Cellular distribution of transferrin immunoreactivity in the developing rat brain

The cellular distribution of immunoreactive transferrin has previously been studied in adult rat brain with the conclusion that transferrin is largely confined to oligodendrocytes, although studies in the developing rodent brain indicated some staining of neurons. Detailed investigations of a wider range of ages than studied earlier have shown that in the developing rat brain, transferrin is not only present in oligodendrocytes but also in other glial cell types and in neurons and endothelial cells. The preponderant cell type which is positive for transferrin and its regional distribution are, however, related to the age of the animal except in most circumventricular organs in which strong transferrin immunoreactivity was seen throughout the developmental period studied.     

Cellular localization of prolactin-releasing peptide messenger RNA in the rat brain.

Prolactin-releasing peptide (PrRP), a novel peptide identified as the endogenous ligand for an orphan receptor isolated from the pituitary, is a potent stimulator of prolactin release. To get a clue of the functional roles of the peptide, we performed in situ hybridization histochemistry for PrRP mRNA to define the cellular localization of PrRP-producing cells in the brain of the cycling adult female rat during diestrus. The PrRP mRNA-containing cells were located in the caudal part of the dorsomedial nucleus of the hypothalamus. In the brainstem, the cells were found in the caudal part of the solitary tract nucleus and in the caudal ventrolateral medulla (ventrolateral intermediate reticular field). Specific signals for PrRP mRNA were not detected in other brain regions. Although PrRP is a candidate for being a hypophysiotropic specific releasing factor, the discrete distribution of PrRP in the extrahypothalamic area suggests that the peptide has other physiological functions in the central nervous system.     

Immunohistochemical localization of TRPC6 in the rat substantia nigra

Transient receptor potential channels (TRPC) are plasma membrane, nonselective cationic channels and have been proposed as candidates involved in the regulation of cellular Ca2+ influx [D.E. Clapham, L.W. Runnels, C., Strubing, The TRP ion channel family, Nat. Rev. Neurosci. 2 (2001) 387-396; A. Martorana, C. Giampa, Z. DeMarch, M.T. Viscomi, S. Patassini, G. Sancesario, G. Bernardi, F.R. Fusco, Distribution of TRPC1 receptors in dendrites of rat substantia nigra: a confocal and electron microscopy study, Eur. J. Neurosci. 24 (2006) 732-738]. Studies on regional localization patterns of TRPCs are necessary to provide helpful guidelines for correlating current types with particular channels. In this study, we examined the distribution of one particular member of TRPC superfamily, namely, TRPC6, in the substantia nigra of normal rat brain. Single and double label immunohistochemistry were employed to perform both light and confocal microscopy observations. Our single label studies showed that, in the substantia nigra, TRPC6 labeled the perikarya with a diffuse and intense immunoreaction product distributed throughout cell cytoplasm whereas only a light immunostaining was observed in the cell nuclei. No labeling of axon or terminals was observed, although TRPC6 was evenly distributed in the neuropil. Our dual label studies showed a TRPC6 immunoreactivity pattern that was localized into the proximal dendrites and axon hillock of the large dopaminergic neurons identified by TH immunoreaction. Furthermore, our double label immunofluorescence study for TRPC6 and mGluR1 showed a complete co-localization of the two markers in the substantia nigra. Moreover, TRPC6 did not co-localize with synaptophysin. Thus, our study shows the postsynaptic localization of TRPC6 and its association with mGluR1 in the midbrain dopamine neurons.     

Autoradiographic localization of [3H]reserpine in rat brain: Correlation with distribution of monoaminergic neurones

The in vivo localization of [³H]reserpine in rat brain was studied by autoradiography using unfixed, frozen sections prepared 18 h after intravenous injection of the radiolabel. Radioactivity was localized in those areas of the brain where monoaminergic cell bodies and nerve terminals have been describes. The radiolabel found was chromatographically identical with reserpine. Treatment of animals with the unlabelled drug 4 h before administration of [³H]reserpine prevented the specific localization of radioactivity in the brain.     

Minocycline alleviates hypoxic-ischemic injury to developing oligodendrocytes in the neonatal rat brain

The role of minocycline in preventing white matter injury, in particular the injury to developing oligodendrocytes was examined in a neonatal rat model of hypoxia-ischemia. Hypoxia-ischemia was achieved through bilateral carotid artery occlusion followed by exposure to hypoxia (8% oxygen) for 15 min in postnatal day 4 Sprague-Dawley rats. A sham operation was performed in control rats. Minocycline (45 mg/kg) or normal phosphate-buffered saline was administered intraperitoneally 12 h before and immediately after bilateral carotid artery occlusion+hypoxia and then every 24 h for 3 days. Nissl staining revealed pyknotic cells in the white matter area of the rat brain 1 and 5 days after hypoxia-ischemia. Hypoxia-ischemia insult also resulted in apoptotic oligodendrocyte cell death, loss of O4+ and O1+ oligodendrocyte immunoreactivity, and hypomyelination as indicated by decreased myelin basic protein immunostaining and by loss of mature oligodendrocytes in the rat brain. Minocycline significantly attenuated hypoxia-ischemia-induced brain injury. The protective effect of minocycline was associated with suppression of hypoxia-ischemia-induced microglial activation as indicated by the decreased number of activated microglia, which were also interleukin-1beta and inducible nitric oxide synthase expressing cells. The protective effect of minocycline was also linked with reduction in hypoxia-ischemia-induced oxidative and nitrosative stress as indicated by 4-hydroxynonenal and nitrotyrosine positive oligodendrocytes, respectively. The reduction in hypoxia-ischemia-induced oxidative stress was also evidenced by the decreases in the content of 8-isoprostane in the minocycline-treated hypoxia-ischemia rat brain as compared with that in the vehicle-treated hypoxia-ischemia rat brain. The overall results suggest that reduction in microglial activation may protect developing oligodendrocytes in the neonatal brain from hypoxia-ischemia injury.     

The regional distribution and cellular localization of iron in the rat brain

The regional distribution and cellular localization of iron throughout the rat brain was determined with iron histochemistry. Densitometry was used to measure the intensity of stain of 51 iron-concentrating sites. Among the areas of highest iron content are the circumventricular organs, islands of Calleja, globus pallidus, ventral pallidum, substantia nigra pars reticulata, interpeduncular nucleus, dentate nucleus, and interpositus nucleus. Iron occurs most commonly in oligodendrocytes and in the fibrous network of the neuropil, but is also found in the interstitial spaces of circumventricular organs and in the tanycytes of the organum vasculosum of the lamina terminalis, median eminence, and walls of the third ventricle. In diverse areas throughout the brain—among them, the islands of Calleja, dentate gyrus of the hippocampal formation, lateral septal nucleus, and central amygdala—iron is found in association with the perikarya and neuronal processes of nerve cells.The overlapping distribution patterns of iron and γ-aminobutyric acid, enkephalin, and luteinizing hormone-releasing hormone suggest that the distribution of iron is related to its association with the metabolism of one or more neurotransmitters or neuroactive compounds.     

Cellular distribution of the NMDA-receptor activated synapto-nuclear messenger Jacob in the rat brain

In previous work, we found that the protein messenger Jacob is involved in N-methyl-d-aspartate receptor (NMDAR) signaling to the nucleus and cAMP response element-binding protein (CREB) mediated gene expression in hippocampal primary neurons. Particularly, extrasynaptic NMDAR activation drives Jacob efficiently into the nucleus where it then induces gene expression that promotes neurodegeneration. However, the protein also translocates to the nucleus in CA1 neurons after Schaffer collateral long-term potentiation (LTP) but not long-term depression (LTD), suggesting that Jacob might be involved in hippocampal and LTP-dependent learning and memory processes. Not much is known about the cellular and subcellular distribution of the protein in brain. In this paper, we provide an overview of the expression of Jacob in rat brain with special emphasis on the hippocampus. We show that Jacob is abundant in hippocampal pyramidal neurons and interneurons but absent from astrocytes and microglia. Interestingly, we found that Jacob is also present in mossy fiber axons. Double immunofluorescence confocal laser scans with presynaptic markers demonstrate that Jacob is indeed found at excitatory but not inhibitory presynaptic sites. Accordingly, we found no substantial co-localization of Jacob with a postsynaptic marker of inhibitory synapses, gephyrin. In contrast, almost all postsynaptic density protein 95 (PSD-95) positive excitatory postsynaptic sites also exhibited strong Jacob-immunofluorescence. Taken together, these data support a synaptic and nuclear role of Jacob that implicates long-distance NMDAR signaling to the nucleus in excitatory neurons.     

Cellular localization and tissue distribution of polycystin-1

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid-filled cysts in both kidneys, in addition to a variety of extra-renal manifestations. The PKD1 gene product, polycystin-1, encodes a novel protein with a putative role in cell–cell/cell–matrix interactions. The present study we focused on the (sub)cellular localization of polycystin-1 in cultured cells, and on its tissue distribution in various organs. In Madin Darby canine kidney (MDCK) cells, several polyclonal antibodies showed intense staining at the sites of interaction between adjacent cells, which remained after Triton extraction. Weak cytoplasmic staining was observed. No signal was detected at the free borders of cell aggregates, supporting a role for polycystin-1 in cell–cell interactions. At the tissue level, polycystin-1 expression was observed in specific cell types in tissues with known manifestations of the disease, but also in tissues of organs which have not been reported to be affected in ADPKD. Expression was frequently seen in epithelia, but also in endocrine cells (pancreatic islets, parathyroid-producing cells, clusters in the adenohypophysis, clusters in the adrenal gland, and Leydig cells in the testis). In addition, expression was observed in myocardium and more weakly in myocytes of cardiac valves, of the cerebral arteries, and of skeletal muscles. Copyright © 1999 John Wiley & Sons, Ltd.     

Cellular localization of nerve growth factor-like immunoreactivity in hippocampus and septum of adult rat brain.

The cellular localization of the nerve growth factor-like immunoreactivity (NGF-LIR) has been studied in the intact adult rat brain at the level of the hippocampus and the septum. Immunolabelling for NGF combined with counterstaining with cresyl violet and double immunostaining technique, which allowed simultaneous localization of NGF-LIR and that of astroglial marker -GFAP, were used. The data indicate neuronal localization of NGF-like immunoreactivity and a lack of colocalization of NGF-LIR with the immunoreactivity of GFAP in the hippocampus. These data are consistent with in situ hybridization results for NGF and immunocytochemical results for pro-NGF localization obtained by others. At the septal level, apart from neuronal localization of NGF-LIR, single NGF-like immunoreactive astrocytes have been observed. This suggests that, although to a very small extent, in vivo intact brain astrocytes may, just as astrocytes growing in vitro, synthesize NGF-like molecules. This finding may be of importance in better understanding the trophic support for NGF responsive cholinergic neurones in the brain.     

Cellular and subcellular localization of an octadecaneuropeptide derived from diazepam binding inhibitor: immunohistochemical studies in the rat brain

Immunocytochemical methods, both light and electron microscopic, were used to identify the cellular and subcellular locations of octadecaneuropeptide-like immunoreactivity (ODN-LI) in rat brains serially sectioned in total. ODN-LI includes a newly discovered family of rat brain neuropeptides that are processing products of a common endogenous neuropeptide precursor, diazepam binding inhibitor (DBI). The members of this neuropeptide family have been shown to displace benzodiazepines and beta carbolines from their specific recognition sites located on the allosteric modulatory centers of GABAA receptors. We have previously examined the distribution of DBI-LI in rat brain. The anti-ODN antiserum used in this study does not cross-react with rat DBI, and thus allows a distinct analysis of ODN-LI as opposed to DBI-LI, in rat brain. Neuronal perikarya with ODN-LI were located in many brain nuclei, such as the pontine n., reticular thalamic n., subgeniculate n., supraoptic n. and suprachiasmatic n., and also in brain areas such as cerebral and cerebellar cortex, hippocampus, inferior colliculus, olfactory bulb and subiculum. In addition to perikaryal labelling, a punctate or diffuse immunostaining with ODN antibodies was detected in many brain regions such as cerebellum, hippocampus, amygdaloid area, olfactory tubercle, some of the deep cerebellar nuclei and some circumventricular organs. At the electron microscopic level ODN-LI was identified in neuronal perikarya, processes and terminals. In the axon terminals, ODN-LI appears to be associated with synaptic vesicles. Whenever ODN-LI was detected within neurons, DBI-LI was also found in identical cells. In addition to neurons, DBI-LI was found in glia or glial-like cells, while ODN-LI was not found in these cells. Our findings are consistent with the hypothesis that ODN may be a neuron-specific processing product of DBI and that ODN-like peptides may act as putative endogenous allosteric modulators of various GABAA receptor subtypes.     

The semi-quantitative distribution and cellular localization of angiotensinogen mRNA in the rat brain.

The present study describes the regional distribution and cellular localization of angiotensinogen-mRNA in the rat brain as investigated by means of in situ hybridization also in combination with immunocytochemistry for glial fibrillary acidic protein. The angiotensinogen gene expression seemed to be restricted to astroglia and showed marked regional differences. In some areas angiotensinogen-mRNA was present in almost all astrocytes with a strong signal (e.g. hypoglossal nucleus), whereas in other areas the angiotensinogen gene was expressed only in a certain population of glial cells. Some areas like the lateral septum were devoid of any detectable angiotensinogen-mRNA. A semi-quantitative atlas of the regional distribution of brain angiotensinogen-mRNA was obtained by using computer-assisted microdensitometry and revealed considerable rostro-caudal fluctuations of the angiotensinogen-mRNA content of certain regions (e.g. the subfornical organ). Furthermore, a semi-quantitative analysis on the cellular level of angiotensinogen gene expression was performed showing a correlation of the angiotensinogen gene expression to the glia content of the regions examined. It was also demonstrated that the angiotensinogen gene expression had its highest levels in several distinct areas of the brain (e.g. the preoptic region and the hypothalamus), whereas other areas showed only low to moderate levels (e.g. the thalamus). The expression of the angiotensinogen gene in the rat brain was not only restricted to areas involved in cardiovascular and neuroendocrine control, but was also present in functionally different regions. Our data thus indicate that, based on the regional distribution of angiotensinogen-mRNA, angiotensin peptides may have other functions besides participation in cardiovascular and neuroendocrine control.     

Corticotropin releasing factor: Immunocytochemical localization in rat brain

Using antiserum generated against the synthetic CRF_1–41 we have immunocytochemically localized perikarya and processes in rat brain. Areas observed to have particularly dense accumulation of CRF-ir cells were in the extra-hypothalamic areas of nucleus accumbens septi, nucleus of the stria terminalis, the medial preoptic region, and the central amygdaloid nucleus. Within the hypothalamus cell bodies were scattered throughout the anterior hypothalamic region and densely packed in the paraventricular nucleus. Fibers appear most dense in the lateral septal region and throughout the external layer of the median eminence.     

Nogo-A在大鼠脑组织中的亚细胞定位

目的Nogo-A在中枢神经系统的髓鞘和神经元中广泛表达,并且有多种亚分布形式。在以往研究的基础上,将通过检测Nogo-A/B(N-18)在大鼠脑组织中的亚细胞定位,进一步获得Nogo-A在细胞核中表达的有力证据。方法通过差速离心法和NP-40处理得到大鼠皮层和小脑样本的纯化细胞核亚组分,利用Westernblot来检测各亚细胞成分中Nogo-A的表达。通过免疫荧光双标染色,共聚焦显微镜观察Nogo-A在大鼠皮层和小脑组织中的亚细胞定位。结果WesternBlot在纯化细胞核中可检测到清晰的Nogo-A条带。免疫荧光染色显示胞核内有免疫阳性物质表达,并且和Hoechst复染的胞核着色有广泛重合。结论Nogo-A在大鼠皮层和小脑细胞核中有明确表达,并且很有可能以全长形式入核。     

Coexpression of connexin45 and -32 in oligodendrocytes of rat brain

Connexin proteins are the subunits of gap junction channels, and are encoded by a gene family. Although several connexin mRNAs were detected in brain, only a few connexin-proteins have been localized to specific cell types in this tissue. Here we describe expression of connexin45 protein in oligodendrocytes in rat hippocampus. Double immunofluorescent staining using specific antibodies to connexin45 and connexin32 paired with cell-type specific marker proteins revealed that connexin45 and connexin32 were co-expressed and colocalized in oligodendrocytes. Each of the connexin antibodies gave rise to the same pattern of punctate fluorescence in the plasma membrane of cell bodies and proximal processes of oligodendrocytes. Connexins in the plasma membrane of oligodendrocytes may form gap junctions between oligodendrocytes, or between oligodendrocytes and astrocytes. Expression of connexin45 in oligodendrocytes may prevent dysmyelinating effects of connexin32 mutations in the central nervous system of Charcot-Marie-Tooth (X-type) patients.     

Cellular localization of angiotensin type 1 receptor and angiotensinogen mRNAs in the subfornical organ of the rat brain

The cellular localization of angiotensin type 1 receptor (AT 1) and angiotensinogen mRNA expression in the subfornical organ (SFO) of the rat brain has been studied by means of non-radioactive in situ hybridization combined with immunocytochemistry for glial fibrillary acidic protein (GFAP) and Neutral red staining. The AT 1 receptor mRNA expression is shown to be within putative nerve cells without any association with the glial fibrillary acidic protein (GFAP)-immunoreactive (IR) cells. In contrast the angiotensinogen cRNA expression is associated predominantly with GFAP-IR cells. The results demonstrate that a neuronal AT 1 receptor mediates the actions of circulating angiotensin II on the SFO and that the angiotensinogen mRNA is predominantly expressed in the SFO astroglial cells.     

Autoradiographic localization of [3H]buspirone binding sites in rat brain

In the C-neurons of rabbit nodose ganglion there is a persistent slow outward current at Vm levels positive to -80 mV. This current was detectable in Na+-free Ringer and disappeared in Ca2+-free medium. Therefore it may be the Ca2+-activated K+ current. This K+ current shows a unique time and voltage dependency, suggesting that it may have a regulatory role on the excitability of C-neurons. Two other types of current also observed in C-neurons were IQ- and IA-like currents. In A-neurons, however, a Ca2+-activated K+ current was not observed at all.