Herman Bachelard

Trafficking of Amino Acids Between Neurons and Glia In Vivo. Effects of Inhibition of Glial Metabolism by Fluoroacetate

Glial-neuronal interchange of amino acids was studied by 13C nuclear magnetic resonance spectroscopy of brain extracts from fluoroacetate-treated mice that received [1,2-13C]acetate and [1-13C]glucose simultaneously. [13C]Acetate was found to be a specific marker for glial metabolism even with the large doses necessary for nuclear magnetic resonance spectroscopy. Fluoroacetate, 100 mg/kg, blocked the glial, but not the neuronal tricarboxylic acid cycles as seen from the 13C labeling of glutamine, glutamate, and γ-aminobutyric acid. Glutamine, but not citrate, was the only glial metabolite that could account for the transfer of 13C from glia to neurons. Massive glial uptake of transmitter glutamate was indicated by the labeling of glutamine from [1-13C]glucose in fluoroacetate-treated mice. The C-3/C-4 enrichment ratio, which indicates the degree of cycling of label, was higher in glutamine than in glutamate in the presence of fluoroacetate, suggesting that transmitter glutamate (which was converted to glutamine after release) is associated with a tricarboxylic acid cycle that turns more rapidly than the overall cerebral tricarboxylic acid cycle.

Metabolism of [U-13C5] glutamine in cultured astrocytes studied by NMR spectroscopy: first evidence of astrocytic pyruvate recycling.

Abstract: Metabolism of [U‐13C5]glutamine was studied in primary cultures of cerebral cortical astrocytes in the presence or absence of extracellular glutamate. Perchloric acid extracts of the cells as well as redissolved lyophilized media were subjected to nuclear magnetic resonance and mass spectrometry to identify 13C‐labeled metabolites. Label from glutamine was found in glutamate and to a lesser extent in lactate and alanine. In the presence of unlabeled glutamate, label was also observed in aspartate. It could be clearly demonstrated that some [U‐13C5]glutamine is metabolized through the tricarboxylic acid cycle, although to a much smaller extent than previously shown for [U‐13C5]glutamate. Lactate formation from tricarboxylic acid cycle intermediates has previously been demonstrated. It has, however, not been demonstrated that pyruvate, formed from glutamate or glutamine, may reenter the tricarboxylic acid cycle after conversion to acetyl‐CoA. The present work demonstrates that this pathway is active, because [4,5‐13C2]glutamate was observed in astrocytes incubated with [U‐13C5]glutamine in the additional presence of unlabeled glutamate. Furthermore, using mass spectrometry, mono‐labeled alanine, glutamate, and glutamine were detected. This isotopomer could be derived via the action of pyruvate carboxylase using 13CO2 produced within the mitochondria or from labeled intermediates that had stayed in the tricarboxylic acid cycle for more than one turn.

Measurement of human tricarboxylic acid cycle rates during visual activation by 13C magnetic resonance spectroscopy

Measurement by 13C magnetic resonance spectroscopy (MRS) of the incorporation of label from [1‐13C] glucose, initially into C4 of glutamate, allows the regional tricarboxylic acid (TCA) cycle flux (FTCA) to be determined in the human brain. In this study, a direct 13C MRS approach was used at 3T, with NOE enhancement and 1H decoupling with WALTZ16, to determine basal FTCA in six volunteers. The values found in the visual cortex are similar to those reported in previous 13C MRS studies, and consistent with PET measurements of the cerebral metabolic rate for glucose, CMRglc. In two preliminary activation studies using light emitting diode (LED) goggles flashing at 8 Hz, compared to darkness as control, increases in FTCA were found from 0.60 ± 0.10 to 0.94 ± 0.03 μmol/min/g (56%) and from 0.34 ± 0.14 to 0.56 ± 0.07 μmol/min/g (65%). These are upper estimates, but they are similar to the increases in CMRglc reported in PET studies, and strongly suggest, in contrast to these PET studies, that cerebral glucose is metabolized oxidatively, even during intense visual stimulation. This is supported by the observation that very little 13C label is incorporated into C3 lactate, as would be expected if glucose were metabolized anaerobically. There is evidence for incorporation of glucose into cerebral glycogen, but this is a relatively minor component of cerebral glucose metabolism.

NMR Spectroscopy in Neurochemistry

Although major developments have been achieved in applications of NMR spectroscopy to cerebral function and metabolism over the past decade, there seems to have been little recognition of this powerful technique by neuroscientists in general and neurochemists in particular. Thus, the topic is only rarely included in programs of international meetings of societies such as the International Brain Research Organization, the International Society for Neurochemistry, the European Society for Neurochemistry, and regional or national societies, e.g., the American Society for Neurochemistry or the Brain Research Association. Also, a brief scan of the contents pages of associated journals (including this one) reveals how rarely articles on NMR appear. This is surprising because its major potential lies in the ability to study novel aspects of brain metabolism noninvasively, with high resolution and especially with unique chemical specificity. Indeed, there are many intriguing investigations that are only feasible using this technique. The reasons for this relative lack of appreciation to date by the neuroscientific community are reasonably clear. The early major impact of NMR was in the superbly highly resolved “anatomical” pictures produced by ‘H NMR imaging, whereas similar resolution is not yet possible from spectroscopy. NMR spectroscopy suffers, with some exceptions, the major disadvantage of relative insensitivity-molecules that occur in small amounts cannot normally be detected unless indirect approaches are used. Also, research on intermediary metabolism (real biochemistry?) is much less fashionable than it was-the emphasis is now much more on topics such as gene cloning and receptor function. Another likely reason for its slow acceptance as a powerful tool is that biochemists may be daunted by its complex concepts in physics and mathematics, so to exploit it effectively requires close collaboration between biochemists and physicists-othenvise, it can suffer from the image of merely confirming results from simpler techniques (“reinventing the wheel”). However, it is its absolute chemical specificity that provides fascinating novel opportunities to explore biochemical function in the living intact brain, including that of humans. It allows for continuous monitoring of selected metabolites, for following rates of flux through the intermediates of metabolic pathways, and unique possibilities of studying enzyme kinetics. By the use of interleaved spectra of different nuclei, virtually simultaneous measurement of the above with intracellular cations (pH, calcium, magnesium, sodium) can be performed. By exploiting differences in emphasis of metabolism between neurones and glial cells we now have the potential means of investigating metabolic relationships and interdependencies of these cell types. The future benefits for clinical investigations of numerous cerebral disorders are immense. In this review we hope to explain briefly the basic principles of NMR spectroscopy and how it can be applied to research on cerebral function and metabolism. We also intend to highlight its advantages and limitations, with emphasis on the future for human studies.

Landmarks in the Application of 13C-Magnetic Resonance Spectroscopy to Studies of Neuronal/Glial Relationships

The development of the use of carbon isotopes as metabolic tracers is briefly described. 13C-labelled precursors (13CO2, 13CH4) first became available in 1940 and were studied in microorganisms, but their use was limited by very low enrichments and lack of suitable analytical equipment. More success was achieved with 11C and especially 14C, as these radioactive tracers did not need to be highly enriched. Although the stable 13C isotope can be used at a low percentage enrichment in mass spectrometry, its application to magnetic resonance spectroscopy (MRS) requires very highly enriched precursors, due to its low natural abundance and low sensitivity. Despite such limitations, however, the great advantage of 13C-MRS lies in its exquisite chemical specificity, in that labelling of different carbon atoms can be distinguished within the same molecule. Effective exploitation became feasible in the early 1970s with the advent of stable instruments, Fourier transform 13C-MRS, and the availability of highly enriched precursors. Reports of its use in brain research began to appear in the mid-1980s. The applications of 13C isotopomer analysis to research on neuronal/glial relationships are reviewed. The presence of neighbouring 13C-labelled atoms affects the appearance of the resonances (splitting due to C–C coupling), and so allows for unique quantification of rates through different and possibly competing pathways. Isotopomer patterns in resonances labelled from a combination of [1-13C]glucose and [1,2-13C2]acetate have revealed aspects of neuronal/glial metabolic trafficking on depolarization and under hypoxic conditions in vitro. This approach has now been applied to in vivo studies on inhibition of glial metabolism using fluoroacetate. The results confirm the glial specificity of the toxin and demonstrate that it does not affect entry of acetate. When the glial TCA cycle is inhibited, the ability of the glia to participate in the glutamate/glutamine cycle remains unimpaired, in that labelling of glutamine, which can only be derived from neuronal metabolism of glucose, persists. The results also confirmed earlier evidence that part of the GABA transmitter pool is derived from glial glutamine.

Effects of Hypoglycaemia and Hypoxia on the Intracellular pH of Cerebral Tissue as Measured by 31P Nuclear Magnetic Resonance

Abstract: Changes in high‐energy phosphate metabolites and the intracellular pH (pHi) were monitored in cerebral tissue during periods of hypoglycaemia and hypoxia using 31P nuclear magnetic resonance spectroscopy. Superfused brain slices were loaded with deoxyglucose at a concentration shown not to impair cerebral metabolism, and the chemical shift of the resulting 2‐deoxyglucose‐6‐phosphate (DOG6P) peak was used to monitor the pHi. In some experiments with low circulating levels of Pi, the intracellular Pi was visible and indicated a pH identical to that of DOG6P, an observation validating its use as an indicator of pHi in cerebral tissue. The pHi was found to be unchanged during moderate hypoglycaemia; however, mild hypoxia (Po2= 16.4 kPa) and severe hypoglycaemia produced marked reductions from the normal of 7.2 to 6.8 and 7.0, respectively. Hypoglycaemia caused a fall in the level of both phosphocreatine (PCr) and ATP, whereas hypoxia affected PCr alone, as shown previously. However, the fall in pHi was similar during the two insults, thus indicating that the change in pH is not directly linked to lactate production or to the creatine kinase reaction.

Threshold Requirements for Oxygen in the Release of Acetylcholine from, and in the Maintenance of the Energy State in, Rat Brain Synaptosomes Ian R. Park.

Abstract: An oxystat was designed to enable maintenance of very low, predetermined oxygen tensions (below 1 μM) in incubated suspensions of synaptosomes. The oxygen thresholds for the energy state (ATP and creatine phosphate levels), for lactate production, and for acetylcholine release were compared. The approximate thresholds (μM O2) in veratridine‐stimulated preparations were: oxygen consumption, 10; ATP, 10; creatine phosphate, 15; lactate release, 20; and acetylcholine release, 25. The results for release of total acetylcholine and of the acetylcholine newly synthesized from [14C]glucose were indistinguishable. The results from this study are discussed in relation to hypoxia and to reported in vivo observations.

Neither Moderate Hypoxia nor Mild Hypoglycaemia Alone Causes Any Significant Increase in Cerebral [Ca2±i: Only a Combination of the Two Insults Has This Effect.: A 31P and 19F NMR Study

(1) The energy state and free intracellular calcium concentration ([Ca2±i) of super‐fused cortical slices were measured in moderate hypoxia (∼65 μM O2), in mild hypoglycaemia (0.5 mM glucose), and in combinations of the two insults using 19F and 31P NMR spectroscopy. (2) Neither hypoxia nor hypoglycaemia alone caused any significant change in [Ca2±i. Hypoxia caused a 40% fall in phosphocreatine (PCr) content but not in ATP level, and hypoglycaemia produced a slight fall in both (as expected from previous studies). These changes in the energy state recovered on return to control conditions. (3) A combined sequential insult (hypoxia, followed by hypoxia plus hypoglycaemia) produced a 100% increase in [Ca2±, and a decrease in PCr level to ∼25% of control. The reverse combined sequential insult (hypoglycaemia, followed by hypoglycaemia plus hypoxia) had the same effect. On return to control conditions there was some decrease in [Ca2±i and a small increase in PCr content, but neither recovered to control levels. (4) Exposure of the tissue to the combined simultaneous insult (hypoxia plus hypoglycaemia) immediately after the control spectra had been recorded resulted in a fivefold increase in [Ca2±i and a similar decrease in PCr level to 20–25% of control. There was little if any change of [Ca2±i or PCr level on return to control conditions. (5) These results are discussed in terms of metabolic adaptation of some but not all of the cortical cells to the single type of insult, which renders the tissues less vulnerable to the combined insult.

The regulation of intracellular pH studied by 31P- and 1H-NMR spectroscopy in superfused guinea-pig cerebral cortex slices.

(1) The intracellular pH (pHi) of superfused slices of guinea-pig cerebral cortex was measured in 31P-NMR spectra using the chemical shifts of intracellular inorganic phosphate (Pi) and of 2-deoxyglucose 6-phosphate (DOG6P). The pHi was found to be 7.30 +/- 0.04 (SD, n = 15) in bicarbonate-buffered medium and 7.20 +/- 0.05 (n = 10, P < 0.001) in bicarbonate-free HEPES buffer of the same pH (7.4). (2) Decreases in pHe below 7.05 resulted in pHi falling to similar values, with a decrease in the energy state. There was no change in intracellular lactate as assessed by 1H-NMR. (3) The tissues showed an ability to buffer higher pH: increasing pHe to 8.0 had no effect on pHi, PCr or lactate. (4) In order to characterize possible mechanisms of pH regulation in the tissue, the recovery from acid insult was investigated under various conditions. Initially pHi was decreased to 6.44 +/- 0.15 (n = 15) by exposure to media containing 6 mM bicarbonate gassed with O2/CO2, 80:20 (pHe 6.4). When this medium was replaced by normal bicarbonate buffer (pH 7.4) there was full recovery of pHi to 7.31 +/- 0.05 (n = 15), whereas replacing the buffer with HEPES resulted in incomplete recovery of pHi to 6.88 +/- 0.15 (n = 15, P < 0.001). (5) In the presence of the carbonic anhydrase inhibitor, acetazolamide (1 mM), or the sodium/proton exchange inhibitor, amiloride (1 mM), there was an incomplete return of pHi to the control value (pHi 6.90 +/- 0.20, n = 5, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ammonium on energy metabolism and intracellular pH in guinea pig cerebral cortex studied by 31P and 1H nuclear magnetic resonance spectroscopy

Abstract 31 P and 1 H nuclear magnetic resonance spectroscopy were used to study the effects of ammonium on high-energy phosphates, intracellular pH and lactate in guinea pig cerebral cortex in vitro . In the presence of glucose, 1 mM ammonium caused an intracellular acidification by 0.2–0.3 pH units without a change in phosphocreatine/ATP (PCr/ATP) ratio, lactate concentration or oxygen uptake. At concentrations of 5 mM or greater, NH 4 + caused an energy failure and an increase in tissue lactate, together with a drop in intracellular pH. A split in the inorganic phosphate resonance was observed during the exposure to both 20 mM NH 4 + and 20 mM K + indicating heterogeneity of the volume-averaged intracellular pH. Cortical brain slices incubated in the presence of 10 mM lactate maintained PCr/ATP ratio and intracellular pH at similar levels as in the presence of glucose, but 1 mM NH 4 + caused a fall in PCr/ATP. Both 20 mM NH 4 + and 20 mM K + stimulated oxygen uptake of the preparation with glucose or lactate as substrate. These results show that the only acute effect of 1 mM NH 4 + in the presence of glucose is an intracellular acidification whereas energetic consequences develop at high levels of this neurotoxic agent.