Gaia Rocchitta

In this chapter we have described the development of a distributed sensor NET for biotelemetric monitoring of brain neurochemistry using amperometric microsensors and biosensors. The implantable biotelemetric units, based on simple and inexpensive components, have been successfully used for accurate transduction of the electrochemical signals generated on the surface of the sensors implanted in the striatum of freely-moving rats. At this stage of development the system exhibits high stability and excellent linear response in the nanoampere current range. The choice of low-power CMOS technology makes the project suitable of further improvements such as the reduction of the power

ABSTRACT Ceria nanoparticles with controlled size have been studied as antioxidant agents for the in vitro protection of catecholaminergic cells (PC12) exposed to manganese, which is responsible for an occupational form of Parkinson-like disease. The nanoparticle internalization has been followed by Raman and confocal microscopy while the effect of nanoceria concentration in the cell metabolism has been assessed by MTT and trypan blue assay. With the perspective to develop an innovative combined treatment, nanoceria has been tested either alone or in association with L-DOPA showing a significant effect in reducing the oxidative stress due to manganese chloride. Finally, to study the protective role of nanoceria on the metabolism of catecholamines, the intracellular concentration of dopamine and its metabolites have been monitored by liquid chromatography with electro-chemical detection in control and nanoparticle-exposed cells as a function of the nanoceria dosing. The results show a protective role of nanoceria both on PC12 cells survival and dopamine metabolism, which makes this class of nanoparticles a potential candidate for the treatment of Parkinson-like diseases induced by chronic manganese intoxication.

Ethanol is one of the most widespread psychotropic agents in western society. While its psychoactive effects are mainly associated with GABAergic and glutamatergic systems, the positive reinforcing properties of ethanol are related to activation of mesolimbic dopaminergic pathways resulting in a release of dopamine in the nucleus accumbens. Given these neurobiological implications, the detection of ethanol in brain extracellular fluid (ECF) is of great importance. In this study, we describe the development and characterization of an implantable biosensor for the amperometric detection of brain ethanol in real time. Ten different designs were characterized in vitro in terms of Michaelis-Menten kinetics (V(MAX) and K(M)), sensitivity (linear region slope, limit of detection (LOD), and limit of quantification (LOQ)), and electroactive interference blocking. The same parameters were monitored in selected designs up to 28 days after fabrication in order to quantify their stability. Finally, the best performing biosensor design was selected for implantation in the nucleus accumbens and coupled with a previously developed telemetric device for the real-time monitoring of ethanol in freely moving, untethered rats. Ethanol was then administered systemically to animals, either alone or in combination with ranitidine (an alcohol dehydrogenase inhibitor) while the biosensor signal was continuously recorded. The implanted biosensor, integrated in the low-cost telemetry system, was demonstrated to be a reliable device for the short-time monitoring of exogenous ethanol in brain ECF and represents a new generation of analytical tools for studying ethanol toxicokinetics and the effect of drugs on brain ethanol levels.

A new telemetry system for simultaneous detection of extracellular brain glucose and lactate and motion is presented. The device consists of dual-channel, single-supply miniature potentiostat-I/V converter, a microcontroller unit, a signal transmitter, and a miniaturized microvibration sensor. Although based on simple and inexpensive components, the biotelemetry device has been used for accurate transduction of the anodic oxidation currents generated on the surface of implanted glucose and lactate biosensors and animal microvibrations. The device was characterized and validated in vitro before in vivo experiments. The biosensors were implanted in the striatum of freely moving animals and the biotelemetric device was fixed to the animal's head. Physiological and pharmacological stimulations were given in order to induce striatal neural activation and to modify the motor behavior in awake, untethered animals.

Abstract:  We previously showed, using microdialysis, that autoxidation of exogenous L-dihydroxyphenylalanine (l-DOPA) occurs in vivo in the extracellular compartment of the freely moving rat, with a consequent formation of l-DOPA semiquinone (l-DOPA-SQ). In the present study, intrastriatal infusion of l-DOPA (1.0 μm for 200 min) increased dialysate l-DOPA concentrations (maximum increases up to 116-fold baseline values); moreover, l-DOPA-SQ was detected in dialysates. Individual dialysate concentrations of l-DOPA were negatively correlated with those of l-DOPA-SQ. Co-infusion of N-acetylcysteine (100 μm) or melatonin (50 μm) increased l-DOPA (up to 151- and 246-fold, respectively) and decreased l-DOPA-SQ (by about 53% and 87%, respectively) dialysate concentrations. Systemic l-DOPA [25 mg/kg intraperitoneally (i.p.) twice in a 12-h interval] significantly increased striatal baseline dialysate concentrations of l-DOPA and decreased dopamine (DA) and ascorbic acid (AsAc) concentrations, when compared with controls. Following systemic l-DOPA, l-DOPA-SQ was detected in dialysates. Endogenous melatonin was depleted in rats maintained on a 24-h light cycle for 1 wk. In melatonin-depleted rats, systemic l-DOPA induced a smaller increase in dialysate l-DOPA, a greater increase in l-DOPA-SQ formation, and a greater reduction in DA and AsAc dialysate concentrations. Co-administration of melatonin (5.0 mg/kg, i.p., twice in a 12-h interval) with l-DOPA, in control as well as in light-exposed rats, significantly increased dialysate l-DOPA concentrations, greatly inhibited l-DOPA-SQ formation, and restored up to the control values dialysate DA and AsAc concentrations. These findings demonstrate that endogenous melatonin protects exogenous l-DOPA from autoxidation in the extracellular compartment of the striatum of freely moving rats; moreover, systemic co-administration of melatonin with l-DOPA markedly increases striatal l-DOPA bioavailability in control as well as in melatonin-depleted rats. These results may be of relevance to the long-term l-DOPA therapy of Parkinson's disease.

We showed previously, using in vitro microdialysis, that the activation of the soluble guanylate cyclase (sGC)/cyclic GMP pathway was the underlying mechanism of the extracellular Ca(2+)-dependent effects of exogenous NO on dopamine (DA) secretion from PC12 cells. In this study, the co-infusion of the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3] quinoxalin-1-one (ODQ) failed to affect the NO donor 3-morpholinosydnonimine (SIN-1, 5.0 mM)-induced DA increase (sevenfold baseline) in dialysates from the striatum of freely moving rats. Ca(2+) omission from the perfusion fluid abolished baseline DA release but did not affect SIN-1-induced DA increases. The reintroduction of Ca(2+) in the perfusion fluid restored the baseline dialysate DA; however, when Ca(2+) reintroduction was associated with the infusion of either SIN-1 or the NO-donor S-nitrosoglutathione (SNOG), a sustained DA overflow was observed. DA overflow was selectively inhibited by the co-infusion of the store-operated channel blocker 2-aminoethoxydiphenyl borate. The chelation of intracellular Ca(2+) by co-infusing 1,2-bis (o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM, 0.2 mM) greatly potentiated both SIN-1- and SNOG-induced increases in dialysate DA. BAPTA-AM-induced potentiation was inhibited by Ca(2+) omission. We conclude that the sGC/cyclic GMP pathway is not involved in the extracellular Ca(2+)-independent exogenous NO-induced striatal DA release; however, when intracellular Ca(2+) is either depleted (by Ca(2+) omission) or chelated (by BAPTA-AM co-infusion), exogenous NO does promote Ca(2+) entry, most likely through store-operated channels, with a consequent further increase in DA release.

The Ca(2+) sensitizer levosimendan (LEV) improves myocardial contractility by enhancing the sensitivity of the contractile apparatus to Ca(2+). In addition, LEV promotes Ca(2+) entry through L-type channels in human cardiac myocytes. In this study, which was performed using microdialysis, infusion of LEV at 0.25 microM for 160 min increased dopamine (DA) concentrations (up to fivefold baseline) in dialysates from the striatum of freely moving rats. Ca(2+) omission from the perfusion fluid abolished baseline DA release and greatly decreased LEV-induced DA release. Reintroduction of Ca(2+) in the perfusion fluid restored LEV-induced DA release. Chelation of intracellular Ca(2+) by co-infusing 1,2-bis (o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM, 0.2 mM) did not affect basal DA release and scarcely affected LEV-induced increases in dialysate DA. In addition, co-infusion of the L-type (Ca(v) 1.1-1.3) voltage-sensitive Ca(2+)-channel inhibitor nifedipine failed to inhibit LEV-induced increases in dialysate DA, which, in contrast, was inhibited by co-infusion of the N-type (Ca(v) 2.2) voltage-sensitive Ca(2+)-channel inhibitor omega-conotoxin GVIA. We conclude that LEV promotes striatal extracellular Ca(2+) entry through N-type Ca(2+) channels with a consequent increase in DA release.

A new embedded telemetry system for amperometric biosensor application is presented. The device consists of a single-supply miniature potentiostat-I/V converter, a microcontroller unit (MCU), a signal transmitter, and a stabilized power supply. The sensor current is ...

Biosensors for glutamate (Glu) were fabricated from Teflon-coated Pt wire (cylinders and disks), modified with the enzyme glutamate oxidase (GluOx) and electrosynthesized polymer PPD, poly(o-phenylenediamine). The polymer/enzyme layer was deposited in two configurations: enzyme before polymer (GluOx/PPD) and enzyme after polymer (PPD/GluOx). These four biosensor designs were characterized in terms of response time, limit of detection, Michaelis-Menten parameters for Glu (J max and K(M)(Glu)), sensitivity to Glu in the linear response region, and dependence on oxygen concentration, K(M)(O2). Analysis showed that the two polymer/enzyme configurations behaved similarly on both cylinders and disks. Although the two geometries showed different behaviors, these differences could be explained in terms of higher enzyme loading density on the disks; in many analyses, the four designs behaved like a single population with a range of GluOx loading. Enzyme loading was the key to controlling the K(M)(O2) values of these first generation biosensors. The counterintuitive, and beneficial, behavior that biosensors with higher GluOx loading displayed a lower oxygen dependence was explained in terms of the effects of enzyme loading on the affinity of GluOx for its anionic substrate. Some differences between the properties of surface immobilized GluOx and glucose oxidase are highlighted.

Abstract:  A capillary apparatus for in vitro microdialysis was used to investigate melatonin and ascorbic acid effects on dopamine (DA) autoxidation or nitric oxide (NO)-mediated oxidation in suspended PC12 cells. Following high K+ (KCl 75 mm) infusion, secreted DA underwent a partial autoxidation or peroxynitrite-mediated oxidation when the potential peroxynitrite generator 3-morpholinosydnonimine (SIN-1, 1.0 mm) was co-infused with KCl. Ascorbic acid was supplied to the medium by means of intracellular reduction of infused dehydroascorbic acid (DHAA) (5.0 mm). Melatonin (50 μm) and DHAA showed a synergistic effect in inhibiting DA autoxidation and peroxynitrite-mediated DA oxidation. Moreover, melatonin increased dialysate recovery of ascorbic acid released from PC12 cells. Endogenous melatonin was depleted in rats maintained on a 24-hr light cycle for 1 wk. In melatonin-depleted rats, baseline levels of dialysate ascorbic acid were lower than controls, while those of DA were unaffected. In these rats, intrastriatal infusion of 5.0 mm SIN-1 induced DA increases significantly lower than in controls; in addition, dialysate ascorbic acid concentrations exhibited significant decreases. Melatonin co-infusion restored SIN-1 effects on dialysate DA and antagonized SIN-1-induced ascorbic acid decreases. Melatonin-depleted rats were allowed to recover. In these rats, striatal baseline ascorbic acid, as well as SIN-1-induced increases in dialysate DA did not differ from controls. Taken together, these findings suggest that endogenous melatonin is an active component of the striatal extracellular antioxidant pool, as it maintains endogenous ascorbic acid in its reduced status and co-operates with ascorbic acid in protecting extracellular DA from exogenous NO-mediated oxidation.

ABSTRACT Amperometric polymer/enzyme composite (PEC) biosensors, incorporating a poly(o-phenylenediamine) ultra-thin permselective barrier, possess a variety of characteristics that make them suitable for monitoring brain energy and neurotransmitter dynamics in vivo. This review highlights PEC sensitivity and selectivity parameters, which allow development of the basic design in a systematic way in order to improve their performance and to diversify the analyte range of these novel probes of brain function.

We showed previously that interaction between NO and iron(II), both released following decomposition of sodium nitroprusside (SNP), accounted for the late SNP-induced dopamine (DA) increase in dialysates from the striatum of freely moving rats.In this study, intrastriatal infusion of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) (0.2 mM for 180 min) induced a moderate increase in dialysate DA and decreases in ascorbic acid dialysate concentrations; in contrast, SNAP 1 mM infusion induced a long-lasting decrease in both DA and ascorbic acid dialysate concentrations. 3-Methoxy-tyramine (3-MT), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and uric acid levels were unaffected.Co-infusion of ferrous sulphate [iron(II), 1 mM for 40 min] with SNAP either 1 or 0.2 mM (for 180 min), produced a significant increase in both DA and 3-MT dialysate concentrations, but it did not affect decreases in dialysate ascorbic acid levels. All other dialysate neurochemicals were unaffected.Co-infusion of ascorbic acid (0.1 mM) with SNAP (1 mM) for 180 min did not modify SNAP-induced decreases in dialysate DA levels. In contrast, co-infusion of uric acid (1 mM) reversed SNAP-induced decreases in dialysate DA; co-infusion of a superoxide dismutase mimetic delayed SNAP-induced DA decreases for a short period, while co-infusion of the antioxidant N-acetylcysteine (NAC, 0.1 mM) significantly increased dialysate DA.The results of this study show that SNAP induces concentration-related changes in DA dialysate levels. At higher concentrations, SNAP induces non-enzymatic DA oxidation, which is inhibited by uric acid and NAC; ascorbic acid failed to protect dialysate DA from oxidation, probably owing to its promoting effect on SNAP decomposition; exogenous iron(II) may react with NO generated from SNAP decomposition, with a consequent increase in dialysate DA and 3-MT, therefore mimicking SNP effects on striatal DA release.We showed previously that interaction between NO and iron(II), both released following decomposition of sodium nitroprusside (SNP), accounted for the late SNP-induced dopamine (DA) increase in dialysates from the striatum of freely moving rats.In this study, intrastriatal infusion of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) (0.2 mM for 180 min) induced a moderate increase in dialysate DA and decreases in ascorbic acid dialysate concentrations; in contrast, SNAP 1 mM infusion induced a long-lasting decrease in both DA and ascorbic acid dialysate concentrations. 3-Methoxy-tyramine (3-MT), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and uric acid levels were unaffected.Co-infusion of ferrous sulphate [iron(II), 1 mM for 40 min] with SNAP either 1 or 0.2 mM (for 180 min), produced a significant increase in both DA and 3-MT dialysate concentrations, but it did not affect decreases in dialysate ascorbic acid levels. All other dialysate neurochemicals were unaffected.Co-infusion of ascorbic acid (0.1 mM) with SNAP (1 mM) for 180 min did not modify SNAP-induced decreases in dialysate DA levels. In contrast, co-infusion of uric acid (1 mM) reversed SNAP-induced decreases in dialysate DA; co-infusion of a superoxide dismutase mimetic delayed SNAP-induced DA decreases for a short period, while co-infusion of the antioxidant N-acetylcysteine (NAC, 0.1 mM) significantly increased dialysate DA.The results of this study show that SNAP induces concentration-related changes in DA dialysate levels. At higher concentrations, SNAP induces non-enzymatic DA oxidation, which is inhibited by uric acid and NAC; ascorbic acid failed to protect dialysate DA from oxidation, probably owing to its promoting effect on SNAP decomposition; exogenous iron(II) may react with NO generated from SNAP decomposition, with a consequent increase in dialysate DA and 3-MT, therefore mimicking SNP effects on striatal DA release.British Journal of Pharmacology (2001) 132, 941–949; doi:10.1038/sj.bjp.0703887

We showed previously that interaction between NO and iron (II), both released following the decomposition of sodium nitroprusside (SNP), accounted for the late SNP-induced dopamine (DA) increase in dialysates from the striatum of freely moving rats; in addition, we showed that co-infusion of iron (II) with the NO-donor S-nitroso-N-acetylpenicillamine mimicked SNP effects on striatal DA release.In the present study, intrastriatal co-infusion of iron (II) (given as FeSO4, 1 mM for 40 min) with the NO-donor and potential peroxynitrite generator 3-morpholinosydnonimine (SIN-1) (0.2, 0.5, 1.0 or 5.0 mM for 180 min), potentiated the SIN-1-induced increase in DA concentration in dialysates from the striatum of freely moving rats. Neither alone nor associated with iron (II) did SIN-1 induce changes in dialysate ascorbic acid or uric acid concentrations.Neither co-infusion of a superoxide dismutase mimetic nor uric acid affected SIN-1-induced increases in dialysate DA concentration.Infusion of the iron chelator deferoxamine (0.2 mM for 180 min) decreased dialysate DA and attenuated SIN-1-induced increases in dialysate DA concentrations.These results suggest that iron plays a key role in SIN-1-induced release of striatal DA and do not support any role for either peroxynitrite or superoxide anion in SIN-1-induced release of striatal DA.We showed previously that interaction between NO and iron (II), both released following the decomposition of sodium nitroprusside (SNP), accounted for the late SNP-induced dopamine (DA) increase in dialysates from the striatum of freely moving rats; in addition, we showed that co-infusion of iron (II) with the NO-donor S-nitroso-N-acetylpenicillamine mimicked SNP effects on striatal DA release.In the present study, intrastriatal co-infusion of iron (II) (given as FeSO4, 1 mM for 40 min) with the NO-donor and potential peroxynitrite generator 3-morpholinosydnonimine (SIN-1) (0.2, 0.5, 1.0 or 5.0 mM for 180 min), potentiated the SIN-1-induced increase in DA concentration in dialysates from the striatum of freely moving rats. Neither alone nor associated with iron (II) did SIN-1 induce changes in dialysate ascorbic acid or uric acid concentrations.Neither co-infusion of a superoxide dismutase mimetic nor uric acid affected SIN-1-induced increases in dialysate DA concentration.Infusion of the iron chelator deferoxamine (0.2 mM for 180 min) decreased dialysate DA and attenuated SIN-1-induced increases in dialysate DA concentrations.These results suggest that iron plays a key role in SIN-1-induced release of striatal DA and do not support any role for either peroxynitrite or superoxide anion in SIN-1-induced release of striatal DA.British Journal of Pharmacology (2001) 134, 275–282; doi:10.1038/sj.bjp.0704232

In vitro microdialysis was used to investigate the mechanism of nitric oxide (NO) donor-induced changes in dopamine (DA) secretion from PC12 cells. Infusion of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 1.0 mm) induced a long-lasting increase in DA and 3-methoxytyramine (3-MT) dialysate concentrations. SNAP-induced increases were inhibited either by pre-infusion of the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4] oxadiazolo[4,3]quinoxalin-1-one (ODQ, 0.1 mm) or by Ca2+ omission. Ca2+ re-introduction restored SNAP effects. SNAP-induced increases in DA + 3-MT were unaffected by co-infusion of the l-type Ca2+ channel inhibitor nifedipine. The NO-donor (+/–)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3, 1.0 mm) induced a short-lasting decrease in dialysate DA + 3-MT. Ascorbic acid (0.2 mm) co-infusion allowed NOR-3 to increase dialysate DA + 3-MT. ODQ pre-infusion inhibited NOR-3 + ascorbic acid-induced DA + 3-MT increases. Infusion of high K+ (75 mm) induced a 2.5-fold increase in dialysate DA + 3-MT. The increase was abolished by NOR-3 co-infusion. Conversely, co-infusion of ascorbic acid (0.2 mm) with NOR-3 + high K+ restored high K+ effects. Co-infusion of nifedipine inhibited high K+-induced DA + 3-MT increases. These results suggest that activation of the NO/sGC/cyclic GMP pathway may be the underlying mechanism of extracellular Ca2+-dependent effects of exogenous NO on DA secretion from PC12 cells. Extracellular Ca2+ entry may occur through nifedipine-insensitive channels. NO effects and DA concentrations in dialysates largely depend on both the timing of NO generation and the extracellular environment in which NO is generated.

The effects of intrastriatal infusion of 3-morpholinosydnonimine (SIN-1) or sodium nitroprusside (SNP) on dopamine (DA), 3-methoxytyramine (3-MT), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), L-dihydroxyphenylalanine (L-DOPA), ascorbic acid and uric acid concentrations in dialysates from the striatum of freely moving rats were evaluated using microdialysis.SIN-1 (1 mM) infusion for 180 min increased microdialysate DA and 3-MT concentrations, while L-DOPA, DOPCA+HVA, ascorbic acid and uric acid levels were unaffected. Co-infusion with ascorbic acid (0.1 mM) inhibited SIN-1-induced increases in DA and 3-MT dialysate concentration.SNP (1 mM) infusion for 180 min increased greatly the dialysate DA concentration to a peak (2950% of baseline) at the end of the infusion, while increases in 3-MT were negligible. In addition, SNP decreased ascorbic acid and L-DOPA but increased uric acid concentration in the dialysate. Co-infusion with deferoxamine (0.2 mM) inhibited the late SNP-induced increase in DA dialysate concentration, but did not affect the decrease in ascorbic acid and increase uric acid dialysate concentrations.SNP (1 mM) infusion for 20 min moderately increased uric acid, DA and 3-MT, but decreased L-DOPA levels in the dialysate. Ascorbic acid concentration increased at the end of SNP infusion. Co-infusion with ascorbic acid (0.1 mM) inhibited the SNP-induced increase in DA and 3-MT, but did not affect the decrease in L-DOPA and increase in uric acid dialysate concentrations.These results suggest that NO released from SIN-1 may account for the increase in the dialysate DA concentration. NO released following decomposition of SNP may account for the early increase in dialysate DA, while late changes in microdialysate composition following SNP may result from an interaction between NO and the ferrocyanide moiety of SNP. Exogenous ascorbic acid inhibits the effect of exogenous NO on DA release probably by scavenging NO, suggesting that endogenous ascorbic acid may modulate the NO control of DA release from 300 striatal dopaminergic terminals.The effects of intrastriatal infusion of 3-morpholinosydnonimine (SIN-1) or sodium nitroprusside (SNP) on dopamine (DA), 3-methoxytyramine (3-MT), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), L-dihydroxyphenylalanine (L-DOPA), ascorbic acid and uric acid concentrations in dialysates from the striatum of freely moving rats were evaluated using microdialysis.SIN-1 (1 mM) infusion for 180 min increased microdialysate DA and 3-MT concentrations, while L-DOPA, DOPCA+HVA, ascorbic acid and uric acid levels were unaffected. Co-infusion with ascorbic acid (0.1 mM) inhibited SIN-1-induced increases in DA and 3-MT dialysate concentration.SNP (1 mM) infusion for 180 min increased greatly the dialysate DA concentration to a peak (2950% of baseline) at the end of the infusion, while increases in 3-MT were negligible. In addition, SNP decreased ascorbic acid and L-DOPA but increased uric acid concentration in the dialysate. Co-infusion with deferoxamine (0.2 mM) inhibited the late SNP-induced increase in DA dialysate concentration, but did not affect the decrease in ascorbic acid and increase uric acid dialysate concentrations.SNP (1 mM) infusion for 20 min moderately increased uric acid, DA and 3-MT, but decreased L-DOPA levels in the dialysate. Ascorbic acid concentration increased at the end of SNP infusion. Co-infusion with ascorbic acid (0.1 mM) inhibited the SNP-induced increase in DA and 3-MT, but did not affect the decrease in L-DOPA and increase in uric acid dialysate concentrations.These results suggest that NO released from SIN-1 may account for the increase in the dialysate DA concentration. NO released following decomposition of SNP may account for the early increase in dialysate DA, while late changes in microdialysate composition following SNP may result from an interaction between NO and the ferrocyanide moiety of SNP. Exogenous ascorbic acid inhibits the effect of exogenous NO on DA release probably by scavenging NO, suggesting that endogenous ascorbic acid may modulate the NO control of DA release from 300 striatal dopaminergic terminals.British Journal of Pharmacology (2000) 131, 836–842; doi:10.1038/sj.bjp.0703635