NEUROSCIENCE:
Let There Be (NADH) Light

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Oxidation of glucose is thought to provide almost all of the energy needed by neurons to support brain activity. Indeed, glucose consumption is tightly linked to neuronal activity in the brain. For decades, these two principles have driven the study of brain energy metabolism and its relation to brain activity. They also provide the foundation for a brain imaging method called 2-deoxyglucose autoradiography that maps regional brain activation as animals undertake different tasks. This method, developed by Sokoloff (1), has been used in combination with another imaging technique, positron emission tomography (PET), to study brain activation under different conditions in human subjects. However, the traditional view that glucose is consumed directly and solely by neurons and that glucose consumption directly reflects neuronal activity is under challenge. In vitro, ex vivo, and in vivo experiments have shown that astrocytes, a type of glial cell in the central nervous system, respond to neuronal activity mediated by the neurotransmitter glutamate by consuming more glucose as well as producing more lactate (2). In parallel, neurons preferentially oxidize lactate present in the extracellular space rather than glucose to meet their energy demands (see the figure). The overall process has been designated the astrocyte-neuron lactate shuttle hypothesis (3). Despite growing interest in this hypothesis, there has not been a clear demonstration that activation of glycolysis (anaerobic glucose metabolism) in the cytoplasm and of oxidative phosphorylation (production of ATP) in the mitochondria are effectively segregated between astrocytes and neurons. On page 99 of this issue (4), Kasischke and co-workers now provide illuminating evidence to support this view.

Brain energetics in the limelight. Separate activation of oxidative phosphorylation (respiration) in neurons (brown) and glycolysis in astrocytes (gray), as revealed by two-photon fluorescence imaging of NADH (4). (1) Stimulation of excitatory (glutamatergic) neurons activates postsynaptic AMPA receptors and induces an excitatory postsynaptic potential (EPSP) in the dendritic spine of the neuron. (2) The depolarization propagates from the dendritic spine to the dendrite, where it may cause further opening of voltage-gated sodium channels and activation of the Na+/K+ ATPase, leading to an increased demand for energy (ATP). (3) In response, oxidative phosphorylation is rapidly activated, causing a decrease in mitochondrial NADH content (the so-called “dip” in the fluorescent signal). (4) Recovery of mitochondrial NADH in dendrites is accomplished by stimulation of the TCA cycle, fueled largely by lactate from the extracellular pool. (5) In parallel, but delayed in time, glutamate reuptake in astrocytes (gray) activates the glial Na+/K+ ATPase. (6) The increased energy demand leads to a strong enhancement of glycolysis in the cytoplasm of astrocytes, as indicated by the large increase in cytosolic NADH fluorescence (the so-called “overshoot”). (7) To maintain the high glycolytic flux, NAD+ must be regenerated via the conversion of pyruvate to lactate through the activity of the enzyme lactate dehydrogenase. Release of lactate into the extracellular space not only replenishes the extracellular pool, but also may sustain the late phase of neuronal activation. In vivo, glucose is delivered from the blood to both the extracellular space and to astrocytes (via astrocytic protrusions called end-feet that are in close contact with the blood vessel wall). AMPAR, -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors; GLU, glutamate; LAC, lactate; PYR, pyruvate; TCA, tricarboxylic acid.CREDIT: LUC PELLERIN AND KATHARINE SUTLIFF/SCIENCE

These authors studied changes in NADH (the reduced form of nicotinamide adenine dinucleotide) in rat hippocampal slices using two-photon fluorescence imaging and confocal microscopy. Alterations in NADH concentration–the major contributor to the fluorescent signal–provide a signature of glycolysis or oxidative phosphorylation activity in the cytoplasm or mitochondria of astrocytes and neurons in the hippocampal brain slices (see the figure). With confocal microscopy, the authors were able to identify the fluorescent signal as emanating from either the cytoplasm or mitochondria of astrocytes or neurons.The authors stimulated the Schaffer collaterals of CA3 neurons in the hippocampal slices and monitored the intrinsic fluorescence signal in the CA1 hippocampal area. At low magnifications of the confocal microscope, the tissue presented a biphasic response: An initial “dip” in the NADH level followed by an “overshoot” that reproduced previous in vivo measurements. At higher magnifications, however, the authors were able to resolve both spatially and temporally the response in the two components. The early “dip” in the fluorescent signal and its recovery to baseline was restricted to the dendrites of neurons in a small area of the hippocampus. From the mitochondrial origin of the fluorescent signal, the authors deduced that the response represented first an increase in oxidative phosphorylation (respiration) in which NADH is consumed, followed by activation of the tricarboxylic acid (TCA) cycle to replenish NADH. In contrast, the late “overshoot” fluorescent signal was located in the cytoplasm of the processes of astrocytes. This signal corresponded to a strong activation of glycolysis in which large amounts of cytoplasmic NADH are generated before being converted back to NAD⁺ as lactate is produced. These two metabolic processes were not only segregated temporally and spatially, but could be also separated pharmacologically. The oxidative response in neuronal dendrites could be blocked by inhibiting the activation of AMPA/kainate receptors, indicating that the oxidative response was postsynaptic. The glycolytic response was not blocked by such treatment, indicating that it was most likely triggered by a presynaptic event. Overall, the data of Kasischke et al. provide strong evidence for the existence of an astrocyte-neuron lactate shuttle.

The elegant work of Kasischke and colleagues reconciles a number of contradictory results obtained with different brain imaging techniques in vivo. Their elegant technique combining imaging with confocal microscopy has provided results that answer the question of whether neuronal activity in the brain is supported by oxidative or nonoxidative metabolism (5). PET and certain other techniques require long sampling times to obtain a sufficient signal and so are more likely to detect long-lasting signals–such as the accumulation of fluorodeoxyglucose (FDG) in response to glucose uptake–rather than early or brief events such as the activation of cellular respiration. Consequently, images generated by FDG-PET primarily reflect astrocytic activity rather than neuronal activity, even though the activation of both cell types is correlated in the majority of cases. Similarly, magnetic resonance spectroscopy (MRS), which monitors changes in glucose and lactate levels with low temporal resolution, is more likely to reveal the late component of the metabolic response associated with astrocytes (6, 7). Thus, lactate “peaks” corresponding to an increase in lactate concentration were often reported in these studies. This would correspond to the enhanced glycolysis in astrocytes and its associated sustained production of lactate. In contrast, use of MRS with higher temporal resolution (8) or of biosensors (9) allows much faster sampling times enabling detection of the early lactate “dip.” The detected “dip” is consistent with the rapid activation of oxidative phosphorylation followed by lactate oxidation via the TCA cycle in neurons, before lactate production by astrocytes replenishes the extracellular lactate pool.

Although the present study clarifies a number of issues in both brain energetics and functional brain imaging, it also holds the promise of finding a solution to other unanswered questions. For example, the strategy of Kasischke et al. may resolve whether excitatory (glutamatergic) and inhibitory (GABAergic) activities have the same impact on brain energetics and elicit similar imaging signals. The remarkable findings of Kasischke and co-workers leave us eager to see the next answer that comes to light.

References

1. L. Sokoloff et al., J. Neurochem. 28, 897 (1977) [Medline].
2. L. Pellerin, P. J. Magistretti, Proc. Natl. Acad. Sci. U.S.A. 91, 10625 (1994) [Medline].
3. P. J. Magistretti, L. Pellerin, D. L. Rothman, R. G. Shulman, Science 283, 496 (1999).
4. K. A. Kasischke, H. D. Vishwasrao, P. J. Fisher, W. R. Zipfel, W. W. Webb, Science 305, 99 (2004).
5. P. T. Fox, M. E. Raichle, M. A. Mintun, C. Dence, Science 241, 462 (1988) [Medline].
6. J. Prichard et al., Proc. Natl. Acad. Sci. U.S.A. 88, 5829 (1991) [Medline].
7. J. Frahm, G. Kruger, K. D. Merboldt, A. Kleinschmidt, Magn. Reson. Med. 35, 143 (1996) [Medline].
8. S. Mangia et al., Neuroscience 118, 7 (2003) [Medline].
9. Y. Hu, G. S. Wilson, J. Neurochem. 69, 1484 (1997) [Medline].

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The authors are at the Institut de Physiologie, 1005 Lausanne, Switzerland. E-mail: luc.pellerin@iphysiol.unil.ch<!–
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