Endogenous melatonin protects L-DOPA from autoxidation in thestriatal extracellular compartment of the freely moving rat:potential implication for long-term L-DOPA therapy in Parkinson’sdisease

Introduction

Parkinson’s disease (PD) is characterized by a selective lossof dopaminergic neurons in the substantia nigra (SN), with

a consequent decrease in neostriatal dopamine (DA)content and impairment of the functioning of the nigro-striatal dopaminergic system. A major problem for

researchers and clinicians is that, by the time patients�symptoms become apparent, about 70–80% of their dop-aminergic neurons may have already died [1]. Althoughcellular and molecular pathways leading to neuronal death

in PD are still unknown, major biochemical processes suchas oxidative stress and impaired energy metabolism may beinvolved. Current concepts also suggest a genetic predis-

position to a toxic process involving oxidative stress andmitochondrial dysfunction [2]. In addition, evidence isaccumulating for the involvement of microglial activation

[3]. In PD, activated microglia are present in proximity todamaged nigral cells, suggesting their possible role intriggering or amplifying neuronal injury as well as inremoving the debris of injured cells [4].

In the past two decades a key role for DA has beenemphasized in the PD pathogenesis [5]. DA neurotoxicitymay result both from its autoxidation and monoamine

oxidase-mediated oxidation. DA autoxidation generatesfree radicals, melanin, and catechol-quinones. Quinoniccompounds are toxic intermediates capable of reacting with

various nucleophilic groups in the cell. DA-derived

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 lm 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 lm) or melatonin (50 lm)

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.

Gaia Rocchitta1,2, RossanaMigheli1, Giovanni Esposito1,Bianca Marchetti1,2, Maria S.Desole1, Egidio Miele1 and PierAndrea Serra1

1Department of Pharmacology, University of

Sassari, Sassari; 2OASI Institute for Research

and Care on Mental Retardation and Brain

Aging (IRCCS), Neuropharmacology Section,

Troina, Italy

Key words: antioxidant melatonin, ascorbic

acid, autoxidation, L-DOPA, Parkinson’s

disease, striatum

Address reprint requests to Pier Andrea Serra,

MD, Department of Pharmacology, University

of Sassari, viale S.Pietro 43B, 07100 Sassari,

Italy.

E-mail: pharmaco@uniss.it

Received August 16, 2005;

accepted October 25, 2005.

J. Pineal Res. 2006; 40:204–213Doi:10.1111/j.1600-079X.2005.00299.x

� 2005 The AuthorsJournal compilation � 2005 Blackwell Munksgaard

Journal of Pineal Research

204

quinones can act as oxidants, producing toxic hydroxyradicals, and can act as electrophiles, covalently binding toand inhibiting cellular sulphydryl-containing compounds

[6]. The most suggestive data showing an involvement ofDA oxidation products in PD is the presence of the specificdopaminergic toxin 6-hydroxydopamine (6-OHDA) in theurine of parkinsonian patients treated with l-DOPA [7].

l-DOPA is the drug of choice in PD therapy. Parkinsoniansymptoms are relieved by administration of l-DOPA,which is converted by neuronal aromatic l-amino acid

decarboxylase (EC 4.1.1.28) into DA, hence restoring DAlevels in surviving neurons, which, however, continue to diedespite the l-DOPA treatment [8]. l-DOPA therapy dra-

matically improves parkinsonian symptoms. However,after years of therapy, disabling motor complicationsdevelop greatly and limit the effectiveness of the drug [9].

l-DOPA, as a catechol-containing compound, can

undergo autoxidation [8, 10] to generate an o-semiquinone(l-DOPA-SQ) which, after disproportionation, gives rise tothe corresponding o-quinone and reactive oxygen species

(ROS), which might further load the pre-existing conditionof oxidative stress at nigro-striatal sites [8]. The quinonesgenerated by l-DOPA oxidation may react with cysteine to

form 5-S-cysteinyl-DOPA. Indeed, Spencer et al. [11] haveshown that cysteinyl-conjugates of DA and l-DOPA in PDare higher than in normal SN. In a previous study [12], we

showed that systemic l-DOPA underwent autoxidation inthe striatal extracellular compartment of freely moving rats,with a consequent formation of l-DOPA semiquinone(l-DOPA-SQ). Moreover, systemic l-DOPA decreased

baseline levels of DA and ascorbic acid (AsAc). Followingsystemic l-DOPA, intrastriatal infusion of the antioxidantN-acetylcysteine (NAC) decreased l-DOPA-SQ formation,

increased dialysate recovery of l-DOPA, restored baselinelevels of DA, but failed to restore baseline AsAc concen-trations.

Melatonin is a serotonin derivative which is synthesized inthe pineal gland during the night [13]. Its lipophilicity ensuresthat melatonin rapidly crosses cellular membranes [14].

Many in vitro and in vivo studies have shown that melatoninis a powerful and broad-spectrum free radical scavenger [15].For instance, melatonin proved to be more active thanvitamin E orAsAc in inhibiting iron-catalyzedDAoxidation

[16] or copper-catalyzed DA oxidation [17]. Moreover,melatonin protected the nigro-striatal system against oxida-tive stress caused by the neurotoxins 1-methyl-4-phenyl-

1,2,3,6-tetrahydropyridine (MPTP) in the mouse [18] and6-OHDA in the rat [19]. In a previous study, performed usingmicrodialysis, we demonstrated that endogenous melatonin

actively co-operates with endogenous AsAc in maintainingthe oxidative homeostasis of the extracellular striatal com-partment of the freely moving rat [20]. Moreover, endo-genous melatonin protected extracellular endogenous DA

and l-DOPA from oxidation. In light of these findings, wedeemed it of interest to assesswhether endogenousmelatoninwould protect exogenous l-DOPA from autoxidation in the

extracellular striatal compartment of the freely moving rats.In addition, we looked into the effects of melatoninco-administration with systemic l-DOPA on both l-DOPA

autoxidation and l-DOPA-induced changes in baselinelevels of DA and AsAc.

Materials and methods

Sources of compounds

l-DOPA, melatonin and NAC were purchased from Sigma-

Aldrich (Milano, Italy).

Animals

Male Wistar rats (Morini, R. Emilia, Italy), weighingbetween 280 and 330 g were used in all experiments. The

rats were maintained under standard animal care condi-tions (12:12 hr light/dark cycle, lights coming on at07:00 hr; room temperature 21�C), with food and waterad libitum. Prior to the start of any experiment, the health

of each rat was assessed according to published guidelines[21]. All procedures were specifically licensed under theEuropean Community directive 86/609 included in

Decreto No. 116/1992 of the Italian Ministry of PublicHealth.

Drug administration

Systemic l-DOPA treatment schedule [25 mg/8.0 mL/kg

intraperitoneally (i.p.) twice in a 12-h interval] was chosenaccording to the previous study [12]. Melatonin wasdissolved in 5% ethanol in saline and administered i.p. at5.0 mg/3 mL/kg twice in a 12-h interval. Melatonin was

injected 5 min before each l-DOPA administration. Intra-striatal NAC and melatonin concentrations were chosenaccording to previous studies [12, 20].

Striatal microdialysis probe

The striatal probe, which combines two independentmicrodialysis probes of concentric design with two separateinlets and two corresponding outlets, was previouslydescribed in detail [20, 22]. The two inlets with two

corresponding separate outlets permit separate co-infusionof drugs and separate dialysate sample collection from thesame intrastriatal site. In the present study, the determin-

ation of both l-DOPA and l-DOPA-SQ in dialysates fromthe outlet contralateral to the inlet of l-DOPA infusionallow us to affirm that detected concentrations reflect those

in the extracellular compartments. On the contrary, thedetermination in dialysates from the ipsilateral outlet wouldinclude also the nondialyzed quota of infused l-DOPA.

Moreover, separate sample collection is useful when one ormore drugs which may have either pro-oxidant or anti-oxidant properties are infused. Briefly, the probe wasconstructed using two sections of plastic-coated silica

tubing (diameter 0.15 mm; Scientific Glass Engineering,Milton Keynes, UK) each placed in the center of semi-permeable polyacrylonitrile dialysis fibers (molecular cut-

off weight of 12 kDa; Filtral 16 Hospal Industrie, MeyzieuCedex, France). Each probe had a final diameter of0.22 mm. The tips of the dialysis fibers were sealed and

joined using quick-drying epoxy glue. The two sections ofsilica tubing served as inlets; the outlets were also madewith a section of plastic-coated silica tubing, positioned inthe center of the polythene tubing. The semi-permeable

Melatonin and in vivo striatal l-DOPA autoxidation

205

membrane was coated with epoxy leaving an active lengthof 4 mm. The diameter of the final probe was approxi-mately 0.50 mm.

Stereotaxic surgery

Stereotaxic surgery was performed under chloral hydrate

(400 mg/kg, i.p.) anesthesia. The microdialysis probes wereimplanted in the right striatum using the following co-ordinates from the atlas of Paxinos and Watson [23]:

A/P + 0.5 mm from bregma, )2.5 mm M/L, and )6.0 mmD/V from dura. Body temperature during anesthesia wasmaintained at 37�C by means of an isothermal-heating pad

(Harvard Apparatus, Kent, UK). Following surgery theanimals were placed in large plastic bowls (50 · 55 cm),and maintained in a temperature- and light-controlledenvironment, with free access to food and water. Experi-

ments were carried out 24 hr after probe implantation withthe animal in its home bowl. This arrangement allowed therats free movement.

Light-exposed rats

Rats were maintained under constant light for 6 days [24].Early in the morning of the sixth day, animals underwentsurgery, which lasted no more than 1 hr. Following

surgery, the animals were placed in large plastic bowls(50 · 55 cm), and maintained in a temperature- and light-controlled environment, with free access to food and water.Light was kept on overnight. Experiments were carried out

24 hr after probe implantation with the animal in its homebowl. Experiments were carried out as above. Baselinedialysates were collected after 60 min of stabilization.

Microdialysis procedure

The composition of the Ringer solution used was as follows(in mm): NaCl 147.0, KCl 4.0, CaCl2 1.2, MgCl2 1.0 (pH6.0). A microinfusion pump (CMA/100; Microdialysis,Solna, Sweden) pumped Ringer solution at a flow rate of

1.5 lL/min using two separate syringes connected to theinlets by a length of polythene tubing; every 20 min, two30 lL dialysate samples were collected manually in 250 lLmicro-centrifuge tubes (Alpha Laboratories, Eastleigh,UK) attached to the outlets. Subsequently, a 20-lL aliquotof each collected dialysate was injected into each of two

parallel analytical systems. Drugs were added to the Ringersolution and infused via the striatal probe implanted in thestriatum.

Chromatographic analysis

l-DOPA-SQ was quantified by high-performance liquid

chromatography with electrochemical detection (HPLC-EC) according to the procedure previously described indetail [12]. The peak, identified as l-DOPA-SQ, appeared

within 20 min from the start of l-DOPA infusion. We werenot able to follow the fate of l-DOPA-SQ (furtheroxidation to l-DOPA-3,4-o-quinone) as our HPLC appar-

atus was not suitable for the detection of the latter l-DOPAoxidation product. Analogously, DA (detection limit

0.1 nm), l-DOPA (detection limit 0.2 nm), and AsAc(detection limit 0.05 lm) were quantified by HPLC-EC aspreviously described [20, 22], using an Alltech 426 HPLC

pump equipped with a Rheodyne injector (mod. 7725),column 15 cm · 4.6 mm i.d. (Toso Haas ODS80TM C18),electrochemical detector BAS mod. LC4B and a PC-basedanalog-to-digital converter system (Varian Star Chromato-

graphic Workstation, Valnut Creek, CA, USA). The mobilephase was citric acid 0.1 m, sodium acetate 0.1 m, ethylen-ediaminetetraacetic acid 1.0 mm, MeOH 9% and sodium

octylsulphate 50 mg/L (pH ¼ 2.9); the flow rate was1.3 mL/min. The first sample was collected after 60 minof stabilization (time 0), then dialysates were collected,

at 20-min intervals, for 40 min prior to the start ofexperiments. In rats given systemic l-DOPA orl-DOPA + melatonin, the second i.p. dose was given15 min before the start of stabilization. As shown previ-

ously [12], following l-DOPA 1.0 lm intrastriatal infusion,HPLC-EC chromatograms of the striatal dialysate revealeda peak (retention time 5.9 min) which was not present in the

striatal dialysate of untreated rats.

Histology

Following the experiments, rats were killed with anoverdose of chloral hydrate (800 mg/kg, i.p.). The location

of each microdialysis probe was confirmed by postmortemhistology. Brains were fixed in formal saline and 50 lmcoronal sections were made with a cryostat. The slices werestained with cresyl violet and examined under a microscope.

Statistical analysis

Concentrations of neurochemicals in dialysates wereexpressed in nm (DA l-DOPA, l-DOPA-SQ) or lm (AsAc)and given as mean ± S.E.M. Drug effects on neurochem-

icals were statistically evaluated in terms of changes inabsolute dialysate concentrations. Statistical significancewas assessed using ANOVA for difference between groupsand over time. Difference within or between groups were

determined by paired or unpaired t-tests with eitherBonferroni multiple comparison adjustment or Student–Newman–Keuls t-test post hoc analysis. Pearson’s correla-

tion coefficient between individual concentrations ofl-DOPA and l-DOPA-SQ was calculated in some instancesThe null hypothesis was rejected when P < 0.05.

Results

l-DOPA 1.0 lm (n ¼ 4) was infused through the ipsilateralinlet for 200 min. l-DOPA concentrations in dialysatesfrom the contralateral outlet increased up to 116-foldbaseline levels 20 min after the start of infusion; thereafter,

l-DOPA concentrations showed a trend to decrease,despite continuous l-DOPA infusion (Fig. 1). l-DOPA-SQ detection in dialysates from the contralateral outlet

occurred about 20 min after the start of l-DOPA infusion.l-DOPA-SQ concentrations increased over-times (Fig. 1).Individual dialysate concentrations of l-DOPA were neg-

atively correlated with those of l-DOPA SQ [r values rangebetween )0.786 (P < 0.005) and )0.842 (P < 0.0001),

Rocchitta et al.

206

d.f. ¼ 8]. l-DOPA infusion induced a short-lasting increasein DA concentrations in dialysates from the contralateraloutlet (maximum increase 107% of baseline after 40 min)and did not affect AsAc dialysate concentrations (Fig. 2).

A concentration of 100 lm NAC (n ¼ 4) was infusedthrough the contralateral inlet. The infusion started 5 minbefore l-DOPA 1.0 lm infusion through the ipsilateral inlet

for 200 min. NAC co-infusion induced the following:(i) significant increases in l-DOPA concentrations (up to151-fold baseline levels) in dialysates from the contralateral

outlet, when compared with l-DOPA group (Fig. 1);(ii) significant decreases (by about 53%) of l-DOPA-SQconcentrations at the end of drug infusion (Fig. 1); (iii) fur-

ther and more sustained increases in dialysate DA (up to153% of baseline after 40 min) (Fig. 2); (iv) slight decrease

in AsAc dialysate concentrations, when compared withbaseline values (Fig. 2).

A concentration of 50 lm melatonin (n ¼ 4) was infusedthrough the contralateral inlet. The infusion started 5 minbefore l-DOPA 1.0 lm infusion through the ipsilateral inlet

for 200 min. Melatonin co-infusion induced the following:(i) greater and significant increases in l-DOPA concentra-tions (up to 246-fold baseline levels) in dialysates from the

contralateral outlet, when compare with both l-DOPA andl-DOPA + melatonin groups (Fig. 1); (ii) greater andsignificant decreases (by about 87%) of l-DOPA-SQconcentrations at the end of drug infusion (Fig. 1);

(iii) greater and more sustained increases in dialysate DA(up to 232% of baseline after 40 min) (Fig. 2); (iv) slightincreases in AsAc dialysate concentrations, when com-

Fig. 1. Detection of l-DOPA-SQ in dialysates from the striatum offreely moving rats following intrastriatal infusion of l-DOPA(n ¼ 4) and effects of N-acetylcysteine (NAC, n ¼ 4) or melatonin(n ¼ 4) co-infusion on dialysate l-DOPA (A) and l-DOPA-SQ (B)concentrations. Dialysates were collected, at 20-min intervals,during continuous intrastriatal infusion of l-DOPA through theipsilateral inlet. NAC or melatonin co-infusion through the con-tralateral inlet started 5 min before l-DOPA infusion. Values aregiven as mean ± S.E.M. and refer to the concentrations in dialy-sates from the contralateral outlet. §P < 0.05 compared withl-DOPA group (A); +(thin horizontal bar) P < 0.05 comparedwith both l-DOPA and l-DOPA + NAC groups (A); +(thinhorizontal bar) P < 0.05 compared with both l-DOPA + NACand l-DOPA + melatonin groups (B); §(thin horizontal bar)P < 0.05 compared with l-DOPA + NAC group (B). Bonferronimultiple comparison adjustment test.

Fig. 2. Effects of intrastriatal infusion of l-DOPA on dopamine(DA, A) and ascorbic acid (AsAc, B) concentrations in dialysatesfrom the striatum of freely moving and effects of N-acetylcysteine(NAC) or melatonin co-infusion on l-DOPA-induced changes.Same groups as in Fig. 1. Dialysates were collected, at 20-minintervals, during continuous intrastriatal infusion of l-DOPAthrough the ipsilateral inlet. NAC or melatonin co-infusionthrough the contralateral inlet started 5 min before l-DOPAinfusion. Values are given as mean ± S.E.M. and refer to theconcentrations in dialysates from the contralateral outlet.*P < 0.05 compared with pertinent baseline values (A); +(thinhorizontal bar) P < 0.05 compared with both l-DOPA andl-DOPA + NAC groups (A); §P < 0.05 compared with l-DOPAgroup (A); §P < 0.05 compared with l-DOPA group (B); +(thinhorizontal bar) P < 0.05 compared with l-DOPA + melatoningroup (B). Bonferroni multiple comparison adjustment test.

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207

pared with baseline levels. Dialysate AsAc concentrations,however, were significantly higher than concentrations inboth l-DOPA and l-DOPA + NAC groups during last 80

and 160 min, respectively, of drug infusion (Fig. 2).As shown previously [12], l-DOPA given systemically

significantly increased striatal dialysate baseline levels ofl-DOPA and decreased those of DA and AsAc. Moreover,

l-DOPA-SQ was detected in dialysates.l-DOPA 25 mg/8.0 mL/kg was given i.p. twice in a 12-hr

interval to three groups of four rats. Baseline concentra-

tions of DA, l-DOPA, l-DOPA-SQ and AsAc weredetermined 1 hr after last l-DOPA administration indialysates from both outlets, as l-DOPA was given

systemically. The data were pooled in order to calculatebaseline values for each rat. The results are given inTable 1. Systemic l-DOPA significantly decreased baselineDA (by about 39%) and AsAc (by about 43%) and

increased baseline l-DOPA (by about 11-fold). Inuntreated rats, l-DOPA-SQ was not detectable in dialy-sates; however, following systemic l-DOPA, l-DOPA-SQ

was detected in concentrations even greater (by about 40%)than l-DOPA ones.In the first group of systemic l-DOPA-treated rats,

dialysate concentrations of DA, l-DOPA, l-DOPA-SQ andAsAc were monitored for 200 min after baseline samplescollection. Neurochemicals were determined in the dialysate

from the outlet conventionally indicated as contralateral.Dialysate l-DOPA concentrations progressively declined(maximum decreases by about 51% at the end of themonitoring period), while those of l-DOPA-SQ progres-

sively increased (by about 77% at the end of the monitoringperiod) (Fig. 3). Dialysate concentrations of both DA andAsAc did not show significant changes, when compared

with baseline values (Fig. 4).In the second group, 100 lm NAC was infused intrastri-

atally through the ipsilateral inlet for 200 min. Dialysates

were collected from the contralateral outlet. NAC infusionincreased dialysate l-DOPA (maximum increase by about101% after 40 min) and decreased dialysate l-DOPA-SQ

concentrations by about 57% at the end of the druginfusion. NAC infusion restored dialysate DA concentra-tions (maximum increase 101% after 40 min), but failed torestore dialysate AsAc concentrations (Fig. 4).

In the third group, 50 lm melatonin was infused intra-striatally through the ipsilateral inlet for 200 min. Dialy-sates were collected from the contralateral outlet.

Melatonin infusion induced a long-lasting increase indialysate l-DOPA (maximum increase by about 207%after 40 min) and a great decrease in dialysate l-DOPA-SQconcentrations (by about 96% at the end of the infusion)

(Fig. 3). Melatonin infusion induced a long-lasting increasein dialysate DA concentrations (maximum increase byabout 269% after 80 min) and fully restored dialysate AsAc

concentrations (Fig. 4).To evaluate the effects of systemic co-administration of

melatonin on systemic l-DOPA-induced changes in dialy-

sate concentrations of striatal neurochemicals, melatonin5.0 mg/3.0 mL/kg i.p. was co-administered with l-DOPA25 mg/8.0 mL/kg i.p. twice, at 12-hr interval, in a group offour rats. We could not evaluate the effects of systemic

co-administration of NAC, as NAC, although readilycrosses the blood–brain barrier when given into the carotidartery, it does not reach the brain when given systemically,

owing to the its rapid body clearance [25].Melatonin was injected 5 min before each l-DOPA

administration. Baseline concentrations of DA, l-DOPA,

l-DOPA-SQ and AsAc were determined 1 hr after lastl-DOPA administration in dialysates from both outlets. Thedata were pooled in order to calculate baseline values for

each rat and are given in Table 1. Melatonin co-administra-tion significantly increased dialysate DA concentrations,when compared with both untreated and systemic l-DOPA-treated rats (by about 29% and 111%, respectively). More-

over, melatonin co-administration significantly increasedbaseline l-DOPA by about 75% and decreased baselinel-DOPA-SQ concentration by about 81%, when compared

with systemic l-DOPA-treated rats. Finally, melatonin co-administration fully restored baseline AsAc concentrations.After baseline samples collection, dialysate concentrations of

neurochemicals were monitored for 200 min. Dialysatel-DOPA progressively increased, while l-DOPA-SQ pro-gressively decreased; l-DOPA increases reached statistical

significance during the last 60 min of monitoring, whencompared with baseline levels (Fig. 5). Dialysate DA con-centrations showed a progressive significant increase thatreached statistical significance during the last 140 min of

Table 1. Effects of systemic l-DOPA on baseline striatal dialysate concentration of neurochemicals in control, light-exposed and systemicmelatonin-treated freely moving rats. See text for detail

Neurochemical

Treatment

None(n ¼ 12)

l-DOPA(n ¼ 12)

l-DOPA + melatonin(n ¼ 4)

l-DOPA +light exposure

(n ¼ 4)l-DOPA + melatonin +light exposure (n ¼ 4)

DA (nm) 4.24 ± 0.32 2.59 ± 0.22a ()38.9) 5.47 ± 0.81ab (+29.0) 1.36 ± 0.37ab ()67.9) 4.84 ± 0.95bd (+14.1)l-DOPA (nm) 2.81 ± 0.36 30.84 ± 1.28a (+1098) 53.96 ± 5.76ab (+1920) 18.15 ± 2.28ab (+646) 50.88 ± 5.21abd (+1811)l-DOPA-SQ (nm) ND 43.06 ± 3.17 8.11 ± 1.61b ()81.1) 63.45 ± 9.57b (+47.4) 9.96 ± 2.48bd ()76.9.0)AsAc (lm) 9.45 ± 0.48 5.03 ± 0.26a ()43.4) 8.49 ± 1.08b ()8.2) 2.38 ± 0.26ab ()74.3) 7.97 ± 0.63bd ()13.8)

ND, not detectable; DA, dopamine; AsAc, ascorbic acid.Data are given as mean ± S.E.M. Values in parentheses are percentage.aP < 0.05 compared with untreated rats; bP < 0.05 compared with l-DOPA-treated rats; cP < 0.05 compared with l-DOPA + mela-tonin-treated group; dP < 0.05 compared with l-DOPA + light-exposed group. Student–Newman–Keuls test.

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monitoring (maximum increase by about 96%); AsAcconcentrations showed only a slight increase (Fig. 6).

Two groups of four rats were exposed to light for 7 days

(see Materials and methods for details). The first group wasgiven l-DOPA 25 mg/8.0 mL/kg i.p. twice in a 12-hrinterval. Baseline concentrations of DA, l-DOPA,l-DOPA-SQ and AsAc were determined 1 hr after last

l-DOPA administration in dialysates from both outlets.The data were pooled in order to calculate baseline valuesfor each rat. The results are given in Table 1. Baseline levels

of l-DOPA were significantly lower (by about 41%) thanrats given systemic l-DOPA, while baseline levels of l-DOPA-SQ were significantly higher (by about 47%).

Baseline DA and AsAc concentrations were significantlylower than rats given systemic l-DOPA, by about 47% and53%, respectively. After baseline samples collection, dial-ysate concentrations of neurochemicals were monitored

for 200 min. Dialysate l-DOPA progressively decreased,while l-DOPA-SQ progressively increased; l-DOPA-SQincreases reached statistical significance during the last60 min of monitoring, when compared with baseline levels

(Fig. 5). Dialysate DA and AsAc concentrations did notshow significant changes (Fig. 6).In the second group of light-exposed rats, melato-

nin (5.0 mg/3.0 mL/kg, i.p.) was co-administered withl-DOPA 25 mg/8.0 mL/kg i.p. twice at 12-hr interval.Melatonin was injected 5 min before each l-DOPA

administration. Baseline concentrations of DA, l-DOPA,l-DOPA-SQ, and AsAc were determined 1 hr after lastl-DOPA administration in dialysates from both outlets. The

data were pooled in order to calculate baseline values foreach rat. The results are given in Table 1. Baseline levels ofl-DOPA were significantly higher (by about 379%) thanbaseline values in light-exposed group, while l-DOPA-SQ

were significantly lower (by about 84%). Both baseline

Fig. 3. Effects of systemic l-DOPA on l-DOPA (A) and l-DOPA-SQ (B) concentrations in dialysates from the striatum of freelymoving and effects of N-acetylcysteine (NAC) or melatonin intra-striatal infusion on systemic l-DOPA-induced changes. NAC(100 lm) or melatonin (50 lm) was infused for 200 min (solidhorizontal bar a) through the ipsilateral inlet. N ¼ 4 for eachgroup. Dialysates were collected at 20-min intervals. Values aregiven as mean ± S.E.M. and refer to concentrations in dialysatesfrom the contralateral outlet. *P < 0.05 compared with pertinentbaseline values (A, B); +(thin horizontal bar) P < 0.05 comparedwith systemic l-DOPA group (A), systemic l-DOPA group (B) andsystemic l-DOPA + melatonin group (B); §(thin horizontal bar)P < 0.05 compared with systemic l-DOPA + NAC group (A);#P < 0.05 compared with systemic l-DOPA group (B). Bonferronimultiple comparison adjustment test.

Fig. 4. Effects of systemic l-DOPA on dopamine (DA) andascorbic acid (AsAc) concentrations in dialysates from the striatumof freely moving rats, and effects of N-acetylcysteine (NAC) ormelatonin intrastriatal infusion on systemic l-DOPA-inducedchanges. NAC (100 lm) or melatonin (50 lm) was infused for200 min (solid horizontal bar a) through the ipsilateral inlet. Samegroups as in Fig. 3. Dialysates were collected at 20-min intervals.Values are given as mean ± S.E.M. and refer to concentrations indialysates from the contralateral outlet. *P < 0.05 compared withpertinent baseline values (A, B): +(thin horizontal bar) P < 0.05compared with both systemic l-DOPA and systemic l-DO-PA + NAC groups (A, B); §(thin horizontal bar) P < 0.05 com-pared with systemic l-DOPA + NAC groups (A). Bonferronimultiple comparison adjustment test.

Melatonin and in vivo striatal l-DOPA autoxidation

209

levels of l-DOPA and l-DOPA-SQ did not statisticallydiffer from baseline levels in l-DOPA + melatonin-treated

group (Table 1). Baseline levels of DA and AsAc weresignificantly higher than baseline values in light-exposedgroup, by about 356% and 335%, respectively. Both

baseline levels of DA and AsAc did not statistically differfrom baseline levels in l-DOPA + melatonin-treatedgroup (Table 1). After baseline samples collection, dialysateconcentrations of neurochemicals were monitored for

200 min. Dialysate l-DOPA progressively increased, whilel-DOPA-SQ progressively decreased; l-DOPA increasesreached statistical significance during the last 60 min of

monitoring, when compared with baseline levels (Fig. 5).Dialysate DA concentrations showed a progressive signifi-cant increase, that reached statistical significance during the

last 140 min of monitoring (maximum increase by about

113%); AsAc concentrations showed only a slight increase(Fig. 6).

Discussion

The key findings in the present study are the following: (i)both intrastriatally and systemically administered l-DOPAundergo autoxidation in the striatal extracellular compart-

ment of the freely moving rat, with a consequent formationand detection of l-DOPA-SQ; (ii) systemic administrationof l-DOPA decreases striatal baseline dialysate concentra-tions of DA and AsAc; (iii) systemic administration of

l-DOPA in melatonin-depleted rats induces formation ofl-DOPA-SQ and decreases in baseline levels of DA andAsAc significantly greater than in control rats; (iv) systemic

co-administration of melatonin with l-DOPA, in control aswell as in light-exposed rats, significantly increased bothbaseline and over-times concentrations of dialysate

Fig. 5. Effects of systemic l-DOPA on l-DOPA and l-DOPA-SQconcentrations in dialysates from the striatum of freely movingcontrol or light-exposed rats and effects of systemic melatonin co-administration on systemic l-DOPA-induced changes. N ¼ 4 foreach group. Dialysates were collected, at 20-min intervals, 1 hrafter last systemic administration. Values are given as mean± S.E.M. and refer to concentrations in dialysates from bothoutlets. *P < 0.05 compared with pertinent baseline values (A, B);+(thin horizontal bar) P < 0.05 compared with pertinent systemicl-DOPA groups (A, B); +(thin horizontal bar) P < 0.05 comparedwith pertinent systemic l-DOPA and systemic l-DOPA + lightexposure groups (A, B); §(thin horizontal bar) P < 0.05 comparedwith systemic l-DOPA group (A, B). Bonferroni multiple com-parison adjustment test.

Fig. 6. Effects of systemic l-DOPA on dopamine (DA) andascorbic acid (AsAc) concentrations in dialysates from the striatumof freely moving control or light-exposed rats and effects ofsystemic melatonin co-administration on systemic l-DOPA-in-duced changes. Same groups as in Fig. 5. Dialysates were collected,at 20-min intervals, 1 hr after last systemic administration. Valuesare given as mean ± S.E.M. and refer to concentrations in dialy-sates from both outlets. *P < 0.05 compared with pertinentbaseline values (A, B); +(thin horizontal bar) P < 0.05 comparedwith pertinent systemic l-DOPA groups (A, B); +(thin horizontalbar) P < 0.05 compared with pertinent systemic l-DOPA andsystemic l-DOPA in light-exposed groups (A, B); §P < 0.05compared with systemic l-DOPA group (A, B). Bonferroni mul-tiple comparison adjustment test.

Rocchitta et al.

210

l-DOPA, greatly inhibited l-DOPA autoxidation and theconsequent formation of l-DOPA-SQ, and fully restoredboth DA and AsAc dialysate concentrations.

Intrastrial infusion of l-DOPA induced a short-lastingincrease in dialysate DA. Co-infusion of the antioxidantNAC with l-DOPA increased dialysate DA concentra-tions, which were further significantly increased when

melatonin was co-infused. Moreover, l-DOPA autoxida-tion was inhibited by NAC co-infusion and, to a greaterextent, by melatonin co-infusion, with a consequent

increase in dialysate recovery of l-DOPA. The findingthat antioxidant drugs increased DA dialysate recoverystrongly suggest that DA released following l-DOPA

infusion undergoes oxidation in the extracellular compart-ment. Systemically administered l-DOPA undergoes bio-transformation to DA in the nigro-striatal system of therat [26]. In a previous study [12], we showed that

stimulation of striatal dopaminergic endings inducedincreases in dialysate DA in rats given systemic l-DOPAmuch greater than in untreated rats. However, newly

synthesized and released DA, unless appropriately shiel-ded, easily undergoes autoxidation or ROS and/or reactivenitrogen species (RNS)-mediated oxidation and/or nitra-

tion in the extracellular compartment, mainly when DA isreleased in excess [20, 27, 28]. Thus, autoxidation ofl-DOPA in the striatal extracellular compartment, with a

consequent formation of l-DOPA-SQ, most likely pro-motes DA nonenzymatic oxidation. Spencer et al. [11]have shown that an acceleration of l-DOPA/DA oxidationoccurs in PD, probably related to therapy with l-DOPA.

Autoxidation of l-DOPA and DA generates quinones andsuperoxide anion (O��

2 ), which is scavenged by AsAc [29]and melatonin [13].

Intrastriatal l-DOPA infusion did not induce significantchanges in dialysate AsAc. Dialysate AsAc concentrationsreflect those found in vivo (0.2–0.4 mm) in the extracellular

striatal compartment of the rat [30]. Moreover, an efficientin vivo homeostatic mechanism keeps constant striatalextracellular AsAc concentrations [30]. However, following

co-infusion of NAC, dialysate concentrations of AsAc weresignificantly lower than those detected following melatoninco-administration. This finding may be explained on thebasis of the activity of the respective oxidation products of

NAC and melatonin. When NAC undergoes oxidation, itgenerates, like glutathione, a thiyl active free radical [31,32], which needs to be scavenged in order to prevent

hydroxyl radical formation [31]. On the contrary, melatoninoxidation products are also known to have antioxidantproperties [33]. Endogenous AsAc [34, 35] and endogenous

melatonin [20] cooperate in scavenging endogenous anti-oxidant (vitamin E, glutathione, uric acid)-derived activeradicals, such as a-tocopheroxyl, thiyl/sulfenyl and urateradical.

When given systemically, l-DOPA decreased dialysatebaseline levels of both DA and AsAc. In systemic l-DOPA-treated rats, intrastriatal infusion of NAC increased over-

times dialysate concentrations of DA and l-DOPA,decreased l-DOPA-SQ generation, restored DA concen-trations, but failed to restore those of AsAc. On the

contrary, intrastriatal infusion of melatonin restored bothDA and AsAc dialysate concentrations, greatly increased

over-times dialysate l-DOPA concentrations and almostfully inhibited l-DOPA-SQ generation. These findingsfurther demonstrate that following exogenous l-DOPA

striatal loading, the l-DOPA autoxidation in the striatalextracellular compartment, with a consequent formation ofl-DOPA-SQ, promotes nonenzymatic oxidation of releasedDA. As a consequence, AsAc and melatonin, the main

components of the striatal extracellular antioxidant system[20, 34], are most likely markedly involved in maintainingoxidative homeostasis. The antioxidant NAC did protect

exogenous l-DOPA and released DA from autoxidation,but the protection needed the cooperation of endogenousAsAc, which was probably involved in scavenging thiyl

radical, the NAC oxidation product. On the contrary,co-infusion of melatonin not only afforded a greaterprotection, but fully restored dialysate AsAc.In a previous study [20], we suggested that endogenous

melatonin might play an active role in maintaining theoxidative homeostasis in the extracellular compartment ofthe striatum of freely moving rats. The results of the present

study confirm this hypothesis. In fact, systemic l-DOPAinduced decreases in baseline DA and AsAc levels inmelatonin-depleted rats significantly greater than in control

rats; moreover, baseline levels of l-DOPA were signifi-cantly lower, while those of l-DOPA-SQ were significantlyhigher. Thus, following exogenous l-DOPA striatal loading

in melatonin-depleted rats, the rate of l-DOPA and DAautoxidation in the striatal extracellular compartment wassignificantly increased, with a consequent greater involve-ment of endogenous AsAc in maintaining the oxidative

homeostasis.The question arises as to whether the result of the present

study might be of relevance to the l-DOPA long-term

therapy of PD. Inhibitors of enzymatic l-DOPA metabo-lism, which do not cross the blood–brain barrier, aresuccessfully administered with l-DOPA with the aim of

increasing l-DOPA bioavailability at nigro-striatal site [36].However, despite the drug-induced increase in l-DOPAbioavailability, some years after the start of therapy

l-DOPA loses its beneficial effects as evidenced by motorfluctuations. The clinical effectiveness of dopaminergicagonists in controlling l-DOPA-associated motor fluctu-ation [37] allow us to speculate that, despite the l-DOPA

loading, the postsynaptic dopaminergic input is greatlydiminished. In this regard, an increase in l-DOPA/DAnonenzymatic oxidation [11] in the extracellular compart-

ment, facilitated by an impaired oxidative homeostasis [38],might assume great relevance. However, no clinical data areavailable on the effectiveness of antioxidant drugs in

controlling l-DOPA/DA nonenzymatic oxidation, whichmost likely occurs at nigro-striatal site in PD [11]. In thepresent study, systemic co-administration of melatoninwith l-DOPA, in control as well as in light-exposed

rats, significantly increased both baseline and over-timesconcentrations of dialysate l-DOPA, greatly inhibitedl-DOPA autoxidation and the consequent formation of

l-DOPA-SQ, and restored both DA and AsAc dialysateconcentrations. Therefore, melatonin appears to be themost suitable antioxidant drug to be used as adjunctive

drug with the aim of protecting l-DOPA and DA from

Melatonin and in vivo striatal l-DOPA autoxidation

211

nonenzymatic oxidation in the striatal extracellular com-partment.We showed previously [12] that transition metals (man-

ganese and iron) greatly increased l-DOPA autoxidation inthe extracellular striatal compartment of the freely movingrats. Dysregulation of iron metabolism and iron-inducedoxidative stress are widely believed to be important

pathogenetic mechanisms of neuronal death in PD [38,39]. Indeed, O��

2 releases iron from storage proteins andenzymic [4Fe-4S] clusters [40]. Thus, the hypothesis that

endogenous iron might increase extracellular l-DOPA/DAoxidation in PD seems to be logical and further supportthe rationale of melatonin use as adjunctive drug to the

l-DOPA therapy of PD. Clinical studies have shown thatlong-term administration of melatonin at pharmacologicaldosage, in PD [41] as well as in other neurologic disorders[42], is devoid of side effects.

It is still claimed that melatonin is not an antioxidantbecause it must be given in what is referred to aspharmacological doses to repair the breach of antioxidant

defenses by ROS, RNS, or toxic reactants leading todamage of critical cellular structures (DNA, lipids, pro-teins) with a consequent disruption of the cellular physiol-

ogy [43]. The results of the present as well as of a previousstudy [12] not only demonstrate that endogenous melatoninactively cooperates with endogenous AsAc in maintaining

the striatal oxidative/nitrosative homeostasis, but alsoindicate, on the basis of endogenous melatonin activity,when and how melatonin might be usefully used astherapeutic drug at pharmacological doses.

Acknowledgment

The research was supported in part by the University ofSassari (ex 60% fund).

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