Normal Adult Rat Brain Research Articles

Regional and cellular codistribution of interleukin 1 beta and nerve growth factor mRNA in the adult rat brain: possible relationship to the regulation of nerve growth factor synthesis.

We have found a regional distribution of IL 1 beta mRNA and IL 1 activity in the normal adult rat brain, which reveals at least partially a colocalization with nerve growth factor (NGF). The predominantly neuronal signal patterns were found over the granule cells of the dentate gyrus, the pyramidal cells of the hippocampus, the granule cells of the cerebellum, the granule and periglomerular cells of the olfactory bulb, and over dispersed cells of the ventromedial hypothalamus and of the frontal cortex. In these areas also the highest levels of IL 1 activity were observed. In the striatum and septum much lower levels of IL 1 beta mRNA and IL 1 activity (shown for the striatum), most likely synthesized by glial cells, could be determined. IL 1 beta-expressing cells were mainly found in brain regions that also synthesize NGF mRNA as shown by in situ hybridization. NGF mRNA could be demonstrated over pyramidal cells of the hippocampus, granule cells of the dentate gyrus, periglomerular cells of the olfactory bulb and over prefrontal cortex neurons. These data indicate that IL 1 beta, among other factors, might also play a regulatory role in the synthesis of NGF in the CNS, as has been demonstrated in the peripheral nervous system (Lindholm, D., R. Heumann, M. Meyer, and H. Thoenen. 1987. Nature (Lond.). 330:658-659).

Two different RNA polymerase II initiation complexes can assemble on the rat brain creatine kinase promoter.

The rat brain creatine kinase (CKB) gene has a structurally complex promoter. Although it contains a consensus RNA polymerase II TATA box, this is located at -60 relative to the main transcription start site, an unprecedented distance for mammalian TATA boxes. Steady-state messenger RNA for CKB is extremely abundant in brain, yet barely detectable in liver. Using a nuclear run-on analysis, we show that this difference is due, at least in part, to a difference in the rate at which CKB is transcribed in these tissues. We also demonstrate a tissue-specific hypersensitive site in the promoter region of the CKB gene. In vitro transcription experiments using nuclear extract isolated from liver, brain, and HeLa cells show a marked difference in the way these extracts recognize the CKB promoter. Two different preinitiation complexes can assemble on the CKB promoter, one which initiated at the same downstream start-site used in normal adult rat brain and one which initiated 35 base pairs further upstream. Extracts from brain form exclusively the downstream initiating complex. However, liver extracts favor formation of the upstream initiating complex, but formation of this complex can be blocked if brain or HeLa extract is added first. We demonstrate that the upstream consensus TATA box can function as a TATA box to mediate upstream initiation, but is not required for downstream initiation. We have shown previously that brain and HeLa cell nuclear extracts contain an abundant factor, TARP, which binds to the upstream consensus TATA box and have proposed that this factor is distinct from the classical TATA box binding factor TFIID. We propose that binding of TARP to the upstream consensus TATA box may block formation of the upstream initiating complex.

Isolation and Characterization of Brain Macrophages from the Central Nervous System of Newborn and Adult Rats and of Rats with Experimental Allergic Encephalomyelitis

A method has been developed to isolate brain macrophages (MΦ) from normal neonatal and adult rat brain cell suspensions, as well as from brain cell suspensions of rats with experimental allergic encephalomyelitis (EAE), by making use of the ability of MΦ to adhere to plastic surfaces.The isolated adherent cells were immuno- and enzyme-cytochemically identified.Phagocytic activity and the presence of Fc-IgG receptors were also examined.Approximately 30%–40% of the isolated adherent cells from neonatal rat brain are phagocytic and can be stained with macrophage-specific monoclonal antibodies, suggesting that these cells belong to the monocyte/macrophage lineage.From normal adult rat brain, only a small number of brain MΦ could be isolated. A highly purified population of brain MΦ was obtained from EAE rat brain.The isolated brain MΦ are phagocytic, possess Fc-IgG receptors and rat MΦ-associated antigens.Besides these features, the isolated brain M(D also express MHC class II antigens (Ia-antigens), which suggests that MΦ may be involved in the regulation of immunological disorders of the CNS. The method reported here for rapidly isolating a large number of blood-monocyte-derived brain MΦ from neonatal and adult brain allows an investigation of the precise role of MΦ in inflammatory diseases of the central nervous system.

The transfer coefficients for L-valine and the rate of incorporation of L-[1-14C] valine into proteins in normal adult rat brain.

An autoradiographic method for the measurement of the rate of valine incorporation into brain proteins is described. The transfer coefficients for valine into and out of the brain and the rate of valine incorporation into normal rat brain proteins are given. The valine incorporation and the transfer constants of valine between different biological compartments are provided for 14 gray matter and 2 white matter structures of an adult rat brain. The rate of valine incorporation varies between 0.52 +/- 0.19 nmol/g/min in white matter and 1.94 +/- 0.47 in inferior colliculus (gray matter). Generally, the rate of valine incorporation is about three to four times higher in the gray matter than in the white matter structures.

Intracellular and extracellular spaces of normal adult rat brain determined from the proton nuclear magnetic resonance relaxation times.

The nuclear magnetic resonance method was used to investigate the state of water molecules in normal rat brain tissue in vitro. The transverse magnetization decay curve (TMDC) of the fresh brain tissue of adult rats (8- or 10-weeks-old) was biexponential, which could be interpreted in terms of two distinct transverse relaxation times (T2). Several factors that may affect the TMDC are discussed. It was concluded that the fast and slow components of T2 correspond to those of the water molecules of the intracellular and the extracellular spaces of normal rat brain tissue, respectively.

The distribution of growth-hormone-releasing factor (GRF) immunoreactivity in the central nervous system of the rat: an immunohistochemical study using antisera directed against rat hypothalamic GRF.

Immunohistochemical methods have been used to chart the distribution of rat hypothalamic growth-hormone-releasing factor (rhGRF) immunoreactivity in the brains of normal and colchicine-treated adult albino rats. The results suggest the existence of at least two distinct rhGRF-containing systems: one responsible for delivery of the peptide to portal vessels in the median eminence, and one whose relationship, if any, to hypophysiotropic function is less direct. A dense plexus of rhGRF-stained fibers was found throughout the external lamina of the median eminence that is the route by which the peptide is delivered to the anterior pituitary. This projection appears to arise primarily from a group of rhGRF-immunoreactive neurons centered in the arcuate nucleus. Some 1,000-1,500 rhGRF-positive neurons were counted on each side of the brain in rats pretreated with colchicine. Colocalization studies, using a sequential double staining technique, indicated that a subset of rhGRF-immunoreactive neurons in the arcuate region contain neurotensin immunoreactivity. No evidence was obtained for colocalization of rhGRF with either of two pro-opiomelanocortin-derived peptides (alpha-melanocyte-stimulating hormone, adrenocorticotropic hormone (1-24)) in individual neurons in the arcuate nucleus. Much smaller groups of neurons were localized in the parvicellular division of the paraventricular nucleus of the hypothalamus and in the dorsomedial nucleus, and it is unclear whether they contribute to the plexus of rhGRF-stained fibers in the median eminence. The only other region in the rat brain in which rhGRF-stained cells were found reliably was in the area that roughly encapsulates the caudal aspect of the ventromedial nucleus of the hypothalamus. Because cells in this region are not known to project to the median eminence, they may be assumed to contribute to the extrahypophysiotropic rhGRF-stained projections outlined below. From the level of the arcuate and ventromedial nuclei, rhGRF-immunoreactive fibers could be traced along the base of the brain and through the periventricular system to discrete terminal fields limited almost exclusively to the hypothalamus and adjoining parts of the basal telencephalon. All parts of the periventricular region of the hypothalamus receive an input, including the preoptic and anterior parts in which somatostatin-containing neurons that project to the median eminence are clustered. Other prominent terminal fields were localized in discrete parts of the dorsomedial, paraventricular, suprachiasmatic, and premammillary nuclei, and in the medial preoptic and lateral hypothalamic areas.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunocytochemical mapping of 1B236, a brain-specific neuronal polypeptide deduced from the sequence of a cloned mRNA

The 318-amino acid, carboxy-terminal sequence of the putative brain-specific polypeptide 1B236 was deduced from the nucleotide sequence of its cloned brain-specific mRNA. Antisera raised against selected synthetic peptide fragments of this protein were used to map the cellular location of the presumptive gene product in the brains of normal or colchicine-pretreated adult rats. Antisera directed against any of three C-terminally located, but nonoverlapping, nonhomologous, synthetic peptide segments (P5, P6, or P7) produced virtually identical maps of intensely immunoreactive neuropil staining. The immunoreactivity was distributed heterogeneously and was most pronounced within olfactory, somatosensory, and limbic systems, and was more modest in certain motor and auditory structures. In colchicine-pretreated rats, large, multipolar perikarya were observed within the amygdala, caudate-putamen, cingulate, parietal, and piriform cortices, as well as in particular diencephalic and pontine nuclei. Smaller immunoreactive neurons with more limited dendritic extensions were observed in the olfactory bulb, the cerebellar cortex, and the dorsal horn and intermediolateral cell columns of the spinal cord. No immunoreactivity was observed in visceral structures innervated by the autonomic nervous system or in non-neural tissues. In addition to the virtually superimposable maps produced by antisera to all three synthetic fragments selected from the C-terminus of 1B236, some uniquely reactive sites were seen. Antisera to the most N-terminal of the three synthetic immunogens (P5) were reactive with neurons of the medial trapezoid nucleus and in nerve terminals surrounding the deep cerebellar nuclei. Antisera against the most C-terminal synthetic immunogen (P7) were reactive with neurons of the paraventricular and supraoptic hypothalamic nuclei. These data demonstrate that the 1B236 protein is located within selected neuronal elements within functionally related cellular circuits established more formally by other methods. Our data show that protein 1B236-immunoreactive cells share at least the expression of this protein and suggest that these cells may also be related epigenetically or evolutionarily. These data, together with other subcellular, ultrastructural, and electrophysiological properties of 1B236, suggest that this protein could be considered as a prohormone capable of yielding several final candidate transmitter products.

Immunocytochemical characterization of cloned cells from normal adult rat brain

A cell clone, isolated from normal adult rat brain and maintained in culture for many passages, has been previously characterized by electron microscopy. Immunohistochemistry has now been shown that it possesses glutamine synthetase (GS) which supports its astrocytic origin. Cells failed, however, to express glial fibrillary acidic protein (GFAP) but did express the intermediate filament protein vimentin. The presence of fibronectin confirms the normal derivation of these cells and excludes the possibility of malignant transformation.

Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study.

The distribution of corticotropin-releasing factor (CRF)-immunoreactive cells and fibers has been examined in the brains of normal adult rats, and in the brains of animals that had been pretreated with intraventricular injections of colchicine, or had been adrenalectomized 3-60 days before perfusion. The results suggest that CRF immunoreactivity is localized in at least three functionally distinct systems. First, most of the CRF-stained fibers in the neurohemal zone of the median eminence, which presumably modulate the release of ACTH and beta-endorphin from the pituitary, appear to arise in the paraventricular nucleus of the hypothalamus (PVH). About 2,000 CRF-stained cells are distributed throughout all eight parts of the PVH, although a majority (80%) of the cells are concentrated in the parvocellular division, and a smaller number (about 15%) are found in parts of the magnocellular division in which oxytocinergic cells predominate. This appears to be the only CRF-stained pathway in the brain that is affected (increased staining intensity) by adrenalectomy. Second, a series of cell groups in the basal telencephalon, hypothalamus, and brain stem that are known to play a role in the mediation of autonomic responses contain CRF-stained neurons. These areas, which are interconnected by stained fibers in the medial forebrain bundle and the periventricular system, include the central nucleus of the amygdala, substantia innominata, bed nucleus of the stria terminalis, medial and lateral preoptic areas, lateral hypothalamic area, central gray, laterodorsal tegmental nucleus, locus ceruleus, parabrachial nucleus, dorsal vagal complex, and regions containing the A1 and A5 catecholamine cell groups. And third, scattered CRF-stained cells are found throughout most areas of the cerebral cortex. Most such cells are confined to layers II and III in the neocortex, and their bipolar shape suggests that they are interneurons. These cells are most common in limbic regions including prefrontal areas, the cingulate gyrus, and areas bordering the rhinal fissure. Scattered immunoreactive cells are also found in dorsal parts of the dentate gyrus and Ammon's horn. These results suggest that the PVH plays a critical role in the modulation of ACTH and beta-endorphin release from the pituitary, and that CRF-containing pathways in the brain are involved in the mediation of autonomic responses.

Extrahypothalamic distribution of CRF-like immunoreactivity in the rat brain

CRF-like immunoreactivity was measured by radioimmunoassay in the brains of normal adult rats and found to be widely distributed in extrahypothalamic areas (e.g., thalamus, amygdala, hippocampus, frontal cerbral cortex, striatum, midbrain, pons-medulla and cerebellum) at levels approximately 10% of the hypothalamus. Sephadex G-50 gel filtration reveals that CRF-like immunoreactivity in the hypothalamus coelutes with synthetic ovine CRF and is also present in the void volume. However, in the extrahypothalamic areas of the rat brain, only CRF-like immunoreactivity that coelutes with synthetic ovine CRF was detected. High performance liquid chromatography revealed equal amounts of immunoreactivity coeluting with CRF and methionine sulfoxide CRF in hypothalamic extracts.

Immunofluorescence studies of the monocytes in the injured rat brain.

Rat brain was obtained on the 4th day after its damage by a stab wound. The injured and the normal control brain tissues were stained by the immunofluorescence technique using anti-granulomonocytic rabbit serum. After the fluorescence observation the same tissues were further stained by hematoxylin and eosin (HE) and studied comparatively by light microscopy. The following results were obtained: (1) The normal adult rat brain lacks the cells which react with the antiserum, thus the resting microglia occurring in the normal adult brain are antigenically different from the cells of the monocyte-macrophage system. (2) In the injured brain tissues monocytes extravasate, enter-brain parenchyma, and take ameboid forms or become macrophages. (3) Among the reactive cells in the injured brain, all of the brain macrophages and most of the ameboid cells were reactive with the antiserum thereby indicating their monocytic origin.

The fine structure of cloned cells from normal adult rat brain.

A cell clone was isolated from a normal rat brain culture and maintained in vitro for many passages. It possessed glial characteristics; in particular ultrastructural examination revealed astrocytic features including the presence of filaments 9--11 nm in diameter.

Subependymal glycosaminoglycan networks in adult and developing rat brain

Histochemical studies of normal adult rat brain indicate two types of glycosaminoglycans in the subependymal region of the lateral ventricle. One network is characterized by an affinity for the cationic dyes alcian blue, aldehyde fuchsin and colloidal iron. These reactions occur at pH 1.0 and at 0.5-0.3 M concentration of MgCl2, which suggests that this material is chondroitin sulfate. The other system is identified by metachromasia with toluidine blue and a loss of PAS staining following sulfation. These findings are consistent with non-sulfated and non-anionic acid mucopolysaccharides. In developing rat brain the differential development of these networks enhances their separate identity. The metachromatic network is present at least by the 10th postnatal day but the polyanionic electrolytes cannot be identified until the 16th to the 22nd days. The possible functional importance of these systems is discussed.

Glial cells from normal adult rat brain established in continuous culture

Glial cells from normal adult rat brain established in continuous culture

Fibrinolytic activity of cultured cells derived during ethylnitrosourea-induced carcinogenesis of rat brain.

Using a fibrin-agarose-overlay technique, high levels of plasminogen-dependent fibrinolytic activity have been demonstrated in cell lines derived from an ethylnitrosourea-induced glioma of the rat brain. Cell lines derived from normal adult rat brain showed only low levels of activity. The degree of lysis produced by a cell line was dependent on the average number of cells per colony, and a different pattern of response was observed for tumour and normal cell lines. A good positive correlation existed between the level of fibrinolytic activity, growth in agar and tumourigenicity of a cell line. Fibrinolytic activity was associated with cell lines derived at various times in the latent period, before the appearance of a visible tumour. Many cell lines derived from rat brains at 57-60 (E7), 90-91 (E8) and 111-112 (E6) days after transplacental exposure to ethylnitrosourea showed fibrinolytic activity, and in the latter group the close association with growth in agar and tumourigenicity was also demonstrated. Results from cell lines derived in the E7 and E8 experiments indicated that the possession of fibrinolytic activity preceded the ability of cells to form colonies in agar.

Cholesterol ester metabolism in the brain: Properties and subcellular distribution of cholesterol-esterifying enzymes and cholesterol ester hydrolases in adult rat brain

1. 1. The properties and subcellular distribution of the enzymes directly related to the formation and degradation of cholesterol esters were investigated in the young adult rat brain. They were a cholesterol-esterifying enzyme, lecithin-cholesterol acyltransferase, and two distinct cholesterol ester hydrolases. 2. 2. The cholesterol-esterifying enzyme incorporated exogenous free [1- 14C]-oleic acid into cholesterol ester. The pH optimum was 5.5–5.6. Exogenous ATP or CoA did not stimulate the activity, and the presence of an excess amount of unlabelled oleyl-CoA did not diminish the incorporation of the radioactive oleic acid. Various bile acids and Tween 20 were inhibitory, particularly at higher concentrations. Under the optimum conditions, the total activity of the brain was approximately 40 μg cholesterol esterified per h per g. Nearly half of the activity was found in the crude mitochondrial fraction. Purified myelin contained no activity. 3. 3. All attempts failed to demonstrate the activity of lecithin-cholesterol acyltranferase in the adult rat brain, using the specific substrate, lecithin labelled at the β-position with [1- 14C]oleic acid. The contribution of this enzyme to the formation of cholesterol ester in normal adult rat brain appears to be, if not nil, negligible. 4. 4. Two distinct cholesterol ester hydrolases were present. One had the pH optimum of 4.2, was activated by deoxycholate, taurocholate, and cholate, and moderately inhibited by Tween 20. Under the optimum condition in the presence of taurocholate, the total activity of this enzyme was approximately 30μg of cholesteryl oleate hydrolyzed per h per g. This enzyme was primarily localized in the crude mitochondrial fraction, and the subfractionation of the crude mitochondrial fraction suggested that this enzyme might be localized in mitochondria, rather than in lysosomes. Purified myelin was devoid of the pH 4.2 cholesterol ester hydrolase. 5. 5. The other cholesterol ester hydrolase had the pH optimum of 6.6. It was strongly inhibited by deoxycholate and activated by taurocholate. Cholate activated the enzyme at a low concentration, but was moderately inhibitory at a higher concentration. At the optimum condition with added taurocholate, the total activity of the brain was about 170 μg cholesteryl oleate hydrolyzed per h per g. The bulk of this enzyme appeared to be localized in microsomes. However, the highly purified myelin fraction was also enriched with this enzyme by more than 3-fold, compared to the starting homogenate, and it retained 20% of the total activity of the pH 6.6 cholesterol ester hydrolase in the homogenate.

Induction of brain D(--)-beta-hydroxybytrate dehydrogenase activity by fasting.

D(m)-beta-hydroxybutyrate dehydrogenase activity is very low in normal adult rat brain; but during fasting it increases severalfold in parallel with the ketosis. The increase may represent part of a mechanism by which the brain adapts to changing patterns of substrate supply during starvation.

Normal oligodendrocytes in tissue culture: A preliminary report on the pulsatile glial cells in tissue cultures from the corpus callosum of the normal adult rat brain

1. 1. Cells believed to be oligodendrocytes have been obtained in outgrowths from explants in vitro of normal adult rat corpus callosum. These cells show a characteristic rhythmic pulsatility apparently identical with that described in 1935 by Canti, Bland and Russell in the case of oligodendrocytes obtained in culture from oligodendroglioma of the human brain. We believe also that we have seen similar cells in a few tissue culture preparations from the cortex cerebri of the normal adult human brain. 2. 2. The morphology of this cell in the living state in culture has been described and some analysis has been attempted of its pulsation as studied by timelapse ciné-photomicrography. 3. 3. Some possible physiological and pathological implications of this pulsatile activity of oligodendrocytes are discussed.