COMPARISON OF THE FATTY ACIDS OF LIPIDS OF SUBCELLULAR BRAIN FRACTIONS

Abstract— Rat brain grey and white matter were fractionated to yield myelin, nerve terminal, synaptic vesicle, nerve terminal 'ghost', and microsomal fractions of white and grey matter. Ester-type glycolipids were found in all fractions except myelin, while cerebrosides occurred in significant concentrations only in myelin and white microsomes. Comparison of the fatty acid profile of the ethanolamine- and serine-containing phospholipids showed marked differences between myelin and the particles from grey matter, while the microsomes of white matter were of intermediate composition. Docosahexaenoic acid, a minor acid in myelin, was a major fatty acid in microsomes of grey and white matter. The fatty acid composition of sphingomyelin was distinctly different in the fractions derived from grey and white matter, clustering about stearate and nervonate in the latter, but only about stearate in the grey. Marked differences in the positional distribution of fatty acids were seen within phosphatidyl choline from myelin and nerve terminals. Ribonucleic acid was found in nerve terminal and synaptic vesicle fractions. The sphingosine found in the ganglioside from microsomes of both grey and white matter was similar with respect to distribution of the C₁₈ and C₂₀ homologues.
The possibility is discussed that microsomes furnish characteristic lipids for the synthesis or renewal of specific membranes, and that these lipids are accumulated somewhat before being released.     

THE INCORPORATION OF RADIOACTIVE AMINO ACIDS INTO BRAIN SUBCELLULAR PROTEINS DURING TRAINING

—Total proteins, free amino acids, tritiated water and subcellular proteins of mouse brain were examined for changes in radioactivity during operant conditioning after subcutaneous administration of labelled amino acids. The conditioning was based on appetitive learning, using sweetened milk as a reward. During training and incorporation for 20-30 min, both [³H]leucine and [1-¹⁴C]leucine underwent a significant increase in catabolism, resulting in a decreased radioactivity in the free amino acids. [2-²H]Methionine underwent a rapid loss of isotope, so that 90% of the radioactivity was in the form of tritiated water at the end of training, and this phenomenon masked any possible effect of training. The brain uptake of [³⁵S]methionine increased during the training, resulting in an increased radioactivity in the proteins. Uptake of [³H]lysine increased slightly during training only after 1 h incorporation and not after 20 or 30 min, as judged from a time course of radioactivity in the free amino acids. Incorporation into nuclear proteins increased selectively during 20 min, and into nuclear and cytosol proteins after 60 min incorporations. It is concluded that changes in the observed rate of incorporation of a precursor into brain subcellular proteins under the influence of behaviour might be the result of changes in precursor catabolism or uptake, or both, and that each amino acid behaves in a different way. Even the same amino acid gives different results depending on the isotope and its position in the amino acid.     

COMPOSITION OF CEREBRAL LIPIDS IN MURINE LEUCODYSTROPHY: THE QUAKING MUTANT

The composition of sphingolipids and phospholipids of mouse brain during myelination was determined in the Quaking mutant, which manifests a genetic disorder of myelin formation, and in littermate controls. The biochemical changes during myelination in the brains of the controls corresponded quantitatively with previous findings in a different strain of mice. The Quaking mutant exhibited concentrations of sphingolipids and phospholipids in brain which were comparable to those of controls in the early stage of myelination but the tissue content failed to increase with maturation. The greatest differences occurred in the cerebrosides which at 65 days of postnatal age were only 10 per cent of control levels. During development the pattern of cerebral levels of sphingomyelin, plasmalogen and total phospholipid in the mutants tended to resemble that of the cerebrosides. The defect in the Quaking mutant is compatible with a failure in maturation of myelin. These findings have been compared with those in the Jimpy mutant, a different genetic disorder of myelin in the mouse previously studied in a similar fashion. The Jimpy mutant is characterized by a quantitatively more pronounced deficiency of myelin lipids and a decline in cerebrosides during brain development.     

THE INCORPORATION OF LABELLED ACETATE INTO CEREBROSIDE AND OTHER LIPIDS OF THE DEVELOPING MOUSE BRAIN

—Cerebroside in the brain is highly localized in myelin and has a relatively slow turnover rate. The aim of this study was to evaluate the true cerebroside biosynthetic activity under conditions in which the degradation and reutilization of brain lipids were as small as possible. The 3-week-old mice were decapitated at 0·5, 1, 2·5, 5 and 15 min after the intraperitoneal injection of labelled acetate and the incorporation of radioactivity into each lipid class was examined. Even at 0·5 min, a considerable amount of radioactivity was found in simple lipids, especially in the free fatty acid fraction, and in the course of time the radioactivity of complex lipids increased. On the other hand, the incorporation of radioactivity into cerebrosides was extremely small throughout the experimental period. Results indicated that the low radioactivity of cerebroside might be due to its high content of long-chain fatty acids which were weakly labelled. The radioactivity of the sphingosine moiety was also low. In short, one of the rate-limiting steps of cerebroside synthesis in brain might exist in long-chain fatty acid and sphingosine synthesis. In addition, the incorporation curves of each component of cerebroside were compared with each other and the difference of the incorporation pattern of non-hydroxy fatty acids of cerebroside was noted.     

INCORPORATION OF AXONALLY TRANSPORTED SUBSTANCES INTO MYELIN LIPIDS

Abstract— The possibility that axonally transported lipids and/or proteins might undergo transaxonal migration and become incorporated into surrounding myelin lamellae was studied by isolating myelin from optic tracts of myelinating rabbits at various times following intraocular injection of [3-¹⁴C]-serine and [2-³H]glycerol. Myelin isolated by a procedure employing ethylene glycol-bis(β-aminoethyl ether)-.N,N'-tetraacetic acid had relatively constant specific radioactivity with respect to both isotopes over a 21 day period. Myelin lipids showed a gradual increase in ¹⁴C specific radioactivity, attributed to reutilization of [¹⁴C]serine from the axon by a compartment of the oligodendrocyte. Free serine is postulated to arise in the axon from catabolism of axonally transported proteins (and possibly lipids) and to migrate transaxonally into the neighboring oligodendroglia. This reutilization mechanism resulted in synthesis of myelin cerebrosides, sphingomyelin, ethanolamine phosphoglycerides and possibly sulfatides, but not gangliosides or serine phosphoglycerides. The data for choline- and inositol-phosphoglycerides are inconclusive. [³H]Glycerol-labeled myelin lipids decreased slowly in ³H specific radioactivity with time, indicating either that [2-³H]glycerol does not participate in the reutilization pathway or that the label is lost in the process. Evidence is presented that ³H- and ¹⁴C-labeled lipids are true myelin constituents. Lipids from the myelin, axolemma- and axon-enriched fractions tended to converge in specific radioactivity over the 21 days, especially the former two fractions. These results together with isotope ratio changes point to an equilibration process whereby lipids are able to transfer. (or exchange) between the 3 compartments. Protein radioactivity in isolated myelin was suggested to arise from residual axon/axolemma contamination, and no evidence was found for transaxonal migration of protein into myelin. The 2 mechanisms elucidated here are believed to account for a quantitatively small portion of myelin lipid and are considered to represent a form of axon-glia interaction.     

METABOLISM OF ARACHIDONOYL PHOSPHOGLYCERIDES IN MOUSE BRAIN SUBCELLULAR FRACTIONS

Abstract— Mouse brain subcellular fractions were prepared at 1, 12, and 24 h and 3 and 8 days after intracerebral injections of [1-¹⁴C]arachidonate. Initially, radioactivity was mainly distributed in the microsomal and synaptosomal fractions, but the proportion of radioactivity in the myelin increased from 5 to 16% within 8 days. Radioactivity of the microsomal lipids started to decline at 1 h after injection, and the decay was represented by two pools with half-lives of 19 h and 10 days, respectively. Radioactivity in the synaptosomal and myelin fractions did not reach a maximum until 24 h after injections. The half-life for turnover of synaptosomal lipids was 9 days.
The decline of radioactivity measured in the microsomal fraction was due mainly to diacyl-GPC and diacyl-GPI, since radioactivity of other phosphoglycerides (diacyl-GPS, diacyl-GPE and alkenyl-acyl-GPE) continued to increase for 12-24 h. In this fraction, half-lives of 10-14 h were obtained for the fast turnover pools of diacyl-GPC and diacyl-GPI, and slow turnover pools with half-lives of 7 days for diacyl-GPI and 10-14 days for other phosphoglycerides were also present. Among the synaptosomal phosphoglycerides, radioactivity of diacyl-GPI declined in a biphasic mode, thus exhibiting half-lives of 5 h and 5 days. Incorporation of labelled arachidonate into diacyl-GPE and diacyl-GPS in the synaptosomal fractions was observed for a period of 24 h. The half-lives for these phosphoglycerides ranged from 8 to 12 days. Results of the study have demonstrated the presence of small pools of arachidonoyl-GPI in synaptosomal and microsomal fractions which were metabolically more active than other arachidonoyl containing phosphoglycerides.     

FUCOSE INCORPORATION INTO GLYCOPROTEINS OF MOUSE BRAIN

—Radioactive fucose was incorporated into glycoproteins of brain in vivo. After intracerebral administration of this precursor, radioactive glycoproteins were the sole detectable product. The glycoproteins formed appeared to have a slow turnover but this was due, at least in part, to re-utilization of fucose released from degraded glycoproteins. Incorporation of fucose into glycoproteins differed from that of glucosamine, since a much smaller proportion of the radioactive fucose was incorporated into soluble glycoproteins. Fucose was rapidly incorporated into glycoproteins of nerve endings, although there was relatively little incorporation into glycoproteins associated with the soluble component of the nerve-ending fraction. As found in previous studies with glucosamine, soluble glycoproteins of nerve endings turned over relatively rapidly. Pretreatment with acetoxycycloheximide markedly inhibited incorporation of fucose into glycoproteins of brain. In contrast to the results with glucosamine, comparable inhibition was observed for fucose in all subcellular fractions of brain including the particulate and soluble components derived from the nerve-ending fraction.     

DEVELOPMENT OF LIPOGENESIS IN RAT BRAIN CORTEX: THE DIFFERENTIAL INCORPORATION OF GLUCOSE AND ACETATE INTO BRAIN LIPIDS IN VITRO

—The oxidation to CO₂ and the incorporation of [U-¹⁴C]glucose and [U-¹⁴C]acetate into lipids by cortex slices from rat brain during the postnatal period were investigated. The oxidation of [U-¹⁴C]glucose was low in 2-day-old rat brain, and increased by about two-fold during the 2nd and 3rd postnatal weeks. The oxidation of [U-¹⁴C]acetate was increased markedly in the second postnatal week, but decreased to rates observed in 2-day-old rat brain at the time of weaning. Both labeled substrates were readily incorporated into non-saponifiable lipids and fatty acids by brain slices from 2-day-old rat. Their rates of incorporation and the days on which maximum rates occurred were different, however, maximum incorporation of [U-¹⁴C]glucose and [U-¹⁴]acetate into lipid fractions being observed on about the 7th and 12th postanatal days, respectively. The metabolic compartmentation in the utilization of these substrates for lipogenesis is suggested. The activities of glucose-6-phosphate dehydrogenase, cytosolic NADP-malate dehydrogenase, cytosolic NADP-isocitrate dehydrogenase, ATP-citrate lyase and acetyl CoA carboxylase were measured in rat brain during the postnatal period. All enzymes followed somewhat different courses of development; the activity of acetyl CoA carboxylase was, however, the lowest among other key enzymes in the biosynthetic pathway, and its developmental pattern paralleled closely the fatty acid synthesis from [U-¹⁴C]glucose. It is suggested that acetyl CoA carboxylase is a rate-limiting step in the synthesis de novo of fatty acids in developing rat brain.     

ACYL GROUP COMPOSITION OF METABOLICALLY ACTIVE LIPIDS IN BRAIN: VARIANCES AMONG SUBCELLULAR FRACTIONS AND DURING POSTNATAL DEVELOPMENT

Using a combination of preparative TLC and GLC technique, the content and acyl group composition of diacyl-glycerophosphoinositols, diacyl-glycerophosphates, diacylglycerols and triacyl-glycerols in brain tissue were determined. The level of diacyl-glycerophosphoinositols in 40 day-old mouse brain was 2.7 μmol/g tissue as compared to 40–170 nmol/g for other minor lipids. The acyl groups of diacyl-glycerophosphoinositols were enriched in 18:0 and 20:4 (n-6). This characteristic acyl group profile was found in microsomes, synaptosomes, and in myelin. The acyl groups of diacyl-glycerophosphates and diacylglycerols were comprised mainly of 16:0, 18:0, 18:1 and 20:4 (n-6). In rat brain subcellular fractions, the acyl groups of diacylglycerols and diacyl-glycerophosphates in the microsomal fraction had a higher proportion of 22:6 (n-3) than those in the myelin and synaptosomal fractions. The acyl groups of the myelin lipids were higher in 18:l and lower in 20:4 (n-6) as compared to those in the microsomal and synaptosomal fractions. The triacylglycerols in brain exhibited an unusual acyl group profile which included small proportions of 14:0, 16:1, 20:4 (n-6), 22:4 (n-6) and 22:6 (n-3). Except for an increase in 18:1 and a corresponding decrease in 16:0 which was found in diacyl-glycerophosphoinositols, no apparent acyl group change was observed in other metabolically active lipids during postnatal brain development.     

FRACTIONATION OF RNA FROM BRAIN SYNAPTOSOMES AND CYTOPLASMIC SUBCELLULAR FRACTIONS

—RNA from rat brain synaptosomes, mitochondria and microsomes was analysed by gel electrophoresis under conditions allowing good resolution in three different molecular weight ranges: 4s-16s, 16s-28s and >28s. Two synaptosome specific RNA bands were found, one with comparatively low molecular weight (8-9 × 10⁴ Daltons) and another very large (s_E > 60s). RNA species with electrophoretic characteristics similar to those reported for liver mitochondrial RNA were found in brain mitochondria. From the electrophoretic data their mean geometric radii were determined.     

FATTY ACIDS OF LECITHIN IN SUBCELLULAR FRACTIONS DURING MATURATION OF BRAIN

Abstract— A study has been made of the fatty acyl profiles of lecithin in subcellular fractions of the brain in rat, guinea pig and rabbit. It was found that cerebral lecithins consisted of at least two groups with dissimilar fatty acyl profiles. The group obtained from myelin showed little variation with age of the rats or among two other species examined. The changes in lecithin fatty acyl composition of brain homogenates were in agreement with a progressively greater contribution of myelin lecithins to brain homogenate lecithins with increasing age.     

TRIPHOSPHOINOSITIDE PHOSPHODIESTERASE IN DEVELOPING BRAIN OF THE RAT AND IN SUBCELLULAR FRACTIONS OF BRAIN

Abstract— An assay system for the measurement of triphosphoinositide phosphodiesterase in homogenates of rat brain is described. With triphosphoinositide (TPI) as substrate, and in the presence of 0·1 m -KCI and saturating amounts of diethyl ether, the activity of phosphodiesterase in myelinated brain was 400–500 μmoles of TPI hydrolysed per g wet wt. per hr. One quarter of the adult level of the enzyme was present in rat brain one day after birth, with the remainder being added prior to and during the early stages of myelination. On subfractionation of brain homogenates, substantial activity of the enzyme was located in the soluble portion and in the paniculate fractions enriched in myelin and synaptosomes. The enzyme associated with the particulate fractions could not be detached from the membranes by any of several methods employed. There was a rough correlation between distribution of phosphodiesterase and that of 5'-nucleotidase, an enzyme associated with plasma membrane in a number of tissues. Some implications of the results are discussed.     

INCORPORATION OF [1-14C]OLEIC ACID AND [1-14C]ARACHIDONIC ACID INTO LIPIDS IN THE SUBCELLULAR FRACTIONS OF MOUSE BRAIN

Abstract— The distribution of radioactivity among lipids of subcellular membrane fractions was examined after intracerebral injections of [1-¹⁴C]oleic and [1-¹⁴C]arachidonic acids. Labelled free fatty acids were distributed among the synaptosomal-rich, microsomal, myelin and cytosol fractions at 1 min after injection. However, incorporation of the fatty acids into phospholipids and trïacylglycerols after pulse labelling occurred mainly in the microsomal and synaptosomal-rich fractions. With both types of labelled precursors, there was a higher percentage of radioactivity of diacyl-glycerophosphoryl-inositols in the synaptosomal-rich fraction as compared to the microsomal fraction. Radioactivity of [1-¹⁴C]oleic acid was effectively incorporated into the triacylglycerols in the microsomal fraction whereas radioactivity of the [1-¹⁴C]arachidonic acid was preferentially incorporated into the diacyl-glycerophosphorylinositols in the synaptosomal-rich fraction. Result of the study indicates that synaptosomal-rich fraction in brain is able to metabolize long chain free fatty acids in vivo and to incorporate these precursors into the membrane phosphoglycerides.     

CALCIUM METABOLISM IN ISOLATED BRAIN CELLS AND SUBCELLULAR FRACTIONS

Abstract— The accumulation of calcium ions by brain mitochondria and microsomes and by fractions containing neuronal or glial cells has been studied in vitro with techniques involving ⁴⁵Ca and ultramicro-flame photometry. ATP and substrate-supported calcium accumulation by brain mitochondria was of the same magnitude as for mitochondria from other organs. Brain microsomes accumulated calcium approximately 15 times less than brain mitochondria. Variations in Na⁺/K⁺ ratios and in ATP/ADP ratios had a more marked influence on microsomal uptake than on mitochondrial uptake. The passive Ca²⁺ binding by glial cells was higher than neuronal perikarya and synaptosomes. Also the calcium accumulation ability in cell suspensions was slightly higher for glial cells as compared to neuronal perikarya. The calcium uptake by glial cells was stimulated by high external K⁺ concentration, which also was the case for nerve endings. The uptake in neuronal perikarya was unaffected by variations in K⁺ concentration. A comparison between neuronal and glial mitochondria showed that both reach a steady state level of similar magnitude, but that the rate of initial accumulation was greater for glial mitochondria. A high glial calcium accumulation was also observed for the microsomal fraction.     

EFFECT OF ELECTROSHOCK ON THYMIDINE INCORPORATION INTO RAT BRAIN DNA

Administration of electroconvulsive shocks in rats induces a marked inhibition of the process of thymidine incorporation into cortical DNA. The inhibitory effect reaches its maximum after 10–15 min. Percentage incorporation returns to normal values within one hour of the treatment. These findings may suggest the participation of a metabolically active fraction of DNA in neural functions.     

INCORPORATION OF PHOSPHATE INTO RAT BRAIN DURING SLEEP AND WAKEFULNESS

Abstract— Labelled inorganic phosphate (³²P₁) was administered intraventricularly to unrestrained sleeping and waking adult rats. After about 20 min of sleep or a comparable period of wakefulness, as monitored by EEG and EMG, the animals were frozen in liquid nitrogen and the brains were analysed. One group of animals (A) was not previously acclimatized to the apparatus. A second group (B) was acclimatized. The specific radioactivity of a phosphoprotein fraction was elevated during sleep in group A but not in group B. The specific radioactivity of the phosphatides of group B was depressed in sleeping as compared with waking animals. This effect was not observed in group A. No significant difference was detected between the EEG patterns of sleeping animals in groups A and B, as evaluated by standard criteria. These observations suggest that the physiological conditions attributable to environmental, emotional or other determinants can influence shifts in brain metabolism during the sleep-wakefulness cycle.     

THE EFFECTS OF LITHIUM ON ATPase ACTIVITY IN SUBCELLULAR FRACTIONS FROM RAT BRAIN

Abstract— The effects of lithium chloride in vitro and in vivo were investigated on Na-K ATPase and Mg ATPase activities in synaptic plasma membrane, mitochondrial and synaptic vesicle fractions prepared from rat brain. In vitro , lithium chloride (10⁻³-10⁻⁸ m ) had no effect on ATPase activity in any of the fractions studied. Lithium chloride given chronically by i.p. injection (30 mg/rat/day) for 9 days had little effect on synaptic plasma membrane ATPases. Dietary administration of lithium chloride (60 mmol/kg food) produced a small but significant increase in synaptic plasma membrane Mg ATPase activity after 3 weeks administration and mitochondrial Mg ATPase activity after 1 week. There was no effect on synaptic plasma membrane Na-K ATPase activity. Salt supplementation reduced the toxic effects of lithium administration and it is suggested that toxicity may account for some of the previously reported changes in synaptic membrane ATPases produced by lithium.