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Michael AC, Borland LM, editors. Electrochemical Methods for Neuroscience. Boca Raton (FL): CRC Press/Taylor & Francis; 2007.

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Electrochemical Methods for Neuroscience.

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Chapter 9From Interferant Anion to Neuromodulator: Ascorbate Oxidizes its Way to Respectability



Ascorbate, the deprotonated form of ascorbic acid, or vitamin C, is an ideal substance for detection by in vivo voltammetry. As an antioxidant, ascorbate readily gives up electrons, a fundamental requirement for voltammetric detection. In addition, the principal target of voltammetric studies is the extra-cellular fluid of the brain where ascorbate exists in plentiful amounts. In fact, the extracellular concentration of brain ascorbate is much higher than that of most other molecules of interest, making it relatively easy to detect and to monitor over time. A related point is that the level of extracellular brain ascorbate is not static but fluctuates with behavior, and there is mounting evidence that these fluctuations are related to the release of glutamate, an amino acid transmitter responsible for most neuronal excitations. In short, an understanding of ascorbate fluctuations is likely to reveal important information about how the brain operates.

From the very beginning, however, voltammetry was aimed at studying the catecholamine and indoleamine transmitters. Not only were they, like ascorbate, easily oxidizable but also they appeared to play critical roles in such fundamental functions as attention, cognition, mood, motivation, and motor control. Indeed, these transmitters, collectively known as the mono-amines, have been the center of neurochemical attention since the 1950s. And so, when Ralph Adams and colleagues first used voltammetric techniques to study brain chemistry, the monoamines were a key target [1,2]. Ascorbate, far from being a molecule of interest, gained recognition, instead, as an interferent anion. Its high extracellular concentration, at more than 1000 times that of the monoamines, complicated monoamine detection. Strategies soon were developed to improve monoamine selectivity and, when applied carefully and appropriately, as described in other chapters in this volume, they helped launch a new appreciation of the complex role that the monoamines play in shaping brain function.

But ascorbate, too, has had a renaissance in neuroscience because of voltammetry. Improvements in electrochemical detection have made it possible to study ascorbate independently of other oxidizable molecules and have suggested functions well beyond those of a simple antioxidant. Other experimental approaches have begun to confirm an expanded role for brain ascorbate. Slowly but steadily, ascorbate has been gaining respect as a versatile molecule linked to the extracellular modulation of glutamate and, coming full-circle, perhaps the monoamines as well. This chapter focuses on how voltammetry helped to shape the ascorbate renaissance.

Basic Chemistry

Isolation and Tissue Availability

Ascorbate is a hexuronic sugar acid, first isolated from adrenal glands and cabbages by Szent- Györgyi in 1928. A few years later, King identified the same molecule in orange juice. It was named ascorbate when it became clear that in humans this molecule could prevent scurvy, a condition whose symptoms include weak and swollen limbs, bleeding gums, hemorrhages under the skin, and a failure of wounds to heal. Scorbutic is the term used to describe scurvy, and thus an ascorbutic molecule (i.e., ascorbate) prevents or counteracts this condition. Although the symptoms of scurvy have been known for at least a thousand years, the underlying cause was first studied systematically in eighteenth century British sailors who had no access to fresh fruits and vegetables during long sea voyages [3]. The introduction of lemon juice into the diet of the British Navy virtually eliminated scurvy in this population.

Humans are vulnerable to this disease because, unlike most animals, they are unable to synthesize ascorbate from glucose. The problem is a non-functional gene for gulonolactone oxidase, the enzyme that catalyzes the final step in this process. Without ascorbate, collagen, the most abundant fibrous protein in the body, becomes malformed and gives rise to fragile connective tissue and blood vessels. The subsequent identification of scurvy in the guinea pig, another animal that fails to synthesize ascorbate, allowed various foods to be tested for ascorbutic potency. The most effective foods, citrus fruits and red and green vegetables, are rich sources of ascorbate.

Although humans and guinea pigs belong to a select group of animals, including other primates, fruit bats, and passeriform birds, that must forage for ascorbate, its concentration in various tissues is strikingly similar to that reported for animals that synthesize it. Dietary ascorbate is absorbed from the gut, but animals equipped with the appropriate synthetic machinery produce ascorbate either in the liver (mammals) or kidneys (reptiles). Regardless of the source, ascorbate enters the bloodstream where it can be taken up and stored by all relevant tissues. Apart from the adrenals, the brain contains the highest tissue level of ascorbate. Blood-borne ascorbate enters brain tissue from the ventricular system [4]. A selective transport mechanism in the choroid plexus ensures a high ventricular concentration even when blood ascorbate levels are low, making it virtually impossible to create an ascorbate deficiency in an otherwise healthy animal. Even in the scorbutic guinea pig, brain ascorbate declines very slowly relative to ascorbate in other organs. On the other hand, when blood ascorbate levels are unusually high, ascorbate can enter the brain by simple diffusion [5]. Neurons accumulate ascorbate via a sodium-dependent transporter [6] subsequently identified as SVCT2 [7]. Because glial cells lack this transporter [8], they may not be a major ascorbate storage site [9], but a glial role in ascorbate release is still possible (see below).

The distribution of ascorbate in adult mammalian brain is remarkably heterogeneous. In fore-brain structures such as the striatum, a region of the basal ganglia that integrates information from the entire cortical mantle, the tissue level of ascorbate is as much as 50% higher than in some midbrain and hindbrain areas [10]. Most ascorbate is found intracellularly, but the level of ascorbate in extra-cellular fluid is maintained at the expense of intracellular stores [11]. In striatum, interestingly, extracellular ascorbate is highest in dorsal and ventral regions, suggesting heterogeneity in release [12].


Because it has two dissociable protons with pKa values of 4.2 and 11.8, ascorbate is a monovalent anion at physiological pH. The most striking chemical property of ascorbate is ease of oxidation, which arises from its enediol structure conjugated to a carbonyl. This capacity to donate electrons allows ascorbate to function as a co-factor for several enzymes that contain either copper or iron at their active sites. Two of these iron-containing enzymes, proline hydoxylase and lysine hydroxylase, are essential for the synthesis of collagen [13]. A well-known copper-containing enzyme, dopamine-β-hydroxylase, converts dopamine into norepinephrine [14].

As an electron donor, ascorbate also protects against the damaging effects of free radicals generated by normal biological activity [15–17]. Most of these free radicals have one unpaired electron and thus can be easily neutralized by the one-electron oxidation of ascorbate. This reaction forms semi-dehydroascorbate or the ascorbyl radical. Loss of a second electron forms dehydroascorbate, the final oxidation product. Both forms of oxidized ascorbate, semi-dehydroascorbate and dehydroascorbate, can be returned to their original, reduced form by glutathione (GSH), a low-molecular- mass, thiol-containing tri-peptide, and by dehydroascorbate reductase, a GSH-dependent enzyme found in brain. Because ascorbate is water soluble, it works in the aqueous phase of tissue to scavenge hydroxyl- and peroxyl-radicals, peroxynitrite, singlet oxygen, and superoxide. Ascorbate also can stop the oxidation of cellular membranes by recycling or preventing the oxidation of vitamin E (α-tocopherol), which is lipid soluble. Ascorbate, therefore, appears to be a critical part of the cellular antioxidant network.

It is also the case, however, that ascorbate combined with copper or iron will promote free-radical production and thus act as a pro-oxidant. Combining a high level of ascorbate with either of these metals in the stomach, for example, generates hydroxyl radicals. Thus, ascorbate in some vitamin pills may have a pro-oxidant effect if they dissolve in the presence of copper or iron ions [18]. A key issue, therefore, is the availability of these ions in vivo. Fortunately, in most cellular systems, these ions are largely sequestered and unable to catalyze free radical reactions. In fact, the presence of ascorbate may actually facilitate iron sequestration. Moreover, extracellular fluid, which contains abundant amounts of ascorbate, is almost completely devoid of copper and iron ions. Thus, although an increase in the normal extracellular level of either ascorbate or one of these reactive metals could elevate the risk of free radical damage, this outcome is unlikely under normal conditions. It could occur, however, in certain pathological states such as iron-overload or tissue injury, both of which increase the availability of transition metal ions and thus increase the likelihood of an interaction with ascorbate [19].

Ascorbate Electrochemistry

In essence, electrochemistry is a form of chemical measurement that occurs at a surface-solution interface. Voltammetry is a special case in which the variable of interest is the current recorded at the surface of a working electrode. The current is generated by an applied potential, which causes oxidation or reduction of a chemical species in solution. The amount of recorded current is directly proportional to the concentration of the chemical species. The identification of ascorbate is based on current-voltage curves and their position along the voltage axis. This section focuses on the oxidative voltammetry of ascorbate in brain extracellular fluid and what this and other experimental approaches have revealed about the mechanisms underlying ascorbate release.


Classical electroanalytical methodology uses various types of carbon-based electrodes to measure oxidation current in response to an applied potential. Separating the ascorbate oxidation signal from that generated by the monoamines became a significant challenge when voltammetry was first used to study brain chemistry [2]. Signals attributed to dopamine, for example, likely included a major contribution from ascorbate. Only when ascorbate was detected independently of other oxidizable substances and shown to fluctuate with behavior did it become clear that ascorbate deserved further study in its own right [20]. The first step in this direction came with development of carbon-paste electrodes that could distinguish ascorbate from other easily oxidized compounds in the brain [21]. Used in conjunction with linear sweep voltammetry, these electrodes revealed a distinct oxidation peak at ~ +50 mV when a potential was applied from −100 to +500 mV versus a Ag/AgCl reference. Ascorbate appeared to be the main component of this peak since the infusion of additional ascorbate in the immediate vicinity of the carbon-paste electrode selectively increased the signal. Confirmation came from evidence that local infusions of ascorbate oxidase, a dimeric copper-requiring enzyme that removes ascorbate by oxidizing it to dehydroascorbate, had the opposite effect. In fact, because dehydroascorbate is electrochemically inert, ascorbate oxidase significantly attenuated the signal.

Carbon-paste electrodes are made by packing carbon paste into a Teflon® (E.I. Du Pont de Nemours & Company, Inc., Wilmington, DE) tube or pulled-glass capillary. A similar but slightly more robust electrode is made from carbon epoxy. In either case, diameters typically range from 50 to 200 μm. Although recipes for carbon-paste and carbon-epoxy mixtures are available [22,23], the relatively large size of these electrodes presents certain electrochemical and physiological complications that can be avoided with the use of electrodes whose active recording area is the exposed tip of a small-diameter carbon fiber [24].

Formed from the high-temperature pyrolysis of polyacrylonitrile (PAN) or, in some cases, pitch, carbon fibers come in large bundles for use in a wide range of commercial applications. PAN-based fibers, for example, are used in composites to make extremely strong yet lightweight materials, such as fishing rods and airplane wings. Pitch-based fibers are not used for critical structural applications because of their low mechanical properties. Fiber diameters of 5–30 μm are preferred for voltammetry because such a small size minimizes perturbation of the brain environment and also permits a high degree of spatial resolution. Thornel P-55 carbon fibers, which are PAN-based and supplied by BP Amoco Chemicals, have individual diameters of ~10 μm and are perhaps the most commonly used. For electrochemistry, a single fiber is inserted in a pulled-glass capillary and positioned to extend beyond the glass tip by 150–200 μm. Sealing the fiber in the glass can be accomplished by injecting an epoxy resin into the non-pulled end of the capillary; micro-filaments in the glass draw the resin into the pulled tip where it hardens and forms an effective seal. The resin is made of Shell EPON Resin 828 (distributed by Miller-Stephenson) mixed with a small amount of ortho-phenylenediamine (Mallinckrodt, St. Louis, MO) to ensure reasonably rapid hardening; if the resin sets too rapidly, which occurs when meta-phenylenediamine is used, the seal will not reach the tip, thus exposing a large segment of the fiber within the glass capillary to extracellular fluid. After the fiber is sealed in the glass tip, conductive metal is inserted into the non-pulled end of the glass to establish electrical contact between the fiber and the recording equipment. Although mercury has been used for this purpose, safety concerns have largely replaced this metal with a bismuth alloy (Small Parts, Inc.), which melts at >70°C. Thus, heating the glass capillary liquefies the alloy and allows a connecting wire to be inserted. As the capillary cools, the alloy hardens, and the electrode is ready for use. A typical carbon-fiber electrode is shown schematically in Figure 9.1.

FIGURE 9.1. Schematic representation (not to scale) of the working electrode carbon fiber installed in the head-mounted microdrive assembly (left) and an expanded view of the electrode tip (right).


Schematic representation (not to scale) of the working electrode carbon fiber installed in the head-mounted microdrive assembly (left) and an expanded view of the electrode tip (right). The microdrive assembly mates with a teflon hub secured to the skull (more...)

To become selective for ascorbate, the exposed fiber requires electrochemical pretreatment in phosphate-buffered saline, a procedure pioneered by Gonon et al. [25]. Although several variations of the pretreatment protocol are now in use, the basic procedure is to apply a 70-Hz triangular wave from zero to up to +2.5 V. The result is a shift in the oxidation potential of ascorbate to a lower value distinct from that for catechols such as dopamine and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC). The higher the triangular-wave voltage, the greater the ascorbate shift away from the catechols, but a voltage >+2.5 V appears to move ascorbate oxidation toward other interfant signals. A related point is that the longer the duration of the triangular wave application during pretreatment, the longer the electrode appears to maintain its selectivity for ascorbate and DOPAC in vivo. In light of these considerations, a recently developed protocol includes two applications of the 70-Hz triangular wave (versus a saturated calomel reference and stainless steel auxiliary): first, for 140 s from zero to +2.1 V and then for 5 s from zero to 2.5 V. Each application, moreover, is followed by 20 s of a constant potential at 1.5 V. In striatum, where most assessments of extracellular ascorbate have taken place, DOPAC is the dominant catechol, accounting for >95% of the catechol peak [26]. Thus, for electrode testing prior to in vivo use, ascorbate and DOPAC are added to the buffer at concentrations that reflect their relative physiological proportions. Buffer content also may affect the extent to which the electrochemical pretreatment separates the ascorbate and DOPAC signals. Phosphate-buffered saline, for example, may require slightly altered time and voltage applications.

The effects of electrochemical pretreatment are evident in Figure 9.2, which depicts voltammograms obtained in citrate-phosphate buffer containing 100 μM ascorbate and 20 μM DOPAC. Note that the untreated electrode is unable to distinguish ascorbate from DOPAC such that both are included in a single oxidation peak; the small size of the peak, moreover, indicates minimal sensitivity. When the same electrode is tested again after application of the triangular wave— even for a few seconds—sensitivity improves and the previously homogeneous oxidation peak moves to a lower potential as separate peaks for ascorbate and DOPAC begin to form. Very distinct peaks emerge when the fiber is exposed to the full pretreatment protocol; a difference of at least 150 mV is evident between each peak. Although it is unclear why electrochemical pretreatment has this effect, a change in the surface properties of the carbon is a likely explanation [27].

FIGURE 9.2. Individual voltammograms obtained in citrate-phosphate buffer containing 100 μM ascorbate and 20 μM DOPAC.


Individual voltammograms obtained in citrate-phosphate buffer containing 100 μM ascorbate and 20 μM DOPAC. Voltammograms were obtained before electrochemical pretreatment (Unzapped), after a brief pretreatment exposure (Partial zap: 3 (more...)

Variations on the electrochemical pretreatment protocol have been used to improve the selectivity of carbon-paste and carbon-epoxy electrodes [27,28]. For each electrode type, the goal of electrochemical pretreatment is to shift the oxidation potential of ascorbate away from that for catechols. Another effect of electrochemical pretreatment is the emergence of a third in vivo peak at applied potentials >200 mV that represents the combined oxidation of serotonin, 5-hydroxyindoleacetic acid (5-HIAA, the serotonin metabolite), and uric acid.

Although the irreversible nature of ascorbate oxidation allows it to be distinguished from catechols at untreated carbon-fiber electrodes used for fast-scan cyclic voltammetry [29], electrochemical monitoring of ascorbate in brain extracellular fluid is typically performed with electrochemically pretreated carbon fibers scanned at a relatively slow rate (<100 mV/s). The applied potential typically sweeps from −200 to +600 mV for 30 s at a rate of 20–30 mV/s. Slow scanning provides a stable oxidation signal for extracellular ascorbate that can be monitored continuously on a minute-by-minute basis for more than two hours without significant loss of sensitivity. A common procedure for slow-scan voltammetry is to apply the potential in a stepwise or staircase fashion while calculating a local differentiation of the oxidation wave by recording the difference of the oxidation currents sampled just before and after each step. The entire scan lasts for 60 s as the potential is applied for 30 s in each direction. For in vivo recording, the reference electrode is typically a Ag/AgCl wire in contact with brain extracellular fluid.

Extracellular Changes

Rodents have been the animals of choice for voltammetric monitoring of brain extracellular ascorbate, largely because they have been so valuable in revealing the extracellular dynamics (i.e., the release, reuptake, and diffusion properties) of the monoamine transmitters. In fact, because the monoamines appear to play such a key role in behavior, it was a natural extension of this line of research to make parallel assessments of extracellular ascorbate. Most of these investigations, moreover, have focused on striatum. Although dopamine and glutamate are known to play key roles in striatal function, it is difficult to ignore evidence that the extracellular level of striatal ascorbate changes with behavioral state. In resting rats, for example, the level typically hovers at ~300 μM but may double during periods of motor activation such as the nocturnal phase of the circadian cycle [30] or following administration of amphetamine or other dopamine agonists [31,32]. Such data, however, do not establish a causal link between ascorbate release and behavioral activation. Although additional support comes from evidence that scorbutic guinea pigs suffer from behavioral depression, this effect is confounded by the poor health of such animals [33]. To make a direct assessment of the relationship between endogenous striatal ascorbate release and behavior, we infused ascorbate oxidase or vehicle bilaterally into the striatum of rats tested in an open-field arena equipped with novel objects and populated with other rats [34]. Motor activation patterns were assessed along with the frequency of approach of novel objects and social interactions. The extent of ascorbate depletion was monitored simultaneously with slow-scan voltammetry, which also was used to measure DOPAC.

Intra-striatal infusion of ascorbate oxidase caused a rapid and pronounced decline in extra-cellular ascorbate. Within 20 min after infusion onset, a >50% decline was evident in all tested animals, and this deficit persisted throughout the recording session, which lasted for 60 min. Vehicle infusions had no effect on striatal ascorbate. The amplitude of the DOPAC signal was largely unchanged after infusion of either ascorbate oxidase or vehicle. Because ascorbate oxidase uses ascorbate as a cofactor in the enzymatic conversion of molecular oxygen to water, there is the possibility of oxygen depletion and subsequent cell loss due to hypoxic injury. In some cases, therefore, histological assessment of striatal tissue was carried out one or two days after ascorbate oxidase or vehicle infusion. No evidence of cell damage other than that typically observed along the infusion tract was found.

Loss of striatal ascorbate led to a near-total inhibition of all recorded behavior, including open-field locomotion, approach of novel objects, and social interaction with other rats. Episodes of simple movement, when they occurred, were limited to brief (<5 s) bouts of head bobbing or turning. No locomotion or rearing was observed in any of these animals as soon as 25 min after ascorbate oxidase infusion. Vehicle controls, in contrast, maintained baseline levels of responsiveness throughout the recording period. Because ascorbate oxidase may disrupt striatal functioning as a result of its relatively large size as a glycoprotein (140,000 kDa), an inactive form of the enzyme was infused as a second control. The inactive enzyme mimicked the effect of vehicle, ruling out an effect of molecular size. To determine if a loss of striatal ascorbate simply changed the dynamics of extracellular ascorbate throughout the brain and thus altered behavior through an extra-striatal mechanism, some rats received bilateral infusions of ascorbate oxidase into dorsal hippocampus, another forebrain area with a high level of extracellular ascorbate. These animals, however, maintained normal behavioral activity, further implicating the loss of striatal ascorbate in the behavioral deficit.

Systemic administration (sc) of 1.0 mg/kg D-amphetamine immediately after the intra-striatal infusion of ascorbate oxidase in separate animals reversed the decline in ascorbate and restored the extracellular level to pre-infusion values. Concomitantly, behavioral activation in these animals returned to baseline and surpassed it by the end of the recording session. Although removal of ascorbate from striatal extracellular fluid may increase susceptibility to oxidative damage, this effect cannot explain the behavioral deficits induced by ascorbate oxidase given their rapid onset and their equally rapid reversal by amphetamine. In fact, our behavioral results with amphetamine rule out the possibility that ascorbate oxidase inhibits behavior non-selectively by binding to dopamine receptors or the dopamine molecule. A related point is that ascorbate oxidase failed to alter striatal DOPAC, arguing against a loss of dopamine. It appears, therefore, that normal behavioral output depends on a critical level of ascorbate in striatal extracellular fluid [34]. Insight into the neural mechanisms controlling the release of ascorbate has emerged from work on glutamate transmission.

Glutamate-Related Mechanisms of Striatal Ascorbate Release

The glutamate projection to the striatum arises primarily from cerebral cortex [35–37]. All cortical areas contribute to this projection, which originates bilaterally from both supragranular and infragranular layers. Corticostriatal neurons comprise the major source of extracellular ascorbate. Cortical ablations, for example, lower the level of extracellular ascorbate in striatum by >70% [38,39], whereas intra-striatal infusion of kainic acid, which destroys intrinsic cells, has no such effect [40]. Conversely, increases in striatal glutamate transmission enhance the release of ascorbate [41,42]. Taken together, these lines of evidence suggest that the level of extracellular ascorbate is closely linked to activity in glutamate afferents arising from cerebral cortex. Evidence also suggests that ascorbate is released by a heteroexchange mechanism involving the uptake of glutamate [20,21,43]. According to this model, glutamate uptake triggers the outward transport of ascorbate. Thus, stimuli that activate corticostriatal neurons and release glutamate also activate glutamate transport, which, in turn, increases ascorbate release. The model also suggests that a high level of extracellular ascorbate would oppose further ascorbate release and thus prevent glutamate uptake. Indeed, a 500 μM increase in striatal ascorbate not only elevated basal extracellular glutamate but also significantly prolonged the disappearance of glutamate release evoked by cortical stimulation [44]. Because glia, like neurons, express glutamate transporters [45–47], the heteroexchange model also may apply to one or more types of glial cells, but this is uncertain [41]. Regardless of the mechanism, however, activation of the corticostriatal pathway is mainly responsible for ascorbate release in striatum.

Although drugs that enhance dopamine transmission also promote striatal ascorbate release [32,48,49], this effect is due to activation of corticostriatal afferents rather than a direct change in striatal dopamine signaling. This conclusion comes mainly from evidence that neurotoxic loss of striatal dopamine terminals has little or no effect on striatal ascorbate release, whereas prevention of cortical activation by disruption of thalamo-cortical afferents blocks this process [50–53]. It also is interesting that other dopamine-rich forebrain areas, such as the nucleus accumbens and medial prefrontal cortex, show relatively little behavior- or drug-related change in ascorbate release, even though the basal level of ascorbate in these areas matches that in striatum [54,55]. Ascorbate release from corticostriatal afferents, therefore, may play a unique behavioral role. Identifying the functional significance of this release is likely to shed new light on basal ganglia operations in health and disease.

Ascorbate Modulation of Glutamate-Evoked Neuronal Signaling

In general terms, the striatum processes cortical information and routes it via both mono- and multi-synaptic connections to basal ganglia output structures to influence motor control. Thus, the striatum is strategically positioned to shape motor output, and, not surprisingly, the striatum plays key roles in motor memory as well as habit learning and retrieval [56–58]. As the main information- processing unit of the striatum, the medium spiny projection neuron responds to cortical activation with a glutamate-mediated excitation. In fact, cortical input is necessary to maintain striatal neurons in a depolarized or “up” state required for impulse activity [59]. Dopamine-containing afferents, arising from the ventral midbrain, modulate cortical input by adjusting the strength of the glutamate response. When applied iontophoretically at low concentrations in resting rats, for example, dopamine enhances glutamate-induced excitations by increasing the magnitude of the glutamate signal relative to background activity [60]. From striatum, information is routed via direct and indirect projections to the entopeduncular nucleus (internal globus pallidus in primates) and substantia nigra reticulata (SNr), which together form the basal ganglia output to thalamo-cortical and downstream motor pathways.

Preliminary experiments with ascorbate iontophoresis also showed a potentiation of the excitatory effects of glutamate on striatal neurons [61,62]. To determine if this effect, like that of dopamine, could be explained by a change in the glutamate signal-to-noise ratio, a detailed assessment of the ascorbate-glutamate interaction was performed [63]. First, the effects of ascorbate iontophoresis were tested on spontaneously active units as well as on silent or sporadically active units driven by glutamate. To control for antioxidant effects, D-iso-ascorbate was substituted for the naturally occurring isomer in some cases. The data revealed that brief (20 s) applications of either ascorbate or D-iso-ascorbate at low ejection currents (5–40 nA) typically had no effect on either spontaneously active or glutamate-driven units. When ascorbate was applied at these currents for two to four minutes, however, glutamate-induced excitations were altered dramatically. For example, prolonged applications of 5–40 nA ascorbate, which often failed to alter basal activity, significantly increased the excitatory response to brief pulses of glutamate. Further analysis revealed that ascorbate enhanced both the onset and absolute magnitude of the glutamate response. In contrast, prolonged application of D-iso-ascorbate at the same ejection currents never potentiated the action of glutamate. In fact, D-iso-ascorbate often attenuated the glutamate response. Interestingly, ascorbate had an attenuating effect only at high ejection currents (>80 nA), which have been shown by concomitant voltammetric measurements to increase extracellular concentrations above 1.0 mM [61]. Thus, at extremely high concentrations, ascorbate appears to antagonize glutamate transmission through a non-specific antioxidant effect [64]. This interpretation is supported by recent evidence that systemic injection of high ascorbate doses (500 mg/kg and 1000 mg/kg) in freely behaving rats not only elevates striatal ascorbate beyond 1.0 mM but also suppresses the responsiveness of striatal neurons to sensorimotor stimulation [65]. In fact, treatment with 1000 mg/kg ascorbate reversed many stimulation-evoked excitations to inhibitions.

It also is interesting that the modulatory role of ascorbate differs from that reported for dopamine. For example, although iontophoretic dopamine can enhance the glutamate response, this effect is manifest as an increase in the glutamate-evoked signal relative to background activity rather than an increase in the absolute magnitude of the signal [66]. Ascorbate, therefore, appears to play a unique role in modulating the strength of glutamate-induced excitations. Because behavior-related increases in striatal ascorbate parallel the ascorbate increases achieved with low-to-moderate iontophoretic doses, behaviorally relevant fluctuations in extracellular ascorbate are likely to exert a critical influence on the flow of glutamate-mediated information through striatal circuits.

Ascorbate and Huntington’s Disease

In an interesting extension of evidence that striatal ascorbate is essential for normal behavioral output [34], mice that model Huntington’s disease (HD), a genetic disorder characterized by striatal degeneration and severe behavioral deficits, suffer from a loss of ascorbate in striatal extracellular fluid [67]. Even more intriguing is evidence that some of the neurological motor signs that these mice display improve upon reversal of the ascorbate deficit [68]. Elevating striatal ascorbate also reverses abnormal electrophysiological activity in HD striatum [69]. This section reviews emerging evidence implicating abnormal striatal ascorbate in HD genetic mouse models.

HD and Transgenic Mice

HD is an autosomal dominant condition caused by an expanded tri-nucleotide (CAG) repeat, which results in the increased length of a poly-glutamine (polyQ) tract in the huntingtin protein [70]. Although normal huntingtin function can tolerate up to thirty-five or thirty-six repeats, just one more is enough to cross the pathogenic threshold. Like other polyQ disorders, juvenile-onset HD is associated with longer polyQ tracts than adult-onset cases. Typical onset occurs in middle-age. Huntingtin is widely expressed throughout the nervous system, and, accordingly, all brain regions show some atrophy in HD. The most pronounced neuronal pathology, however, strikes the medium spiny neurons of the striatum.

The function of the huntingtin protein is far from understood, but it may have anti-apoptotic properties. In HD, mutated huntingtin appears to acquire a neurotoxic function, which may be related to the formation of intra-nuclear inclusions, a disruption of gene transcription, or abnormal interactions with other proteins [71,72]. At the synaptic level, the polyQ expansion is likely to result in aberrant transmission that causes toxic insults to vulnerable neuronal populations. These and other insights into HD pathology have come from genetic mouse models. The most extensively characterized are the R6 lines, which contain an exon-1 transgene [73]. The behavioral phenotype and neuropathology have been studied most thoroughly in the R6/2 line, which carries ~150 CAG repeats. These animals develop motor abnormalities (e.g., tics, stereotyped and choreiform movements, and tremor) as early as five or six weeks of age [74] and show intra-nuclear inclusions of huntingtin conjugated to ubiquitin [75]. Although the R6/2 striatum does not show marked cell loss, there is evidence of late-onset neuronal degeneration, striatal atrophy, and decreased expression of striatal signaling genes [71,76]. We have been using this line to assess possible HD-related abnormalities in the regulation of extracellular striatal ascorbate [67].

Ascorbate Dysregulation in HD Striatum

Recording voltammetric signals from behaving mice requires some modification of the head-mounted assembly that has been used successfully in studies of behaving rats [77–79]. A key component is a lightweight (1.5 g) bilateral micro-drive system fashioned from two nylon rods as described elsewhere [80]. The bilateral design allows for a working electrode to be lowered into either the left or right target structure (e.g., striatum), while a reference electrode is positioned on the brain surface of the contralateral hemisphere. As shown in Figure 9.3, the recording system fits conveniently on the head to permit noise-free voltammetry. Because the working electrode is lowered acutely on the recording day, we avoid the loss of sensitivity and other complications associated with chronically implanted voltammetric electrodes [81].

FIGURE 9.3. Digital photograph of a freely behaving mouse prepared for voltammetric recording.


Digital photograph of a freely behaving mouse prepared for voltammetric recording. The bilateral microdrive system allows for lowering the working electrode into the target structure on the recording day, while the reference electrode is positioned at (more...)

At the beginning of each voltammetry session, all mice are anesthetized to allow electrode placement in striatum. While under anesthesia (30–60 min), both wild-type and transgenic mice show no difference in signal amplitude for either ascorbate or DOPAC. As recording continues over the next 60–90 min, however, clear ascorbate differences emerge as both groups of mice become behaviorally active. Thus, whereas wild-type animals show the expected behavior-related increase in ascorbate release, HD mice respond with an ascorbate decrease that in some cases represents a decline of >50% from anesthesia baseline [67]. Figure 9.4 illustrates this difference recorded from representative wild-type and HD mice during and after recovery from anesthesia. This behavior-related divergence in the ascorbate signal is evident at six weeks of age—the earliest recording age to date—and is present two or three weeks later, emphasizing the persistence of the effect [67].

FIGURE 9.4. Individual voltammograms obtained from the striatum of a transgenic (HD) and a wild-type (WT) mouse during anesthesia and subsequent waking (behaving).


Individual voltammograms obtained from the striatum of a transgenic (HD) and a wild-type (WT) mouse during anesthesia and subsequent waking (behaving). Note the typical increase in the ascorbate signal associated with behavioral arousal in the WT mouse (more...)

Behavioral assessments indicate that relative to wild-type mice, HD transgenics show a more restricted range of motor responses [68]. HD mice, for example, move in fixed, stereotypic patterns and spend less time interacting with the environment. These animals also show hind-paw flicks, which appear as an aborted grooming maneuver but are considered an HD neurological sign [74]. Repeated daily injections of ascorbate (300 mg/kg, ip), however, restore the loss of behavior-related striatal ascorbate release and improve motor behavior [68]. Ascorbate-treated HD mice, for example, significantly increase turning probability in a plus maze, a measure of motor flexibility, and decrease hind-paw flicks compared to vehicle-treated transgenics. These groups did not differ, however, on total locomotion arguing against an ascorbate-induced suppression of overall motor activity. It also is interesting that treatment of wild-type mice with ascorbate had no significant effect on either striatal ascorbate release or behavior indicating that under normal conditions this dose of ascorbate does not easily penetrate brain tissue.

At the neuronal level, HD striatum appears hyperactive. An assessment of the distribution of striatal firing rates in R6/2 and wild-type mice revealed that transgenics had significantly more fast-firing units [69]. Although both groups modulated unit activity with behavioral state such that anesthesia slowed and subsequent behavioral recovery elevated unit firing, R6/2 neurons consistently discharged above the wild-type rate. Again, however, multiple injections of ascorbate (300 mg/kg, ip), which restore extracellular ascorbate levels in R6/2 striatum, attenuated this difference.

Although these lines of evidence suggest that a deficit in striatal ascorbate release plays a key role in HD, the mechanism underlying this effect is unclear. In view of evidence that ascorbate release is linked to glutamate uptake (see above), one could argue that low extracellular ascorbate simply indicates a glutamate uptake problem. In fact, there is evidence for decreased mRNA levels of the major astroglial glutamate transporter (GLT1 or EAAT2) accompanied by decreased glutamate uptake in the striatum of transgenic mice [82]. Thus, a defect in astrocytic glutamate uptake may contribute to striatal neuropathology. This hypothesis is consistent with rapidly firing striatal neurons in the R6/2 line because over-activation of glutamate receptors can result in excitotoxicity. As the most abundant glutamate transporter in the brain, GLT1 is critically important for maintaining low resting levels of extracellular glutamate (<1 μM) by concentrating the transmitter across the cell membrane. Because excitotoxicity includes the production of cell-damaging free radicals, e.g. [83–85], an accompanying low level of extracellular ascorbate may simply exacerbate the problem by lowering anti-oxidant capacity.

Although a failure of glutamate uptake and the accompanying loss of extracellular ascorbate may comprise critical components of the neuropathological cascade in HD, it is important to recognize that a restoration of striatal ascorbate attenuates both behavioral and firing-rate abnormalities in R6/2 mice. Conceivably, therefore, impaired ascorbate release could play a primary role in HD pathology. One of these roles may involve an anti-oxidant effect. In fact, GLT1, like other glutamate transporters, is vulnerable to the action of biological oxidants [86]. Thus, without the oxidant-fighting capacity of extracellular ascorbate, a progressive positive feedback loop may develop in which glutamate uptake declines, leading to less ascorbate release and further impairing glutamate uptake. The addition of ascorbate may lower the oxidant load on GLT1 and resume the removal of glutamate. The result would be a decline in striatal firing rate and, thus, improved striatal processing of behavior-related neuronal signals. Alternatively, restoring extracellular ascorbate could alter striatal function directly by interfering with the binding of glutamate to the N-methyl-D-aspartate (NMDA) receptor. At high extracellular concentrations (~1 mM), for example, ascorbate occupies an NMDA redox site that antagonizes the action of glutamate [64]. Because striatal NMDA receptors appear to be abnormally sensitive to glutamate activation in HD mice [87], ascorbate treatment may stabilize striatal physiology and, thus, behavior by preventing NMDA receptor over-activation. It is clear that pre- and postsynaptic modulation of glutamate transmission by ascorbate deserves further assessment in the ongoing effort to understand and treat HD.

Summary and Conclusion

The ability of ascorbate to give up electrons not only accounts for most of its physiological functions but also makes it accessible to voltammetric detection. Although ascorbate has never approached the level of interest generated by the monoamines, voltammetry has been largely responsible for evidence implicating ascorbate release in the striatum as a modulator of neuronal information processing and behavior. This modulatory role seems closely tied to glutamate through a direct influence on both its uptake and postsynaptic action. In fact, a deficit in striatal ascorbate release in mouse genetic models of HD suggests several ways in which glutamate may contribute to this and, perhaps, other neurodegenerative diseases. Pursuit of these suggestions may identify key targets in the search for treatment strategies. As research on ascorbate continues, it is likely that voltammetry will remain a critical part of this effort.


Portions of the research described in this chapter were supported by U.S. Public Health Service grants from the National Institute on Drug Abuse, (DA02451), and the National Institute of Neurological Disease and Stroke (NS35663). Support from the Hereditary Disease Foundation is also acknowledged. Mr. Scott J. Barton and Mr. Paul E. Langley provided expert technical support; Ms. Faye Caylor assisted with editing and manuscript organization.


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