Ascorbate regulation and its neuroprotective role in the brain

REVIEW Ascorbate regulation and its neuroprotective role in the brain Margaret E. Rice Ascorbic acid (vitamin C) occurs physiologically as the ascorbate anion:a water-soluble antioxidant that is found throughout the body. However, despite the high, homeostatically regulated levels of brain ascorbate, its specific functions in the CNS are only beginning to be elucidated. Certainly, it acts as part of the intracellular antioxidant network, and as such is normally neuroprotective. There is also evidence that it acts as a neuromodulator. A possibly unique role it might have is as an antioxidant in the brain extracellular microenvironment, where its concentration is modulated by glutamate–ascorbate heteroexchange at glutamate uptake sites. Ongoing studies of ascorbate and glutamate transporters should lead to rapid progress in understanding ascorbate regulation and function. Trends Neurosci. (2000) 23, 209–216

O

VER the past 25 years, there has been increasing evidence that ascorbate (see Box 1) is an important antioxidant, enzyme co-factor and neuromodulator in the brain. Despite evidence for many roles, its specific functions remain obscure. In order to learn about the function of ascorbate in the CNS, it is necessary to understand how it is compartmentalized. Ascorbate enters the CNS primarily by active transport at the choroid plexus. It diffuses from CSF to brain extracellular fluid (ECF), where its concentration is regulated homeostatically. Extracellular ascorbate levels are also dynamically modulated by glutamate-mediated activity, via glutamate–ascorbate heteroexchange. From the ECF, ascorbate is taken up into brain cells, where its concentration is increased up to 20-fold. It is therefore predominantly intracellular in brain tissue, with further compartmentalization between neurons and glia.

Ascorbate levels in plasma, CSF, ECF and brain tissue The brain, spinal cord and adrenal glands have the highest ascorbate concentrations of all the tissues in the body, as well as the greatest retention capacities1. Under normal conditions, turnover of ascorbate in brain is about 2% per hour2. Under conditions of ascorbate deficiency (see Box 1), however, brain ascorbate content is retained tenaciously, with decreases of less than 2% per day3. Thus, total brain ascorbate levels are under strong homeostatic regulation2,4. Ascorbate uptake from the blood into cerebrospinal fluid (CSF) involves active, stereospecific, Na1-dependent transport at the choroid plexus4. In rat brain, this process accumulates ascorbate in CSF to a concentration of about 500 mM (Ref. 5), which is tenfold higher than the concentration typically found in plasma in a variety of mammalian species1,2,6 (Fig. 1). Ascorbate can also enter the ECF by carrier-mediated uptake and by simple diffusion across brain capillaries at the blood–brain barrier8 (Fig. 1). However, detectable levels of the recently described Na1-dependent ascorbate transporters, SVCT1 and SVCT2, are found in the choroid plexus, but not in brain capillaries7. An alternative mechanism for ascorbate entry across the blood–brain barrier has been pro0166-2236/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.

posed to be transport of its neutral oxidation product, dehydroascorbate, by a facilitative glucose transporter, GLUT1, with subsequent reduction of dehydroascorbate to ascorbate once it is in the brain (Ref. 9). The fact that ascorbate is found almost exclusively in the reduced form in plasma10, however, suggests that this route is minor7. From the CSF, ascorbate equilibrates with brain ECF, in which its concentration is 200–400 mM (Refs 5,11,12; Fig. 1), and is then transported from the ECF into brain cells4,13–15. Overall brain-tissue ascorbate concentration is several millimolar16,17, with the average concentration in neurons estimated to be 10 mM, but only 1 mM in glia18. Brain-tissue content of ascorbate is regionally dependent: higher levels are found in anterior regions, such as the cerebral cortex and hippocampus, with progressively lower levels in more-posterior regions, such as the brainstem and spinal cord16,17. This pattern primarily reflects the increasing white-matter content of the posterior regions of the CNS, because the ascorbate content of white matter is much lower than that of neuron-rich gray matter17. Regional and subregional variation in neuron-to-glia ratio should also contribute to tissuespecific levels. Finally, brain ascorbate levels are gender dependent, with lower, estrogen-regulated levels in the female brain than in the male brain19.

Homeostasis and regulation of extracellular ascorbate in brain Extracellular ascorbate concentration, [Asc]o, is also maintained homeostatically11. This was first shown in brain slices in vitro: isolated mammalian brain tissue loses ascorbate rapidly when incubated in ascorbate free-media. Up to 80% of tissue ascorbate is lost from brain slices after only brief incubation11,15,20, possibly via reversal of the ascorbate transporter21. In early studies of rat brain slices, McIlwain and colleagues found that slices lost ascorbate in media that contained less than 200 mM ascorbate, but concentrated it in the presence of levels of ascorbate that were higher than this concentration20. Subsequently, Schenk and colleagues11, who used voltammetric microelectrodes to monitor [Asc]o in rat striatal slices, found that [Asc]o remained constant PII: S0166-2236(99)01543-X

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Margaret E. Rice is at the Depts of Neurosurgery, and Physiology and Neuroscience, New York University School of Medicine, NY 10016, USA.

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Box 1.Ascorbate chemistry and biosynthesis Ascorbic acid is a water-soluble, hexonic sugar acid, with a molecular weight of 176.13. It has two dissociable protons, with pKa values of 4.2 and 11.8 (Ref. a), and, thus, is a monovalent anion, ascorbate, at physiological pH (Fig. I). Its enediol structure enables it to be an electron donor, via loss of two electrons to form its final oxidation product, dehydroascorbate (Fig. I). Most oxidizing free radicals (substances that have one unpaired electron) generated by biological systems can lead to the one-electron oxidation of ascorbate to form semi-dehydroascorbate, which is also referred to as the ascorbyl radical. Detection of ascorbyl radical formation using electron-spin resonance can be used as a measure of oxidative stressb. This radical intermediate is also formed in enzymatic reactions that involve ascorbate as an electrondonating co-factor c. CH2OH

CH2OH

HCOH

HCOH O

O−

O

O OH

Ascorbate

O

O

+ 2e− + H+

O

Dehydroascorbate

Fig. I. Molecular structure of L-ascorbic acid as the monovalent anion, ascorbate, and its oxidation product, dehydroascorbate (formed by the loss of two electrons and a proton). The best-known functions of ascorbate, as an antioxidant and free radical scavenger, are due to its properties as an electron donor. Given its low redox potential, ascorbate is a broad-spectrum radical scavenger that is effective against peroxyl- and hydroxyl-radicals, superoxide, singlet oxygen, and peroxynitrited–g. Although ascorbate reactions occur in the aqueous phase, this can prevent oxidation of lipidsoluble vitamin E (a-tocopherol), which in turn stops peroxidation of cell membranes h,i. Oxidized ascorbate, both semi-dehydroascorbate and dehydroascorbate, can be reduced and thus recycled by glutathione (GSH) and other intracellular thiols j,k, and in some cells by a GSH-dependent dehydroascorbate reductase l,m. The presence of this intracellular enzyme in brain was confirmed recently, with regionally distinct levels confined to gray matterm. Oxidized GSH, in turn, is recycled by GSH reductase j. As part of the predominantly intracellular antioxidant network, ascorbate acts in concert with other low-molecularweight substances, including GSH and vitamin E, as well as antioxidant enzymes, such as superoxide dismutase and glutathione peroxidasen. The distinct compartmentalization of ascorbate, as well as other novel characteristics, suggests that it might have other unique functions, as discussed elsewhere. One well-defined function of ascorbate is to serve as an electron-donating enzyme co-factor. The most important ascorbate-dependent enzyme processes are collagen biosynthesis, via hydroxylation reactionso, and noradrenaline–adrenaline synthesis by dopamine-b-hydroxylasec. Why loss of ascorbate synthesis in humans was not a fatal mutation Ascorbic acid is synthesized from glucose in most animals via the following simplified pathway: → D-glucuronic acid → L-gulonic acid → L-gulono-g-lactone → L-ascorbic acid.

D-glucose

during a 30 min incubation, even though tissue content fell by 75% during that time. This suggested that [Asc]o was maintained homeostatically at the expense of intracellular stores. The mechanisms involved in determining the ‘set point’ for [Asc]o are not yet understood. 210

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Humans, nonhuman primates and guinea pigs have lost the ability to synthesize ascorbate because they carry a nonfunctional gene for the enzyme, L-gulono-g-lactone oxidase, which is required for the last step of ascorbate biosynthesisp. Importantly, mutations in this gene are not fatal for several reasons. First, ascorbate is available from dietary sources and, equally important, is readily absorbed from the gutq. In addition, even in animals that can synthesize ascorbate, this occurs only in the liver (mammals) or kidneys (reptiles)r, with subsequent distribution to all other tissues via plasma. Consequently, all other tissues have mechanisms for uptake and storage of ascorbate. In humans, the effects of ascorbate deficiency were noticed first during long sea voyages, away from fresh fruit and vegetabless. The major symptoms of scurvy derive from inhibition of collagen synthesis, although depression is also an early symptoms. References a Davies, M.B. et al. (1991) Vitamin C: Its Chemistry and Biochemistry, Royal Society of Chemistry b Buettner, G.R. and Jurkiewicz, B.A. (1993) Ascorbate free radical as a marker of oxidative stress: an EPR study. Free Radic. Biol. Med. 14, 49–55 c Diliberto, E.J., Jr et al. (1987) Adrenomedullary chromaffin cells as a model to study the neurobiology of ascorbic acid: from monooxygenation to neuromodulation. Ann. New York Acad. Sci. 498, 28–53 d Nishikimi, M. (1975) Oxidation of ascorbic acid with superoxide anion generated by the xanthine-xanthine oxidase system. Biochem. Biophys. Res. Commun. 63, 463–468 e Bodannes, R.S. and Chan, P.C. (1979) Ascorbic acid as a scavenger of singlet oxygen. FEBS Lett. 105, 195–196 f Machlin, L.J. and Bendich, A. (1987) Free radical tissue damage: protective role of antioxidant nutrients. FASEB J. 1, 441–445 g Vatassery, G.T. (1996) Oxidation of vitamin E, vitamin C, and thiols in rat brain synaptosomes by peroxynitrite. Biochem. Pharmacol. 52, 579–586 h Seregi, A. et al. (1978) Protective role of ascorbic acid content against lipid peroxidation. Experientia 34, 1056–1057 i Niki, E. (1991) Action of ascorbic acid as a scavenger of active and stable oxygen radicals. Am. J. Clin. Nutr. 54, 1119S–1124S j Meister, A. (1994) Glutathione-ascorbic acid antioxidant system in animals. J. Biol. Chem. 269, 9397–9400 k Winkler, B.S. et al. (1994) The redox couple between glutathione and ascorbic acid: a chemical and physiological perspective. Free Radic. Biol. Med. 17, 333–349 l Rose, R. C. (1993) Cerebral metabolism of oxidized ascorbate. Brain Res. 628, 49–55 m Fornai, F. et al. (1999) Localization of a glutathione-dependent dehydroascorbate reductase within the central nervous system of the rat. Neuroscience 94, 937–948 n Cohen, G. (1994) Enzymatic/nonenzymatic sources of oxyradicals and regulation of antioxidant defenses. Ann. New York Acad. Sci. 738, 8–14 o Barnes, M.J. (1975) Function of ascorbic acid in collagen metabolism. Ann. New York Acad. Sci. 258, 264–277 p Nishikimi, M. et al. (1994) Cloning and chromosomal mapping of the human nonfunctional gene for L-gulono-g-lactone oxidase, the enzyme for L-ascorbic acid biosynthesis missing in man. J. Biol. Chem. 269, 13685–13688 q Kallner, A. et al. (1977) On the absorption of ascorbic acid in man. Int. J. Vitam. Nutr. Res. 47, 383–388 r Chatterjee, I.B. et al. (1975) Synthesis and some major functions of vitamin C in animals. Ann. New York Acad. Sci. 258, 24–47 s Carpenter, K.J. (1986) The History of Scurvy and Vitamin C, Cambridge University Press

More recently, Meile and Fillenz reported that [Asc]o is also regulated homeostatically in vivo, in awake, behaving animals12. In these studies, 100 –1000 mM ascorbate was perfused through a microdialysis probe, with voltammetric detection of [Asc]o in the tissue adjacent to

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the probe. Concentrations that were lower than 400 mM caused [Asc]o to fall, whereas higher concentrations caused an increase in [Asc]o. After each perturbation, [Asc]o recovered within a few minutes, which demonstrates homeostasis. The concentration at which there was no net change in [Asc]o was 400 mM (Ref. 12). Homeostatic regulation of [Asc]o suggests that the extracellular compartment of brain tissue might be an important site of action for ascorbate.

Dynamic regulation of [Asc]o by glutamate–ascorbate heteroexchange Although brain [Asc]o is regulated homeostatically, dynamic activity-dependent changes in [Asc]o also occur. Indeed, [Asc]o shows marked circadian variation22. In rats, which are nocturnal, average [Asc]o is 20–60% higher throughout the brain during the night than in the daytime. These variations are closely linked with motor behavior, such that within a given animal, striatal [Asc]o can vary over twofold between active and inactive states22. Moreover, the behavioral stimulant amphetamine causes an increase in [Asc]o in the striatum that is attenuated, but not abolished, in anesthetized animals compared with freely moving ones23. Selective activation of dopaminergic pathways in the basal ganglia by amphetamine, however, does not cause [Asc]o to increase in other structures24,25. Taken together, these findings indicate that motor behavior per se is not involved in modulation of [Asc]o. Rather, increases in [Asc]o have been linked to activation of specific glutamatergic pathways, and subsequent glutamate release and uptake26. In a series of careful experimental studies, Fillenz and colleagues have determined that the primary mechanism leading to increases in [Asc]o is its heteroexchange with glutamate24–28. As glutamate is taken up after release, intracellular ascorbate is released from cells by a glutamate–ascorbate heteroexchange mechanism. A key experiment in this series was the in vitro demonstration that ascorbate was released during Na1-dependent, stereospecific uptake of glutamate by synaptosomes from several brain regions, although not from the cerebellum27. Subsequently, others showed that local injection of glutamate in vivo caused a dose-dependent increase in [Asc]o that was prevented by glutamate uptake inhibitors, but not by glutamate-receptor antagonists29, which further implicated glutamate transporters in the process. Consistent with this mechanism, both circadian22 and amphetamine-induced30 increases in striatal [Asc]o were attenuated after cortical lesions that removed the major glutamatergic input to striatum. The amphetamine-induced response appears to require dopamine release, which then activates a loop that includes substantia nigra, thalamus, cortex and striatum25. Two important questions about glutamate–ascorbate heteroexchange remain unanswered: what glutamate transporters are involved, and are they located on neurons or glia? So far, five glutamate, or excitatoryamino-acid, transporters have been identified (for a review, see Refs. 31,32). Of these, EAAT1 (also known as GLAST) and EAAT2 (also known as GLT1) are localized predominantly on glial cells, with higher levels of EAAT1 in the cerebellum than in other regions and lower levels of EAAT2 in the cerebellum than elsewhere31,33,34. By contrast, EAAT3 (also known as EAAC1) is found predominantly in neurons and on both glutamatergic and non-glutamatergic cells throughout the brain35. EAAT4 is found at high levels on cerebellar Purkinje cells36. The

Blood–brain barrier Plasma 50 µM Neurons 10 mM Asc Glu Asc ?

Choroid plexus Plasma 50 µM Asc SVCT2 ECF

[Asc]o 200–400 µM

CSF 500 µM Asc SVCT2

Glu Asc Glia 1 mM

Asc ?

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Fig. 1. Ascorbate compartmentalization and regulation in the CNS. Whether ascorbate is synthesized in liver or kidney or acquired from the diet, it is distributed to all organs in the body via the blood supply, with plasma levels that are typically around 50 µM (Refs 1,2,6). Active transport at the choroid plexus, via the SVCT2 isoform of the ascorbate transporter7, concentrates ascorbate in ventricular cerebrospinal fluid (CSF) (Ref. 4). This pool of ascorbate is in equilibrium with the extracellular fluid (ECF) compartment of the brain5, with diffusion of CSF ascorbate to ECF (broken arrow). Ascorbate is taken up from ECF into neurons and glia: in neurons, ascorbate is transported via SVCT2, however, glia do not have detectable levels of either SVCT1 or SVCT2 (Ref. 7), so that the mechanism of uptake into glial cells is not yet known. Ascorbate can enter brain ECF at the blood–brain barrier at the level of brain capillaries8, although capillaries do not have SVCT1 or SVCT2 (Ref. 7). One possible pathway might be facilitated transport of the ascorbate oxidation product, dehydroascorbate, by glucose transporters at the blood–brain barrier9; whether this occurs under normal physiological conditions is uncertain. Extracellular ascorbate concentration, [Asc]o , is regulated homeostatically in the brain, although it is also modulated dynamically by glutamate release, with increases in [Asc]o caused by heteroexchange with glutamate as glutamate is taken up by one or more transporters. Whether the source is neurons or glia, or both, is not yet known. Illustrated concentrations and pathways represent general features of ascorbate regulation in the brain that would be expected to occur in most regions of the CNS. Abbreviations: Asc, ascorbate; Glu, glutamate.

most recently described transporter, EAAT5, is found in the retina32. Given that glutamate-uptake-dependent release of ascorbate has been demonstrated in synaptosomes from forebrain regions, but not from the cerebellum27, this might implicate EAAT2 and EAAT3, rather than cerebellum-enriched EAAT1 or EAAT4, in mediating glutamate–ascorbate heteroexchange. It is relevant in this regard, that release from synaptosomes per se does not necessarily mean that glutamate– ascorbate heteroexchange accompanies uptake into neurons only, because synaptosomes can also contain astroglial membranes31. Determination of which transporters and cell types are involved in glutamate–ascorbate heteroexchange has been limited by the relative lack of specificity of currently available glutamate-transport inhibitors. Cammack and colleagues found that increases in [Asc]o induced by local glutamate injection or during perforant path stimulation in the hippocampus could be blocked by D,L-threo-b-hydroxy-aspartic acid and to TINS Vol. 23, No. 5, 2000

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(b) 5.0

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Ascorbate content (µmol g–1)

6 Neuron:glia ratio

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Fig. 2. Dependence of ascorbate content in adult cerebral cortex on neuron density and developmental changes in ascorbate content during gliogenesis in the rat cortex. (a) Linear increase in ascorbate content with increasing neuronal density in adult cerebral cortex across species. (Neuron density data are taken from Ref. 41.) The intercept of the y-axis was used to estimate the concentration of ascorbate in glia, which would be the theoretical cell population when neuron density was zero. Appropriate intracellular and extracellular volume fractions were used to calculate an intracellular concentration of 0.9 mM in glia18. Data are mean 6SEM; n 5 9–61 samples per mean; r2 5 0.997. (b) Actual and calculated ascorbate content in developing rat cerebral cortex. Squares are experimental data and circles are calculated ascorbate contents. Cortical ascorbate content at postnatal day 3 (P3) and cerebellar content at P15 were used with appropriate intracellular and extracellular volume fraction data to calculate an ascorbate concentration in neurons of 10 mM (Ref. 18). In a test of the reliability of these calculated values for ascorbate concentration in neurons and in glia [from (a)], these data were used to predict the pattern of changes in tissue ascorbate content in developing rat cerebral cortex. In this model, known values of neuron-to-glia ratios during development42 [see inset in (b)] were used, together with estimated developmental changes in intracellular and extracellular volume fractions in rat cortex, to calculate ascorbate content from P3–P90. Full details of the calculations and specific assumptions are given elsewhere18. As can be seen, the model fits the experimental data very well: actual and calculated values for ascorbate cannot be distinguished statistically. The overall goodness of fit from the early postnatal period to adulthood (P90) supports the validity of these intracellular concentrations for ascorbate in neurons and in glia. Inset indicates the decreasing neuron-to-glia ratio in rat cortex during gliogenesis42, which is reflected in the concomittant fall in ascorbate content (b). Because glutathione (GSH) is more concentrated in glia than in neurons, cortical GSH content increases during this same period18. Modified, with permission, from Ref. 18.

lesser extent by the nominally glia-selective stilbene derivative, SITS (Ref. 29). Similarly, Miele and colleagues showed that the [Asc]o increase that accompanies tailpinch stress was largely inhibited by local infusion of 28 L-trans-pryrrolidine-2,4,-dicarboxylate (L-trans-PDC) . These data are consistent with release from neurons, given that both D,L-threo-b-hydroxy-aspartic acid and L-trans-PDC have been shown to have higher affinities for synaptosomal transporters compared with astrocytic transporters37. This pattern must again be interpreted cautiously, given that levels of GLT1 are decreased in cultured astrocytes and that synaptosomes can contain glial membranes31.

Compartmentalization of ascorbate between neurons and glia As already discussed, ascorbate is regulated homeostatically between the intracellular and extracellular compartments. What about compartmentalization between neurons and glia? At first glance, it might seem that this question could best be addressed using isolated populations of neurons and glia in culture. As noted above, however, ascorbate is readily lost from mammalian cells in vitro. Indeed, the situation is even more extreme in isolated cells than in brain slices, with ascorbate levels that are below detection limits in cells cultured in ascorbate-free media14,38,39. Several lines of evidence from whole-tissue studies, however, indicate that ascorbate is localized preferentially in neurons, with much lower concentrations being found in glia. Forty years ago, Shimizu and colleagues identified ascorbate in the cytosol of neurons in the locus coeruleus40, which use ascorbate as a co-factor for 212

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noradrenaline synthesis21, with lower levels in surrounding glia. It should be noted that there is little correlation between tissue levels of ascorbate and noradrenergic innervation per se16. More recently, studies that quantified tissue levels of ascorbate, using HPLC with electrochemical detection, found that the ascorbate content of adult rat cortex was roughly fourfold higher in neuron-rich cerebral cortex than in the essentially neuron-free optic nerve, again consistent with predominant localization of ascorbate in neurons17. The best quantitative information on neuron versus glial compartmentalization has come from studies of the ascorbate content of brain tissue with known neuron density or neuron-to-glial ratio. For example, the neuron density of the cerebral cortex varies across mammalian species, with an inverse dependence on brain size41. Across species, cortical ascorbate content increases linearly with increasing neuron density: human ,rabbit,guinea pig,rat,mouse18 (Fig. 2a). The y-axis intercept of this plot of ascorbate content versus neuron density can be used to estimate ascorbate levels in tissue with zero neuron density, that is, a pure glial population. With appropriate values for the volume fractions of the intra- and extracellular compartments, an intracellular glial ascorbate concentration was calculated to be about 1 mM (Ref. 18). Another natural system in which defined changes in neuron density occur is the developing rat brain. During development, regionally distinct changes in neuron-toglia ratio occur during the first three postnatal weeks42,43. In cerebral cortex (Fig. 2b), tissue ascorbate content is highest shortly after birth (postnatal day 3, P3), when cortical tissue is nearly a pure neuronal population

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with only few immature glial cells. (a) (b) Over the next few weeks, ascorbate levels fall, consistent with the timecourse of cortical gliogenesis42 (Fig. 2b). In the cerebellum, by contrast, ascorbate levels are relatively constant until P9, when they begin to increase markedly, coincident with the onset of cerebellar granule-cell (c) (d) proliferation43. A maximum ascorbate content in developing cerebellum is reached by P15, which then declines gradually to adult levels18. The tissue ascorbate contents of rat cortex at P3 and cerebellum at P15 therefore represent levels in predominantly neuronal populations. Again using the appropriate intracellular and extracellular volume fractions, intracellular ascorbate Fig. 3. Localization of SCVT2 mRNA in rat brain detected by in situ hybridization. Bright-field micrographs of cryosections hybridized to digoxigenin-labeled antisense cRNA probes of SCVT2. (a) shows the whole brain, note neuronal staining concentration in neurons was calcuthroughout, including cerebral cortex. (b) shows the choroid plexus, (c) shows the hippocampus and (d) shows the lated to be 10 mM (Ref. 18). Because cerebellum. Tissue sections hybridized to sense cRNA probes of SCVT2 showed no label (not illustrated). Scale bars, 2 mm this concentration was obtained in (a), 50 µm in (b), 200 µm in (c) and 100 µm in (d). Modified, with permission, from Ref. 7. from two distinct neuron populations, high intracellular ascorbate levels appear to be a general neuronal characteristic. plexus (Fig. 3b) would be half-saturated at normal plasma ascorbate concentrations (50 mM), whereas the neuronal New transporter data – SCVT2 in neurons uptake carrier would always be saturated at normal brain The study of ascorbate regulation in the CNS has been [Asc]o (200–400 mM) (Fig. 1). Both conditions will contribimpeded by the lack of molecular information about ute to the strong homeostasis of CNS ascorbate levels2. the transporter(s) responsible for ascorbate uptake into Ascorbate as an antioxidant and oxygen-radical the brain and into brain cells. Importantly, however, scavenger in the brain Tsukaguchi and colleagues7 recently described the first 1 members of a new family of Na -dependent vitamin C High levels of ascorbate and of SVCT2 in a variety of (ascorbate) transporters (SVCT1 and SVCT2), isolated neuronal cell types imply a function for neuronal ascorfrom rat cDNA libraries. These isoforms share 65% bate beyond its actions as a cell-specific enzyme coamino-acid homology. Both facilitate electrogenic, Na1- factor. Importantly, the tenfold difference between dependent uptake of ascorbate with similar affinities ascorbate levels in neurons and glia18 is consistent with for this substrate7. The major difference between the the estimated tenfold higher rate of oxidative metabotransporter isoforms appears to be their complementary lism in neurons compared with glial cells45. The imporlocalization: SVCT1 is present in kidney, liver and lung, tance of ascorbate as an intracellular antioxidant is for example, and SVCT2 is present in neural, neuro- further supported by the finding that brain ascorbate endocrine, exocrine and endothelial tissues. Only SVCT2 (but not glutathione) levels in pond turtles are two to is found in the brain7. Subsequently, Rajan and col- three times higher than in mammals17. These diving leagues44 cloned and characterized SVCT2 from a hu- animals have a remarkable tolerance of hypoxia, owing man placental cDNA library. Human SVCT2 shares a to a variety of mechanisms46,47. High levels of ascorbate 95% sequence homology with rat SVCT2 and is also could represent a specific adaptation to prevent oxifound in the brain44. dative damage during reoxygenation after a hypoxic Consistent with higher concentrations of ascorbate dive17. Interestingly, turtle brain tissue also maintains its in neurons than glia, in situ hybridization in the rat ascorbate content in vitro48, unlike isolated mammalian brain indicates that SVCT2 is localized at high levels in tissue. neurons, but not in glial cells7 (Fig. 3). Glia accumulate Although ascorbate can act as a pro-oxidant in vitro, ascorbate by active uptake in culture13,39; however, the the occurrence of naturally high levels of ascorbate in transporter involved has not yet been identified. These neuronal cytosol, taken with other data discussed below, recently discovered ascorbate uptake transporters are argues strongly against normally pro-oxidant actions distinct from the glutamate transporters that promote in vivo. This conclusion was also reached by Halliwell ascorbate release by glutamate–ascorbate hetero- in a recent review of the antioxidant versus pro-oxidant exchange. In the same way as glutamate transporters, effects of ascorbate49. Indeed, neuroprotection by ascorhowever, SVCT2 is present in both glutamatergic and bate has been demonstrated in several recent studies, GABAergic neurons7, including glutamatergic pyrami- both in vitro and in vivo. dal cells of the hippocampus (Fig. 3c), glutamatergic Cerebral ischemia granule cells of the cerebellum (Fig. 3d) and GABAergic Under normal circumstances, ascorbate is recycled as cerebellar Purkinje cells (Fig. 3d). it is used, so that antioxidant protection is maintained. The affinity SVCT2 for ascorbate7 is similar to the During anoxic depolarization, however, ascorbate is lost 30–50 mM affinities reported previously for ascorbate from cells and released into extracellular fluid50. With uptake by choroid plexus and brain slices2,4. This affinity continued ischemia, tissue levels of ascorbate and other for ascorbate would mean that transport at the choroid low-molecular-weight antioxidants fall51. Consequently, TINS Vol. 23, No. 5, 2000

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Box 2.The relationship between ascorbate and GSH Ascorbate and glutathione (GSH) are the two most abundant low-molecular-weight antioxidants in mammalian brain tissue. With total tissue contents of 2–3 mM (Refs a–c) for both ascorbate and GSH, these levels are 10 to 20 times higher than those of cysteine, and 500 to 1000 times higher than the levels of uric acida,b. Ascorbate and GSH have similarities and differences in their properties, which suggest that they have complementary but distinct roles in the CNS. GSH is an anionic tripeptide, g-glutamyl-cysteinyl-glycine, that is synthesized in all cells in the CNS (Refs d,e). In contrast to ascorbate, which is more highly concentrated in neuronsf, GSH is localized preferentially in gliae–h, with average intracellular levels of 4 mM in glia and 2.5 mM in neuronsf. A particularly important function of both and ascorbate and GSH might be their ability to neutralize reactive hydroxyl radicals (•OH), because there are no enzymes analogous to superoxide dismutase or GSH peroxidase to scavenge • OH (Ref. i). In addition, ascorbate and GSH can interact as a redox couple j,k. Depletion of either ascorbate or GSH can be compensated for by the continued presence of the other. For example, administration of GSH ester can prevent the onset of scurvy in ascorbate-deficient guinea pigs, whereas ascorbate administration can prevent tissue damage after inhibition of GSH synthesis j. On a cellular level, the distinct ascorbate: GSH ratio in neurons (4:1) and glia (1:4)f might alter the dynamics of ascorbate–GSH interactions, with greater reliance on ascorbate in neurons and on GSH in glia.

when aerobic metabolism resumes, intracellular stores of these agents are no longer adequate to quench reactive species. Thus, one need not postulate excessive radical production (although this can occur) as a cause of ischemic cell death. Rather, loss of antioxidants from the intracellular compartment during ischemia or other injury leaves cells vulnerable to oxidative damage. Increased detection of hydroxyl radical (•OH; see Box 2) during post-ischemic reperfusion52 is consistent with this hypothesis. Conversely, enhanced brain ascorbate content after dietary supplementation can protect cortical mitochondria from in vivo ischemia/reperfusion injury in rats53 and decrease focal ischemia-induced damage in primates54,55. Similarly, ascorbate can prevent mitochondrial hyperoxidation during post-ischemic reoxygenation in vitro in brain slices56.

Excitotoxicity Another important function of ascorbate might be to prevent redox imbalance from reactive oxygen species (ROS) generated by activation of glutamate receptors57–60. Both NMDA and kainate can cause depolarization of mitochondria and increased production of superoxide59,60. In recent studies, ascorbate was found to buffer glutamate-generated ROS and limit consequent cell death in cultured neurons61,62. An intracellular site of action for these effects was suggested by other studies in brain slices, in which both ascorbate and glutamate-receptor antagonists were shown to inhibit edema formation63. For ascorbate to be effective, it had to be accumulated by brain cells; moreover, isoascorbate, which is not a substrate for the stereospecific ascorbate transporter, was not effective in preventing brain-slice edema. There are several possible mechanisms for ascorbate protection against glutamate-induced cell death. Direct scavenging of ROS is likely to have the greatest role, because ascorbate can protect neurons from oxidative 214

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References a Lyrer, P. et al. (1991) Levels of low molecular weight scavengers in the rat brain during focal ischemia. Brain Res. 567, 317–320 b Uemura, Y. et al. (1991) Neurochemical analysis of focal ischemia in rats. Stroke 22, 1548–1553 c Rice, M.E. et al. (1995) High levels of ascorbic acid, not glutathione, in the CNS of anoxia-tolerant reptiles contrasted with levels in anoxia-intolerant species. J. Neurochem. 64, 1790–1799 d Orlowski, M. and Karkowsky, A. (1976) Glutathione metabolism and some possible functions of glutathione in the nervous system. Int. Rev. Neurobiol. 19, 75–121 e Makar, T.K. et al. 1994) Vitamin E, ascorbate, glutathione, glutathione disulfide, and enzymes of oxidative metabolism in cultures of chick astrocytes and neurons: evidence that astrocytes play an important role in oxidative processes in the brain. J. Neurochem. 62, 45–53 f Rice, M.E. and Russo-Menna, I. (1998) Differential compartmentalization of brain ascorbate and glutathione between neurons and glia. Neuroscience 82, 1213–1223 g Slivka, A. et al. (1987) Histochemical evaluation of glutathione in brain. Brain Res. 409, 275–284 h Raps, S.P. et al. (1989) Glutathione is present in high concentrations in cultured astrocytes but not in cultured neurons. Brain Res. 493, 398–401 i Cohen, G. (1994) Enzymatic/nonenzymatic sources of oxyradicals and regulation of antioxidant defenses. Ann. New York Acad. Sci. 738, 8–14 j Meister, A. (1994) Glutathione-ascorbic acid antioxidant system in animals. J. Biol. Chem. 269, 9397–9400 k Winkler, B.S. et al. (1994) The redox couple between glutathione and ascorbic acid: a chemical and physiological perspective. Free Radic. Biol. Med. 17, 333–349

damage64,65 and decrease levels of glutamate-generated ROS (Refs 61,62). In addition, however, glutamate– ascorbate heteroexchange might provide protection by facilitating glutamate uptake or by increasing [Asc]o, either of which could minimize excitotoxic consequences of glutamate release28. Finally, ascorbate has also been suggested to decrease NMDA-mediated currents by acting at the redox modulatory site of the NMDA receptor66. NMDA-receptor-mediated currents are increased by thiol reducing agents and decreased by agents that promote disulfide-bond formation66. Because of the relative redox potentials of ascorbate and thiols67, ascorbate should have no direct effect at this site. If ascorbate becomes oxidized, however, it could promote thiol oxidation, because electron transfer from a reduced thiol to oxidized ascorbate (for example, semi-dehydroascorbate, see Box 1) is favored thermodynamically. Consequently, apparent redox modulation of the NMDA receptor by ascorbate might be a result of in vitro oxidation.

Other actions of ascorbate In addition to its functions as an antioxidant in the CNS, ascorbate has been shown to be a neuromodulator of both dopamine- and glutamate-mediated neurotransmission, as reviewed by Grunewald24, and Rebec and Pierce25. Predominant localization of ascorbate in neurons is consistent with such neuromodulatory functions. In addition, ascorbate is an essential co-factor for noradrenaline synthesis21, and is required for the release of noradrenaline and ACh from synaptic vesicles68. Ascorbate is also an essential co-factor in the synthesis of many neuropeptides69 and at physiological concentrations enhances peptide release70. In addition, ascorbate promotes myelin formation by Schwann cells by enabling these cells to assemble a basal lamina71.

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A number of other actions of ascorbate have been described in the literature, from alterations in neurotransmitter binding affinity to inhibition of Na1–K1ATPase activity. Because most of these studies have been conducted in vitro, often in isolated cells or cell membranes, the potential for pro-oxidant effects of ascorbate is high, which casts doubt on the physiological relevance of some of the findings. This does not necessarily mean that ascorbate never acts as a pro-oxidant in vivo; indeed, under pathological conditions such as ischemia, when ascorbate compartmentalization is disrupted, these actions could occur and contribute to CNS injury. Under normal conditions, however, it is increasingly clear that ascorbate facilitates neuroprotection.

Concluding remarks The high intracellular concentration of ascorbate in neurons suggests that it is has a significant role in normal neuronal physiology. Given the well-established characteristics of ascorbate as an electron donor and free-radical scavenger, it is likely that this role comes from neuroprotective actions as a component of the neuronal antioxidant network (see Box 1). In addition, ascorbate is one of the few antioxidants in ECF. The fact that [Asc]o is regulated homeostatically, but modulated by glutamate-mediated activity, suggests that the extracellular compartment is an important site for ascorbate neuroprotection. Ongoing advances in research on ascorbate and glutamate transporters should lay the groundwork for rapid increases in our understanding of ascorbate regulation in the brain. As new tools to manipulate ascorbate levels become available, we should similarly gain new insights into its specific functions in the brain. Selected references 1 Hornig, D. (1975) Distribution of ascorbic acid, metabolites and analogues in man and animals. Ann. New York Acad. Sci. 258, 103–117 2 Spector, R. (1977) Vitamin homeostasis in the central nervous system. New Engl. J. Med. 296, 1293–1398 3 Hughes, R.E. et al. (1971) The retention of ascorbic acid by guineapig tissues. Br. J. Nutr. 26, 433–438 4 Spector, R. and Lorenzo, A.V. (1973) Ascorbic acid homeostasis in the central nervous system. Am. J. Physiol. 225, 757–763 5 Stamford, J.A. et al. (1984) Regional differences in extracellular ascorbic acid levels in the rat brain determined by high speed cyclic voltammetry. Brain Res. 299, 289–295 6 Chatterjee, I.B. et al. (1975) Synthesis and some major functions of vitamin C in animals. Ann. New York Acad. Sci. 258, 24–47 7 Tsukaguchi, H. et al. (1999) A family of mammalian Na1-dependent L-ascorbic acid transporters. Nature 399, 70–75 8 Lam, D.K.C. and Daniel, P.M. (1986) The influx of ascorbic acid into the rat’s brain. Q. J. Exp. Physiol. 71, 483–489 9 Agus, D.B. et al. (1997) Vitamin C crosses the blood-brain barrier in the oxidized form through the glucose transporters. J. Clin. Invest. 100, 2842–2848 10 Dhariwal, K.R. et al. (1991) Ascorbic acid and dehydroascorbic acid measurements in human plasma and serum. Am. J. Clin. Nutr. 54, 712–716 11 Schenk, J.O. et al. (1982) Homeostatic control of ascorbate concentration in CNS extracellular fluid. Brain Res. 253, 353–356 12 Miele, M. and Fillenz, M. (1996) In vivo determination of extracellular brain ascorbate. J. Neurosci. Meth. 70, 15–19 13 Wilson, J.X. (1989) Ascorbic acid uptake by a high-affinity sodiumdependent mechanism in cultured rat astrocytes. J. Neurochem. 53, 1064–1071 14 Kalir, H.H. and Mytilenou, C. (1992) Ascorbic acid in mesencephalic cultures: effects on dopaminergic neuron development. J. Neurochem. 57, 458–464 15 Rice, M.E. et al. (1994) Ascorbic acid, but not glutathione, is taken up by brain slices and preserves cell morphology. J. Neurophysiol. 71, 1591–1596 16 Milby, K. et al. (1982) Detailed mapping of ascorbate distribution in rat brain. Neurosci. Lett. 28, 15–20 17 Rice, M.E. et al. (1995) High levels of ascorbic acid, not glutathione, in the CNS of anoxia-tolerant reptiles contrasted with levels in

anoxia-intolerant species. J. Neurochem. 64, 1790–1799 18 Rice, M.E. and Russo-Menna, I. (1998) Differential compartmentalization of brain ascorbate and glutathione between neurons and glia. Neuroscience 82, 1213–1223 19 Kume-Kick, J. and Rice, M.E. (1998) Estrogen-dependent modulation of rat brain ascorbate levels and ischemia-induced ascorbate loss. Brain Res. 803, 105–113 20 McIlwain, H. et al. (1956) The composition of isolated cerebral tissues: ascorbic acid and cozymase. Biochem. J. 64, 332–335 21 Diliberto, E.J., Jr et al. (1987) Adrenomedullary chromaffin cells as a model to study the neurobiology of ascorbic acid: from monooxygenation to neuromodulation. Ann. New York Acad. Sci. 498, 28–53 22 O’Neill, R.D. et al. (1983) The effect of unilateral cortical lesions on the circadian changes in rat striatal ascorbate and homovanillic acid levels measured in vivo using voltammetry. Neurosci. Lett. 42, 105–110 23 Gonon, F. et al. (1981) Voltammetry in the striatum of chronic freely moving rats: detection of catechols and ascorbic acid. Brain Res. 223, 69–80 24 Grünewald, R.A. (1993) Ascorbic acid in the brain. Brain Res. Rev. 18, 123–133 25 Rebec, G.V. and Pierce, R.C. (1994) A vitamin as neuromodulator: ascorbate release into the extracellular fluid of the brain regulates dopaminergic and glutamatergic transmission. Prog. Neurobiol. 43, 537–565 26 O’Neill, R.D. (1984) Voltammetrically monitored brain ascorbate as an index of excitatory amino acid releases in the unrestrained rat. Neurosci. Lett. 52, 227–233 27 Grünewald, R.A. and Fillenz, M. (1984) Release of ascorbate from a synaptosomal fraction of rat brain. Neurochem. Int. 6, 491–500 28 Miele, M. et al. (1994) The physiologically induced release of ascorbate in rat brain is dependent on impulse traffic, calcium influx and glutamate uptake. Neuroscience 62, 87–91 29 Cammack, J. et al. (1991) The pharmacological profile of glutamateevoked ascorbic acid efflux measured by in vivo voltammetry. Brain Res. 565, 17–22 30 Basse-Tomusk, A. and Rebec, G.V. (1990) Corticostriatal and thalamic regulation of amphetamine-induced ascorbate release in the neostriatum. Pharmacol. Biochem. Behav. 35, 55–60 31 Gegelashvili, G. and Schousboe, A. (1998) Cellular distribution and kinetic properties of high-affinity glutamate transporters. Brain Res. Bull. 45, 233–238 32 Trotti, D. et al. (1998) Glutamate transporters are oxidant-vulnerable: a link between oxidative and excitoxic neurodegeneration? Trends Pharmcol. Sci. 19, 328–334 33 Torp, R. et al. (1994) Differential expression of two glial glutamate transporters in the rat brain: an in situ hybridization study. Eur. J. Neurosci. 6, 936–942 34 Chaudhry, F.A. et al. (1995) Glutamate transporters in glial plasma membranes: highly differentiated localizations revealed by quantitative ultrastructural immunocytochemistry. Neuron 15, 711–720 35 Conti, F. et al. (1998) EAAC1, a high-affinity glutamate transporter, is localized to astrocytes and GABAergic neurons besides pyramidal cells in the rat cerebral cortex. Cereb. Cortex 8, 108–116 36 Dehnes, Y. et al. (1998) The glutamate transporter EAAT4 in rat cerebellar Purkinje cells: a glutamate-gated chloride channel concentrated near the synapse in parts of the dendritic membrane facing astroglia. J. Neurosci. 18, 3606–3619 37 Rauen, T. et al. (1992) Comparative analysis of sodium-dependent L-glutamate transport of synaptosomal and astroglial membrane vesicles from mouse cortex. FEBS Lett. 312, 15–20 38 Makar, T.K. et al. (1994) Vitamin E, ascorbate, glutathione, glutathione disulfide, and enzymes of oxidative metabolism in cultures of chick astrocytes and neurons: evidence that astrocytes play an important role in oxidative processes in the brain. J. Neurochem. 62, 45–53 39 Siushansian, R. and Wilson, J.X. (1995) Ascorbate transport and intracellular concentrations in cerebral astrocytes. J. Neurochem. 65, 41–49 40 Shimizu N. et al. (1960) Histochemical demonstration of ascorbic acid in the locus coeruleus of the mammalian brain. Nature 186, 479–480 41 Tower, D.B. and Elliott, K.A.C. (1952) Activity of the acetylcholine system in cerebral cortex of various unanesthetized animals. Am J. Physiol. 168, 747–759 42 Parnavelas, J.G. et al. (1983) A quantitative and qualitative ultrastructural study of glial cells in the developing visual cortex of the rat. Philos. Trans. R. Soc. Lond. B Biol. Sci. 301, 55–84 43 Altman, J. (1972) Postnatal development of the cerebellar cortex of the rat: III. Maturation of the components of the granular layer. J. Comp. Neurol. 145, 465–514 44 Rajan, D.P. et al. (1999) Human placental sodium-dependent vitamin C transporter (SVCT2): molecular cloning and transport function. Biochem. Biophys. Res. Comm. 262, 762–768

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Acknowledgements The author gratefully acknowledges the generosity of Matthias A. Hediger and Urs V. Berger in providing photomicrographs for Fig. 3, and appreciates helpful discussions about SVCT2 and properties of ascorbate uptake with Berger. Critical reading of the manuscript by colleagues, including Marat Avshalumov, Duncan MacGregor, Charles Nicholson and Billy Chen, is also gratefully acknowledged. The author’s research is funded by grants from the NIH/NINDS (NS-34115 and NS-36362).

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45 Siesjö, B.K. (1980) Regional metabolic rates in the brain. In Brain Energy Metabolism, pp. 131–150, Wiley 46 Lutz, P.L. (1992) Mechanisms for anoxic survival in the vertebrate brain. Annu. Rev. Physiol. 54, 601–618 47 Sick, T.J. et al. (1992) Maintaining coupled metabolism and membrane function in anoxic brain: A comparison between turtle and rat. In Surviving Hypoxia (Hochachka, P. et al., eds), pp. 351–363, CRC 48 Rice, M.E. and Cammack, J. (1991) Anoxia-resistant turtle brain maintains ascorbic acid content in vitro. Neurosci. Lett. 132, 141–145 49 Halliwell, B. (1996) Vitamin C: antioxidant or pro-oxidant in vivo. Free Radic. Res. 25, 439–454 50 Hillered, L. et al. (1988) Increased extracellular levels of ascorbate in striatum after middle cerebral artery occlusion in the rat monitored by intracerebral microdialysis. Neurosci. Lett. 95, 286–290 51 Lyrer, P. et al. (1991) Levels of low molecular weight scavengers in the rat brain during focal ischemia. Brain Res. 567, 317–320 52 Cao, W. et al. (1988) Oxygen free radical involvement in ischemia and reperfusion injury to brain. Neurosci. Lett. 88, 233–238 53 Sciamanna, M.A. and Lee, C.P. (1993) Ischemia/reperfusioninduced injury of forebrain mitochondria and protection by ascorbate. Arch. Biochem. Biophys. 305, 215–224 54 Ranjan, A. et al. (1993) Ascorbic acid and focal ischaemia in a primate model. Acta Neurochir. 123, 87–91 55 Henry, P.T. and Chandry, M.J. (1998) Effect of ascorbic acid on infarct size in experimental focal cerebral ischemia and reperfusion in a primate model. Acta Neurochir. 140, 977–980 56 Pérez-Pinzón, M.A. et al. (1997) Brain Res. 754, 163–170 57 Dykens, J.A. et al. (1987) Mechanism of kainate toxicity to cerebellar neurons in vitro is analogous to reperfusion tissue injury. J. Neurochem. 49, 1222–1228 58 Coyle, J.T. and Puttfarcken, P. (1993) Oxidative stress, glutamate, and neurodegenerative disorders. Science 262, 689–695

59 Lafon-Cazal, M. et al. (1993) NMDA-dependent superoxide production and neurotoxicity. Nature 354, 535–537 60 Prehn, J.H. (1998) Mitochondrial transmembrane potential and free radical production in excitotoxic neurodegeneration. NaunynSchmiedebergs’s Arch. Pharmacol. 357, 316–322 61 Ciani, E. et al. (1996) Inhibition of free radical production or free radical scavenging protects from the excitotoxic cell death mediated by glutamate in cultures of cerebellar granule cells. Brain Res. 728, 1–6 62 Atlante, A. et al. (1997) Glutamate neurotoxicity in rat cerebellar granule cells: a major role for xanthine oxidase in oxygen radical formation. J. Neurochem. 68, 2038–2045 63 Brahma, B. et al. (2000) Ascorbate inhibits edema in brain slices. J. Neurochem. 74, 1263–1270 64 Sato, K. et al. (1993) Synergism of tocopherol and ascorbate on the survival of cultured neurones. NeuroReport 4, 1179–1182 65 Sharma, P. (1997) Consequences of hypoxia on the cell size of neuropeptide-Y neurons and the role of ascorbate in cultured neurons from chick embryo. Neurochem. Int. 30, 337–344 66 Majewska, M.D. et al. (1990) Regulation of the NMDA receptor by redox phenomena: inhibitory role of ascorbate. Brain Res. 537, 328–332 67 Buettner, G.R. (1993) The pecking order of free radicals and antioxidants: lipid peroxidation, a-tocopherol, and ascorbate. Arch. Biochem. Biophys. 300, 535–543 68 Kuo, C-H. et al. (1978) Subcellular distribution of ascorbic acid in rat brain. Jpn. J. Pharmacol. 28, 789–791 69 Glembotski, C.C. (1987) The role of ascorbic acid in the biosynthesis of the neuroendocrine peptides α-MSH and TRH. Ann. New York Acad. Sci. 498, 54–62 70 Miller, B.T. and Cicero, T.J. (1987) Ascorbate-induced release of LHRH: noradrenergic and opioid modulation. Brain Res. Bull. 19, 95–99 71 Eldridge, C.F. et al. (1987) Differentiation of axon-related Schwann cells in vitro. I. Ascorbic acid regulates basal lamina assembly and myelin formation. J. Cell Biol. 105, 1023–1034

Resonance, oscillation and the intrinsic frequency preferences of neurons Bruce Hutcheon and Yosef Yarom

Bruce Hutcheon is at the Institute for Biological Science, National Research Council of Canada, Ottawa, Canada K1A 0R6, and Yosef Yarom is at the Dept of Neurobiology, and the Center for Neuronal Computation, Hebrew University, Jerusalem, Israel 91904.

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The realization that different behavioural and perceptual states of the brain are associated with different brain rhythms has sparked growing interest in the oscillatory behaviours of neurons. Recent research has uncovered a close association between electrical oscillations and resonance in neurons. Resonance is an easily measurable property that describes the ability of neurons to respond selectively to inputs at preferred frequencies. A variety of ionic mechanisms support resonance and oscillation in neurons. Understanding the basic principles involved in the production of resonance allows for a simplified classification of these mechanisms. The characterization of resonance and frequency preference captures those essential properties of neurons that can serve as a substrate for coordinating network activity around a particular frequency in the brain. Trends Neurosci. (2000) 23, 216–222

T

HE WORKING BRAIN is characterized by the rhythmic activation of large numbers of its neurons on characteristic temporal and spatial scales. These modes of coherent activity appear as the various brain rhythms. A series of firmly established empirical associations with the behavioural states of organisms provides compelling evidence that brain rhythms reflect basic modes of dynamical organization in the brain1. However, the mechanisms that bind neurons into these rhythmical coherent ensembles are not well understood. What determines the characteristic frequency range of each brain rhythm? Broadly speaking, there are two TINS Vol. 23, No. 5, 2000

types of explanation. One invokes patterns of connectivity between neurons and the dynamic properties of the intervening synapses. For example, reverberating activity within re-entrant neural circuits could result in the rhythmic activation of fundamentally nonoscillatory neurons within well-defined frequency bands2. A different explanation states that network rhythmicity arises via the coupling of oscillatory subunits, each of which possesses an intrinsically determined frequency preference3. These two explanations are not mutually exclusive (network connectivity could reinforce the patterns of excitation produced by coupled

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