Showing papers in "Journal of Neurochemistry in 1973"

Myelination in rat brain: method of myelin isolation.

Abstract: — A procedure is described for the preparation from rat brain of myelin having the same degree of purity at all ages. Such a procedure is essential for the study of myelin composition during development. Microsomal contamination was successfully eliminated by adjusting the method to maintain a constant amount of brain per unit volume in the initial density gradient step, and by including two osmotic shocks and two low-speed centrifugation steps. Myelin prepared in this way from animals ranging from 15 days to 14months of age had a total ATPase activity of 0.3-2.0 μmol of Pi.h−1.mg−1 dry wt of myelin, representing 0.1-1.2 per cent recovery of the total homogenate activity; a Na+, K+- ATPase activity of 0.1-1.6 μfnol of Pi.h−1.mg−1 dry wt, representing 0.04-1.5 per cent recovery; a nucleic acid content of 0.2-0.7 per cent of myelin dry wt, representing 0.2-2.0 per cent recovery; and a ganglioside NANA content of 0.04-0.07 per cent of myelin dry wt. representing 0.2-4.6 per cent recovery. The myelin prepared from 20-day animals had the highest content of the first three constituents; otherwise the values of the four constituents were relatively constant per unit weight of myelin. The amounts of nucleic acid and ganglioside recovered in the myelin fractions increased with increasing age and myelin yield.

Catecholamines in fetal and newborn rat brain.

Abstract: The levels of dopamine and norepinephrine were determined in the brains of fetal and newborn rats by means of a sensitive, radiometric-enzymatic assay. Catecholamines were converted to their 3-O-methylated derivatives in the presence of catechol-O-methyl transferase (EC 2.1.1.1) and [3 H-methyl]S-adenosylmethionine; and the [3H]-derivatives were isolated by selective extraction. The assay had a sensitivity for dopamine and norepinephrine of 100 picograms and was linear to at least 30 nanograms of catecholamines. Both amines were present at 15 days of gestation and increased 15-fold in content during the last week of gestation. The regional distribution of these neurotransmitters in the brain of the newborn rat correlated with the distribution of their biosynthetic enzymes. An investigation of the effects of reserpine, pheniprazine, α-methyl-para-tyrosine, diethyldithiocarbamate and l -DOPA on the levels of dopamine and norepinephrine in the brains of the 18-day gestational fetus indicated that the levels of these neurotransmitters are under controls similar to those known to occur in the brain of the adult rat.

Myelination in rat brain: changes in myelin composition during brain maturation

Abstract: — Myelin was isolated from rat brains during development by a procedure giving fractions of constant purity at all ages. The lipid composition of these fractions and of whole brains of littermates was determined. The amount of myelin recovered per brain was a nearly linear function of the logarithm of age from the youngest (15 days) to the oldest (425 days) animals studied. With the exception of the earliest age point, the isolated myelin accounted for approximately 40 per cent of total brain galactolipid, evidence that a constant fraction (calculated to be 60 per cent) of myelin was recovered at all ages. Although the lipid-protein ratio of the myelin was constant with age, marked changes were seen in the amounts of cerebroside, sulphatide, phosphatidylcholine and desmosterol. The total galactolipid increased from 21 per cent of the total lipid at age 15 days to about 31 per cent at maturity. Phosphatidylcholine decreased from 17 to 11 per cent during the same period. Desmosterol decreased from 2.5 per cent of the total sterol to 0.2-0.3 per cent. All of these changes were complete between 2 and 5 months of age; no other ‘lower phase’ lipids showed significant changes with age. Although qualitatively similar to those reported by others, the changes differed in magnitude, with more stability in the levels of cholesterol and phosphatidalethanolamine with development. A sensitive indicator of the maturation of myelin was the mole ratio galactolipid/phosphatidylcholine, which varied from 1.2 at age 15 days to 2.8 at maturity. The maximum rate of myelination occurred at 20 days of postnatal age when myelin was deposited at the rate of 3.5 mg day−1 brain−1. However, at this age the rat brain had only 15 per cent of its eventual complement of myelin. The rate of accumulation of cerebroside in the whole brain paralleled that of myelin, and was the only lipid to show this relationship. Myelin deposition appeared to be almost solely responsible for the continued increase in brain weight after about 100 days of age.

Optimal freezing conditions for cerebral metabolites in rats.

Abstract: — Optimal freezing conditions for metabolites were evaluated in 250-450 g rats As a standard procedure, the brains were frozen in such a way that the blood pressure and arterial oxygenation were upheld during the freezing The progression of the freezing front was determined by means of implanted thermocouples, and the interruption of the circulation by means of injections of carbon particles into the blood stream The freezing gave rise to a rapid interruption of the circulation in the superficial cortical layer first reached by the freezing front well before the temperature reached 0°C In deeper regions the progression of the freezing front was slower and interruption of the circulation occurred simultaneously with the freezing of the tissue Measurements of labile cerebral metabolites, including phosphocreatine, ATP, ADP, AMP and lactate, failed to show signs of autolysis in the part of cortex which became unperfused at temperatures above zero Since the energy state was identical in superficial cortical areas and in areas that did not freeze until after 40–90 s, it is concluded that the freezing technique gives optimal conditions for metabolites also in deep cerebral structures Decapitation of unanaesthetized animals gave rise to large autolytic changes in the cerebral cortex In unanaesthetized animals that were immersed in liquid nitrogen the changes were less marked and mainly affected the concentrations of phosphocreatine, ADP and lactate When paralysed animals that were anaesthetized with N2O were immersed in liquid nitrogen the only significant change from the control was a decrease in phosphocreatine content The virtual absence of autolytic changes in this group of animals was not related to the anaesthesia since more pronounced changes were observed in phenobarbitone-anaesthetized rats immersed in the coolant These differences could be explained by the fact that spontaneously breathing animals immersed in liquid nitrogen developed arterial hypoxia much faster than paralysed animals It is concluded that an optimal metabolite pattern can only be obtained in anaesthetized animals, frozen with a method that was described by Kerr almost 40 years ago (Kerr, 1935) If unanaesthetized animals must be used, greater attention should be paid to the oxygenation of the blood during the freezing than to such factors as speed of freezing or depth of anaesthesia

High affinity transport of choline into synaptosomes of rat brain.

Abstract: —The accumulation of [3H]choline into synaptosome-enriched homogenates of rat corpus striatum, cerebral cortex and cerebellum was studied at [3H]choline concentrations varying from 0.5 to 100 μm. The accumulation of [3H]choline in these brain regions was saturable. Kinetic analysis of the accumulation of the radiolabel was performed by double-reciprocal plots and by least squares iterative fitting of a substrate-velocity curve to the data. With both of these techniques, the data were best satisfied by two transport components, a high affinity uptake system with Km. values of 1.4 μM (corpus striatum), and 3.1 μM (ceμ(cerebral cortex) and a low affinity uptake system with respective Km. values of 93 and 33 μM for these two brain regions. In the cerebellum choline was accumulated only by the low affinity system. When striatal homogenates were fractionated further into synaptosomes and mitochondria and incubated with varying concentrations of [3H]choline, the high affinity component of choline uptake was localized to the synaptosomal fraction. The high affinity uptake system required sodium, was sensitive to various metabolic inhibitors and was associated with considerable formation of [3H]acetylcholine. The low affinity uptake system was much less dependent on sodium, and was not associated with a marked degree of [3H]acetylcholine formation. Hemicholinium-3 and acetylcholine were potent inhibitors of the high affinity uptake system. A variety of evidence suggests that the high affinity transport represents a selective accumulation of choline by cholinergic neurons, while the low affinity uptake system has some less specific function.

Gangliosides of human myelin: sialosylgalactosylceramide (g7) as a major component

Abstract: — Gangliosides were isolated from purified human myelin in a yield of 62 μg of lipid-bound sialic acid per 100 mg of dry myelin. Sialosylgalactosyl ceramide (G7) was found to be a major component of the ganglioside fraction, amounting to 15 per cent of the total sialic acid. It accounted for 10 per cent of lipid-bound sialic acid in adult human white matter, making it the third most abundant ganglioside on a molar basis. These results were obtained with an improved method for isolating total gangliosides in high yield, by employing DEAE-Sephadex column chromatography. Myelin from other mammalian species had considerably less G7, and there were also indications of maturational changes. Both 2-hydroxy and unsubstituted fatty acids were components of the ceramide unit, in a ratio of 3:2, respectively. The overall fatty acid pattern was very similar to that for myelin cerebroside and sulphatide. Long-chain bases included only C18 species, with sphingosine predominating (>90 per cent). These observations suggest a metabolic relationship between G7 and either cerebroside or sulphatide.

Protein composition of myelin of the peripheral nervous system

Abstract: — Myelin was purified from the peripheral nervous system (PNS) of several species. The protein composition of these preparations was examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Proteins characteristic of all samples include, in order of increasing mobility: a series of high molecular weight proteins, the major peripheral nerve protein (P0), two uncharacterized proteins, and two basic proteins (P1 and P2). Quantitative results, obtained by densitometry of gels stained with Fast Green showed differences in protein distribution, both between species, and from different types of nerves obtained from the same animal. The relative amounts of P1 and P2 proteins were the most variable; e.g. myelin from guinea-pig sciatic nerve had little or no P2 protein, whereas 15 per cent of the myelin protein of beef posterior intradural root was Pz protein. P0, P1 and P2 proteins from rabbit sciatic nerve and P0 and P2 proteins from beef dorsal and ventral intradural roots were purified and their amino acid compositions were determined. Our results indicated that the P1 protein is very similar in size and amino acid composition to the basic protein of central nervous system myelin, whereas the P0 and P2 proteins are unique to the PNS.

Choline: selective accumulation by central cholinergic neurons.

Abstract: — Most of the cholinergic input to the hippocampus was destroyed by placement of lesions in the medial septal area. In animals with such lesions we found that hippocampal ChAc activity was reduced by 85–90% and endogenous acetylcholine levels were reduced by more than 80 %. When hippocampal synaptosomes from animals with lesions were incubated with [3H]choline at concentrations of 7.5 nm, 1 μm and 10 μm there was approximately a 60 % reduction in the uptake of [3H]choline, suggesting that cholinergic nerve endings were mainly responsible for [3H]choline uptake. At 0.1 mm concentrations of [3H]choline, there was only a 25 % reduction of choline uptake, suggesting that at higher concentrations of choline there was more nonspecific uptake. The uptake of radiolabelled tryptophan, glutamate and GABA were only slightly or not at all affected by the lesions. There was a significant reduction of uptake of radiolabelled serotonin and norepinephrine, since known monoaminergic tracts were disrupted. Choline uptake was reduced only in brain regions in which cholinergic input was interrupted (i.e. the cerebral cortex and hippocampus) and remained unchanged in other regions (i.e. the cerebellum and striatum). The time course of the reduction in choline uptake was similar to that of the reductions in ChAc activity and endogenous ACh levels; there was no decrease at 1 day, a significant decrease at 2 days, and the maximal decrease at 4 days postlesion. There was a close correlation among choline uptake, ChAc activity and ACh levels in the four brain regions examined (i.e. the striatum, cerebral cortex, hippocampus and cerebellum). Our results suggest that when hippocampal synaptosomes (and perhaps synaptosomes from other brain areas as well) are incubated in the presence of choline, at concentrations of 10 μm m or lower, then cholinergic nerve endings are responsible for the bulk of the choline accumulated by the tissue.

Freeze-blowing: a new technique for the study of brain in vivo.

Abstract: — A new apparatus is described which removes and freezes brains of conscious rats more rapidly than was heretofore possible. The apparatus consists of two probes which are driven simultaneously into the cranial vault of the rat immobilized in a specially constructed restraining cage. When in position, air under pressure enters through one probe and blows the supratentorial portion of the brain tissue (situated between the olfactory bulbs and the superior colliculi) out the other probe and into a thin chamber previously cooled in liquid N2. This method stops brain tissue metabolism more rapidly than the previously-described methods of microwave irradiation, decapitation into liquid N2, or whole-animal immersion into liquid N2, as evidenced by the measurement of labile metabolites and redox states. Thus, samples of freeze-blown brain had higher levels of a-oxoglutarate, creatine phosphate, pyruvate, glucose and glucose-6-phosphate and lower levels of lactate, malate and AMP than brain tissue obtained by the other methods. The free cytoplasmic [NAD+]/[NADH2], [NADP+]/[NADPH2] and [ATP]/[ADP] [HPO42-] ratios were higher in freeze-blown samples. These data indicate that more extensive anoxic metabolism occurred when methods other than freeze-blowing were used. We conclude that the levels of metabolites measured in brain obtained with the freeze-blowing technique more closely resemble those which occur in vivo.

The determination of picomole amounts of acetylcholine in mammalian brain.

Abstract: — In any assay for the determination of acetylcholine based on the conversion of choline to a product, the immediate problem is the removal of endogenous choline. Other published enzymatic assays have taken advantage of electrophoresis to accomplish this goal. In the assay to be described, this is accomplished by the enzymatic phosphorylation of endogenous choline by choline kinase. Once this reaction is complete, endogenous acetylcholine is simultaneously hydrolysed and then phosphorylated with [32P]ATP. The labelled product [32P]phosphorylcholine is separated from the labelled substrate by precipitation of the ATP and further separation is accomplished on microcolumns of ion exchange resin. Using this methodology, picomole amounts of acetylcholine, derived from tissue, can be measured.

AMINO ACIDS AS CENTRAL NERVOUS TRANSMITTERS: THE INFLUENCE OF IONS, AMINO ACID ANALOGUES, AND ONTOGENY ON TRANSPORT SYSTEMS for l‐GLUTAMIC AND l‐ASPARTIC ACIDS AND GLYCINE INTO CENTRAL NERVOUS SYNAPTOSOMES OF THE RAT

Abstract: —The accumulation by synaptosomal fractions from rat central nervous system tissue of transmitter candidate amino acids and non-candidate amino acids was studied with respect to ionic requirements, metabolic inhibitors, structural analogues, and ontogeny. For l-glutamic and l-aspartic acids in cortex and spinal cord and glycine in spinal cord a stringent sodium requirement for high affinity uptake was demonstrated. Detailed kinetic analysis of the sodium requirement for glutamic acid uptake into cortical synaptosomal fractions suggests that: (1) sodium ion acts both competitively and non-competitively in determining the velocity of high affinity uptake; (2) in the absence of sodium ion only the low affinity uptake can be demonstrated; and (3) inhibition of the Na–K-ATPase enzyme system with ouabain reduces the velocity of uptake to 60% of control values in contrast to alanine whose synaptosomal uptake is less dependent on this enzyme system. No potent inhibitors of l-glutamic acid accumulation were found among several structural analogues or derivatives. The kinetic analysis for glycine accumulation was more complex and suggested allosteric interactions between glycine and sodium ion. Developmental studies revealed GABA and l-glutamic acid to have absolute sodium requirements for cortical synaptosomal accumulation from the 16th gestational day through adulthood, with glycine accumulation showing a decreasing sodium requirement as maturation proceeded over this period. The combined evidence suggests that sodium requirement for high affinity uptake is a characteristic of neuroactive substances and may be used as a screening tool to search for other neurotransmitter candidates.

High affinity uptake of transmitters: studies on the uptake of L-aspartate, GABA, L-glutamate and glycine in cat spinal cord.

Abstract: — The uptake of l-aspartate, l-glutamate and glycine each appeared to be mediated by two kinetically distinct systems with apparent Km's of the order of 10 ('high affinity') and 100 μM ('low affinity') in slices of cat spinal cord, whereas the uptake of GABA appeared to be mediated by a single system of high affinity. The high affinity uptake of these amino acids in slices of spinal grey matter was approximately 5 times faster than that in slices of spinal white matter. The high affinity uptake systems in the cord slices survived homogenisation of the tissue under conditions known to preserve nerve terminals. Subcellular fractionation studies indicated that osmotically-sensitive particles of equilibrium density equivalent to that of 1.0 m-sucrose were at least in part responsible for the uptake of these amino acids. Inhibition studies indicated that three structurally specific systems of high affinity transported these amino acids: 1 specific for glycine—not inhibited by GABA or any of the other depressant amino acids found in cat spinal cord; 2 specific for GABA—not inhibited by glycine, taurine, l-aspartate or l-glutamate and (3) specific for l-aspartate and l-glutamate—not inhibited by glycine or GABA but strongly inhibited by various acidic amino acids such as l-cysteate and l-cysteine sulphinate. The high affinity uptake of these amino acids was not inhibited by any of the known antagonists of the postsynaptic actions of these amino acids—strychnine (glycine), bicuculline and benzyl penicillin (GABA), methioninesulphoximine and l-glutamate diethyl ester (l-aspartate and l-glutamate). p-Chloromercuriphenylsulphonate strongly inhibited the high affinity uptake of glycine and GABA but was much less effective as an inhibitor of l-aspartate/l-glutamate high affinity uptake. This is in good agreement with microelectrophoretic studies in which this mercurial was found to potentiate depression of neuronal firing induced by glycine and GABA much more readily than excitation induced by l-aspartate or l-glutamate. These findings suggest the importance of high affinity transport processes in the removal of amino acids from the synaptic environment.

The effect of hypoxia on monoamine synthesis, levels and metabolism in rat brain

Abstract: — Rats were exposed to 5.6% oxygen environments for up to 2 h. The accumulation of brain DOPA and 5-hydroxytryptophan at 30 min after decarboxylase inhibition was used to estimate cerebral tryosine and tryptophan hydroxylase activity, respectively, in vivo. There was a continuing decrease in tryosine hydroxylase activity during the 2 h in whole brain as well as five brain regions. Tryptophan hydroxylase activity declined during the 1st h, but then increased towards control levels during the 2nd h. There was an increase in brain tryptophan during the 2nd h as well. In whole brain and the five brain regions, there was no significant change in the levels of noradrenaline, dopamine or 5-hydroxytrypamine. During a 1 h exposure to 5.6% oxygen, there was decreased accumulation of noradrenaline, dopamine and 5-hydroxytryptamine after MAO inhibition and decreased accumulation of homovanillic acid and 5-hydroxyindoleacetic acid after probenecid administration. The dercreased synthesis and metabolism of the monoamines is most likely attributable to insufficient brain tissue oxygen as a substrate for the two hydroxylase enzymes.

Evidence for the close association of a glycoprotein with myelin in rat brain.

Abstract: — Myelin was purified from rats which had been injected intracerebrally with radioactive fucose in order to label specifically the glycoproteins. Myelin contained a small amount of fucose-labelled glycoproteins in comparison to that in other subcellular fractions, but polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed a unique pattern of radioactive glycoproteins dominated by a major peak. The same glycoprotein was not prominent in the other subcellular fractions which were examined. This major glycoprotein in the myelin fraction was also labelled after injection with [3H]glucosamine or N-[3H]acetylmannosamine. It was the most intensely staining myelin protein when gels were treated with periodic acid-Schiff reagents, an indication that, in terms of protein-bound carbohydrate, it is the major glycoprotein in the myelin fraction. The glycoprotein was present in myelin purified from rats ranging in age from 14 days to 14 months. Extensive recycling of the myelin through the purification procedures did not significantly reduce the amount of glycoprotein in the myelin. Double label experiments with [3H]fucose and [14C]fucose were used to compare glycoproteins in myelin purified from white and grey matter, respectively, and from mixed homogenates of myelinated and unmyelinated brain. The results obtained from these experiments suggested that the glycoprotein is closely associated with myelin and that it is not in an unrelated contaminating structure. Possible locations of the glycoprotein are discussed. They include the myelin membrane itself, the oligodendroglial plasma membrane, and the axolemma of myelinated axons.

A soluble preparation from rat brain that incorporates into its own proteins [14c]arginine by a ribonuclease‐sensitive system and [14c]tyrosine by a ribonuclease‐insensitive system

Abstract: — A 100,000 g supernatant fraction from rat brain that was passed through a column of Sephadex G-25-40 was able, after addition of some factors, to incorporate [I4C]arginine (apparent Km= 5 μM) and [14C]tyrosine (apparent Km= 20 μM) into its own proteins. The factors required for the incorporation of [14C]arginine were: ATP (optimal concentration = 0-25-2 μM) and Mg2+ (optimal concentration 5 mM). For the incorporation of [I4C]tyrosine the required factors were: ATP (apparent Km= 0-75 μM), Mg2+ (optimalconcentration 8-16 mM) and K+ (apparent Km= 16 mM). Addition of 19 amino acids did not enhance these incorporations. Optimal pHs were: for [14C]arginine and [14C]tyrosine, respectively, 7-4 and 7-0 in phosphate buffer and 7–9 and 7-3-8-1 in tris-HCl buffer. Pancreatic ribonuclease abolished the incorporation of [14C]arginine but had practically no effect in the incorporation of [14C]tyrosine. Furthermore, [14C]arginyl-tRNA was a more effective donor of arginyl groups than [14C]arginine, whereas [14C]tyrosyl-tRNA was considerably less effective than [14C]tyrosine. The incorporations of [14C]arginine and [14C]tyrosine into brain proteins were from 25- to 2000-fold higher than for any other amino acid tested (12 in total). In brain [14C]arginine incorporation was higher than in liver and thyroid but somewhat lower than in kidney. In comparison to brain, the incorporation of [14C]tyrosine was negligible in liver, thyroid or kidney. Kinetic studies showed that the macromolecular factor in the brain preparation was complex. The protein nature of the products was inferred from their insolubilities in hot TCA and from the action of pronase that rendered them soluble. [14C]Arginine was bound so that its a-amino group remained free. Maximal incorporation of [14C]tyrosine in brain of 30-day-old rats was about one-third of that in the 5-day-old rat. The changes with postnatal age in the incorporation of [14C]arginine were not statistically significant.

The use of microwave heating to inactivate cholinesterase in the rat brain prior to analysis for acetylcholine.

Abstract: — Heating with 2450 MHz microwave radiation has been investigated as a means for animal sacrifice concurrent with enzyme inactivation. Uniform inactivation of cholinesterase (EC 3.1.1.8) in the entire brain can be effected in the rat within 4 s and in the mouse within 2 s without destruction of acetylcholine. The acetylcholine content in the whole brain of a rat was found to be 25.4 ± 1.5 nmol/g after irradiation, in comparison to 13.8 ± 1.7 nmol/g after standard methods of sacrifice. In the mouse whole brain, the comparable acetylcholine contents were 25.5 ± 2.6 and 13.7 ± 1.7 nmol/g, respectively. The value of this procedure for rapid inactivation of enzymes in the study of acetylcholine turnover is discussed.

Use of catechol O-methyltransferase for the enzyme radiochemical assay of dopamine

Abstract: PREVIOUS reports (SAELENS et al., 1967; IVERSEN and JARROTT, 1970; NIKODIJEVIK et al., 1969) have described the use of the methyl transferase enzymes phenylethanolamine N-methyltransferase and catechol 0-methyltransferase in conjunction with 14Cor 3H-labelled S-adenosyl methionine for the enzyme radiochemical assay of noradrenaline. In the method described by NIKODIJFXIK et aZ. (1969) the noradrenaline content of tissues was assayed with the use of highly purified catechol 0methyltransferase (COMT) prepared from rat liver. The product of the enzyme reaction, normetanephrine, was recovered by extraction into an organic solvent. This method, however,cannot be applied to tissues in which both noradrenaline and dopamine are present, since these amines are substrates for COMT and the methylated products from both amines would be recovered in the organic phase. The present method was devised for the determination of small amounts of dopamine in tissue extracts, in the presence of noradrenaline or other biogenic amines. The high sensitivity of the method makes it possible to measure dopamine in very small samples of tissue. After a series of preliminary studies we have adopted the following procedure: a reaction mixture is freshly prepared at the start of each experiment; it is composed of 300 pl of bovine liver COMT in 0.5 M-sodium phosphate buffer, pH 7.0, containing about 50 mg of protein per ml (for preparation see below); 150 pl of [3H-]methyl S-adenosyl methionine (100 pCi/ml) (S.A. 7.9 Ci/mmol, The Radiochemical Centre, Amersham, England) diluted with non-radioactive S-adenosyl methionine to a specific activity of 600 mCi/mmol or 150 pl of [14C-]methyl S-adenosyl methionine (10 pCi/ml) (S.A. 55 mCi/mmol; The Radiochemical Centre, Amersham, England); 50 pI of a solution containing 80 mM-pargyline and 10% (v/v) 2-mercaptoethanol. Just before use 325 p1 of 1 M-triS base containing 30 m-magnesium chloride is added. This amount of tris is necessary to bring the pH of the final incubation mix to 9.1, the optimum for the COMT reaction. Twenty-five microlitres of this mixture is added to ice-cold 200 p1 microtubes, and 10 pl of 0.1 M-perchloric acid (blanks), or 10 p1 of standard solutions containing 1.25-12.5 ng of dopamine in 0.1 M-perchloric acid or 10 pl of the tissue samples extracted in 0.1 M-perchloric acid, are then added, in addition to some tissue samples a further 2 pl of an aqueous solution containing 5 or 10 ng of dopamine was added as an internal standard. Tissue extracts are prepared in the following way. Dissected brain areas (0.3-35.0 mg wet wt.) were placed in microtubes with 1-20 vol. of 0.1 M-perchloric acid, the volume of acid being adjusted according to the expected range of dopamine concentrations. The tissue is disrupted with the tip of a dissecting needle, frozen and thawed and centrifuged in a microcentrifuge at 6000 g for 5-10 min. Ten microlitres of the resulting supernatant fluid is taken for dopamine determinations. The incubation mix and samples are agitated with a vortex mixer, and incubated at 37°C for 15 min. The reaction is stopped by immersing the tubes in a salted ice bath. Twenty microlitres (4 x 5 pl) are spotted on Whatman No. 1 chromatographic paper. The origin is previously spotted with 5 pl of a solution of 3-methoxytyramine (10 mg/ml in 0.01 M-perchloric acid) as carrier. The chromatograms are developed by the descending technique for 24-48 h with t-amyl alcohol (2-methylbutan-2-01): 25-30 % methylamine (4: 1 v/v). Under these conditions the RF values for 3-methoxytyramine and normetanephrine are sufficiently different to give a clear separation. The 3-methoxytyramine spot is visualized under a U.V. lamp (254 nm), when it produces a weak violet fluorescence, normetanephrine can also be visualized as a yellow spot. The methoxytyramine spots are marked, cut out, shredded and extracted in counting vials for 4-18 h in 3 ml of a solution of ethano1:concentrated ammonia (100:22 v/v). The radioactivity is counted after adding 12 ml of a Triton X-100: toluene (1:2) mix containing 0.4% butyl PBD (CIBA).

The activities of butyrylcholinesterase and carbonic anhydrase, the rate of anaerobic glycolysts, and the question of a constant density of glial cells in cerebral cortices of various mammalian species from mouse to whale

Abstract: — The question of a constant density of glial cells in mammalian cerebral cortex regardless of species was examined by surveying the cortical activities of two enzymes primarily localized to dial cells. The cortical activity of butyrylcholinesterase (EC 3.1.1.8) was essentially constant at a rate of approx. 0.1 μmol of butyrylthiocholine hydrolysed min-1 g-1 over the range of species from rat (brain wt., 1.6 g) to fin whale and sperm whale (brain wt., 6800 and 7800 g, respectively). Over the same range the activity of cortical acetylcholinesterase, a neuronal enzyme, decreases by a factor of 7. Thus, butyrylcholinesterase ranged from < 2 per cent (in small rodent brains) to approximately 10 per cent (in whale brain) of the cortical acetylcholinesterase activity. The cortical activity of carbonic anhydrase (EC 4.2.1.1) was constant at a rate of 6.2 (± 0.25) μmol of CO2 evolved min-1 g-1 over the range of species from guinea-pig (brain wt., 4.75 g) to fin whale (brain wt., 6800 g). These data obtained by assaying the dehydration reaction were confirmed by limited assays of the esterase activity of the enzyme (with p-nitrophenylacetate as substrate) and agreed with limited, previously reported data for the hydration reaction. Thus, the circumstantial evidence strongly favoured a relative constancy of cortical glial cell density regardless of species. The rates of anaerobic glycolysis in the cerebral cortex of various species were also investigated. For six species from mouse (brain wt., 0.4 g) to beef (brain wt., 380 g) cortical anaerobic glycolysis varied only slightly in the range of 50–62 μmol of CO2 evolved h-1 g-l, whereas cortical oxygen consumption for the same range of species decreased by a factor of 3. Previously frozen samples of beef cortex glycolysed at 35 per Cent of the rate of fresh (unfrozen) samples. Since identical rates were obtained for previously frozen samples of fin whale cerebral cortex, we concluded that the relative constancy of cortical anaerobic glycolysis could be extended to the range from mouse to whale and that this aspect of cortical metabolism is probably primarily glial in localization. Some implications of the latter conclusion for the proposed role of astrocytes as modulators of neuronal activity have been discussed.

Studies on rna synthesis in two populations of nuclei from the mammalian cerebral cortex

Abstract: A method is presented for the rapid separation of cell nuclei from the rabbit cerebral cortex into two populations. The first of these consists largely of nuclei with the morphological characteristics of neuronal nuclei, the second almost entirely of nuclei with the morphological characteristics of glial cell nuclei. From studies based upon sensitivity to the toxin α-amanitin, the ratio of incorporation of different bases, ionic requirements and differential sensitivity to actinomycin D, it is concluded that under both the classical low and high salt conditions described by other workers, two enzymes are active in RNA synthesis. The presence of a third enzyme of low activity cannot be excluded. No qualitative difference in the number of enzymes involved in RNA synthesis in neuronal and glial cell nuclei has been found, but there are quantitative differences in activity between the two nuclear populations.

ENZYMIC AND CEREBRAL METABOLIC EFFECTS OF 2‐DEOXY‐d‐GLUCOSE

Abstract: —The time course of effects of 2-deoxy-d-glucose on cerebral glucose metabolism has been studied in vivo and the inhibitory actions of 2-deoxy-d-glucose and 2-deoxy-d-glucose-6-phosphate on cerebral glycolytic enzymes in vitro. Mice were given 2-deoxy-d-glucose 3 g/kg intraperitoneally. Blood 2-deoxy-d-glucose/glucose ratio was 2–3 from 5 to 30 min after injection, the hyperglycaemic response to 2-deoxy-d-glucose having been suppressed with propranolol. Maximal cerebral 2-deoxy-d-glucose uptake observed was 1μ11 μmol/g/min between 5 and 10 min after injection. At 10 min brain concentrations of 2-deoxy-d-glucose and 2-deoxy-d-glucose-6-phosphate were 5·82 and 3·12 μmol/g. Analysis of the fate of d-[U-14C] glucose given subcutaneously 5 min before death showed that glucose uptake was reduced to 40–60 per cent of control from 5 to 30 min after 2-deoxy-d-glucose. However brain glucose concentration rose three to five-fold 20–30 min after 2-deoxy-d-glucose. The majority of glucose entering the brain after 10 min of 2-deoxy-d-glucose treatment was recovered as glucose. Conversion of brain glucose to other acid soluble components was reduced to 1/3 at 10 min and 1/5 at 20–30 min. Glucose-6-phosphate concentration rose from 5 min onwards and was maintained at twice control concentration from 10–30 min. However, because of the rapid entry of 2-deoxy-d-glucose and its conversion to 2-deoxy-d-glucose-6-phosphate, the 2-deoxy-d-glucose 6-P/glucose 6-P ratio was between 19 and 32. Brain adenosine triphosphate concentration did not change, creatine phosphate concentration fell after 25 min. Measurement of enzyme activities in cerebral homogenates (using 1 mivs substrate concentration) showed that hexokinase (EC 2.7.1.1) was 40 per cent inhibited by 5 mm-deoxy-d-glucose (but not by 2-deoxy-d-glucose 6-P). Glucose 6-P dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.43) and phosphoglucomutase (EC 2.7.5.1) were not affected by either 2-deoxy-d-glucose (5 mm) or 2-deoxy-d-glucose 6-P (5 or 20 mm). Hexose-phosphate isomerase (EC 5.3.1.9) was 70 per cent inhibited by 20 mm-d-deoxy-d-glucose 6-P. Phosphofructokinase (EC 2.7.1.11) was inhibited by 17 per cent by 2-deoxy-d-glucose 6-P (20 mm). During the initial impairment of cerebral function by 2-deoxy-d-glucose there is competitive inhibition of glucose transport into the brain; later, glycolysis is more powerfully depressed by the inhibition of isomerase produced by the high intracerebral concentration of 2-deoxyglucose-6-phosphate.

Chemical, enzymatic and ultrastructural characterization of 5-hydroxytryptamine-containing neurons from the ganglia of Aplysia californica and Tritionia diomedia.

Abstract: — Several identified neurons in Aplysia and Tritonia ganglia were shown to contain measurable quantities (4–6 pmol/cell body) of 5-hydroxytryptamine (5-HT). A metabolic correlate for the limited distribution of 5-HT among the neurons of Tritonia is provided by the finding that the enzyme, aromatic acid decarboxylase (AAD), is 500 times more active in nerve cells containing 5-HT than in neurons devoid of the amine. Although all Aplysia neurons have some AAD activity, 5-HT cell bodies in this species are 10-fold more active than cell bodies which do not contain 5-HT. The cytoplasm of 5-HT cell bodies in Aplysia and Tritonia characteristically contains granules that have minimum diameters of approx. 1000 A and eccentric opaque cores. This type of granule was not found in somata which did not contain measurable 5-HT. These data illustrate the metabolic and morphological specialization in 5-HT-containing neurons of molluscs.

A sensitive, rapid and simple method for the simultaneous spectrophotofluorometric determinations of norepinephrine, dopamine, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in discrete areas of brain

Abstract: OVER the last decade the role of biogenic amines and their metabolites in the brain has been the subject of considerable interest with regard to the relationship of these substances to the actions of various drugs (CARISSON, 1964), alterations of behaviour (SCHILDKRAUT and KETY, 1967), and such disease entities as hypertension (SMOOKLER and BUCKLEY, 1969). However, until recently it was necessary to perform one assay for the determination of norepinephrine and dopamine (CHANG, 1964) and another for the measurement of serotonin (5-hydroxytryptamine) and 5-hydroxy-3indoleacetic acid (AHTEE et al., 1970). Combined assay procedures have been developed for the determinations of norepinephrine and serotonin (MAICKEL et al., 1968) and norepinephrine, dopamine and serotonin (ANSELL and BEESON, 1968; SHELLENRERGER and GORDON, 1971 ; WELCH and WELCH, 1969) but only one (WELCH and WELCH, 1969) includes the possibility for determination of 5-hydroxyindoleacetic acid, the chief metabolite of serotonin. The fluorometric procedure developed in our laboratory allows the rapid determination of the levels, in a single sample, of what are considered to be the major biogenic amines-norepinephrine. dopamine, and serotonin, as well as the latter’s major metabolite, 5-hydroxyindoleacetic acid. This assay utilizes the adsorption of the catecholamines onto alumina to remove noncatechol fluorescent substances, as described by CHANG (1964); the extraction of serotonin similar to that described by ANSELL and BEESON (1968); the development of a solvcnt extraction for 5-hydroxyindoleacetic acid; and utilization of the o-phthaldialdchyde reaction described by MAICKEL et al. (1968) for the determination of serotonin and 5-hydroxyindoleacetic acid. The value of obtaining data on discrete areas of the brain rather than from the whole brain has been shown to be of considerable importance (MILLER et al., 1968; HANK and APRISOS, 1971). Our procedure has been designed for application to the brain, but could be applied to other tissues and urine. In addition to measurement of the substances described, this assay could also be used for the determination of epinephrine with the appropriate adjustment of the standards (Cmrici, 1964).

ACCUMULATION OF CYCLIC ADENOSINE 3′, 5′‐MONOPHOSPHATE IN CEREBRAL CORTICAL SLICES FROM RAT AND MOUSE: STIMULATORY EFFECT OF α‐ AND β‐ADRENERGIC AGENTS AND ADENOSINE

Abstract: — Norepinephrine, epinephrine, isoproterenol, and adenosine elicit enhanced accumulations of cyclic AMP in incubated slices of rat cerebral cortex. Combinations of norepinephrine, epinephrine, isoproterenol, or histamine with adenosine have a greater than additive effect on cyclic AMP levels. The effects of isoproterenol appear to be mediated via a classical β-adrenergic receptor whereas the effects of norepinephrine appear due to interactions with both α- and β-adrenergic receptors. The presence of the phosphodiesterase inhibitor, isobutylmethylxanthine, potentiates the effects of the catecholamines and reveals a histamine-mediated increase in cyclic AMP levels. After an initial stimulation of cyclic AMP formation with norepinephrine, followed by washing of the slices, the cyclic AMP-generating system is unresponsive to norepinephrine but does respond to an adenosine-norepinephrine combination. In mouse cerebral cortical slices, catecholamines appear to elicit an accumulation of cyclic AMP primarily via interaction with a β-adrenergic receptor.

Factors affecting the turnover of cerebral glycogen and limit dextrin in vivo.

Abstract: The turnover of cerebral glycogen in mice has been investigated by using [U-14C]glucose as a precursor. The time required for turnover of total glycogen and limit dextrin has been determined in normal animals and animals given phenobarbital or hydrocortisone. In all 3 groups, the turnover time for limit dextrin was twice that of total glycogen. Phenobarbital increased the time for turnover of total glycogen and limit dextrin approximately 2-fold, whereas hydrocortisone diminished the turnover time of both fractions to one-half. The accumulation of glycogen during phenobarbital anesthesia (2·5-fold) is attributed to the decrease in rate of phosphorolysis rather than elevated glycogenesis. The ratio of phosphorylase a to total phosphorylase was significantly decreased in the brains of phenobarbital-treated mice, while the ratio of glycogen synthetase I to total synthetase activity was not affected. The administration of hydrocortisone had no effect on either the phosphorylase or synthetase of mouse brain. A mathematical model was devised to determine the rate constants for incorporation of labelled glucose into brain glycogen and the subsequent loss of radioactivity. Metabolite levels and enzyme activities have been correlated with the observed changes in glycogen turnover.

The regional distribution of the polyamines spermidine and spermine in brain.

Abstract: — The distribution in brain of the polyamines spermidine and spermine is described in the rat, dog, sheep, rabbit and in man. The distribution pattern was about the same in all the species, spermidine concentration being highest in areas rich in white matter. The concentration of spermine was lower than that of spermidine and showed less variation from area to area. Rat brain polyamine content was the same in rats killed by decapitation as in those killed by rapid freezing in liquid nitrogen and was also unchanged up to 48 h after the death of the animal.

Depolarizing stimuli and the release of physiologically active amino acids from suspensions of mammalian synaptosomes.

Abstract: —Potassium causes increased metabolism and release of physiologically active amino acids from suspensions of mammalian synaptosomes. Sheep and rat hypothalamic synaptosomes, and rabbit, sheep and rat cortical synaptosomes show respiratory and glycolytic response to electrical stimulation and show a parallel calcium-dependent differential release of physiologically active amino acids. Attempts were made to eliminate the actions of heating and electrode products as the agents causing the observed effects and the case is made that depolarization is the trigger mechanism.

Amino acids in synaptic vesicles from mammalian cerebral cortex: a reappraisal

Abstract: Synaptic vesicles were prepared from rat cerebral cortex and separated by gel filtration from small molecular weight compounds contaminating this fraction. Electron microscopy of the vesicle suspension showed that vesicles were by far the most abundant morphological entities. The amino acid content of the purified vesicle fraction was examined and the two amino acids appearing in the most significant amounts were found to be taurine and glutamate. This amino acid pool was not osmotically sensitive as is the vesicular pool of ACh and remained attached to the vesicular protein after passage through Sephadex columns equilibrated in water. However, amino acids added to the vesicle fraction prior to passage through Sephadex did not become associated with this pool and this indicated that the vesicular pool was not likely to be an artifact due to the vesicular protein non-specifically adsorbing amino acids. The release of taurine from incubated synaptosome beds was studied and elevated medium K+ (56 mm) was found to cause a small increase (36 per cent) in the amount of the taurine released to the medium. During the same experiments another physiologically active amino acid, glutamate, was released in more significant amounts, increasing in the medium by 186 per cent. The possible significance of the presence of taurine is discussed.

Evidence that the major protein in rat sciatic nerve myelin is a glycoprotein.

Abstract: Evidence is presented that the major protein of rat sciatic nerve myelin is a glycoprotein. When myelin proteins were separated by polyacrylamide gel electrophoresis, the major band which was stained with amido black–Coomassie blue was also stained with periodic acid-Schiff reagents for carbohydrate. Radioactive labelling of myelin in vivo with [3H]leucine and [14C]fucose, followed by electrophoresis of the proteins, indicated that with both isotopes the major labelled peak corresponded to the major stained band. In addition, a second smaller peak of [14C]fucose migrated ahead of the major peak. Delipidated myelin contained galactose, mannose, fucose and sialic acid.

Kinetics of the sodium-dependent transport of gamma-aminobutyric acid by synaptosomes

Abstract: The kinetics of transport of gamma-aminobutyric acid [2,3-3H] by synaptosomes from rat brain was studied by means of a rapid filtration technique. The rate of uptake was proportional to the protein concentration over the range 0.05—0.2 mg of synaptosomal protein per ml. Although apparent allosteric kinetics were observed with sodium, transport followed simple saturation kinetics with respect to GABA and no heterotropic, cooperative effects of GABA on sodium on kinetics were observed. A minimum of three interacting sodium sites is suggested the basis of Hill plots of the sodium data. Both the apparent Km and Vmax for GABA were functions of the sodium ion concentration but the effect of sodium was considerably greater on Vmax than on the apparent Km The Vmax for GABA was 1.1 ± 0.5 nmol.min−1 mg−1 of protein at 95 mm sodium and decreased to 12 per Cent of this value at 19 mm sodium. The apparent Km for GABA increased from 4.0 ± 1.0 μm at 95 mm sodium to 8.4 ± 2.0 μm at 19 mm sodium. Potassium was a noncompetitive inhibitor with respect to GABA and did not affect the apparent cooperativity observed with sodium. These findings are discussed in terms of models of GABA transport.