Serotonergic modulation of receptor occupancy in rats treated with l-DOPA after unilateral 6-OHDA lesioning

Received September 9, 2011; revised manuscript received October 29,2011; accepted November 14, 2011.Address correspondence and reprint requests to Adjmal Nahimi, MD,

PhD student, Department of Nuclear Medicine and PET Centre, Centerof Functionally Integrative Neuroscience, Aarhus University Hospitals,Aarhus University, Norrebrogade 44, Building 10G, 6th Floor, 8000Aarhus C, Denmark.E-mails: [email protected]; [email protected]

Abbreviations used: 5-HT, serotonin; 6-OHDA, 6-hydroxydoapmine;8-OHDPAT, (±)-8-hydroxy-2-(dipropylamino) tetralin hydrobromide;ANOVA, analysis of variance; AP, anterior–posterior; AUC, area undercurve;BPND, binding potential;DA, dopamine;DAT,DA transporter; DV,dorso-ventral; ERLiBiRD, Estimation of Reversible Ligand Binding andReceptor Density; i.p., intraperitoneal; L-DOPA, L-3,4 dihydroxyphenyl-alanine; LID, L-DOPA-induced dyskinesia; ML, medio-lalteral; NA,noradrenaline; PD, Parkinson’s disease; PET, positron emission tomog-raphy; ROI, region of interest; s.c., subcutaneous; VOI, volume of interest.

, ,

,

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*Center of Functionally Integrative Neuroscience, Aarhus University Hospitals, Aarhus University,

Denmark

�Department of Nuclear Medicine and PET Centre, Aarhus University Hospitals, Aarhus University,

Denmark

�Centre for Psychiatric Research, Aarhus University Hospitals, Aarhus University, Denmark

§Department of Neuroscience and Pharmacology, University of Copenhagen, Denmark

Abstract

Recent studies suggest that L-3,4 dihydroxyphenylalanine

(L-DOPA)-induced dyskinesia (LID), a severe complication of

conventional L-DOPA therapy of Parkinson’s disease, may be

caused by dopamine (DA) release originating in serotonergic

neurons. To evaluate the in vivo effect of a 5-HT1A agonist

[(±)-8-hydroxy-2-(dipropylamino) tetralin hydrobromide, 8-

OHDPAT] on the L-DOPA-induced increase in extracellular

DA and decrease in [11C]raclopride binding in an animal

model of advanced Parkinson’s disease and LID, we mea-

sured extracellular DA in response to L-DOPA or a combina-

tion of L-DOPA and the 5-HT1A agonist, 8-OHDPAT, with

microdialysis, and determined [11C]raclopride binding to DA

receptors, with micro-positron emission tomography, as the

surrogate marker of DA release. Rats with unilateral 6-hy-

droxydopamine lesions had micro-positron emission tomog-

raphy scans with [11C]raclopride at baseline and after two

pharmacological challenges with L-DOPA + benserazide with

or without 8-OHDPAT co-treatment. Identical challenge regi-

mens were used with the subsequent microdialysis concomi-

tant with ratings of LID severity. The baseline increase of

[11C]raclopride-binding potential (BPND) in lesioned striatum

was eliminated by the L-DOPA challenge, while the concurrent

administration of 8-OHDPAT prevented this L-DOPA-induced

displacement of [11C]raclopride significantly in lesioned ven-

tral striatum and near significantly in the dorsal striatum. With

microdialysis, the L-DOPA challenge raised the extracellular

DA in parallel with the emergence of strong LID. Co-treatment

with 8-OHDPAT significantly attenuated the release of extra-

cellular DA and LID. The 8-OHDPAT co-treatment reversed

the L-DOPA-induced decrease of [11C]raclopride binding and

increase of extracellular DA and reduced the severity of LID.

The reversal of the effect of L-DOPA on [11C]raclopride bind-

ing, extracellular DA and LID by 5-HT agonist administration is

consistent with the notion that part of the DA increase asso-

ciated with LID originates in serotonergic neurons.

Keywords: 5-HT1A receptor agonist, 6-hydroxydopamine,

L-DOPA induced dyskinesia, micro-positron emission tomog-

raphy and microdialysis, Parkinson’s disease.

J. Neurochem. (2012) 120, 806–817.

JOURNAL OF NEUROCHEMISTRY | 2012 | 120 | 806–817 doi: 10.1111/j.1471-4159.2011.07598.x

806 Journal of Neurochemistry � 2011 International Society for Neurochemistry, J. Neurochem. (2012) 120, 806–817� 2011 The Authors

Parkinson’s disease (PD) is a progressive neurodegenerativedisorder characterized by both motor and non-motorsymptoms. Among the motor symptoms, tremor, rigidity,bradykinesia and postural instability are the most commonthat follow the loss of neurons of the dopaminergicnigrostriatal projection and dopamine (DA) in the striatum.The standard treatment of PD is replacement therapy withthe precursor of DA, L-3,4 dihydroxyphenylalanine (L-DOPA), administered in combination with an inhibitor ofperipheral[extracerebral] aromatic amino acid decarboxylasesuch as carbidopa or benserazide. Although direct DAagonists are often used in the early stages of the disease toreduce or delay the need for L-DOPA, ultimately L-DOPAmust be added to any form of continuing treatment. Themajor drawback of L-DOPA therapy is the eventualdevelopment, within 5–8 years, of L-DOPA-induced motorfluctuations and dyskinesia (Ahlskog and Muenter 2001).Peak-dose dyskinesia is the most common and disablingsymptom, consisting of involuntary, rapid and choreic-typemovements (Jankovic 2005). Although abnormalities in DAturnover and DA transporter function likely play some role,repeated abnormal stimulation of both the DA D2 and D1

receptors, in response to rapid fluctuations in the extracel-lular (Abercrombie and Bonatz 1990) and synaptic (Paveseet al. 2006) and (de la Fuente-Fernandez et al. 2004)concentrations of DA following a therapeutic dose of L-DOPA, are the conventional explanations of L-DOPA-induced dyskinesia (LID) with an emphasis on the role ofprocesses downstream to the DA D1 receptor activation(Berthet and Bezard 2009) and (Cenci and Konradi 2010).Positron emission tomography (PET) with [11C] raclopride,a tracer of the DA D2/3 receptors, the binding of whichresponds to changes in synaptic DA concentrations,revealed a greater rapid increase of DA concentration 1 hafter administration of a clinical dose of L-DOPA indyskinetic patients compared with stable responders (de laFuente-Fernandez et al. 2004).

Although increased DA synthesis coupled with decreasedstorage capacity and fewer DA transporters in dopaminergicterminals may tentatively explain the non-physiologicalconcentrations of DA measured in response to a therapeuticdose of L-DOPA in PD patients and animals with severenigrostriatal lesions, a number of reports suggest thatunregulated conversion of L-DOPA into DA and subsequentuncontrolled release can occur in non-DA cells, especiallyfrom serotonergic (Everett and Borcherding 1970) and (Nget al. 1970) and noradrenergic neurons and from glial cells(Cenci and Lundblad 2006). Centrally, aromatic amino aciddecarboxylase or DOPA decarboxylase is active in manycells, including dopaminergic neurons, but also in seroto-nergic and noradrenergic neurons (Cenci and Lundblad2006). L-DOPA is an amino acid that undergoes facilitateddiffusion across cell membranes, and the activity of the

enzymes regulate the fractions of L-DOPA that are convertedto DA in different cellular compartments of the brain (Gjeddeet al. 1993) and (Cumming et al. 1995). When the DOPAdecarboxylase activity is reduced in PD, larger than normalfractions of the amino acid are subject to conversion in cellsother than the dopaminergic neurons of the nigrostriatalpathway.

The existence of the enzyme complement necessary for thesynthesis of DA from L-DOPA in serotonergic terminalsraises the possibility of a role of these neurons in theincreased L-DOPA-induced DA release in advanced PDpatients. Immunohistochemical studies also demonstrate thatL-DOPA treatment can induce the formation and presence ofDA in serotonergic neurons (Yamada et al. 2007). Themechanism of release of DA from serotonergic cells is notknown but there is evidence that this release is inhibited bythe pre-synaptic action of serotonin (5-HT) and stimulationof raphe 5HT1A autoreceptors (Kannari et al. 2001). LID isreduced by co-treatment with 5-HT1A receptor agonists,presumably through autoreceptor-induced decrease in joint 5-HT and DA release from serotonergic neurons (Eskow et al.2009). Lesions to both the dopaminergic and serotonergicsystems reduce fluctuations of extracellular DA in responseto L-DOPA and decrease LID (Tanaka et al. 1999; Cartaet al. 2007). These findings underline the essential role thatserotonergic terminals may play in the striatal metabolism ofL-DOPA and in the induction of LID in patients withadvanced PD as well as in animals with severe dopaminergicnigrostriatal lesions. By means of diffusion-based volumetransmission (Cragg and Rice 2004), DA released atserotonergic terminals after a dose of L-DOPA, in theabsence of DA transporters, could, in theory, diffuse widelyand rapidly throughout the extracellular and synaptic spacesin the striatum, and this could contribute significantly toincreased synaptic DA concentration and receptor stimula-tion.

These mechanistic interactions between serotonergic anddopaminergic neurons in advanced PD cannot easily beinvestigated non-invasively in human subjects. Although invivo imaging with raclopride as a surrogate marker of DArelease can provide some clues to the changes of striatal DAconcentrations after pharmacological challenge, actual mea-surements of DA concentration must confirm the changes ofDA receptor occupancy determined with raclopride imaging.In the present study, we determined the extracellularconcentrations of striatal DA in concert with behaviouralassessment and in vivo PET measurement of synaptic DAoccupancy in rats lesioned unilaterally with 6-hydroxydop-amine (6-OHDA) as an experimental model of PD and LIDin response to L-DOPA challenges with and without a 5-HT1A agonist.

By doing so, we tested two specific hypotheses that revealthe role of serotonergic innervation of the striatum in LID:

� 2011 The AuthorsJournal of Neurochemistry � 2011 International Society for Neurochemistry, J. Neurochem. (2012) 120, 806–817

Dopamine originates from serotonin neurons in LID | 807

(i) Co-administration of (±)-8-hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OHDPAT), a 5-HT1A agonist, withL-DOPA prevents the L-DOPA-induced displacement of[11C]raclopride and the instance of LID, without affecting theL-DOPA-induced motor effect in the unilaterally 6-OHDAlesioned rat.

(ii) The L-DOPA induced decrease of [11C]raclopridebinding is more pronounced in the ventral striatum than inthe dorsal striatum, in reflection of the dorsoventral gradientof serotonergic innervation of the striatum.

Materials and methods

Experimental animalsTwenty-one female Sprague–Dawley rats (Taconic, Sjaelland,Denmark) weighing 250–270 g at the beginning of the experimentwere housed at a 12-h light/dark cycle with free access to food andwater during the entire period. All experiments were performedaccording to the Danish Experimental Animal Inspectorate.

DrugsL-3,4-Dihydroxyphenylalanine methyl ester hydrochloride, bense-razide hydrochloride, (±)-8-hydroxy-2-(dipropylamino) tetralin hyd-robromide, desipramine hydrochloride and 6-hydroxydopaminehydrochloride were purchased from Sigma-Aldrich (Brøndby,Denmark). Dormicum was purchased from Midazolam (Herlev,Denmark); Hypnorm from VetaPharma (Leeds, UK) and Rimadylfrom Aarhus University Hospital Pharmacy (Aarhus, Denmark).

Experimental parkinsonismThe rats were unilaterally lesioned with 6-OHDA, injected into themedial forebrain bundle as described in detail elsewhere (Lee et al.2000). Briefly, Hypnorm (fentanyl, 0.315 mg/mL and fluanisone,10 mg/mL) and Dormicum (midazolam, 5 mg/mL) were separatelydissolved in 1 : 1 solutions in sterile saline. Anaesthesia wasinduced with Dormicum (4.5 mg/kg midazolam) and Hypnorm(9 mg/kg fluanisone and 0.2835 mg/kg fentanyl) administered in avolume of 1.8 mL/kg and was maintained with a third of the initialdose every 30–60 min. Desipramine hydrochloride (25 mg/kg i.p.)was given at least 60 min prior to anaesthesia to protect noradren-ergic terminals. Animals were positioned in a stereotaxic frame(David Kopf, Tujunga, CA, USA) and received two infusions of 6-OHDA of 6 and 7.5 lg 6-OHDA in a concentration of 3 lg/lLdissolved in a solution of 0.05% L(+)-ascorbic acid in sterile salineat the rate of 1 lL/min. Injections were performed into the rightmedial forebrain bundle with a Hamilton syringe directed at thefollowing coordinates: (i) tooth bar at +3.4, anterior–posterior (AP))4.0, medial–lateral (ML) )0.8, dorsal–ventral (DV) )8.0 (6 lg 6-OHDA), and (ii) tooth bar at )2.3, AP )4.4, ML )1.2, DV )7.8(7.5 lg 6-OHDA) in mm from bregma, midline and the duralsurface, respectively. Before cautious removal, the Hamilton syringewas left at the site of injection for an additional 3 min to allow fordiffusion of 6-OHDA. The rats received post-operative analgesiawith Rimadyl (4 mg/kg) and additional saline was administeredsubcutaneously (s.c.) for hydration. The rats were carefullymonitored until fully awake.

Dyskinesia and motor activityAfter a few days of recovery from surgery, the animals started toreceive daily injections of L-DOPA (8 mg/kg, s.c.) and benserazide(15 mg/kg, s.c.) in a volume of 0.3 mL saline. This dose has beenfound to induce stable expression of dyskinetic features in rats withsevere 6-OHDA unilateral lesions (Cenci et al. 1998; Carta et al.2007; Lindgren and Andersson 2010). Of the original group, 13 ratsdeveloped LID within a few days of initiation of L-DOPA + bense-razide treatment and were included in the rest of the study. We ratedthe severity of LID according to three topographical manifestations,using the abnormal involuntary movement rating scale (Cenci et al.1998) with small modifications. Briefly, we observed and scoredLID for 2 min every 20th minute for a total period of 300 min afterL-DOPA or L-DOPA + 8-OHDPAT administration. The topograph-ical manifestations included: (i) limb: choreiform limb movements,stereotypic and repetitive movements on the side contralateral to thelesion; (ii) axial: deviation or torsion of the head, neck and trunkcontralateral to the lesion; (iii) oral: repetitive chewing movementsand tongue protrusion. L-DOPA-induced motor effects wereconsidered to be drug-induced rotations in the form of full bodyturns contralateral to the lesioned striatum and were counted withthe same schedule as LID. Each of the three LID manifestations andthe drug-induced rotations were rated as: 0 = absent; 1 = occa-sional, that is, present during less than 50% of the observation time;2 = frequent, that is, present during more than 50% of theobservation time; 3 = present during the entire observation timebut could be disrupted by strong external stimulation (sound forexample) and 4 = continuous and not interrupted by strong sensorystimuli. The final motor score and LID score in this study, for thepurpose of comparison to micro-PET and microdialysis data, wereobtained during the performance of the microdialysis study. Thefinal LID score used for analysis consists of the average of the threetopographic manifestations.

Positron emission tomographyThe rats underwent micro-PET after 1.5–3 weeks of daily L-DOPAtreatment and the identification of reproducible LID as the state ofrats with stable dyskinesia. Each rat underwent three micro-PETsessions with [11C]raclopride, at baseline before pharmacologicalchallenge, 25–68 min after a dose of L-DOPA + benserazide(50 mg/kg + 25 mg/kg s.c.), and 26–63 min after injection of L-DOPA + benserazide immediately followed by injection of 8-OHDPAT (0.6 mg/kg i.p.). The high dose of 50 mg/kg of L-DOPAfor the PET challenge was chosen to permit comparison with earlierwork (Abercrombie and Bonatz 1990; Miller and Abercrombie1999; Tanaka et al. 1999; Kannari et al. 2001). The dose of 0.6 mg/kg of 8-OHDPAT was selected as an intermediate to the doses usedby Kannari et al. (2001), to induce a significant effect, measurablewithin the timeframe of our micro-PET design, while avoidingunwanted side effects of 5-HT agonism. The baseline and onechallenge session with either L-DOPA or L-DOPA + 8-OHDPATwere performed on day one, and the session with the secondchallenge was performed within a week, that is, always on differentdays to reduce possible interactions between the two challenges. Theanimals did not receive its routine dose of L-DOPA at the regulartime on the day of the scan as the drug injections were done asneeded prior to injection of the radiotracer. Thus, for the baselinescan and the second and third scan of the series completed on

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808 | A. Nahimi et al.

different days, the animal received the last dose of L-DOPAapproximately 26–28 h prior to the onset of the session. We used thesame small animal tomograph (micro-PET R4; CTI Concorde,Knoxville, TN, USA) for all scans. Anaesthesia was induced in achamber filled with 5% isoflurane in a mixture of oxygen (O2)(0.4 L/min) and air (1.5 L/min). After the induction of anaesthesia,we positioned the head of the animal in a custom-built Plexiglashead holder and maintained the anaesthesia with a cone maskdelivering isoflurane (1.8–2.0%) in O2 (0.4 L/min) and air (1.5 L/min) fitted to the head holder. A catheter was inserted transcuta-neously into the tail vein for injection of [11C]raclopride in saline.Rectal temperature was maintained close to 36.5�C with a heat lampregulated by a rectal thermometer. The saturation and heart rateswere monitored with the ABL520 system (Radiometer, Copenha-gen, Denmark). At the beginning of each PET session, we obtaineda 10-min attenuation scan with a 68Ge point source. Dynamicemission recordings were initiated upon injection of a dose of[11C]raclopride, between 7 and 8 MBq/100 g body weight, followedby a 60 (in three scans 90) min long emission recording of 23 framesincreasing in duration from 15 s to 10 min. After the PET sessions,animals woke up spontaneously and were returned to the housingcage. Data processing and data analysis procedures are described indetail elsewhere (Pedersen et al. 2007). Briefly, attenuation-cor-rected dynamic emission images were reconstructed by 3-D-filteredback projection resulting in a 128 · 128 · 63 matrix. Summedemission recordings were manually registered to a digital high-resolution atlas of the rat brain (Toga et al. 1995; Rubins et al.2003). Time activity curves were extracted by the resampling of fivevolumes of interest composed of left and right dorsal striatum(40 mm3), left and right ventral striatum (7 mm3) and cerebellum(314 mm3) templates in the native space. We calculated the bindingpotential BPND relative to ‘non-displaceable’ (i.e. non-saturable)radioligand in the tissue by means of the Logan reference tissuemodel (Logan et al. 1996) with cerebellum as the non-displaceablebinding reference tissue in the period between 30 and 60 min. Forcomparison, we also used the non-linear regression method,Estimation of Reversible Ligand Binding and Receptor Density(ERLiBiRD) method using the time frames between 1 and 60 min(Møller et al. 2007). Only the results from the Logan referencetissue model are shown here, as the results with ERLiBiRD analysiscompletely supported the results with Logan reference tissue model.Three [11C] raclopride doses accidently were injected interstitiallyand hence the corresponding results were not used for furtheranalysis.

MicrodialysisFollowing the completion of all micro-PET sessions, a subset of theanimals (N = 7–8) were used to measure extracellular DA in parallelwith the rating of the severity of LID. The animals were allowed afew days of recovery after the completion of the PET studies. Theywere then moved to the animal housing area of the microdialysislaboratory and were allowed a few days to acclimatize to the newenvironment. They did not receive L-DOPA during this period (10–14 days) after which daily L-DOPA was re-initiated. Microdialysisstudies were performed about a week after the expression of stableabnormal involuntary movements was reinstated, that is, about 3.0–3.5 weeks after the last PET study. The microdialysis proceeded asdescribed by Wegener et al. (2000). Under anaesthesia (Hypnorm0.05 mL/100 g s.c. and Midazolam 0.5 mg/100 g i.p.), two micro-

dialysis probes (concentric probes of rigid design with 3 mm activemembrane, Goodfellow) were implanted stereotactically in bothdorsal striata through a burr hole in the skull (AP + 0.5, ML ± 3.0,DV )6.0 from the dural surface and bregma, respectively, tooth bar)3.3). The probes were fastened by dental cement (GC Fuji PLUS,Alsip, IL, USA) and the rats were allowed to recover for 2 daysbefore the microdialysis experiments were conducted. Approxi-mately 24 h prior to the dialysis experiment, the rats were brought tothe testing cage and connected to the wires in the microdialysissetup and to a swivel joint allowing unrestricted movement withinthe testing cage and the probes were perfused continuously withdistilled water (Maxima) at a rate of 2 lL/min overnight. On the dayof the experiment, the probes were perfused continuously withRinger¢s solution (147 mM NaCl, 4 mM KCl, 2.3 mM CaCl2) at aflow rate of 2 lL/min at least half an hour prior to collecting samplesto make sure that the wires were filled with Ringers solution. Duringthe experiment, dialysate was collected at 20-min intervals (40 lL/sample). After collection, 5 lL of perchloric acid (0.005 M) wasadded to 30 lL of each sample to reduce further metabolism anddegradation. After centrifugation, the samples were stored at )80�Cuntil analysis. On the day of analysis, we used high pressure liquidchromatography with electrochemical detection (ESA 5014BCoulometric and Coulochem III, ESA, Chelmsford, MA, USA) toanalyze the samples. The mobile phase (NaH2PO4 13,40 g/L,Na2EDTA 15.0 mg/L, octane sulfonic acid 400 mg/L and acetonitrile90 mL/L, pH 2.6 adjusted by phosphoric acid) was delivered at a flowrate of 400 lL/min. Using Chromeleon chromatographic software(Dionex, Chelmsford, MA, USA), the samples were analyzed for thecontent of DA. The detection limit was 0.1 nM.We acquired baselinedata for 3 h, after which L-DOPA + benserazide (50 mg/kg+ 25 mg/kg s.c.) was administered and sampling continued for afurther 5 h. On the following day, after acquisition of new baselinedata for 3 h to ensure that the previous dose of L-DOPA did not affectthe results, the rats received L-DOPA + benserazide (50 mg/kg + 25 mg/kg s.c.) + 8-OHDPAT (0.6 mg/kg i.p.) and samplingcontinued for 5 h. The order of challenge drugs to be administered onday 1 or on day 2 were randomly chosen. Following the end of themicrodialysis study the rats were rapidly decapitated with a guillotinefor verification of lesion severity with autoradiography and probelocation.

AutoradiographyThe brains were removed, immediately frozen in isopentane andstored at )80�C until processing. The brains were cut into 20 lmcoronal sections on the Vibratome ULTRApro 5000, mounted onpoly-L-lysine slides (Menzel Glazer Polysine, TM) and again storedat )80�C until further processing. We assessed the abundance of DAtransporters by autoradiography of the DA transporter ligand[3H]WIN 35,428 to evaluate the ipsilateral loss of DA terminals bycomparison with the contralateral side. Briefly, the slides weredefrosted at 25�C for 30 min and pre-incubated for 5 min in buffer 1(50 mM Tris–HCl, 120 mM NaCl and 5 mM KCl dissolved indistilled water at pH 7.9 and temperature 4�C) followed byassessment of the total DA transporter (DAT) binding by incubationof the slides with the DAT-tracer [3H]WIN 35,428 (15 nM) in buffer2 (50 mM Tris–HCl, 300 mM NaCl and 5 mM KCl dissolved indistilled water pH 7.9 at 4�C) for 40 min. We assessed non-specificbinding on adjacent slides by the addition of a NA-DA reuptakeinhibitor (Nomifensine 10 mM) to buffer 2, of course with the DA

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Dopamine originates from serotonin neurons in LID | 809

transporter ligand [3H]WIN 35,428 (15 nM), in a separate tray. Afterthe incubation, the slides were rinsed from excess incubation bufferby a post-wash twice for 1 min in buffer 2 and then dipped indistilled water at 4�C. The slides were dried under a stream of coldair, placed in a vacuum desiccator with paraformaldehyde for 24 hand exposed to tritium-sensitive imaging phosphor screens (Fuji-film) for 5 days. After exposure, the imaging phosphors werescanned with the BAS-5000 scanner. The autoradiograms wereanalyzed using Image Gauge 4.03 (FujiFilm). We measured digitallight units by placing regions of interest on the sections based onthree criteria: most intact tissue, darkest binding and outlines of thestriatum. An average of non-specific binding, in units of digital lightunits, was measured from the slides that had been incubated withboth [3H ]WIN 35,428 and nomifensine. The specific binding wascalculated by subtracting the non-specific binding from the totalbinding.

Statistical analysisStatistical analyses used Graphpad Prism version 5.0. Results arereported as means with the standard error of mean indicated by thesymbol (±). Probability of less than 0.05 was considered statisticallysignificant.

The severity of the lesion was calculated as the percentbinding of [3H]WIN 35,428 in the lesioned compared with theintact striatum. Two-way analysis of variance (ANOVA) followedby Bonferroni’s post-tests were used in the comparisonsof [11C]raclopride binding at baseline and after L-DOPA orL-DOPA + 8-OHDPAT treatment, using lesion and challenge asfactors. The changes in DA receptor occupancy after L-DOPAand L-DOPA + 8-OHDPAT challenges compared with baselinewere estimated from the changes of [11C]raclopride BPNDaccording to conventions (Gjedde and Wong 1987; Wong et al.1997; Innis et al. 2007):

DBPND ¼ ½1� ðBPND=Challenge=BPND=BaselineÞ� � 100

One-tailed, paired t-tests compared the changes of radioligandbinding at baseline and during challenge conditions.

We used the average of orolingual, limb and axial dyskinesiascores as a measure of total dyskinesia, while the drug inducedrotations reflected the motor effect of L-DOPA and hence a separateindex of therapeutic efficacy. Using time and challenge withL-DOPA or L-DOPA + 8-OHDPAT as factors, two-way ANOVA

separately assessed the roles of total dyskinesia (as index ofserotonergic mechanism) and drug-induced rotation (as index ofdopaminergic mechanism).

We calculated the area under curve (AUC) to quantify theeffects of drug challenge on extracellular DA concentrations -following challenge with L-DOPA or L-DOPA + 8-OHDPAT.The microdialysis data only illustrate the effects of challenge drugson extracellular DA as only a subset of animals that were used inthe micro-PET study were also used for microdialysis.

Results

DenervationCompared with the intact striatum, DA transporter densityevaluated by [3H]WIN 35,428 declined by 97% (95% CI94.08–99.61) on the lesioned side, indicating a high degreeof lesion and severe loss of nigrostriatal dopaminergicprojections. Two-way ANOVA showed significant increase(p < 0.0001) of [11C]raclopride BPND in dorsal and ventralstriatum on the 6-OHDA injected side, determined witheither method of analysis (Fig. 1), compared with thecontralateral striatum.

(a)

(b)

Fig. 1 [11C] Raclopride BPND at baseline

and after L-DOPA or L-DOPA + 8-OHDPAT

challenge, calculated with Logan reference

tissue model, in the dorsal (a) and ventral

(b) striatum. The significance of the differ-

ence between baseline and challenge was

tested with two-way ANOVA’s, seperately for

dorsal and ventral striatum (see text)

(**p < 0.01; ***p < 0.001).

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810 | A. Nahimi et al.

Dyskinesia and motor activityTwo-way ANOVA showed significant effects of challengedrugs (p < 0.0001) and of time of administration ofchallenge drugs (p = 0.0004) and a significant interaction(p < 0.0001) between challenge drugs and of time ofadministration of challenge drugs on the severity of expres-sion of LID. Bonferroni post-tests showed that dyskinesiaseverity significantly declined with co-administration of 8-OHDPAT until 180 min post-administration (Fig. 2a). Therewas a significant effect of time of administration of thechallenge drugs (p < 0.0001) and of the challenge drugsthemselves (p = 0.0072) but no significant interaction(p = 0.3763) between the time of administration and thekind of challenge drugs on the magnitude of drug-inducedrotations. Bonferroni post-tests revealed significant differ-ence in drug-induced rotations between the L-DOPA andL-DOPA + 8-OHDPAT drug challenges during the first

20 min post-administration (p < 0.001). In the following280 min, the drug-induced rotations were similar forL-DOPA and L-DOPA + 8-OHDPAT treatment (Fig. 2b).

Dopamine receptor availabilityTo take into account the possible effect of the injected massof raclopride on [11C]raclopride BPND, the total injected doseof raclopride per body weight (kg) was calculated separatelyfor the three PET-sessions and tested for correlation to[11C]raclopride BPND in all volumes of interest in the threePET sessions (the mass was known in 24 scans). The meaninjected raclopride mass per bodyweight (kg) was3.067 nmol/kg (± 0.4211) in the baseline session,5.414 nmol/kg (± 0.9364) in the L-DOPA session and4.203 nmol/kg (± 0.8234) in the L-DOPA + 8-OHDPATchallenge session. There was no significant correlationbetween the injected raclopride mass and [11C]racloprideBPND in the three PET sessions, except, after L-DOPA + 8-OHDPAT challenge, in the lesioned ventral striatum(p = 0.0313).

Two-way ANOVA revealed significant effect of lesion on[11C]raclopride binding at baseline in both ventral and dorsalstriatum (p < 0.0001) and significant effect of drug challengein the ventral striatum (p < 0.0001) and close to significanteffect in the dorsal striatum (p = 0.0996). The interactionbetween lesion and drug challenge was significant in both thedorsal and ventral striatum (p = 0.0444 and 0.0409, respec-tively). Bonferroni post-tests revealed a significant decreaseof baseline [11C]raclopride binding after the L-DOPAchallenge (p < 0.01 and p < 0.001 in the dorsal and ventralstriatum, respectively). Co-administration of 8-OHDPATreversed the L-DOPA-induced decrease of [11C]raclopridebinding in the ventral striatum (p < 0.01) and showed a trendin the dorsal striatum, although the latter did not reachsignificance (p > 0.05) as shown in Fig. 1 of the [11C]raclo-pride-binding potentials. The ERLiBiRD non-linear methodshowed similar results (not shown here). However, as inPedersen et al. (2007), [11C]raclopride BPND were somewhatlower with ERLiBiRD than with the Logan reference tissuemodel.

Dopamine receptor occupancyWe calculated the DA receptor occupancy from the changeof baseline [11C]raclopride binding after the drug challenge,with one-tailed t-tests of the significance of random changeof DA receptor occupancy after L-DOPA or L-DOPA + 8-OHDPAT drug challenge. The dorsal striatum of the intacthemisphere sustained 7% and 5% increases in DA receptoroccupancy after L-DOPA and L-DOPA + 8-OHDPAT treat-ments, respectively (non-significant p = 0.2811). In thedorsal striatum of the lesioned hemisphere, the L-DOPAchallenge increased the DA receptor occupancy by 18%,while the DA receptor occupancy marginally rose by 4%with the L-DOPA + 8-OHDPAT treatment (p = 0.0682). In

Dyskinesia scores

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Fig. 2 Total dyskinesia scores were calculated from mean of orolin-

gual, limb and axial dyskinesia scores. Co-administration of 8-OHD-

PAT significantly reduced total dyskinesia from 20 to 100 min

(p < 0.001), 120 to 140 min (p < 0.01) and 160 to 180 min (p < 0.05)

after drug challenge (a). Co-administration of 8-OHDPAT only reduced

drug induced rotations in the first 20 min (p < 0.0001) after drug

challenge. L-DOPA challenge (circles) and L-DOPA + 8-OHDPAT

challenge (squares) (b).

� 2011 The AuthorsJournal of Neurochemistry � 2011 International Society for Neurochemistry, J. Neurochem. (2012) 120, 806–817

Dopamine originates from serotonin neurons in LID | 811

the intact ventral striatum, L-DOPA treatment raised the DAreceptor occupancy by 14%, and 8-OHDPAT co-treatmentreversed the change to 0 (p = 0.213). In the lesioned ventralstriatum, L-DOPA treatment raised DA receptor occupancyby 29% that significantly declined to 3% with 8-OHDPATco-treatment (p = 0.0165), as shown in Figs 3 and 4.

Micro-dialysis measurements of dopamineBy placing microdialysis probes bilaterally in both the rightand left dorsal striatum, we were able to measure extracellularDA concentrations, before and after challenge, in both the intactand lesioned striata. Following administration of L-DOPA, theAUC of the DA concentrations on the lesioned side was muchlarger, than that of the AUC of the intact side, magnitudesaveraging 5854 units and 1179 units, respectively for lesionedand intact hemispheres. The co-administration of 8-OHDPATdramatically reduced DA concentrations in both lesioned andintact striatum, to 110.5 and 32.54 units, respectively, for thelesioned and intact sides, as shown in Fig. 5.

Discussion

In this animal model of advanced PD and LID, we combinedin vivo micro-PET and microdialysis to assess the contribu-tions of serotonergic neurons to the effects of exogenous L-DOPA in the lesioned brain. The unilaterally lesioned ratshad three PET sessions with the DA receptor radioligand[11C]raclopride, once at baseline and then twice after both L-DOPA and L-DOPA + 8-OHDPAT challenges, accompaniedby microdialysis of extracellular DA and rating of LID. Aspredicted on the basis of previous results, the co-administra-tion of the 5-HT agonist, 8-OHDPAT, did significantly

inhibit the increase of extracellular DA in the lesionedhemisphere and the displacement of the DA receptorradioligand from its receptors, and the effect was greater inventral than in dorsal striatum, as predicted from the anatomyof serotonergic innervation of the striatum.

BaselineThe baseline of parkinsonism showed elevated [11C]raclo-pride binding in both dorsal and ventral striatum of thelesioned hemisphere, irrespective of analysis method. Thisfinding is consistent with the abundant evidence fromuntreated symptomatic PD patients (Rinne et al. 1993),symptomatic MPTP-lesioned monkeys (Doudet et al. 2002)and 6-OHDA-lesioned rodents (Sossi et al. 2009). In thesesubjects, the increase of [11C]raclopride binding appears toreflect the combined changes of increased number ofreceptors (Bmax) and decreased DA receptor occupancy(Rinne et al. 1995) and (Doudet et al. 2002) and is consistentwith the emergence of motor deficits from loss of nigrostri-atal DA innervation where the loss exceeds 75% of baseline(Sossi et al. 2009). In this study, we had more than 90% lossof terminal density, confirmed by autoradiography of DAtransporters.

In PD patients, chronic L-DOPA treatment reverses theincrease of [11C]raclopride binding (Turjanski et al. 1997),such that no significant differences of [11C]raclopridebinding are found in patients with advanced PD comparedwith age-matched control subjects. However, in this model ofthe disease in rats, the increase of [11C]raclopride bindingpersisted with the daily treatments with L-DOPA + bense-razide at the time of the baseline PET session. Thediscrepancy may relate to the different frequency of

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(a)

(b)Fig. 3 DA receptor occupancy after drug

challenge relative to baseline in dorsal (a)

and ventral (b) striatum was calculated

according to conventions. One tailed t-tests

were performed to test the significance of

the difference in DA receptor occupancy

between the two challenge condi-

tions[(*p < 0.05; and a trend towards sig-

nificance is indicated with (*)].

Journal of Neurochemistry � 2011 International Society for Neurochemistry, J. Neurochem. (2012) 120, 806–817� 2011 The Authors

812 | A. Nahimi et al.

treatment, as human subjects receive L-DOPA four timesevery day, while the animals were treated only once a day.The short half-life of L-DOPA and more rapid metabolism inrodents compared with primates may reduce the DA receptor

stimulation and hence attenuate the compensatory down-regulation of DA receptors in response to L-DOPA in theanimal model. Also, the PET session required that the ratsreceived the last dose of L-DOPA at least 24 h prior to thetomography.

L-DOPA challengeRaclopride is a benzamide antagonist of the DA D2/3

receptors. As for other benzamides, it is believed thatchanges in radioligand binding in the challenge situationreflect changes of synaptic DA, that is, an increase insynaptic DA inhibits radioligand binding (Cumming et al.2002; Ginovart 2005), although other mechanisms affectingreceptor availability may also be involved (Doudet andHolden 2003; Ginovart 2005; Gjedde et al. 2005).

The L-DOPA challenge had no significant effect on the[11C]raclopride binding in the intact striatum, and themicrodialysis revealed much smaller increases of the extra-cellular DA in this hemisphere than in the lesionedhemisphere after the L-DOPA challenge, as observed inprevious studies with microdialysis (Abercrombie andBonatz 1990) and PET (Sossi et al. 2009). The acute L-DOPA challenge significantly inhibited the [11C]raclopridebinding in both dorsal and ventral striatum of the lesionedhemisphere, which is consistent with the prediction ofincreased extracellular DA after the challenge, and with theevidence from microdialysis. With micro PET, the increaseof DA receptor occupancy in dorsal and ventral striatum ofthe lesioned hemisphere approximated 18% and 29%,respectively. Similar increases in DA occupancy have beenreported in the striatum of patients with advanced PD andLID (de la Fuente-Fernandez et al. 2004; Pavese et al. 2006)and in previous micro PET studies of 6-OHDA lesioned rats(Sossi et al. 2009). The change in radioligand binding afterthe L-DOPA challenge was more pronounced in patients withLID than in stable responders (Tedroff et al. 1996; de laFuente-Fernandez et al. 2004; Pavese et al. 2006).

With respect to the possible metabolism of L-DOPA in,and release of DA from, serotonergic neurons (Cenci andLundblad 2006; Carta et al. 2007), we predicted that the L-DOPA-induced DA release, as measured with micro-PET,would be larger in the lesioned ventral striatum than in thelesioned dorsal striatum, as suggested by the increasingdensity of serotonergic innervation from dorsal to ventralstriatum. The prediction was upheld by the observation thatthe L-DOPA-induced increase of DA receptor occupancy inthe lesioned hemisphere numerically was larger in ventralstriatum (29%) than in dorsal striatum (18%), although thedifference was not statistically significant. The effect ofpartial voluming in this study also needs to be taken intoconsideration: It has been suggested that in humans 30% ofthe raclopride radioactivity in the ventral striatum originatesfrom putamen and caudate (Mawlawi et al. 2001). Partialvoluming may play an even greater role in the small rat brain

(a)

(b)

Fig. 4 We defined volumes of interest (VOIs) for the dorsal and

ventral striatum in the digital rat atlas (a). Blue and green VOIs indicate

the intact, left, dorsal and ventral striata, respectively. Yellow and

white VOIs indicate the lesioned, right dorsal and ventral straita,

respectively. (b) Parametric images of [11C] raclopride BPND at

baseline (upper image) and after L-DOPA (middle image) or

L-DOPA + 8-OHDPAT (lower image) were created using the Logan

reference tissue model (arrow shows lesioned side). As seen in the

upper and middle image and in support of our hypothesis, L-DOPA

administration reduces the increase in baseline [11C] raclopride bind-

ing. As seen in the lower image, co-treatment with 8-OHDPAT

reverses the effect of L-DOPA on [11C] raclopride binding.

� 2011 The AuthorsJournal of Neurochemistry � 2011 International Society for Neurochemistry, J. Neurochem. (2012) 120, 806–817

Dopamine originates from serotonin neurons in LID | 813

and the poor resolution scanner used and thus, the greaterserotonergic DA release in ventral than in dorsal striatumcould be under-estimated.

In support of a gradient of serotonergic release of DA inadvanced PD, recent microdialysis data demonstrated that thedensity of serotonergic innervation in- and outside striatumpredicted the magnitude of L-DOPA-induced changes inextracellular DA concentration (Navailles et al. 2011). Wecould not test this observation in the present microdialysisstudy, as we only placed one probe in the middle of eachdorsal striatum.

L-DOPA + 8-OHDPAT challengeThe possible involvement of the serotonergic system in PDand LID has been the focus of considerable attention, and hasled to the suggestion of adjunctive 5-HT-selective drugs tothe treatment of PD patients with LID (Cenci and Lundblad2006; Carta et al. 2007; Fox and Chuang 2008). However,despite this special focus on the role of 5-HT1A receptors inthe management of LID, few imaging studies have actuallyaddressed the interactions between 5-HT and DA inadvanced PD with LID (Esposito and Di Matteo 2008).

The 5-HT1A receptors are located post-synaptically inforebrain regions and pre-synaptically on the soma anddendrites of the serotonergic cell bodies in the raphe nuclei.The 8-OHDPAT agonist of 5-HT1A receptors is highlyselective and potent (Stamford et al. 2000). By stimulation ofpre-synaptic 5-HT1A autoreceptors, the agonist lowers 5-HTsynthesis and neuronal firing as well as the subsequentrelease of 5-HT from terminals in the striatum of neurons thatoriginate in the dorsal raphe nucleus (Barnes and Sharp1999). Pharmacological inhibition of the activity of 5-HTneurons or lesioning of the 5-HT system in animal models ofadvanced PD both limit the L-DOPA-induced increase ofextracellular DA in striatum and the severity of theaccompanying LID (Tanaka et al. 1999; Kannari et al.2001; Carta et al. 2007; Lindgren and Andersson 2010).The non-physiological release of DA from serotonergic

neurons would lead to non-physiological stimulation of DAreceptors. Hence, we tested the receptor occupancy of DAreceptors in response to L-DOPA co-administered with 8-OHDPAT, and the co-administration attenuated the L-DOPA-induced changes both of extracellular DA and DAreceptor occupancy in the lesioned striatum as measured bymicrodialysis and microPET with raclopride as the surrogatemarker of DA occupancy. When 8-OHDPAT was co-administered, [11C] raclopride significantly returned tobaseline in the ventral striatum but only showed a trend inthe dorsal striatum, which is in contrast with our microdi-alysis results that show a dramatic decrease in extracellularDA in the dorsal striatum following 8-OHDPAT co-admin-istration. It is however in line with previous studies(reviewed by Laruelle 2000) that show that the binding ofbenzamides, such as raclopride, more readily respond toincreases in intrasynaptic DA compared with increases inextrasynaptic/extracellular DA. Breier et al. (1997) showedthat an increase in extracellular DA by 1365%, afteramphetamine challenge, decreased [11C] raclopride bindingwith 21%. However, Kim et al. (1998) showed that achallenge with nicotine that increases DA neuronal activity,without blocking DAT, increased extracellular DA by only29% but reduced [11C] raclopride binding by 21%. Laruelle(2000) suggests that this apparent discrepancy betweenchanges in extracellular DA on one hand and [11C] raclopridebinding on the other hand support the hypothesis that thebinding of benzamides reacts more readily to changes inintrasynaptic DA (Kim et al. 1998) compared with changesin extracellular DA (Breier et al. 1997). Although ourdialysis data suggest a minimal effect of L-DOPA co-administered with 8-OHDPAT on the extracellular concen-trations of DA, it is possible that this effect was intrasynap-tically sufficient to induce some competition betweenraclopride and the endogenous DA released. In our dialysisstudy, an average 600% increase in L-DOPA inducedextracellular DA led to about 18% change in raclopridebinding in the dorsal striatum. The extracellular DA

Dopamine average

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Fig. 5 Measurements of extracellular DA at baseline and after L-

DOPA or L-DOPA + 8-OHDPAT challenge (time of administration of

drug challenge is indicated by the dotted line). After L-DOPA admin-

istration the increase in extracellular DA was larger in the lesioned side

compared with the intact side. 8-OHDPAT reduced the release of DA

in both the intact and lesioned striata.

Journal of Neurochemistry � 2011 International Society for Neurochemistry, J. Neurochem. (2012) 120, 806–817� 2011 The Authors

814 | A. Nahimi et al.

concentrations after L-DOPA + 8-OHDPAT showed only amarginal increase and the change in raclopride bindingfollowing L-DOPA + 8-OHDPAT in the dorsal striatum wasin fact very close to the test–retest reproducibility value ofthe method in our hands (6%), suggesting that the apparentlack of significance may result from variability in a smallsample of animals. It is however remarkable that the reversalof L-DOPA induced release of DA when co-administeed with8-OHDPAT can be measured non-invasively in a smallsubject sample, especially when taking into consideration thetechnical limitations of small animal imaging and the effectof partial voluming in small brain regions. This suggests thatsimilar studies may be performed successfully in a reason-able subset of human subjects, arguing favourably for thetranslational application of such pharmacological challenges.

In addition to the inhibition in serotonergic neurons of L-DOPA metabolism and DA release, stimulation of 5-HT1A

receptors also may affect motor activity in animals withimpaired dopaminergic neurotransmission. This effect maybe mediated by stimulation of post-synaptic 5-HT1A recep-tors which modulate the glutamatergic cortico-striatal inner-vation that affects general activity (Reith et al. 1998; Mignonand Wolf 2005).

Dyskinesia and therapeutic motor effectsIn this study, we scored three manifestations of dyskinesia(limb, axial and oral dyskinesia) and one of motor activity(rotation). We averaged the dyskinesia scores for all threemanifestations as we observed no significant differences inthe effect of the pharmacological challenges on the individ-ual scores. In contrast, there were marked differencesbetween the effects of the challenges on the dyskinesia onthe one hand and the motor score on the other. As expected,the effects of L-DOPA challenge occurred rapidly and wereobserved within 20 min of administration, most likelybecause of an immediate increase of extracellular DA andwere followed by emergence of LID and rotational behaviorand decline of [11C]raclopride binding. In the interval thatmatched the duration of a micro-PET session, total LIDscores fell significantly with the L-DOPA + 8-OHDPATtreatment, compared with the L-DOPA treatment alone.While co-administration of 8-OHDPAT significantly reducedthe total dyskinesia throughout the entire 5-h post-challengeperiod, L-DOPA + 8-OHDPAT administration only delayedthe onset of drug-induced rotations by up to 40 min.

Co-treatment with 8-OHDPAT not only reduced LID asshown by others (Carta et al. 2007) but it also altered thetemporal development of the LID. While maximum LIDappeared within 20 min of the injection and remainedmaximal for the next 4 h, slowly decreasing by the end ofthe experiment, observable dyskinesia after L-DOPA + 8-OHDPAT did not emerge until an hour post-injection andprogressed slowly towards a peak during the last hour of thestudy, never reaching the intensity observed after L-DOPA

administration. This pattern suggests that co-administrationof 8-OHDPAT may lower the firing of serotonergic neuronssufficiently to delay the DA release. As measured withmicro-PET, co-treatment with 8-OHDPAT significantlyprevented or reduced the L-DOPA induced increase of DAoccupancy that lowered the binding of [11C] raclopride in theventral striatum.

It is generally accepted that the beneficial and detrimentaleffects of L-DOPA are present with the same frequency inpatients with advanced PD that receive long-term L-DOPAtreatment. This relationship between positive and negativeeffects of L-DOPA complicates the treatment of PD since it isvery difficult to limit the LID without losing the beneficialeffects of L-DOPA. The present study suggests that non-physiological activation of D2 receptors by DA, inappropri-ately released from serotonergic neurons, may play animportant role in the development of LID.

The present study also suggests that adjunct therapy with a5HT1A receptor agonist, optimized for dose and time ofadministration, may help maintain the beneficial motoreffects of L-DOPA therapy, at least in some patients as itreduces the occurrence of the unwanted dyskinesia.

In conclusion, this study demonstrates that evaluating theeffect(s) of pharmacological challenges by one or multipledrugs may be a feasible goal in human subjects. Althoughstudies similar to ours, demonstate the effects of co-adminis-tration of L-DOPA with 5HT1A agonist on DA synthesis andrelease have been published, most have used a combination ofbehavioral assessment with an invasive method such as post-mortem examination or microdialysis, making them ethicallynot acceptable for a human population. This study add asignificant translational component to earlier studies as wedemonstrate that PET, using a specific tracer of DA release (inour case raclopride but it could be feasible with tracers ofextrastriatal DA function such as FLB457 or fallypride) mayhelp investigate the potential corrective effect of adjuncttherapies as well as their mechanism of action in subjectssuffering from Parkinson’s Disease.

Acknowledgements

This study received financial support from Aarhus PET-Centre,Aarhus University Hospitals, Aarhus University, Denmark and froma grant from the Danish Medical Research Council. The authorshave no conflicting interests. We are most grateful to Dr ElissaStrome for teaching us how to carry out the surgical procedures.

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