AMINO ACID INCORPORATION IN VITRO INTO PROTEIN OF NEURONAL AND GLIAL CELL-ENRICHED FRACTIONS

Slices of rabbit cerebral cortex were incubated in the presence of labelled amino acids. Following incubation, neuron- and gliaenriched fractions were obtained by density gradient centrifugation and the TCA-insoluble radioactivity determined. The protein-bound radioactivity was five to six times higher in the neuronal-enriched fraction than in the glial-enriched fraction after incubation with tritiated leucine. The neuronal fraction incorporated also a number of other amino acids to a higher extent than the glial fraction (neuron/glia ratio 2·5-6). A definite dependence of incorporation on the rate of oxygenation was demonstrated. The suppression of amino acid incorporation was more marked for the neuronal fraction than for the glial fraction during incubation in relative hypoxia. An increase of potassium concentration in the incubation medium enhanced the amino acid incorporation in both fractions. Low sodium levels decreased the incorporation. Puromycin inhibited incorporation to approximately 30 per cent of control for both fractions. Addition of cycloheximide and dinitrophenol resulted in greater inhibition of incorporation in the neuronal fraction than in the neuroglial fraction. Actinomycin D did not markedly affect the incorporation in any fraction. These results are discussed in relation to in vivo and in in vitro differences for transport and incorporation of amino acids.     

RIBOSOMAL RNA PRECURSORS IN NEURONAL AND GLIAL RAT BRAIN NUCLEI

Abstract– The method of T hompson (1973) for isolation and fractionation of brain nuclei was modified by the introduction of 12mM-Mg²⁺ in the isolating media. This technique gives a good yield of pure (85-90%) neuronal and glial rat brain nuclei, with minimal disruption of nuclei and degradation or processing of nuclear RNA. The RNA/DNA ratio of neuronal nuclei is about 3-fold higher than that of glial nuclei. Analysis of nucleolar RNA fractions by urea-agar gel electrophoresis allows the identification of 45S, 41S, 39S, 36S, 32S and 21S pre-rRNA components. The pattern of nucleolar pre-rRNA and rRNA species in neuronal and glial nuclei is identical. These results demonstrate the existence in brain nuclei of multiple pre-rRNA processing pathways qualitatively similar to those observed in other animal tissues.     

PROTEIN TURNOVER IN CELL-ENRICHED FRACTIONS FROM RABBIT BRAIN1

Abstract— A modification of the available methods was used for preparation of nerve and glia cell-enriched fractions from rabbit brain and spinal cord. The rate of incorporation of tritiated leucine and the turnover rates of protein during 10 days was studied in the bulkprepared cell fractions. The rate of incorporation into the nerve cell fraction was approximately three times greater than in the glia fraction. The nerve cells had one rapid and one slow phase in decline of radioactivity, while the glial cells were characterized by a more uniform decline. The soluble radioactivity was followed in whole tissue of brain and spinal cord and certain differences between the two were observed.     

NEURONAL AND NEUROPIL FRACTIONS FROM DEVELOPING RAT BRAIN

Abstract— A method is described for the preparation of enriched fractions containing isolated neuronal and glial cells from brains derived from 1 to 20-day-old rats. The method is based on mechanical disaggregation in a medium containing Ficoll-PVP followed by centrifugation on a single-stage two-step gradient at 13,000 g for 30min. The neuronal and neuropil (glial) fractions are approx 70–80% pure in cellular terms.
The cells showed well-preserved cytoplasmic and nuclear morphology at the light and electron microscope level and between 70 and 80% excluded trypan blue. Despite changes in the total cell population with age due to glial proliferation, the proportionate recovery of cells in the separated fractions was fairly constant: based on DNA determination, 23 and 29% of all neurons and 15 and 17% of glia were recovered in the purified fractions from Day 1 and Day 20 animals respectively.
Changes in neuronal cell size with age were reflected in a 2.5-fold increase in protein recovered in the neuronal fraction per mg DNA. Protein and RNA levels/mg DNA in the neuropil fraction reached a maximum at Day 10. It is concluded that the method produces a defined and reliable purification of cells in the separated fractions throughout the studied age range and therefore provides a sound basis for studies on the distribution of biochemical systems between cell types during post-natal development.     

THE BIOSYNTHESIS AND CONCENTRATION OF GALACTOSYL DIGLYCERIDE IN GLIAL AND NEURONAL ENRICHED FRACTIONS OF ACTIVELY MYELINATING RAT BRAIN

—In continuation of our studies on the association of the galactosyl diglycerides of brain with myelination, we have measured the biosynthesis and concentration of these glyceride glycolipids, in oligodendroglial, astroglial, neuronal, and myelin enriched fractions from brains of rats of postnatal age 16, 19 and 29 days. The relative purity of cell fractions and myelin derived from 50 to 60 brains of each age-group was checked by phase contrast microscopy and 2′,3′-cyclic nucleotide-3′-phosphohydrolase activity. The relative purity was comparable to that reported by other investigators for cell fractions from bovine brain. Of the three cell types, the oligodendroglia had the highest and the neurons had the lowest capacity to enzymatically synthesize and to accumulate monogalactosyl diglyceride. The amount of monogalactosyl diglyceride found in myelin compared to that found in oligodendroglial fraction greatly increased during development between 16 and 29 days of age. The biosynthesis of galactosyl ceramide but not glucosyl ceramide was highest in oligodendroglial enriched cell fraction. However, ceramide glucosyl-transferase activity, which was greatly affected by the method used for cellular separation, was highest in a microsomal fraction derived from grey matter. Our results support the contention that the oligodendroglial cells are the site of synthesis of myelin constituents of the central nervous system, and that there is a temporal relationship between this site of synthesis and the site of deposition (myelin).     

PROTEIN SYNTHESIS IN ISOLATED NUCLEI FROM ADULT RAT BRAIN

Nuclei from adult rat brains isolated with isotonic sucrose were incubated with [³H]leucine and later purified by centrifugation through hypertonic sucrose solutions. It was found that under these conditions, tritiated leucine was incorporated into TCA precipitable material. Protein synthesis was impaired if the nuclei were treated with the nonionic detergent Triton X-100 or hypertonic sucrose. The presence of puromycin or cycloheximide markedly inhibited the incorporation of the radioactive amino acid. Actinomycin D and RNase did not have any effect on the incorporation. Autoradiography indicated the presence of labelled material within the nuclei and not in cytoplasmic contaminants. Glial nuclei were more actively involved in protein synthesis than neuronal nuclei.     

DISC ELECTROPHORETIC SEPARATION OF PROTEINS IN NEURONAL, GLIAL AND SUBCELLULAR FRACTIONS FROM CEREBRAL CORTEX

Abstract— Soluble proteins were studied in preparations from rabbit brain cortex enriched in neuronal or glial cells and in subcellular cortical fractions. Analytical polyacrylamide gels were used for acidic (pH 9-5) and basic (pH 4-3) proteins and qualitative and quantitative differences are described. The isozymes of lactic dehydrogenase, brain specific proteins and radioactive labelling patterns were used to characterize some soluble proteins.     

PATTERNS AND LABELLING CHARACTERISTICS IN NEURONAL AND GLIAL RNA

Abstract— Rabbit cortex slices were incubated in a medium containing [³H]-uridine for various periods of time. Following incubation, neuronal and glial cell fractions were prepared on a discontinuous Ficoll-sucrose gradient. RNA was extracted from neurons and glia with a tris-sodium dodecyl sulphate-phenol solution and fractionated on a composite agarose-polyacrylamide gel. The stained gel showed major bands corresponding to 28 s , 18 s , 5 s and 4 s fractions and additional minor bands at the position of 24 s , 21 s and 13 s. Neuronal and glial RNA had the same general RNA pattern but the 5 s fraction was more pronounced in neuronal RNA and 4 s more pronounced in glial RNA. After 30 min labelling both neuronal and glial RNA had maximum activities in fractions higher than 28 s with a peak corresponding to 45 s . In the lower mol. wt. region the labelling was essentially poly-disperse. With increasing incubation time, peaks corresponding to 38 s and 32 s appeared as well as to ribosomal and soluble fractions. Incorporation of activity into total RNA expressed as d.p.m/ μ g of nucleic acids, showed similar labelling in neurons and glia after 30 and 60 min and a 3-4 times higher incorporation into neuronal RNA after 180 min. The possible implications of these results are discussed.     

GLUCOSE AND AMINO ACID METABOLISM IN ISOLATED NEURONAL AND GLIAL CELL FRACTIONS IN VITRO

Abstract—

• 1 The metabolism of three substrates, [U-¹⁴C]glucose, [U-¹⁴C]pyruvate and [U-¹⁴C]glutamate has been studied in vitro in neuronal and glial cell fractions obtained from rat cerebral cortex by a density gradient technique.
• 2 The mixed cell suspension, after washing, metabolized glucose and glutamate in a manner essentially similar to the tissue slice. Exceptions were a reduced ability to generate lactate from glucose and alanine from glutamate, and a lowered effect of added glucose in suppressing the production of aspartate from glutamate.
• 3 After 2 hr incubation with [U-¹⁴C]glucose, the concentration of the amino acids glutamate, glutamine, GABA, aspartate and alanine were raised in the neuronal, compared to the glial fraction to 234 per cent, 176 per cent, 202 per cent, 167 per cent and 230 per cent respectively although both were lower than in the tissue slice. Incorporation of radio-activity was absolutely lower in the neuronal fraction, however, and the specific activities of the amino acids were: glutamate 12 per cent, GABA 18 per cent, aspartate 34 per cent, and alanine 33 per cent of those in the glial fraction.
• 4 After the incubation with [U-¹⁴C]pyruvate, the pool size of the amino acids were higher than after incubation with glucose, except for GABA, which was reduced to one-third. The concentrations of the amino acids glutamate, glutamine, GABA, aspartate, and alanine in the neuronal fraction were respectively 46 per cent, 143 per cent, 105 per cent, 97 per cent, and 57 per cent of those in the glial. Thus, with the exception of alanine, the specific activity of the neuronal amino acids compared to the glial was little increased when pyruvate replaced glucose as substrate.
• 5 After 2 hr incubation with [U-¹⁴C]glutamate in the presence of non-radioactive glucose, the pool sizes of all the amino acids were increased in both neuronal and glial fractions, with the exception of neuronal alanine and glial glutamine. The concentrations of the amino acids glutamine, GABA, aspartate and alanine were raised in the neuronal fraction, compared to the glial, to 425 per cent, 187 per cent, 222 per cent, and 133 per cent respectively. The specific activities of all the amino acids were higher than with glucose alone with the exception of alanine, and neuronal GABA. Neuronal glutamine and aspartate had specific activities respectively 102 per cent and 84 per cent of glial.
• 6 An unidentified amino acid, with R_F comparable to that of alanine and specific activity close to that of glutamate, was also present after incubation. It was relatively concentrated in the neuronal fraction.
• 7 The distribution of the enzymes glutamate dehydrogenase, aspartate aminotransferase, glutamate decarboxylase and glutamine synthetase between the cell fractions was studied. With the exception of glutamine synthetase, none of the enzymes was lost from the cell fractions during their preparation. Only 14 per cent of the glutamine synthetase, compared with 75 per cent of total protein, was recovered in the fractions. Of the enzymes, glutamate dehydrogenase activity was 406 per cent, and glutamate synthetase activity 177 per cent in the neuronal fraction compared to the glial in the absence of detergent. In the presence of detergent, glutamate dehydrogenase control was 261 per cent, aspartate aminotransferase activity 237 per cent is the neuronal as compared to the glial fraction.
• 8 Incorporation of radioactivity into acid-insoluble material from either glutamate or pyruvate was twice as high into the neuronal as the glial fraction.
• 9 The extent to which these differences may be extrapolated back to the intact tissue is considered, and certain correction factors calculated. The significance of the observations for an understanding of the compartmentation of amino acid pools and metabolism in the brain, and the possible identification of such compartments, is discussed.

     

DNA POLYMERASES IN FRACTIONATED RAT BRAIN NUCLEI

Abstract— —Adult rat brain nuclei were separated by discontinuous sucrose gradient centrifugation into astrocyte enriched, neuron enriched, and oligodendrocyte/microglia fractions. Nuclear fractions were subjected to velocity sucrose gradient centrifugation and gradient fractions assayed using relatively specific reaction mixtures for DNA polymerase-α, -β and TdT. NEM resistant DNA polymerase activity (DNA polymerase-β) was detected in equivalent amounts in all nuclear fractions. High molecular weight NEM sensitive activity (DNA polymerase-α) was found primarily in the neuron enriched fraction. The significance of the presence of DNA polymerase-α, an enzyme thought to be involved in DNA replication, in a cell incapable of cell division is unknown. TdT was detected in all fractions with increased activity in the neuron enriched fraction. The finding of TdT in thymocytes and neurons further supports the hypothesis that this enzyme is involved in the storage of noninherited information.     

NEURONAL CELL BODY ENRICHED AND GLIAL CELL ENRICHED FRACTIONS FROM YOUNG AND ADULT RAT BRAINS: PREPARATION AND MORPHOLOGICAL AND BIOCHEMICAL PROPERTIES

—A technique for separating neuronal and glial cell fractions in bulk from rat brain has been developed which does not use any digestive enzymes. Carbonic anhydrase, S-100 protein and 2′,3′-cyclic nucleotide 3′-phosphohydrolase were associated with the glial fraction. Glutamate and aspartate were the most abundant free amino acids; the neuronal fraction was richer in GABA and glycine than the glial fraction. Both d -glutamate and potassium ions were actively accumulated in each fraction during aerobic incubation. Na,K-ATPase was higher in the glial fraction; its activity, especially in the glial fraction, increased rapidly from the 10th to the 20th day after birth. Lactate dehydrogenase showed higher activity in the neuronal than in the glial fraction during early development; its isozyme pattern in each fraction was similar to that of whole brain. Acetylcholine esterase had its maximum activity around the 15th day in both fractions. Limitations of this bulk separation technique are discussed.     

PROTEIN SYNTHESIS BY HIGHLY AGGREGATED AND PURIFIED POLYSOMES FROM YOUNG AND ADULT RAT BRAIN

The state of aggregation and the activity of polyribosomes as well as the activity of the pH 5 enzyme fraction were studied at two stages of postnatal brain development, 9 and 50 days after birth. When the polyribosomes were prepared at 0°C in the presence of 5 mm -Mg²⁺, more than 85 per cent of the polyribosome material exhibited a sedimentation coefficient higher than 110 S. High Mg²⁺ concentrations are, therefore, unnecessary to obtain highly aggregated brain polyribosomes. The basal amino acid incorporating activity of both 9- and 50-day-old rat brain preparations is at least equal to that of rat liver. When prepared by the same procedure as above, 9-day-old rat brain polyribosomes seem to be more active (20 per cent) than those of adult brain. However, this difference in activity depends on the presence of a non-ribosomal inactive contaminant which is always present in higher amounts in adult brain preparations. When purified from this contaminant, the preparations do not differ in activity. High Mg²⁺ concentrations are also not necessary for optimal protein synthetic activity and, in fact, are inhibitory. When assayed with both types of highly aggregated polyribosomes, the pH 5 enzyme fraction from adult brain is clearly less active than that of 9-day-old rats. These results suggest that the loss of brain protein synthesis during development does not depend on the stability of the messenger RNA-ribosome complex but only on the soluble pH 5 enzyme fraction.     

SYNTHESIS OF RNA IN DEVELOPING RAT BRAIN IN VITRO

—Incorporation of [8-¹⁴C]adenine into a rapidly-labelled fraction of RNA derived from the nucleus, and into a cytoplasmic RNA of high molecular weight was studied in brain slices from new born rats. The kinetic behaviour of the two fractions of RNA was compatible with a precursor-product relationship between them. The change in the specific activity of adenine and the reduction of radioactivity in prelabelled RNA of brain slices in the presence of actinomycin D, suggest that the observed degradation of nuclear RNA is not due to random changes, but is limited to a relatively small fraction, presumably messenger RNA.     

IN VIVO INHIBITION OF RAT BRAIN PROTEIN SYNTHESIS BY l-DOPA

Abstract— A study has been made of the effect of a single intraperitoneal dose of l -DOPA on the in vivo metabolism of [¹⁴C]leucine and [¹⁴C]lysine by the brain, and on their uptake into brain protein. Administration of 500 mg DOPA/kg to 40-g rats raised the concentrations of several free amino acids; the only amino acid which underwent a statistically significant increment was alanine. Intracisternally-injected [U-¹⁴C]leucine was rapidly metabolized to other labelled compounds; DOPA administration did not influence significantly the rate of its metabolism. No similar metabolic change was observed after administering [U-¹⁴C]lysine intracisternally.
Incorporation of [¹⁴C]leucine and [¹⁴C]lysine into total brain protein was significantly reduced 45 min after DOPA administration. There was also depression of the uptake of labelled amino acid into a supernatant fraction, obtained by high speed centrifugation of the brain homogenate, and into brain microtubular protein (tubulin). Reduced amino-acid incorporation into brain proteins observed 45 min after l -DOPA injection coincided with extensive disaggregation of brain polyribosomes. At 120 min after DOPA treatment, disaggregation was no longer significant and there was a smaller depression in labelled amino aicd incorporation, which disappeared completely 240 min after l -DOPA injection. It is concluded that disaggregation of brain polysomes following DOPA treatment is an accurate reflection of a change in the intensity of brain protein synthesis in vivo.     

MEASUREMENTS OF RATES OF PROTEIN SYNTHESIS IN RAT BRAIN SLICES

The use of tracer concentrations of labelled amino acids to measure incorporation in incubated slices of brain results in wide fluctuations with time in the specific activity of the precursor. Using concentrations of about 1 mm of labelled amino acid facilitates the accurate measurement of rates of synthesis. These higher precursor levels in the medium decrease the fluctuations in free amino acid specific activity due to dilution by endogenous amino acid and the production of amino acid by protein degradation, and decrease the lag in incorporation due to transport phenomena. Concentrations of 1 mm amino acid in the medium did not inhibit protein synthesis; with valine, leucine, phenylalanine, lysine and histidine, incorporation rates were similar when measured at trace concentrations and at 1 mm medium levels. The source of amino acid for protein synthesis appears to be intracellular. No evidence could be found for the preferential use of extracellular medium amino acid. The rate of incorporation of amino acids in incubated slices of rat brain was 0.087 per cent of the protein amino acid/h.     

IN VIVO INHIBITION OF RAT BRAIN PROTEIN SYNTHESIS BY d-AMPHETAMINE

Abstract— Between 1 and 4 h after rats received a single injection of d-amphetamine (15 mg/kg)(when brain polysomes are known to be disaggregated), the in vivo incorporation of [¹⁴C]lysine into trichloroacetic acid-precipitable brain protein was reduced by 28–48%. Incorporation of the ¹⁴C label into the protein present in a 100,000 g supernatant extract of whole brain was similarly reduced (by 44%). Amphetamine administration suppressed protein synthesis in rat cerebral cortex, cerebellum, hypothalamus, striatum, and brainstem to an equivalent extent. The drug did not significantly affect lysine pool sizes measured in these brain regions; thus the reduced incorporation of labeled lysine was not the result of an isotope dilution effect. We therefore conclude that the brain polysome disaggregation resulting from amphetamine administration is associated with decreased in vivo synthesis of some brain proteins.     

THE EFFECTS OF TETRAPHENYLBORON ON THE ISOLATION OF NEURONAL AND GLIAL CELLS FROM GUINEA-PIG BRAIN

Abstract— The addition of 20 mM-tetraphenylboron to the tissue-softening medium of a neuronal and glial cell isolation procedure results in a 2-fold and 5-fold increase in the yield of neuronal and glial-enriched material respectively. The purity of the isolated fractions appears unaltered. While tetraphenylboron strongly inhibits protein synthesis, carbonic anhydrase activity and structural integrity appear unaffected by tetraphenylboron. The potential use of this compound in cell isolations is discussed.     

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.