the K8 protein was required for the colocalization with PML. We also demonstrated that the K8 protein bound to p53 in vivo and in vitro, and that high expression of the K8 protein caused the accumulation of p53 to the PML bodies in CHO cells, suggesting that the K8 protein functions in the recruitment of p53 to the PML bodies. These data suggest that the K8 protein may be associated with the functional modulation of p53 in the nucleus during the lytic phase of HHV-8.

Animals↗
PubMed1994

The distribution and colocalization of neuropeptides and catecholamines in nerves supplying the gall bladder of the toad, Bufo marinus.

The indirect immunofluorescence technique was used to determine the distribution of peptide-containing axons in the gall bladder of the cane toad, Bufo marinus. In addition, the adrenergic innervation of the gall bladder was examined by use of immunoreactivity to the catecholamine-synthesizing enzyme, tyrosine hydroxylase, and glyoxylic acid-induced fluorescence. On the basis of peptide coexistence, two intrinsic populations of neurones and their projecting fibres could be distinguished substance P neurones and vasoactive intestine peptide neurones. Neither of these two types of neurones contained any other colocalized neuropeptides. Four populations of nerve fibres arising from cell bodies outside the gall bladder were identified: nerves containing colocalized galanin, somatostatin and vasoactive intestinal peptide; nerves containing colocalized calcitonin gene-related peptide and substance P; adrenergic nerves containing neuropeptide Y; and nerves containing only adrenaline.

Animals↗
PubMed1993

Origin of galanin in nerves of cat airways and colocalization with vasoactive intestinal peptide.

Galanin is a 29 amino acid residue neuropeptide. In mammalian airways, galanin is found in nerve fibers associated with airway smooth muscle, bronchial glands, and blood vessels, and in nerve cell bodies of airway ganglia. The present study was conducted to determine if galanin-containing fibers in the walls of feline airways originate from the nerve cell bodies of airway ganglia. The colocalization of galanin with vasoactive intestinal peptide was also investigated. Organotypic cultures of cat airways were held in culture for 0 (nonculture control), 3, 5, and 7 days. After each culture period, the distribution of galanin and the colocalization of galanin with vasoactive intestinal peptide were determined by immunocytochemistry. Galanin-containing fibers were found in bronchial smooth muscle, around bronchial glands and in the walls of bronchial arteries and arterioles throughout the culture period. Nerve fibers and cell bodies containing both galanin and vasoactive intestinal peptide were observed after all culture periods. Nerve fibers and cells bodies that contained galanin frequently contained vasoactive intestinal peptide as well, but nerve fibers with only galanin or vasoactive intestinal peptide were also observed. Galanin- and vasoactive intestinal peptide-containing nerve fibers and cell bodies were both well maintained throughout the culture period. The findings show that galanin-containing nerve fibers associated with bronchial smooth muscle, bronchial glands, and bronchial arteries, originate from nerve cell bodies of intrinsic airway ganglia, and that galanin and vasoactive intestinal peptide are frequently colocalized in these neurons.

Animals↗
PubMed1993

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.

Arginine Vasopressin↗
PubMed1993

The distribution and colocalization of neuropeptides and 5-hydroxytryptamine in pelvic nerves supplying the posterior large intestine of the toad, Bufo marinus.

The distribution and colocalization of neuropeptides and 5-hydroxytryptamine in the posterior portion of the large intestine of the toad was studied using single- and dual-label immunohistochemistry. Neurons containing colocalized galanin/somatostatin or vasoactive intestinal peptide alone were observed along intramural pelvic nerves. Some of the galanin/somatostatin neurons also contained 5-hydroxytryptamine. Synaptic boutons containing colocalized calcitonin gene-related peptide/vasoactive intestinal peptide were associated with the galanin/somatostatin neurons. The muscle of the large intestine was also innervated by axons containing galanin/somatostatin, vasoactive intestinal peptide/calcitonin gene-related peptide or vasoactive intestinal peptide alone. Nerve fibres containing calcitonin gene-related peptide/substance P, probably representing primary afferent nerves, were also associated with muscle bundles. Submucosal blood vessels carried dense plexuses of fibres containing vasoactive intestinal peptide alone or and calcitonin gene-related peptide/substance P. Adrenergic perivascular nerves also contained galanin and neuropeptide Y.

Adrenergic Fibers↗
PubMed1994

Colocalization of NADPH-diaphorase activity and certain neuropeptides in the esophagus of opossum (Didelphis virginiana).

Nitric oxide and various neuropeptides in the myenteric plexus regulate esophageal motility. We sought colocalization of nitric oxide synthase and neuropeptides in frozen sections of mid-portion of smooth-muscled opossum esophagus using NADPH-diaphorase activity to mark the synthase and immunoreactivity to detect peptides. The peptides, all with demonstrated physiological activity in this organ, were calcitonin gene-related peptide, galanin, neuropeptide Y, substance P, and vasoactive intestinal polypeptide. The ExtrAvidin Peroxidase immunostain for each peptide was carried up to the final peroxidase reaction with 3-amino-9-ethyl-carbazole. The NADPH-diaphorase reaction was applied with short incubation to provide light staining just before the peroxidase reaction was performed. We examined sections for the proportions of singly and dually labeled nerve cells in the myenteric plexus. NADPH-diaphorase activity was highly colocalized with calcitonin gene-related peptide (59%), galanin (54%), and vasoactive intestinal polypeptide (53%). It showed little colocalization with neuropeptide Y (10%) and substance P (8%). The proportions of all nerve cells containing each of the substances were: NADPH-diaphorase--33%, calcitonin gene-related peptide--30%, galanin--55%, neuropeptide Y--16%, substance P--35%, and vasoactive intestinal polypeptide--58%. We conclude that the nerves responsible for peristalsis in the esophagus may act by releasing nitric oxide along with other inhibitory substances, calcitonin gene-related peptide, galanin, and vasoactive intestinal polypeptide, but not excitatory substances, neuropeptide Y and substance P.

Animals↗
PubMed1992

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

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 FMRF 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 FMRF amide-like substances occur in the same neurons in some brain regions.

Animals↗
PubMed1993

Colocalization of neurotransmitters in the deep cerebellar nuclei.

An abundance of glycine and glycine receptor immunoreactivities was found in all three parts of the deep cerebellar nuclei. Glycine immunoreactivity was restricted to small neurons throughout most of the deep cerebellar nuclei except for a few large positive neurons in the ventral part of the fastigial nuclei. In addition, glycine immunoreactivity was found in boutons outlining somata of large glycine negative neurons. Complementary to the glycine positive boutons was an intense glycine receptor immunoreactivity on large deep cerebellar nuclei neurons. Comparisons of immunoreactivities for glycine, GABA and aspartate in consecutive one micron sections revealed that many small neurons colocalized glycine and GABA, while some large neurons in the fastigal region colocalized glycine and aspartate. Ultrastructural investigations revealed glycine receptors on postsynaptic sites of dendrites and somata. Most boutons, which were presynaptic to glycine receptor sites, were filled with small flattened vesicles; however, a small percentage of boutons had round clear or dense core vesicles. Frequently, each bouton apposed multiple active zones on the dendrite or soma. One of these active zones was positive for glycine receptor and another was negative. This study supports: (1) glycine as a neurotransmitter in deep cerebellar nuclei, and (2) glycine and GABA colocalization in the same cell and bouton, but releasing to different receptor sites on the target neuron. Furthermore, the coexistence of glycine with GABA in the same deep cerebellar neuron may play an important role in controlling the conset and duration of signal transmission.

Animals↗