THE EFFECT OF FASTING ON THE METABOLISM OF 5-HYDROXYTRYPTAMINE AND DOPAMINE IN THE BRAIN OF THE MOUSE

Abstract— The turnover of 5-hydroxytryptamine in the forebrain and of dopamine in the striatum was studied in mice fasted for 20 h. Such mice showed an increased tissue concentration of 5-hydroxyindoleacetic acid in the forebrain and an increased accumulation of this acid after probenecid. Fasted mice also showed a higher concentration of homovanillic acid in the striatum than fed mice. However, the administration of probenecid produced a smaller increase in homovanillic acid concentration in fasted than in fed mice. The decay of dopamine following α-methyl- p -tyrosine was reduced in fasted mice at 2 h, but not at 1 h or 6 h after administration of the inhibitor. The possibility that fasting increases the activity of some dopaminergic neurones while decreasing the activity of others is considered. The existence of a pool of homovanillic acid at a site within the striatum where the probenecid-sensitive transport is not effective is postulated.     

POLYAMINE METABOLISM IN THE BRAIN AND LIVER OF THE DEVELOPING MONKEY

Abstract— The concentrations of putrescine and the polyamines, spermidine and spermine, along with the specific activities of the enzymes involved in their biosynthesis, ornithine decarboxylase, S -adenosylmethionine decarboxylase and spermidine synthase have been measured in brain and liver of the developing Rhesus monkey from mid-gestation, through birth and neonatal life to maturity. The results suggest that it is an increased concentration of putrescine and an increased specific activity of ornithine decarboxylase which are associated with the rapid growth of fetal brain during the middle of gestation. By the end of two-thirds of gestation, both of these parameters have attained values similar to those found in mature brain. The concentration of spermidine in brain and the specific activities of S -adenosylmethionine decarboxylase and spermidine synthase are lower in fetal brain than adult brain and increase slowly after birth to reach values similar to those of the adult only after several months. These results provide additional evidence that in the mature brain spermidine serves some function other than one associated with rapid growth.
Fetal liver at mid-gestation was characterized by increased concentrations of both putrescine and spermidine and increased specific activities of the enzymes which synthesize them. By two-thirds of gestation, values similar to those found in adult liver had been attained. Liver has thus reached maturity with regard to polyamine metabolism by this time.     

EFFECTS OF SODIUM FLUOROACETATE ON THE METABOLISM OF N-ACETYLASPARTATE AND ASPARTATE IN MOUSE BRAIN

A subconvulsant dose of sodium fluoroacetate inhibited the metabolic utilization of intracerebrally-administered N-acetyl-l -[U-¹⁴C]asparticacid and the labelling of glutamine from this precursor in mouse brain, but not the labelling of glutamate or aspartate. A convulsant dose also inhibited the utilization of l -[U-¹⁴C]aspartic acid. When intraperitoneal injection of a convulsant dose of sodium fluoroacetate was followed by intracerebral injection of N-acetyl-l -[U-¹⁴C]asparticacid, the levels of N-acetylaspartate, aspartate and glutamate in brain were lowered, while the glutamine content was increased. The specific radioactivity of glutamine relative to that of glutamate was much lower when these compounds were labelled from l -[U-¹⁴C]aspartic acid than when N-acetyl-l -[U-¹⁴C]aspartic acid was used as the precursor. Intracerebral injection of tracer amounts of l -[U-¹⁴C]aspartic acid reduced the content of N-acetylaspartate in brain and raised the glutamine content. Sodium fluoroacetate had no additional effect on the relative specific radioactivity of glutamine or the content of N-acetylaspartate, aspartate, glutamate or glutamine when l -[U-¹⁴C]aspartic acid was the precursor. We consider the results to be consistent with a selective inhibition both by sodium fluoroacetate and by exogenous aspartic acid of the tricarboxylic acid cycle in brain associated with the biosynthesis of glutamine. We suggest that the activity of this pathway may regulate the metabolism of N-acetylaspartate and aspartate.     

THE EFFECTS OF BOTULINUM TOXIN ON ACETYLCHOLINE METABOLISM IN MOUSE BRAIN SLICES AND SYNAPTOSOMES

The effects of Type A botulinum toxin on acetylcholine metabolism were studied using mouse brain slice and synaptosome preparations. Brain slices that had been incubated with the toxin for 2h exhibited a decreased release of acetylcholine into high K⁺ media. Botulinum toxin did not affect acetylcholine efflux from slices in normal K⁺ media. When labeled choline was present during the release incubation, a‘newly-synthesized’pool of acetylcholine was formed in the tissue. In toxin-treated slices exposed to high K⁺, both the production and the release of this‘newly-synthesized’acetylcholine were depressed. A possible explanation for these actions of botulinum toxin would be via an inhibition of the high affinity uptake of choline. This hypothesis was tested by measuring the high affinity uptake of [³H]choline into synaptosomes prepared from brain slices. Previous exposure of slices to botulinum toxin caused a significant reduction in the accumulation of label by the synaptosomes. These data are discussed in terms of our current understanding of the mechanism of action of botulinum toxin and the toxin's interaction with the mechanisms regulating acetylcholine turnover.     

THE EFFECT ON GABA METABOLISM IN BRAIN OF ISONICOTINIC ACID HYDRAZIDE AND PYRIDOXINE AS A FUNCTION OF TIME AFTER ADMINISTRATION

—The effect of intramuscularly administered INH on brain levels of GABA in chicks was dependent on the amount injected. A subconvulsant dose of INH (1·1 mmol/kg) produced a slow steady decline in the level of GABA, whereas a convulsant dose (2·19 mmol/kg) brought about a sequential fall and rise in GABA level. This sequence of events reflected changes in the relative activities of GAD and GABA-T brought about by the hydrazide. The administration of pyridoxine together with the INH (2·19 mmol/kg) prevented the onset of seizures and lessened the effect of the INH on GABA levels and GAD activity but not on GABA-T activity. The possibility that a deranged GABA metabolism is responsible for hydrazide-induced seizures is discussed.     

THE EFFECT OF ELECTROCONVULSIVE SHOCK ON PROTEIN SYNTHESIS IN MOUSE BRAIN

The effect of a single electroconvulsive shock on protein synthesis in mouse brain cortex was studied by observing the incorporation into protein of intraperitoneally injected [³H]- or [¹⁴C]leucine. When the precursor was injected immediately after the electroshock there was a 50 per cent inhibition of the incorporation which was not seen with injections at times later than 10 min. To investigate a possible specificity, the cerebral cortices of experimental and sham control animals which had been injected with different isotopes were homogenized together and fractionated by differential centrifugation. Cell fractions were then separately extracted with phosphate buffer and with Triton X-100. The ratio of ³H to ¹⁴C in each fraction was compared with that of the total homogenate to reveal any specific effects due to the electroconvulsive shock. The treatment produced a slight inhibition of the incorporation of the isotope into the heavier particulate fractions (i.e. nuclei, mitochondria, synaptosomes) relative to that in the microsome and cell sap fractions. A possible explanation of these results is given with a discussion of the limitations of the technique.     

THE EFFECT OF ELEVATED AMINO ACIDS ON AMINOTRANSFERASE LEVELS IN BRAIN AND LIVER OF THE MOUSE

Abstract— Adult mice were fed standard diets that were enriched with selected amino acids, i.e. 3% methionine, 6% valine, or 8% lysine. These diets caused the following changes in the amino acid pool of the brain measured at 7 and 21 days. The high methionine diet resulted in 50-fold higher levels of methionine and cysteine and somewhat lower levels of serine and glutamine. The valine and lysine-enriched diets also caused 2- to 4-fold increases in valine and lysine contents of brain, respectively. In spite of the large changes in amino acid levels, however, there were essentially no changes in aspartate: α-ketoglutarate, alanine: α-ketoglutarate, ornithine: α-ketoglutarate, methionine: α-ketoglutarate, and the branched chain aminotransferase activities of brain 3, 10, and 21 days after the onset of the dietary regimen. In contrast, these diets produced significant changes in some of these enzyme activities in liver. Changes in liver included a 2-fold increase in ornithine and alanine aminotransferase activities with the methionine-enriched diet. Liver ornithine aminotransferase activity also increased slightly in animals fed the valine-enriched or lysine-enriched diet.     

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.     

METABOLISM OF POLYAMINES IN NORMAL AND SCRAPIE-AFFECTED MOUSE BRAIN AND SPLEEN

Abstract— Following intracerebral inoculation of mouse adapted scrapie agent into mice, polyamine concentration in the brain decreases to about 75 per cent of the normal level during the first 2 months after intracerebral inoculation of the agent. Between 2 and 4 months after infection thelevel of spermidine and spermineincreased by 80 and 40 percent respectively to reach concentrations of 25 and 20 per cent higher than controls of the same age. During the same period the rate of incorporation of [¹⁴C]putrescine into spermidine is increased four-fold as compared with controls. The changes in polyamine levels correlate well with the pattern of astrocyte hypertrophy and are similar to those reported for human brain tumours. The concentration of polyamines in spleen increases soon after inoculation. Whilst changes in brain polyamines might be referred to the hypertrophic growth of astrocytes those in spleen are perhaps due to an increased metabolic activity of spleen cells associated with the replication of the agent. These results are derived from experimental mouse scrapie and not naturally occurring disease in sheep.     

EFFECT OF COPPER STATUS ON BRAIN NEUROTRANSMITTER METABOLISM IN THE LAMB

Abstract— Ataxic and non-ataxic lambs reared under field conditions which gave rise to low copper status were treated with copper intravenously. Untreated ataxic animals served as controls. The neurotransmitter amines, dopamine, norepinephrine and serotonin, were determined in the anterior and posterior regions of the brain stem. Dopamine levels in the anterior region, including the corpus striatum, were significantly lower in the untreated animals than in those treated with copper. Norepinephrine levels were also lower but serotonin concentrations were not different. Plasma amine oxidase activity was markedly higher in the copper treated animals but monoamine oxidase activity in brain stem homogenates was not significantly affected. The monoamine oxidase activity in cortical and cerebellar homogenates was significantly lower in the treated animals than in the untreated animals.     

THE METABOLISM OF PALMITIC ACID IN THE PHOSPHOLIPIDS, NEUTRAL GLYCERIDES AND GALACTOLIPIDS OF MOUSE BRAIN

Abstract— Following intracerebral injection, [¹⁴C]palmitic acid was rapidly incorporated into a variety of brain lipids. After 12 hr, 78 per cent of the lipid radioactivity was in phospholipids, 15 per cent was in triacylglycerols, 1 per cent each was in free fatty acids and galactolipids, and the remainder was in other neutral glycerides. Over 65 per cent of the phospholipid radioactivity was found in the choline phosphoglycerides but this proportion decreased substantially with time. At later times, increasing portions of the radioactivity were present in the monounsaturated acyl groups and the alkenyl groups but no radioactivity was detected in cholesterol or polyunsaturated acyl groups. These results indicate that most of the extensive recycling of radioactivity took place without oxidative degradation of the palmitoyl groups. The relative rates of incorporation of radioactivity were compared at 12 hr after injection. The specific radioactivities of the serine, ethanolamine, and choline phosphoglycerides had ratios of 6:3:2 based on the palmitoyl group content and 1:2:4 based on their phosphorus content. The specific radioactivities of galactolipids with O -acyl groups were higher than the specific radioactivitiesof cerebrosides or cerebroside sulphates. A new solvent mixture for thin-layer chromatography of brain galactolipids was described (chloroform-acetone-methanol-water, 60:20:20:1, by vol.).     

EFFECT OF OXOTREMORINE ON THE APPARENT REGIONAL TURNOVER OF ACETYLCHOLINE IN MOUSE BRAIN

The effect of oxotremorine (1 mg kg^-1 i.p.) on the steady state concentration of acetylcholine (ACh) and choline (Ch) and the transformation of radioactive choline ([³H]Ch) was studied in different brain regions of the mouse following death by microwave irradiation of the head. Oxotremorine significantly increased the concentration of endogenous ACh in the cortex and hippocampus and of endogenous Ch in the cortex. Pretreatment with atropine (5 mg kg^-1 i.p.) prevented the increase in ACh. The biosynthesis of radioactive ACh ([³H]ACh) was decreased in all brain regions. Atropine (5 mg kg^-1) pretreatment counteracted this effect of oxotremorine (1 mg kg^-1), while methylatropine (5 mg kg^-1) had no effect except in the striatum. A calculation of the apparent turnover rate of ACh showed that oxotremorine (1 mg kg^-1) decreased the turnover in the cortex, hippocampus, midbrain. and striatum.     

THE EFFECT OF l-DOPA AND AN INHIBITOR OF PERIPHERAL DECARBOXYLATION ON GLUCOSE METABOLISM IN BRAIN1

Abstract– The effect of the administration of l -DOPA plus an inhibitor of peripheral l -aromatic amino acid decarboxylase (aromatic-l -amino-acid carboxy-lyase; EC 4.1.1.28) on the metabolism of glucose in brain was studied by administering [U-^I4C]glucose (20μCi) to three groups of rats: (1) rats that had been injected with l -DOPA (200mg/kg) 28min earlier; (2) rats that had been similarly injected with l -DOPA and also with N-(d,l -seryl)-N′-(2,3,4-trihydroxybenzyl)hydrazine (50 mg/kg), an inhibitor of l -aromatic amino acid decarboxylase, 30min before the l -DOPA; and (3) appropriate controls. The flux of ¹⁴C from glucose in plasma to those amino acids that are in equilibrium with the tricarboxylic acid cycle intermediates was reduced by treatment with l -DOPA and reduced further by treatment with l -DOPA and the decarboxylase inhibitor. Concentrations of glucose in brain and in plasma were increased after treatment with l -DOPA; these increases were attenuated if the inhibitor was given before the l -DOPA. After treatment with l -DOPA, there were decreases in the concentration of aspartate, tryptophan, and tyrosine in brain. After the administration of l -DOPA and the decarboxylase inhibitor, the concentrations in brain of alanine, glutamate, tyrosine, and phenylalanine were greater, and the concentrations of aspartate, leucine, lysine, histidine, arginine, and tryptophan were less than in control rats.     

THE EFFECT OF BILIRUBIN ON THE ENERGY METABOLISM OF BRAIN MITOCHONDRIA

Abstract— Unconjugated bilirubin caused uncoupling of oxidative phosphorylation in brain mitochondria prepared from mature or weanling rats. Human serum albumin in an amount sufficient to bind 100 per cent of the added bilirubin was able to protect the mitochondria from the inhibitory effects of bilirubin. Bilirubin inhibited both the endogenous and DNP-activated ATPase activities but had no effect upon the Mg²⁺-stimulated ATPase; albumin prevented the inhibition.     

THE MEASUREMENT OF TRIGLYCERIDE IN BRAIN AND THE METABOLISM OF BRAIN TRIGLYCERIDE IN VITRO

Abstract—

• 1 Triglyceride has been isolated from brain by thin-layer chromatography and determined by absorption of the carbonyl group at 1740 cm^?1. The means of yields from whole mouse brain, whole rat brain, rat brain grey matter, rat brain stem, and incubated slices of rat brain cortex were 0.15–0.17 μmole/g tissue.
• 2 The distribution of fatty esters varied from preparation to preparation. Palmitate, stearate and oleate usually occurred in greatest amounts. Hydrolysis of a preparation of triglyceride from whole rat brain with pancreatic lipase indicated that palmitate was equally distributed between the α and β esters.
• 3 [1-¹⁴C]Acetate was rapidly incorporated into triglyceride of slices of incubated rat brain cortex. When the resulting triglyceride was hydrolysed with pancreatic lipase the distribution of radioactivity amongst the hydrolysis products was consistent with both the α and β esters of the triglyceride having been radioactively labelled.

     

PITFALLS IN THE USE OF RAPID FREEZING FOR STOPPING BRAIN AND SPINAL CORD METABOLISM IN RAT AND MOUSE

The rate at which the metabolism is stopped by means of freezing in Freon-12 (–150°C) was studied in various areas of the rat and mouse CNS, using changes in temperature and levels of glucose and lactate as parameters for this rate. The rat cerebral cortex was frozen after 0.5 min while the hypothalamus reached 0°C after more than 1.5 min. The skin on the skull was found to be the most important temperature isolator for the cortex. Substrate levels can be studied in this area only if this piece of skin is removed previously. In the mouse, the cerebral cortex was frozen after 6 s, the hypothalamus after 0.5 min. The lumbar level of the mouse spinal cord was frozen after 15 s, the cervical level only after 47 s. Liquid nitrogen alone cooled the mouse cerebral cortex at least as fast as did Freon at its melting point. A gradual decrease from dorsal to ventral was observed in the glucose level of the molecular layer of the mouse cerebellum. The existence of a freezing front, moving slowly from dorsal to ventral, and its consequences for the measured levels of biologically labile substrates, are discussed.     

γ-VINYL GABA: EFFECTS OF CHRONIC ADMINISTRATION ON THE METABOLISM OF GABA AND OTHER AMINO COMPOUNDS IN RAT BRAIN

Rats were given γ-vinyl GABA (4-amino-hex-5-enoic acid), a new irreversible inhibitor of GABA aminotransferase (GABA-T), by daily subcutaneous injection (100mgkg) for 11 days. Amino acids were quantitated in the brains of the γ-vinyl GABA-treated and control animals 24 h after the last injection, and enzyme activities of GABA-T and glutamic acid decarboxylase (GAD) were measured. Chronic administration of γ-vinyl GABA produced a 150% increase in brain GABA content, along with marked increases in the contents of B-alanine and homocarnosine. Brain GABA-T activity was reduced by 26%, and GAD activity was reduced by 22%. In addition, γ-vinyl GABA caused a marked increase in hypotaurine content in rat brain, suggesting that it acts as an inhibitor of hypotaurine dehydrogenase, and it produced significant decreases in brain contents of glutamine and threonine. Although it is an effective GABA-T inhibitor, γ-vinyl GABA apparently affects several other brain enzymes as well, and it may not be an ideal drug for elevating brain GABA levels in man.