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*Institut National de la Sante et de la Recherche Medicale, Unite Mixte de Recherche S679, Paris, France

�Universite Pierre et Marie Curie-Paris 6, Paris, France

�INSERM, Unite Mixte de Recherche S677, Paris, France

§Centre de Recherche Pierre Fabre, Castres, France

¶Assistance Publique-Hopitaux de Paris, Hopital de la Salpetriere, Paris, France

Parkinson’s disease (PD) is a debilitating disorder charac-terized by severe motor deficits resulting from the degen-eration of the nigrostriatal dopaminergic (DA) pathway(Agid 1991; Fahn and Przedborski 2000). The cellular andmolecular mechanisms that cause PD remain largelyunknown, but several lines of evidence support the ideathat impairment of the mitochondrial respiratory chain atthe level of complex I plays a pivotal role in thepathomechanism: (i) Mitochondrial complex I activity isreduced in autopsied brains from PD patients (Schapira2008). (ii) Cybrids that contain mtDNA from PD patientsexpress the complex I defect that is also present in donorplatelets (Orth and Schapira 2002). (iii) The complex Iinhibitor MPP+ selectively kills DA neurons in mice andnon-human primates after systemic administration of itsprodrug MPTP (Dauer and Przedborski 2003). (iv) Acci-dental self-administration of MPTP in humans leads to an

irreversible neurodegenerative condition that is clinicallyalmost indistinguishable from PD (Langston et al. 1999).

Consequently, mouse models of MPTP intoxication havebeen used extensively to explore the molecular mechanismsof PD and to assess the effects of molecules that could haltdisease progression. However, these models present somelimitations: (i) DA cell death occurs rapidly in contrast to thepresumably slow evolution of the disease process; (ii) Some

Received July 4, 2008; revised manuscript received July 31, 2008;accepted August 7, 2008.Address correspondence and reprint requests to Patrick P. Michel,

UMR S679, Bat. Pharmacie, Hopital de la Salpetriere, 47, bd del’hopital, 75013 Paris, France. E-mail: patrick-pierre.michel@upmc.frAbbreviations used: a-SYN, a-synuclein; DA, dopamine or dopami-

nergic; GFAP, glial fibrillary acidic protein; LP, large pumps; PD, Par-kinson’s disease; SN, substantia nigra; SNc, SN pars compacta; SP, smallpumps; TH, tyrosine hydroxylase.

Abstract

Mouse models of MPTP intoxication have been used exten-

sively to explore the molecular mechanisms of Parkinson’s

disease. However, these models present some limitations

since; (i) Dopaminergic (DA) cell death occurs rapidly in

contrast to the presumably slow evolution of the disease

process. (ii) Some of the key histological features of the dis-

ease such as Lewy body like inclusions and long-term

inflammatory changes are lacking. Fornai et al. [Proc. Natl

Acad. Sci. USA 102 (2005), 3413] suggested that continuous

delivery of MPTP with Alzet osmotic minipumps may possibly

circumvent these problems. Our results show, however, that

MPTP infusion via Alzet osmotic minipumps (40 mg/kg/day)

produces only a transient depletion in striatal dopamine (DA)

without causing dopaminergic cell loss in the substantia nigra.

Neuronal cell loss occurred, however, if MPTP was infused

concomitantly with probenecid, an uricosuric agent which

potentiates the effects of the toxin injected via the i.p. route.

Even under these conditions, dopaminergic cell loss was

moderate ()25%) and other neurodegenerative changes

characteristic of Parkinson’s disease remained undetectable.

Keywords: dopaminergic (DA) neurons, microgliosis, MPTP,

neurodegeneration, osmotic minipumps, Parkinson.

J. Neurochem. (2008) 107, 701–711.

JOURNAL OF NEUROCHEMISTRY | 2008 | 107 | 701–711 doi: 10.1111/j.1471-4159.2008.05651.x

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 701–711 701

of the key histological features of the disease are lacking(Fornai et al. 2005). In particular, inflammatory processesinvolving microglial cells are present only transiently inMPTP-intoxicated mice whereas they persist up to the latestages of the degenerative process in PD (McGeer et al.2003). In addition, ubiquitin- and a synuclein (a-SYN)positive inclusion bodies, the Lewy bodies, are not observedin MPTP-treated mice (Shimoji et al. 2005). It was suggestedthat some features of PD are not reproduced in the MPTPmouse model because the mode of administration of thetoxin is sporadic and not continuous (Fornai et al. 2005).

To address this point, Fornai et al. (2005) have usedosmotic minipumps to deliver MPTP over several weeks,thereby generating mild, continuous and prolonged insults toDA neurons. Interestingly, they reported that this mode ofadministration not only caused severe lesions of the DAnigrostriatal pathway but also triggered the formation ofnigral inclusions that were reminiscent of Lewy bodies.Thus, the current investigation was designed with thefollowing aims: (i) to further develop and possibly improvethe model of MPTP intoxication using osmotic minipumps,with the intention of establishing lesions of the DAnigrostriatal system that incorporate the formation of inclu-sion bodies, and (ii) to determine whether neurodegenerationis associated with long-term inflammatory events mediatedby glial cells, since this has not been previously documentedin the MPTP mouse model.

Our results show that MPTP infusion via osmotic mini-pumps produces a transient depletion in striatal DA withoutcausing DA cell loss in the substantia nigra (SN). DA cell losswas obtained, however, if MPTP was infused concomitantlywith probenecid as adjuvant. Even under these conditions,however, ubiquitin-positive inclusions and inflammatorychanges involving microglial cells were not detectable.

Materials and methods

Pharmacological reagentsMPTP hydrochloride (MPTP HCl) and probenecid were purchased

from Sigma-Aldrich (St Quentin Fallavier, France). MPTP HCl was

dissolved in 0.9% saline to a final concentration of 200 mg/mL for

delivery via osmotic minipumps, or to a final concentration of

2 mg/mL for administration by repeated i.p. injections. When used

as an adjuvant molecule for some of the MPTP treatments,

probenecid was first solubilized in 2 N NaOH and then further

diluted to 50 mg/mL using 0.1 M Tris buffer (pH 8) and adjusted

to a final pH of 7.3 with 2 N HCl. This solution served as a solvent

for MPTP when the toxin was prepared for delivery via the osmotic

minipumps. For i.p. injections, the probenecid solution was further

diluted in saline to a concentration of 12.5 mg/mL and injected

separately.

Treatment protocolsAnimal handling and surgical procedures were carried out

according to ethical regulations and guidelines from the Guidefor the Care and Use of Laboratory Animals (National Research

Council 1996) and the European Communities Council Directive

86/609/EEC. Adult male C57Bl/6J mice (9–10 weeks of age;

Janvier Breeding Center, Le Genest-St Isle, France) were treated

with MPTP alone or in combination with probenecid, using

multiple intraperitoneal (i.p.) injections or osmotic minipumps

(Alzet, Charles River Laboratories, L’Arbresle, France). MPTP

administration via the i.p. route consisted in acute (MPTPa,

4 · 20 mg/kg, 2 h apart)(Jackson-Lewis et al. 1995; Vila et al.2000; Przedborski et al. 2004) or chronic (MPTPc, 10 · 25 mg/kg

combined with 250 mg/kg probenecid, 3.5 days apart) (Petroske

et al. 2001; Przedborski et al. 2004) treatment regimens. When the

treatments were administered via the pumps, dose regimens

corresponded to 40–80 mg/kg/day and 10–20 mg/kg/day for

MPTP and probenecid, respectively. Pump specifications by the

manufacturer indicated a fluid delivery rate of 0.25 lL/h for a

period of two (Alzet model 1002, small pumps: SP) or four weeks

Table 1 Protocols of MPTP intoxication

Treatments Administration n Dosage regimens

Duration of

treatments

Additional time without

treatment (washout)

aMPTPa i.p. (2 h apart) 6/4 20 mg/kg (4·) 6 h 1 weekd and 6 weeksbMPTPc (+ Prob.) i.p. (every 3.5 days) 8 25 mg/kg + 250 mg/kg (10·) 5 weeks 1 weekcMPTPLP LP 5/5 40 mg/kg/day 3 weekse, 4 weeks nonee, 1 week

(MPTP, Prob.)LP LP 5 40 mg/kg/day + 10 mg/kg/day 4 weeks 1 week

(MPTP, Prob.)2SP 2 SP 5/5 80 mg/kg/day + 20 mg/kg/day 2 weeks 1 week and 3 weeks

Each control group consisted of mice (n = 5–6) receiving repeated i.p. injections of saline either acutely or chronically. Where mentioned in the text,

control animals (n = 5) in the LP experiments received saline infusions via the pumps.aJackson-Lewis et al. 1995; Vila et al. 2000.bPetroske et al. 2001.cFornai et al. 2005.dAdditional mice were killed at earlier time points for the detection of MAC-1+ and GFAP+ cells.eMice were killed before the delivery duration of the pumps had terminated.

(MPTP, Prob.), MPTP diluted with probenecid; MPTPc, MPTP and probenecid injected, sequentially; LP, 2004 Alzet minipump; SP, 1002 Alzet

minipump.

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 701–711� 2008 The Authors

702 | D. Alvarez-Fischer et al.

(Alzet model 2004, large pumps: LP). To evaluate the influence of

pump placement, we initially performed i.p. and s.c. implantations.

In all subsequent experiments, we used the i.p. placement site.

When SP were used, two pumps of the same model were

implanted in each animal. All treatment paradigms are depicted in

detail in Table 1.

ImmunohistochemistryFree-floating coronal sections of the right striatum and the whole

midbrain were immunostained as described (Hoglinger et al. 2004,2007) with the following primary antibodies: rabbit anti-tyrosine

hydroxylase (TH; 1/1000; Pel Freez, Paris, France); rabbit anti-glial

fibrillary acidic protein (GFAP; 1/2000; DAKO, Glostrup, Denmark);

rat anti-CD-11b (MAC-1; 1/250; Serotec, Oxford, UK); rabbit anti-

ubiquitin (1/2000; DAKO); guinea-pig anti-a SYN (1/8000; Calbio-

chem, San Diego, CA, USA); The following secondary antibodies

were used: anti-rabbit (1/200, Vector Lab, Peterborough, UK); anti-

mouse (1/250, Vector Lab); anti-guinea pig (1/250, Vector Lab); anti-

rat (1/250, Serotec). Brainstem tissue sections from transgenic

mice over-expressing the human [A30P] a-SYN mutation

(Kahle et al., 2000) were used as positive controls for ubiquitin

immunoreactivity.

Cell countingTyrosine hydroxylase TH+ neurons revealed with the chromogen

diaminobenzidine were analysed by bright field microscopy using a

Nikon Optiphot-2 microscope (Nikon France, Champigny/Marne,

France) equipped with the Explora Nova VisioScan T4.18 software

(La Rochelle, France) as previously described (Hoglinger et al.2003). TH+ cell bodies were quantified stereologically on regularly

spaced sections covering the whole mesencephalon from the rostral

pole of the substantia nigra to the locus coeruleus using the

VisioScan stereology tool. The substantia nigra was identified

according to established anatomical landmarks (Franklin and

Paxinos 1997). The investigator performing the quantification was

blinded to the treatment groups during analysis. The total number of

SN TH+ neurons varied from 12 800 to 13 400 in saline-treated

mice. For semi-quantitative assessment of the increase in glial cell

numbers, five grades were used: (++++) massive, (+++) strong, (++)

moderate, (+) small, (0) no increase.

Optical densityAll analysed striatal sections from a given experiment were

immunostained in parallel for TH. Sections corresponding to two

different sagittal striatal levels were analysed for each brain.

Digitized images from striatal tissue sections were acquired using

a desktop illuminator and optical densities were quantified using

the Simple PCI software from Compix Inc. (Sewickley, PA, USA).

The investigator performing the quantification was blinded to the

treatment groups during analysis.

HPLC detection of striatal dopamine and MPP+

For dopamine (DA) quantification, mice were killed at indicated

time points. The left striatum was dissected and placed into 250 lL0.1 N perchloric acid containing 0.05% disodium EDTA and 0.05%

sodium metabisulfite. Brain tissue pieces were then sonicated for

10 s and the resulting homogenates were centrifuged at 13 000 g for20 min at 4�C. Supernatants were filtered through a 0.2 lm

membrane and filtrates were stored at )80�C until further analysis.

Before injection, 10 lL phosphate buffer (2 M, pH 7.5) and 5 lLascorbic acid (0.3 mg/mL) were added to 100 lL of each test

sample. Elution was performed at a rate of 1 mL/min and the sample

injection volume was 10 lL. The potential for electrochemical

detection was set at + 0.65 V and the column temperature

maintained at 19�C. Eluates were delivered via a mobile phase

consisting of a buffered aqueous solution containing 15.9%

methanol, 1.25 mM octane-1 sulfonic acid sodium salt and 76%

of a buffer containing 0.7 M KH2PO4, 1 mM EDTA, 31 mM

triethylamine, at pH 3, into a reversed phase C18 column

(250 · 4.6 mm, bonded silica, Sunfire, Waters, Guyancourt,

France). Striatal levels of DA ranged from 6.2 to 9.7 ng/mg wet

tissue in control mice.

For striatal MPP+ measurements, mice were killed 5 days after

implantation of a LP containing MPTP alone or MPTP and

probenecid. Striatal MPP+ was also measured 2, 4 and 6 h after a

single i.p. injection of 25 mg/kg MPTP with or without a

subsequent i.p. injection of 250 mg/kg probenecid. Tissue pieces

were placed into 500 lL 0.1 N HClO4 and were processed as

described for DA. Eluates were delivered at a rate of 1.0 mL/min

via a mobile phase consisting of 2.7 g potassium dihydrogen

sulphate dissolved in 697 mL acetonitrile per liter (adjusted to pH

2.5 with H3PO4) onto a reversed phase C18 column

(250 · 4 mm, pre-column 5 · 4 mm) filled with Nucleosil 100

C18 (Knauer, Berlin, Germany). The sample injection volume

was 10 lL. MPP+ was assessed by UV detection, at a wavelength

of 295 nm.

Statistical analysisData were expressed as mean ± SEM in percentage of controls.

Experimental data were compared by one-way ANOVA followed by a

post-hoc Student Neuman-Keul’s test for all pairwise comparisons.

p < 0.05 was considered significant.

Results

Lesions of the nigrostriatal pathway following continuousinfusion of MPTP with osmotic minipumpsMPTP delivered at a rate of 40 mg/kg/day via osmoticminipumps implanted i.p. (LP, Alzet model 2004, fillingvolume �200 lL) caused a 37.1% decrease in striatal DA inmice that were killed 3 weeks after pump placement (Fig.1a). In contrast, the number of TH+ cell bodies in the SNremained unaffected within the same time frame (Fig. 1b),thus suggesting that the effects of the toxin were restricted toDA nerve terminals under these experimental conditions.Similar results were obtained in mice where the pumps wereimplanted s.c. (Fig. 1a). However, we used the i.p. implan-tation site exclusively in all subsequent experiments since itwas better tolerated by the mice. It is worth noting that miceimplanted i.p. with the LP recovered normal levels of striatalDA five weeks after implantation, i.e., one week aftercompletion of toxin delivery (Fig. 1b). As expected becauseof the missing striatal DA loss, no DA cell loss was

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 701–711

Modelling Parkinson’s disease in mice | 703

detectable at this time point in the SN. Note that continuousinfusion of saline in the sham groups did not cause anysignificant loss of striatal DA or TH+ cell bodies in the SNwhen compared to i.p. saline controls.

Since we were intrigued by the absence of TH+ cell lossin the SN, we used conventional i.p. injection protocols(4 · 20 mg/kg separated by 2 h intervals) to verify theefficacy of the batch of MPTP used in the pumpexperiments. At 1 week following this i.p. injection proto-col, striatal DA levels were reduced by 81.6%, and thenumber of nigral TH+ neurons in the same animals wasreduced by 38.8%, thus confirming the toxic activity of oursupply of MPTP (Fig. 1).

Since MPTP delivered via the minipumps caused onlyminor and reversible damage to DA neurons, we wished tocompare the concentrations of its active metabolite MPP+ inthe striatum of mice receiving the toxin via a LP (40 mg/kg/day; day 5 after pump placement) or via a single i.p. injection(25 mg/kg). As expected, after i.p. injection of mice withMPTP, the levels of MPP+ peaked after 2 h and decreasedprogressively thereafter. Most noticeably, striatal levels ofMPP+ at 2 and 4 h after the i.p. injection of MPTP were,respectively, five and three times higher than in micereceiving the toxin via the minipumps (Fig. 2).

Lesions of the nigrostriatal system following MPTP/probenecid co-infusion using osmotic minipumpsProbenecid potentiates the neurotoxicity of repeated i.p.injections of MPTP possibly by reducing the clearance of thetoxin or that of its metabolites from kidney and brain(Petroske et al. 2001). Given that MPTP alone failed to exertlong-term toxic effects when administered by osmoticminipump, we wished to determine whether probeneciddelivered concomitantly with the toxin via the pumps couldgenerate DA cell loss (Fig. 3). In mice receiving MPTP(40 mg/kg/day) together with probenecid (10 mg/kg/day) viaa single large pump (Alzet model 2004), we found that thelevels of DA (measured by HPLC) and the density of DAnerve terminals (evaluated by optical density measures of THimmunoreactivity) were decreased by 28.6% and 32.7%,respectively, five weeks after pump implantation, i.e. 1 weekafter termination of drug delivery (Fig. 3a and b). THimmunostaining revealed that striatal deficits were associated

Fig. 2 Striatal MPP+ after MPTP treatments. Striatal MPP+ levels

were measured by HPLC in striatal tissue extracts of mice that had

received MPTP alone or together with probenecid in a LP (40 mg/kg/

day) or via a single i.p. injection (25 mg/kg) with or without a sub-

sequent i.p. injection of probenecid. Times of kill are indicated as the

number of hours (h) or days following initiation of MPTP intoxications.

n = 4–6 for each group. *p < 0.05 compared to untreated mice.

#p < 0.05 compared to mice implanted with the LP.

Saline i

.p.

Saline

LP i.p., 5

wee

ks

MPTPLP i.p

., 3 w

eeks

MPTPLP s.

c., 3

weeks

MPTPLP i.p

., 5 w

eeks

MPTPa , 1

wee

k

Fig. 1 Lesions of the nigrostriatal DA pathway after osmotic minipump

infusion of MPTP. (a) Striatal DA content and (b) counts of TH+ cells

within the SN after different treatments with MPTP administered

continuously using a LP or sporadically through the i.p. route. Data are

expressed as percentage of saline i.p.-injected mice. Times of kill after

initiation of the treatments are indicated as the number of weeks in

the figure labels. Note that MPTP infusion with the pumps caused a

transient depletion of striatal DA but no loss of SN TH+ neurons.

*p < 0.05, compared to saline i.p.-injected mice.

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 701–711� 2008 The Authors

704 | D. Alvarez-Fischer et al.

with a decrease of 24.5% in the number TH+ neurons in theSN (Fig. 3c and d). SN tissue sections of mice receivingMPTP without probenecid in the LP are also presented inFig. 3(d) for comparative purposes. If the same quantity ofMPTP/probenecid was administered using two smaller pumpsbut over a shorter exposure period of two weeks (SP, Alzet

model 1002 model; 80 mg/kg/day MPTP + 20 mg/kg/dayprobenecid), lesions of the nigrostriatal system were alsoobserved. Specifically, the levels of DA and the density ofTH+ nerve terminals in the striatum were reduced by 52.7%and 58.1%, respectively, at 3 weeks after implantation of thepumps, i.e. 1 week after termination of drug delivery (Fig. 3a

MPTPC, 6 weeks

MPTPa, 1 week

(MPTP, Prob.)LP, 5 weeks

MPTPLP, 5 weeks

Control

(a) (d)

(b)

(c)

Fig. 3 Lesions of the nigrostriatal DA pathway after osmotic minipump

infusion of MPTP and probenecid. (a) Striatal DA content, (b) optical

densities of striatal TH stained fibers and (c) counts of TH+ cells in the

SN, following treatments with MPTP and probenecid administered

continuously with osmotic minipumps or sporadically via i.p. injections.

Data are expressed as percentage of saline i.p.-treated mice (con-

trols). *p < 0.05 compared to controls. (d) Digitized images of repre-

sentative TH-stained sections of the SN in mice receiving MPTP alone

or MPTP + probenecid using i.p. injections or osmotic minipump

delivery. Note that when DA neurons were lost in the SN, DA neurons

in the VTA appeared also affected to some extent by MPTP treat-

ments. Scale bar: 400 lm. Treatment protocols are indicated in the

figure labels. Times of kill are indicated as the number of weeks fol-

lowing the initiation of MPTP intoxication.

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 701–711

Modelling Parkinson’s disease in mice | 705

and b). At this time point, however, there was no detectableloss of TH+ neurons in the substantia nigra (Fig. 3c). Twoweeks later (i.e. 5 weeks after implantation of the pumps), thedecrease in striatal DA persisted although there was atendency for recovery ()26.9%); the reduction in striatalDAwas also reflected by lower optical densities of TH stainedcontralateral striata ()53.8%) (Fig. 3a and b). Interestingly, atthis 5-week post-implantation time point, a moderate butsignificant loss of TH+ neurons ()14.3%) was observed(Fig. 3c). Applying MPTP and probenecid separately in twoSP caused DA lesions of similar intensity (not shown).

When MPTP and probenecid were injected i.p. using thechronic treatment protocol described by Petroske et al.(2001), we observed a substantial reduction in DA levels()66.4%) and DA nerve terminal density ()67.0%) in thestriatum, at 1 week following the treatments. Consistent withthese observations, the number of SN TH+ neurons was alsoreduced by �40% in this group of mice (Fig. 3c and d). DAcell loss obtained in the acute paradigm of treatment withMPTP is also illustrated in Fig. 3(d) for comparison.

It is noteworthy here that MPP+ levels in the striatum werenot significantly higher when MPTP-treated mice receivedprobenecid either via the pump or via the i.p. injection route(Fig. 2).

Characterization of the glial reaction following MPTP/probenecid treatments administered via osmoticminipumpsIt has been established that inflammatory processes mediatedby astrocytes and microglial cells may contribute actively toDA cell demise in conventional models of MPTP intoxica-tion and in PD as well (McGeer and McGeer 2008). For this

reason, we wished to examine the presence of reactiveastrocytes (GFAP+) and that of microglial cells (MAC-1+) inthe different treatment paradigms we used throughout thisstudy. Results are summarized in Table 2.

An increase in the number of GFAP+ cells was detectablein chronic treatment regimens of MPTP/probenecid intoxi-cation using osmotic minipumps (Table 2; Fig. 4). Theincrease was moderate in the SN pars compacta (SNc) andthe striatum when the administration was carried out usingtwo SP. It was smaller and restricted to the SNc when theadministration was made with a single LP. Astrogliosis wasalso present in mice receiving repeated i.p. injections ofMPTP and probenecid (Petroske protocol), principally in theSN and to a lesser extent in the striatum. Finally, the astroglialreaction was massive in the SNc, the substantia nigra parsreticulata (SNr) and the striatum of mice exposed to the acutetreatment with MPTP alone (Jackson-Lewis protocol).

Activated microglial cells remained undetectable in treat-ment paradigms combining MPTP and probenecid adminis-tration (Table 2; Fig. 5). However, following the acutetreatment with MPTP alone (Jackson-Lewis protocol), thedensity ofMAC-1+ cells increased dramatically at day 2 and 3,in the striatum and SN, respectively. The presence of activatedmicroglial cells was, however, transient since MAC-1+ cellswere virtually absent at day 7 following MPTP injections(Table 2; Fig. 5). Finally, in a last attempt to produce long-term activation of microglial cells, we performed an acutetreatment with MPTP followed by an administration of MPTPand probenecid either via the pumps or repeated i.p. injections.Detailed treatment protocols and assessment of DA cell deathare presented in Supporting information (Fig. S1). Also in thatcase, activated microglial cells were virtually absent from theSN and striatum of MPTP intoxicated mice.

Detection of aggregation processes following MPTP/probenecid treatments administered via osmoticminipumpsIn mice receiving MPTP and probenecid continuously (byminipumps) or sporadically (by i.p. injection), we failed todetect aggregates using an antibody that identifies both freeubiquitin residues and ubiquitinated proteins (Fig. 6). As apositive control, this antibodywas highly effective in detectingpathological lesions in transgenic mice over-expressing thehuman [A30P] a-SYN mutation (Frasier et al. 2005). Specif-ically, immunostaining was intense in distorted neuriticextensions and occasionally in cell bodies (Fig. 6). Weobserved that a-SYN immunocytochemistry also failed toidentify aggregates inMPTP-treatedmice as well (not shown).

Discussion

Our study demonstrates that MPTP caused a transientdepletion in striatal DA but no DA cell loss when admin-istered with Alzet osmotic minipumps. A moderate but

Table 2 Increase in the density of astroglial and microglial cells

following MPTP intoxication

Intoxication protocol

GFAP+ cells MAC-1+ cells

SNc SNr Striatum SN Striatum

MPTPa, 2 days ++++ +++ ++++ +++a ++++

MPTPa, 7 days +++ ++ +++ 0 0

(MPTP, Prob.)2SP, 3 weeks ++ + ++ 0 0

(MPTP, Prob.)LP, 5 weeks + 0 0 0 0

MPTPc, 6 weeks ++ ++ + 0 0

Density of GFAP+ astrocytes and MAC-1+ microglial cells in the SN

and striatum of mice receiving MPTP treatments either via i.p. injec-

tions or via osmotic minipump delivery. For semi-quantitative

assessment of glial reactivity, five grades of density were used; (++++)

massive, (+++) strong, (++) moderate, (+) small, (0) no increase. Note

that the density of GFAP+ astrocytes was substantial in the substantia

nigra pars reticulata (SNr) in saline treated control mice. Times when

animals were killed are indicated in the Intoxication Protocol column as

the number of days or weeks after the start of the intoxication.aPeak effect observed after 3 days in the SN.

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 701–711� 2008 The Authors

706 | D. Alvarez-Fischer et al.

significant loss in TH+ cell bodies was obtained, however, ifMPTP was co-administered with an adjuvant molecule,probenecid. Reactive microglial cells and ubiquitin immu-noreactive inclusions, two other key histological hallmarks of

PD were, however, absent from the brain of mice receivingMPTP/probenecid via minipumps.

Fornai et al. (2005) reported that continuous administrationof MPTP via Alzet minipumps caused severe lesions of the

SN

Co

ntr

ols

MP

TP

a , 2

day

sM

PT

Pa ,

7 d

ays

(MP

TP,

Pro

b)2

SP, 3

wee

ksM

PT

Pc ,

6 w

eeks

Str.

Fig. 4 Astrogliosis in MPTP-treated mice.

Digitized images of GFAP+ astrocytes in the

SN and striatum of mice receiving MPTP

treatments either via osmotic minipumps or

by i.p. injections. Treatment protocols are

indicated in the figure labels. Times of kill

are indicated as the number of days or

weeks following the initiation of MPTP

intoxication. Arrows point to GFAP+ cells in

the SNc. Scale bar: 500 lm (left panel) and

50 lm (right panel).

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 701–711

Modelling Parkinson’s disease in mice | 707

nigrostriatal system in mice. Using a comparable protocol ofadministration with a LP, we failed, however, to detect asignificant decrease in the number of TH+ cell bodies in theSN. These discrepancies may possibly be related to differencesin MPTP susceptibility that could be strain-, sex- or age-dependent (Hamre et al. 1999). However, male C57 BL6Jmice of similar age were used in both studies (Fornai et al.2005). Our data may also be explained by variability in thetoxicity of different batches of MPTP, although this is unlikelysince the neurotoxic potency of MPTP was always tested inparallel using conventional i.p. intoxication protocols. It isworth noting that striatal DA levels were reduced transientlywhile the delivery of MPTP with the LP was still in progress.This suggests that the toxin caused temporary metabolicimpairment of DA nerve terminals via its active metaboliteMPP+. One may also deduce from this observation that the

concentrations of MPP+ within the striatum did not reachlevels sufficient to promote DA cell death. Accordingly, weestablished that striatal concentrations of MPP+ in micereceiving MPTP from Alzet minipumps implanted i.p. wereonly 1/5 and 1/3 of those detected 2 and 4 h following a singlei.p. injection of 25 mg/kg MPTP, respectively. This explana-tion may be only partially valid, however, since similar ratioswere also reported by Fornai et al. (2005) when comparingstriatal levels of MPP+ following administration of MPTPeither via the pumps or via the i.p. route.

Because probenecid has been shown to potentiate the toxiceffects of MPTP administered chronically by repeated i.p.injections and to render these effects irreversible (Petroskeet al. 2001; Novikova et al. 2006), we expected that thiscompound could also increase the toxicity of MPTP ifadministered simultaneously via a LP. Consistent with our

SN

Control

MPTPa, 2 days

(MPTP, Prob)LP, 5 weeks

MPTPc, 6 weeks

Striatum

Fig. 5 Microglial reaction in MPTP-treated

mice. Digitized images of MAC-1+ cells in

the SN and striatum of mice receiving

MPTP treatments either via osmotic mini-

pumps or by i.p. injections. Treatment pro-

tocols are indicated in the figure labels.

Times of kill are indicated as the number of

days or weeks following the initiation of

MPTP intoxication. Scale bars: 250 lm (left

panel) and 500 lm (right panel).

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 701–711� 2008 The Authors

708 | D. Alvarez-Fischer et al.

hypothesis, a significant loss of DA cell bodies was observedin these conditions after termination of the pump treatments.It is worth noting that replacing the large 2004 Alzet pump,which has a 4-week delivery duration, by two smaller 1002pumps that deliver the same total quantity of toxin, but over ashorter 2-week period of time, also generated a loss of DAneurons which was, however, more limited. Importantly, DAneuronal loss was detected in both paradigms after a periodof washout ranging from 1 to 3 weeks with the LP and the2SP, respectively, suggesting that the lesions did not simplyreflect a transient down-regulation of TH. Also consistentwith these results, the density of striatal DA nerve terminalsand the levels of DA were significantly reduced at corre-sponding time points in both paradigms of treatment.Surprisingly, probenecid did not increase striatal MPP+

levels suggesting that this compound did not operate simplyby increasing the central bioavailability of the pyridinium forDA nerve terminals but via another mechanism that remainsto be elucidated.

Inflammatory processes mediated by glial cells are thoughtto participate actively in the progression of PD either insporadic or familial forms of the disease (Hirsch et al. 2003;Miklossy et al. 2007). Microglial cells, the resident innateimmune cells of the brain, and astrocytes to a lesser extent arebelieved to play an important role in the inflammatory processeven up to the late stages of neurodegeneration (Henze et al.2005; Jenner and Olanow 2006; Kim and Joh 2006; Miklossyet al. 2006). Astrogliosis was of weak intensity and restrictedto the SNc following a treatment with MPTP/probenecidusing the single LP, but was more intense in both the SNc andthe striatum when the treatments were performed using two

SP or repeated i.p. injections. This observation is also in linewith previous studies reporting the presence of reactiveastrocytes in the chronic protocol of MPTP/probenecidintoxication via the i.p. route (Dervan et al. 2004).

Microglial cells are supposed to become activated bydysfunctional or dying SN DA neurons in PD or in animalmodels of the disease (Dauer and Przedborski 2003; Hunotand Hirsch 2003). The presence of reactive astrocytes mayalso amplify this process (Henze et al. 2005). Yet, activatedMAC-1+ cells remained undetectable in any of the testparadigms of MPTP/probenecid administration via mini-pump delivery or i.p. injections, which indicates thatinflammatory processes mediated by microglial cells werenot involved in DA cell demise in these conditions. Thepresence of MAC-1+ microglial cells was, however, evidentin the brain of mice acutely intoxicated with MPTP in boththe SN and striatum, thereby demonstrating the validity ofour detection protocol. Even in this experimental situation,MAC-1+ cells were induced only transiently, and hadcompletely disappeared from the SN and striatum at 7 daysfollowing MPTP administration. It is worth noting that thepresence of reactive microglial cells after an acute treatmentwith MPTP could not be maintained in mice that weresubsequently treated with MPTP/probenecid using eitherpump infusion or repeated i.p. injections. This indicates thatthe delivery of MPTP/probenecid via the pumps does notcreate experimental conditions that are favourable formicroglial-dependent inflammatory processes.

The absence of microglial reaction in chronic protocolsof MPTP intoxication using osmotic minipumps mayhave several explanations: (i) Inflammatory processes in

Controls MPTPc, 6 weeks

(MPTP, Prob.)LP, 5 weeks [A30P] α-SYN Omit

Fig. 6 Lack of ubiquitin-positive aggre-

gates in the SN of MPTP-treated mice.

Ubiquitin-stained SN tissue sections in mice

in which MPTP and probenecid were

administered continuously using osmotic

minipumps or sporadically using repeated

i.p. injections. A diffuse and faint staining

was observed in the SN of both control and

MPTP-treated mice. In contrast, distorted

neuritic extensions and occasional cell

bodies were intensely stained in brainstem

tissue sections from transgenic mice over-

expressing the human [A30P] a-synuclein

mutation. Omit, negative controls per-

formed by omitting the primary antibody.

Scale bar: 50 lm.

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 701–711

Modelling Parkinson’s disease in mice | 709

MPTP-intoxicated mice may essentially result from cumu-lative insults caused by high concentrations of MPP+ overrelatively short periods of time; (ii) Microgliosis which isbelieved to increase as a function of age (Sugama et al.2003) may have been limited in our study since we usedyoung adult mice. (iii) It cannot be excluded that the immuneresponses in rodents and humans or non-human primatesoccur by different mechanisms and/or follow differentkinetics (Gandy and Walker 2004). Indeed, activatedmicroglial cells remain present in monkeys injected withlow amounts of MPTP and in humans exposed accidentallyto the toxin, long after the initial intoxication (Langston et al.1999; McGeer et al. 2003; Barcia et al. 2004).

The presence of misfolded proteins forming intracytoplas-mic filamentous inclusions, known as Lewy bodies, repre-sents another neuropathological hallmark of sporadic andhereditary forms of PD (Trojanowski et al. 1998; Miklossyet al. 2007). Lewy bodies may contribute mechanistically tothe degeneration of neurons in PD, although this is still aquestion of debate. Lewy bodies are usually identified bytheir content in ubiquitin or a-SYN, and are not typicallyobserved in acute and subacute models of MPTP intoxica-tion. Yet, Fornai et al. (2005) reported the presence of Lewybody-like inclusions that were immunopositive for ubiquitinand a-SYN when MPTP was continuously administered withminipumps, suggesting that the aggregation process may befavoured under conditions of a low-level, continuous andlong-term intoxication paradigm. Using the paradigms ofMPTP/probenecid administration with the pumps, we failed,however, to detect ubiquitin-positive inclusions, indicatingthat abnormal processing of proteins may not be prominentin this experimental model. The same antibody allowed us,however, to detect the ubiquitin pathology characteristic oftransgenic mice over-expressing a human mutated form ofa-SYN (Frasier et al. 2005), thereby confirming the validityof our detection protocol. In addition, we also failed to detectpositive aggregates after repeated i.p. injections of MPTPand probenecid, despite a prior report showing the presenceof lipofuscin granules that accumulate a-SYN in thisintoxication paradigm (Meredith et al. 2002). Our resultsare, however, in agreement with a more recent study carriedout using the same protocol of intoxication (Shimoji et al.2005). Also consistent with our observations are those ofseveral other studies that have failed to demonstrate thepresence of classical Lewy bodies both in non-humanprimates treated with MPTP and in humans years after theyhad been exposed accidentally to the toxin (Eslamboli 2005),thus suggesting that the process of protein misfolding andaggregation may not be a mere consequence of mitochondrialdysfunction induced by MPP+.

In summary, we have shown that probenecid can substan-tially increase the toxic effects of the PD neurotoxin MPTPadministered in mice via Alzet minipumps. The loss of SNDA neurons remained, however, more limited than in

conventional (repeated i.p. injections) models of intoxicationwith MPTP. Other degenerative events typically associatedwith PD, such as microglial cell activation and intracellularprotein aggregation, were not observed in the process of DAcell degeneration. Nonetheless, this model may be advanta-geous for testing molecules for a therapeutic potential in PDfor two reasons: (i) MPTP does not need to be injectedrepeatedly, and (ii) this mode of administration may permitthe detection of drugs active in the early (presymptomatic)phase of neurodegenerative processes relevant to PD.

Acknowledgements

This work was supported by Institut de Recherche Pierre Fabre

(Castres, France), Institut National de la Sante et de la Recherche

Medicale (INSERM) and Universite Pierre et Marie Curie (UPMC),

Univ. Paris 6. D. Alvarez-Fischer was supported by a fellowship

from the German Academic Exchange Service (DAAD). Transgenic

mice over-expressing the human [A30P] a-SYN mutation were

kindly provided by Margot Fournier and Olga Corti. We are grateful

to Michel Hamon for his comments on some results and to Marie-

Paule Muriel and Vanessa Brochard for technical assistance.

Supporting information

Additional Supporting information may be found in the online

version of this article:

Fig. S1 Lesions of the nigrostriatal DA pathway following

treatments combining acute and chronic administration of MPTP.

Please note: Wiley-Blackwell are not responsible for the content

or functionality of any supporting materials supplied by the authors.

Any queries (other than missing material) should be directed to the

corresponding author for the article.

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