endogenous morphine-like compound immunoreactivity increases in parkinsonism

BRAINA JOURNAL OF NEUROLOGY

Endogenous morphine-like compoundimmunoreactivity increases in parkinsonismGiselle Charron,1,2 Evelyne Doudnikoff,1,2 Alexis Laux,3 Amandine Berthet,1,2 Gregory Porras,1,2

Marie-Helene Canron,1,2 Pedro Barroso-Chinea,1,2 Qin Li,4 Chuan Qin,4 Marika Nosten-Bertrand,5

Bruno Giros,5 Francois Delalande,6 Alain Van Dorsselaer,6 Anne Vital,1,2 Yannick Goumon3 andErwan Bezard1,2,4

1 University of Bordeaux, Institut des Maladies Neurodegeneratives, UMR 5293, F-33000 Bordeaux, France

2 CNRS, Institut des Maladies Neurodegeneratives, UMR 5293, F-33000 Bordeaux, France

3 Institut National de la Sante et de la Recherche Medicale, INCI, CNRS UPR3212, Strasbourg, France

4 Institute of Laboratory Animal Sciences, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100021, China

5 Universite Pierre et Marie Curie, Institut National de la Sante et de la Recherche Medicale – Centre National de la Recherche Scientifique,

CNRS UMRS-952, UMR 7224, Paris, France

6 Laboratoire de spectrometrie de masse BioOrganique, IPHC-DSA, ULP, CNRS, UMR7178, Strasbourg 67087, France

Correspondence to: Erwan Bezard,

CNRS UMR 5293,

146 rue Leo Saignat,

33076 Bordeaux cedex,

France

E-mail: [email protected]

Morphine is endogenously synthesized in the central nervous system and endogenous dopamine is thought to be necessary for

endogenous morphine formation. As Parkinson’s disease results from the loss of dopamine and is associated with central pain,

we considered how endogenous morphine is regulated in the untreated and L-DOPA-treated parkinsonian brain. However, as

the cellular origin and overall distribution of endogenous morphine remains obscure in the pathological adult brain, we first

characterized the distribution of endogenous morphine-like compound immunoreactive cells in the rat striatum. We then studied

changes in the endogenous morphine-like compound immunoreactivity of medium spiny neurons in normal, Parkinson’s

disease-like and L-DOPA-treated Parkinson’s disease-like conditions in experimental (rat and monkey) and human Parkinson’s

disease. Our results reveal an unexpected dramatic upregulation of neuronal endogenous morphine-like compound immunor-

eactivity and levels in experimental and human Parkinson’s disease, only partially normalized by L-DOPA treatment. Our data

suggest that endogenous morphine formation is more complex than originally proposed and that the parkinsonian brain ex-

periences a dramatic upregulation of endogenous morphine immunoreactivity. The functional consequences of such endogenous

morphine upregulation are as yet unknown, but based upon the current knowledge of morphine signalling, we hypothesize that

it is involved in fatigue, depression and pain symptoms experienced by patients with Parkinson’s disease.

Keywords: immunohistochemistry; medium spiny neuron; astrocyte; dopamine; mouse; rat; monkey; endogenous morphine; human

Abbreviations: DAT = dopamine transporter; MPTP = 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

doi:10.1093/brain/awr166 Brain 2011: of 18 | 1

Received January 30, 2011. Revised June 9, 2011. Accepted June 12, 2011.

� The Author (2011). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.

For Permissions, please email: [email protected]

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IntroductionIt is now soundly established that morphine is endogenously

synthesized in the CNS of vertebrates (Gintzler et al., 1978;

Stefano et al., 1996, 2000; Muller et al., 2008; Goumon et al.,

2009). However, its biosynthesis was debated until a recent report

using mice that are unable to synthesize dopamine due to a gen-

etic deletion of tyrosine hydroxylase specifically in dopaminergic

neurons, showed that endogenous dopamine is necessary for en-

dogenous morphine formation in normal mammalian brain (Neri

et al., 2008). Those dopamine-null mice displayed basal hyper-

algesia, further supporting a role for dopamine in endogenous

morphine biosynthesis (Hnasko et al., 2005). The neurological dis-

order classically associated with dopamine depletion is Parkinson’s

disease (Hornykiewicz, 1966). Apart from parkinsonian motor

symptoms, Parkinson’s disease is less widely appreciated as a dis-

ease that causes a large variety of pain syndromes, with a preva-

lence of �40% (Chaudhuri and Schapira, 2009). Pain syndromes

are attenuated by the most effective symptomatic therapy repre-

sented by the dopamine precursor, L-3,4-dihydroxyphenylalanine

(L-DOPA), suggesting that the dysfunction may occur in

dopamine-dependent centres (Schestatsky et al., 2007; Ford,

2010). For instance, the pain threshold for cold is substantially

lower in patients with Parkinson’s disease who were withdrawn

from treatment compared with controls, and is then normalized

after L-DOPA therapy (Brefel-Courbon et al., 2005). Patients with

Parkinson’s disease with primary central pain displaying hyperalge-

sia and the absence of habituation of sympathetic sudomotor re-

sponse to repetitive pain are improved by L-DOPA treatment

(Schestatsky et al., 2007).

Although endogenous morphine distribution has been studied in

the normal brain (Gintzler et al., 1978; Bianchi et al., 1993; Muller

et al., 2008; Laux et al., 2011), its cellular origin and distribution

remains obscure in parkinsonism despite earlier findings reporting

morphine level modulation in urine of patients with Parkinson’s

disease OFF and ON medication (Matsubara et al., 1992). We

therefore first characterized the endogenous morphine-like com-

pound immunoreactive cell distribution in the rat striatum and

second, studied the changes in the endogenous morphine-like

compound immunoreactivity of the medium spiny neurons in

normal, Parkinson’s disease-like and L-DOPA-treated Parkinson’s

disease-like conditions in experimental and human Parkinson’s dis-

ease. Our results reveal an unexpected dramatic regulation of

neuronal and glial endogenous morphine immunoreactivity in ex-

perimental and human Parkinson’s disease, somewhat normalized

by L-DOPA treatment.

Materials and methods

Rodent experimentsExperiments were performed in accordance with French (87-848,

Ministere de l’Agriculture et de la Foret) and European Communities

Council Directive of 24 November 1986 (86/609/EEC) for care of

laboratory animals and were approved by the Ethical Committee of

Centre National de la Recherche Scientifique, Region Aquitaine.

Wild-type rats

Twelve adult male Sprague Dawley rats (Charles River Laboratories)

weighing 175–200 g at the beginning of the experiment were used.

On Day 0 of the protocol, unilateral dopamine deprivation of the stri-

atum was obtained by 6-hydroxydopamine (3 mg/ml) injection in the

right medial forebrain bundle (2.5 ml at anteroposterior = �3.7 mm,

mediolaterial = + 1.7 mm and dorsoventral = �8 mm, relative to

bregma) as previously described (Schuster et al., 2008, 2009;

Berthet et al., 2009). Animals displaying a 495% loss of tyrosine

hydroxylase-immunopositive fibres in the striatum (Schuster et al.,

2008, 2009; Berthet et al., 2009), as assessed after completion of all

experiments (not shown), were retained for final analysis. From Day

21 to Day 30 post-surgery, rats were treated once daily with benser-

azide [15 mg/kg, intraperitoneally (i.p.)] and either vehicle (n = 4) or

L-DOPA (6 mg/kg, i.p.) (n = 4). The benserazide and L-DOPA treated

mice were dyskinetic at the time of death. On Day 30, all animals

received the last vehicle injection � L-DOPA and were sacrificed

60 min later.

Wild-type mice

Experiments were performed on 21 45-day-old laboratory-bred adult

male C57BL/6 mice weighting 30 � 3 g. Dopamine depletion was

achieved by injecting either saline (controls) or a combination of

2.5 mg/kg reserpine i.p. (Sigma Aldrich) at 24 h and 100 mg/kg of

�-methyl-p-tyrosine (Sigma Aldrich) 24, 16, 4 and 1 h before sacrifice,

as previously described (Thomas et al., 2008). Thirteen mice were used

for neurochemistry and eight for immunohistochemistry.

Dopamine transporter knock-out mice

Dopamine transporter (DAT) mutant mice were generated by in vivo

homologous recombination as previously described (Giros et al.,

1996). Twelve female mice between the ages of 2 and 4 months

were used for immunohistochemistry: four wild-type mice (DAT+ / + ),

four heterozygous mice (DAT�/ + ) and four homozygous mice

(DAT�/�).

Tissue preparation

Terminal procedure was identical for mice and rats as previously

described (Berthet et al., 2009). Animals were deeply anaesthetized

with chloral hydrate (VWR), 150 mg/kg (i.p.). Brains of saline and

reserpine-treated mice for neurochemical experiments were collected

fresh, freeze–thawed in isopentane and immediately stored at �80�C

until further use. All other animals were transcardially perfused with a

mixture of 2% paraformaldehyde and 0.2% glutaradehyde in 0.1 M

phosphate-buffer at pH 7.4. Brains were removed and post-fixed over-

night in 2% paraformaldehyde at 4�C. Coronal sections were cut at

60 mm on a Vibratome (Leica, VT 1000 S) and collected in phosphate-

buffered saline (pH 7.4). To enhance the penetration of immunorea-

gents, the sections were equilibrated in a cryoprotectant solution,

phosphate-buffered saline with 25% saccharose, freeze–thawed in iso-

pentane and stored in phosphate-buffered saline with 0.03% sodium

azide at 4�C until use.

Non-human primate experimentsAll experiments were carried out in accordance with the European

Communities Council Directive of 24 November 1986 (86/609/EEC)

for care of laboratory animals in an AAALAC accredited facility

and were approved by the Institute of Lab Animal Science IACUC.

Animals were housed in individual primate cages under controlled con-

ditions of humidity (50%�5%), temperature (24 � 1�C) and light

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(12 h light/12 h dark cycle, time lights on 08:00 a.m.), food and water

were available ad libitum and animal care supervised by veterinarians

skilled in the healthcare and maintenance of non-human primates. The

six female cynomolgus monkeys (Macaca fascicularis, Xierxin)

involved in this study have been previously presented (Guigoni

et al., 2005, 2007; Nadjar et al., 2006). Briefly, two macaques were

left untreated (control group); four were administered with

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) hydrochloride.

Once a bilateral parkinsonian syndrome had stabilized (i.e. unchanged

disability score over several weeks), two macaques were kept without

any dopaminergic supplementation (MPTP-lesioned group) and two

macaques were treated chronically with twice daily administration of

levodopa (Modopar; L-DOPA/carbidopa, ratio 4:1) for 6–8 months at

a tailored dose designed to produce a full reversal of parkinsonian

condition (L-DOPA-treated MPTP-lesioned group) as previously

described (Guigoni et al., 2005, 2007; Nadjar et al., 2006). The

L-DOPA-treated MPTP-lesioned group were dyskinetic at the time of

death. Animals were deeply anaesthetized with sodium chloral hydrate

(150 mg/kg) 1 h after the last vehicle/L-DOPA dose and perfused

transcardially with a mixture of 2% paraformaldehyde and 0.2% glu-

taradehyde in 0.1 M phosphate buffer. Brains were removed, bisected

along the midline, stored in 2% paraformaldehyde overnight and cut

into 60 mm frontal sections with a vibratome (Leica, VT1000S).

Sections were collected in phosphate-buffered saline, cryoprotected

in phosphate-buffered saline with 25% saccharose, freeze–thawed in

isopentane and stored in phosphate-buffered saline with 0.03%

sodium azide until use. The clinical assessments and the characteriza-

tion of the extent of nigrostriatal denervation have been previously

published (Guigoni et al., 2005, 2007; Nadjar et al., 2006) in order to

ensure that all the MPTP-treated animals displayed comparable lesion

of the nigrostriatal pathway. Indeed, the number of tyrosine-

hydroxylase immunopositive neurons in the substantia nigra pars com-

pacta and the striatal tyrosine-hydroxylase immunostaining were iden-

tical between the two MPTP-treated groups and dramatically low

compared with control animals (loss of terminals 495%) as previously

shown (Guigoni et al., 2005).

Post-mortem human samplesThe observations on human tissue were based on the analysis of

formalin-fixed and paraffin-embedded human specimens from an

archival collection (Comite Protection des Personnes N� CEBH 2009/

03; Ministere Enseignement Superieur et Recherche: DC-2008-337)

declared and approved by the ethics committee (Comite de

Protection des Personnes Sud-Ouest et Outre Mer III) of Bordeaux

University Hospital. Striatum material was available for both neuro-

pathologically confirmed cases of Parkinson’s disease (n = 3; two

males, one female; 62 � 7 years old at death) and controls with no

detectable CNS disease (n = 3; two males, one female; 63 � 8.5 years

old at death). The post-mortem delay was similar for all cases (524 h)

but the delay for the female Parkinson’s disease case was 60 h. The

striatum was studied within a coronal 5-mm thick section demonstrat-

ing connections between the head of the caudate and the putamen

across the internal capsule.

Immunocytochemical procedures

Immunofluorescent single labelling

Vibratome-cut, free-floating sections were rinsed in 0.1 M phosphate-

buffered saline (pH 7.4) and incubated in 0.3% H2O2/phosphate-

buffered saline to inhibit endogenous peroxidases. After preincubation

in 3% normal goat serum, 3% bovine serum albumin in phosphate-

buffered saline to minimize non-specific labelling, the tissue sections

were incubated with the primary antibody (diluted in phosphate-

buffered saline 0.1 M, 3% bovine serum albumin, 3% normal goat

serum) overnight at room temperature. The nature, origin and dilution

of the primary antibodies are listed in Table 1. A goat anti-rabbit IgG

conjugated to alexa 488 fluorochrome (Molecular Probes) or a goat

anti-mouse IgG conjugated to alexa 568 fluorochrome (Molecular

Probes) were used as secondary antibodies (1:400). Sections were

then washed in phosphate-buffered saline and mounted in

Vectashield mounting medium with DAPI (Vector Laboratories) and

examined using a Zeiss Axioplan 2 fluorescent microscope.

Fluorescence was visualized at 488 nm (green), 568 nm (red) and

350 nm (blue). The specificity of the anti-endogenous morphine-like

compound antibody has been reported elsewhere (Muller et al., 2008;

Glattard et al., 2010; Laux et al., 2011). When sections were incu-

bated without the primary antibody, no labelling was observed.

Similarly, incubation of endogenous morphine primary antibody with

an excess of morphine, completely abolished staining, confirming the

specificity for endogenous morphine (Fig. 1).

Immunofluorescent double-labelling

To further characterize the striatal endogenous morphine-like com-

pound positive cells, we performed a double immunohistochemistry

Table 1 List of antibodies

Antibody Species Dilution Source

Morphine Mouse 1:2000 Aviva Systems Biology

D1 dopamine receptor Rat 1:500 Sigma

D2 dopamine receptor Rabbit 1:500 Santa Cruz

Glutamate acid decarboxylase 65-67 Rabbit 1:1000 Millipore

Dopamine and cAMP-regulated phosphoprotein, 32 kDa Rabbit 1:500 Cell Signalling

S100 Rabbit 1:1000 Abnova

NG2 Rabbit 1:500 Millipore

Glial fibrillary acidic protein Mouse 1:1000 Millipore

CYP2D1 Rabbit 1:1000 ENZO Life Sciences

Tyrosine hydroxylase Rabbit 1:2000 Incstar

Choline acetyltransferase Goat 1:200 Millipore

Parvalbumin Guinea pig 1:1000 Synaptic System

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procedure against endogenous morphine and against: (i) markers of

medium spiny neurons (dopamine and cAMP-regulated phosphopro-

tein, 32 kDa, D1 dopamine receptor, D2 dopamine receptor and glu-

tamate acid decarboxylase 65/67); (ii) the rate-limiting enzyme for

endogenous morphine synthesis, CYP2D1; and (iii) markers of inter-

neurons, (choline acetyltransferase and parvalbumin). Immunohisto-

chemical procedure was as described above except that two primary

antibodies derived from different animal species were applied together.

Similarly, two secondary antibodies raised in different species and

labelled with two different fluorochromes, alexa 488 conjugated

against rabbit and alexa 568 against mouse, were applied.

Immunoperoxidase labelling in experimental models

The number of striatal endogenous morphine-like compound immuno-

positive neurons was defined after immunolabelling with the peroxid-

ase technique. Brain sections were incubated overnight in endogenous

Figure 1 Distribution of endogenous morphine-like compound immunoreactive structures. (A) Sagittal section of rat brain stained with

anti-endogenous morphine antibody showing main endogenous morphine-like compound positive midbrain and forebrain regions. Scale

bar = 3 mm. (B) Absence of staining when anti-endogenous morphine antibody was preadsorbed with the immunizing alkaloid before

incubation. (C–J) Magnifications of selected endogenous morphine-like compound positive regions boxed in A: cortex (C),

caude-putamen (D), Calleja islets (E), substantia nigra pars reticulata (F), pons (G), interposed cerebellar nuclei (H), cerebellum (I), inferior

colliculus (J); scale bar = 50 mm. (K–L) Immunocytochemical localization of endogenous morphine-like compounds on striatal coronal

section. Inset in L shows that both weakly (open circle) and strongly (asterisk) immunoreactive neurons are present as well as glial cells

(arrow). (M) Control immunohistochemistry with omission of primary antibody. Scale bar = 2 mm (K), 25 mm (L–M), 10 mm (L inset).


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morphine primary antibody, as described above. Sections were first

incubated in biotinylated secondary antibody and then in avidin–

biotin complex (Vectastain� Elite ABC kit) prepared according to the

manufacturer’s recommendations. Morphine immunoreactivity was re-

vealed with NovaREDTM kit (Vector Laboratories). After washing with

phosphate-buffered saline, sections were mounted on microscope

slides in Eukitt (Merck).

Immunoperoxidase labelling on post-mortemhuman samples

Four micrometre-thick paraffin-embedded striatal sections were pro-

cessed as previously described (Vital et al., 2009). Briefly sections were

dewaxed and pressure cooked in sodium citrate buffer (0.01 M, pH

6.0) for antigen retrieval. The sections were washed, blocked with a

universal blocking buffer (Biogenex) and incubated with endogenous

morphine antibody overnight. Control sections were incubated with

blocked antiserum. After transfer in 0.3% H2O2/phosphate-buffered

saline to block the endogenous peroxidase reaction, the sections were

treated with biotinylated anti-rabbit IgG (diluted 1:200 in

phosphate-buffered saline) and incubated with Vectastain� Elite ABC

reagent. The colour was developed using NovaREDTM kit. Sections

were counterstained with Mayer’s Hemalun and mounted with an

aqueous agent for microscopy Aquatex� (Merck).

Immunogold labelling

Subcellular endogenous morphine-like compound distribution was ana-

lysed at electron microscopic level in the rat striatum. The endogenous

morphine was detected by the pre-embedding immunogold technique

as previously described (Dumartin et al., 1998, 2000; Guigoni et al.,

2007; Berthet et al., 2009). Sections were incubated in 4% normal

donkey serum for 45 min and then in a mixture of endogenous mor-

phine antibody (1:2000 in phosphate-buffered saline supplemented

with 1% normal donkey serum) overnight at room temperature.

After washing in phosphate-buffered saline and PBS-BSAcTM

(Aurion), the sections were incubated for 3 h at room temperature in

donkey anti-mouse IgGs conjugated to ultra-small gold particles

(0.8 nm; Aurion) diluted 1:100 in PBS-BSAcTM gel. After post-fixation

in 1% glutaraldehyde in phosphate-buffered saline and several

washes, the immunogold signal was intensified using a silver enhance-

ment kit (HQ silverTM; Nanoprobes) for 6 min at room temperature in

the dark. The sections were then post-fixed in 0.5% osmium tetroxide

and dehydrated in ascending series of ethanol dilutions that also

included 70% ethanol containing 1% uranyl acetate. The sections

were post-fixed, dehydrated and embedded in resin (Durcupan

ACM; Fluka). Serial ultra-thin sections were cut with a Reichert

Ultracut S, contrasted with lead citrate and examined with Hitachi

H-7650 microscope.

Image analysisFluorescent images were captured on a confocal Leica microscope DM

2500 TCS SPE. Digital images were acquired separately for each wave-

length [488 nm (green) and 568 nm (red)] and then merged. Adobe

Photoshop CS3 was used to further process digital images. Any ad-

justments to brightness and contrast were made uniformly to all parts

of the image.

Cell counting was performed using a computerized image analysis

system (Mercator V6.50, Explora Nova) linked to a Leica microscope

type DM 6000B (�20 objective). The number of immunoreactive

medium spiny neurons (round or oval shape with a cell body diameter

of 10–15 mm) was counted for two populations and classed according

to labelling intensity, from weak ( + ) to strong labelling ( + + + )

(Fig. 1L).

Quantitative analysis was carried out on the whole striatum area

and results were expressed as an average density of endogenous mor-

phine cells per mm2. To minimize the inherent variability in the immu-

nochemical procedures, comparisons were made in tissue sections that

were processed simultaneously. Only those neurons that were on the

surface of the sections, where antibody penetration was guaranteed,

were counted.

Neurochemical procedures

Dopamine quantification by reverse phasehigh-performance liquid chromatography

Mouse brains were homogenized in 1 ml of 1 M ClHO4 and centri-

fuged for 30 min at 14 000 g. Supernatants were analysed using an

Akta PurifierTM high-performance liquid chromatography system

(GE Healthcare Bioscience) with reverse phase column (250/4

Nucleosil 100-5-C18HD; Macherey Nagel). Buffer A was 5 mM

H3PO4, 5 mM hexanesulphonic acid in water. Buffer B was 100%

acetonitrile. Detection of dopamine was monitored with a fluorescence

detector (RF-10AXL; Shimadzu; excitation at 270 nm and emission at

320 nm) as previously described (Muzzi et al., 2008).

Morphine-specific enzyme-linked immunosorbentassay

Homogenized brains were sonicated at 4�C (3 � 10 s) in phosphate-

buffered saline. Extracts were centrifuged (30 min, 10 000 g at 4�C).

Forty microlitres of supernatants (tested in duplicates) were used for

enzyme-linked immunosorbent assay analysis (Glattard et al., 2010).

The morphine-specific enzyme-linked immunosorbent assay kit

from Immunalysis Corporation (provided by AgriYork 400 Limited)

was used for the quantification of morphine present in brain tissue

extracts (control mice, n = 4; �-methyl-p-tyrosine–reserpine mice,

n = 5) (Muller et al., 2008). The specificity of the test for morphine

was confirmed by testing different amounts of dopamine, adrenaline,

noradrenaline, norlaudanosoline/tetrahydropapaveroline, morphine,

morphine-6-glucuronide, morphine-3-glucuronide and codeine (0.01–

25 ng/ml, data not shown) (Muller et al., 2008; Glattard et al., 2010).

For all tests, morphine standards were diluted in the appropriate

buffer. Samples with a higher coefficient of variation value were

retested to obtain a coefficient of variation 48%. The calculated

methodological detection limit of the batch of enzyme-linked immuno-

sorbent assay kit used for the study was 0.01 ng/ml of morphine.

Morphine-like compound extraction from mousebrain and striatum

Mouse brain and striatum were homogenized in 1 ml of methanol.

After centrifugation (14 000 g, 20 min at 4�C), the supernatant was

recovered and dried with a SpeedVac evaporator. The sample was

suspended in 300ml of urea buffer (1 M, pH 8.4) and submitted to

heat treatment (100�C, 30 min). After a centrifugation (14 000 g,

20 min at 4�C), alkaloids present in the supernatant were purified

into OasisTM HLB 96-well plates (60mm, 60 mg, Waters) that had

been preconditioned with 500 ml of methanol, followed by 500 ml

(NH4)2CO3 (10 mM, pH 8.8). Samples were loaded onto the

OasisTM HLB plate and the SPE was rinsed with 500ml (NH4)2CO3

(10 mM, pH 8.8). Alkaloids were eluted with 300 ml of acetonitrile/

isopropanol/formic acid (40/59/1 v:v:v). Extracted samples were

dried prior to liquid chromatography-tandem mass spectrometry

analysis.


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High-performance liquid chromatography-tandemmass spectrometry instrumentation and analyticalconditions

Liquid chromatography separations were carried out with an Agilent

LC 1100 binary pump, autosampler, vacuum degasser and column

oven coupled with an Agilent 6410 Triple Quad LC/MS (Agilent

Technologies). The dry samples were dissolved in 10 ml acetonitrile

(70%), vortex-mixed for 1 min and injected onto an acrylamido-type

column (TSKgel Amide-80, TOSOH) at 25�C. The solvent system con-

sisted of 100% water, 0.15% formic acid and 5 mM ammonium acet-

ate (solvent A) and 100% acetonitrile (solvent B). Elution was

performed at a flow rate of 220 ml/min with a 70–40% linear gradient

(solvent B) over the first 8 min, followed by an 80% stage (solvent B)

over 2 min before the reconditioning of the column at 70% of

solvent B. The system was fully controlled by MassHunter software

(Agilent Technologies). Electrospray ionization was achieved in the

positive mode with the spray voltage set at 4000 V. Nitrogen was

used as nebulizer gas and nebulizer pressure was set at 20 psi with a

source temperature of 100�C. Desolvation gas (nitrogen) was heated

to 350�C and delivered at a flow rate of 10 l/min. Qualification was

performed in selected reaction monitoring mode with the following

transitions: morphine, m/z (mass-to-charge ratio) 286.1–165.1, and

codeine, m/z 300.2–214.8, and m/z 300.2–164.8.

Statistical analysisComparisons between groups were carried out using either one- or

two-factor (ANOVA) followed by Bonferroni post hoc test. Unpaired

t-test was used for monkey experiments in which a low n was used or

for comparisons between two groups. The level of statistical signifi-

cance was set at P5 0.05.

Results

Endogenous morphine-like compoundimmunoreactivity in the rat brainWe first characterized the distribution of the endogenous

morphine-like compound immunoreactivity (morphine, mor-

phine-6-glucuronide, morphine-3-glucuronide and codeine) on sa-

gittal rat brain sections (Fig. 1A and B). The endogenous

morphine-like compound staining was heterogeneously distributed

between brain areas (Fig. 1C–J; Table 2). The cortex (Fig. 1C),

the caude-putamen (Fig. 1D) and the substantia nigra pars

reticulata (Fig. 1F) are clearly stained, whereas the islands of

Calleja (Fig. 1E), the pons (Fig. 1G), the interposed cerebellar

nuclei (Fig. 1H), the cerebellum (Fig. 1I) and the inferior colliculus

(Fig. 1J) were strongly stained. Interestingly, most of these nuclei

play a role in motor control.

Phenotype of striatal endogenousmorphine-like compoundimmunopositive neuronsThe striatum plays a major role in the pathophysiology of

Parkinson’s disease, being the primary target of nigrostriatal dopa-

mine neurons (Parent et al., 1995; Prensa and Parent, 2001).

We therefore focused our attention on the striatum that displays

endogenous morphine-like compound immunopositivity (Fig. 1D

and K) in both neuronal and non-neuronal labelling (Fig. 1L).

Two types of endogenous morphine-like compound positive neu-

rons were observed in the rat striatum: the vast majority is con-

stituted by the oval-shaped medium spiny neurons (�15 mm in

size) while a few larger neurons (�20 mm) were also labelled. In

the medium spiny neurons, endogenous morphine-like compound

immunostaining was present in the cytoplasm and in proximal

processes (Fig. 2A–D). Endogenous morphine-like compound

immunolabelling was observed in 45% of dopamine and cAMP-

regulated phosphoprotein, 32 kDa-immunopositive medium spiny

neurons (Fig. 2A). Those endogenous morphine-like compound

immunoreactive medium spiny neuron-like neurons were glutam-

ate acid decarboxylase positive (Fig. 2B) and either D1 dopamine

receptor positive (Fig. 2C) or D2 dopamine receptor positive (Fig.

2D). We investigated if these medium spiny neurons also ex-

pressed CYP2D1, the rat enzyme homologue of human CYP2D6

that is considered a key enzyme in endogenous morphine synthe-

sis (Boettcher et al., 2005; Stefano et al., 2008). Varying degrees

of CYP2D1 immunoreactivity were found but of the majority of

endogenous morphine-like compound positive, medium spiny neu-

rons were also CYP2D1-positive (Fig. 2E). Finally, we defined the

phenotype of the larger neurons using either choline acetyltrans-

ferase or parvalbumin immunohistochemistry (Parent et al., 1995).

None of the choline acetyltransferase-immunopositive neurons

were endogenous morphine positive (Fig. 2F), while the

parvalbumin-immunopositive neurons displayed a clear endogen-

ous morphine immunopositivity (Fig. 2G).

The electron microscopy analysis confirmed the light microscopy

observations, i.e. that endogenous morphine-like compounds are

Table 2 List of endogenous morphine-like compoundimmunoreactive brain areas

Brain area Immunoreactivity

Cerebral cortex + +

Caudate putamen + +

Accumbens nucleus + +

Islands of Calleja + + +

Substantia nigra pars reticulata + +

Subthalamus nucleus + +

Hippocampus + + +

Red nucleus +

Interstitial nucleus of Cajal +

Darkschwitsh nucleus +

Inferior colliculus + + +

Superior colliculus +

Pontine nuclei + + +

Cerebellum (Purkinje layer) + + +

Cerebellum (molecular layer) +

Interposed cerebellar nuclei + + +

Vestibular nucleus (lateral) + +

Immunostaining intensity was visually scored as follows: + weak; + + moderate;+ + + strong.


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expressed by medium spiny neurons (Fig. 3). Indeed, the most

abundant immunopositive neurons are circular, medium-sized

(�10–15 mm in diameter), display an unindented nucleus and

scant somatic cytoplasm containing few subcellular organelles

(Dumartin et al., 1998, 2000; Berthet et al., 2009) (Fig. 3A).

Within the cytoplasm, endogenous morphine-like immunoreactiv-

ity was present at the inner face of the plasma membrane and at

the outer face of vesicles (Fig. 3A and B). Dendrites were also

found to be immunoreactive (Fig. 3C and D), with reaction prod-

ucts being located on microtubules (Fig. 3C).

Interestingly, only one-third of the endogenous morphine-like

compound positive medium spiny neurons were D1 dopamine

receptor positive (Fig. 4A), while two-thirds were D2 dopamine

receptor positive (Fig. 4B). Furthermore, one can distinguish two

categories of medium spiny neurons according to their immunor-

eactivity. Most medium spiny neurons with weak labelling ( + ),

where moderate endogenous morphine-like immunoreactivity

was found, were D1 dopamine receptor positive (Fig. 4A) or D2

dopamine receptor negative (Fig. 4B), while those with strong

labelling ( + + + ), where very intense immunoreactivity was

observed, were D1 dopamine receptor negative (Fig. 4A) or D2

dopamine receptor positive (Fig. 4B). Altogether, these observa-

tions suggest a differential influence upon the so-called ‘direct’

and ‘indirect’ striatofugal pathways (Alexander et al., 1986;

Albin et al., 1989) although physiological investigations are

needed to characterize the respective impact.

Striatal endogenous morphine-likecompound immunopositivenon-neuronal cellsBesides clear immunoreactivity in neurons, stellate cells with smal-

ler cell bodies and numerous processes resembling astrocytes were

also immunopositive for endogenous morphine-like compounds.

Astrocytes were identified by their immunoreactivity for glial fibril-

lary acidic protein and S100b (Kacem et al., 1998; Raponi et al.,

2007). While glial fibrillary acidic protein signal preferentially

stained astrocyte processes rather than cell bodies, S100b staining

strongly labelled both the cytoplasm and the processes (Fig. 5A).

Endogenous morphine-like compound immunoreactivity was de-

tected in the S100b-positive cells, which have a ramified form,

their numerous cell processes sometimes coiling up around blood

vessels and in close vicinity of neurons (Fig. 5B). Electron micros-

copy localization of S100b (immunoperoxidase) and endogenous

morphine-like (immunogold) revealed clear examples of such tri-

adic associations (Fig. 5D). In contrast, endogenous morphine-like

immunoreactivity was not observed in NG2-positive glial cells

(Fig. 5C).

Increase in striatal endogenousmorphine-like immunoreactivity in therat model of Parkinson’s diseaseAs the medium spiny neurons are the primary targets of the dopa-

mine terminals originating from the substantia nigra pars compacta

(Parent et al., 1995; Prensa and Parent, 2001), we investigated

Figure 2 Phenotype of endogenous morphine-like compound

immunopositive striatal medium spiny neurons. Double

immunofluorescence was conducted for endogenous

morphine-like compounds (eM, red) and dopamine and

cAMP-regulated phosphoprotein, 32 kDa (DARPP-32, A),

glutamate acid decarboxylase 65-67 (GAD, B), D1 dopamine

receptor (D1R, C), D2 dopamine receptor (D2R, D), CYP2D1

(E), choline acetyltransferase (ChAT, F) and parvalbumin (PV, G)

(all in green). Arrows and asterisks indicate immunopositivity for

the considered marker. Scale bars = 10 mm.


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the change in endogenous morphine-like compound immunoreac-

tivity of these neurons in the three experimental conditions. The

pattern of distribution of endogenous morphine-like compound

immunopositive elements varied in number and in staining inten-

sity (Fig. 6A). We distinguished two categories of medium spiny

neurons according to their immunoreactivity as defined in Fig. 4:

those with weak labelling ( + ), where moderate endogenous

morphine-like compound immunoreactivity was found, and those

with a strong labelling ( + + + ), where very intense immunoreac-

tivity was observed. A qualitative analysis reveals that in the stri-

atum of sham animals, weakly labelled ( + ) medium spiny neurons

were observed among a very lightly immunoreactive neuropil (Fig.

6A). Lesioning the nigrostriatal pathway in 6-hydroxydopamine

rats led to an increased immunostaining with an obvious increase

in the number of strongly labelled ( + + + ) medium spiny neurons

(Fig. 6A) and of the neuropil. While such an increase was more

pronounced on the lesioned side, it was also present on the unle-

sioned side (Fig. 6A). L-DOPA treatment of 6-hydroxydopamine-

lesioned rats led to a decrease in intensity of neuronal and neuropil

immunostaining on both sides (Fig. 6A), without however, return-

ing to the immunostaining observed in sham animals.

Quantitative analysis shows that the total number of immuno-

positive medium spiny neurons is not statistically different after

unilateral 6-hydroxydopamine lesion and/or further L-DOPA treat-

ment (Fig. 6B). This result suggests that (i) the overall number of

endogenous morphine-like compound immunopositive medium

spiny neurons does not change across experimental conditions;

and (ii) lesioning one hemisphere affects endogenous morphine-

like compound immunoreactivity in both hemispheres. When dis-

tinguishing between the strongly labelled and weakly labelled

medium spiny neurons (Fig. 6C), these two categories show

opposing variations. The number of strongly labelled medium

spiny neurons dramatically increases after 6-hydroxydopamine

lesion (P50.05), on both sides of the brain, although the mag-

nitude is greater on the lesioned side (Fig. 6C). The number of

weakly labelled medium spiny neurons decreases on the lesioned

side of 6-hydroxydopamine-lesioned rats (P50.05; Fig. 6C).

On the lesioned side, the number of strongly labelled medium

Figure 3 Immunogold detection of endogenous morphine-like compound at electron microscopic level in the rat striatal medium spiny

neurons. (A) An endogenous morphine-like compound immunoreactive soma with the size and cytological features of a medium spiny

neuron. Gold particles are associated with the outer face of vesicles and with the inner face of the plasma membrane. (B) Detail from a

Golgi apparatus. Gold particles are associated with vesicles at the outer face. (C) An endogenous morphine-like compound immunor-

eactive dendrite. The inset shows a higher magnification with gold particles located on microtubules. (D) An axodendritic synapse (arrow)

between an endogenous morphine-like compound labelled dendrite (D2) and an unlabeled axon terminal (A1). Scale bar = 1 mm (A);

0.5 mm (B and C); 0.2 mm (D); 0.2 mm (C inset).


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spiny neurons decreased after L-DOPA treatment (P50.05 versus

6-hydroxydopamine) but remained higher than in sham animals

(P50.05).

Altogether these data suggest that dopamine depletion in the

rat leads to an upregulation of striatal endogenous morphine-like

compounds that is subsequently downregulated, though not nor-

malized, by treatment with L-DOPA.

Increase in striatal endogenousmorphine-like compoundimmunoreactivity in the macaquemodel of Parkinson’s diseaseUnilateral lesion of the nigrostriatal pathway in the rat is known to

induce changes on both sides of the brain (e.g. Johansson et al.,

2001; Konradi et al., 2004) although the most prominent changes

generally occur on the dopamine-depleted side. As a clinically rele-

vant bilateral model was therefore needed, we used the gold-

standard non-human primate model of Parkinson’s disease, the

MPTP-treated macaque monkey (Bezard et al., 2003a; Gold

et al., 2007; Berton et al., 2009; Ahmed et al., 2010). The distri-

bution of endogenous morphine-like compound immunoreactive

medium spiny neurons in the caudate nucleus and the putamen,

the two forming the monkey striatum, was very similar to the

pattern described in the rat model (see above), i.e. uniform

distribution throughout the striatum and with two categories, i.e.

the weakly labelled and the strongly labelled medium spiny neu-

rons (Fig. 7A). While no significant change was observed in the

caudate nucleus (Fig. 7B), the total number of endogenous mor-

phine-like compound immunopositive medium spiny neurons in

the putamen was greater in the MPTP-lesioned group compared

with the control group (unpaired t-test, P5 0.01; Fig. 7B).

Chronic L-DOPA treatment led to a normalization of the total

number of endogenous morphine-like compound immunopositive

medium spiny neurons in the putamen (P50.05 versus

MPTP-lesioned; non-significant versus control group; Fig. 7B).

When distinguishing between the strongly labelled and weakly

labelled medium spiny neurons (Fig. 7C and D), these two cate-

gories show opposing variations. The number of strongly

labelled-like-immunolabelled medium spiny neurons dramatically

increased in the MPTP-lesioned group in both caudate and puta-

men (P50.01), while weakly labelled-like-immunolabelled

medium spiny neurons decreased (P50.05; Fig. 7C and D).

Chronic L-DOPA treatment decreased the number of strongly

labelled medium spiny neurons and increased the number of

weakly labelled medium spiny neurons with no more significant

difference with either the control or the MPTP-lesioned animals

(Fig. 7C and D).

Altogether these data suggest that striatal dopamine depletion

in the macaque model of Parkinson’s disease also leads to an

upregulation of striatal endogenous morphine-like compounds

Figure 4 Differential endogenous morphine-like compound labelling of striatal medium spiny neurons based upon their dopamine

receptor immunoreactivity. (A) D1 dopamine receptor immunoreactivity (arrows) is located on weakly endogenous morphine-positive

medium spiny neurons ( + ). (B) Only strongly endogenous morphine-like compound positive medium spiny neurons ( + + + ) express D2

dopamine receptor immunoreactivity (arrowheads). Scale bar = 10 mm. D1R = D1 dopamine receptor; D2R = D2 dopamine receptor;

eM = endogenous morphine.


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Figure 5 Phenotype of endogenous morphine-like compound immunopositive striatal glial cells. (A) S100b-positive glial cells (green)

co-localize with endogenous morphine-like compounds (red) as indicated by arrow. Asterisk indicates an endogenous morphine-like

compound positive medium spiny neuron. (B) S100b-positive cell (red) colocalizes with glial fibrillary acidic protein (GFAP, green).

(C) NG2-postive cells (green, arrow), however, do not co-localize with endogenous morphine-like compound immunopositivity (red).


(continued)

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that are later downregulated, though not normalized, by L-DOPA

treatment.

Increased endogenous morphine-likecompound immunoreactivity in thestriatum of human Parkinson’s diseaseEndogenous morphine-like compound immunoreactivity is faint

in post-mortem control samples (Fig. 8A) with only a few medium

spiny neurons displaying a clear staining. In patients with Parkinson’s

disease, however, endogenous morphine-like compound immuno-

positive medium spiny neurons were far more numerous with, as

before, two populations: one with strongly labelled perikarya and

the other with a weak staining (Fig. 8B). Endogenous morphine-like

compound immunopositive astrocytes became observable in pa-

tients with Parkinson’s disease (Fig. 8B). Even though these patients

have been exposed to dopaminergic drugs in the course of their

disease, these observations support the experimental data collected

in the rat and macaque models of Parkinson’s disease, i.e. that par-

kinsonism is associated with a dramatic increase in endogenous

morphine-like compound immunoreactivity.

Figure 5 Continued(D) Triadic association unravelled at electron microscopy level with combined endogenous morphine (immunogold particles) and S100b(black, peroxidase reaction) staining. An endogenous morphine-like compound positive medium spiny neuron (MSN; highlighted in green)

is surrounded by an endogenous morphine-like compound and a S100b-positive astrocyte (A; highlighted in red) which process apposes to

the basal membrane of endothelial cell lining a blood vessel (V; highlighted in blue). Green box is further magnified to better show

endogenous morphine-like immunogold particles in the medium spiny neuron, while the red box shows that astrocyte stroma was S100blabelled with peroxidase and endogenous morphine-like labelled with immunogold particles. Scale bar = 10 mm (A–C), 1mm (D). V = blood

vessel.

Figure 6 Dopamine depletion increases the number of strongly labelled endogenous morphine-like compound immunopositive striatal

medium spiny neurons in the rat. (A) (Top) Examples of endogenous morphine-like compounds immunostaining (brown) in the striatum of

sham-operated, 6-hydroxydopamine-lesioned and L-DOPA-treated 6-hydroxydopamine-lesioned rats. (Middle) Examples of topo-

graphical counting maps. Weakly labelled medium spiny neurons are indicated by blue dots while strongly labelled medium spiny neurons

are indicated by red dots. (B) Total number of endogenous morphine-like compound positive medium spiny neurons (MSN) per mm2

(mean � SEM) on both lesioned (L) and unlesioned (UL) sides in sham-operated, 6-hydroxydopamine-lesioned and L-DOPA-treated

6-hydroxydopamine-lesioned rats. Two-way ANOVA; lesion effect: F(1,23) = 0.28; L-DOPA effect: F(1,23) = 0.03; interaction:

F(1,23) = 1.52. (C) Number of weakly and strongly labelled medium spiny neurons per mm2 (mean � SEM) on both lesioned (L) and

unlesioned (UL) sides in sham-operated, 6-hydroxydopamine-lesioned and L-DOPA-treated 6-hydroxydopamine-lesioned rats. Two-way

ANOVAs followed by Bonferroni. Strongly immunolabelled: lesion effect: F(1,23) = 7.65, P5 0.05; L-DOPA effect: F(1,23) = 1.13;

interaction: F(1,23) = 11.17, P50.01. Weakly immunolabelled: lesion effect: F(1,23) = 11.15, P5 0.01; L-DOPA effect: F(1,23) = 1.78;

interaction: F(1,23) = 7.81, P50.05. *P50.05 with sham values on same side; **P50.05 with 6-hydroxydopamine-lesioned values.

Scale bar = 70 mm (A). eM = endogenous morphine.


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Increase of endogenous morphine levelsin mouse brain after a-methyl-p-tyrosine–reserpine treatmentSuch consistency across models and human disease supports

the proposal that dopamine depletion leads to an increased pro-

duction of endogenous morphine-like compounds. These results

are contrary to the expectation based on the demonstration that

dopamine-null mice do not express endogenous morphine (Neri

et al., 2008), i.e. that dopamine is required for the synthesis of

endogenous morphine. Further neurochemical evidence supporting

our anatomical data was therefore mandatory to demonstrate our

case and we selected first the �-methyl-p-tyrosine–reserpine-

treated mouse model of Parkinson’s disease. As expected, there

was a striking increase in the total number of endogenous

Figure 7 Increased number of endogenous morphine-like compound immunopositive striatal medium spiny neurons in the

MPTP-lesioned macaque. (A) Examples of endogenous morphine-like compound immunostaining (brown) in the striatum of control,

MPTP-lesioned and L-DOPA-treated MPTP-lesioned macaque monkeys. (B) Total number of endogenous morphine-like compound

positive medium spiny neurons per mm2 (mean � SEM) in the caudate nucleus and the putamen of control, MPTP-lesioned and

L-DOPA-treated MPTP-lesioned macaque monkeys. Number of weakly and strongly labelled medium spiny neurons per mm2

(mean � SEM) in the caudate nucleus (C) and the putamen (D) of control, MPTP-lesioned and L-DOPA-treated MPTP-lesioned

macaque monkeys. (B–D): *P5 0.05 with control value; **P50.05 with MPTP-lesioned value. Scale bar = 50 mm (A).

Figure 8 Striatal endogenous morphine-like compound immunoreactivity increases in human Parkinson’s disease. Representative

microphotographs of differential endogenous morphine-like compound immunostaining in the striatum of a control human subject (A)

and a Parkinson’s disease subject (B). + and + + + refer to staining intensity classification. Inset in (B) shows that no endogenous

morphine-like compounds immunostaining was observed in subjects with Parkinson’s disease when the primary antibody was preabsorbed

with exogenous morphine. Asterisk indicates a medium spiny neuron and the arrow an astrocyte. Scale bar = 25 mm.


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morphine-like compound positive medium spiny neurons in

�-methyl-p-tyrosine–reserpine-treated mice compared with

saline-treated mice (299.3 � 17.0 versus 173.1 � 20.3, unpaired

t-test, P5 0.0001). Accordingly, there was a huge significant

rise in the number of strongly labelled, associated with a signifi-

cant decrease in weakly labelled medium spiny neurons (Fig. 9A).

The extent of dopamine depletion induced in �-methyl-p-tyrosine–

reserpine-treated mice (P50.05; Fig. 9B) correlated with a

striking increase in endogenous morphine brain levels in

�-methyl-p-tyrosine–reserpine treated mice (morphine-specific

enzyme-linked immunosorbent assay; P50.05; Fig. 9C)

compared with saline-treated animals. These neurochemical data

suggest that dopamine depletion induced by �-methyl-p-tyrosine–

reserpine treatment in the mouse model induces an upregulation

of true endogenous morphine.

To confirm the presence of morphine in brain extracts of mice,

liquid chromatography–tandem mass spectrometry analyses were

performed in selected reaction monitoring mode. The specific tran-

sition m/z 286.2–165.1 was used to qualify the presence of mor-

phine. The results obtained unambiguously confirm the presence

of morphine in mouse brain (Fig. 9D for standard of morphine and

for mouse brain). We then micro-dissected the whole striatum of

two control and two �-methyl-p-tyrosine–reserpine-treated mice

and searched for endogenous morphine and codeine, its direct

precursor (specific transition of codeine m/z 300.2–214.8, and

m/z 300.2–164.8). In such low amounts of tissue, neither codeine

nor endogenous morphine was detected in control mice, whereas

codeine was present in �-methyl-p-tyrosine–reserpine-treated

mice (21.9 � 8.5 fmol/mg of tissue). The endogenous morphine

level was still below sensitivity, reflecting the current limits of mass

spectrometry detection of endogenous alkaloids in small, specific

brain areas. However, the detectable level of the direct endogen-

ous morphine precursor codeine in dopamine-depleted striatum

supports the dramatic increase in endogenous morphine-like com-

pound immunoreactivity observed in our rat and monkey models

of Parkinson’s disease.

Lack of endogenous morphine-likecompound regulation in striatum ofhyperdopaminergic DAT�/� miceNeurochemical data unequivocally demonstrate opposing dopa-

mine/endogenous morphine regulation in parkinsonism instead

of a parallel regulation (Neri et al., 2008). What holds true in

parkinsonism might be wrong in animals with an intact nigrostrial

pathway and yet hyperdopaminergic. Would dopamine be

required for the synthesis of endogenous morphine? One can

expect that hyperdopaminergia in a non-dopamine-depleted stri-

atum would be linked to an increased endogenous morphine-like

immunoreactivity. Endogenous morphine-like compound

Figure 9 Endogenous morphine levels increase in dopamine-depleted mice. (A) Number of weakly and strongly labelled medium spiny

neurons per mm2 in the caudate-putamen of control (n = 4) and �-methyl-p-tyrosine–reserpine (AMPT/Res) treated mice (n = 4). Results

are presented as mean � SEM relative to controls. Unpaired t-test: *P5 0.05. (B and C) Amounts of dopamine (B) and morphine (C)

present in brains of control (n = 4) and �-methyl-p-tyrosine–reserpine treated mice (n = 5). Results are presented as nmol/g of wet

tissue � SEM. Unpaired t-test: *P50.05. (D) Characterization of morphine in mouse brain extracts performed in selected reaction

monitoring (SRM) mode with the following transitions: m/z 286.2! 165.1. Selected reaction monitoring trace of the specific transition

for morphine in standard samples of morphine (Top: 50, 100, 500 and 1000 fmol) and mouse brain (Bottom).


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immunoreactivity in the striatum was therefore measured in a

mouse model of hyperdopaminergia without degeneration of the

nigrostriatal pathway, namely the dopamine transporter knock-out

mouse (DAT�/�). Indeed DAT�/� mice are known to exhibit a

500% increase in dopamine extracellular content compared with

DAT + / + mice (Jones et al., 1998; Benoit-Marand et al., 2000).

As no change in endogenous morphine-like compound immunor-

eactivity was observed in the striatum of DAT�/� mice compared

with both DAT + / + and DAT + /� (Fig. 10A–C), and because of its

presence in a dopamine-depleted state, we propose that endogen-

ous morphine synthesis is not linked to dopamine content, sug-

gesting that alternate synthesis pathways must exist.

DiscussionThe present data demonstrate an upregulation of endogenous

morphine-like immunoreactivity, both in number of immunoposi-

tive cells and intensity of immunostaining, in the dopamine-

depleted striatum of the 6-hydroxydopamine-lesioned rat model,

the �-methyl-p-tyrosine–reserpine treated mouse, the MPTP-

treated macaque model and of human Parkinson’s disease. Such

consistency across models and human disease supports the pro-

posal that dopamine depletion leads to an increased production

of endogenous morphine. In addition, L-DOPA treatment in ex-

perimental parkinsonism consistently decreased the number of

endogenous morphine-like compound immunopositive medium

spiny neurons.

These results are contrary to expectations based on the demon-

stration that dopamine-null mice do not express endogenous

morphine-like compounds (Neri et al., 2008), i.e. that dopamine

is required for the synthesis of endogenous morphine. Endogenous

morphine-like immunoreactivity increase is likely to be due to an

overproduction of dopamine metabolites in the brain, such as

tetrahydropapaveroline that can be used by non-dopaminergic

cells to synthesize morphine-like compounds (Boettcher et al.,

2005; Goumon et al., 2009). The morphine precursor tetrahydro-

papaveroline represents a degradation product of dopamine that is

found in extracellular brain spaces, CSF and urine. Interestingly,

the presence of a high quantity of endogenous morphine is re-

ported in the urine and serum of L-DOPA-treated patients with

Parkinson’s disease (Matsubara et al., 1992; Arun et al., 1998) as

well as in patients with alcohol addiction. The model is invalidated

by the present data set as: (i) endogenous morphine-like com-

pounds increase occurs in a Parkinson’s disease-like state; and

(ii) partial normalization of endogenous morphine-like compound

immunoreactivity occurs in animals treated with L-DOPA. It thus

raises the questions of the cellular origin and synthesis pathway of

endogenous morphine.

In patients with Parkinson’s disease, endogenous morphine-like

compounds are overproduced compared with non-pathological

individuals. Tetrahydropapaveroline (or norlaudanosoline) is

Figure 10 Endogenous morphine-like compound immunoreactive medium spiny neurons in DAT�/� mice striatum. (A) Similarly to the

rat and the monkey, endogenous morphine-like compound immunopositive medium spiny neurons were observed in DAT + / + , DAT + /�

and DAT�/� with either moderate or intense staining. (B) The total number of endogenous morphine-like compound immunopositive

medium spiny neurons was not affected by genotype. One-way ANOVA: F(2,20) = 2.32. (C) Distinguishing strongly-stained from the

weakly-stained medium spiny neurons in the medial (M) or lateral (L) part of the striatum did not reveal further difference.

HO = homozygous; HT = heterozygous; WT = wild-type. Scale bar = 50 mm (A).


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supposedly related to Parkinson’s disease development and also

may be derived from its treatment with L-DOPA (Soto-Otero

et al., 2006). Tetrahydropapaveroline is easily found in the

plasma, urine and brain of parkinsonian patients treated with

L-DOPA (Matsubara et al., 1992; Cashaw, 1993a, b). It was pro-

posed that tetrahydropapaveroline is spontaneously synthesized by

a condensation of dopamine and 3,4-dihydroxyphenylacetalde-

hyde (metabolic degradation of dopamine by monoamine oxi-

dase), but recent data suggest that tetrahydropapaveroline is

probably converted to reticuline (next intermediate in morphine

biosynthesis) by a specific enzyme (Soto-Otero et al., 2004,

2006). While sinister-tetrahydropapaveroline is involved in mor-

phine biosynthesis in plants, recent studies have demonstrated

that only rectus-tetrahydropapaveroline is converted to reticuline

by the amine N-methyltransferase into endogenous morphine in

mammals (Grobe et al., 2011). This exclusive selectivity for the

rectus-tetrahydropapaveroline enantiomer is of great importance

since high amounts of the sole sinister-tetrahydropapaveroline

have been found in the human brain (Sango et al., 2000).

Because rectus-tetrahydropapaveroline was not detected and con-

sidering the recent discovery of its pivotal role in endogenous

morphine synthesis, we hypothesize that the rectus-tetrahydropa-

paveroline enantiomer is present but rapidly processed into

endogenous morphine. Accordingly, L-DOPA administration,

which leads to sinister-tetrahydropapaveroline formation, does

not induce endogenous morphine formation in mouse brain

(Grobe et al., 2010). These data explain, at least in part, the ap-

parent discrepancy of our results with literature as the enantiomer

issue explains both the lack of further endogenous morphine in-

crease after L-DOPA treatment in our models and the increase of

tetrahydropapaveroline in the urine of L-DOPA-treated patients

with Parkinson’s disease.

Endogenous morphine is expressed in various cell types, i.e. in

neuronal and non-neuronal cells (Laux et al., 2011). Two hypoth-

eses may explain the presence of endogenous morphine or related

compounds in neurons. One possibility is that endogenous mor-

phine and/or endogenous morphine-like compounds (morphine

glucuronides and/or codeine) could be taken up from the extra-

cellular space by active or passive transport. Indeed, several re-

ports suggest that morphine-like compounds could be synthesized

by non-dopaminergic or non-catecholaminergic cells such as the

pancreatic cells, hepatocytes, placenta-derived cells (Weitz et al.,

1987; Molina et al., 1995; Poeaknapo et al., 2004). In our

Parkinson’s disease models, biosynthesis of endogenous morphine

is obviously not completed in dopaminergic neurons. Endogenous

morphine-like compounds present in non-dopaminergic brain cells

may thus be synthesized after uptake of precursors (e.g. dopamine

and sinister-tetrahydropapaveroline). Such a hypothesis implies co-

operation between different cell types to ensure complete biosyn-

thesis of endogenous morphine.

Our results show the presence of endogenous morphine-like

compounds in a large number of astrocytes. Astrocytes do not

express tyrosine hydroxylase (Jaeger, 1985) and thus are unable

to de novo synthesize dopamine and thus endogenous morphine.

Two hypotheses can be proposed to explain the presence of

endogenous morphine-like compounds in these cells. Firstly,

astrocytes may achieve morphine biosynthesis after precursor

uptake. Secondly, astrocytes are known to be involved in reuptake

and the catabolism of neurotransmitters (Iversen, 2006).

Interestingly, astrocytes express multidrug-resistance like protein

1, 3, 4 and 5 as well as glutamate transporter, which correspond

to transporters that are described to be potentially involved

in morphine/morphine-6-glucuronide/morphine-3-glucuronide/

codeine uptake/efflux (Hirrlinger et al., 2002, 2005; Bourasset

et al., 2003; Somogyi et al., 2007). In addition, presence of

UDP-glucuronosyltransferase 1A6 enzyme (UGT1A6) in rat astro-

cytes (Suleman et al., 1998; Heurtaux et al., 2006) suggests that

after morphine uptake, astrocytes may catabolize morphine in

morphine-glucuronides. Moreover, the presence of morphine

and/or morphine glucuronides in astrocytic foot processes

around blood vessels may suggest a possible uptake from the

blood or release into the blood (Brix-Christensen et al., 1997,

2000). The fact that high levels of endogenous morphine are

found in urine of patients with Parkinson’s disease (Matsubara

et al., 1992) strongly suggests that this endogenous morphine is

mainly produced by peripheral cells as endogenous morphine

levels in the brain, even increased in Parkinson’s disease, would

hardly explain such a rise. It is likely that biosynthesis of endogen-

ous morphine is different in brain and peripheral cells (Laux et al.,

2011). While endogenous morphine-like compound (morphine-6-

glucuronide) is fully synthesized in catecholaminergic chromaffin

cells (Goumon et al., 2006), CNS endogenous morphine accumu-

lates in �-aminobutyric acid-ergic neurons and astrocytes that may

have taken up a specific precursor (Laux et al., 2011). This differ-

ence in the endogenous morphine synthesis pathway between

brain and periphery is of importance since the peripheral endogen-

ous morphine may be taken into account for urinary excretion

independently of the brain. We cannot exclude, however, that

endogenous morphine synthesis could occur in non-dopamine-

depleted brain regions, thus acting as a compensatory mechanism

(Bezard and Gross, 1998). Altogether our results suggest that

astrocytes may be responsible for morphine catabolism in the

CNS, a mechanism that now requires our attention because of

the extensive use of morphine in clinics and as a drug of abuse.

A central unresolved question is the role of endogenous mor-

phine and of its regulation in the striatum. Although the vast

majority of the striatal neurons express endogenous morphine-like

compounds, there is a striking pattern with moderate endogenous

morphine-like immunoreactivity in D1 dopamine receptor-positive

medium spiny neurons and intense immunoreactivity in the D2

dopamine receptor-positive medium spiny neurons. This result

suggests a differential influence upon the so-called ‘direct’ and

‘indirect’ striatofugal pathways (Alexander et al., 1986; Albin

et al., 1989), although physiological investigations would be

needed to characterize the respective impact. It remains that the

large increase in immunoreactivity after dopamine depletion

should thus mostly occur in the D2 dopamine receptor-positive

medium spiny neurons of the indirect pathway. These neurons

co-express encephalin with �-aminobutyric acid as neurotransmit-

ters. Encephalin levels are tightly regulated by dopamine tone as

the rise in encephalin expression occurs even before the symptom

appearance in progressive models of Parkinson’s disease (Bezard

et al., 2003b). This spatial and temporal analogy would support

the hypothesis of a compensatory role for the rise in endogenous


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morphine-like compounds in Parkinson’s disease. A similar ration-

ale can be proposed for interneurons. While no choline

acetyltransferase-immunopositive neurons were found to be en-

dogenous morphine positive, the parvalbumin-immunopositive

neurons (also called the fast-spiking interneurons), displayed

clear endogenous morphine immunopositivity. Those fast-spiking

�-aminobutyric acid interneurons provide potent feed-forward in-

hibition of striatal projection neurons (Mallet et al., 2005).

Endogenous morphine might thus play a role in the regulation

of their physiological output towards medium spiny neurons

(Mallet et al., 2006).

Since the functional consequences of endogenous morphine in-

crease have not been investigated in the present study, we should

limit ourselves to highlighting a few functional possibilities. A rise

in morphine levels is classically associated with an analgesic effect

but effects could be far more widespread. Endogenous morphine

has a high affinity for the m-opiate receptor that displays

high-affinity binding for morphine-related morphinan alkaloids

such as morphine-6-glucuronide, endomorphins, endorphins and

dynorphins, as well as the clinically established antagonists nalox-

one and naltrexone (Stefano et al., 1996). Different splicing vari-

ants of the m-opiate receptor are expressed in neural tissues and

mediate the inhibition of adenylate cylcase and induce nitric oxide

production and release (Ferreira et al., 1991; Pan, 2005;

Pasternak, 2007). Nitric oxide, as a morphine second messenger,

has been associated with antinociception (Przewlocki et al., 1993)

as well as tolerance and dependence (Majeed et al., 1994). In

relation to the immune system, morphine, but not the other

opioid peptides, stimulates constitutive nitric oxide release in

macrophages, granulocytes, various types of human and rat endo-

thelial cells, invertebrate neurons and immunocytes, and in rat

median eminence fragments, all in a naloxone antagonizable

manner (Stefano et al., 1996, 2008). Effects of opiate alkaloid

signalling upon the immune system could be seen as moderately

immunosuppressive. However, one can predict that such a phe-

nomenon should be of low magnitude in Parkinson’s disease based

upon the local paracrine mode of transmission, but could be put in

parallel with the immunodepressed profile of patients with

Parkinson’s disease that has been suggested to be associated

with fatigue (Katsarou et al., 2007) and depression (Kummer

and Teixeira, 2008). Pain is a possibly related symptom but, as

morphine is antinociceptive, one can posit that endogenous mor-

phine rise is a non-motor compensatory mechanism that fails over

time as do the motor compensatory mechanisms (Bezard and

Gross, 1998; Bezard et al., 2003b), at least in a subpopulation

of patients. We thus have a variety of non-motor symptoms

(Chaudhuri and Odin, 2010) whose pathophysiology could be

related to enhanced endogenous morphine levels, paving the

way for experimental and clinical investigations and target identi-

fication for future treatments.

AcknowledgementsWe thank A. Estager and M.L. Thiolat for excellent technical

assistance. E.B. designed and organized the experiments; G.C.,

E.D., A.B., G.P., A.L., F.D., A.V.D., M.-H.C., P.B.-C., Q.L., C.Q.,

M.N.-B and A.V. performed experiments; G.C., B.G., Y.G. and E.B.

analysed the data and wrote the article.

FundingThe Universite Victor-Segalen Bordeaux 2, the Universite de

Strasbourg and the Centre National de la Recherche Scientifique

provided the infrastructural support. The present work was funded

by MAPKinDYSK (to E.B.) and TRAFinLID (to E.B.) Agence

Nationale de la Recherche grants, a Conseil Regional d’Aquitaine

fellowship (to A.B.) a French Ministere delegue a la Recherche et a

l’Enseignement Superieur fellowship ( to A.L.) and a Fondation

pour la Recherche Medicale grant (to Y.G.).; The funders had

no role in study design, data collection and analysis, decision to

publish or preparation of the article.

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