The distribution of transferrin immunoreactivity in the rat central nervous system

Recent studies have demonstrated receptors in the nervous system for transferrin, the iron binding and transport protein in the blood. This study using immunohistochemistry at the light and electron microscopic levels demonstrates that transferrin (Tf) is found predominantly in oligodendrocytes in both the gray and white matter of the cerebral cortex, cerebellum and spinal cord. Within the cerebral cortex, layer V has more Tf-labeled cells than the other cortical layers. In the spinal cord, lamina VII has the highest density of Tf-positive cells. Based on location, 3 types of oligodendrocytes can be described: perineuronal, interfascicular and perivascular. In addition to oligodendrocytes, endothelial cells and possibly some neuronal membranes of layer V pyramidal and anterior horn cells label with Tf antiserum. Ultrastructurally, Tf reaction product is homogeneously distributed throughout the perinuclear cytoplasm of both oligodendrocytes and endothelial cells. The importance of iron in motor and behavior function is well established although the mechanism of action of iron in the CNS is not well understood. The presence of Tf in oligodendrocytes implies that these neuroglia are involved in iron mobilization and storage in the CNS. Stored quantities of iron and the ability to mobilize the iron through stored transferrin may be the reason for the extreme dietary restrictions necessary to induce iron-deficient CNS disorders.     

Immunohistochemical localization of the sigma1-receptor in oligodendrocytes in the rat central nervous system

By using a new polyclonal antibody raised against a 21-amino acid peptide sequence corresponding to the fragment 138-157 of the cloned rat sigma(1)-receptor, we demonstrated by immunoperoxidase and double immunofluorescence techniques, that rat oligodendrocytes express the sigma(1)-receptor. Experiments in vivo and in vitro showed that sigma(1)-receptor colocalized with specific markers of progenitor (A2B5) and mature oligodendrocytes (GalC, RIP). These results suggest that sigma(1)-receptor in oligodendrocytes might be involved in myelination by direct implication in cholesterol biosynthesis or by interaction with endogenous ligands such as neurosteroids.     

Nitric oxide-mediated cGMP synthesis in oligodendrocytes in the developing rat brain

We investigated the nature of cGMP-synthesizing cells in the developing rat forebrain using cGMP-immunocytochemistry in combination with in vitro incubation of brain slices. When brain slices of immature rats, aged between 1 and 4 weeks, were incubated with sodium nitroprusside (SNP), a nitric oxide (NO) donor compound, in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), small round cells with a few processes in and around the corpus callosum were visualized with the cGMP-antibody. The morphology and the distribution of the cGMP-positive cells were consistent with the criteria for oligodendrocytes. Furthermore, the cGMP-positive cells expressed 2′3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) and gelsolin, which are marker proteins for oligodendrocytes. Therefore, we concluded that the cGMP-positive cells were oligodendrocytes. A subpopulation of the oligodendrocyte was found to be cGMP-immunoreactive also when slices were incubated in the absence of SNP. Furthermore, incubation of the slice in the presence of L-NAME, an inhibitor of NO synthase, but in the absence of SNP abolished cGMP immunostaining. In addition, some populations of neurons and astrocytes in restricted brain areas produced cGMP in response to the incubation with SNP as previously reported, whereas both ameboid and ramified microglial cells did not respond to the treatment. Atrial natriuretic peptide, a stimulator of particulate guanylyl cyclase, enhanced cGMP synthesis in astrocytes in some brain regions but not in oligodendrocytes. These findings indicate that oligodendrocytes in the immature rat brain express soluble guanylyl cyclase. No cGMP-positive oligodendrocytes were found in the mature rat brain, suggesting that cGMP may mediate signals related to myelinogenesis in the rat brain. GLIA 19:286–297, 1997. © 1997 Wiley-Liss, Inc.     

Distribution of big tau in the central nervous system of the adult and developing rat

The diversity of neuronal morphology and function is correlated with specific expression of various microtubule associated proteins (MAPs). One of the major neuronal MAPs, tau, has multiple isoforms formed as a result of alternative splicing and phosphorylation that are differentially expressed during development. Big tau is a high molecular weight isoform that contains an additional large exon (4a) and is expressed primarily by neurons in the peripheral nervous system (PNS). We cloned the complete 4a exon in an expression vector, isolated the recombinant protein and produced antibodies specific to Big tau that were used to localize Big tau in the developing spinal cord and in the adult central nervous system (CNS). In developing spinal cord, Big tau is first expressed in the central projections of the dorsal root ganglia neurons and in motor neurons at embryonic day 18 and postnatal day 2, respectively. In the adult rat CNS, almost all neurons that extend processes into the PNS express Big tau, including all cranial nerve motor nuclei and central processes of most sensory ganglia; of these ganglia, only the bipolar neurons of the olfactory, Vestibular and spiral ganglia did not express Big tau. Retinal ganglion cells are the only CNS neurons, whose processes remain entirely within the CNS, that express high levels of Big tau. The limited and specific distribution of Big tau is consistent with a role in stabilizing microtubules in axons that are subjected to great shear forces. © 1995 Wiley-Liss, Inc.     

Mts1 protein expression in the central nervous system after injury

We recently showed that Mts1 is expressed in white matter astrocytes in the rat brain and spinal cord from the first postnatal day. Its expression level declined in the adult CNS, but its topographical localization was maintained. Only white matter astrocytes in the cerebellum did not express Mts1. After dorsal root or sciatic nerve injury, we observed a marked upregulation of Mts1 in the area of the dorsal funiculus undergoing Wallerian degeneration. Here we show that upregulation of Mts1 is a consistent feature of astrocytes in white matter undergoing Wallerian degeneration. In addition, Mts1 is upregulated in astrocytes outlining the lesion site of a penetrating injury to the forebrain, or cerebellum. Gray matter astrocytes did not express Mts1, even after direct injury. In injured brain, we consistently noted a close relationship between Mts1-expressing astrocytes and ED1-positive microglia/macrophages, which are known to be highly motile cells. Mts1 was expressed in the periventricular area and the rostral migratory stream, i.e., sites of ongoing neuroplasticity in adulthood, and was upregulated in these areas after injury. These data suggest that Mts1-expressing astrocytes play a significant role in degenerative events in the mature white matter, interact with phagocytic microglia/macrophages and regulate cell migration and differentiation in areas of the adult brain with a high degree of plasticity.     

Immunohistochemical study of myelin sheaths formed by oligodendrocytes interacting with dissociated dorsal root ganglion neurons in culture

The addition of central nervous system (CNS) glial cells to dissociated networks of rat dorsal root ganglion neurons in tissue culture provided a useful system for the study of CNS myelin sheath formation. The CNS myelin basic proteins (BP) and proteolipid protein (PLP) were demonstrable in these cultures by immunoperoxidase techniques. Both BP and PLP were detectable in myelinating oligodendrocytes and CNS myelin sheaths. Anti-BP serum and anti-PLP serum were useful immunohistochemical staining reagents for the identification of myelinating oligodendrocytes and CNS myelin sheaths in tissue culture.     

Voltage-dependent potassium currents in cultured trout oligodendrocytes.

Ionic currents were recorded in cultured oligodendrocytes from the brain of trout using the whole-cell configuration of the patch-clamp technique. Outward currents were evoked at membrane potentials more positive than -40 mV, which could be separated into two components according to their kinetic parameters and their sensitivity to the holding potential: a fast inactivating current which was completely suppressed by 4-aminopyridine and reduced by tetraethylammonium and a slow steady-state conductance which was similarly sensitive to both potassium channel blockers. The current reversal potential was close to the potassium equilibrium potential. In contrast to mammalian oligodendrocytes but in similarity with cultured Schwann cells, trout oligodendrocytes did not exhibit any inwardly rectifying currents at hyperpolarized membrane potentials.     

Beta IV tubulin is selectively expressed by oligodendrocytes in the central nervous system

Oligodendrocyte differentiation and myelination involve dramatic changes in cell signaling pathways, gene expression patterns, cell shape, and cytoskeletal organization. In a pilot study investigating CNS angiogenesis, oligodendrocytes were intensely labeled by antisera directed against the C-terminal of Tie-2, a 140-kDa transmembrane receptor for angiopoietin. Immunoprecipitation of rat brain proteins with Tie-2 C-terminal antisera, however, produced a single spot of approximately 55-kDa pI approximately 5 by two-dimensional (2D) electrophoresis, which was identified as beta-tubulin by mass spectrometry. Isotype-specific antibodies for beta(IV) tubulin selectively labeled oligodendrocytes. First detected in premyelinating oligodendrocytes, beta(IV) tubulin was abundant in myelinating oligodendrocyte perinuclear cytoplasm and processes extending to and along developing myelin internodes. Beta(IV) tubulin-positive MTs were diffusely distributed in oligodendrocyte perinuclear cytoplasm and not organized around the centrosome. Beta(IV) tubulin may play a role in establishing the oligodendrocyte MT network, which is essential for the transport of myelin proteins, lipids, and RNA during myelination.     

Palladin is expressed preferentially in excitatory terminals in the rat central nervous system.

Palladin is a recently described intracellular protein associated with the actin cytoskeleton and cell adhesion in fibroblasts. In Western and Northern blot analyses, palladin expression is ubiquitous in embryonic mice, but it is down-regulated dramatically in most adult tissues. Significant amounts of palladin persist in the brain of adult rodents, as assessed by Western blot analysis. With this work, we extend preliminary observations and determine the overall distribution and subcellular location of palladin throughout the rat brain. In sagittal and coronal sections of the central nervous system, immunostain for palladin is present throughout the brain and spinal cord, but not uniformly. The densest regions of immunostain include the olfactory bulb, cerebral and cerebellar cortex, hippocampus, amygdala, superior colliculus, and superficial laminae of the spinal dorsal horn. Because immunostain characteristically is punctate, we performed double staining for palladin and the presynaptic marker synaptophysin. Confocal microscopy showed that palladin-immunopositive puncta are also immunopositive for synaptophysin; the proportion of synaptophysin-immunopositive puncta that also stained for palladin ranged from 100% of mossy fiber terminals in field CA3 of the hippocampus and in the cerebellar cortex to 60--70% of terminals in the cerebral cortex, striatum, and spinal dorsal horn. The presence of palladin in synaptic terminals was confirmed by electron microscopy. Because immunostained terminals commonly establish asymmetric synapses, the selectivity of palladin expression in synaptic terminals was tested by double staining for palladin and gamma-aminobutyric acid. The modest level of colocalization in this material at both the light microscopic and electron microscopic levels suggests a selectivity of palladin for terminals that release excitatory neurotransmitters. As concomitant work in cell cultures has shown that palladin participates in axonal development and migration, the present results suggest that palladin persists at excitatory synapses of the adult nervous system.     

Aldosterone-sensitive neurons in the rat central nervous system

The purpose of this study was to identify brain sites that may be sensitive to the adrenal steroid aldosterone. After a survey of the entire brain for mineralocorticoid receptor (MR) immunoreactivity, we discovered unique clusters of dense nuclear and perinuclear MR in a restricted distribution within the nucleus of the solitary tract (NTS). These same cells were found to contain the glucocorticoid-inactivating enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), a signature of aldosterone-sensitive tissues. Immunoreactivity for various other NTS marker molecules failed to colocalize with HSD2 in these putative aldosterone target neurons, so they may represent a unique neuronal phenotype. Finally, the entire rat CNS was examined for evidence of HSD2 protein expression. Outside the NTS, HSD2-immunoreactive neurons were found in only two other sites: the ventrolateral division of the ventromedial hypothalamic nucleus and a few scattered neurons in the medial vestibular nucleus, just rostral to the NTS. HSD2 immunoreactivity was also found in the ependymal cells that form the subcommissural organ. In summary, few brain sites contain neurons that may be aldosterone sensitive, and only one of these sites, the NTS, contains neurons that express HSD2 and contain dense nuclear MR. The HSD2 neurons in the NTS may represent an important target for aldosterone action in the brain.     

Phosphohippolin expression in the rat central nervous system

The FXYD family is a small single-span membrane protein family; recently, we have identified a novel member of this family from the cDNA library of the rat hippocampus and named phosphohippolin (Php) (Mol. Br. Res. vol. 86, 2001). The deduced amino acid sequence of this novel Php comprises 93 residues with a core motif of FXYD and a single transmembrane domain. This indicates that Php belongs to FXYD6 subfamily of the seven FXYD subfamilies (FXYD1-7). Php shows a 48.1% homology with rat phospholemman (FXYD1), a transmembrane family protein. In this study, polyclonal antibodies against the carboxyl-terminal sequence of rat Php were raised and purified. The spatial expression of the Php protein was in the neuronal fibers of the medial part of lateral habenula nucleus, thalamus, hypothalamus, stria terminalis, zona incerta, amygdaloid body and cingulum, olfactory bulb, hippocampus, cerebral cortex and cerebellum. A unique Php distribution was identified in the cerebellum, with a predominant expression pattern in the granule layer of lobules VI-IX of the posterior lobe. Developmental studies demonstrated that the highest level of Php expression was seen in the postnatal (PN) 3-week-old rat brain, and a significant amount of Php still existed in the adult brain. These findings suggest that Php may play an important role in the excitability of neurons in the central nervous system during postnatal development, as well as those in the adult brain.     

Iron localization in superficial siderosis of the central nervous system

Originally conceived as an uncommon disorder, with the advent of MRI, CNS superficial siderosis has been observed more frequently. We present histologic, histochemical, immunohistochemical, immunofluorescent and ultrastructural evaluation of a 56‐year‐old woman with superficial siderosis. Iron was concentrated in macrophages, superficial astrocytes and gray matter oligodendroglia deep within the cord. While spatially associated with dystrophic glial and neuronal spheroids, iron did not colocalize with mitochondria. Neurotoxic effects were observed despite selective iron localization only within a variety of non‐neuronal cell types.     

Stimulation of oligodendrocytes by extracts from astrocyte-enriched cultures

A soluble, heat-labile, trypsin-sensitive factor present in extracts of astrocyte-enriched cultures enhances the expression of myelination-associated events in rat brain cultures enriched in oligodendrocytes. Both the number of oligodendrocytes detected by immunofluorescence microscopy and the levels of biochemical activities associated with oligodendrocyte differentiation increased in the cultures in response to the extract in a time- and concentration-dependent fashion. The possible significance of these studies for normal oligodendrocytes function, and thus myelination, is discussed.     

Increased proliferation of oligodendrocytes in the hypomyelinated mouse mutant-jimpy

Previous studies of the hypomyelinated mouse mutant jimpy have shown that the number of oligodendrocytes are reduced about 50%. To determine the cause of the cellular reduction, light and electron microscopy were combined with thymidine autoradiographic techniques. The number of neuroglial cells which incorporate radioactive thymidine in the mutants is increased severalfold over control values. Electron microscopic autoradiograms indicate the majority of the labeled cells are oligodendroblasts. However, the total number of glia in the white matter of jimpy and control animals is the same during development and even up to the time of the animal's death. The presence of mitotic cells suggests that the oligodendrocytes undergo division but the abundance of dying cells suggests that they die sometime afterwards. The results of the quantitative autoradiographic studies in combination with our other data strongly suggest that the immediate failure of these cells to form myelin shealths is due to a shortened life span and/or continued cell proliferation.     

Extrahypothalamic neurohypophysical peptides in the rat central nervous system

Vasopressin (VP) and oxytocin (OT) have been identified in a number of extrahypothalamic areas, both by immunohistochemistry and by radioimmunoassay. Because of the incomplete nature of the data available, we have conducted a survey of the VP and OT concentrations in the rat central nervous system. VP and OT were readily detectable in all areas studied. With the exception of the amygdala, OT concentrations were generally 2-4 times those of VP. The physiological function of neurohypophysial hormones in these extrahypothalamic areas is essentially unknown.