Brain Interstitial Nociceptin/Orphanin FQ Levelsare Elevated in Parkinson’s Disease

Matteo Marti, PhD,1,2 Silvio Sarubbo, MD,3 Francesco Latini, MD,3 Michele Cavallo, MD,3

Roberto Eleopra, MD,4 Sara Biguzzi, MD,4 Christian Lettieri, MD,4 Carlo Conti, MD,5

Michele Simonato, MD,1,2 Silvia Zucchini, PhD,1,2 Rocco Quatrale, MD,6 Mariachiara Sensi, MD,6

Sanzio Candeletti, PhD,7 Patrizia Romualdi, PhD,7 and Michele Morari, PhD1,2*

1Department of Experimental and Clinical Medicine, Section of Pharmacology, University of Ferrara, Ferrara, Italy2Center for Neuroscience and National Institute of Neuroscience, University of Ferrara, Ferrara, Italy

3Division of Neurosurgery, S. Anna University Hospital of Ferrara, Ferrara, Italy4Clinical and Neurological Department, Angel’s Hospital of Mestre, Venice, Italy

5Department of Neurosurgery, Angel’s Hospital of Mestre, Venice, Italy6Clinical and Neurological Department, S. Anna University Hospital of Ferrara, Ferrara, Italy

7Department of Pharmacology, University of Bologna, Bologna, Italy

Abstract: Expression and release of nociceptin/orphanin FQ(N/OFQ) are elevated in the substantia nigra reticulata of 6-hydroxydopamine-hemilesioned rats, suggesting a pathogenicrole for N/OFQ in Parkinson’s disease. In this study, weinvestigated whether elevation of N/OFQ expression in 6-hy-droxydopamine-hemilesioned rats selectively occurs in sub-stantia nigra and whether hypomotility following acute halo-peridol administration is accompanied by a rise in nigral N/OFQ levels. Moreover, to prove a link between N/OFQ andidiopathic Parkinson’s disease in humans, we measured N/OFQ levels in the cerebrospinal fluid of parkinsonian patientsundergoing surgery for deep brain stimulation. In situ hybrid-ization demonstrated that dopamine depletion was associatedwith increase of N/OFQ expression in substantia nigra (com-pacta 1160%, reticulata 1105%) and subthalamic nucleus(145%), as well as reduction in caudate putamen (220%).No change was observed in globus pallidus, nucleus accum-

bens, thalamus, and motor cortex. Microdialysis coupled tothe bar test allowed to demonstrate that acute administrationof haloperidol (0.8 and 3 mg/kg) increased nigral N/OFQ lev-els (maximally of 147% and 153%, respectively) in parallelwith akinesia. A correlation with preclinical studies wasfound by analyzing N/OFQ levels in humans. Indeed, N/OFQlevels were found to be �3.5-fold elevated in the cerebrospi-nal fluid of parkinsonian patients (148 fmol/ml) comparedwith nonparkinsonian neurologic controls (41 fmol/ml).These data represent the first clinical evidence linking N/OFQ to idiopathic Parkinson’s disease in humans. Theystrengthen the pathogenic role of N/OFQ in the modulationof parkinsonism across species and provide a rationale fordeveloping N/OFQ receptor antagonists as antiparkinsoniandrugs. � 2010 Movement Disorder SocietyKey words: cerebrospinal fluid; haloperidol; human; noci-

ceptin/orphanin FQ; 6-OHDA; Parkinson’s disease

Nociceptin/orphanin FQ (N/OFQ) is an opioid-like

neuropeptide that activates the N/OFQ peptide (NOP)

receptor. N/OFQ and its receptor are widely expressed

throughout the rodent and primate central nervous sys-

tems and modulate a number of biological functions

such as pain, mood, reward, and locomotion.1,2 N/OFQ

has been proposed to play a role in Parkinson’s disease

(PD) based on the findings that NOP receptor anta-

gonists attenuated motor deficit in models of parkin-

sonism, such as the haloperidol-treated rat and

mouse,3–5 the 6-hydroxydopamine (6-OHDA)-hemile-

sioned rat,4,6,7 or the 1-methyl-4-phenyl-1,2,5,6-tetrahy-

dropyridine (MPTP)- intoxicated mouse and nonhuman

primate.8,9 Moreover, mice carrying a deletion of the

gene encoding for the N/OFQ precursor (ppN/OFQ)

were found resistant to the MPTP-induced degenera-

Potential conflict of interest: The authors declare no conflict of in-terest.

Received 12 January 2010; Revised 19 April 2010; Accepted 30April 2010

Published online 29 June 2010 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/mds.23271

*Correspondence to: Michele Morari, Department of Experimentaland Clinical Medicine, Section of Pharmacology, University of Fer-rara,via Fossato di Mortara 17-19, 44100 Ferrara, ItalyE-mail: m.morari@unife.it

1723

Movement DisordersVol. 25, No. 11, 2010, pp. 1723–1732� 2010 Movement Disorder Society

tion of SN compacta (SNc) dopamine (DA) cells, sug-

gesting that N/OFQ contributes to both the parkinso-

nian symptoms and neurotoxicity associated with the

disease.4 To further endorse a pathogenic role of N/OFQ

in PD, the parkinsonian toxins 6-OHDA4,10 and

MPP110,11 promoted ppN/OFQ expression in rat SN.

Indeed, such upregulation resulted in increased extracel-

lular N/OFQ levels in the SNr of 6-OHDA-lesioned

rats4 possibly leading to exacerbation of the physiologi-

cally inhibitory influence of endogenous N/OFQ on ni-

grostriatal DA transmission and motor behavior.12 De-

spite such compelling preclinical evidence, no relation-

ship between N/OFQ and PD in humans has been

demonstrated so far.2 Nonetheless, lower ppN/OFQ and

NOP expression was found in the hippocampus and cen-

tral amygdala, respectively, of alcoholics,13 and lower

N/OFQ binding was observed in patients with temporal

lobe epilepsy,14 indicating that changes in N/OFQ trans-

mission may be associated with neurologic diseases. To

possibly support this view, N/OFQ plasma levels were

found elevated in Wilson’s disease,15 an autosomal re-

cessive disorder related to defective copper metabolism

in the liver and copper overload in tissues, leading,

among others, to basal ganglia degeneration.16

The aims of this study were 3-fold: (i) to investigate

whether the increase in ppN/OFQ expression following

6-OHDA lesioning is selective for SN or involves

other brain areas, (ii) to demonstrate that functional

parkinsonism is associated with an increase in nigral

N/OFQ levels, and (iii) to provide clinical evidence

that N/OFQ may be relevant for PD in humans. To

pursue these aims, in situ hybridization was employed

to study ppN/OFQ expression in SN, primary (M1)

and secondary (M2) motor cortex, caudate-putamen

(CPu), nucleus accumbens (Ac), globus pallidus (GP),

and subthalamic nucleus (STN) of 6-OHDA-hemile-

sioned rats in comparison with sham-operated rats.

Moreover, microdialysis coupled to the bar test (a test

for akinesia) was used in awake rats to simultaneously

monitor SNr N/OFQ levels and immobility time fol-

lowing haloperidol administration. Finally, N/OFQ lev-

els were measured in brain cerebrospinal fluid (CSF)

samples obtained from PD patients undergoing surgery

for deep brain stimulation and compared with those

measured in nonparkinsonian neurologic controls.

MATERIALS AND METHODS

Animals

Male Sprague-Dawley rats (Harlan, S. Pietro al Nati-

sone, Italy) were used for this study. Four animals were

housed for cage, with free access to food and water, and

kept under environmentally controlled conditions (12-

hour light/dark cycle with light on between 07:00 AM

and 07:00 PM). The experimental protocols were

approved by the Italian Ministry of Health (licence no

194/2008-B) and the Ethical Committee of the Univer-

sity of Ferrara, and adequate measures were taken to

minimize animal pain and the number of animals used.

6-OHDA Lesion

Unilateral lesion of dopaminergic neurons was

induced in isoflurane-anesthetized male rats as previ-

ously described.6 Eight micrograms of 6-OHDA (dis-

solved in 4 ll of saline containing 0.02% ascorbic

acid) were stereotaxically injected into the right medial

forebrain bundle according to the following coordinates

from bregma: anteroposterior-4.4 mm, mediolateral-1.2

mm, dorsoventral-7.8 mm below dura.17 Control rats

(sham operated) were injected with vehicle. Two

weeks after 6-OHDA injection, successfully lesioned

rats (hemiparkinsonian) were selected using the rota-

tional model.18 Rats showing a turning behavior >7

turns/min in the direction ipsilateral to the lesion fol-

lowing intraperitoneal administration of amphetamine

(5 mg/kg) were enrolled in the study. This behavior

has been associated with >95% loss of striatal DA ter-

minals6 and extracellular DA levels.19 Sham-operated

rats were also injected with amphetamine.

In Situ Hybridization

Rats were killed by decapitation under light diethyl-

ether anesthesia. Their brains rapidly removed, frozen

in isopentane, cooled in a dry ice/methanol bath, and

stored at 2708C until use. Probes were prepared from

a full-length cDNA insert cloned in a pBluescript

SK(1) plasmid (proN/OFQ from C. Mollereau, Tou-

louse, France). The proN/OFQ plasmid was linearized

with BamH1 and transcribed with T7 RNA polymerase

to obtain antisense riboprobes or linearized with Xho1

and transcribed with T3 RNA polymerase to obtain

sense riboprobes. All riboprobes were obtained by run-

ning the transcription assays in the presence of a[33P]-rUTP, and hydrolyzed to fragments of approximately

200 base pairs with sodium carbonate at 608C.20

Twenty-micrometer coronal sections were cut at

three different brain levels from bregma: 10.70 to

11.20, 20.30 to 20.92, and 22.56 to 23.60.17 Sec-

tions were thaw mounted onto polylysine-coated slides,

fixed in 4% paraformaldehyde, soaked in 33 phos-

phate buffered saline, rinsed in a graded ethanol series,

dried, and stored at 2208C until use. Immediately

before in situ hybridization, they were pretreated with

proteinase K (1 lg/ll, 10 minutes, 378C) and with ace-

1724 M. MARTI ET AL.

Movement Disorders, Vol. 25, No. 11, 2010

tic anhydride (0.25% vol/vol, 10 minutes, and room

temperature). In situ hybridization was performed as

previously described.21 Briefly, sections were incubated

overnight at 528C with 40 ll hybridization mixture:

50% deionized formamide, 23 STE (Sodium-Tris-

EDTA), 53 Denhart’s solution, 100 lg/ml ssDNA,

100 lg/ml tRNA, 0.05% sodium pyrophosphate, and

60 ng/ml [33P]-riboprobe. They were then rinsed in 43SSC, treated with RNAase A (20 lg/ml, 30 minutes,

378C), washed in 13 SSC for 10 minutes, 0.13 SSC

at 528C for 30 minutes, and 0.13 SSC at room tem-

perature for 10 minutes, and dehydrated. Autoradio-

grams were generated by apposing these dried sections

alongside [33P]-riboprobe standards to Kodak BioMax

MR film at 2708C for 30 days.

The mean total optical density within an area of in-

terest was calculated by multiple sampling of that area

in two sections taken from each animal using a digital

analysis system (RBR Altair, Firenze, Italy). Back-

ground optical density in film areas not exposed to sec-

tions was subtracted from the total optical density.

Finally, data have been expressed as percent of optical

density in the control (noninjected) side.

Microdialysis in Haloperidol-Treated Rats

One probe of concentric design (1-mm dialyzing

membrane; AN69; Hospal, Bologna, Italy) was

implanted in SNr (anteroposterior 25.5, mediolateral

6 2.2, VD 28.3) under isoflurane anaesthesia. After

surgery, rats were allowed to recover, and experiments

were run 48 hour after probe implantation. Microdialy-

sis probes were perfused (3 ll/minutes) with a modi-

fied Ringer solution (for composition see above) added

with polypep 0.1%, captopril 300 lM, and BSA

0.3%.22 Haloperidol was administered intraperitoneally

after a 270-minute washout period, at doses causing

akinesia in both rats and mice (0.8 and 3 mg/kg,

respectively), according to our previous studies.3,4

Microdialysis samples were collected every 60

minutes, starting 180 minutes before haloperidol

administration. Samples were mixed with an equal vol-

ume of trifluoroacetic acid (TFA; 1% vol:vol) and

loaded onto C18 cartridges (SEPCOL-1; Bachem Ltd.,

St Helens, United Kingdom). Microcolumns were

washed twice with 3 ml 1% TFA and then eluted with

3 ml 60% acetonitrile in 1% TFA. Eluates were lyoph-

ilized and stored at 2708C until radioimmunoassay.

The adopted extraction procedure was validated by

measuring the recovery of [3H-Leucyl]-N/OFQ (Amer-

sham Biosciences Europe, Freiburg, Germany) under

identical conditions. More than 95% of the tritiated N/

OFQ added to a sample was recovered in the eluate

(90–92% in the first milliliter).

Analysis of Endogenous N/OFQ

N/OFQ-like immunoreactivity (N/OFQ-LI) present

in microdialysis fractions was measured 7 weeks after

6-OHDA lesion by a specific radioimmunoassay (RIA)

according to the method described by Ploj et al.23 Ly-

ophilized samples, reconstituted in 25 ll methanol in

0.1% HCl (1:1), were mixed with 100 ll [125I]-N/OFQ(Bachem Ltd.) and 100 ll N/OFQ antiserum (anti-

serum 96:21, kindly supplied by Dr I. Nylander, Upp-

sala, Sweden). The antiserum, raised against N/OFQ,

shows 0.5% cross-reactivity with the N/OFQ fragment

N/OFQ (1–13) and less than 0.1% cross-reactivity with

nocistatin and the following opioid peptides: dynorphin

A (DYN A), dynorphin B (DYN B), their truncated

fragments or elongated forms DYN A (1–6), DYN A

(1–32), and DYN B (1–29), or with Met-enkephalin,

Met-enkephalinArg6Phe7, Leu-enkephalin, and b-endorphin. The antiserum was used at the appropriate

dilution to give 30% to 34% binding of the [125I]-N/

OFQ added (4,800–5,000 cpm). The labelled peptide

and the antiserum were diluted in a gelatin buffer con-

taining 0.15 M NaCl, 0.02% sodium azide, 0.1% gela-

tin, 0.1% Triton X-100, and 0.1% BSA in a 0.05-M so-

dium phosphate buffer (pH 7.4). RIA tubes were incu-

bated at 48C for 24 h. Charcoal slurry (1 ml/tube) was

used to separate free and antibody bound peptide (15%

horse serum, 3% charcoal, and 0.3% dextran in RIA

buffer). Bound peptide was separated by centrifugation

(5,000g at 48C), and 1 ml aliquots of the supernatants

were counted for 1 minute on a Beckman 5500 gamma

counter (Beckman, Fullerton, CA). The detection limit

of the RIA assay was 1 fmol/tube. RIA curves and

data were analyzed using the GraphPad Prism 4 soft-

ware for Windows (GraphPad Software, San Diego,

CA). The limit of detection of the assay was 1 fmol,

and N/OFQ in vitro recovery was 2.0 6 0.1%.

Behavioral Testing During Microdialysis

To correlate changes of N/OFQ dialysate levels with

motor activity, rats undergoing microdialysis were

challenged in the bar test, a test for akinesia24 as previ-

ously described.3,7 Behavioral testing was performed

every 60 minutes. The bar test was performed essen-

tially as described above. Rat forepaws were placed

alternatively on blocks of increasing heights (3, 6, and

9 cm), and the immobility time was recorded (cut-off

time of 20 seconds).

1725N/OFQ LEVELS IN PARKINSON’S DISEASE

Movement Disorders, Vol. 25, No. 11, 2010

Subjects

Experiments were undertaken with the understanding

and written consent of each subject, with the approval

of the local Ethical Committees of the Hospitals of

Ferrara and Mestre, and in compliance with national

registration and the Code of Ethical Principles for

Medical Research Involving Human Subjects of the

World Medical Association (Declaration of Helsinki).

Two groups of patients were enrolled in the study:

patients suffering from idiopathic PD (n 5 20) and

nonparkinsonian neurologic controls (n 5 17). Patients

with idiopathic PD were diagnosed by neurologists at

the two Hospitals involved in the study using the Diag-

nostic Criteria of Gelb et al.25 Unified Parkinson’s dis-

ease rating scale (UPDRS, Part III: Motor section) was

used to quantify parkinsonian disabilities. CSF (usually

�0.5 ml) was obtained from PD patients subjected to

craniotomy (under local anaesthesia) performed to

implant an electrode for deep brain stimulation. All PD

patients have been off-treatment (L-dopa and dopami-

nomimetics) for at least 24 hours before surgery. In

nonparkinsonian neurologic controls, CSF (�0.5 ml)

was obtained by either lumbar puncture (n 5 7) or cra-

nial drainage (n 5 10). Subjects were affected by dif-

ferent pathologies: tumors, brain haemorrhage, normo-

tensive hydrocephalus, head trauma, paraplegy and

amyotrophic lateral sclerosis. Brain CSF was collected

during craniotomy or, in a fewer cases, via ventricular

catheter to reduce intracranial pressure.

Analysis of Endogenous N/OFQ

CSF samples were mixed with an equal volume of

TFA (1% vol:vol) and stored at 2808C until use. Sam-

ples were then lyophilized and processed for RIA as

described above.

Data Presentation and Statistical Analysis

In microdialysis studies, N/OFQ release was

expressed as N/OFQ concentration in a 60-minute dial-

ysate fraction and reported as N/OFQ-like immunore-

activity (N/OFQ-LI fmol/fraction). N/OFQ-like immu-

noreactivity in CSF sample was expressed as femto-

moles per milliliter of analyzed CSF sample. Statistical

analysis was performed by the Student’s t test for

unpaired data (Figs 2 and 4) or by repeated-measure

one-way ANOVA (Fig. 3). If ANOVA yielded a sig-

nificant F score, then post hoc analysis was performed

by contrast analysis to determine group differences. If

a significant time 3 treatment interaction was found,

the sequentially rejective Bonferroni test was used

(implemented on excel spreadsheet) to determine spe-

cific differences (i.e., at the single time-point level)

between groups. P values <0.05 were considered to be

statistically significant.

Materials

Haloperidol was purchased from Tocris Neuramin

(Bristol, United Kingdom), amphetamine and 6-OHDA

bromide from Sigma Chemical Company (St Louis,

MO). All drugs were freshly dissolved in isosmotic sa-

line solution just before use.

RESULTS

In Situ Hybridization

In situ hybridization analysis revealed distribution

patterns of ppN/OFQ mRNA consistent with those

described in other studies.26,27 ppN/OFQ gene expres-

sion was studied 7 weeks after unilateral injection of

vehicle or 6-OHDA. In sham-operated rats, constitutive

ppN/OFQ gene expression was found in all areas

examined (Fig. 1), highest levels being found in motor

cortex whereas lowest in STN. The rank order of

expression was M1 5 M2 > thalamus > GP 5 SNr 5SNc > CPu 5 Ac > STN. No changes were observed

between the injected and noninjected side in vehicle-

injected animals. Conversely, in the lesioned side of 6-

OHDA-treated rats, reduced expression (220%) was

observed in the dorsolateral, central, and ventrolateral

portions of the CPu (Fig. 2A, B) whereas significant

increases (Fig. 2C) were observed in STN (145%),

SNc (1160%) and SNr (1105%).

N/OFQ Levels in the SNr of

Haloperidol-Treated Rats

Haloperidol caused significant increases in immobil-

ity time compared with saline-treated animals. Immo-

bility time was almost maximally elevated (50.4 6 0.6

seconds; Fig. 3A) 1 hour after intraperitoneal adminis-

tration of 0.8 mg/kg haloperidol, then stabilized above

threshold levels (60 seconds). A similar response was

observed following haloperidol 3 mg/kg (Fig. 3A),

with the difference that immobility time was already

maximal after 1 hour. In line with akinesia values, N/

OFQ levels rose in the first hour after haloperidol (0.8

mg/kg; Fig 3B), peaked (147%) after 2 hours, and

then displayed a tendency to decline. A quali-quantita-

tively similar response was observed following halo-

peridol 3 mg/kg (Fig. 3B).

1726 M. MARTI ET AL.

Movement Disorders, Vol. 25, No. 11, 2010

N/OFQ Levels in Human CSF

Nonparkinsonian neurologic controls (n 5 17)

ranged in age between 24 and 86 years (mean 6 SEM,

55.1 6 5.2 years), with no difference between men

FIG. 1. Representative coronal sections (A–E) showing brain levels(AP from bregma) at which ppN/OFQ expression was investigated.AcDC, nucleus accumbens dorsal core; AcDS, nucleus accumbensdorsal shell; AcLC, nucleus accumbens lateral core; AcMC, nucleusaccumbens medial core; AcVC, nucleus accumbens ventral core;AcVS, nucleus accumbens ventral shell; CPu, caudate-putamen;CPuCe, caudate-putamen central; CPuCM, caudate-putamen centro-medial; CPuDL, caudate-putamen dorsolateral; CPuDM, caudate-puta-men dorsomedial; CPuVL, caudate-putamen ventrolateral; CPuVM,caudate-putamen ventromedial; GP, globus pallidus; M1, primarymotor cortex; M2 secondary motor cortex; PO, posterior thalamic nu-clear group, SNc, substantia nigra compacta; SNr, substantia nigrareticulata; STN, subthalamic nucleus; VL, ventrolateral thalamic nu-cleus; VM, ventromedial thalamic nucleus; VPL, ventral posterolateralthalamic nucleus; VPM, ventral posteromedial thalamic nucleus. FIG. 2. Changes of ppN/OFQ expression following 6-OHDA lesion.

Relative optical density values of ppN/OFQ mRNA hybridization toriboprobes 7 weeks after injection of vehicle (sham operated; openbars; n 5 5) or 6-OHDA (hemiparkinsonian; solid bars; n 5 6) inthe right medial forebrain bundle of rats. The results are expressedas mean 6 SEM, percent of control signal (in the noninjected side).*P < 0.05, **P < 0.01, versus sham-operated rats (Student t test forunpaired data).

1727N/OFQ LEVELS IN PARKINSON’S DISEASE

Movement Disorders, Vol. 25, No. 11, 2010

(55.0 6 8.2, n 5 8) and women (54.8 6 6.2; n 5 9;

Table 1). N/OFQ levels in the CSF of control patients

were 41.1 6 5.6 fmol/ml. Higher levels were found in

women (50.1 6 8.0 fmol/ml) than in men (30.9 6 6.3

fmol/ml), but this difference did not reach statistical

significance (P 5 0.084). Samples were obtained from

brain drainage (10 patients) or lumbar puncture (7

patients). N/OFQ levels in brain (33.0 6 4.9 fmol/ml)

and lumbar (52.6 6 10.5 fmol/ml) samples were not

statistically different. PD patients (n 5 20; 11 men and

9 women) ranged in age from 43 to 71 years (mean

60.1 6 1.9 years; Table 1). Male and female subjects

were homogeneous for age (60.5 6 2.5 and 60.6 6 2.6

years, respectively) and age at PD onset (49.2 6 2.3

and 48.2 6 2.3 years, respectively), both being

affected by PD by more than 11 years (Table 1). They

had advanced and severe PD according to both the

UPDRS III (46.1 6 4.1) and the Hoehn and Yahr

(2.9 6 0.1) scales, with no difference between men

(47.5 6 4.6 and 2.9 6 0.1, respectively) and women

(45.0 6 4.5 and 2.8 6 0.1, respectively). N/OFQ lev-

els were about 3.5-fold higher (148.5 6 14.6 fmol/ml)

in the CSF of PD patients than controls (41.1 6 5.6

fmol/ml), with no significant gender difference (Table

1 and Fig. 4).

DISCUSSION

6-OHDA lesioning has been previously reported to

stimulate ppN/OFQ expression in SN.4,10,27 This study

FIG. 3. Haloperidol simultaneously elevated extracellular N/OFQ levels and akinesia in naive rats. Microdialysis coupled to the bar test wasused to simultaneously monitor immobility time (in seconds; A) and extracellular N/OFQ levels in substantia nigra reticulata (SNr; B) of naiverats following administration of haloperidol (0.8 and 3 mg/kg, intraperitoneal) or saline. Behavioral testing and sample collection was performedevery hour. Data (mean 6 SEM of 9 determinations) are expressed as seconds of immobility (A) and N/OFQ-like immunoreactivity (N/OFQ-LI)in fmol/fraction (B). **P < 0.05, versus saline. ##P < 0.05 versus haloperidol 3 mg/kg (repeated-measure ANOVA followed by contrast analysisand the sequentially rejective Bonferroni’s test).

TABLE 1. Clinical features and N/OFQ levels in the cerebrospinal fluid of subjects enrolled in the study

Nonparkinsonian neurologic controls Parkinson’s disease

Total Male Female Total Male Female

N 17 8 9 20 11 9Age (yr) 55.1 6 5.2 55.0 6 8.2 54.8 6 6.2 60.6 6 1.8 60.5 6 2.5 60.7 6 2.6Age at onset (yr) – – – 48.7 6 1.6 49.2 6 2.3 48.2 6 2.3UPDRS III – – – 46.1 6 4.1 47.5 6 4.6 45.0 6 4.5Hoehn & Yahr stage – – – 2.9 6 0.1 2.9 6 0.1 2.8 6 0.1Disease duration (yr) – – – 11.8 6 1.1 11.3 6 1.4 12.5 6 1.6N/OFQ (fmol/ml) 41.1 6 5.6 30.9 6 6.3 55.1 6 8.2 148.5 6 14.6 158.1 6 23.5 136.7 6 15.8

Data are expressed as mean 6 SEM.

1728 M. MARTI ET AL.

Movement Disorders, Vol. 25, No. 11, 2010

reveals that 6-OHDA also promotes ppN/OFQ expres-

sion in STN, other areas being unaffected (M1, M2,

GP, Ac, and thalamus) or showing a slight decrease

(CPu). Therefore, considering that SNc DA neurons in-

nervate CPu, GP, and STN and arborize extensively in

SNr, elevation of ppN/OFQ expression is not a gener-

alized consequence of DA loss. The phenotype of ppN/

OFQ expressing cells has been investigated only in

mesencephalic DA areas. In SNc, a large number

(�50%) of GAD1 neurons but only a minimal portion

(�10%) of TH1 neurons express ppN/OFQ, suggesting

that GABA interneurons but not DA neurons synthe-

size and release N/OFQ.27 These neurons are under

GABAergic control and do not respond to DA.28,29

Thus, increased expression following 6-OHDA lesion-

ing may be due to a reduction of GABAergic inputs

from the striatonigral GABAergic pathway, which

becomes hypoactive in PD,30 and/or to an increased

excitatory glutamatergic drive from STN, which

becomes overactive after 6-OHDA.31

Increase of ppN/OFQ expression in STN may also

underlie the rise in SNr N/OFQ levels observed follow-

ing 6-OHDA.4 Indeed, despite the STN contains light

N/OFQ immunoreactivity, its target areas are highly

immunoreactive,26 overall suggesting that N/OFQ may

be released from glutamatergic nerve terminals arising

from STN. Although there is no evidence for N/OFQ

being co-released with glutamate in CNS, this hypothe-

sis cannot be ruled out since an opioid peptide, dynor-

phin (the endogenous ligand of kappa opioid recep-

tors), is released from hippocampal glutamatergic ter-

minals.32 This possibility is further corroborated by the

finding that haloperidol-evoked nigral N/OFQ levels in

parallel with akinesia. Indeed, haloperidol promoted

subthalamic activity33 and glutamate release in SNr,3,5

as a consequence of blockade of D2 receptors in stria-

tum and, possibly, extrastriatal areas.24 N/OFQ levels

measured 7 weeks after 6-OHDA4 were about three

times higher than those evoked by acute haloperidol.

This may be explained on the basis of a greater subtha-

lamic activation under parkinsonian conditions and/or

contribution of additional sources of N/OFQ release

during long-term plasticity associated with DA deple-

tion. In particular, N/OFQ levels may also derive from

astrocytes, which synthesize N/OFQ34 and are acti-

vated following 6-OHDA.35 The selective activation of

ppN/OFQ expression along the STN-SN axis and the

elevation of SNr N/OFQ levels observed following 6-

OHDA or haloperidol strengthen the role of nigral en-

dogenous N/OFQ in the modulation of parkinsonian

hypokinesia. Previous studies have demonstrated that

endogenous N/OFQ inhibits motor activity via overin-

hibition of nigrothalamic projection neurons. Indeed,

the antiakinetic effect evoked by local administration

of NOP receptor antagonists in SNr was associated

with reduction of glutamate and increase of GABA

release in SNr, as well as reduction of GABA release

in ventromedial thalamus.7 Consistent with pharmaco-

logic studies, NOP receptor knockout mice were resist-

ant to the akinesiogenic action of haloperidol,4 a phe-

nomenon associated with the loss of haloperidol ability

to elevate glutamate and reduce GABA release in

SNr.5

Few studies have attempted to correlate human neu-

rologic diseases with changes in N/OFQ transmission

and N/OFQ levels in plasma or CSF.2 Lower ppN/

OFQ and NOP expression was found in the brain of

alcoholics13 while lower N/OFQ binding was observed

in the brain of epileptic patients14 (see Introduction).

N/OFQ plasma levels were also found elevated in Wil-

son’s disease,15 a disorder of copper metabolism asso-

ciated with basal ganglia degeneration.16 Therefore,

the demonstration that N/OFQ levels were about 3.5-

fold higher in the CSF of PD patients than awake con-

trols represents the first clinical evidence linking N/

OFQ to idiopathic PD in humans. To the best of our

FIG. 4. Cerebrospinal fluid (CSF) levels of N/OFQ are elevated inParkinson’s disease (PD). CSF samples were obtained from patientswith idiopathic PD (n 5 20) during surgical procedures to implantelectrodes for deep brain stimulation. As a control, N/OFQ levelswere measured in the CSF of patients (n 5 17) suffering fromtumors, brain haemorrhage, normotensive hydrocephalus, headtrauma, paraplegy, and amyotrophic lateral sclerosis. Data (means 6SEM) are expressed as N/OFQ-like immunoreactivity (N/OFQ-LI) infmol/ml. **P < 0.01, versus control (Student t test for unpaireddata).

1729N/OFQ LEVELS IN PARKINSON’S DISEASE

Movement Disorders, Vol. 25, No. 11, 2010

knowledge, it is also the first measure of N/OFQ lev-

els in the CSF obtained at the brain level. N/OFQ lev-

els in lumbar samples of nonparkinsonian neurologic

controls (52.6 fmol) were similar to those reported in

a previous study (44.7 fmol)36 and in the same order

of magnitude of those measured in brain samples

(33.0 fmol). However, cranial and lumbar sampling

was never performed in the same subject, making it

difficult to unequivocally prove that N/OFQ levels in

CSF do not have a rostrocaudal gradient as consis-

tently demonstrated for monoamines and their metabo-

lites.37–39 Nevertheless, the lack of difference in N/

OFQ levels between brain and lumbar samples sug-

gests that this is not the case, in line with what has

been observed for opioids (b-lipotropin, b-endor-phin,40 and other neuropeptides such as NPY,41 sub-

stance P42 or ACTH).40 However, the possibility that

lumbar CSF samples are enriched with N/OFQ derived

from spinal cord neurons should be considered due to

the high density of N/OFQ immunoreactivity in this

area.26

Human CSF is produced by choroid plexus and is

in equilibrium with brain interstitial fluid, reflecting

its composition.43 Thus, N/OFQ levels in CSF reflect

neuronal activity/metabolism. ppN/OFQ expression in

the human and rodent brains substantially over-

laps,13,26,44 although, possibly relevant to this study,

highest ppN/OFQ mRNA levels were found in human

STN.44 N/OFQ content was also measured in a num-

ber of brain structures,45 among which the SN (�7.9

fmol/mg tissue), CPu (�4.6 fmol/mg tissue), and GP

(�2.2 fmol/mg tissue). Therefore, enhanced N/OFQ

levels in the CSF of PD patients may be consistent

with increased N/OFQ expression and release in the

basal ganglia complex, as predicted on the basis of

studies in rodent models of parkinsonism. On a quan-

titative basis, the observed increase in N/OFQ levels

(3.5-folds) was quite remarkable, also considering the

CSF volume (�160 ml) and formation rate (0.40 ml/

minutes, leading to complete renewal every 6

hours).46 Although a reduction in CSF turnover occur-

ring with age46 may amplify such difference, the

degree of such increase may further emphasize the

pathologic relevance of this phenomenon. The influ-

ence of N/OFQ in motor control in humans is still

unknown. Studies with NOP receptor antagonists in

naive and parkinsonian nonhuman primates8 have pro-

vided preliminary evidence that endogenous N/OFQ

may act as a physiologic constraint of movement, as

shown in rodents.12,47,48 On this basis, an increase in

N/OFQ levels in human CSF may be considered as

pathogenic, leading to a worsening of motor deficit

and, possibly, also contributing to neurodegeneration

associated with PD.4,11

CONCLUSIONS

Enhancement of ppN/OFQ expression has been

observed in the STN and SN of 6-OHDA-hemilesioned

rats. Moreover, elevation of SNr extracellular N/OFQ

levels was observed following administration of akine-

siogenic doses of haloperidol (a functional model of

parkinsonism) in line with previous observations made

in 6-OHDA-hemilesioned rats4 (a neurodegeneration

model of parkinsonism). The finding that higher N/

OFQ levels were found in the CSF of PD patients

nicely complemented these preclinical data, strengthen-

ing the notion that elevated N/OFQ levels are a feature

of both experimental parkinsonism and idiopathic PD.

Further studies will be undertaken to evaluate the role

of subthalamonigral pathways in generating nigral N/

OFQ levels and, particularly, whether N/OFQ levels

will be modified by deep brain stimulation of STN.

Although the role of N/OFQ in movement control in

humans remains unknown, the striking correlation

between N/OFQ dynamics in PD patients and parkinso-

nian rodents strengthens the idea that endogenous N/

OFQ plays a pathogenic role in parkinsonism across

species and provides a strong rationale for developing

NOP receptor antagonists for PD therapy.4,6–8

Acknowledgments: This work has been supported bygrants from the Italian Ministry of the University (FIRBInternazionalizzazione no RBIN047W33) to M Morari.

Full financial disclosure: Grants, Employment.

Author Roles: 1. Research project: A. Conception, B. Or-ganization, C. Execution; 2. Statistical Analysis: A. Design,B. Execution, C. Review and Critique; 3. Manuscript: A.Writing of the first draft, B. Review and Critique; MatteoMarti: 1A, 1B, 1C, 2B, 2B, 3B; Silvio Sarubbo: 1B, 1C, 3B;Francesco Latini: 1B, 1C; Michele Cavallo: 1A, 1B, 1C, 3B;Roberto Eleopra: 1A, 1B, 1C, 3B; Sara Biguzzi: 1B, 1C;Christian Lettieri: 1B, 1C; Carlo Conti: 1B, 1C; MicheleSimonato: 1A, 1B, 1C, 3B; Silvia Zucchini: 1A, 1B, 1C, 3B;Rocco Quatrale: 1B, 1C; Mariachiara Sensi: 1B, 1C; SanzioCandeletti: 1A, 1B, 1C, 3B; Patrizia Romualdi: 1A, 1B, 1C,3B; Michele Morari: 1A, 1B, 2A, 2B, 2C, 3A, 3B.

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