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Behavioural Brain Research 189 (2008) 364–372

Research report

Hemiparkinsonian rats rotate toward the side with theweaker dopaminergic neurotransmission

Claudio Da Cunha a,∗, Evellyn Claudia Wietzikoski a, Marcelo Machado Ferro b,Glaucia Regina Martinez b, Maria Aparecida Barbato Frazao Vital a,

Debora Hipolide c, Sergio Tufik c, Newton Sabino Canteras d

a Laboratorio de Fisiologia e Farmacologia do Sistema Nervoso Central, Departamento de Farmacologia,UFPR, C.P. 19.031, 81.531-980 Curitiba PR, Brazil

b Departamento de Bioquımica e Biologia Molecular, UFPR, Curitiba, Brazilc Departamento de Psicobiologia, UNIFESP, Sao Paulo, Brazil

d Departamento de Anatomia, Instituto de Ciencias Biomedicas-3, USP, Sao Paulo, Brazil

Received 4 December 2007; received in revised form 23 January 2008; accepted 24 January 2008Available online 5 February 2008

bstract

Rats with unilateral lesion of the substantia nigra pars compacta (SNpc) have been used as a model of Parkinson’s disease. Depending on theesion protocol and on the drug challenge, these rats rotate in opposite directions. The aim of the present study was to propose a model to explainow critical factors determine the direction of these turns. Unilateral lesion of the SNpc was induced with 6-hydroxydopamine (6-OHDA) or-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Separate analysis showed that neither the type of neurotoxin nor the site of lesion alonghe nigrostriatal pathway was able to predict the direction of the turns these rats made after they were challenged with apomorphine. However, theombination of these two factors determined the magnitude of the lesion estimated by tyrosine-hydroxylase immunohistochemistry and HPLC-EDeasurement of striatal dopamine. Very small lesions did not cause turns, medium-size lesions caused ipsiversive turns, and large lesions caused

ontraversive turns. Large-size SNpc lesions resulted in an increased binding of [3H]raclopride to D2 receptors, while medium-size lesions reduced3

he binding of [ H]SCH-23390 D1 receptors in the ipsilateral striatum. These results are coherent with the model proposing that after challenged

ith a dopamine receptor agonist, unilaterally SNpc-lesioned rats rotate toward the side with the weaker activation of dopamine receptors. Thisctivation is weaker on the lesioned side in animals with small SNpc lesions due to the loss of dopamine, but stronger in animals with large lesionsue to dopamine receptor supersensitivity.

2008 Elsevier B.V. All rights reserved.

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eywords: Substantia nigra pars compacta; Turning behaviour; Dopamine; Bas

. Introduction

In 1968, Ungerstedt proposed that the turning behaviour ofats with unilateral lesion of the substantia nigra pars compactaSNpc) induced by intracerebral infusion of 6-hydroxydopamine6-OHDA) which are challenged with dopaminergic drugs cane used as a model of Parkinson’s disease (PD). Since then, it

as been one of the most popular models to study drugs poten-ially useful to treat this disease [4,16,41,49]. However, there iso consensual explanation for the direction of the turns made

∗ Corresponding author. Tel.: +55 41 3361 1717; fax: +55 41 3266 2042.E-mail address: dacunha@ufpr.br (C. Da Cunha).

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166-4328/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.bbr.2008.01.012

glia; Dopamine receptor; Parkinson’s disease; MPTP; 6-Hydroxydopamine

y these rats. Most studies have shown that rats with unilat-ral lesions induced by 6-OHDA present a contraversive turningehaviour (in the direction opposite to the lesioned side) whenhallenged with apomorphine. However, ipsiversive or few turnsn both directions have also been reported in animals with uni-ateral lesions induced by 6-OHDA [2,7,15,35,38,44,46], andther neurotoxins (e.g., rotenone, MPP+) [22,24,42]. Turningehaviour seems to be caused by a misbalance between the rightnd left side drive to initiate movements due to an asymmetricalodulation of midbrain dopaminergic neurons on corticostriatal

ynapses [12,32].The site of infusion of 6-OHDA has been reported to be crit-

cal to explain the direction of the turns [38,40,42]. The medialorebrain bundle (MFB) has been indicated as the infusion

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ite that causes more contraversive turns after an apomorphinehallenge. In contrast, ipsiversive behaviour is observed whenhese animals are challenged with amphetamine [46]. Dopamineeceptor supersensitivity in the striatum has also been suggesteds an important factor inducing contraversive turns [44].

In the present study, rather than simply describing the criticalactors that determine the occurrence and direction of the turn-ng behaviour in unilaterally SNpc-lesioned rats, we propose aational model to explain this phenomenon. The hypothesis thathe direction of the turns is determined by the site of lesion in theNpc, its size or magnitude or by dopamine receptor supersensi-

ivity in the striatum was tested in 6-OHDA- and MPTP-lesionedats. These neurotoxins were chosen to induce large and smallesions in the SNpc, respectively [9,35]. The hypothesis that a

isbalance between dopamine receptor activation in the rightnd left striatum determines the direction of the turns was alsoested. This hypothesis does not rule out the site of the lesion andts magnitude or dopamine receptor supersensitivity as importantactors determining the direction of turning behaviour. Instead,t proposes that the right/left striatal dopaminergic neurotrans-

ission misbalance is determined by the combination of theseactors.

. Materials and methods

.1. Animals

Adult male Wistar rats from our own breeding stock weighing 280–310 g athe beginning of the experiments were used. The animals were maintained in aemperature-controlled room (22 ± 2 ◦C) on a 12-h dark:12-h light cycle (lights

n at 7:00 a.m.), with food and water available ad libitum. All experimentalrocedures were in compliance with the guidelines of the National Institutesf Health and the Brazilian Society for Neuroscience and Behaviour and werepproved by the Institutional Animal Care and Use Committee of the Federalniversity of Parana State.

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ig. 1. Schematic drawing of coronal slices of the rat brain showing the sites of injectioorebrain bundle; SNpc-L, SNpc-C, and SNpc-M = lateral, central, and medial portio

n Research 189 (2008) 364–372 365

.2. Chemicals

The drugs and other fine chemical compounds used in these experimentsere purchased from the following sources: chloral hydrate (Reagen, Brazil),

thanol, acetonitrile, tetrahydrofuran and perchloric acid (Merck, Germany),enicillin G-procaine (Bristol-Myers Squibb, USA), sodium thiopental (Abbottaboratories, USA), [3H]SCH-23390 and [3H]raclopride (PerkinElmer Lifecience, USA), and atropine sulfate, acetaldehyde, apomorphine, citric acid,opamine, desipramine, DOPAC, EDTA, HVA, MPTP, HCl, heptanesulfoniccid, butaclamol, sulpiride and 6-OHDA (Sigma Chemical Co., USA). The mon-clonal antibody against TH raised in mice was purchased from Incstar Corp.USA), and the ABC Elite kit to localize the antigen–antibody complex wasurchased from Vector Laboratories (USA). The Synergi C-18 reverse-phaseolumn was purchased from Phenomenex (Torrance, CA, USA).

.3. Surgeries

The animals received atropine sulfate (0.4 mg/kg, i.p.) to suppress salivationnd penicillin G-procaine (20,000 U in 0.1 ml, i.m.) to prevent infection, andere anesthetized with 3 ml/kg equitesin (1% sodium thiopental, 4.25% chloralydrate, 2.13% magnesium sulfate, 42.8% propylene glycol, and 3.7% ethanol inater). Animals lesioned with MPTP received three i.p. injections of 120 mg/kg

cetaldehyde 10 min before, at the beginning, and immediately after surgery, toncrease the effectiveness of the neurotoxin. Animals lesioned with 6-OHDAeceived 25 mg/kg desipramine (i.p.) 10 min before surgery to prevent the lossf noradrenergic neurons. MPTP (100 �g/�l in saline) or 6-OHDA (8 �g/�l inaline supplemented with 0.2% ascorbic acid) was infused through a 30-gaugetainless needle at 0.25 �l/min. The needle was maintained in place for an addi-ional 2 min to avoid reflux. The following volumes and coordinates were usedor neurotoxin infusion: medial part of the SNpc (SNpc-M): 1 �l, anteroposte-ior (AP), −5.9 mm from the bregma; mediolateral (ML), ±1.2 mm from theidline; dorsoventral (DV), −8.6 mm from the skull; nose bar, −3.3 mm from

he interaural line; central part of the SNpc (SNpc-C): 1 �l, AP, −5.0 mm; ML,2.1 mm; DV, −7.7 mm; lateral part of the SNpc (SNpc-L): 1 �l, AP, −6.1 mm;L, ±2.6 mm; DV, −7.3 mm. Fig. 1 illustrates the sites of MPTP or 6-OHDA

nfusion. The following parameters were used for animals that received MPTPnto the three sites of the SNpc (SNpc-M,C,L): the same coordinates as describedbove, 1 �l into the medial, 2 �l into the central, and 2 �l into the lateral partf the SNpc. MFB: 2 �l: AP, −1.9 mm; ML, +1.9 mm; DV, −7.2 mm. Thesetereotaxic coordinates were adapted from the Atlas of Paxinos and Watson

n of MPTP or 6-OHDA. Adapted from Paxinos and Watson [36]. MFB = medialns of the substantia nigra pars compacta, respectively.

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36]. Sham-operated animals were submitted to the same surgical procedure,ncluding the hole in the skull, but the infusion needle was not lowered into therain. Another set of animals (Saline) was submitted to the same procedure butaline was infused instead of the neurotoxins. After surgery, the animals werellowed to recover from the anesthesia in a temperature-controlled chamber andere then returned to their home cage. The animals were fed a pasty diet con-

isting of a mixture of crumbled chow and water during the first 5 postoperativeays. This procedure reduced body weight loss and mortality.

.4. Turning behaviour

One week after surgery the animals were challenged by injecting 1 mg/kgpomorphine, s.c. Immediately after the injection, the rats were individuallylaced in a round plastic container measuring 28 cm in diameter and 25 cm ineight, and the number of 360◦ turns toward the lesioned side (ipsiversive) andoward the opposite side (contraversive) were video-recorded over a period ofh [47].

.5. Tyrosine hydroxylase immunohistochemistry

After the behavioural test, the animals were killed by decapitation and theirorsal striata were removed for the determination of dopamine concentrationsee below). The posterior part of the rat brain was preserved in formalin for 1eek and then placed in 20% sucrose formalin 48 h before sectioning. Four seriesf 30-�m thick sections were cut with a sliding microtome on the frontal planend collected from the caudal diencephalon to the caudal midbrain. The sectionsere immunostained for tyrosine hydroxylase (TH) with a monoclonal antibody

gainst TH (1:5000 dilution). The antigen–antibody complex was localized withn ABC Elite kit (Vector, UK). Slides were then dehydrated and coverslippedith DPX. The number of TH-immunoreactive neurons was determined using

he 40× objective of an Axiophot Stereo HB050 (Zeiss, Germany) microscopequipped with a camera lucida. The UTHSCSA Image Tool version 2.0 softwareUniversity of Texas, San Antonio, USA) was used to count these neurons onoth sides of the midbrain, including the ventral tegmental area, SNpc, andetrorubral nucleus in eight series of coronal sections based on the Atlas ofaxinos and Watson [37]: −4.8 mm, −5.2 mm, −5.6 mm, −5.8 mm, −6.04 mm,6.3 mm, −6.72 mm, and −7.04 mm from the bregma.

.6. Determination of dopamine and metabolites by HPLC-ED

Endogenous levels of dopamine and its non-conjugated metabolites,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)ere assayed by reverse-phase HPLC with electrochemical detection (ED).he system consisted of a Synergi Fusion-RP C-18 reverse-phase column

150 × 4.6 mm i.d., 4-�m particle size, Phenomenex, USA), an L-ECD-6A elec-rochemical detector (Shimadzu, Japan), and an LC-10AD pump (Shimadzu).he column was maintained inside a temperature-controlled oven (30 ◦C, Shi-adzu). The oxidation potential was fixed at +0.80 V using an Ag/AgCl working

lectrode. The tissue samples were homogenized with an ultrasonic cell disrupterSonics, USA) in 0.1 M perchloric acid. After centrifugation at 15,000 × g for0 min, 20 �l of the supernatant was injected into the chromatograph. The mobilehase, used at a flow rate of 1 ml/min, had the following composition: 15.7 gitric acid, 471.5 ml HPLC-grade water, 78 mg heptane sulfonic acid, 20 mlcetonitrile, and 10 ml tetrahydrofuran, pH 3.0. The peak areas of the externaltandards were used to quantify the sample peaks.

.7. Autoradiographic binding assays

Seven days after surgery, rats from the saline-M,C,L (n = 8), saline-MFBn = 8), MPTP-M,C,L lesion (n = 8) and 6-OHDA-MFB (n = 8) groups wereacrificed. The brains were removed quickly, frozen over dry ice and stored at80 ◦C. Twenty-micrometer coronal sections were cut with a Leica cryostat at

20 ◦C and mounted on washed gelatin-coated slides at approximately 0.3 mm

ntervals along the anterior–posterior extent of the brain. The slides were storedt −80 ◦C until assayed. Binding assays of [3H]SCH-23390 to D1 receptors [34]nd of [3H]raclopride to D2 receptors [39] were performed as described previ-usly. Briefly, slices were brought to room temperature and then preincubated

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n Research 189 (2008) 364–372

n 50 mM Tris–HCl, 120 mM NaCl, 4 mM MgCl2, 1 mM EDTA, and 1.5 mMaCl2 2H2O buffer, pH 7.4, for 30 min (D1) or 15 min (D2). Sections were then

ncubated in fresh buffer containing 2 nM [3H]SCH-23390 (85.0 Ci/mmol) for0 min at room temperature (D1), or in buffer containing 2 M [3H]raclopride60.1 Ci/mmol) for 120 min at room temperature (D2). Additional sets of slicesere incubated in the presence of 2 �M butaclamol (D1) or 10 �M sulpiride

D2) for the determination of nonspecific binding. Sections were then washedn buffer and allowed to dry at room temperature. The slices were exposed toodak BioMax MR films in tungsten cassettes together with calibrated stan-ards for 4 weeks. The films were developed and densitometric analyses wereerformed using an M2 MCID system (Imaging Research, Canada). Anatomicalegions were defined according to Paxinos and Watson [36]. Multiple readingsere obtained by an investigator who was unaware to which group the animalselonged and averaged over serial sections for each brain. Density values ofifferent regions of the striatum region were first averaged within each section,hen across sections within each brain, and then across subjects within eachroup.

.8. Statistics

All results are reported as the mean ± S.E.M. Differences among groupsere analyzed by one-way ANOVA, followed by the Newman–Keuls test. Data

egarding the time course of apomorphine-induced turns were scored as the num-er of ipsiversive–contraversive turns and were analyzed by two-way ANOVAor repeated measures, followed by the Newman–Keuls test. Differences wereonsidered to be statistically significant when p ≤ 0.05.

. Results

.1. Effects of the unilateral infusion of 6-OHDA or MPTPnto different sites of the nigrostriatal pathway on theurning behaviour of rats

The effects of these treatments on apomorphine-induced turn-ng behaviour are shown in Fig. 2. The infusion of saline per sento the SNpc induced ipsiversive turns compared to the shamroup (Fig. 2A; F(6,65) = 37.99, p ≤ 0.001, one-way ANOVA;≤ 0.05, Newman–Keuls post hoc test). The infusion of MPTP

nto the central, lateral, or central + medial + lateral sites of theNpc, but not into the medial part of the SNpc or into the MFB,

nduced more ipsiversive turns compared to the saline groupFig. 2A; p ≤ 0.05, Newman–Keuls test). Equivalent results wereound when comparing the time-course curves of the effect ofpomorphine (Fig. 2B). Two-way ANOVA showed significantffects for site of infusion (F(6,65) = 37.99, p ≤ 0.001), timeF(11,715) = 17.43, p ≤ 0.001), and site versus time interactionF(66,715) = 3.80, p ≤ 0.001).

As can be seen in Fig. 2A′, rats lesioned with 6-OHDAnfused into the MFB or into the medial part of the SNpc pre-ented contraversive turns significantly different from the salineroup (F(5,52) = 14.65, p ≤ 0.001, one-way ANOVA; p ≤ 0.05,ewman–Keuls test). Contraversive turning behaviour was alsobserved in most, but not all, animals that received 6-OHDAnto the central part of the SNpc (Fig. 2A′); the scores ofhis group did not differ significantly from those obtained forham or saline animals (p > 0.2, Newman–Keuls test). More-

ver, infusion of 6-OHDA into the lateral part of the SNpcid not cause contraversive behaviour (p > 0.2, Newman–Keulsest), but ipsiversive turns that differed significantly from shamnimals (Fig. 2A′; F(5,52) = 3.43, p ≤ 0.01, one-way ANOVA;

C. Da Cunha et al. / Behavioural Brain Research 189 (2008) 364–372 367

Fig. 2. Turning behaviour of unilaterally MPTP- and 6-OHDA-lesioned rats challenged with apomorphine. Data are expressed as the mean ± S.E.M. number ofipsiversive (positive scale) and contraversive turns (negative scale) counted over 1 h after the drug challenge (A and A′) or at intervals of 5 min (B and B′) (N = 8–12animals per group). *p ≤ 0.05 compared to the sham group; +p ≤ 0.05 compared to the saline group (Newman–Keuls test after one-way ANOVA). MFB = medialforebrain bundle; SNpc-L, SNpc-C, and SNpc-M = lateral, central, and medial portions of the substantia nigra pars compacta, respectively. Sham = operated animalss infust

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ubmitted to the same surgical procedure, including the hole in the skull, but theo the same procedure but saline was infused instead of the neurotoxins.

≤ 0.05, Newman–Keuls test). Equivalent results were foundhen comparing the time-course curves of the effect of apomor-hine (Fig. 2B′). Two-way ANOVA showed significant effectsor site of infusion (F(5,52) = 13.44, p ≤ 0.001), but not for timeF(11,572) = 0.46). There was a significant site versus time inter-ction (F(55,572) = 1.43, p ≤ 0.05).

.2. Effect of the size of the SNpc lesion on turningehaviour

The results presented above showed that MPTP-inducedesions caused ipsiversive or no turning behaviour (Fig. 2A and). On the other hand, 6-OHDA caused contraversive, ipsiver-

ive or no turning behaviour (Fig. 2A′ and B′). The predominantffect depended on the site of injection, but the administra-ion of 6-OHDA into different sites of the SNpc caused turnsn opposite directions. Therefore, we claim that the size of theesion, roughly determined by the neurotoxin type and its sitef injection, is the critical factor for the induction of ipsiversiver contraversive behaviour. We tested this hypothesis by group-ng 6-OHDA or MPTP rats according to their turning behaviouripsiversive, contraversive or none), irrespective of the site ofnjection into the SNpc (Fig. 3).

The following conclusions can be drawn based on the num-

er of remaining midbrain TH-immunostained neurons, striatalopamine and its metabolites after these treatments, as shown inig. 3: (1) very small SNpc lesions caused no turning behaviour;2) small lesions caused ipsiversive turns; (3) large lesions

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ion needle was not lowered into the brain. Saline = operated animals submitted

aused contraversive turns. These observations were made irre-pective of the site into which the neurotoxins were infused.

One-way ANOVA followed by the Newman–Keuls tested to this conclusion based on the data regarding the lossf TH-immunostained neurons in MPTP-lesioned rats, asllustrated in Fig. 3A (F(2,10) = 8.99, p ≤ 0.01,) and in 6-HDA-lesioned rats, as illustrated in Fig. 3A′ (F(3,19) = 11.89,≤ 0.001). The same conclusion can be drawn from theata regarding the loss of dopamine and its metabolitesn the striatum. Dopamine after MPTP treatment: Fig. 3B:(2,23) = 24.00, p ≤ 0.001; dopamine after 6-OHDA treatment:ig. 3B′: F(3,23) = 52.45, p ≤ 0.001; DOPAC after MPTP treat-ent: Fig. 3C: F(2,23) = 5.10, p ≤ 0.05; DOPAC after 6-OHDA

reatment: Fig. 3C′: F(3,23) = 27.92, p ≤ 0.001. Significant dif-erences between groups were also observed for HVA after-OHDA treatment (Fig. 3D′: F(3,23) = 40.64, p ≤ 0.001), butot after MPTP treatment (Fig. 3D: F(2,23) = 1.16, p = 0.33).

.3. Effect of the size of the SNpc lesion on the binding ofadioligands to D1 and D2 dopamine receptors in thetriatum

Even animals that received MPTP into the three sites ofhe SNpc presented medium-size SNpc lesions and ipsiversive

urns (Fig. 3). On the other hand, all animals that received-OHDA into the MFB presented large SNpc lesions and con-raversive turning behaviour. As can be seen in Fig. 4, largeigral lesions induced by the infusion of 6-OHDA into the MFB

368 C. Da Cunha et al. / Behavioural Brain Research 189 (2008) 364–372

Fig. 3. Number of mesencephalon TH-immunostained neurons (A and A′) and levels of DA (B and B′), DOPAC (C and C′), and HVA (D and D′) in the ipsilateralstriatum of unilaterally MPTP- and 6-OHDA-lesioned or sham rats. Data are expressed as the mean ± S.E.M. The animals were grouped according to their turningbehaviour as indicated by the color of the bars (N = 4–9 animals per group). *p ≤ 0.05 compared to the sham group, +p ≤ 0.05 compared to the asymptomaticgroup, #p ≤ 0.05 compared to the ipsiversive group (Newman–Keuls test after one-way ANOVA). DA = dopamine; DOPAC = 3,4-dihydroxyphenylacetic acid;H SNpr re, incb infus

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VA = homovanillic acid. MFB = medial forebrain bundle; SNpc-L, SNpc-C, andespectively. Sham = operated animals submitted to the same surgical procedurain. Saline = operated animals submitted to the same procedure but saline was

aused a significant increase in the binding of [3H]raclopride to2 receptors in the ipsilateral striatum (Fig. 4A′). On the otherand, the smaller lesions induced by MPTP caused a significanteduction in the binding of [3H]SCH-23390 to D1 receptors inhe ipsilateral striatum (Fig. 4B). One-way ANOVA followed

y the Newman–Keuls test: Fig. 4A′, [3H]raclopride bindingn 6-OHDA-treated rats (F(3,144) = 3.24, p ≤ 0.05); Fig. 4B,3H]SCH-23390 binding in MPTP-treated rats (F(3,140) = 2.99,≤ 0.05). No significant effect was found for [3H]raclopride

ed

c-M = lateral, central, and medial portions of the substantia nigra pars compacta,luding the hole in the skull, but the infusion needle was not lowered into theed instead of the neurotoxins.

inding in MPTP-treated animals (Fig. 4A, F(3,153) = 1.58,= 0.19) or for [3H]SCH-23390 binding in 6-OHDA animals

Fig. 4B′, F(3,166 = 1.90, p = 0.13).

. Discussion

The results of the present study do not support the hypoth-sis that the site of infusion or the type of neurotoxin aloneetermines the direction of turns induced by apomorphine in

C. Da Cunha et al. / Behavioural Brain Research 189 (2008) 364–372 369

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Npc-hemilesioned rats. It is true that the infusion of a specificeurotoxin into some specific sites along the nigrostriatal path-ay causes a predominant pattern of turning behaviour (Fig. 2).owever, the direction of the turns cannot be predicted only by

he site of infusion. The present findings clearly demonstrate thathe infusion of 6-OHDA, but not of MPTP, into the MFB and intohe medial part of the SNpc caused contraversive turns. MPTPlways caused ipsiversive or no turning behaviour, whereas 6-HDA caused predominantly contraversive turns. However, the

atter may also cause ipsiversive turns, like in rats infused intohe lateral part of the SNpc. In addition, even when infused with-OHDA into the medial or central part of the SNpc, some 6-HDA rats presented ipsiversive behaviour. This inconsistent

urning behaviour of 6-OHDA rats has been reported in sometudies [7,20,35,38,41,44]. In order to obtain a more homoge-eous sample, many authors excluded 6-OHDA rats that did notresent contraversive turns [26,41,43].

The apparently chaotic pattern of results described aboveakes sense if explained not as a result of the site of lesion, but

ather as a function of the loss of midbrain dopaminergic neu-ons and striatal depletion of dopamine, irrespective of the sitef lesion (Fig. 3). Before discussing the evidence in favor of thisecond hypothesis, we will see whether the pattern of lesion canrovide a clue regarding the neurotoxic mechanisms of MPTPnd 6-OHDA. When 6-OHDA was infused into a site with aigh density of dopaminergic fibers, the neurotoxin caused largeesions in neurons from which dopaminergic fibers originate.his was the case of infusion of 6-OHDA into the MFB. In thisase, most of the midbrain dopaminergic neurons were lost [seelso 28, 45, 49]. The fibers that arise from neurons of the medialnd central parts of the SNpc leave it by passing through the

ost medial and ventral parts of the SNpc and then enter theFB [14,30]. Therefore, the more medial and ventral the site

f 6-OHDA infusion, the more the neurotoxin will be in con-act with dopaminergic fibers. The present study and previous

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atal slices of rat brain one week after the administration of MPTP or 6-OHDA. *p ≤ 0.05 compared to the non-lesioned side (right striatum).

nvestigations have found that infusion of 6-OHDA into medialnd ventral parts of the SNpc caused a more marked and robustontraversive turning behaviour than infusion into lateral andorsal parts of the SNpc [7,35,38,41]. These findings suggesthat 6-OHDA is mainly taken up by dopaminergic fibers.

On the other hand, MPTP did not cause a large loss of mid-rain dopaminergic neurons when it was infused into sites richn dopaminergic fibers, such as the MFB and the ventromedialart of the SNpc. It is unlikely that the toxic metabolite of MPTP,PP+, is taken up preferentially by dopaminergic neuron bod-

es/dendrites compared to axons, since it is taken up by the samerotein that also takes up 6-OHDA, i.e., the dopamine trans-orter (DAT) [27]. It is also possible that monoamine oxidase(MAO-B), the enzyme that transforms MPTP into MPP+, is

ifferentially expressed in fibers and bodies of dopaminergiceurons. In addition, the brain capillaries of rats contain excep-ionally high levels of MAO-B compared to primates [23]. Asconsequence, rats are more resistant to the neurotoxic effectsf MPTP than primates [5,19]. Therefore, even the simultane-us infusion of a highly concentrated solution of MPTP intohree sites throughout the SNpc did not cause the same loss of

idbrain dopaminergic neurons than the infusion of 6-OHDAnto the MFB. This treatment did not mimic the magnitude andpreading loss of midbrain dopaminergic neurons that resultedrom the infusion of 6-OHDA into the MFB. The ipsiversiveurning behaviour of rats infused with MPTP into the three sites,n contrast to the contraversive turning behaviour of MFB 6-HDA rats, strongly suggests that it was not the site of infusionut the degree of dopaminergic neuron loss and the consequenttriatal dopamine depletion which determined the direction ofhe turns.

This hypothesis was further tested by grouping animalseceiving 6-OHDA or MPTP into different sites according toheir turning behaviour (Fig. 3). This criterion suggested thathere are two critical thresholds that determine the direction of

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urning behaviour: Animals with midbrain dopaminergic neu-on loss below a certain level did not present turning behaviour,hereas those with higher, but partial, loss of dopaminergiceurons presented ipsiversive turning behaviour and those thatost almost all midbrain dopaminergic neurons presented con-raversive turning behaviour. Only 6-OHDA rats showed thisehaviour.

The evidence presented above describes, but not necessarilyxplains why the magnitude of the loss of midbrain dopaminer-ic neurons determines the direction of turning behaviour inats. Our findings suggest that this explanation comes from

he dopaminergic synaptic transmission in the striatum. Par-ial depletion of striatal dopamine reduced the binding to D1eceptors in MPTP rats (Fig. 4B), while severe dopamine deple-

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ig. 5. Schematic drawing of rats with partial (A and C) or total (B and D) loss of nr amphetamine (C and D). The animal rotates toward the site that presents the weakeceptor agonist (dopamine + apomorphine) and on the density of dopamine receptor

n Research 189 (2008) 364–372

ion increased the binding to D2 receptors in 6-OHDA ratsFig. 4A′). It is reasonable to suppose that the modulation of1 and D2 receptors can alter dopaminergic neurotransmission

n the striatum, thus determining ipsiversive or contraversiveurns in rats challenged with apomorphine. Dopamine receptorctivation in the striatum deinhibits the basothalamic output thattimulates cortical motor areas [12]. Turning behaviour is causedy a misbalance between ipsiversive and contraversive striatalopaminergic stimulation [41].

The model to explain the direction of turning behaviour inNpc-hemilesioned rats is schematically presented in Fig. 5.

n MPTP rats, a decrease of dopamine levels and D1 receptorensitivity was observed on the lesioned side. Therefore, the non-esioned side was more stimulated by endogenous dopamine

igrostriatal dopamine neurons after a challenge with apomorphine (A and B)er synaptic signal. The synaptic efficacy depends on the amount of dopamine

s in striatal neurons (see Section 4 for details).

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lus the exogenous dopamine receptor agonist apomorphineFig. 5A). 6-OHDA rats presented D2 receptor supersensitiv-ty on the lesioned side, which compensates for the decreasef dopamine and, therefore, the lesioned side was more stimu-ated than the non-lesioned side in the presence of apomorphineig. 5B). Similar findings have been reported in previous stud-

es on 6-OHDA rats [13,21,31,33,44,48] and MPTP monkeys1]. Therefore, the balance of dopaminergic neurotransmittersetween the lesioned and non-lesioned sides was the oppositen MPTP and 6-OHDA rats, which would explain the oppositeirections of their turning behaviour.

The ipsiversive turning behaviour of 6-OHDA and MPTPats challenged with amphetamine can also be explained byhis model. Since amphetamine induces a higher release ofopamine on the non-lesioned side in MPTP and 6-OHDA rats,his side is more stimulated in both cases, a fact explaininghy amphetamine induces ipsiversive turning behaviour in bothPTP- and 6-OHDA-hemilesioned rats (Fig. 5C and D).In summary, the present study suggests that rats with unilat-

ral lesion of the SNpc rotate toward the side with the weakertriatal dopaminergic neurotransmission. The dopaminergiceurotransmission misbalance is determined by the magni-ude of dopamine depletion and by up- or down-regulation ofopamine receptors. When challenged with apomorphine, ani-als with small unilateral lesion/depletion of dopamine present

psiversive turning behaviour due to a weaker stimulation ofhe lesioned side (caused by the loss of dopamine), whereasnimals with an almost total unilateral loss of midbrain dopamin-rgic neurons/striatal dopamine present contraversive turningehaviour due to dopamine receptor supersensitization.

It is possible that the mechanisms that convert ipsiversive intoontraversive turning behaviour in SNpc-hemilesioned rats areimilar to those that support the motor abnormalities observeduring the early and late phases of PD. Nowadays, some studiesuggest that one of the most marked disabilities observed duringhe late phase of the disease, i.e., dyskinesia, is closely related toontraversive turns in 6-OHDA rats [25,26]. Previous investiga-ions from our group showed that MPTP rats are a good modelor the study of cognitive impairments that occur during the earlyhase of PD [3,6,8–11,17,18,29,37]. Therefore, we suggest thatt would be worth studying MPTP and 6-OHDA rats to betternderstand the mechanisms that underlie the characteristics andimits of the early and late stages of PD.

cknowledgements

This research was supported in part by CNPq, CAPES,APESP, Instituto do Milenio, and Fundacao Araucaria. Thekillful assistance of Mariza Bortolanza, Vinicius A. Lima, Jeffhandler, Scheila M. G. Takayasu, and Michele L. Gregorio iscknowledged.

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