Alterations in metabolism and gene expression in brain regions during cuprizone-induced demyelination and remyelination

Exposure of mice to the copper chelator, cuprizone, results in CNS demyelination. There is remyelination after removal of the metabolic insult. We present brain regional studies identifying corpus callosum as particularly severely affected; 65% of cerebroside is lost after 6 weeks of exposure. We examined recovery of cerebroside and ability to synthesize cerebroside and cholesterol following removal of the toxicant. The temporal pattern for concentration of myelin basic protein resembled that of cerebroside. We applied Affymetrix GeneChip technology to corpus callosum to identify temporal changes in levels of mRNAs during demyelination and remyelination. Genes coding for myelin structural components were greatly down-regulated during demyelination and up-regulated during remyelination. Genes related to microglia/macrophages appeared in a time-course (peaking at 6 weeks) correlating with phagocytosis of myelin and repair of lesions. mRNAs coding for many cytokines had peak expression at 4 weeks, compatible with intercellular signaling roles. Of interest were other genes with temporal patterns correlating with one of the three above patterns, but of function not obviously related to demyelination/remyelination. The ability to correlate gene expression with known pathophysiological events should help in elucidating further function of such genes as related to demyelination/remyelination.     

Heat production and lipid metabolism in broiler and layer chickens during embryonic development

We compared heat production (HP) and lipid metabolism in broiler and layer chickens (Gallus gallus) during embryonic development. To investigate HP and respiratory quotient (RQ), oxygen (O2) consumption and carbon dioxide (CO2) production were measured using an open-circuit calorimeter system. HP consistently had a tendency (P = 0.06) to be lower in broilers than in layers during embryonic development, and HP gradually decreased with developmental stage in both strains. RQ values of both strains were approximately 0.7 at every embryonic stage investigated. These results suggest that chicken embryos mainly use lipid for energy, and the RQ was significantly lower in broilers than in layers during embryonic development. Consumption of the yolk sac as a lipid source was faster in broilers than in layers. Plasma D-3-hydroxybutyrate (D3HB) and glycerol concentrations, associated with fatty acid oxidation, were lower in broiler than layer embryos. These results demonstrate that HP and lipid metabolism are different between the strains during embryonic development, and may be one factor for the growth difference between broiler and layer embryos.     

Nucleolar and nucleoplasmic RNA polymerase activity in different regions of rat brain during postnatal development.

RNA polymerase activities of whole nuclei, of isolated and purified nucleoli and of the nucleoplasmic fractions obtained from cerebral hemispheres, cerebellum and brain stem of rat at different days of postnatal development have been determined. In the whole nuclei the fraction of RNA polymerase which is sensitive to alpha-amanitin, is strongly affected by salt concentration; at low ionic strength most of the activity is resistant to the drug while at high ionic strength the enzymatic activity shows a greater sensitivity to the drug. In isolated nucleoli RNA synthesis is not inhibited at all by alpha-amanitin. The biosynthesis of RNA, at low ionic strength, is inhibited by low doses of actinomycin D, whereas at high ionic strength it is remarkably inhibited only by higher doses of the drug. The sensitivity of the reaction to alpha-amanitin and actinomycin D provide good evidence that UTP or GTP incorporation into RNA in purified nuclei and nucleoli, is dependent on RNA polymerases acting on DNA template and is not dependent on homopolymer formation. These results show that in the whole brain nuclei at low ionic strength there is a preferential synthesis of rRNA, whereas at high ionic strength the synthesis of heterogenous RNA predominates. In isolated nucleoli the synthesis of RNA is restricted to rRNA.     

Effect of essentiale on pulmonary edema and changes in the lipid metabolism during adrenaline administration

The experiments on white rats have confirmed that the development of lung edema following epinephrine infusion is characterized by considerable changes in phospholipid and cholesterol blood and lung metabolism. Essentiale preinjection prevented the decrease in phospholipid lung content and blood plasma cholesterol, which was accompanied by less prominent lung edema.     

Bioelectrical activity and neuromediator metabolism in different brain structures during chronic haloperidol administration]

First haloperidol administration is followed by the reorganization of evoked potentials in visual cortex. During haloperidol administration (10-12 days after the beginning) variations of evoked potentials is visual cortex and in subcortical structures uniform evoked potentials took place.     

Enzyme activities involved in lipid metabolism during embryonic development of Macrobrachium borellii

The activities of the enzymatic systems involved in the activation and degradation of fatty acids, and in the synthesis of triacylglycerols and phospholipids were studied in vitro using total cellular homogenate and subcellular fractions of eggs of the shrimp Macrobrachium borellii at different developing stages. Egg development was divided into seven stages based on morphological features of the embryo. Palmitoyl-CoA ligase activity increased as the embryo developed and showed its maximum at stage V. An increase in the synthesis of triacylglycerols and diacylglycerols was also observed at this stage. Diacylglycerylethers were synthesized more actively during the first stages of development. The higher specific activity observed in total homogenate than in microsomal fraction suggested that their synthesis was not exclusively microsomal. Phospholipid synthesis was very active all along development, reflecting active membrane biosynthesis. The highest activity of the cytosolic triacylglycerol lipase was observed at stage V. Fatty acid degradation, measured as mitochondrial beta-oxidation activity, did not vary significantly during development. We conclude that both the anabolic and catabolic processes concerning lipid metabolism are very active, with values similar to those described for adult hepatopancreas, revealing the major role of lipids during shrimp embryogenesis energetics, and that the highest activities of lipid synthesis-hydrolysis take place at stage V when embryos are under active organogenesis. J. Exp. Zool. 286:231-237, 2000.     

Changes in energy metabolism in several brain regions and vegetative responses of white rats during neurotization

Changes of vegetative reactions and cytochrome oxidase (CChO) activity in various brain structures were studied in rats during neurotization. One week neurotization led to an increase of arterial blood pressure, respiration rate, cardiac stroke volume and heart rate. In three weeks of neurotization there was a decrease of stroke volume accompanied by an increase of heart rate and some decrease or respiration rate leading to a reduction of oxygen consumption. Neurotization during one and especially three weeks elicited an enhancement of CChO activity in various brain areas, more pronounced in the cerebral cortex. A four week "rest" after neurotization during three weeks normalized the CChO activity. CChO activation during neurotization is supposed to be one of the mechanisms of adaptation to hypoxia accompanying neurosis.     

The deposition and metabolism of polyphosphoinositides in rat and guinea-pig brain during development

1. The deposition of triphosphoinositide and diphosphoinositide in rat and guinea-pig cerebral hemispheres during growth was measured. 2. The maximum increase in concentration of both of these phospholipids occurs during the period of myelination, but in the rat some di- and tri-phosphoinositide is present before significant myelination begins. 3. In guinea-pig cerebral hemispheres the polyphosphoinositides remaining after post-mortem breakdown are selectively enriched in dissected white matter compared with grey matter. 4. The polyphosphoinositides in the cerebral hemispheres of rats were labelled with injected (32)P very rapidly; the specific radioactivities were in the order triphosphoinositide>diphosphoinositide>monophosphoinositide>total lipid phosphorus. 5. The synthesis of triphosphoinositide in rat forebrain occurs at an appreciable rate before, and at the start of, myelination, but the amount formed per gram of tissue is four to five times greater in adult rat brains, thus maintaining a constant turnover time (about 1hr.) for the whole triphosphoinositide fraction. This indicates that the rapid turnover of triphosphoinositide is independent of myelin deposition. 6. The specific radioactivity of the brain acid-soluble phosphorus pool referred to a constant dose of (32)P/g. body wt. falls rapidly with age, reaching a minimum at 13-14 days, and then rises again. The specific radioactivities of the polyphosphoinositides reflect this change. 7. Part of the polyphosphoinositides in rat and guinea-pig cerebral hemispheres is rapidly hydrolysed post mortem leaving a stable portion resistant to further breakdown. 8. The rate and extent of post-mortem hydrolysis of the polyphosphoinositides in both species decrease with age. 9. After (32)P labelling, the specific radioactivity of the triphosphoinositide remaining in the cerebral hemispheres of the rat after post-mortem breakdown is lower than the original triphosphoinositide fraction, suggesting two metabolically distinct pools.     

Cellular energy metabolism during fetal development. VI. Fatty acid oxidation by developing brain.

We have investigated fatty acid oxidation and development profiles of palmitoyl-CoA synthetase and carnitine palmitoyltransferase in homogenates of developing rat brain. Palmitate showed a peak rate of oxidation between 10 days and the time of weaning, after which activity declined to adult levels. Acetate oxidation increased until Day 10, plateaued until Day 18 when it increased sharply and remained elevated through Day 25 before declining to the adult level. Leucine oxidation also showed a late peak as compared with palmitate. Palmitoyl-CoA synthetase activity was highest in late fetal development and in the newborn after which activity declined gradually to adult levels. Carnitine palmitoyltransferase activity peaked at 10–15 days of age similar to the profile obtained for long chain fatty acid oxidation. During the period of peak fatty acid oxidation, cytochrome oxidase activity increased twofold but the developmental increase in fatty acid oxidation and enzyme levels was much greater than the increase in mitochondrial number. These data suggest that during periods of high fat intake in the suckling rat the brain has an increased capacity for long chain fatty acid oxidation and that in addition to ketone bodies and leucine, fatty acids may be utilized as an alternative substrate in developing brain.     

Molecular cloning of the mouse CCK gene: expression in different brain regions and during cortical development.

In this paper we describe experiments that address specific issues concerning the regulation of the mouse cholecystokinin gene in brain and intestine. The mouse cholecystokinin gene was cloned and sequenced. Extensive homology among the mouse, man and rat genes was noted particularly in the three exons and the regions upstream of the RNA start site. RNAse protection assays for each of the three exons were used to demonstrate that CCK is expressed in only a subset of tissues and that the same cap site and splice choices are used in brain, intestine as well as in cerebellum, cortex, midbrain, hypothalamus and hippocampus. CCK RNA was also noted to be detectable in kidney. Thus the same gene using the same promoter is expressed in subsets of cells that differ in their biochemical, morphologic and functional characteristics. The level of expression of CCK was also monitored during mouse cortical development and the appearance of CCK RNA was compared to glutamate decarboxylase (GAD), enkephalin and somatostatin. It was noted that each of these cortical markers was first expressed at different times during cortical development. The appearance of CCK RNA during intestinal development was also measured and found to precede appearance in cortex by several days.     

Myelin subfractions isolated from mouse brain. Studies of normal mice during development, quaking mutants, and three brain regions.

Myelin isolated from three areas of mouse brain, from whole brain at several ages in normal mice, and from whole brain of adult quaking mutant mice was separated into seven bands and a pellet on discontinuous density gradients using 0.32, 0.45, 0.55, 0.60, 0.70, 0.75 and 0.85 M sucrose. The distribution of myelin in the subfractions was independent of homogenization and shocking conditions employed to isolate the myelin preparations, but was related to the type of myelin applied to the gradient. Compared to myelin isolated from older animals, myelin isolated from 18-24 day old mice displayed a distribution pattern with greater proportions of material banding at lesser sucrose densities. Similarly, myelin obtained from hindbrain contained proportionately more material layering at lesser sucrose densities compared to myelin isolated from cerebral cortex. Myelin subfraction patterns observed for 8-12 day old control mice and quaking mutants were unlike each other or any other myelin preparation examined. In the 18-90 days old animals, the markers studied were not uniformly distributed among the myelin subfractions. The pellet and the layer banding at the 0.75/0.85 M sucrose interface contained the highest specific concentrations of sialic acid, nucleic acid, and total adenosine triphosphatase activity. In contrast, the specific activity of 2',3'-cyclicnucleotide-3'-phosphohydrolase was lowest in the pellet as well as the three bands obtained above 0.60 M sucrose and was highest in the fraction banding at the 0.65/0.70 M sucrose interface. The results obtained were not consistent with an artifactual origin of the myelin subfractions, but instead suggested that the subfraction have physiological significance. One explanation for the different banding patterns observed between young and mature myelin may be the different amount of myelin in various brain regions during development.     

Glycosaminoglycans of brain during development.

The concentration of hyaluronic acid, chondroitin sulfate, and heparan sulfate was measured in rat brain at 2-day intervals from birth to 1 month of age, and in 40-day-old and adult animals. The levels of all three glycosaminoglycans increased after birth to reach a peak at 7 days after which they declined steadily, attaining by 30 days concentrations within 10% of those present in adult brain. The greatest change was seen in hyaluronic acid, which decreased by 50% in 3 days, and declined to adult levels (28% of the peak concentration) by 18 days of age. Only heparan sulfate showed a significant change in metabolic activity during development (a fourfold increase in the relative specific activity of glucosamine), most of which occurred after 1 week of age. In 7-day-old rats almost 90% of the hyaluronic acid in brain is extractable by water alone, as compared to only 15% in adult animals, and this large amount of soluble hyaluronic acid in young rat brain is relatively inactive metabolically. On the basis of our data we propose that the higher amounts of hyaluronic acid found in very young brain may be responsible for the higher water content of brain at these ages, and that the hydrated hyaluronic acid serves as a matrix through which neuronal migration and differentiation may take place during early brain development.