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PubMed1995

Odorant receptors and desensitization proteins colocalize in mammalian sperm.

BACKGROUND: The identification of transcripts encoding putative olfactory receptors in mammalian germ cells (1) has generated the hypothesis that olfactory receptors may serve a chemosensory role in sperm chemotaxis during fertilization. We have sought to identify and localize these receptors and their regulatory machinery in rat sperm in order to gain further insight into mammalian sperm chemotaxis and odorant receptor physiology. MATERIALS AND METHODS: We conducted reverse transcription-polymerase chain reaction (RT-PCR) using degenerate primers directed against sequences conserved across members of the known odorant receptor family to identify transcripts from testis and round spermatids. Western analysis and immunohistochemistry were performed using antibodies raised against two peptide sequences conserved among odorant receptors and using fusion protein antibodies to G-protein receptor kinase 3 (GRK3/beta ARK2) and beta-arrestin2. RESULTS: We detected transcripts encoding putative odorant receptors in both testis and round spermatids of the adult rat. Restriction digests of the PCR products demonstrated the existence of multiple gene products. Two anti-odorant receptor antibodies specifically recognized a 64 kD band in rat sperm preparations by Western blot. The proteins GRK3 and beta-arrestin2, implicated in olfactory desensitization, were detected in sperm cytosolic extracts using Western analysis. Immunohistochemistry colocalized putative odorant receptors, GRK3 and beta-arrestin2 to elongating spermatids in the testis and to the midpiece of mature sperm. CONCLUSIONS: The specific localization of odorant receptors to the respiratory center of mature sperm is consistent with a role for these proteins in transducing chemotactic signals. Based on the colocalization, it is plausible that GRK3 and beta-arrestin2 function in sperm to regulate putative chemoreceptor responses.

Animals↗

PubMed1992

Colocalization of thrombospondin and syndecan during murine development.

Thrombospondin is an adhesive glycoprotein that is thought to play a role in tissue genesis and repair. We have used a monoclonal anti-thrombospondin antibody, designated 5G11, to localize thrombospondin in paraformaldehyde fixed, paraffin-embedded sections of developing mouse embryos. Thrombospondin expression is observed in uterine smooth muscle, endometrial glands, the decidua, and trophoblastic giant cells during the initial phase of post-implantation development in the embryo. Cardiac myocytes and neuroepithelial cells show positive staining for thrombospondin at day 8.5 of gestation, and this expression continues throughout the development of the myocardium and central nervous system. Strong staining for thrombospondin is seen in developing bone and in the liver. Thrombospondin is also observed in developing smooth muscle and skeletal muscle, as well as in a variety of epithelia, including the epidermis, small intestinal epithelium, lens epithelium, renal tubular epithelium, and the epithelium of the developing tooth. Comparison of thrombospondin staining with that of two known cell surface receptors for thrombospondin, syndecan and the vitronectin receptor, reveals remarkable colocalization of thrombospondin and syndecan in all tissues, but almost no coexpression with the vitronectin receptor. Coexpression of thrombospondin and syndecan may play an important role in cell-cell or cell-matrix interactions during development.

Animals↗

PubMed1990

Immunohistochemical study of cerebral amyloid angiopathy. III. Widespread Alzheimer A4 peptide in cerebral microvessel walls colocalizes with gamma trace in patients with leukoencephalopathy.

Brain tissue from 11 patients with cerebral amyloid angiopathy, changes of Alzheimer's disease, and variable degrees of subcortical leukoencephalopathy was examined by immunohistochemical methods, using primary antibodies to peptide segments representing portions of the Alzheimer A4 (beta-) peptide or gamma-trace peptide (seen most commonly in Icelandic patients with cerebral hemorrhage (hereditary cerebral hemorrhage with amyloidosis [HCHWA-I]). Variable A4 immunostaining was seen within cortical (and rarely white matter) parenchyma in the form of senile plaques (with or without central cores), and within capillary and arteriolar walls. Within individual patients, A4 deposits were often primarily parenchymal or vascular, and when they were vascular they tended to be more prominent in arteriolar than in capillary wall segments. Perivascular A4 deposits were often detected around strongly immunoreactive microvessels. Gamma-trace immunoreactivity was noted in many A4-positive microvessel walls, but staining was always less intense than with the anti-A4 antibody. We conclude that patients with severe cerebral amyloid angiopathy may show wide variation in the severity and topography of A4 deposits within brain parenchyma. A4 may colocalize with gamma-trace peptide, suggesting that A4 and gamma-trace forms of cerebral amyloid angiopathy may not be as biochemically distinctive as has been suggested. Other proteases or protease inhibitors may contribute to the pathogenesis of cerebral amyloid angiopathy or cerebral amyloid angiopathy-related stroke syndromes.

Amyloid↗

PubMed1985

The renin-angiotensin system in the rat anterior pituitary: colocalization of renin and angiotensin II in gonadotrophs.

Discovery of components of the renin-angiotensin system (RAS) in the adenohypophysis of several species has prompted speculation concerning the location and possible function of a pituitary RAS. Although both renin and angiotensin II have been localized within the rat adenohypophysis, their colocalization has not been previously demonstrated within the same cells. In the present study, immunohistochemical staining by the avidin-biotin-peroxidase complex technique was used to demonstrate the coexistence of renin and angiotensin II in adenohypophyseal cells identified morphologically and immunocytochemically as gonadotrophs. These results support the existence of an adenohypophyseal RAS, at least part of which is under intracellular control. The influence of this system on control of fluid balance, blood pressure, and the secretion of other hypophyseal hormones is discussed.

Angiotensin II↗

PubMed1986

Colocalization of luteinizing hormone and serotonin in secretory granules of mammalian gonadotrophs.

Previous studies have demonstrated the uptake of exogenous and storage of endogenous 5-hydroxytryptamine (5-HT) in parenchymal cells of the anterior pituitary. The present experiments were undertaken to test the hypothesis that the endogenous 5-HT of the anterior lobe of the pituitary is costored with beta-luteinizing hormone (beta-LH) within the same secretory granules of gonadotrophs. Electron microscope immunocytochemistry was used to detect 5-HT and beta-LH immunoreactivities in the anterior pituitary glands of mice and bats. Primary antisera generated in different species of animals to these two antigens were localized with appropriate species-specific secondary antisera coupled to colloidal gold particles of different sizes. This enabled 5-HT and beta-LH immunoreactivities to be demonstrated simultaneously on ultrathin sections of fixed anterior lobe tissue mounted on electron microscope (EM) grids. In both bats and mice 5-HT immunoreactivity, identified by immunostaining of beta-LH, was found in gonadotrophs, and in no other cell type. Within gonadotrophs about 25% of the secretory granules were labeled by antisera to both 5-HT and beta-LH, although 100% of granules reacted with the antiserum to B-LH. No secretory granules were found that were immunostained only by the antiserum to 5-HT. It is concluded that endogenous 5-HT may be a normal constituent of mammalian gonadotrophs and that it is colocalized with beta-LH in at least a subset of the secretory granules of these cells. It cannot yet be concluded that gonadotrophs synthesize 5-HT as well as taking it up from the ambient medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Distribution and colocalization of NADPH-diaphorase activity, nitric oxide synthase immunoreactivity, and VIP immunoreactivity in the newly hatched chicken gut.

BACKGROUND: The distribution and colocalization of nitric oxide synthase and NADPH-diaphorase have been investigated quite extensively in the mammalian gut; however, no such study has been undertaken in the avian gut. In the present report, we have therefore studied the distribution and coexpression of nitric oxide synthase (NOS), NADPH-diaphorase, and vasoactive intestinal polypeptide (VIP) in enteric neurons of the newly hatched chicken gut. METHODS: Immunohistochemical methods were used to detect NOS immunoreactivity (NOS-IR) and VIP immunoreactivity (VIP-IR). NADPH-diaphorase activity was detected using a histochemical technique. RESULTS: Neurons expressing NADPH-diaphorase activity, NOS-IR, and VIP-IR were detected in both the myenteric and submucous plexus of all regions of the gastrointestinal tract examined. All NADPH-diaphorase positive neurons were also NOS-IR and all NOS-IR neurons were NADPH-diaphorase positive, in both plexuses, indicating that NADPH-diaphorase can be used as a marker for NOS containing neurons in the chicken gut. The majority of VIP-IR neurons also expressed NADPH-diaphorase activity. Only few neurons that expressed NADPH-diaphorase activity did not express VIP-IR. The proportion of VIP immunopositive neurons that were NADPH-diaphorase negative increased anally and these neurons were more prominent in the submucous than the myenteric plexus ganglia. NADPH-diaphorase positive, NOS-IR, and VIP-IR nerve fibres were detected in the circular muscle, but very few, if any, were present in the longitudinal muscle. VIP-IR, but not NOS-IR or NADPH-diaphorase activity, was detected in mucosal fibres, in contrast to the situation in the mammalian gut. CONCLUSIONS: These results indicate that in birds, as in mammals, nitric oxide may play a role in the neural control of the gut musculature, but that it is unlikely to be involved in the nervous control of mucosal activity.

Animals↗

PubMed1983

Gamma actin, spectrin, and intermediate filament proteins colocalize with vinculin at costameres, myofibril-to-sarcolemma attachment sites.

Localization of vinculin at the sarcolemma of striated muscle fibers defines an orthogonal lattice. The costameres of the lattice are the riblike bands of vinculin that run perpendicular to the long axis of the fiber, repeat in register with I bands of the subjacent myofibrils, and seem to couple the myofibril to the sarcolemma [Pardo et al 1982, 1983a]. The colocalization studies presented in this paper show that gamma actin, spectrin, and intermediate filament antigens are additional components of this lattice of costameres. In addition, the results show that gamma actin and spectrin are also components of the internal network of collars, first visualized with antibody to desmin [Granger and Lazarides, 1978], that connects the myofibrils to each other at the level of the Z line.

Actins↗

PubMed1988

Somatostatin-like immunoreactivity and glycine high-affinity uptake colocalize to an interplexiform cell of the Xenopus laevis retina.

Antibodies directed against somatostatin have been used to label a population of interplexiform cells (IPCs) in the Xenopus laevis retina. These cells have spherical soma which lie in the inner nuclear layer (INL), adjacent to or one cell distal to the inner plexiform layer (IPL). Processes from these cells project throughout the IPL, with a fairly dense accumulation of labeled dendrites in the upper two-fifths of the IPL and a dense, narrow band of labeled dendrites adjacent to the ganglion cell layer. These cells also have finer processes, originating at the cell body, that traverse the INL and ramify in the outer plexiform layer (OPL). Double label experiments show that all of the cells that contain somatostatin-like immunoreactivity (SOM-LI) in the INL are also labeled by high-affinity uptake with 3H-glycine. Immunocytochemistry of retinal whole mounts shows that these cells are evenly distributed across the retina at a density of 542 +/- 65 cells/mm2. On the basis of the colocalization experiments and the morphological homogeneity of these cells, we suggest that they represent a single cell type. Interplexiform cell processes were further characterized by electron microscopy after immunocytochemistry or 3H-glycine autoradiography. In the IPL, IPC processes are seen to be postsynaptic at both ribbon and conventional synapses. This input is found almost entirely in the distal two-fifths of the IPL. Interplexiform cell processes are presynaptic to unlabeled processes in both the distal and proximal IPL. In the OPL, labeled processes are found near or contiguous with photoreceptor bases, and are often presynaptic to small-diameter processes. The postsynaptic processes have been identified as bipolar cell dendrites in six cases. Interplexiform cell processes may also contact horizontal cell processes in the OPL.

Animals↗

PubMed1988

Nerve growth factor receptor immunoreactivity in the nonhuman primate (Cebus apella): distribution, morphology, and colocalization with cholinergic enzymes.

A monoclonal antibody raised against the receptor for nerve growth factor (NGF) was used to examine the distribution and morphology of NGF receptor-containing neurons within the central nervous system of Cebus apella monkeys. Most somata demonstrating positive immunoreactivity were localized within the Ch1-4 regions of the basal forebrain. Neurons in the Ch1 region displayed morphological features typical of cholinergic medial septal neurons. These perikarya were primarily vertically oriented (40-50 micron along the vertical axis) with both apical and basal neuritic processes. Magnocellular (40-50 micron) neurons within the Ch2 (vertical limb of the diagonal band), Ch3 (horizontal limb of the diagonal band) and Ch4 (nucleus basalis of Meynert) regions were multipolar and had rounded perikarya that often displayed an eccentric nucleus. Fibers presumably originating from the Ch1-2 regions were observed throughout the fimbria-fornix system and were found to terminate preferentially within the CA1 and CA3 regions of the hippocampal formation and within the dentate gyrus of the hippocampus. An intense fiber network was also observed in the olfactory tubercle and other rhinencephalic structures, presumably originating from the Ch3 region of the basal forebrain. Beaded processes emanating from the Ch4 region primarily coursed within the external capsule and terminated preferentially within layers I, II, and IV of the cerebral cortex. In a pattern similar to that of cortical acetylcholinesterase (AChE) staining, NGF receptor immunopositive fibers were oriented in a tangential plane within the molecular layer of the cortex and in both a radial and tangential fashion within the cortical granular cell layers. In addition to neural innervation, there was an extensive vascular apposition by NGF receptor-containing neurites on both large caliber vessels and microcapillaries. NGF receptor immunoreactivity was extensively, but not exclusively, colocalized with choline acetyltransferase (ChAT) and AChE in the basal forebrain. A small population of cholinergic neurons were observed that were not NGF receptor-immunoreactive. Conversely, a few NGF receptor-containing neurons that were noncholinergic were also observed in this brain region. NGF receptor-containing somata were also identified in the putamen. The number of immunoreactive neurons observed in this structure, however, would not appear to be sufficient to account for the homologous NGF receptor binding densities described in rodents.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗

PubMed1989

Nerve growth factor receptor and choline acetyltransferase colocalization in neurons within the rat forebrain: response to fimbria-fornix transection.

Although it is well known that magnocellular cholinergic basal forebrain neurons are trophically responsive to nerve growth factor (NGF) and contain NGF receptors (NGFr), the exact distribution of forebrain NGFr-immunoreactive neurons and the degree to which cholinergic neurons are colocalized with them have remained in question. In this study we employed a very sensitive double-labelling method and examined in the same tissue section the distribution and cellular features of NGFr-positive and choline acetyltransferase (ChAT)-immunolabelled neurons within the rat basal forebrain. Throughout this region the majority of magnocellular basal forebrain neurons were immunoreactive for both NGFr and ChAT. However, a small percentage of neurons in the ventral portion of the vertical limb of the diagonal band of Broca were immunoreactive only for NGFr, whereas a larger population of magnocellular neurons in the substantia innominata exhibited only ChAT immunoreactivity. No NGFr-immunoreactive cells were found associated with ChAT-positive neurons in the striatum, neocortex, or hippocampus, and no single-labelled NGFr-immunoreactive neurons were found outside the basal forebrain area, except for a large number of positive-labelled cells along the ventricular walls of the third ventricle. In addition to its function in maintaining the normal integrity of the basal forebrain and cholinergic, peripheral sympathetic, and neural-crest-derived sensory neurons, NGF may also have a role in the growth of these neurons after damage to the nervous system. To examine this postulate the hippocampus was denervated of its septal input and examined 8 weeks later. Two populations of neurons were found to have undergone collateral sprouting--namely, the midline magnocellular cholinergic neurons of the dorsal hippocampus and the sympathetic noradrenergic neurons of the superior cervical ganglion. Both of these neuronal populations also stained strongly for NGFr. In contrast, the small intrinsic cholinergic neurons of the hippocampus exhibited neither sprouting response nor staining for NGFr. In view of these results, we suggest that the differing sprouting responses demonstrated by these three neuronal populations may be due to their responsiveness to NGF, as indicated by the presence or absence of NGF receptors.

Animals↗

PubMed1989

Enkephalin-like immunoreactivity in the cat superior colliculus: distribution, ultrastructure, and colocalization with GABA.

The distribution of enkephalin (ENK) immunoreactivity has been examined in the cat superior colliculus (SC) by means of light and electron microscope immunocytochemistry. The antisera were directed against leucine enkephalin but also recognized methionine enkephalin. Colocalization of ENK with gamma aminobutyric acid (GABA) was studied with a two-chromagen double-labeling technique. Enkephalin antiserum labeling was highly specific. Dense neuropil labeling was found only in a thin band 75-100 microns wide within the upper superficial gray layer of SC. Negligible neuropil labeling was seen deeper, except for patches of label within the intermediate gray layer. Intensely labeled neurons also had a specific distribution. Forty-seven percent were located within the upper 200 microns of SC, 40% within the deep superficial gray layer, 11% in the optic layer, and only 2% below that layer. Almost all ENK-labeled cells were small (mean area of 117 microns2). Some of these had horizontal fusiform cell bodies and horizontally oriented dendrites. Others had small round somata and thin, obliquely oriented dendrites. In double-labeling experiments, 18% of anti-ENK-labeled cells were also immunoreactive for GABA. Four distinct types of ENK-labeled profile were identified with the electron microscope. Presynaptic dendrites (PSD) with loose accumulations of synaptic vesicles were densely labeled with the antiserum. Conventional dendrites were also labeled. Both types of labeled profile received input from unlabeled synaptic terminals, including those from the retina that contained pale mitochondria and round synaptic vesicles and formed asymmetric synaptic contacts. Retinal terminals were never labeled with the antisera. However, some axon terminals with round synaptic vesicles, dark mitochondria, and symmetric synaptic densities were labeled by the antisera, as were some thinly myelinated axons. These results show that there is a small population of enkephalinergic neurons in the cat SC, some of which also contain GABA. Because not all cells with identical morphologies were double labeled, it appears that neurons of like morphology are chemically heterogeneous.

Animals↗

PubMed1989

Nerve growth factor receptor immunoreactive profiles in the normal, aged human basal forebrain: colocalization with cholinergic neurons.

A monoclonal antibody raised against the receptor for nerve growth factor (NGF) has been used to map the distribution of NGF receptor-containing profiles within the human basal forebrain of four male and three female elderly patients without neurologic or psychiatric illness. Immunohistochemically processed tissue reveals a continuum of NGF receptor-positive neurons located within the medial septum, vertical and horizontal limb nuclei of the diagonal band, and nucleus basalis. NGF receptor-containing neurons are also found within the bed nucleus of the stria terminalis, the anterior commissure, the internal capsule, and the internal and external medullary laminae of the globus pallidus. Virtually all (greater than 95%) NGF receptor-containing neurons colocalize with the specific cholinergic marker choline acetyltransferase (ChAT) or the nonspecific marker acetylcholinesterase (AChE). Conversely, a few cholinergic perikarya are found which are not NGF receptor positive (and vice versa). These findings demonstrate that human basal forebrain neurons on which NGF receptor immunoreactivity is detected are primarily cholinergic and analogous to the nonhuman primate Ch1-Ch4 subgroups of Mesulam et al. (J. Comp. Neurol., 214:170-197, '83). NGF receptor-containing fiber tracts are observed emanating from the medial septum and vertical limb nucleus of the diagonal band coursing medially within the fornix. Another fascicle originating mainly from the nucleus basalis and travelling within the external capsule enroute to the cortex is observed innervating all cortical layers. Comparison of NGF receptor- and ChAT-containing neurons reveals cholinergic perikarya within the striatal complex, whereas virtually no NGF receptor-containing neurons are found in these structures. An occasional displaced NGF receptor-containing neurons is seen in the ventrolateral portion of the putamen and the white matter underlying the nucleus accumbens. These data are discussed in terms of the relationship of NGF receptor- and ChAT-containing neurons within the basal forebrain and in terms of the possible functional significance of NGF in normal and diseased brain.

Acetylcholinesterase↗

PubMed1991

SCPB-and FMRFamide-like immunoreactivities in lobster neurons: colocalization of distinct peptides or colabeling of the same peptide(s)?

Virtually all of the SCPB-like immunoreactive neurons (ca. 60 cells) in the lobster Homarus americanus also contain FMRFamide-like immunoreactivity. Control experiments reveal that SCPB-and FMRFamide-like immunoreactivities are successfully preadsorbed with their specific antigens, while the normal staining pattern is retained following preadsorption of each antibody with the alternate peptide. These experiments potentially lead to the conclusion that the anti-SCPB and anti-FMRFamide antibodies are labeling distinct compounds that are colocalized in lobster neurons. The lobster nervous system does not, however, contain authentic FMRFamide, but rather several FMRFamide-like compounds (Trimmer et al., J. Comp. Neurol. 266:16-26, 1987). The most abundant of these is the octapeptide TNRNFLRFamide. Experiments demonstrate that SCPB-like immunoreactivity is completely preadsorbed with synthetic TNRNFLRFamide, while there is a significant or complete loss of staining after preadsorption of the FMRFamide antibody with this molecule. Met-enkephalin-Arg-Phe-amide (YGGFMRFamide), an extended opioid peptide containing the FMRFamide sequence, also preadsorbs SCPB- and FMRFamide-like immunoreactivities, while Met-enkephalin-Arg-Phe (YGGFMRF) has no effect on the staining properties of these antibodies. These results suggest that the SCPB antibody can bind to extended forms of FMRFamide-like molecules, and that anti-SCPB and anti-FMRFamide antibodies may be colabeling one or more FMRFamide-like molecules in lobster neurons.

Amino Acid Sequence↗

PubMed1991

Ultrastructural localization of neurotensin-like immunoreactivity within dense core vesicles in perikarya, but not terminals, colocalizing tyrosine hydroxylase in the rat ventral tegmental area.

Within the rat ventral tegmental area (VTA), the parabrachial pigmentosus and paranigral subdivisions are known to differ in their functional responses to injected neurotensin. These subdivisions also vary in their connections with other brain regions and in their number of neurotensin-containing perikarya as seen by light microscopy. In both subdivisions, there may be intracellular as well as synaptic relations between dopamine and neurotensin. Dopaminergic neurons are known to be physiologically activated by neurotensin (NT) and may also contain this peptide. To characterize further the cellular relationships in each subdivision, we examined the ultrastructural immunocytochemical localization of a rat antiserum against NT and a rabbit antiserum against the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) in single sections. The NT antiserum was raised against the entire peptide sequence. Immunoblots showed that the antiserum recognized the original antigen as well as the related peptides neuromedin N and lysine 8- arginine 9- neurotensin 10-13 (LANT-6). In both the parabrachial pigmentosus and paranigral subdivisions, neurotensin-like immunoreactivity (NTLI) was localized predominantly in the large (80-100 nm) dense core vesicles using the peroxidase anti-peroxidase (PAP) method. In tissue labeled for NT by the PAP method and for TH by immunoautoradiography, serial section analysis revealed that all perikarya containing NTLI (n = 19) were also TH-positive. Three times as many perikarya colocalized NTLI and TH in the parabrachial pigmentosus subdivision (n = 15) as in the paranigral subdivision (n = 4). Occasionally, a perikaryon containing TH and NTLI could be found in direct apposition to a TH-labeled perikaryon without glial separation. In contrast to perikarya and dendrites, terminals showing NTLI (38 in parabrachial pigmentosus, 29 in paranigral) lacked detectable TH labeling. Of the terminals containing NTLI whose synaptic junctions could be identified, 48% were symmetric and 10% were asymmetric. The targets of these terminals included perikarya and dendrites lacking detectable immunoreactivity (69% in parabrachial pigmentosus, 55% in paranigral), immunolabeled for TH (26% in parabrachial pigmentosus, 38% in paranigral) or containing both NTLI and TH (5% in parabrachial pigmentosus, 7% in paranigral). Single terminals containing NTLI sometimes contacted more than one neuronal target, some of which were apposed to each other without glial separation. TH-labeled terminals synapsed onto double-labeled perikarya in the paranigral subdivision, but were not observed to do so in the parabrachial pigmentosus subdivision.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Colocalization of prosomatostatin-derived peptides in the caudate-putamen of the rat.

In the striatum of rat, somatostatin 14, somatostatin 28, and somatostatin 28(1-12) have previously been localized within a small population of medium aspiny local circuit neurons. Because all three peptide fragments are generated through the cleavage of prosomatostatin by different converting enzymes, the possibility for differential expression of these peptides exists. In order to investigate this possibility, frozen sections were collected from the brains of adult female Wistar rats fixed with 4% paraformaldehyde and double labelled using immunocytochemistry and in situ hybridization. Sections were first processed for somatostatin 14, somatostatin 28, or somatostatin 28(1-12) by using the avidin-biotin complex immunocytochemical technique followed by in situ hybridization using 35S-labelled antisense riboprobes to somatostatin mRNA. The results of such analysis revealed that somatostatin 28 and somatostatin mRNA are 100% colocalized. Somatostatin 14 and somatostatin 28(1-12), in contrast, are only present within 66% of the neurons that express somatostatin mRNA. Examination of the anatomical distribution of neurons that express both somatostatin mRNA and somatostatin 14 or somatostatin 28(1-12) protein reveals that these neurons are present throughout the caudate-putamen of rat but are more prevalent in the ventromedial regions. Neurons that express somatostatin mRNA but not somatostatin 14 or somatostatin 28(1-12) are also present throughout the caudate-putamen but are most numerous within a dorsolateral strip just beneath the corpus callosum. These results suggest that the somatostatin neuron population within the rat caudate-putamen is actually composed of two smaller subpopulations based on neuropeptide content. The first subpopulation contains somatostatin 28 and constitutes one-third of the total somatostatin population, whereas the other contains somatostatin 28, somatostatin 14, and somatostatin 28(1-12) and represents the remaining two-thirds of the cells that express somatostatin mRNA.

Animals↗

PubMed1994

Size-related colocalization of glycine and glutamate immunoreactivity in frog and rat vestibular afferents.

Presence and distribution of glutamate, glycine, GABA and beta-alanine in VIIIth nerves of frogs and rats were investigated with postembedding immunocytochemical methods on serial semithin sections. In Scarpa's ganglion of the frog, all cell bodies were glutamate immunoreactive. About 17% of the cells per section were also glycine immunoreactive, but none were GABA or beta-alanine immunoreactive. The mean diameter of glycine-positive cell bodies (26.7 +/- 6.9 microns; N = 130) was significantly (P < 0.0001) larger than that of glycine-negative cell bodies (15.7 +/- 5.4 microns; N = 272). The intensity of glutamate immunostaining decreased with cell diameter, whereas the intensity of glycine immunostaining increased with cell diameter. As a result, the staining intensities for glutamate and glycine in a given cell were negatively correlated. Glycine immunoreactivity was also present in a size-related manner in distal and proximal afferent fibers. The majority of thin fibers (< 4 microns) was glycine negative, whereas most of the thick fibers (> 10 microns) were glycine positive. Glycine-positive fibers were observed in the sensory epithelial of all end organs in the inner ear. The saccular macula and its nerve, however, contained only few glycine immunoreactive structures. In Scarpa's ganglion of the rat, all cells were immunoreactive for glutamate, about 12% for colocalized glycine, and none for GABA or beta-alanine. Glycine-positive cell bodies were significantly (P < 0.0001) larger (32.2 +/- 5.2 microns; N = 82) than glycine-negative cell bodies (25.1 +/- 5.3 microns; N = 274). Cell bodies in the spiral ganglion were only glutamate immunoreactive, whereas staining for glutamate, glycine, and GABA was dense in the ventral cochlear nucleus. These results demonstrate that thicker vestibular afferent fibers represent a particular subpopulation that differs from the majority of thinner afferents due to their glycine immunoreactivity.

Alanine↗

PubMed1994

TrkA-immunoreactive profiles in the central nervous system: colocalization with neurons containing p75 nerve growth factor receptor, choline acetyltransferase, and serotonin.

The present investigation used an antibody directed against the extracellular domain of the signal transducing nerve growth factor receptor, trkA, to reveal immunoreactive perikarya or fibers within the olfactory bulb and tubercle, cingulate cortex, nucleus accumbens, striatum, endopiriform nucleus, septal/diagonal band complex, nucleus basalis, hippocampal complex, thalamic paraventricular and reuniens nuclei, periventricular hypothalamus, interpeduncular nucleus, mesencephalic nucleus of the fifth nerve, dorsal nucleus of the lateral lemniscus, prepositus hypoglossal nucleus, ventral cochlear nucleus, ventral lateral tegmentum, medial vestibular nucleus, spinal trigeminal nucleus oralis, nucleus of the solitary tract, raphe nuclei, and spinal cord. Colocalization experiments revealed that virtually all striatal trkA-immunoreactive neurons (> 99%) coexpressed choline acetyltransferase (ChAT) but not p75 nerve growth factor receptor (NGFR). Within the septal/diagonal band complex virtually all trkA neurons (> 95%) coexpressed both ChAT and p75 NGFR. More caudally, dual stained sections revealed numerous trkA/ChAT (> 80%) and trkA/p75 NGFR (> 95%) immunoreactive neurons within the nucleus basalis. In the brainstem, raphe serotonergic neurons (45%) coexpressed trkA. Sections stained with a pan-trk antibody that recognizes primarily trkA, as well as trkB and trkC, labeled neurons within all of these regions as well as within the hypothalamic arcuate, supramammilary, and supraoptic nuclei, hippocampus, inferior and superior colliculus, substantia nigra, ventral tegmental area of T'sai, and cerebellular Purkinje cells. Virtually all of these other regions with the exception of the cerebellum also expressed pan-trk immunoreactivity in the monkey. The widespread expression of trkA throughout the central neural axis suggests that this receptor may play a role in signal transduction mechanisms linked to NGF-related substances in cholinergic basal forebrain and noncholinergic systems. These findings suggest that pharmacological use of ligands for trkA could have beneficial effects on the multiple neuronal systems that are affected in such disorders as Alzheimer's disease.

Animals↗

PubMed1994

Colocalization of the p185HER2 oncoprotein and integrin alpha 6 beta 4 in Calu-3 lung carcinoma cells.

Anti-p185HER2 monoclonal antibodies often show intense reactivity with the basement membrane of tumor cells that overexpress the HER2/neu gene product (p185HER2). To evaluate a possible interaction between p185HER2 and adhesion molecules or their receptors, the polarity of p185HER2 was tested in lung carcinoma cell line Calu-3, which overexpresses this protein, in cultures grown as confluent monolayers or as aggregates. MAb immunostaining patterns indicated that p185HER2 is concentrated on the baso-lateral membrane of cells and that it colocalizes with the integrin alpha 6 beta 4 at the cell-cell junctions where laminin is also found. The same membrane region showed intense reactivity with antiphosphotyrosine antibodies. Furthermore, integrin clustering induced by the specific antibody was accompanied by the clustering of p185HER2, as indicated by immunoelectron microscopy, and by a subsequent increase in p185HER2 tyrosine phosphorylation. Treatment with exogenous laminin also resulted in increased basal levels of p185HER2 phosphorylation. These data suggest a physical interaction between the integrin and the oncoprotein that might be functionally relevant in directly controlling the tyrosine phosphorylation of the catalytic domain of p185HER2.

Antibodies, Monoclonal↗