Effect of hypoxia on the incorporation of [2-3H]glycerol and [1-14C]-palmitate into lipids of various brain regions

The lipid metabolism in guinea pig brain after intermittent hypoxia, prolonged for 80 hrs, was markedly impaired. The in vivo incorporation of [2-³H]glycerol and [1-¹⁴C]palmitate into lipids of microsomes, mitochondria, myelin, and synaptosomes, purified from cerebral hemispheres, was significantly lower in the hypoxic animals than in the controls. The same effect was observed on the incorporation of labeled precursors into lipids of mitochondria purified from cerebellum and brainstem. In particular, the labeling of the major phospholipids present – ie, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) – in the mitochondria of the three brain regions examined decreased after hypoxic treatment.     

Changes in enzyme activities of glycerolipid metabolism of guinea-pig cerebral hemispheres during experimental hypoxia

Hypoxic treatment causes changes of some enzymatic activities involved in the glycerolipid metabolism in subcellular fractions of guinea pig cerebral hemispheres. The activity of lysophosphatidylcholine acyltransferase, choline phosphotransferase, glycerol-3-phosphate acyltransferase(s), as well as the activity of triacylglycerol lipase significantly decreased in the microsomes of cerebral hemispheres of animals intermittently exposed to hypoxic treatment for eighty hours. At the same time, a marked activation of microsomal and mitochondrial phospholipase A₂ occurred. The changes of the above-mentioned enzymatic activities after the hypoxic treatment correlated well with the increase in the level of brain and blood free fatty acids. The changes also correlated with the decrease of labeled lipid precursors incorporated into lipids of the cerebral hemispheres, observed during oxygen insufficiency.     

RNA synthesis and processing in the gerbil brain after transient hindbrain ischaemia

Ribonucleic acid (RNA) synthesis was investigated in gerbils subjected to 15 min transient hindbrain ischaemia using [2-¹⁴C]uridine autoradiography. Distribution of synthesized RNA in the subcellular fraction of the tissue was detected by differential centrifugation and density gradient separation using Whittaker's method. In [2-¹⁴C]uridine autoradiography, uptake of the tracer into the RNA fraction was not reduced after transient ischaemia. Distributional analysis of [2-¹⁴C]uridine in the subcellular fractions revealed that tracer activity in the P₃ (microsomes) fraction decreased in the ischaemic regions and tended to decrease in the P₄ (ribosomes) fraction, although not significantly. Tracer activity in the P₁ (nuclei and cell debris) and P₂ (mitochondria, myelin and nerve ending particles) fractions did not decrease. These results indicate that RNA synthesis in the nuclei is not inhibited by ischaemia, but RNA processing is disturbed by the level of the transport. Modification of RNA synthesis and processing by transient ischaemia may influence protein synthesis.     

RNA metabolism in subcellular fractions from rat cerebral cortex and from the visual cortex of rats with retinal degeneration

The metabolism of RNA in subcellular fractions from rat cerebral cortex was studied. The following fractions were prepared initially: nuclei (purified through hypertonic sucrose), myelin, mitochondria, synaptosomes, microsomes, and supernatant. The proportion of RNA in each fraction was approximately 10, 3.4, 9.5, 10.1, 33.5, and 24.9%, respectively. This contrasted with the distribution of protein which was 4, 4.4, 20.1, 13.8, 19.5, and 26.5%, respectively. DNA was primarily nuclear but 1–2% was in the mitochondrial fraction. The synaptosomes were further fractionated by osmotic shock and gradient centrifugation and it was found that over 40% of the RNA was associated with the synaptosomal mitochondria and the bulk of the remainder associated with the membrane fraction (characterized by their high Na⁺-K⁺-ATPase, glutamine synthetase, and p-nitrophenyl phosphatase activity). Labeling studies using [6-¹⁴C] orotic acid revealed that the nuclear fraction was the most rapidly labeled, reaching a maximum RNA specific activity within 2 hr and decreasing thereafter. The cytoplasmic fractions all reached a maximum specific activity within 48 hr and had the same specific activity as the nuclear fraction α after 72 hr. The supernatant fraction was the most rapidly labeled of the cytoplasmic fractions, the synaptosome fraction the least. However there was no delay in the labeling of the synaptosome fraction. Initially, labeled synaptosomal RNA was labeled in the synaptosomal supernatant but after 48 hr the bulk of the label was associated with the RNA in the synaptosomal membranes. When the nuclear fraction was further separated into its neuronal and glial components it was found that the incorporation of [¹⁴C] orotic acid was approximately three times greater in the neuronal fraction after 2–4 hr but difference was apparent before 0.5 hr or after 24 hr. The metabolism of RNA in the subcellular fractions of the visual cortex of blind and sighted rats was found to differ significantly, the former having a slower rate of transfer of RNA from nucleus to cytoplasm. The difference in the incorporation rates of [¹⁴C] orotic acid into neuronal and glial nuclei was also less in the blind visual cortex. The RNA metabolism in the subcellular fractions of the frontal cortex of blind and sighted rats did not differ.     

Pool size of CDP-choline in the brain,heart, and lung of normal and hypoxic guinea pigs

The pool size of CDP-choline in the brain, heart, and lung of normal and hypoxic animals was measured by radioactive isotopic dilution. The effect of exogenous CDP-choline administration on the pool size in these experimental groups was also examined. The hypoxic treatment caused a significant decrease of endogenous CDP-choline in the brain and lung only; no significant effect was observed in the examined organs after CDP-choline treatment.     

Hormonal regulation of brain development. VI. Kinetic studies of the incorporation in vivo of ( 3 H)orotic acid into RNA of brain subcellular fractions of 10-day-old normal and hypothyroid rats

The time course incorporation of the precursor into RNA from brain subcellular fractions has been studied after the intra-arachnoidal injection of [5-³H]orotic acid in 10-day-old normal and neonatally radio-thyroidectomized rats.     

Hereditary protein C deficiency associated with riboflavin-responsive lipid storage myopathy

We report a 14 year old patient who presented lung emboli and deep vein thrombosis in relationship to protein C deficiency. He had a carnitine-deficient lipid myopathy. Fresh muscle homogenate showed low activities in oxidizing [1-¹⁴C]-butyrate, [1-¹⁴C]-octonoate and [1-¹⁴C]-palmitate. A deficient short chain butyryl-CoA dehydrogenase (SCAD) was found in isolated muscle mitochondria. The patient improved dramatically with daily therapy of 200 mg riboflavin, 2 g carnitine and anticoagulation with Coumadin. The treatment was found to restore fatty acid oxidation in fresh muscle homogenate, deficient acylCoA-dehydrogenases in mitochondria and decrease lipid droplets. These results suggest that in this type of lipid myopathy riboflavin supplementation may be effective. The link with protein C deficiency is discussed.     

Effects of CDP-choline on neurologic deficits and cerebral glucose metabolism in a rat model of cerebral ischemia

The effects of cytidine 5'-diphosphocholine (CDP-choline) on neurologic deficits and cerebral glucose metabolism were studied in a rat model of transient cerebral ischemia. Cerebral ischemia was induced by occluding both common carotid arteries for 20 or 30 minutes 24 hours after the vertebral arteries were permanently occluded by electrocautery. CDP-choline was administered intraperitoneally twice daily for 4 days after reestablishing carotid blood flow. CDP-choline at two dosages (50 and 250 mg/kg) shortened the time required for recovery of spontaneous motor activity in a dose-related manner; recovery time was measured early after reperfusion. Neurologic signs were observed for 10 days. High-dose CDP-choline improved neurologic signs in the rats within 20-30 minutes of ischemia. When cerebral glucose metabolism was assessed on Day 4, increases in the levels of glucose and pyruvate were accompanied by decreases in the synthesis of labeled acetylcholine from uniformly labeled [14C]glucose measured in the cerebral cortex of rats with 30 minutes of ischemia. High-dose CDP-choline also attenuated changes in these variables. CDP-[1,2-14C]choline injected intravenously 10 minutes after reperfusion was used for membrane lipid biosynthesis. These results indicate that CDP-choline has beneficial effects on brain dysfunction induced by cerebral ischemia, which may be due in part to the restorative effects of CDP-choline on disturbed cerebral glucose metabolism, probably by stimulating phospholipid biosynthesis.     

Effect of inhibition of β-oxidation on incorporation of [U-C]palmitate and [1-C]arachidonate into brain lipids

The purpose of the present study was to determine the effect of inhibiting the mitochondrial β-oxidation of free fatty acids on the incorporation of radiolabeled free fatty acids into brain lipids. To this end, methyl 2-tetradecylglycidate (MEP), an irreversible inhibitor of carnitine palmitoyltransferase I, was given orally to male rats 2, 4, and 6 h prior to an intravenous infusion of the saturated fatty acid [U-¹⁴C]palmitic acid (PA) or the polyunsaturated fatty acid [1-¹⁴C]arachidonate (AA). With [U-¹⁴C]PA, MEP (10–25 mg/kg) increased brain organic radioactivity 2-fold and decreased brain aqueous radioactivity 3- to 5-fold relative to control values at all pretreatment times. The effect was due to prolongation of the plasma integral of [U-¹⁴C]PA due to peripheral inhibition of β-oxidation, and to direct inhibition of β-oxidation of the tracer within the brain. MEP had no effect on brain organic radioactivity after infusion of [1-¹⁴C]AA. Increasing the interval between MEP administration and [U-¹⁴C]PA infusion from 2 to 6 h resulted in a dramatic redistribution of [U-¹⁴C]PA within brain lipids. The percentage of radioactivity in phospholipids decreased from 65 to 33%, whereas that in the free fatty acid fraction increased from 10 to 47% and that in triglycerides was elevated 2–3 fold over control levels. These results indicate that MEP may facilitate the use of radiolabeled PA as an in vivo probe of brain lipid metabolism using quantitative autoradiography or position emission tomography.     

Dose response of ethanol ingestion on antioxidant defense system in rat brain subcellular fractions

This study investigated the response of the antioxidant defense system in brain subcellular fractions after oral graded doses of ethanol to rat. Four groups of male Fischer-344 rats were orally administered saline, ethanol 2 g, 4 g, and 6 g/kg, respectively, and sacrificed 1 hour post treatment. Brain cytosol, synaptosomes, microsomes and mitochondria were separated by density gradient differential centrifugation and assayed for antioxidant system. A significant and dose-dependent-decrease in superoxide dismutase (SOD) activity was observed in all brain subcellular fractions. Catalase (CAT) activity was significantly decreased in brain mitochondria (67% and 80% of control) at higher doses of ethanol; whereas, CAT activity was significantly increased in cytosol, synaptosomes and microsomes. Glutathione peroxidase (GSH-Px) activity was significantly increased in all brain subcellular fractions except in cytosol at higher dose of ethanol. Malondialdehyde (MDA) content was significantly increased in all brain subcellular fractions showing dose response of ethanol-induced oxidative stress. The increase in MDA levels in the brain synaptosomes and microsomes were higher at 6 g dose of ethanol (155% and 163% of control) when compared to mitochondria and cytosol. Glutathione (GSH) levels were significantly increased in brain cytosol and microsomes at higher dose of ethanol (164% and 159% of control); whereas, the GSH concentration was significantly decreased in brain synaptosomes and mitochondria. The antioxidant enzyme (AOE) activity ratios (GSH-Px/SOD and GSH-Px + CAT/SOD) were dose dependently increased in all brain subcellular fractions, particularly in synaptosomes. The GSH/GSSG ratio was dose dependently increased in brain microsomes. The perturbations in the antioxidant defense system and enhanced lipid peroxidation following graded doses of ethanol ingestion indicate a dose-dependent-oxidative 2133stress response in brain subcellular compartments of rats.     

Protein pattern alterations in hippocampal and cortical cells as a function of training in rats

This study reports changes in the protein pattern and incorporation of l-[U-¹⁴C]-leucine in brain cells of the hippocampus and sensorimotor cortex of rats. The following subcellular fractions were analyzed by SDS-acrylamide gel electrophoresis: plasma membranes, synaptosomal membranes and synaptic mitochondria.Recurring reversal training gave an increased synthesis of synaptosomal membrane proteins with mol. wt. 35,000–45,000 and 60,000 and 100,000 in trained animals compared to active controls. Lesser changes were observed in plasma membrane and synaptic mitochondria fractions. Of the brain areas studied, the hippocampal synaptosomal fraction showed an initial, temporary response, and the cortical cell fractions responded subsequently. Judged from the time sequence of the protein response, it seems that recurrent reversal training induces a change in synaptic protein towards higher molecular weights, suggesting that these changes reflect a modification of the distribution of synaptic protein.     

Glycerol oxidation in rat brain: Subcellular localization and kinetic characteristics

The oxidation of [1,3-¹⁴C]glycerol to ¹⁴CO was measured in slices, whole homogenates, and subcellular fractions of rat brain. In all of these tissue preparations, the Lineweaver-Burk plots of glycerol oxidation were biphasic, yielding two apparent K_m and V values. Similar kinetic characteristics were obtained with brain homogenates from guinea pig, mouse, rabbit, monkey, and pig. In other tissues of the rat, including heart, kidney, liver, and skeletal muscle, the Lineweaver-Burk plots for glycerol oxidation were not biphasic but were linear. Heating the brain homogenates for five minutes at 5°C caused a 50% decrease in the rate of oxidation of glycerol without a change in the biphasic double reciprocal plot. The addition of purified glycerol kinase (EC 2.7.1.30) to the homogenate caused an increase in the rate of oxidation and resulted in a linear Lineweaver-Burk plot. Brain mitochondria were prepared by two different methods, both of which yielded an enrichment of glycerol oxidation. In contrast, the rate of glucose oxidation was higher in homogenates than in mitochondria, and glucose competed with glycerol as substrate only extramitochondrially. The effects of various metabolic inhibitors suggested the participation of intact, coupled mitochondria, of glycolytic enzymes, and of electron transport in the oxidation of glycerol. The data support the primary localization of glycerol oxidation in nonsynaptic mitochondria in brain and the presence in that organelle of enzymes of the Embden-Meyerhoff pathway or an as yet unidentified system for oxidizing this compound.     

Imaging cerebral 2-ketoisocaproate metabolism with hyperpolarized (13)C magnetic resonance spectroscopic imaging

The branched chain amino acid transaminase (BCAT) has an important role in nitrogen shuttling and glutamate metabolism in the brain. The purpose of this study was to describe the cerebral distribution and metabolism of hyperpolarized 2-keto[1-¹³C]isocaproate (KIC) in the normal rat using magnetic resonance modalities. Hyperpolarized KIC is metabolized to [1-¹³C]leucine (leucine) by BCAT. The results show that KIC and its metabolic product, leucine, are present at imageable quantities 20 seconds after end of KIC administration throughout the brain. Further, significantly higher metabolism was observed in hippocampal regions compared with the muscle tissue. In conclusion, the cerebral metabolism of hyperpolarized KIC is imaged and hyperpolarized KIC may be a promising substrate for evaluation of cerebral BCAT activity in conjunction with neurodegenerative disease.     

Delayed post-ischemic administration of CDP-choline increases EAAT2 association to lipid rafts and affords neuroprotection in experimental stroke

Glutamate transport is the only mechanism for maintaining extracellular glutamate concentrations below excitotoxic levels. Among glutamate transporters, EAAT2 is responsible for up to 90% of all glutamate transport and has been reported to be associated to lipid rafts. In this context, we have recently shown that CDP-choline induces EAAT2 translocation to the membrane. Since CDP-choline preserves membrane stability by recovering levels of sphingomyelin, a glycosphingolipid present in lipid rafts, we have decided to investigate whether CDP-choline increases association of EAAT2 transporter to lipid rafts. Flotillin-1 was used as a marker of lipid rafts due to its known association to these microdomains. After gradient centrifugation, we have found that flotillin-1 appears mainly in fractions 2 and 3 and that EAAT2 protein is predominantly found colocalised with flotillin-1 in fraction 2. We have also demonstrated that CDP-choline increased EAAT2 levels in fraction 2 at both times examined (3 and 6 h after 1 g/kg CDP-choline administration). In agreement with this, [(3)H] glutamate uptake was also increased in flotillin-associated vesicles obtained from brain homogenates of animals treated with CDP-choline. Exposure to middle cerebral artery occlusion also increased EAAT2 levels in lipid rafts, an effect which was further enhanced in those animals receiving 2 g/kg CDP-choline 4 h after the occlusion. Infarct volume measured at 48 h after ischemia showed a reduction in the group treated with CDP-choline 4 h after occlusion. In summary, we have demonstrated that CDP-choline redistributes EAAT2 to lipid raft microdomains and improves glutamate uptake. This effect is also found after experimental stroke, when CDP-choline is administered 4 h after the ischemic occlusion. Since we have also shown that this delayed post-ischemic administration of CDP-choline induces a potent neuroprotection, our data provides a novel target for neuroprotection in stroke.     

Effect of in vitro ischemic or hypoxic treatment on mitochondrial electron transfer activity in rat brain slices assessed by gas–tissue autoradiography using [O] molecular oxygen

We have investigated the effect of in vitro ischemic or hypoxic treatment on mitochondrial electron transport function in brain slices using gas–tissue autoradiography technique with [¹⁵O]O₂. Brain slices were preincubated in Krebs-Ringer phosphate medium bubbled with 100% O₂ for 30 min at 37°C. (1) Control culture was incubated in the same medium bubbled with 100% O₂ for 5–40 min at 37°C, then for another 30 min under the same conditions. (2) In vitro ischemia was induced by placing the culture in the medium deprived of glucose and bubbled with 100% N₂ for 5–40 min, then returning it to control conditions and culturing for another 30 min. (3) In vitro hypoxia was induced by placing the culture in the medium with glucose and bubbled with 100% N₂ for 5–40 min, then returning it to the control conditions for 30 min. After the three different treatments, the [¹⁵O]O₂ fixation by brain slices reflect to mitochondrial electron transport function was determined using gas–tissue autoradiography technique with [¹⁵O]O₂. The fixation of [¹⁵O]O₂ by striatum, cerebral cortex and hippocampus was reduced dependent upon the period of in vitro ischemic treatment. In contrast, the [¹⁵O]O₂ fixation by those brain regions was only slightly reduced by hypoxia treatment. The reduction in [¹⁵O]O₂ fixation induced by ischemic treatment was prevented by an antioxidant: glutathione, glutathione monoethyl ester or acetylsalicylic acid. The preventive effect of antioxidants on the mitochondrial damage induced by ischemia was more remarkable in the striatum than in the cerebral cortex and hippocampus. In the comparison of [¹⁵O]O₂ fixation between ischemia-treated young and senescent brain slices, reduction of ¹⁵O fixation by every brain region examined was more prominent in senescence than in the young. These results suggest that gas–tissue autoradiography using [¹⁵O]O₂ is useful to assess mitochondrial electron transport dysfunction induced by ischemia treatment in brain slices and that the oxidative stress participates in the mechanism of ischemia-induced dysfunction in mitochondria.     

Sigma-1 and Sigma-2 sites in rat brain: Comparison of regional,ontogenetic, and subcellular patterns

Radioligand binding assay conditions were established for the selective labeling of sigma-1 and sigma-2 sites in membrane homogenates of rat brain. Selective sigma-1 assays were conducted using 5 nM (+)[³H]SKF-10,047 in the presence of 300 nM dizocilpine (MK-801). Selective sigma-2 assays were conducted using 5 nM [³H]DTG in the presence of 1 μM (+)SKF-10,047. Distributions of sigma-1 and sigma-2 binding among brain regions were found to differ. While the brain stem yields the highest level of sigma-1 binding, it yields among the lowest levels of sigma-2 binding. The reverse is true in hippocampal membranes. Different ontogenetic patterns were also observed. Sigma-2 binding decreases substantially during brain development, whereas sigma-1 binding does not vary significantly. Patterns of distribution among subcellular fractions of rat brain homogenates were found to be similar. Both sigma-1 and sigma-2 sites are most enriched in microsomal fractions, and neither is enriched in synaptosomal or mitochondrial fractions. The present results suggest that sigma-1 and sigma-2 sites are distinct entities; they do not appear to be located on a common macromolecule, and they do not represent two different affinity states of a single type of binding site. While the precise subcellular locations (if sigma-1 and sigma-2) sites remain to be determined, we conclude that localization of either type of binding site to synaptic regions of plasma membrane or to mitochondria is highly unlikely © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such is in the public domain in the United States of America.

     

In Vivo and In Vitro Effects of Phenytoin (PHT) on ATPases and [14C]-PHT Binding in Synaptosomes and Mitochondria from Rat Cerebral Cortex

Summary: The effect of phenytoin (PHT) on Na⁺-K⁺-ATPase and Mg²⁺-ATPase activities and on [¹⁴C]-PHT binding in vitro to synaptosomal and mitochondrial sub cellular fractions from rat cerebral cortex was studied after chronic PHT treatment. Synaptosomal and mitochondrial fractions were characterized with plasma membrane and mitochondrial enzymatic markers. Synaptoso-mal Na⁺-K⁺-ATPase was not affected in vitro by PHT 1–200 μM or by chronic treatment with 2–50 mg/kg/day of the unlabeled drug for 8 days. Mitochondria1 Mg²⁺-ATPase was significantly stimulated by PHT after chronic treatment with 5 mg/kg/day for 8 days; reaching maximal effect (76%), at 10–25 mg/kg. PHT had no effect on mitochondrial Mg²⁺-ATPase when added in vitro. [¹⁴C]-PHT binding in vitro to the subcellular fractions was determined by dialysis to assess in vivo binding of the unlabeled PHT during chronic treatment. Indeed, [¹⁴C]-PHT bound to synaptosomes was significantly reduced by chronic PHT treatment from 218 ±10 to 119 & 11 pmol/mg protein after 1 week of treatment; a similar effect was obtained after 2–3 weeks with 10 mg/kg/day. Mitochondrial fraction bound 117 ±10 pmol/mg protein labeled PHT. Chronic treatment with unlabeled PHT also reduced the amount of [¹⁴C]-PHT bound to 19.9 ± 2.2 pmol/mg protein. These results show slow reversible PHT in vivo binding to synaptosomes and mitochondrias from rat cerebral cortex, supporting the idea that the modulatory action of PHT on Na+ and Ca2+ permeabilities are mediated through these slow reversible binding proteins. The data also suggest a possible role of intra synaptosomal mitochondria in [Ca²⁺]_i buffering.     

Enhancement of free fatty acid incorporation into phospholipids by choline plus cytidine

Cytidine and choline, present in cytidine 5'-diphosphate choline (CDP-choline), are major precursors of the phosphatidylcholine found in cell membranes and important regulatory elements in phosphatide biosynthesis. Administration of CDP-choline to rats increases blood and brain cytidine and choline levels; this enhances the production of endogenous CDP-choline which then combines with fatty acids (as diacylglycerol), to yield phosphatidylcholine. We examined the effect of providing cytidine and choline on incorporation of free fatty acids into phosphatidylcholine and other major phospholipids in PC12 cells. Addition of equimolar cytidine and choline (100-500 microM) to [3H]-arachidonic acid (50 microM, 0.2 microCi, bound to bovine serum albumin) dose-dependently increased the accumulations of [3H]-phosphatidylcholine (PtdCho), [3H]-phosphatidylinositol (PtdIno) and [3H]-phosphatidylethanolamine (PtdEtn) (by up to 27+/-3%, 16+/-3% and 11+/-3%, respectively, means+/-S.E.M.). This effect was seen with 8-18 h of incubation. The incorporation of [3H]-oleic acid into [3H]-PtdCho was even more enhanced (by up to 42+/-3%) as were the incorporations of [14C]-choline and [3H]-glycerol. The effects of choline and cytidine were enhanced by 12-O-tetradecanoylphorbol-13-acetate (TPA, 1 microM), which activates CTP:phosphocholine cytidylyltransferase (CT) and facilitates choline uptake. Replacing choline by ethanolamine also enhanced the incorporation of [3H]-arachidonic acid into [3H]-PtdEtn, [3H]-PtdIno and [3H]-PtdCho. Arachidonic acid (10-200 microM) alone failed to affect the incorporation of [14C]-choline into phosphatidylcholine. We suggest that the increases in phospholipid synthesis caused by concurrent cytidine and choline supplementation enhance the incorporation of arachidonic acid and certain other fatty acids into the major glycerophospholipids. Removing these fatty acids as source of potentially toxic oxidation products could contribute to the beneficial effects of CDP-choline in treating stroke or other brain damage.     

Biosynthesis of DNA and RNA in neuronal and glial cells from various regions of developing rat brain

Slices of cerebral hemispheres, brain stem, and cerebellum from rats 5–30 days old were used for in vitro incorporation of [methyl-³ H] thymidine and [6-¹⁴ C] orotic acid into DNA and RNA, respectively. The rates of DNA and RNA synthesis decreased markedly during development, with the most marked decrease observed for DNA. The different brain regions showed specific patterns of decline of DNA and RNA synthesis. Following incubation of slices, the tissues were fractionated to obtain fractions enriched in neuronal cells and in glial cells. In cerebellum, the granule neurons were separated from the Purkinje neurons. The glial: neuronal ratio of DNA specific activity was different in the three regions examined: in cortex it decreased from 6 at 10 days to 3 at 20–30 days; in brain stem it was 3 throughout 10–30 days; in the cerebellum (glia:granule neuron ratio) it was also 3 at 30 days but only 0.3 at 10 days. Concerning the RNA incorporation, small differences were found between neuronal and glial cells.     

Detection of reduced GABA synthesis following inhibition of GABA transaminase using in vivo magnetic resonance signal of [C]GABA C1

Previous in vivo magnetic resonance spectroscopy (MRS) studies of gamma-aminobutyric acid (GABA) synthesis have relied on ¹³C label incorporation into GABA C2 from [1-¹³C] or [1,6-¹³C₂]glucose. In this study, the [¹³C]GABA C1 signal at 182.3 ppm in the carboxylic/amide spectral region of localized in vivo ¹³C spectra was detected. GABA-transaminase of rat brain was inhibited by administration of gabaculine after pre-labeling of GABA C1 and its metabolic precursors with exogenous [2,5-¹³C₂]glucose. A subsequent isotope chase experiment was performed by infusing unlabeled glucose, which revealed a markedly slow change in the labeling of GABA C1 accompanying the blockade of the GABA shunt. This slow labeling of GABA at elevated GABA concentration was attributed to the relatively small intercompartmental GABA-glutamine cycling flux that constitutes the main route of ¹³C label loss during the isotope chase. Because this study showed that using low RF power broadband stochastic proton decoupling is feasible at very high field strength, it has important implications for the development of carboxylic/amide ¹³C MRS methods to study brain metabolism and neurotransmission in human subjects at high magnetic fields.