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PubMed1992

Immunocytochemical colocalization of hypothalamic progestin receptors and tyrosine hydroxylase in steroid-treated monkeys.

Progesterone (P)-induced PRL secretion in estradiol (E)-primed monkeys is not due to direct pituitary stimulation, because lactotropes do not express progestin receptors (PR). However, the hypothalamus, particularly the tuberoinfundibular dopaminergic system (TIDA), plays a major role in the regulation of PRL secretion. To determine whether hypothalamic dopamine neurons are progestin target cells, the colocalization of PR and tyrosine hydroxylase (TH), a phenotypic marker of dopaminergic neurons, was examined with double immunocytochemistry. Two methods for visualizing the antigens were applied; the first was a dual peroxidase method, and the second was a peroxidase-alkaline phosphatase method. In addition, the question of whether E induces PR in dopamine neurons was explored. Spayed female monkeys were treated with empty Silastic capsules, E-filled capsules for a period of 28 days, or E capsules supplemented with P capsules for the last 14 days of E treatment. Only the E- plus P-treated monkeys exhibited an increase in serum PRL during the P treatment period. Frontal sections at the level of the optic chiasm and arcuate nucleus were examined for the colocalization of TH and PR. After E treatment, hypothalamic PR-positive cells increased in both intensity and number. Neurons expressing both TH and PR were detected in the rostral hypothalamus, lateral to the third ventricle (A11-rostral) and in a discrete subventricular population (A11-subvent). The lateral population continued caudally (A11-caudal). The A11-subvent population exhibited little steroid regulation. Of the remaining A11 TH neurons, approximately 20% exhibited PR in the spayed and E-treated groups. Addition of P doubled the percentage of PR-containing TH neurons in this group. Although very few TH-positive neurons in the ventral arcuate nucleus contained PR (A12-ventral), many double labeled neurons were observed in the dorsal arcuate region (A12-dorsal). Ventral arcuate TIDA neurons were not regulated by steroids, but E plus P increased PR expression in A12-dorsal. Double labeled cells were rarely seen in the zona incerta (A13) or the emerging ventral tegmental area (A10). In summary, P probably does not act directly on ventral arcuate TIDA neurons to stimulate PRL secretion. However, the frequency of PR-positive dopamine neurons in the A11-rostral, A11-caudal, and A12-dorsal groups increased with E and P treatment. Therefore, the contribution of the PR-positive periventricular dopamine neurons to progestin-stimulated PRL secretion may be important.

Animals↗

PubMed1993

Colocalization of immunoreactive endothelin-1 and neurohypophysial hormones in the axons of the neural lobe of the rat pituitary.

The intracellular localization of immunoreactive endothelin (ET)-1, and its colocalization with vasopressin (VP) and oxytocin (OT) in the rat neural lobe were investigated by immunogold techniques using specific antisera raised against ET-1, VP, and OT. There were two types of axons: the first contained VP-immunolabeled neurosecretory granules, and the second contained OT-immunolabeled neurosecretory granules. A considerable number of the neurosecretory granules in both types of axon were immunolabeled with antibodies against ET-1, although the VP-immunolabeled granules were more heavily labeled with anti-ET-1 antiserum than OT-immunolabeled ones. Double immunogold labeling clearly demonstrated the intragranular colocalization of immunoreactions for ET-1 and VP and that of immunoreactions for ET-1 and OT. These results suggest that ET in the neural lobe may be released concomitantly with neurohypophysial hormones. Its biological significance remains to be elucidated.

Animals↗

PubMed1993

Ultrastructural colocalization of growth hormone binding protein and pituitary hormones in adenohypophyseal cells of the rat.

GH receptor immunoreactivity is widely distributed within the rat pituitary gland, although apart from somatotrophs the cell types with GH receptor immunoreactivity have yet to be identified. It is also unknown whether this immunoreactivity reflects the presence of GH binding proteins (GHBPs) or authentic receptors. The possible colocalization of GHBPs and pituitary hormones in somatotrophs, lactotrophs, gonadotrophs, thyrotrophs, and corticotrophs was therefore examined using immunogold electron microscopy. Pituitary sections were indirectly immunostained with antibodies for GH, PRL, LH, FSH, TSH, or ACTH using gold-labeled immunoglobulin G. The same grids were also immunostained with a polyclonal antibody raised against rat GHBP using protein A conjugated to gold particles of a different size. Some sections were gold-labeled using a monoclonal antibody (MAb 4.3) raised against the unique hydrophobic tail of the GHBP, using gold-labeled immunoglobulin G. The cellular and ultrastructural distribution of immunoreactivity within the pituitary gland was similar after labeling with either GHBP antiserum. In all immunoreactive cells the labeling was most intense in secretory granules. Specific staining was not, however, demonstrated in the nucleus, in contrast with earlier findings using MAb 263. Moreover, while staining with MAb 263 appeared to be ubiquitous, some pituitary cells were not labeled by the polyclonal GHBP antiserum or by MAb 4.3. GHBP immunoreactivity was, however, colocalized with hormones in all pituitary cell-types, although in some cells GHBP immunoreactivity was not present in all secretory granules. These results clearly demonstrate the presence of GHBPs in adenohypophyseal cells, in which they appear to be stored or secreted with GH, PRL, LH, FSH, TSH, and ACTH. The function of GHBPs in the pituitary gland is, however, uncertain.

Adrenocorticotropic Hormone↗

PubMed1992

Subcellular distribution of the Rap1A protein in human neutrophils: colocalization and cotranslocation with cytochrome b559.

Rap1A, a low molecular weight guanosine triphosphate-binding protein (LMWG), has been shown previously by us to be associated with purified cytochrome b from stimulated human neutrophils. In the present studies, we show that Rap1A is also associated with affinity-purified cytochrome b from unstimulated neutrophils and use specific anti-Rap1 peptide antibodies to biochemically and immunocytochemically determine the subcellular distribution of Rap1A in resting and activated human neutrophils. Analysis of the subcellular fractionation of unstimulated cells by Western blotting of isopycnic sucrose density gradient fractions with anti-Rap1 peptide antibodies indicated that Rap1A colocalized with cytochrome b in the plasma membrane as well as in the specific granule membranes and that it was translocated, along with cytochrome b, to the plasma membrane when the cells were stimulated with phorbol myristate acetate (PMA). No evidence for a cytosolic localization of Rap1A was found in our studies; however, if the cells were disrupted by sonication, rather than N2 cavitation, a fraction of the Rap1A was released from the membrane. Electron microscopy of thin sections of cryofixed, molecular-distillation dried neutrophils labeled with anti-Rap1 antibody alone or double-labeled with anti-Rap1 and anti-cytochrome b peptide antibodies confirmed our biochemical localization, and quantitation showed that more than half of the specific granule-associated Rap1A was translocated to the plasma membrane in PMA-stimulated cells. Ultrastructural analysis of neutrophils phagocytosing Staphylococcus aureus also demonstrated the translocation of Rap1A with cytochrome b. Approximately 70% of the total Rap1A labeling was associated with the phagolysosomal membrane, the site of assembly of the superoxide-generating system. The colocalization and cotranslocation of Rap1A with cytochrome b in resting and activated neutrophils is consistent with a functional association of these two molecules in the intact cell and provides further evidence for a role of this LMWG in the structure or function of the neutrophil superoxide-generating system.

Biological Transport↗

PubMed1992

Colocalization of a large heterodimeric proteoglycan with basement membrane proteins in cultured cells.

A novel large heterodimeric dermatan sulfate proteoglycan with core proteins of 460 and 300 kDa, respectively, had been described as a secretory product of human fetal skin fibroblasts (Breuer et al., J. Biol. Chem. 266, 13224-13232 (1991)). Pulse-chase experiments showed a preferential association of the proteoglycan with the cell membrane. Immunogold labeling indicated its localization in fibrils on the cell surface as well as in fibrillar extensions from the cell body. Immunofluorescence studies yielded a fibrillar and punctate staining pattern which was also seen in cultured human and porcine endothelial cells. Dot-like structures were observed in transformed human keratinocytes. Various immunocytochemical double-labeling experiments indicated a remarkable colocalization of the proteoglycan with fibronectin, laminin, perlecan, and type IV collagen whereas only occasionally a colocalization with chondroitin-6-sulfate was found. No evidence for an enrichment of the proteoglycan in vinculin-containing structures was obtained. These results suggest that the proteoglycan is a widely distributed macromolecule which can associate with basement membrane components. Preliminary findings in rat cornea supported this conclusion.

Basement Membrane↗

PubMed1991

Substance P- and enkephalin-like immunoreactivities are colocalized in certain neurons of the substantia gelatinosa of the rat spinal cord: an ultrastructural double-labeling study.

The finding that certain cells of the substantia gelatinosa of the rat spinal cord contain both substance P (SP)- and enkephalin (ENK)-like immunoreactive material offers new insights into the mechanisms of action of these peptides in the processing of nociceptive sensory information. The simultaneous detection of these immunoreactivities was obtained in the superficial dorsal horn of the rat spinal cord at the ultrastructural level using monoclonal antibodies. An internally radiolabeled monoclonal antibody (against SP or ENK) was used to recognize one antigenic site, while the other antigenic site was identified by either a bispecific monoclonal antibody (for SP) or a monoclonal antibody (for ENK). The bispecific anti-SP antibody recognized HRP, whereas a secondary bispecific antibody recognized both the IgG of the anti-ENK monoclonal antibody and HRP. In laminae I-III, SP-like immunoreactivity (SP-LI) and ENK-like immunoreactivity (ENK-LI) were colocalized in a significant number of axonal varicosities, which contained round or pleomorphic synaptic vesicles. Such double-labeled varicosities, however, were not found to be components of synaptic glomeruli. Most of the immunostained boutons of lamina I were SP-like immunoreactive only. In rats pretreated with colchicine, SP-LI and ENK-LI were colocalized in small perikarya of lamina II and in some lamina I cells. These findings indicate that SP and ENK occur in a significant population of interneurons of the superficial dorsal horn. It is suggested that some of these neurons may correspond to stalked cells and release one or the other substance depending on physiological conditions.

Animals↗

PubMed1991

Immunohistochemical colocalization of amyloid precursor protein with cerebrovascular amyloid of Alzheimer's disease.

Molecular cloning and cDNA sequencing have indicated that the fibril-forming, amyloidogenic beta/A4 peptide of cerebrovasculature and plaque core in AD is encoded as part of a larger precursor, amyloid precursor protein (APP). A panel of antibodies directed against synthetic peptides, which correspond to distinct domains of this putative APP molecule (i.e., amino acid residues 45-62, 587-596, 597-606, 597-638 [beta/A4 peptide], 638-658 and 653-661), were used to probe immunohistochemically serial sections of formalin-fixed, paraffin-embedded Alzheimer's disease (AD) brains for the presence of APP and/or its derivatives. Histochemical staining of adjacent sections with Bielschowsky's silver impregnation and with Congo red or thioflavin S-staining techniques was also done to identify the structures with amyloid deposition. All these antibodies exhibited intense immunoreactivity with amyloidotic cerebral vessels, including meningeal and parenchymal. This observation indicates that the amyloidotic vasculature of AD brain contains, in addition to the fibril-forming beta/A4 protein, nonamyloidogenic APP and/or its derivatives. More importantly, this APP immunoreactivity colocalized with angiopathic amyloid, which is characterized by phenol-resistant, birefringent congophilia. Parallel analyses with a dual SABC/silver impregnation procedure further confirmed that APP and/or its derivatives, including the amyloidogenic beta/A4, colocalized with argentophilic amyloid in the cerebrovasculature of AD.

Alzheimer Disease↗

PubMed1991

Interaction of colocalized neuropeptides: functional significance in the circadian timing system.

The suprachiasmatic nucleus (SCN), which appears to act as a circadian clock, contains a subpopulation of local circuit neurons in which vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), and gastrin releasing peptide (GRP) are colocalized. To determine whether VIP, PHI, and GRP interact within the SCN to produce a signal important for circadian control, the behavioral and cellular effects of coadministration of these neuropeptides were investigated. Coadministration of VIP, PHI, and GRP within the SCN mimicked the phase-delaying effects of light on circadian control following in vivo microinjection and activated SCN single units recorded in vitro. These behavioral and cellular effects of coadministration of VIP, PHI, and GRP were significantly greater than administration of VIP, PHI, or GRP alone or coadministration of any 2 of these peptides. These data illustrate a new mechanism whereby multiple, colocalized neuropeptides interact in a functionally significant manner, and indicate that the interaction of VIP, PHI, and GRP may be involved in the regulation of circadian rhythms by the SCN.

Animals↗

PubMed1991

Identification of tubulin-binding proteins of the chicken erythrocyte plasma membrane skeleton which colocalize with the microtubular marginal band.

The plasma membrane of nucleated erythrocytes contains a microtubular marginal band which appears to be associated with the plasma membrane skeleton. In this report, we identify two families of cytoskeletal proteins which may be involved in such an association. These proteins, of molecular mass 78 kDa and 48 kDa on SDS-PAGE, are shown to bind tubulin based on a 125I-labeled tubulin binding assay. Solubilization of isolated chicken erythrocyte plasma membranes in Triton X-100 shows that these proteins centrifuge with the pellet, indicating that they are bound to the membrane skeleton. Finally, immunofluorescence studies using antisera raised against the 78 kDa and 48 kDa proteins show that they colocalize with the marginal band in intact cells. Colocalization of cytoskeletal tubulin-binding proteins with the marginal band favors a hypothesis suggesting that the 78 kDa and 48 kDa proteins are involved in the association of the two molecular superstructures.

Animals↗

PubMed1990

Immunoreactive A4 and gamma-trace peptide colocalization in amyloidotic arteriolar lesions in brains of patients with Alzheimer's disease.

Cerebral amyloid angiopathy (CAA) defines a biochemically heterogeneous entity that manifests as effacement of cerebral microvessel walls by a fibrillar material with characteristic tinctorial properties. In biochemical terms, the amyloid that infiltrates blood vessels in CAA is composed of the A4 or beta peptide of Alzheimer's disease (AD), a molecule related to gamma trace or cystatin C (seen in patients with hereditary cerebral hemorrhage with amyloidosis in Iceland, HCHWA-I), or the PrP characteristic of spongiform encephalopathy and scrapie. Using antibodies to synthetic peptides representing portions of the 4.2-kd Alzheimer A4 peptide and the gamma-trace peptide, we immunostained sections of brain from patients with AD, senile dementia of Alzheimer's type, and CAA with associated leukoencephalopathy. Immunohistochemical studies demonstrated colocalization of the A4 and gamma-trace peptides within arteriolar walls, but only rarely in A4 amyloidotic capillaries or senile plaque cores of amyloid. When gamma-tracelike reactivity was noted in capillary walls, it was sometimes noted within the cytoplasm of pericytes. Immunostaining was always more intense when the anti-A4 antibody was used as the primary antibody. Gamma-trace immunostaining was more prominent on the adventitial component of arteriolar walls, whereas A4 staining was usually seen more diffusely throughout the blood vessel wall, especially in the media. Rarely individual pericytelike cells showed prominent gamma-trace immunoreactivity. These findings suggest that A4 and gamma-tracelike molecules may colocalize within arteriolar walls within the brains of patients with AD, and highlight the fact that CAA identified with AD and HCHWA-I are not as biochemically distinct as was assumed previously. Furthermore these findings suggest that other peptidases or protease inhibitors may be found within amyloidotic microvessel walls and may contribute to senile brain change and CAA-related strokes, including hemorrhage and encephalomalacia.

Aged↗

PubMed1989

Dopamine and neurotensin storage in colocalized and noncolocalized neuronal populations.

The effects of reserpine on dopamine (DA) and neurotensin (NT) levels were studied in four different brain regions of the rat. Reserpine (0.5-5.0 mg/kg i.p., 6, 18, 48 and 72 hr) produced a dose- and time-dependent decrease in both DA and NT levels in the prefrontal cortex, a brain region innervated by a mixed DA/NT projection. The effect of reserpine was not mimicked by alpha-methylparatyrosine (200 mg/kg i.p.) pretreatment. Furthermore, the reserpine-induced decline in prefrontal cortex DA and NT levels occurred after gamma-butyrolactone (GBL)-induced inhibition of impulse flow (750 mg/kg i.p.). In contrast, in the nucleus accumbens and striatum, regions which contain colocalized (nucleus accumbens) and intrinsic (striatum and nucleus accumbens) neurotensin perikarya, reserpine produced declines in DA and increases in NT levels. alpha-Methylparatyrosine decreased striatal and nucleus accumbens DA levels without altering NT levels in these structures. GBL produced an increase in DA levels in the nucleus accumbens and striatum while decreasing nucleus accumbens and striatal NT levels. Reserpine attenuated the decline in nucleus accumbens and striatal NT levels produced by GBL. In the periaqueductal grey, a brain region densely innervated by NT which has a small population of DA perikarya, reserpine had no effect on NT levels. Because there is no known colocalization of DA and NT in the striatum, the increases in striatal NT levels after depletion of DA may indicate that striatal DA afferents control the release and/or synthesis of NT within NT cells in the striatum, thus leading to alterations in striatal tissue levels.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Butyrolactone↗

PubMed1988

Calmodulin colocalization with cold-stable and nocodazole-stable microtubules in living PtK1 cells.

To investigate the association of calmodulin (CaM) with microtubules (MTs) in the mitotic apparatus (MA), the distributions of both CaM and tubulin were examined in mitotic PtK1 cells in which MT subclasses had been selectively removed or altered by treatment with cold or with the MT inhibitor, nocodazole. A fluorescent CaM conjugate with tetramethylrhodamine isothiocyanate (CaM-TRITC) was microinjected into living cells, and the CaM distribution in the living cell was compared to the distribution of MTs indicated by tubulin immunofluorescence. In cells which had been treated for 2 h at 0 to 4 degrees C or with a low (0.03 micrograms/ml) dose of nocodazole, the only MTs remaining appeared to be kinetochore MTs (kMTs). The distribution of microinjected CaM-TRITC in these cells was indistinguishable from that found in untreated cells and appeared to be colocalized with the kMTs. In cells which were treated with a high (3.0 micrograms/ml) dose of nocodazole, only short MTs remained. When CaM-TRITC was injected into these cells, it formed a somewhat punctate distribution near the chromosomes and, after tubulin immunofluorescence processing, colocalized with what appeared to be remnants of kMTs. We believe that these observations support the hypothesis that CaM exists in the MA in a structural association with kMTs.

Animals↗

PubMed1988

Colocalization of taurine- and cysteine sulfinic acid decarboxylase-like immunoreactivity in the cerebellum of the rat with monoclonal antibodies against taurine.

Two monoclonal antibodies against fixative-modified taurine, Tau1 and Tau2, were produced, characterized, and used in the present study to analyze the distribution of taurine in the cerebellum of the rat. In addition, immunohistochemical colocalization experiments were performed to determine whether cerebellar neurons contain both taurine and its synthesizing enzyme, cysteine sulfinic acid decarboxylase (CSADC). In ELISAs, both Tau1 and Tau2 displayed high affinities for taurine conjugated to various carrier proteins and possessed some cross-reactivity for other amino acids which are present in lower concentrations in the brain than taurine. Tau2 was found to recognize only taurine and hypotaurine when paraformaldehyde was used to fix the amino acids to carrier proteins. With the use of glutaraldehyde fixation, Tau1 cross-reacted with conjugates of beta-alanine and hypotaurine and Tau2 cross-reacted strongly with conjugates of cysteic acid and hypotaurine and weakly with cysteine sulfinic acid. Despite different cross-reactivities, Tau1 and Tau2 exhibited almost identical patterns of neuronal staining in bands of Purkinje cells in the cerebellum. Staining of Purkinje cell dendrites was more prominent than staining of the soma. Light immunoreactivity was present in Golgi, stellate, and basket cells. A scattered population of granule cells displayed taurine-like immunoreactivity at the electron microscopic level. Immunostaining was identified in some terminals in the Purkinje cell layer and in a limited number of mossy fibers. Tau2-like immunoreactivity was colocalized with CSADC-like immunoreactivity in the cerebellar neurons described above. These immunoreactive cells may represent a subpopulation of neurons that contain a higher concentration of taurine than neighboring cells due to their ability to synthesize taurine. The intense immunoreactive staining of Purkinje cell dendrites provides support for the hypothesis that calcium-dependent release of taurine in the cerebellum may originate primarily from dendritic rather than synaptic processes and suggests a neuromodulator role for taurine in the cerebellum.

Animals↗

PubMed1993

Macromolecules that are colocalized with deposits of beta 2-microglobulin in hemodialysis-associated amyloidosis.

BACKGROUND: Common elements in many different types of amyloid may have important roles in amyloidogenesis. The proteinaceous tissue deposits have a common appearance in polarized light and other similar features. The present investigation describes for the first time the relation between beta 2-microglobulin (beta 2-M)-type amyloidosis and colocalized materials, as demonstrated using specific antibodies and hyaluronan-binding protein. EXPERIMENTAL DESIGN: Amyloid-rich carpal tunnel synovium was obtained surgically from 28 patients who were being treated by maintenance hemodialysis. Serial sections were examined using a hyaluronan (hyaluronic acid)-binding protein and antibodies against heparan sulfate-glycosaminoglycan, chondroitin sulfate-proteoglycan, dermatan sulfate-proteoglycan, alpha 1-antichymotrypsin, alpha 1-antitrypsin, inter-alpha-trypsin inhibitor, haptoglobin, and ubiquitin. RESULTS: Accumulation of hyaluronan was of three types, namely, localization around beta 2-M deposits, colocalization with deposition of beta 2-M itself and localization at a small distance from beta 2-M deposits. Immunostaining for heparan sulfate glycosaminoglycan was demonstrated at the sites of beta 2-M plaques. Chondroitin sulfate-proteoglycan did not show specific patterns of immunostaining, resembling hyaluronan rather than heparan sulfate. The other materials tested, alpha 1-antichymotrypsin, alpha 1-antitrypsin, inter-alpha-trypsin, haptoglobin and ubiquitin, were not immunostained at sites of beta 2-M plaques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed that the molecular weight of heparan sulfate-glycosaminoglycan was 16,000. CONCLUSIONS: These results suggest that HS has an important role in hemodialysis-associated amyloidosis as it does in other types of amyloidosis. Moreover, accumulation of hyaluronan may be an indication of inflammation of the carpal synovium.

Amyloidosis↗

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↗

PubMed1996

Nuclear colocalization of the Ro 60 kDa autoantigen and a subset of U snRNP domains.

The Ro 60 kDa protein is an RNA binding molecule present both in the cytoplasm and in the nucleus. Cytoplasmic Ro 60 kDa is complexed to other proteins and to certain RNAs denoted hYRNAs. This RNA-protein complex is also known as the Ro/SSA antigen recognized by sera from patients with certain autoimmune disorders. Components interacting with the nuclear Ro 60 kDa protein fraction in mammalian cells have not been identified. To look for an association with previously known nuclear structures, rabbit antisera to the amino- and carboxy-terminal parts of the Ro 60 kDa protein were used in immunomorphological studies on HeLa cells. A strong speckled nuclear pattern and a weak cytoplasmic staining were detected. Double immunofluorescence staining with affinity purified anti-Ro 60 kDa antibodies and monoclonal antibodies recognizing the Sm and RNP antigens of the U snRNPs, displayed colocalization. Another U snRNP containing nuclear compartment, the coiled bodies, did not contain any Ro 60 kDa protein. Cells infected with a toga virus demonstrated redistribution of both U snRNP antigens and the Ro 60 kDa protein with retained colocalization. These results indicate a role for the nuclear fraction of the Ro 60 kDa protein in RNA processing.

Animals↗

PubMed1996

Glutamate and aspartate immunoreactive neurons of the rat basolateral amygdala: colocalization of excitatory amino acids and projections to the limbic circuit.

The basolateral amygdala has projections to several structures that take part in the limbic cortico-striato-pallido-thalamic circuit, including the prefrontal cortex, ventral striatum, and mediodorsal thalamic nucleus. The present investigation used a technique that combines retrograde tract tracing with immunohistochemistry for glutamate and aspartate to determine if amygdaloid neurons projecting to different targets in the limbic circuit can be distinguished on the basis of their content of excitatory amino acids. Cell counts revealed that at least 85-95% of the neurons in the basolateral nucleus projecting to the prefrontal cortex or ventral striatum were pyramidal cells that exhibited glutamate or aspartate immunoreactivity. Colocalization studies indicated that 94-100% of aspartate-immunoreactive neurons in the basolateral nucleus were also glutamate positive and that 92-94% of glutamate-immunoreactive neurons were also aspartate positive. A small number of glutamate-positive pyramidal neurons in the anterior subdivision of the cortical nucleus were found to project to the mediodorsal thalamic nucleus. However, the great majority of amygdaloid neurons with projections to the mediodorsal nucleus did not exhibit glutamate or aspartate immunoreactivity. The absence of glutamate and aspartate immunoreactivity in these cells suggests that these neurons do not use excitatory amino acids as neurotransmitters. The finding of high levels of glutamate and aspartate in basolateral amygdaloid neurons projecting to the prefrontal cortex and ventral striatum is consistent with previous reports indicating that these neurons may use excitatory amino acids as neurotransmitters, but is not a definitive criterion for this determination.

Amygdala↗

PubMed1996

Colocalization of ionotropic glutamate receptor subunits with NADPH-diaphorase-containing neurons in the rat mesopontine tegmentum.

Tegmental cholinergic neurons vary their discharge patterns across the sleep-wake cycle, and glutamate is suggested to play an important role in determining these firing patterns. Cholinergic and noncholinergic neurons in the mesopontine tegmentum have different susceptibilities to various excitotoxins, presumably because of heterogeneity in the expression of glutamate receptor subtypes in this area. By using a double-labeling procedure that combines nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) histochemistry and avidin-biotin-peroxidase immunocytochemistry with diaminobenzidine as the chromogen, we compared the colocalization of AMPA receptor subunits GluR1, GluR2/3, and GluR4, kainate receptor subunits GluR5/6/7, and an NMDA receptor subunit NMDAR1 on NADPH-diaphorase-positive (cholinergic) neurons in the mesopontine tegmentum. Throughout the brainstem, neurons immunoreactive for GluR2/3 and NMDAR1 were most numerous, whereas neurons labeled for GluR1, GluR4, and GluR5/6/7 were less common. Specifically within the mesopontine tegmentum, the proportion of double-labeled neurons in the diaphorase-containing cell population was highest with GluR1 (43%) and lowest with GluR5/6/7 (12%). Regardless of the receptor subunit type, the greatest numbers of double-labeled neurons were observed in the pedunculopontine tegmental nucleus pars compacta and the fewest in the dorsal aspect of the laterodorsal tegmental nucleus. In addition, there were regional differences in the relative expression of receptor subunits and diaphorase-positive neurons across the subdivisions of the tegmental cholinergic column. Because each ionotropic subunit confers distinctive properties to a receptor channel, the present results suggest that mesopontine cholinergic neurons have nonuniform responses to glutamate and are also discriminable from basal forebrain cholinergic neurons in terms of glutamate receptor configuration.

Animals↗