Localization of urokinase-type plasminogen activator mRNA in the adult mouse brain

Urokinase-type plasminogen activator (uPA) is an inducible serine protease, secreted by a variety of cell types, that functions in fibrinolysis and has been implicated also in events such as cell migration and tissue remodeling and repair. To explore the role of uPA in the adult brain, we have now screened the whole mouse brain for cells expressing the uPA gene through in situ hybridization using ³⁵S-complementary RNA. uPA mRNA was visualized predominantly in three regions: (1) the subicular complex, (2) the entorhinal cortex, (3) the parietal cortex, where the signal was somewhat lower and confined to layers IV and VI. Weaker signals were seen in the basolateral nucleus of the amygdala and in the anterodorsal thalamic nucleus, and also in the hilus of the dentate gyrus where labeling was slightly over background. Cells exhibiting uPA mRNA signaling were large neurons according to morphological criteria. These results support the view of uPA being involved in neuronal functions of the adult brain, specifically in the hippocampal formation and the parietal cortex.     

Localization of tissue plasminogen activator mRNA in adult rat brain

The distribution of tissue plasminogen activator (tPA) messenger RNA in rat brain was studied using in situ hybridization with ³⁵S UTP-labeled RNA probes derived from a fulllength tPA cDNA. Sense strand controls produced low, even backgrounds, with small elevations in the hippocampus. Full-length antisense probes produced strong signals over cerebral ventricular ependyma (including ependyma of the subcommissural organ), meninges, blood vessels, and Purkinje cell layer of the cerebellum, as well as strong signals over scattered cells throughout the brain. Some of these scattered labeled cells were large with lightly stained nuclei, while others were small with darkly stained nuclei. The large labeled cells, which were probably neurons, constituted 8% and 8% of cells in the brain stem and neocortex, respectively, and 100% of Purkinje cells. The small cells, which were present in all areas of the brain, constituted 3–11 % of cells in individual brain areas.     

Localization of mRNA for Ca/calmodulin-dependent protein kinase I in the brain of developing and mature rats

The gene expression of Ca²⁺/calmodulin-dependent protein kinase I (CaM kinase I) in the brain of developing and adult rats was examined by in situ hybridization histochemistry. During the development, CaM kinase I showed two chronological expression patterns; the persistent and relative high expression as observed in the olfactory bulb and cerebellar cortex, and the gradual decrease in the expression during the postnatal development as observed in most other brain regions. The gene expression was not detected in the germinal ventricular zone and cerebellar external granular layer. In the mature brain, CaM kinase I mRNA was expressed widely, though weakly in general, in almost all neurons, except for the olfactory bulb, cerebellum and hippocampus expressing at high intensity. These findings suggest that CaM kinase I may play a variety of neuronal Ca²⁺/calmodulin-mediated signaling processes in the developing and mature brains.     

Localization of the ras-like rab3A protein in the adult rat brain.

Rab3A is a small GTP-binding synaptic vesicle protein, shown to dissociate from synaptic vesicle membranes upon depolarization-induced exocytosis. Using an antiserum raised against rab3A, we found that the antigen was localized to the neuropil of specific brain regions, but was not present in major fiber tracts or most cell bodies. For example, the neuropil of several thalamic nuclei (i.e, dorsal lateral geniculate nucleus, lateral posterior nucleus, ventroposterior nucleus), cerebral cortex, upper layers of the superior colliculus and matrix zones of the neostriatum, were strongly immunoreactive, while the anterior commissure, corpus callosum, optic tract and internal capsule were devoid of staining. The hippocampus, dark staining was observed in the stratum oriens, stratum radiatum and molecular layer of the dentate gyrus, while the pyramidal, stratum lacunosum moleculare and dentale granule layers were not stained. In cerebellum the molecular layer and to a lesser extent, the underlying granule cell layer showed enhanced immunoreactivity. Seven days after excitotoxic lesions of the cerebral cortex, rab3A immunoreactivity was diminished in the mirror locus in the contralateral coritcal hemisphere and in certain thalamic nuclei ipsilateral to the injection site. These results show that rab3A is localized to a number of specific regions. Its absence from other areas suggests that this synaptic vesicle protein is not universal to all neuronal terminals and pathways. In addition, our lesion studies indicate that for some brain regions, much of the antigen originates in cortical neurons and is distributed within specific axonal projections.     

Localization of mRNA for c-kit receptor and its ligand in the brain of adult rats: an analysis using in situ hybridization histochemistry.

Localization of mRNA for the c-kit receptor and its ligand (Sl factor) in the brain of adult rats was studied using in situ hybridization histochemistry. The mRNA for the c-kit receptor was detected in the forebrain, the lower brain stem and the cerebellum. In the forebrain, the c-kit mRNA signals were detected in the olfactory bulb, the caudate-putamen, throughout the superficial cortex, the accumbens nucleus, the nucleus of vertical limb diagonal band, the bed nucleus of anterior commissure, Ammon's horn, the entopeduncular nucleus, the subthalamic nucleus, the dorsal raphe nucleus, the parasubiculum, the presubiculum, the ventricular nucleus of lateral lemniscus, and the entorhinal cortex. In the lower brain stem, the signals were detected in the inferior colliculus, the spinal vestibular nucleus, the spinal tract nucleus of trigeminal nerve, and the pyramidal tract. In the cerebellum, the signals were detected in the molecular layer of the cortex and cerebellar nuclei. By contrast, the signals of mRNA for Sl factor were detected in the forebrain and the cerebellum. In the forebrain, the signals were detected in the olfactory bulb, the endopiriform nucleus, the septohippocampal nucleus, the habenular nuclei, and most of the thalamic nuclei. In the cerebellum, the signals were detected in Purkinje cells. Several pairs of structures were found in which mRNA of either the c-kit receptor or the Sl factor was expressed and between which the synaptic connection had been reported, suggesting that the interaction between the c-kit receptor and the Sl factor may play some roles in the development of such synaptic connections.     

Localization of protein Ser/Thr phosphatase 5 in rat brain

Protein phosphatase 5 is a recently discovered Ser/Thr phosphatase that is structurally related to calcineurin and protein phosphatases 1 and 2. Northern blot and in situ hybridization studies have shown that protein phosphatase 5 mRNA is present at high levels in brain and is localized to discrete regions. In the present study, we used immunocytochemistry and immunoblot analyses to examine the regional and subcellular distribution of this enzyme in brain. Our work demonstrates that protein phosphatase 5 is widely expressed throughout brain, but is not uniformly distributed. The most intense staining occurred in neurons of the cerebellum, cerebral cortex, and the supraoptic nucleus of the hypothalamus. Other areas also contained immunoreactive cell bodies, including the globus pallidus, hippocampus, thalamus, lateral preoptic area of the hypothalamus, substantia nigra and other brainstem nuclei. Staining in these cells was observed primarily in perikarya and proximal processes.     

Localization of zip1 and zip4 mRNA in the adult rat brain

The localization of two members of the Slc39a (zip1 and zip4) family of zinc transporters was examined in the brains of adult mice. Zip1 was highly enriched in brain regions with high densities of neuronal cell bodies, including the hippocampus, thalamus, and perifontal cortex. Zip1 was also expressed in commissural fiber tracts such as the corpus callosum and anterior commissure, but little was found in the internal and external capsules. Also, very low amounts of zip1 mRNA were detected in resting astrocytes and reactive astrocytes that were examined at 14 days after inflicting a stab wound. Zip1 mRNA was detected in ependymal cells lining the third and lateral ventricles and epithelium cells in the choroid plexus. Interestingly, zip4 mRNA was detected in the choroid plexus but not in the ependymal cells or other neural elements. Zip4 mRNA was also detected in brain capillaries, but zip1 mRNA was not. In zip4 knockout heterozygotes that express green fluorescent protein regulated by the zip4 promoter, green fluorescent protein was detected in brain capillaries. Because zip4 levels are regulated by dietary Zn, our studies suggest that the brain has the potential of adapting to changes in Zn status. © 2009 Wiley‐Liss, Inc.     

Localization of mRNA for Dri 42, subtype 2b of phosphatidic acid phosphatase, in the rat brain during development

The expression of a gene termed Dri 42, a differentiation-related gene originally identified from intestine and a gene encoding phosphatidic acid phosphatase 2b isoform, was localized in developing and matured rat brains by in situ hybridization histochemical analysis. The gene expression was dominant in the ventricular germinal zone without significant expression in the intermediate, mantle and marginal zones throughout embryonic brain and spinal cord. The dominant expression in the ventricular germinal zone was maintained at P0 and P7, but it markedly decreased at later postnatal stages, while persistently high expression was detected in Bergmann glial cells of the cerebellar Purkinje cell layer throughout the postnatal development.     

Localization of mRNA for CHRNA7 in human fetal brain.

The aim of this study was to determine the regional distribution in situ of the mRNA for the alpha 7 subunit of the neuronal nicotinic acetylcholine receptor in human fetal brain. We found high levels of alpha 7 gene expression in nuclei that receive sensory information, such as those of the neocortex and hippocampus, the thalamic nuclei, the reticular thalamic nucleus, the pontine nuclei and the superior olive complex. These data support a possible regulatory function for alpha 7-containing receptors in sensory processing, which may be involved in the pathological physiology of schizophrenia and autism. Early alpha 7 gene expression is also consistent with a morphogenetic role for alpha 7 receptors in central nervous system development.     

Localization of mRNA for SHIP2, SH2 domain-containing inositol polyphosphate 5-phosphatase, in the brain of developing and mature rats

The localization of mRNA for SHIP2, SH2 domain containing inositol 5-phosphatase SHIP isozyme, was examined by in situ hybridization histochemistry in the brain of developing and mature rats. SHIP2 mRNA was first detected in the ventricular germinal zone at embryonic stages. As the postnatal development proceeded, the expression signal was evident in cell of the white matters, presumptive oligodendrocytes, and no significant expression was seen in neurons throughout the development.     

Localization of the ras-like rab3A protein in the adult rat brain

Rab3A is a small GTP-binding synaptic vesicle protein, shown to dissociate from synaptic vesicle membranes upon depolarization-induced exocytosis. Using an antiserum raised against rab3A, we found that the antigen was localized to the neuropil of specific brain regions, but was not present in major fiber tracts or most cell bodies. For example, the neuropil of several thalamic nuclei (i.e, dorsal lateral geniculate nucleus, lateral posterior nucleus, ventroposterior nucleus), cerebral cortex, upper layers of the superior colliculus and matrix zones of the neostriatum, were strongly immunoreactive, while the anterior commissure, corpus callosum, optic tract and internal capsule were devoid of staining. The hippocampus, dark staining was observed in the stratum oriens, stratum radiatum and molecular layer of the dentate gyrus, while the pyramidal, stratum lacunosum moleculare and dentale granule layers were not stained. In cerebellum the molecular layer and to a lesser extent, the underlying granule cell layer showed enhanced immunoreactivity. Seven days after excitotoxic lesions of the cerebral cortex, rab3A immunoreactivity was diminished in the mirror locus in the contralateral coritcal hemisphere and in certain thalamic nuclei ipsilateral to the injection site. These results show that rab3A is localized to a number of specific regions. Its absence from other areas suggests that this synaptic vesicle protein is not universal to all neuronal terminals and pathways. In addition, our lesion studies indicate that for some brain regions, much of the antigen originates in cortical neurons and is distributed within specific axonal projections.     

Influence of immobilization stress on the expression and phosphatase activity of protein phosphatase 2A in the rat brain.

BACKGROUND: Protein phosphatase 2A (PP2A) is a major kinase phosphatase that plays an important role in regulating the activities of protein kinase cascades. It has been revealed that stress changes neuronal gene expression by activating these cascades. We examined the expression of the catalytic subunit C and serine and threonine phosphatase activity of PP2A in the rat frontal cortex and hippocampus following various immobilization stress paradigms. METHODS: Immunoblot and immunohistochemical analyses were performed to examine the expression of PP2A. The level of phosphatase activity of PP2A was determined as the amount of free phosphate generated from a synthetic phosphopeptide. RESULTS: Immunoblot analysis revealed no significant change in the level of PP2A immunoreactivity in response to either a single or repeated stress. Immunohistochemical analysis revealed that neither a single nor repeated stress changed PP2A immunoreactivity in the hippocampus; however, the levels of serine and threonine phosphatase activity in the frontal cortex and hippocampus were significantly upregulated in response to a single or repeated stress. CONCLUSIONS: These results demonstrated that both a single and repeated immobilization stress upregulated the activity of PP2A in the rat brain, suggesting that PP2A may be involved, at least in part, in the downregulation of protein kinase activation induced by stress.     

Localization and response of the 14-3-2 brain protein.

The 14-3-2 acidic protein specific for nerve tissue has been localized to bands 4 and 5 counted from the anodal front band in a polyacrylamide-gel electrophoretic pattern. The material consisted of cut-out layers of pyramidal nerve cells of the CA3 hippocampal region of the rat. Extracted protein samples were subjected to immunoprecipitation, Coons' double-layer immunofluorescence, microcomplement fixation, and a localization experiment using microdensitometry on a 400 muphi gel electrophoresis pattern with and without extra added 14-3-2 protein. The protein fractions 4 and 5, now shown to contain 14-3-2 protein, have recently been found to incorporate increased precursors at training as compared to controls.     

Localization of trkB mRNA in postnatal brain development

We investigated the localization of trkB mRNA, which encodes a putative component of high-affinity brain-derived neurotrophic factor (BDNF) or the neurotrophin-3 (NT-3) receptor, in the postnatal rat brain by in situ hybridization histochemistry. At birth, trkB mRNA was strongly expressed in various regions with the thalamus and cerebral cortex showing the strongest expression. As the rat grows, expression generally persisted or declined in most regions with the exception of the hippocampus where trkB mRNA expression increased during postnatal development. In the adult brain, trkB mRNA was detected in the olfactory system, cerebral cortex, hippocampal formation, amygdala, and cerebellar cortex. These findings, together with the developmental profiles of BDNF and NT-3 mRNA expressions, suggest that trkB product (gp145^trkB) mainly transduces NT-3 signals early in the postnatal period, and BDNF signals later in the period. © 1993 Wiley-Liss, Inc.     

Localization of cannabinoid receptor mRNA in rat brain

Cannabinoid receptor mRNA was localized in adult rat brain by ³⁵S-tailed oligonucleotide probes and in situ hybridization histochemistry. Labelling is described as uniform or non-uniform depending on the relative intensities of individual cells expressing cannabinoid receptor mRNA within a given region or nucleus. Uniform labelling was found in the hypothalamus, thalamus, basal ganglia, cerebellum and brainstem. Non-uniform labelling that resulted from the presence of cells displaying two easily distinguishable intensities of hybridization signals was observed in several regions and nuclei in the forebrain (cerebral cortex, hippocampus, amygdala, certain olfactory structures). Olfactory-associated structures, basal ganglia, hippocampus, and cerebellar cortex displayed the heaviest amounts of labelling. Many regions that displayed cannabinoid receptor mRNA could reasonably be identified as sources for cannabinoid receptors on the basis of well documented hodologic data. Other sites that were also clearly labelled could not be assigned as logical sources of cannabinoid receptors. The localization of cannabinoid receptor mRNA indicates that sensory, motor, cognitive, limbic, and autonomic systems should all be influenced by the activation of this receptor by either exogenous cannabimimetics, including marijuana, or the yet unknown endogenous “cannabinoid” ligand. © 1993 Wiley-Liss, Inc.     

Localization of nerve growth factor receptor messenger RNA and protein in the adult rat brain

We have used in situ hybridization and immunocytochemistry to map the cellular localization of NGF receptor (NGF-R) mRNA and protein in the adult rat brain. In addition to basal forebrain magnocellular neurons, NGF-R is widely expressed within the CNS, including neurons of the caudate/putamen, ventral premamillary nucleus, mesencephalic trigeminal nucleus, prepositus hypoglossal nucleus, raphe nucleus, nucleus ambiguous, and Purkinje cells of the cerebellum. Cells of the vestibulocochlear ganglion also contain NGF-R mRNA and protein. Ventricular subependymal cells and tanycytes are clearly stained by immunocytochemistry, yet only very weak hybridization is detectable in these cells. Also, greater amounts of NGF-R protein than of mRNA appear to be present in the glomeruli of the olfactory bulb, area postrema, and nucleus tractus solitarius. Areas that contain only NGF-R immunoreactive fibers and terminals can be distinguished from the cellular sites of NGF-R biosynthesis and include the suprachiasmatic nucleus, the principal olivary pretectal nucleus, the superior colliculus, the inferior olive, and the principal and spinal trigeminal nuclei. This study shows that NGF-R is widely expressed within individual neurons in different areas of the rat brain and identifies new potential CNS target sites of endogenous NGF.     

Localization of gene expression of calreticulin in the brain of adult mouse.

The localization of gene expression of calreticulin, a calcium-binding protein in the endoplasmic reticulum, was examined throughout the entire brain of adult mice by in situ hybridization. Calreticulin mRNA is expressed widely and heterogeneously in discrete neurons throughout the brain, but the white matters expressed it weekly or faintly. In the olfactory bulb, the mRNA is expresses moderately in the mitral cells, but weakly in the periglomerular cells and internal granule cells. In the cerebrum, the gene is expressed intensely in the piriform cortex, but weakly in neocortex, the entorhinal cortex and the amygdaloid nuclei. In the hippocampal formation, calreticulin mRNA is expressed intensely in the CA1-CA3 regions but less intensely in the granule cells of the dentate gyrus. The caudate-putamen, thalamic and hypothalamic nuclei, and mammillary nuclei express the mRNA weakly or faintly. In the mesencephalon, pons and medulla, moderate expression of the mRNA is detected in the pontine nuclei and the locus ceruleus. Weak expression of the mRNA is detected in several discrete nuclei and zones such as the substantia nigra, the superior colliculus and the central gray. Expression signals of calreticulin mRNA are faint in the inferior olive. In the cerebellum, calreticulin mRNA is expressed moderately in the Purkinje cells whereas no significant expression is detected in the granule cells. The plexus choroideus of the lateral, third and fourth ventriculi express calreticulin mRNA intensely although no distinct expression of the mRNA is discerned in the ependyma.