Utilization of 3-hydroxybutyrate by chick cerebral hemispheres during postnatal maturation.

1. The utilization of 3-hydroxybutyrate has been studied in the chick telencephalon during its post-hatching maturation. 2. In the 1-day-old chick the blood concentration of 3-hydroxybutyrate appears to be relatively high and its value is 5 times that estimated in the 4- and 30-day-old chicks. 3. The determination of the cerebral arteriovenous differences of 3-hydroxybutyrate shows that the brain of the newly-hatched chick takes up 3 times more actively this ketone body than the brain of the 4-day-old bird does. 4. During incubation in a non-oxygenated and an oxygenated physiological medium, in the presence of 3-hydroxy [3-14C]butyrate, the specific radioactivity of the dicarboxylic amino acids in the 1-day-old chick brain slices is higher than in those of the 30-day-old chick, particularly in the oxygenated medium. 5. Thirty minutes after a subcutaneous injection of 3-hydroxy [3-14C]butyrate, the specific radioactivity of the dicarboxylic amino acids in the 1-day-old chick telencephalon is 3-4 times higher than that in the 4- and 30-day-old chick. 6. In conclusion, in the brain of the newly hatched chick, 3-hydroxybutyrate is an efficient precursor in the biosynthesis of dicarboxylic amino acids, particularly glutamate, and, as glucose, it is metabolically related to the "large compartment" of glutamate. 7. These results have been discussed comparatively to those previously obtained in the developing rodent brain.     

Ultrastructural and biochemical studies of the swelling of developing chick telencephalic slices

Summary An ultrastructural and biochemical study of the importance and localization of tissue swelling was performed on telencephalic slices of 1- and 30-day-old chicks incubated in an oxygenated or a non-oxygenated physiological medium. The swelling of slices is greater for 30-day-old chick material than for that from 1-day-old chicks. It also reaches higher values in the non-oxygenated than in the oxygenated medium. When the 30-day-old chick telencephalic slices are incubated in an oxygenated medium, swelling mainly affects astrocytes, and especially the astrocytic endfeet. When they are incubated in a non-oxygenated medium, the astrocytes and astrocytic endfeet are very swollen and in addition the swelling also affects the neurons and their organelles. Extracellular space is increased. When 1-day-old chick telencephalic slices are incubated in a non-oxygenated medium, the tissue structures are well preserved. Swelling predominantly affects astrocytes and astrocytic endfeet. Neurons are not affected and the extracellular space is reduced. However, when they are incubated in an oxygenated medium, tissue structures are greatly affected showing a high degree of disorganization. Extracellular space is greatly increased. This study thus indicates that the best incubation conditions are an oxygenated medium for 30-day-old chick telencephalic slices which are characterized by an aerobic metabolism, and a non-oxygenated medium for 1-day-old chick telencephalic slices which have a predominantly anaerobic metabolism.     

Effect of acute cyanide intoxication on the active transport of amino acids in brain slices

(1) Acute hypoxia was produced in adult rats by cyanide inhalation and the effect on the active transport of amino acids was studied in brain slices. (2) Initial and steady-state accumulation of amino acids and rates of amino acid exit were identical in brain slices from control and treated animals when a glucose-containing incubation medium was used. (3) When the incubation was carried out in a glucose-free incubation medium, the inhibition of initial and steady-state accumulation and the stimulation of amino acid exit observed in control slices were significantly reduced or abolished in slices from treated animals. (4) Tissue swelling, size of ‘inulin space’ and glucose consumption did not differ in the two groups of animals. (5) Also the respiration rate was identical in slices from control and treated animals incubated in the presence of glucose. In the absence of added substrate, brain slices from treated animals consumed 15-20 per cent more oxygen than control slices. (6) A possible correlation between the effects observed on amino acid transport and on respiration is suggested. The reasons why cyanide given in vivo or added in vitro have different effects on amino acid transport in brain slices are discussed.     

Utilization of acetate by chick brain during postnatal maturation.

1. The study of the compartmentation of glutamate metabolism has been performed in the chick brain in vivo and in vitro in the presence of [U-14C]acetate between day 1 and day 30 of postnatal maturation. 2. The compartmentation of glutamate metabolism in vivo appears between day 1 and day 4 after hatching in the cerebral hemispheres and optic lobes. It is however more precocious in the optic lobes. In the cerebellum, it appears later, at about day 4 after hatching. The compartmentation of glutamate metabolism appears at the same time as the rapid incorporation of glucose into amino acids takes place in the cerebral hemispheres and the optic lobes. 3. In the chick telencephalon in vitro, the compartmentation of glutamate metabolism is visible from day 1 after hatching onwards. This difference is undoubtedly linked to the absence of an interference of glucose metabolism with acetate metabolism in vitro, and to the presence of a third compartment in the cerebral slices.     

Metabolic Stability of Hippocampal Slice Preparations During Prolonged Incubation

Abstract: Hippocampal slices were prepared under three conditions: (1) in medium containing glucose and oxygen at 4°C; (2) as in (1), but at 37°C; (3) in medium devoid of glucose and oxygen at 37°C. The rates of recovery to roughly steady-state levels and through 8 h of incubation were monitored for energy metabolite levels and related parameters. In vitro stable values are compared with in situ hippocampal levels. Regardless of the conditions under which slices were prepared, metabolite levels required up to 3 h to stabilize, and these levels were maintained or improved through 8 h of incubation. Further, the maximal concentrations of metabolites were independent of the conditions of slice preparation. Total adenylates and total creatine levels reached 55% of those in vivo. Lactate decreased from the decapitation-induced high levels, but stabilized at concentrations about twice those in rapidly frozen brain. Cyclic AMP and cyclic GMP exhibited peak levels at 30 min of incubation, and cyclic GMP remained elevated for 3 h. Although all three methods of slice preparation resulted in similar metabolite profiles on incubation, the initial decreases in high energy phosphates were delayed by chilling. Most striking, the slices prepared in the absence of glucose and oxygen exhibited much smaller orthodromic evoked potentials in the dentate gyrus. The presence of glucose and oxygen during preparation of the slices appears to be critical to the electrophysiological response of the tissue.     

AMINO ACID METABOLISM AND AMMONIA FORMATION IN BRAIN SLICES

The formation of ammonia and changes in the contents of free amino acids have been investigated in slices of guinea pig cerebral cortex incubated under the following conditions: (1) aerobically in glucose-free saline; (2) aerobically in glucose-free saline containing 10 mM-bromofuroic acid, an inhibitor of glutamate dehydrogenase (EC 1.4.1.2); (3) aerobically in saline containing 11-1 mM-glucose and (4) anaerobically in glucose-free saline. Ammonia was formed at a steady rate aerobically in glucose-free medium. The formation of ammonia was largely suppressed in the absence of oxygen or in the presence of glucose whereas the inhibitor of glutamate dehydrogenase produced about 50 per cent inhibition. Other inhibitors of glutamate dehydrogenase exerted a similar effect. Ammonia formation was also inhibited by some inhibitors of aminotransferases but not by others. Inhibition was generally more pronounced during the second and third hour of incubation. With the exception of glutamine which decreased slightly, the contents of all amino acids increased markedly during the anaerobic incubation. During aerobic incubation in a glucose-free medium, there was an almost complete disappearance of glutamic acid and GABA. Glutamine also decreased, but to a relatively smaller extent. The content of all other amino acids increased during aerobic incubation in glucose-free medium, although to a lesser extent than under anaerobic conditions. The greater increase of amino acids appearing anaerobically in comparison to the increase or decrease occurring under aerobic conditions corresponded closely to the greater amount of ammonia formed aerobically over that formed anaerobically. This finding is interpreted as indicating a similar degree of proteolysis under anaerobic and aerobic conditions; aerobically, the amino acids are partly metabolized with the concomitant liberation of ammonia. In glucose-supplemented medium, the content of glutamine was markedly increased. The content of glutamate and aspartate remained unchanged, whereas that of some other amino acids increased but to a lesser extent than in the absence of glucose. Proteolysis in the presence of glucose was estimated at about 65 per cent of that in its absence. In the presence of bromofuroate the rate of disappearance of glutamate was unchanged, but there was a larger increase in the content of aspartate and a smaller decrease of GABA and glutamine. Other changes did not differ significantly from those observed in the absence of bromofuroate. We conclude that the metabolism of amino acids in general and of glutamic acid in particular differs according to whether they are already present within the brain slice or are added to the incubation medium. Only the endogenous amino acids appear to be able to serve as precursors of ammonia and as substrates for energy production.     

Lactate prevents the alterations in tissue amino acids, decline in ATP, and cell damage due to aglycemia in retina

Under conditions of energy impairment, CNS tissue can utilize substrates other than glucose to maintain energy metabolism. Retinas produce large amounts of lactate, although it has not been shown that lactate can be utilized by retina to prevent the cell damage associated with hypoglycemia. To investigate this, intact, isolated retinas were subjected to aglycemic conditions in the presence or absence of 20 mM lactate. Retinas incubated in the absence of glucose for 60 min showed a threefold elevation in tissue aspartate and 60% decreases in tissue glutamate and glutamine, demonstrating a mobilization of carbon from glutamine and glutamate to the tricarboxylic acid cycle. Lactate prevented these changes in tissue amino acids, indicating metabolism of lactate with sparing of tissue glutamate and glutamine. Tissue ATP was 20 and 66% of control values with zero glucose or zero glucose plus lactate, respectively. Consistent with previous findings, incubation of retinas in the absence of glucose caused acute swelling of retinal neurons and release of GABA into the medium at 60 min. These acute toxic affects caused by the absence of glucose were completely prevented by the presence of lactate. At 24 h of recovery following 60 min of zero glucose, many pyknotic profiles were observed and lactate dehydrogenase (LDH) release into the medium was elevated sevenfold, indicating the extent of cell death. In contrast, no elevation in LDH was found and histology appeared normal in retinas exposed to zero glucose in the presence of lactate. alpha-Cyano-4-hydroxy cinnamate (4-CIN; 0.5 mM), an inhibitor of the monocarboxylic acid transporter and mitochondrial pyruvate carrier, blocked the ability of lactate to maintain ATP and protect retinas from aglycemia but had no effect on ATP or toxicity per se. Derangements in tissue aspartate, glutamate, and glutamine, which were prevented by lactate during zero glucose incubation, were again observed with lactate plus zero glucose in the presence of 4-CIN. However, 0.5 mM 4-CIN alone in the presence of glucose produced similar increases in aspartate and decreases in glutamate and glutamine as observed with zero glucose while having only modest inhibitory effects on [U-(14)C]lactate uptake, suggesting the mitochondrial pyruvate carrier as the main site of action. The above findings show that lactate is readily utilized by the chick retina during glucose deprivation to prevent derangements in tissue amino acids and ATP and retinal neuronal cell death.     

THE METABOLISM OF TISSUE CULTURES : I. PRELIMINARY STUDIES ON CHICK EMBRYO

1. The metabolism of chick embryo tissues has been followed by analysis of the culture media after various periods of incubation in roller bottles. 2. The initial rate of glucose utilization is increased by increasing glucose in the medium from 100 to 500 mg. per cent. Total glucose used can be increased in the same way or by daily addition of small amounts. Glucose is used in greatest amount when the medium containing 100 mg. per cent is replaced daily. 3. Although glucose consumption appears necessary for survival of cultures it may be used at a rate far in excess of that required for life and maximal growth. Complete blocking of mitosis by colchicine does not alter the rate of glucose utilization. 4. Proteolytic activity of the cultures is shown by an increase in the amino nitrogen of the peptone medium after incubation with tissue. 5. Utilization of nitrogen from an amino acid medium is shown by a decrease in the amino nitrogen of this medium. Cells obtaining their nitrogen from amino acids proliferate as rapidly as those grown in a medium identical except for the substitution of peptone, but the cell type is markedly different, in that embryo muscle forms cells resembling regenerating adult muscle. 6. Lactic acid was formed in both the presence and absence of glucose. Its formation increased with increased glucose utilization. There is some evidence that lactate may be utilized, and that it favors growth in the absence of glucose. 7. Added pyruvate was rapidly metabolized by the tissues. It, too, favors growth slightly in the absence of glucose.     

AMINO ACID METABOLISM IN GLIAL CELLS: HOMEOSTATIC REGULATION OF INTRA- and EXTRACELLULAR MILIEU BY C-6 GLIOMA CELLS

Abstract— The amino acid and carbohydrate metabolism of confluent cultures of C-6 glioma cells has been investigated. It was observed that the presence of glutamine in the incubation fluid was essential to maintain high glutamine levels in the cells during a 2 h incubation. When cells were incubated in a cerebrospinal fluid-like medium glutamate, glutamine, aspartate and γ-aminobutyrate (GABA) levels were comparable to those occurring in whole forebrain of adult rat in vivo. Glucose uptake was high, approx 1 μmol/mg protein/2 h, 50% of which was accounted for by lactate production. Of the remaining glucose uptake a substantial proportion was unaccounted for by known oxygen-coupled citric acid cycle flux, or glycogen or amino acid synthesis. Interestingly, the cells released into the medium significant amounts of the neuroinhibitory amino acids, GABA and glycine, and rapidly cleared the medium of the neuroexcitatory amino acids glutamate and aspartate. Metabolism of [2-¹⁴C]glucose and [³H]acetate by the cells indicated rapid labelling of the glutamate and aspartate pools of the cells by glucose in 1 h, but the relative specific activities of glutamine and GABA were much lower. The metabolism of tracer concentrations of [³H]acetate to glutamate by the cells indicated greater dilution of this isotope compared to that of labelled glucose. However, the ratio of ³H to ¹⁴C radioactivity in glutamate and other amino acids was similar to that in the mixture of glucose and acetate added to the medium. Therefore, some active route of acetate metabolism which communicates metabolically with the route of glucose metabolism to glutamate appears to exist in the cells. Significant acetate activation and fatty acid turnover would explain the present results. Some of the amino acid labelling patterns observed in these studies are not consistent with these glial-like cells behaving as models for the small compartment of amino acid metabolism in brain. Enzyme measurements corroborated the metabolic studies. Glutamate decarboxylase activity was 3–10% of the level found in whole brain. GABA transaminase was also low compared to brain as was glutamine synthetase. Glutamate dehydrogenase was present at levels equal to or higher than those of whole brain.     

Inhibition of Brain Energy Metabolism by the Branched-chain Amino Acids Accumulating in Maple Syrup Urine Disease

In the present work we investigated the in vitro effect of the branched-chain amino acids (BCAA) accumulating in maple syrup urine disease (MSUD) on some parameters of energy metabolism in cerebral cortex of rats. ¹⁴CO₂ production from [1-¹⁴C]acetate, [1-5-¹⁴C]citrate and [U-¹⁴C]glucose, as well as glucose uptake by the brain were evaluated by incubating cortical prisms from 30-day-old rats in the absence (controls) or presence of leucine (Leu), valine (Val) or isoleucine (Ile). All amino acids significantly reduced ¹⁴CO₂ production by around 20–55%, in contrast to glucose utilization, which was significantly increased by up to 90%. Furthermore, Leu significantly inhibited the activity of the respiratory chain complex IV, whereas Val and Ile markedly inhibited complexes II–III, III and IV by up to 40%. We also observed that trolox (α-tocopherol) and creatine totally prevented the inhibitory effects provoked by the BCAA on the respiratory chain complex activities, suggesting that free radicals were involved in these effects. The results indicate that the major metabolites accumulating in MSUD disturb brain aerobic metabolism by compromising the citric acid cycle and the electron flow through the respiratory chain. We presume that these findings may be of relevance to the understanding of the pathophysiology of the neurological dysfunction of MSUD patients.     

Lactate formation by rat small intestine in vitro

The formation of lactic acid by mucosal slices, rings and muscle from rat jejunum has been studied for periods of up to 8 min. Lactate output by mucosal slices incubated in the absence of glucose was characterised by two phases: a rapid, initial phase of release lasting about 1 min, followed by a much slower phase extending over the remainder of the incubation period. Glucose addition at 30 s initiated a second rapid phase of lactate release into the medium which was again followed by a slower rate of lactate output up to 8 min. The time course of lactate output suggested that there was a negative Pasteur effect in mucosal slices, which could not be reversed by the addition of ADP or glucose 6-phosphate. By contrast, the rate of lactate formation by rings and muscle from rat jejunum increased steadily over the incubation period, indicating a positive Pasteur effect. When Na⁺ in the incubating medium were replaced by K⁺, lactate formation by mucosal slices and rings was considerably reduced. Measurements of tissue lactate content before and during incubation revealed that about three-quarters of the lactate released by mucosal slices during the first 30 s of incubation was present initially in the tissue. After the first 30 s the tissue lactate remained constant both in the presence and absence of glucose so that the lactate released into the incubation medium is equivalent to the lactate formed by the slices. The role of the various tissue components of the small intestine in lactate formation is discussed in relation to sites of glucose entry.     

METABOLISM OF GLUCOSE AND GLUTAMATE BY SYNAPTOSOMES FROM MAMMALIAN CEREBRAL CORTEX

—(1) Synaptosomes incubated in high sodium, low potassium media showed high linear respiration in the presence of glucose which was converted into lactate, aspartate, glutamate, glutamine, alanine and GABA during 1 hr incubation periods. (2) Total conversion of glucose into most of these substrates over the incubation period was similar in synaptosomes and cortex slices. Half the lactate and only a small fraction of the glutamine made by slices was formed by synaptosomes. (3) Pool sizes of amino acids in cortex slices after incubation with glucose were, in general, higher than in synaptosomes, glutamate and glutamine being four-fold higher in slices. (4) Most of the amino acids made from glucose by synaptosomes were contained within their structure and not lost to the medium. (5) Glutamate was actively metabolized by synaptosomes to aspartate, glutamine, alanine and GABA. The specific radioactivities of the amino acids (except glutamine) after 1 hr incubation, approached that of the glutamate. (6) Pyridoxal phosphate added to the incubation medium increased GABA production from glutamate but not from glucose.     

Regulation of carbohydrate metabolism in lymphoid tissue. Nature of the endogenous substrates and their contribution to the respiratory fuel of the sliced rat spleen in vitro.

1. Tissue glycogen contributes, maximally, only 10% of the respiratory fuel of the rat spleen slice in the absence of an added carbon source, and makes no significant contribution when glucose (3mM) is added. 2. The reserves of fatty acid in the form of triglyceride (35.5mumol of fatty acid/g dry wt. of tissue) fall by approx. 25% after incubation of spleen slices with or without added glucose for 2h, and , on this basis, account for 32% of the oxidative fuel. 3. In contrast, the total oxidative contribution of fatty acid reserves to the respiratory fuel, determined on the basis of inhibiton of respiration by 2-bromostearate, is 42-52%. This range includes tissue from both starved and well-fed animals and is not significantly altered by the presence of added glycose (3mM). 4. Large quantities of NH3 (31-35mumol//h per g dry wt. of tissue) are produced by spleen slices incubated in the absence of added substrates, and this value is suppressed by approx. 50% on incubation with glucose (3mM). Adenine nucleotide breakdown can account for only 17% of the total ammonia produced. 5. Individual free amino acid concentrations in spleen were determined, both in vivo and in slices before and after 60 min of incubation. Although the total free amino acid pool size increases by 45% during incubation, owing to protein breakdown, the tissue concentrations of aspartate, glutamate, glutamine and alanine do not increase. It is suggested that these amino acids areoxidized in a net sense to CO2 and water with the liberation of free NH3 via transamination reactions, glutaminase, the purine nucleotide cycle and the tricarboxylic acid cycle. 6. It is concluded that the normal endogenous metabolism of sliced rat spleen (43-52% due to lipids, 30% due to amino acids and 10% due to glycogen) is modified by added glycose only to the extent that glycogen oxidation and 50% of the contribtion made by ino acids are suppressed; endogenous lipid metabolism is unaffected.     

Compartmentation of citric acid cycle metabolism in brain: labelling of glutamate, glutamine, aspartate and gaba by several radioactive tracer metabolites

—(1) Compartmentation of the metabolism of amino acids in brain has been studied in slices of cerebral cortex incubated with sodium [1-¹⁴C]acetate, sodium [1-¹⁴C]-bicarbonate, [1-¹⁴C]GABA or l-[1-¹⁴C]glutamate and in samples of brain after injection in vivo of [1-¹⁴C]- or [³H]acetate. (2) The method of treatment of the slices (a) maintained in ice-cold medium prior to incubation; (b) preincubation at 37°C and transfer to fresh medium affected the metabolism of the added, labelled substrate, particularly its labelling of glutamine. (3) The specific activity of glutamine labelled from the above metabolites was greater than that of glutamic acid in experiments of 10–30 minutes duration, whether or not subjected to pretreatment in the cold. (4) Incubation in medium containing 27 mm-K⁺ was associated with a decrease in the relative specific activity (RSA) of glutamine, except for the increase when l-[1-¹⁴C]glutamate was the precursor. (5) The data have been discussed in terms of metabolic compartmentation and their consistency with the concept of the presence in brain of more than one citric acid cycle, one containing the relatively smaller pools of intermediates and associated with synthetic processes; the other containing the relatively larger pools of intermediates and functioning as a homeostatic buffer for energy metabolism.     

Compartmentation and exchangeability of brain amino acids: evidence from studies of transport into tissue slices.

Compartmentation of the free amino acid pool of brain slices was investigated by measuring the approach to isotopic equilibrium between tissue and medium when slices were incubated with traces of radioactive amino acids. Trace quantities were used to minimize the effects of uptake, which could make the detection of slowly equilibrating pools difficult by greatly increasing tissue amino acid levels. Small, sequestered compartments were found. After 2 h in 20 vol of glucose-containing, oxygenated medium, the nonequilibrating compartments for lysine, leucine, tyrosine, histidine, valine, and threonine were 41, 20, 17, 16, 11, and 6% of their final tissue concentrations, respectively. The data for rapidly metabolized, nonessential, amino acids were more difficult to interpret. Considerable mixing of incoming glutamic and aspartic acids with their endogenous pools was observed and tissue glycine reached isotopic equilibrium within 1 h. With higher concentrations of amino acids, equilibration was complete in 30 min with 2 mm glycine in the medium; 83% in 30 min with 2 mm glutamic acid, and 95% in 60 min with 5 mm glutamic acid in the medium. The amino acid composition of protein free extracts of slices and medium was determined. During incubation, despite a large efflux of amino acids into the medium, most tissue amino acids remained close to their initial concentrations. Net increases in essential amino acids were accounted for by the breakdown of 0.7% of total tissue protein during the first hour and 0.3% during the second hour of incubation.     

Viability of some metabolic processes in the isolated toad brain adapted to two osmotic environments

The viability of the isolated toad brain in an aerated Ringer-like medium has been evaluated by the following criteria: 1) amino acid content before and after incubation; 2) accumulation of amino acids in the incubation medium; 3) a comparison of glucose utilization and [U-¹⁴C]glucose metabolism with that occurring in vivo; 4) tissue swelling; and 5) tissue lactate content. On the basis of these criteria, the isolated toad brain, from toads adapted to a fresh-water or a salt-water environment, retains considerable metabolic integrity for at least 2 hr of incubation at 25° C. Specifically, there was no swelling of the tissue, no apparent accumulation of lactate in the tissue, glucose appeared to be utilized at a rate not too different from that calculated for the toad brain in vivo, and the distribution of label from [U-¹⁴C]glucose had an overall pattern which resembled that observed in vivo. The tissue levels of amino acids were generally stable in vitro; however, there was a marked decline in the content of aspartate. The accumulation of amino acids in the medium varied considerably from one amino acid to another. Thus, there was very little net efflux of aspartate, GABA, and glutamate from the tissue but considerable net efflux of glutamine. This efflux of amino acids was greater from brains of hyperosmotically adapted toads than from the brains of toads adapted to fresh water by amounts proportional to their initial tissue contents.     

THE SPONTANEOUS AND ELECTRICALLY EVOKED RELEASE, FROM SLICES OF GUINEA-PIG CEREBRAL CORTEX, OF ENDOGENOUS AMINO ACIDS LABELLED VIA METABOLISM OF d-[U-14C]GLUCOSE

Abstract— In an effort to identify neurotransmitters in slices of guinea-pig cerebral cortex, a study was made of the release of endogenous amino acids which had become labelled via metabolism of d -[U-¹⁴C]glucose. While incorporation of ¹⁴C into endogenous glutamate, aspartate, GABA, alanine and threonine-serine-glutamine (unseparated) was large enough to permit measurement of their release, that into other amino acids was not. In parallel experiments, the release of exogeneous labelled glutamate, aspartate, GABA and α-aminoisobutyrate was examined. Electrical field stimulation evoked a transient increase in the release of all the adequately labelled endogenous amino acids and all the exogenous amino acids. The stimulated ‘increase’ in the release of each of the endogenous ¹⁴C-labelled transmitter candidates (glutamate, aspartate and GABA) was larger than that of any other amino acid (except that of exogenous GABA). When the experiments were performed without the glucose (5 mm ) usually present in the medium bathing the slices, larger amounts of each labelled amino acid were released from the slices than in the presence of glucose. Moreover, the pattern of selective release of the endogenous labelled transmitter candidates was much more pronounced in the absence of glucose. It is likely that in the absence of glucose, release from the tissue was larger because cells in the slice were relatively depolarized and uptake of amino acids into cells was impaired. Because previous evidence suggests that over 90% of glucose consumption occurs in the ‘large metabolic compartment’ which is thought to be composed of neuronal elements, neurons were probably the main site from which the larger release of endogenous ¹⁴C-labelled transmitter candidates was evoked. The exogenous amino acids were probably released from several cellular elements in the slices. It was concluded that the pattern of a selective release of the endogenous labelled transmitter candidates may have been indicative of a transmitter releasing mechanism in nerve terminals.     

Uptake and release of glutamate in cerebral-cortex slices from the rat

1. Cerebral-cortex slices from rat brain, loaded with labelled l-glutamate as a result of aerobic incubation with labelled glucose, lost less than 15% of this glutamate on subsequent incubation in the presence of unlabelled glucose and l-glutamate. This indicates that very little exchange occurs between extracellular l-glutamate and glutamate accumulated in the neurons as a result of glucose metabolism. 2. Slices, loaded with labelled l-glutamate as a result of aerobic incubation in a medium containing unlabelled glucose and labelled l-glutamate, lost more than half of this glutamate on subsequent incubation in the presence of unlabelled l-glutamate. This indicates that exchange occurs between extracellular glutamate and glutamate accumulated in brain slices as a result of its uptake from the incubation medium. 3. Evidence was obtained suggesting that only a part of the glutamate, accumulated in brain slices as a result of its uptake from an incubation medium containing both glucose and l-glutamate, entered the neurons; apparently almost all the rest entered the glia. 4. It is concluded that the slices contain a pool of glutamate, derived from glucose and located in the neurons, which is poorly exchangeable with extracellular glutamate, and another pool of glutamate, derived from extracellular glutamate and located in the glia, which is freely exchangeable with extracellular glutamate.     

Locations of amino acids in brain slices from the rat. Tetrodotoxin-sensitive release of amino acids

1. Amino acids, particularly glutamate, gamma-aminobutyrate, aspartate and glycine, were released from rat brain slices on incubation with protoveratrine (especially in a Ca(2+)-deficient medium) or with ouabain or in the absence of glucose. Release was partially or wholly suppressed by tetrodotoxin. 2. Tetrodotoxin did not affect the release of glutamine under various incubation conditions, nor did protoveratrine accelerate it. 3. Protoveratrine caused an increased rate of formation of glutamine in incubated brain slices. 4. Increased K(+) in the incubation medium caused release of gamma-aminobutyrate, the process being partly suppressed by tetrodotoxin. 5. Incubation of brain slices in a glucose-free medium led to increased production of aspartate and to diminished tissue contents of glutamates, glutamine and glycine. 6. Use of tetrodotoxin to suppress the release of amino acids from neurons in slices caused by the joint action of protoveratrine and ouabain (the latter being added to diminish reuptake of amino acids), it was shown that the major pools of glutamate, aspartate, glycine, serine and probably gamma-aminobutyrate are in the neurons. 7. The major pool of glutamine lies not in the neurons but in the glia. 8. The tricarboxylic cycle inhibitors, fluoroacetate and malonate, exerted different effects on amino acid contents in, and on amino acid release from, brain slices incubated in the presence of protoveratrine. Fluoroacetate (3mm) diminished the content of glutamine, increased that of glutamate and gamma-aminobutyrate and did not affect respiration. Malonate (2mm) diminished aspartate and gamma-aminobutyrate content, suppressed respiration and did not affect glutamine content. It is suggested that malonate acts mainly on the neurons, and that fluoroacetate acts mainly on the glia, at the concentrations quoted. 9. Glutamine was more effective than glutamate as a precursor of gamma-aminobutyrate. 10. It is suggested that glutamate released from neurons is partly taken up by glia and converted there into glutamine. This is returned to the neurons where it is hydrolysed and converted into glutamate and gamma-aminobutyrate.     

GLUCOSE AND AMINO ACID METABOLISM IN OCTOPUS OPTIC AND VERTICAL LOBES IN VITRO

(1) The metabolism of glucose and amino acids in vitro was compared in the rat cerebral cortex and the optic and vertical lobes of the octopus brain. (2) Specific activities and pool sizes of the five amino acids, glutamate, aspartate, glutamine, alanine and γ-aminobutyric acid (GABA), were determined in octopus and rat brain slices after 2 hr incubation with 10 mm -[U-¹⁴C]glucose, 10 mm -L-[U-¹⁴C]glutamate, and 10mm -^L-[U-¹⁴C]glutamate with added 10 mM-glucose. Amino acid pool sizes were similar in rat and octopus brain, with the exception of alanine, which was higher in the octopus. Generally specific activities were from four- to 20-fold higher in rat brain. With [U-¹⁴C]glucose as substrate, specific activities of GABA and glutamate were highest in rat; those of alanine and glutamine highest in octopus brain. With ^L-[U-¹⁴C]glutamate the specific activities of GABA and aspartate were highest in rat, that of aspartate highest and GABA lowest in octopus. The addition of glucose to ^L-[U-¹⁴C]glutamate as substrate had little effect on the specific activities of any of the amino acids. (3) The uptake of some amino acids was determined by incubation with [U-¹⁴C]amino acids for 2 hr, and ¹⁴CO₂ formation was also measured. The amount of label taken up by octopus was uniformly 20-25 per cent of that found for rat brain. The amount of ¹⁴CO₂, however, differed according to the amino acid. Four times as much ¹⁴CO₂ was generated from alanine by octopus optic lobe and twice as much by the vertical lobe than rat cortex, but from glutamate, only 24 per cent in the optic and 15 per cent in the vertical lobe. No ¹⁴CO₂ was generated from [U-¹⁴C]GABA in the octopus, by contrast with the rat. (4) Activity of some of the enzymes involved in amino acid metabolism was determined in homogenates of rat cortex and octopus optic and vertical lobes, with and without activation by Triton X-100. Enzymic activities in the octopus, with the exception of alanine aminotransferase, were lower than in the rat, and glutamate decarboxylase could not be detected in octopus brain, in the absence of detergent.