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Neuroscience 164 (2009) 360–369

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HE NOVEL DELTA OPIOID RECEPTOR AGONIST UFP-512 DUALLYODULATES MOTOR ACTIVITY IN HEMIPARKINSONIAN RATS VIA

ONTROL OF THE NIGRO-THALAMIC PATHWAY

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. S. MABROUK,a,b M. MARTI,a,b S. SALVADORIc AND

. MORARIa,b*

Department of Experimental and Clinical Medicine, Section of Pharma-ology, University of Ferrara, via Fossato di Mortara 17-19, 44100 Fer-ara, Italy

National Institute of Neuroscience and Neuroscience Center, Universityf Ferrara, via Fossato di Mortara 17-19, 44100 Ferrara, Italy

Department of Pharmaceutical Sciences and Biotechnology Center, Uni-ersity of Ferrara, via Fossato di Mortara 17-19, 44100 Ferrara, Italy

bstract—The present study aimed to characterize the abilityf the novel delta opioid peptide (DOP) receptor agonist H-Dmt-ic-NH-CH(CH2–COOH)-Bid (UFP-512) to attenuate motor defi-its in 6-hydroxydopamine (6-OHDA) hemilesioned rats. Loweroses (0.1–10 �g/kg) of UFP-512 administered systemicallyi.p.) stimulated stepping activity in the drag test and overallait abilities in the rotarod test whereas higher doses (100–000 �g/kg) were ineffective or even worsened Parkinsonism.icrodialysis coupled to an akinesia test (bar test) was thensed to determine the circuitry involved in the motor actionsf UFP-512. An antiakinetic dose of UFP-512 (10 �g/kg) de-reased GABA in globus pallidus (GP) as well as GABA andlutamate (GLU) release in substantia nigra reticulata (SNr).n the other hand, a pro-akinetic dose (1000 �g/kg) of UFP-12 increased pallidal GABA, simultaneously producing a de-rease in GABA and an increase in nigral GLU release. More-ver, to test the hypothesis that changes in motor behaviorere associated with changes in nigro–thalamic transmission,mino acid release in ventromedial thalamus (VMTh, a target ofigro–thalamic GABAergic projections) was also measured.he anti-akinetic dose of UFP-512 reduced GABA and increased

halamic GLU release while the pro-akinetic dose increasedABA without affecting thalamic GLU release. Finally, regionalicroinjections were performed to investigate the brain areas

nvolved in motor actions of UFP-512. UFP-512 microinjectionsnto GP increased akinesia whereas UFP-512 microinjectionsnto SNr reduced akinesia. Furthermore, the selective DOP re-eptor antagonist naltrindole (NTD) increased akinesia whennjected into either area, GP being more sensitive. We concludehat UFP-512, depending on dose, improves or worsens motorctivity in hemiparkinsonian rats by acting differentially as aOP receptor agonist in SNr and a DOP receptor antagonist inP, ultimately decreasing or increasing the activity of nigro–

halamic GABAergc output neurons, respectively. © 2009 IBRO.ublished by Elsevier Ltd. All rights reserved.

Corresponding author. Tel: �39-0532-455210; fax: �39-0532-455205.-mail address: m.morari@unife.it (M. Morari).bbreviations: ANOVA, analysis of variance; AP, antero–posterior;A, dopamine; DOP, delta opioid peptide; DV, dorso–ventral; ENK,nkephalins; GLU, glutamate; GP, globus pallidus; ML, medio–lateral;TD, naltrindole; PD, Parkinson’s disease; RM, repeated measure;NC-80, (�)-4-[(�R)-�-(2S,5R)-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-enzyl]-N–N-diethylbenzamide; SNr, substantia nigra reticulata; UFP-512,

s-Dmt-tic-NH-CH(CH2–COOH)-Bid; VMTh, ventromedial thalamus;-OHDA, 6-hydroxydopamine.

306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rightoi:10.1016/j.neuroscience.2009.08.058

360

ey words: delta opioid, microdialysis, naltrindole, 6-OHDA,arkinson’s disease, UFP-512.

pioid transmitter systems have long been studied for theirnvolvement in a number of physiological functions rangingrom nociception to mood and movement. The localization ofpioid receptors and their endogenous ligands within theasal ganglia, a set of midbrain structures critical to motorrogramming and execution, has also led investigators totudy their contribution to neurodegenerative diseases suchs Parkinson’s disease (PD; for a review see Samadi et al.,006). Previous studies were encouraged by the finding that

oss of dopamine (DA) cells in the substantia nigra (SN)ompacta (SNc), a hallmark of PD, results in alterations ofpioid signaling throughout the basal ganglia (Rinne et al.,983; Llorens-Cortes et al., 1984; Gerfen et al., 1990). Forxample, the pathogenic rise in striato–pallidal GABA releasehich follows the loss of striatal D2 receptor stimulation (Ma-euf et al., 1994; Stanford and Cooper, 1999) is compen-ated for by an upregulation of striatal preproenkephalin-ARNA (Bezard et al., 2001) which is presumed to causexcessive release in globus pallidus (GP). Indeed, delta opi-id peptide (DOP) receptor stimulation was shown to reduceallidal GABA release in vitro (Dewar et al., 1987; Maneuf etl., 1994) as well as in vivo (Mabrouk et al., 2008). To confirm

he compensatory nature of such changes in DOP receptorransmission, the selective nonpeptide DOP receptor agonist�)-4-[(�R)-�-(2S,5R)-allyl-2,5-dimethyl-1-piperazinyl)-3-me-hoxy-benzyl]-N–N-diethylbenzamide (SNC-80; Bilsky et al.,995) caused pronounced reversal of motor deficits in-methyl-4-phenyl-1,2,5,6-tetrahydropyridine treated marmo-ets and reserpinized or 6-hydroxydopamine (6-OHDA)emilesioned (hemiparkinsonian) rats (Manueuf et al., 1994;inna and Di Chiara, 1998; Hill et al., 2000; Hille et al.,001). We recently confirmed these findings in hemipar-insonian rats (Mabrouk et al., 2008), showing that, inontrast with the commonly accepted belief, the site of thentiparkinsonian action of SNC-80 was the SN reticulataSNr) rather than GP. Indeed, the antiakinetic effect and theccompanied reduction in pallidal GABA release elicitedy SNC-80 were prevented by intranigral but not intrapal-

idal perfusion of the DOP receptor antagonist naltrindoleNTD). Moreover, injections of SNC-80 into SNr but not GPeplicated the antiparkinsonian effects of systemic SNC-0. These findings prompted us to further evaluate theircuitry underlying the antiparkinsonian effect of DOP re-eptor agonists. In particular, the impact of DOP receptor

timulation on nigro–thalamic transmission was investi-

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O. S. Mabrouk et al. / Neuroscience 164 (2009) 360–369 361

ated since in previous studies we reported that the anti-kinetic effect of nociceptin/orphanin FQ opioid peptideeceptor antagonists was associated with a reduction ofABA release in the ventromedial thalamus (VMTh; Marti etl., 2007, 2008), a main target of nigro–thalamic GABAergicrojections (for review see Parent and Hazrati, 1995). In theresent study, we used a novel DOP receptor agonist,-Dmt-Tic-NH-CH(CH2–COOH)-Bid (UFP-512), a systemi-ally active pseudopeptide which displays high affinity for theuman recombinant DOP receptor as well as high selec-ivity over mu opioid peptide (MOP) and kappa opioideptide (KOP) receptors (Vergura et al., 2007). We exam-

ned the effects of systemic UFP-512 on motor deficits in-OHDA hemilesioned rats using previously validated be-avioral tests: the drag and rotarod tests (Marti et al., 2005,007). These experiments revealed that UFP-512 couldroduce motor facilitation and inhibition depending on theose (lower ones causing facilitation and higher onesausing inhibition). Additionally, to investigate the circuitrynderlying the opposite actions of UFP-512 on motor ac-ivity, we investigated the effects of low and high UFP-512oses on glutamate (GLU) and GABA release in GP, SNrnd VMTh with in vivo microdialysis combined with a testor akinesia (bar test). Indeed an opposite regulation ofkinesia was observed depending on dose which corre-

ated with an opposite regulation of basal ganglia output inMTh. To clarify these effects, we microinjected UFP-512 asell as the selective DOP receptor antagonist NTD into bothP and SNr, to determine the origin of these effects. Overall,

he findings described here offer additional evidence for theifferential role of DOP receptors located in GP and SNr inotor control under parkinsonian conditions, and verify a

omplex mechanism of action for the novel pseudopeptideFP-512, possibly resembling that of a partial agonist

EXPERIMENTAL PROCEDURES

ale Sprague–Dawley rats (150 g; Harlan, Italy; S. Pietro al Nati-one, Italy) were kept under regular lighting conditions (12 h light/ark cycles) and given food and water ad libitum. The experimentalrotocols performed in the present study were approved by thethical Committee of the University of Ferrara and adequate mea-ures were taken to minimize animal pain and discomfort as well asumber of animals used for these studies.

nilateral lesion with 6-OHDA

nilateral lesion of dopaminergic neurons was induced in isoflu-ane-anesthetized male rats as previously described (Marti et al.,007). Eight micrograms of 6-OHDA (dissolved in 4 �l of salineontaining 0.02% ascorbic acid) were stereotaxically injected intohe medial forebrain bundle according to the following coordinatesrom bregma: antero–posterior (AP) �4.4 mm, medio–lateral (ML)1.2 mm, dorso–ventral (DV) �7.8 mm below dura (Paxinos andatson, 1982). In order to select the rats which had been suc-

essfully lesioned, the rotational model was employed (Unger-tedt and Arbuthnott, 1970). Two weeks after 6-OHDA injection,enervation was evaluated with a test dose of amphetamine (5g/kg i.p., dissolved in saline just before use). Rats showing a

urning behavior �7 turns/min in the direction ipsilateral to theesion were enrolled in the study. This behavior has been associ-ted with �95% loss of striatal DA terminals (Marti et al., 2007)nd extracellular DA levels (Marti et al., 2002). Experiments were

erformed approximately 6–8 weeks after lesion. n

ehavioral studies

ifferent behavioral tests were used to evaluate different motorunctions as previously described (Marti et al., 2005): (1) the dragest (modification of the “wheelbarrow test”; Schallert et al., 1979)easures rat ability to balance body posture using the forelimbs in

esponse to an externally imposed dynamic stimulus (i.e. back-ard dragging); (2) the fixed-speed rotarod test (Rozas et al.,997) measures overall motor performance as an integration ofoordination, gait, balance, muscle tone and motivation to run.

The drag and rotarod tests were repeated in a fixed sequencedrag then rotarod) before (control) then 20 and 70 min after drugnjection. Rats were trained for approximately 10 days to the specific

otor tasks until their motor performance became reproducible.

Drag test. Rats were lifted from the tail (allowing the forepawso rest on the table) and dragged backwards at a constant speed�20 cm/s) for a fixed distance (100 cm). The number of steps madey each forepaw was counted by two separate observers.

Rotarod test. The fixed-speed rotarod test was employedsing an established protocol (Marti et al., 2004). Rats wererained for 10 days to a complete motor task on the rotarod (i.e.rom 5 to 55 rpm; 180 s each) until their motor performanceecame reproducible in three consecutive sessions. Rats werehen tested at four increasing speeds (usually 10, 15, 20, and 25pm), causing a progressive decrement of performance to about0% of the maximal response (i.e. the experimental cut-off time).

eurochemicals and behavioral studies

Microdialysis. Two probes of concentric design were ster-otaxically implanted, under isoflurane anesthesia, in SNr (1 mmialysing membrane) and GP (1 mm dialysing membrane, AN69,ospal, Bologna, Italy) or VMTh (1 mm dialysing membrane) all

psilateral to the nigro–striatal lesion according to the followingoordinates from bregma: GP; AP �1.3, ML �3.3, DV �6.5, SNr;P �5.5, ML �2.2, DV �8.3, VMTh; AP �2.3, ML �1.4, DV �7.4.robes were secured to the skull by acrylic dental cement andetallic screws. After surgery, rats were allowed to recover andxperiments were run 24 h after probe implantation. Microdialysisrobes were perfused at a flow rate of 3.0 �l/min with a modifiedinger’s solution (composition in mM: CaCl2 1.2; KCl 2.7, NaCl48 and MgCl2 0.85). Samples were collected every 15 min,tarting 6 h after the onset of probe perfusion. At least four stablealues were obtained before administering the treatments via an.p. injection. At the end of each experiment the placement of therobes was verified by microscopic examination.

Endogenous GLU and GABA analysis. GLU and GABAere measured by high performance liquid chromatography (HPLC)oupled with fluorometric detection as previously described (Marti etl., 2007). Thirty microliters of o-phthaldialdehyde/mercaptoethanoleagent were added to 40 �l aliquots of sample, and 60 �l of theixture was automatically injected (Triathlon autosampler; Sparkolland, Emmen, Netherlands) onto a 5-C18 Chromsep analyticalolumn (3 mm inner diameter, 10 cm length; Chrompack, Middel-urg, Netherlands) flowing at 0.48 ml/min (Beckman 125 pump;eckman Instruments, Fullerton, CA, USA) with a mobile phaseontaining 0.1 M sodium acetate, 10% methanol and 2.2% tetrahy-rofuran (pH 6.5). GLU and GABA were detected by means of auorescence spectrophotometer FP-2020 Plus (Jasco, Tokyo Ja-an) with the excitation and the emission wavelengths set at 370 and50 nm respectively. The limits of detection for GLU and GABA werebout 1 and 0.5 nM, respectively. Retention times for GLU andABA were 3.5�0.2 and 18.0�0.5 min, respectively.

Bar test. The bar test was coupled to microdialysis in ordero characterize the time course of drug effect with respect to

eurochemical changes. This test (Kuschinski and Hornykiewicz,

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O. S. Mabrouk et al. / Neuroscience 164 (2009) 360–369362

972; for a review see Sanberg et al., 1988) measures the ratbility to respond to an imposed static posture. Each forepaw waslaced alternatively on blocks of increasing heights (3, 6 and 9 cm)hile undergoing microdialysis. Amount of time spent on the baras measured every 15 min for 45 min prior to drug and 90 minfter drug administration. Total time (in sec) spent by each paw onhe blocks was recorded (cut-off time 20 s for each block height).

Microinjection. Two microinjection cannulae (outer diame-er 0.55 mm, inner diameter 0.35 mm) were stereotaxically im-lanted under isoflurane anesthesia above SNr and GP ipsilateralo the nigro–striatal lesion according to the following coordinatesrom bregma: SNr; AP �5.5, ML �2.2, DV �7.8, GP; AP �1.3, ML

3.3, DV �6.0. Cannulae were secured to the skull by acrylicental cement and metallic screws. After surgery, rats were al-

owed to recover and experiments were run 72 h after cannulaemplantation. Drugs were prepared and injected in 0.5 �l volumef saline. Rats were then tested according to the same bar testrotocol as used previously (see above).

Data presentation and statistical analysis. Motor perfor-ance has been calculated as number of steps (drag test) or time

pent on rotarod (in sec) and expressed as percent of the controlession. For the bar test, time spent on bar is expressed as percentf the average of the two basal values before treatment. In microdi-lysis studies, GABA and GLU release has been expressed asercentage�SEM (standard error of the mean) of basal values (cal-ulated as mean of the two samples before the treatment). In text andgure legends, amino acid dialysate levels were also given in abso-ute values (in nM).

Statistical analysis has been performed on percent data byne-way repeated measure (RM) analysis of variance (ANOVA).n the case ANOVA yielded a significant F-score, post hoc anal-sis was performed by contrast analysis to determine group dif-erences. In case a significant time�treatment interaction wasound, the sequentially rejective Bonferroni’s test was used (im-lemented on excel spreadsheet) to determine specific differ-nces (i.e. at the single time-point level) between groups. P-alues �0.05 were considered to be statistically significant.

Materials. 6-OHDA hydrobromide and d-amphetamine sul-te were purchased from Sigma (St. Louis, MO, USA) while NTDydrochloride was from Tocris (Bristol, UK). UFP-512 was syn-

hesized at the Department of Pharmaceutical Chemistry of theniversity of Ferrara. All drugs were readily dissolved in saline.

RESULTS

ffects of systemic administration of UFP-512 onotor behavior (drag and rotarod tests)

o investigate whether the pseudopeptide DOP receptorgonist UFP-512 could attenuate motor deficits in 6-OHDAemilesioned rats, the drug was administered systemicallyi.p.) over a wide range of doses (0.1–1000 �g/kg) andotor performance evaluated in the drag and rotarod tests.

As previously reported (Marti et al., 2005, 2007), unilat-ral 6-OHDA lesioning produced hypoactivity which mainlyffected the contralateral (parkinsonian) paw and led to motorsymmetry. Indeed in the drag test, the number of stepsade by the contralateral paw (2.0�0.1; n�44) was reduced

ompared to the ipsilateral one (10.0�0.4; n�44) and motorerformance of the rotarod (499�25 sec in the 5–55 rpmange; n�81) was reduced compared to sham-operated an-mals (1176�64 sec; taken from Marti et al., 2008). RMNOVA on the number of steps at the contralateral paw in

he drag test (Fig. 1A) showed an overall effect of treatment l

F5,35�12.28, P�0.0001) and time (F1,32�5.03, P�0.031)ut not a significant time�treatment interaction (F5,32�1.37,�0.26). Post hoc analysis at 20 min revealed that UFP-512

ncreased the number of steps at 1 and 10 �g/kg (�81% and131%, respectively). This effect was not detected 70 min

ater and no effect was observed with higher doses (i.e. 100r 1000 �g/kg) at either time point. No effect was seen at the

psilateral paw. RM ANOVA on rotarod values (Fig. 1B)howed a significant effect of treatment (F5,30�10.81,�0.0001), but not time (F1,36�1.62, P�0.21), and a signif-

cant time�treatment interaction (F5,36�3.30, P�0.014).ost hoc analysis at 20 min revealed that UFP-512 increasedotor performance at 1 �g/kg (�63%; P�0.001) and inhib-

ted it at 100 �g/kg (�36%; P�0.05). No effect was observedt 70 min following treatment (Fig. 1B).

ffect of systemic administration of UFP-512 oneurotransmitter release and akinesia (bar test)

icrodialysis was used to investigate the circuitry under-

ig. 1. UFP-512 modulated motor activity in 6-OHDA hemilesioned rats.ffect of systemic administration of the pseudopeptide DOP receptorgonist UFP-512 (0.1–1000 �g/kg i.p.) in the drag (A) and rotarod (B)

ests. Each experiment consisted of three different sessions: a controlession followed by two sessions performed 20 and 70 min after vehicler UFP-512 administration (see Experimental Procedures). Data are ex-ressed as percentages of basal motor activity in the control session andre means�SEM of seven determinations per group. Basal motor activityalues in the drag test were (number of steps): 10.0�0.4 (ipsilateral paw)nd 2.0�0.1 (contralateral paw). The time spent on the rod (rotarod test)as 499�25 s (0–55 rpm range). * P�0.05, significantly different fromehicle (RM ANOVA followed by contrast analysis and the sequentiallyejective Bonferroni’s test).

ying motor responses to UFP-512. We selected a lower

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O. S. Mabrouk et al. / Neuroscience 164 (2009) 360–369 363

ose (10 �g/kg) to study the pathways involved in motoracilitation, and a 100-fold higher one (1000 �g/kg) to verifyhether lack of response was associated with receptoresensitization or recruitment of pro-akinetic pathways.he effect of UFP-512 10 and 1000 �g/kg on GLU andABA release in GP and SNr was then measured. Addi-

ionally, we measured amino acid release in VMTh and, inhe same animals, correlated neurochemical changes withhanges of immobility time, as a measure of akinesia (barest).

lobus pallidus

xtracellular GABA levels in GP were 3.4�0.4 nM (n�38).M ANOVA revealed a significant effect of treatment

F2,10�61.84, P�0.0001) but not time (F7,105�0.82, P�.56), and a significant time�treatment interaction (F14,105�.85, P�0.0001). Post hoc analysis revealed that UFP-512 10g/kg caused a long-lasting reduction of GABA levels whichas significant from 45 min after injection onward (maxi-al �35% reductions; Fig. 2A). Conversely, UFP-512000 �g/kg transiently increased GABA levels (maximal

ig. 2. UFP-512 dually modulated GABA release in the GP of 6-OHDAFP-512 (1000 �g/kg i.p.) increased it (A). UFP-512 did not affect GLeven (B) experiments per group and are expressed as percent baseABA levels in the dialysate were 3.4�0.4 nM (A) while basal GLUNOVA followed by contrast analysis and the sequentially rejective B

ig. 3. UFP-512 reduced GABA and dually modulated GLU releaseFP-512 10 and 1000 �g/kg both reduced GABA release in SNr (A). H

1000 �g/kg i.p.) increased it (B). Data are means�SEM of six experi

s the mean of the two samples before the treatment). Basal GABA levels in theM (B). * P�0.05, significantly different from vehicle (RM ANOVA followed by

45%), the effect being significant starting at 30 min afternjection. Extracellular GLU levels in the GP were 62�9.4M (n�46) and were not affected by UFP-512 at eitherose tested (Fig. 2B).

ubstantia nigra reticulata

xtracellular GABA levels in SNr were 4.7�0.46 nM (n�36).M ANOVA revealed a significant effect of treatment

F2,10�7.17, P�0.012), time (F7,105�4.88, P�0.0001), and aignificant time�treatment interaction (F14,105�2.43, P�.005). Post hoc analysis revealed that UFP-512 10 �g/kgaused a rapid and prolonged decrease in GABA levelsmaximal of �34%) starting 15 min after drug injectionFig. 3A). Also, UFP-512 1000 �g/kg caused a long-lastingecrease (maximal �30% reduction) in GABA levels whichas somewhat delayed (significant from 45 min). Extracellu-

ar GLU levels in SNr were 70�6.2 nM (n�44). RM ANOVAevealed a significant effect of treatment (F2,10�15.69, P�.0008), but not time (F7,105�0.35, P�0.93), and a sign-

ficant time�treatment interaction (F14,105�2.76, P�.0016). Post hoc analysis revealed that UFP-512 10 �g/kg

oned rats. UFP-512 (10 �g/kg i.p.) reduced GABA release in GP whilee in GP at either dose tested (B). Data are means�SEM of six (A) orulated as the mean of the two samples before the treatment). Basalre 62�9.4 nM (B). * P�0.05, significantly different from vehicle (RMi’s test).

ubstantia nigra pars reticulata (SNr) of 6-OHDA hemilesioned rats.UFP-512 (10 �g/kg i.p.) reduced GLU release in SNr while UFP-512r group (A and B) and are expressed as percent baseline (calculated

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aused a transient decrease in GLU levels (maximal �41%)t 30 and 45 min whereas UFP-512 1000 �g/kg caused aransient increase (maximal �43%) at 45 after drug injectionFig. 3B).

entromedial thalamus

xtracellular GABA levels in the VMTh were 3.6�0.14 nMn�30). RM ANOVA revealed a significant effect of treat-ent (F2,8�76.42, P�0.0001) but not time (F7,100�0.64,�0.721), and a significant time�treatment interaction

F14,100�3.8, P�0.0001). Post hoc analysis revealed thatFP-512 10 �g/kg caused a decrease in GABA levels

maximal �19% reduction) at 30 and 45 min whereas UFP000 �g/kg increased GABA release (maximal �27% in-rease) at 30 and 45 min after drug injection (Fig. 4A).xtracellular GLU levels in the VMTh were 54.0�4.4 nM

n�30). RM ANOVA revealed a significant effect of treat-ent (F2,8�8.54, P�0.01) but not time (F7,92�1.94,�0.07) and a significant time�treatment interaction

F14,92�2.83, P�0.001). Post hoc analysis revealed thatFP-512 10 �g/kg increased GLU levels (maximal �49%)t 30 min whereas UFP 1000 �g/kg was ineffective, al-hough a trend to decrease was observed (Fig. 4B).

ar test

hen measuring akinesia in the bar test, motor asymmetryecame evident: the immobility time at the ipsilateral paw11.4�1.4 s; n�43) was �60% less than that recorded athe contralateral paw (27.5�1.8 s; n�43).

RM ANOVA on immobility time at the contralateral paw (Fig.A) showed a significant effect of treatment (F2,8�27.52,�0.0003) but not time (F7,92�1.25, P�0.283), and a signif-

cant time�treatment interaction (F14,92�5.03, P�0.0001).ost hoc analysis revealed that UFP-512 10 �g/kgaused a transient attenuation of akinesia (�39% reduc-ion with respect to basal values) 30 and 45 min afternjection while UFP-512 1000 �g/kg inversely caused anncrease (�39%) in immobility time also at 30 and 45 min

ig. 4. UFP-512 dually modulated GABA and differentially affected Geduced GABA release in VMTh while UFP-512 (1000 �g/kg i.p.) incFP-512 (1000 �g/kg i.p.) was without effect (B). Data are means�Saseline (calculated as the mean of the two samples before the treatme

evels were 54�4.4 nM (B). * P�0.05, significantly different from vehonferroni’s test).

ost injection. Similar to the contralateral paw, RM ANOVA c

n immobility time at the ipsilateral paw (Fig. 5B) showed aignificant effect of treatment (F2,8�7.46, P�0.015) but notime (F7,92�1.15, P�0.34), and a significant time�treatmentnteraction (F14,92�2.00, P�0.026). Post hoc analysis re-ealed that UFP-512 10 �g/kg caused a mild and transientttenuation of akinesia (�26%) 45 min after injection whileFP-512 1000 �g/kg inversely caused an increase (�71%)hich was significant 15 min after injection (Fig. 5B).

ffects of regional microinjections of UFP-512

o investigate the brain areas involved in motor actions ofFP-512, different doses of the pseudopeptide were mi-roinjected into GP or SNr. Subjects implanted with micro-

njection cannulae displayed similar motor asymmetryompared to animals undergoing microdialysis: immobilityime was 11.0�1.1 s (n�38) at the ipsilateral paw and3.2�1.5 s (n�38) at the contralateral one.

For the effect of UFP-512 microinjected into GP, RMNOVA on immobility time at the contralateral paw (Fig. 6A)howed a significant effect of treatment (F2,8�11.71, P�.004) but not time (F5,60�0.35, P�0.88), or time�treatment

nteraction (F10,60�1.17, P�0.33). Post hoc analysis revealedhat UFP-512 was ineffective at 0.01 nmol and caused a de-ayed but long-lasting increase (�65%) in akinesia at 0.1mol. Similar to the contralateral paw, RM ANOVA on

mmobility time at the ipsilateral paw (Fig. 6B) showed aignificant effect of treatment (F2,8�23.60, P�0.015) butot time (F5,60�2.35, P�0.051), and a significant

ime�treatment interaction (F10,60�2.57, P�0.01). Postoc analysis revealed that UFP-512 caused a mild andransient increase (�33%) in akinesia at 0.01 nmol and aong-lasting increase (maximal �69%) at the higher dose.

For the effect of UFP-512 microinjected into SNr, RMNOVA on immobility time at the contralateral paw (Fig. 6C)howed a significant effect of treatment (F2,8�30.14, P�.0002) but not time (F5,66�0.62, P�0.68), or time�tre-tment interaction (F10,66�0.49, P�0.89). Post hoc analysisevealed that UFP-512 was ineffective at 0.01 nmol while

se in VMTh of 6-OHDA hemilesioned rats. UFP-512 (10 �g/kg i.p.)(A). UFP-512 (10 �g/kg i.p.) increased GLU release in VMTh whilee to six (A, B) experiments per group and are expressed as percentl GABA levels in the dialysate were 3.6�0.14 nM (A) while basal GLUANOVA followed by contrast analysis and the sequentially rejective

LU releareased itEM of fivnt). Basaicle (RM

ausing a reduction in akinesia (maximal �45%) at 0.1 nmol

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O. S. Mabrouk et al. / Neuroscience 164 (2009) 360–369 365

or the majority of time points tested (30–75 min). RMNOVA at the ipsilateral paw (Fig. 6D) showed a significantffect of treatment (F2,8�11.3, P�0.005) but not timeF5,60�0.71, P�0.62), or time�treatment interactionF10,60�1.76, P�0.09). Post hoc analysis revealed thatFP-512 0.01 nmol caused a mild and transient reduction

n akinesia (�30%) while UFP-512 0.1 nmol caused aong-lasting reduction (�34% at 90 min).

ffects of regional microinjections of NTD

o verify whether the effects of UFP-512 could be attrib-ted to DOP receptor blockade, NTD was microinjected

nto GP and SNr. For the effect of NTD microinjected intoP, RM ANOVA on immobility time at the contralateralaw (Fig. 7A) showed a significant effect of treatmentF2,8�9.49, P�0.008) but not time (F5,66�1.02, P�0.41),r time�treatment interaction (F �0.77, P�0.66). Post

ig. 5. UFP-512 dually modulated akinesia in 6-OHDA hemilesionedats undergoing microdialysis and challenged in the bar test. UFP-51210 �g/kg i.p.) reduced the immobility time at the contralateral (A) andpsilateral (B) paws while UFP-512 (1000 �g/kg i.p.) elevated it. Datare expressed as percent basal motor activity (calculated as the meanf immobility time in the two sessions before treatment) and areeans�SEM of five to six determinations per group. Basal motorctivity values in the bar test were (sec): 11.4�1.4 (ipsilateral paw)nd 27.5�1.8 (contralateral paw). * P�0.05, significantly different fromehicle injected group (RM ANOVA followed by contrast analysis andhe sequentially rejective Bonferroni’s test).

10,66

oc analysis revealed that both NTD 0.1 and 1 nmol p

aused long-lasting increases in akinesia (�59% and9%, respectively). RM ANOVA on immobility time at the

psilateral paw (Fig. 7B) showed a significant effect of treat-ent (F2,8�23.54, P�0.0004) but not time (F5,66�1.40,�0.24), and a significant time�treatment interaction

F10,66�1.56, P�0.14). Post hoc analysis revealed that bothTD 0.1 and 1 nmol caused increases in akinesia (�52%nd 81%, respectively), yet the higher dose was longer last-

ng. For the effect of NTD microinjected into SNr, RM ANOVAn immobility time at the contralateral paw (Fig. 7C) showedsignificant effect of treatment (F2,10�34.69, P�0.0001) and

ime (F5,75�2.41, P�0.04), but no time�treatment interac-ion (F10,75�1.68, P�0.10). Post hoc analysis revealed thatTD 0.1 nmol was slightly and transiently effective whileTD 1 nmol caused a long-lasting increase in akinesia

�56%). At the ipsilateral paw, RM ANOVA (Fig. 7D)howed a significant effect of treatment (F2,10�5.16, P�.03) and time (F5,75�6.92, P�0.00001), but no time�

reatment interaction (F10,75�1.04, P�0.42). Like the con-ralateral paw, NTD 0.1 nmol caused a long-lasting in-rease in akinesia (�56%) while NTD 1 nM was withoutffect.

DISCUSSION

he main finding of the present study is that in hemiparkin-onian rats, the novel DOP receptor agonist UFP-512 causedpposite changes in motor activity which was dose-relatednd mirrored by changes in GABA release in GP and VMThs well as GLU release in SNr. In particular, motor facilitationas observed at low doses and was associated with a re-uction of thalamic GABA release while motor inhibition ap-eared at higher doses and was accompanied by increases

n thalamic GABA release. This suggests that UFP-512 canppositely modulate the activity of nigro–thalamic output neu-ons leading to opposite changes in thalamo–cortical trans-ission and motor output, depending on the dose tested.vidence that these opposite effects are mediated by differ-nt actions in GP and SNr has been presented. Indeed, directicroinjections of UFP-512 into SNr or GP reduced or en-anced akinesia, respectively whereas microinjections ofTD into both areas enhanced akinesia. These data suggest

hat UFP-512, behaving as a partial agonist, attenuatesarkinsonian-like akinesia by stimulating DOP receptors inNr and causes a worsening of parkinsonism by blockingOP receptors in GP.

DOP receptor stimulation has been shown to facilitateocomotion and antiparkinsonian behaviors in a number ofxperimental models of PD (Maneuf et al., 1994; Pinna andi Chiara, 1998; Hill et al., 2000; Hille et al., 2001; Mabroukt al., 2008). In these studies, SNC-80 was preferentiallysed. However, since DOP receptor agonists can lead toifferent degrees of receptor desensitization and signalingathway recruitment (for a review see Varga et al., 2004), it is

mportant to examine the behavioral and neurochemical pro-les of different classes of DOP receptor agonists in order toevelop molecules with antiparkinsonian activity with fewerdverse effects (such as tolerance). UFP-512 is a novel

seudopeptide with high affinitity (pKi�10.2) for the human

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O. S. Mabrouk et al. / Neuroscience 164 (2009) 360–369366

ecombinant DOP receptor as well as high selectivity overOP (160-fold) and KOP (3500-fold) receptors (Vergura et al.,

007). In past studies, UFP-512 displayed antidepressant andnxiolytic properties in the 0.1–1 mg/kg dose range but failedo affect spontaneous locomotion in mice (Vergura et al.,007). Interestingly however, UFP-512 reduced immobilityime in the rat forced swimming test at 0.1 and 0.3 mg/kghile higher doses (1 mg/kg) were ineffective. Analogously in

he current study, UFP-512 improved motor behavior at lowoses (1–10 �g/kg i.p.) and depressed it at higher ones100–1000 �g/kg i.p.). The bell-shaped curve cannot beasily explained as simple receptor desensitization phenom-non since in vitro studies have shown that UFP-512 causes

imited desensitization (as measured by the cAMP pathway)f DOP receptors with respect to other DOP agonists such asNC-80 (Aguila et al., 2007). Moreover, SNC-80 has been

ested up to 60 mg/kg without causing inhibition of movementn the reserpine-treated rat model of PD (Hill et al., 2000). Inine with those studies, SNC-80 dose dependently facilitatedocomotor activity in hemiparkinsonian rats, with no signs ofnhibition even at the highest dose tested (10 mg/kg i.p.;

abrouk et al., 2008). It has been reported that DOP receptorgonists bearing the Dmt-tic pharmacophore, such as UFP-12, undergo metabolism to form diketopiperazine deriva-ives (Marsden et al., 1993) which are endowed with DOPeceptor antagonist characteristics (Balboni et al., 1997). In

ig. 6. Microinjection of UFP-512 in GP or SNr oppositely affected imnd 0.1 nmol) injected into GP increased immobility time at the con

mmobility time at 1 nmol at both contralateral (C) and ipsilateral (D) paf immobility time in the two sessions before treatment) and are meanhe bar test were (sec): 11.1�1.8 (ipsilateral paw) and 24.8�1.6 (contNOVA followed by contrast analysis and the sequentially rejective B

act, extensive structure-activity studies with this family of a

seudopeptides have shown that slight chemical modifica-ions may cause agonists to become antagonists (Balboni etl., 2008). Since we previously showed that systemic admin-

stration of the DOP receptor antagonist NTD caused inhibi-ion of motor activity and a worsening of ParkinsonismMabrouk et al., 2008), it is possible that the inverse correla-ion between dose and effect in the 100–1000 �g/kg range isue to the progressive build-up of extracellular concentra-

ions of a UFP-512 metabolite with antagonist properties athe DOP receptor. The recent finding that UFP-512 behavess partial agonist at recombinant DOP receptor (T. Costaersonal communication) lends further support to the hypoth-sis that the descending portion of the UFP-512 bell-shapedurve is due to progressive blockade of DOP receptors. In-eed, NTD microinjected in GP or SNr enhanced akinesia,

he GP being more sensitive to UFP-512. Conversely, UFP-12 enhanced akinesia when injected in GP and reducedkinesia when injected into SNr. Moreover, the DOP receptor

ull agonists SNC-80 reverted akinesia when injected intoNr but was ineffective when injected into GP (Mabrouk etl., 2008).

In SNr, studies have suggested DOP receptors to bexpressed on nerve terminals of the striatonigral pathwayAbou-Khalil et al., 1984) as well as postsynaptically on cellodies, possibly on GABAergic interneurons (Mansour etl., 1995) as well as on somato-dendritic complex (Cahill et

ime in 6-OHDA hemilesioned rats during the bar test. UFP-512 (0.01(A) and ipsilateral (B) paws. In SNr, UFP-512 caused reductions inare expressed as percent basal motor activity (calculated as the meanof five to six determinations per group. Basal motor activity values inaw). * P�0.05, significantly different from vehicle injected group (RM’s test).

mobility ttralateralws. Datas�SEM

l., 2001) and axon collaterals (Rick and Lacey, 1994) of

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O. S. Mabrouk et al. / Neuroscience 164 (2009) 360–369 367

igro–thalamic projection neurons. In this case, since theeceptors may indeed be localized to different cells anday occur both pre and postsynaptically, it is possible thatifferent dose of UFP-512 may act on different cells within

he same nuclei to cause the observed effects. Neverthe-ess, the different regional effects suggest that the bell-haped dose response curve of systemic UFP-512 coulde interpreted as a combination of alternate mechanismsf action at SNr (DOP receptor stimulation) and GP (DOPeceptor antagonism).

Combined neurochemical and behavioral analysis sup-orted this view. Indeed, motor facilitatory doses of UFP-12 and SNC-80 (Mabrouk et al., 2008) reduced GABAelease in GP (and SNr) whereas motor inhibiting doses ofFP-512 and NTD (Mabrouk et al., 2008) elevated it.nkephalins (ENK) are known to inhibit GABA release

rom striato–pallidal terminals in GP (Maneuf et al., 1994)uggesting that the GABA releasing action of NTD andFP-512 (high doses) may take place at the pallidal level.his action would cause (further) disinhibition of the indi-ect pathway and motor impairment. In line with this view,igh doses of UFP-512 also increased nigral GLU release,lthough it must be noted that such an effect was notbserved following NTD administration (Mabrouk et al.,008). Conversely, the inhibition of GABA release induced

ig. 7. Microinjection of NTD in GP or SNr increased immobility in 6-Onto GP increased immobility time at the contralateral (A) and ipsilaontralateral (C) and ipsilateral (D) at 1 nmol but only slightly at 0.1 nmcalculated as the mean of immobility time in the two sessions beforeotor activity values in the bar test were (sec): 12.1�0.6 (ipsilateral

ehicle injected group (RM ANOVA followed by contrast analysis and

y SNC-80 could only be prevented by selective DOP t

eceptor blockade in SNr, suggesting that SNC-80 andFP-512 (low doses) act at the nigral level.

The reason for such a dual action of UFP-512 may bexplained by the higher ENKergic tone found in GP com-ared to SNr. Indeed, the GP receives a dense ENKergic

nnervation from neurons originating in striatum while SNreceive only very few (Gerfen and Young, 1988). More-ver, 6-OHDA lesioning and other methods of inhibiting DA2 receptors cause an upregulation of striatopallidal ENKxpression (Nisenbaum et al., 1996, for review see Steinernd Gerfen, 1998). The purported partial agonist nature ofFP-512 would cause it to behave as an antagonist in theresence of high ENK tone, as in GP, and as an agonisthere DOP receptors are not or less saturated by theirndogenous ligand (as in SNr).

An important finding of the present study is that UFP-12 motor actions correlated with changes in amino acidelease in VMTh. Indeed, a low dose of UFP-512 reducedhalamic GABA release and attenuated akinesia while aigh dose of UFP-512 caused opposite effects. VMTh isne of the main targets of SNr GABA projection neurons,

.e. the basal ganglia output, and it has been demonstratedhat changes in VMTh GABA release reflect changes inigro–thalamic transmission (Timmerman and Westerink,997; Mark et al., 2004; Marti et al., 2007). According to

ilesioned rats during the bar test. Naltrindole (0.1 and 1 nmol) injectedpaws. In SNr, NTD also caused increased immobility time at bothcontralateral paw. Data are expressed as percent basal motor activityt) and are means�SEM of five to six determinations per group. Basal

27.2�1.0 (contralateral paw). * P�0.05, significantly different fromentially rejective Bonferroni’s test).

HDA hemteral (B)ol at thetreatmen

he tonic inhibitory role of nigro–thalamic projections on the

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O. S. Mabrouk et al. / Neuroscience 164 (2009) 360–369368

halamic filter (Deniau and Chevalier, 1985), it is likely thatnhibition and facilitation of thalamic GABA release resultsn facilitation and inhibition of thalamo–cortical circuits and

otor behavior, respectively. Changes in GABA release inMTh were not mirrored by changes in GLU release. Thus,

ow UFP-512 doses inhibited GABA and facilitated GLUelease while high UFP-512 elevated GABA without affect-ng GLU release. The main source of GLU release in VMThs represented by cortico–thalamic projections (Chevaliernd Deniau, 1982; McFarland and Haber, 2002) and an

ncrease in thalamic GLU release likely reflects activationf cortico–thalamic pathways. Indeed, increased activationf thalamic NMDA receptors has been shown to increase

ocomotion in rats (Klockgether et al., 1986). Alternatively,ncreased GLU release may arise from disinhibition of GLUerve terminals from an inhibitory intrathalamic GABAergicone (possibly mediated by GABAB receptors; Nyitrai et al.,999). The lack of changes in GLU release following an

ncrease in GABA release may be related to the saturationf inhibitory presynaptic GABA receptors caused by tonicctivity of nigro–thalamic neurons. Thus, further increases

n nigro–thalamic activity and GABA release would notause GLU inhibition.

The finding that both low and high doses of UFP-512educed, although with a different time course, SNr GABAelease is unexpected and quite puzzling. However, it isonsistent with that previously reported for SNC-80 andTD (Mabrouk et al., 2008) and possibly related to dy-amic changes in different pools of extracellular GABA.hus, the effect of low doses may reflect a reduction in thectivity of nigro–thalamic neurons through an action atOP receptors expressed on recurrent collaterals (Ricknd Lacey, 1994). Conversely, the effect of high dosesay reflect a reduction in the activity in pallido–nigralrojections due to marked increase in pallidal GABA re-

ease. We speculate that the decrease in GABA originatingrom this specific pool together with the increase in nigralLU would result in increased nigro–thalamic transmissionnd, ultimately, a reduction of motor activity observed inehavioral assays.

CONCLUSION

FP-512 produced opposing motor effects in 6-OHDA hemile-ioned rats depending on dose; low ones causing facilitationnd higher ones causing inhibition. These changes wereccompanied by inhibition and facilitation of GABA release,espectively, in VMTh suggesting that motor changes wereue to opposite regulation of nigro–thalamic GABAergic path-ay and, ultimately, thalamo–cortical transmission and motorutput. Microinjection studies pointed towards a mechanismhereby UFP-512 behaves as partial agonist: at low doses itould acts as an agonist (similar to SNC-80) in stimulatingOP receptors in SNr and in reducing parkinsonism, whereas atigh doses it would act as an antagonist (similar to NTD) inlocking upregulated ENK transmission at DOP receptors

n GP and worsening parkinsonism. DOP receptor ago-ists have been studied for the treatment of movement

isorders such as PD (see Introduction). However, DOP

eceptor antagonists may have some benefit in the treat-ent of dyskinesias brought about by chronic L-DOPA

reatment (Henry et al., 2001) or neuroleptics (McCormicknd Stoessl, 2002), probably via an action in GP (McCor-ick and Stoessl, 2002). The findings here demonstrate

hat UFP-512 has a short-lasting action and a narrowherapeutic window which would limit its use as an antipar-insonian therapeutic agent. Nevertheless, partial agonistsay have a unique place in the treatment of the disease.scalating dose therapy where UFP-512 or another DOP

eceptor partial agonist could be used as an agonist prioro the onset of dyskinesia and as antagonist after dyski-esias have developed could prove to be useful.

cknowledgments—Supported by University of Ferrara (FAR008) and Italian Ministry of University (FIRB Internazionalizzazi-ne) grants to M Morari.

REFERENCES

bou-Khalil B, Young AB, Penney JB (1984) Evidence for the presyn-aptic localization of opiate binding sites on striatal efferent fibers.Brain Res 323:21–29.

guila B, Coulbault L, Boulouard M, Léveillé F, Davis A, Tóth G,Borsodi A, Balboni G, Salvadori S, Jauzac P, Allouche S (2007) Invitro and in vivo pharmacological profile of UFP-512, a novelselective delta-opioid receptor agonist; correlations between de-sensitization and tolerance. Br J Pharmacol 152:1312–1324.

alboni G, Guerrini R, Salvadori S, Tomatis R, Bryant SD, Bianchi C,Attila M, Lazarus LH (1997) Opioid diketopiperazines: synthesisand activity of a prototypic class of opioid antagonists. Biol Chem379:19–29.

alboni G, Fiorini S, Baldisserotto A, Trapella C, Sasaki Y, Ambo A,Marczak ED, Lazarus LH, Salvadori S (2008) Further studies onlead compounds containing the opioid pharmacophore Dmt-Tic.J Med Chem 51:5109–5117.

ezard E, Ravenscroft P, Gross CE, Crossman AR, Brotchie JM (2001)Upregulation of striatal preproenkephalin gene expression occursbefore the appearance of parkinsonian signs in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine monkeys. Neurobiol Dis 8:343–350.

ilsky EJ, Calderon SN, Wang T, Bernstein RN, Davis P, Hruby VJ,McNutt RW, Rothman RB, Rice KC, Porreca F (1995) SNC 80, aselective, nonpeptidic and systemically active opioid delta agonist.J Pharmacol Exp Ther 273:359–366.

ahill CM, McClellan KA, Morinville A, Hoffert C, Hubatsch D,O’Donnell D, Beaudet A (2001) Immunohistochemical distributionof delta opioid receptors in the rat central nervous system: evi-dence for somatodendritic labeling and antigen-specific cellularcompartmentalization. J Comp Neurol 440:65–84.

hevalier G, Deniau JM (1982) Inhibitory nigral influence on cerebellarevoked responses in the rat ventromedial thalamic nucleus. ExpBrain Res 48:369–376.

eniau JM, Chevalier G (1985) Disinhibition as a basic process in theexpression of striatal functions. II. The striato-nigral influence onthalamocortical cells of the ventromedial thalamic nucleus. BrainRes 334:227–233.

ewar D, Jenner P, Marsden CD (1987) Effects of opioid agonist drugson the in vitro release of 3H-GABA, 3H-dopamine and 3H-5HT fromslices of rat globus pallidus. Biochem Pharmacol 36:1738–1741.

erfen CR, Young WS III (1988) Distribution of striatonigral and stria-topallidal peptidergic neurons in both patch and matrix compart-ments: an in situ hybridization histochemistry and fluorescent ret-rograde tracing study. Brain Res 460:161–167.

erfen CR, Engber TM, Mahan LC, Susel Z, Chase TN, Monsma FJ

Jr, Sibley DR (1990) D1 and D2 dopamine receptor-regulated gene

H

H

H

K

K

L

M

M

M

M

M

M

M

M

M

M

M

M

N

N

P

P

P

R

R

R

S

S

S

S

S

T

U

V

V

O. S. Mabrouk et al. / Neuroscience 164 (2009) 360–369 369

expression of striatonigral and striatopallidal neurons. Science250:1429–1432.

enry B, Fox SH, Crossman AR, Brotchie JM (2001) Mu- and delta-opioid receptor antagonists reduce levodopa-induced dyskinesia inthe MPTP-lesioned primate model of Parkinson’s disease. ExpNeurol 171:139–146.

ill MP, Hille CJ, Brotchie JM (2000) �-opioid receptor agonists as atherapeutic approach in Parkinson’s disease. Drug News Perspect13:261–268.

ille CJ, Fox SH, Maneuf YP, Crossman AR, Brotchie JM (2001)Antiparkinsonian action of a � opioid agonist in rodent and primatemodels of Parkinson’s disease. Exp Neurol 172:189–198.

lockgether T, Schwartz M, Turski L, Sontag KH (1986) The rat ven-tromedial thalamus nucleus and motor control: role of N-methyl-D-aspartate-mediated excitation, GABAergic inhibition, and muscarinictransmission. J Neurosci 6:1702–1711.

uschinski K, Hornykiewicz O (1972) Morphine catalepsy in the rat:relation to striatal dopamine metabolism. Eur J Pharmacol 19:119 –122.

lorens-Cortes C, Javoy-Agid F, Agid Y, Taquet H, Schwartz JC(1984) Enkephalinergic markers in substantia nigra and caudatenucleus from Parkinsonian subjects. J Neurochem 43:874–877.

abrouk OS, Volta M, Marti M, Morari M (2008) Stimulation of deltaopioid receptors located in substantia nigra reticulata but not glo-bus pallidus or striatum restores motor activity in 6-hydroxydopa-mine lesioned rats. New insights into the role of delta receptors inparkinsonism. J Neurochem 107:1647–1659.

aneuf YP, Mitchell IJ, Crossman AR, Brotchie JM (1994) On the roleof enkephalin cotransmission in the GABAergic striatal efferents tothe globus pallidus. Exp Neurol 125:65–71.

ansour A, Fox CA, Akil H, Watson SJ (1995) Opioid receptor mRNAexpression in the rat CNS: anatomical and functional implications.Trends Neurosci 18:22–29.

ark KA, Soghomonian JJ, Yamamoto BK (2004) High-dose meth-amphetamine acutely activates the striatonigral pathway to in-crease striatal glutamate and mediate long-term dopamine toxicity.J Neurosci 24:11449–11456.

arsden BJ, Nguyen TM, Schiller PW (1993) Spontaneous degrada-tion via diketopiperazine formation of peptides containing a tetra-hydroisoquinoline-3-carboxylic acid residue in the 2-position of thepeptide sequence. Int J Pept Protein Res 41:313–316.

arti M, Mela F, Bianchi C, Beani L, Morari M (2002) Striatal dopa-mine-NMDA receptor interactions in the modulation of glutamaterelease in the substantia nigra pars reticulata in vivo: opposite rolefor D1 and D2 receptors. J Neurochem 83:635–644.

arti M, Mela F, Veronesi C, Guerrini R, Salvadori S, Federici M,Mercuri NB, Rizzi A, Franchi G, Beani L, Bianchi C, Morari M(2004) Blockade of nociceptin/orphanin FQ receptor signaling in ratsubstantia nigra pars reticulata stimulates nigrostriatal dopaminer-gic transmission and motor behavior. J Neurosci 24:6659–6666.

arti M, Mela F, Fantin M, Zucchini S, Brown JM, Witta J, Di BenedettoM, Buzas B, Reinscheid RK, Salvadori S, Guerrini R, Romualdi P,Candeletti S, Simonato M, Cox BM, Morari M (2005) Blockade ofnociceptin/orphanin FQ transmission attenuates symptoms andneurodegeneration associated with Parkinson’s disease. J Neuro-sci 25:9591–9601.

arti M, Trapella C, Viaro R, Morari M (2007) The nociceptin/orphaninFQ receptor antagonist J-113397 and L-DOPA additively attenuateexperimental parkinsonism through overinhibition of the nigrotha-lamic pathway. J Neurosci 27:1297–1307.

arti M, Trapella C, Morari M (2008) The novel nociceptin/orphanin

FQ receptor antagonist Trap-101 alleviates experimental parkin-

sonism through inhibition of the nigro-thalamic pathway: positiveinteraction with L-DOPA. J Neurochem 107:1683–1696.

cCormick SE, Stoessl AJ (2002) Blockade of nigral and pallidalopioid receptors suppresses vacuous chewing movements in arodent model of tardive dyskinesia. Neuroscience 112:851–859.

cFarland NR, Haber SN (2002) Thalamic relay nuclei of the basalganglia form both reciprocal and nonreciprocal cortical connections,linking multiple frontal cortical areas. J Neurosci 22:8817–8832.

isenbaum LK, Crowley WR, Kitai ST (1996) Partial striatal dopaminedepletion differentially affects striatal substance P and enkephalin mes-senger RNA expression. Brain Res Mol Brain Res 37:209–216.

yitrai G, Kovács I, Szárics E, Skuban N, Juhász G, Kardos J (1999)Role of intracellular Ca(2�) stores shaping normal activity in brain.J Neurosci Res 57:906–915.

arent A, Hazrati LN (1995) Functional anatomy of the basal ganglia.I. The cortico-basal ganglia-thalamo-cortical loop. Brain Res BrainRes Rev 20:91–127.

axinos G, Watson C (1982) The rat brain in stereotaxic coordinates.Sydney, Australia: Academic Press.

inna A, Di Chiara G (1998) Dopamine-dependent behavioural stim-ulation by non-peptide delta opioids BW 373U86 and SNC 80. 3.Facilitation of D1 and D2 responses in unilaterally 6-hydroxydopa-mine-lesioned rats. Behav Pharmacol 9:15–21.

ick CE, Lacey MG (1994) Rat substantia nigra pars reticulata neu-rones are tonically inhibited via GABAA, but not GABAB, receptorsin vitro. Brain Res 659:133–137.

inne UK, Rinne JK, Rinne JO, Laakso K, Tenovuo O, Lönnberg P,Koskinen V (1983) Brain enkephalin receptors in Parkinson’s dis-ease. J Neural Transm Suppl 19:163–171.

ozas G, Guerra MJ, Labandeira-García JL (1997) An automatedrotarod method for quantitative drug-free evaluation of overall mo-tor deficits in rat models of parkinsonism. Brain Res Brain ResProtoc 2:75–84.

amadi P, Bédard PJ, Rouillard C (2006) Opioids and motor complica-tions in Parkinson’s disease. Trends Pharmacol Sci 27:512–517.

anberg PR, Bunsey MD, Giordano M, Norman AB (1988) The cata-lepsy test: its ups and downs. Behav Neurosci 102:748–759.

challert T, De Ryck M, Whishaw IQ, Ramirez VD, Teitelbaum P (1979)Excessive bracing reactions and their control by atropine and L-DOPA in an animal analog of Parkinsonism. Exp Neurol 64:33–43.

tanford IM, Cooper AJ (1999) Presynaptic mu and delta opioid re-ceptor modulation of GABAA IPSCs in the rat globus pallidus invitro. J Neurosci 19:4796–4803.

teiner H, Gerfen CR (1998) Role of dynorphin and enkephalin in theregulation of striatal output pathways and behavior [Review]. ExpBrain Res 123:60–76.

immerman W, Westerink BH (1997) Electrical stimulation of thesubstantia nigra reticulata: detection of neuronal extracellularGABA in the ventromedial thalamus and its regulatory mechanismusing microdialysis in awake rats. Synapse 26:62–71.

ngerstedt U, Arbuthnott GW (1970) Quantitative recording of rota-tional behaviour in rats after 6-hydroxydopamine lesions of thenigrostriatal dopamine system. Brain Res 24:485–493.

arga EV, Navratilova E, Stropova D, Jambrosic J, Roeske WR,Yamamura HI (2004) Agonist-specific regulation of the delta-opioidreceptor. Life Sci 76:599–612.

ergura R, Balboni G, Spagnolo B, Gavioli E, Lambert DG, McDonaldJ, Trapella C, Lazarus LH, Regoli D, Guerrini R, Salvadori S, CalóG (2007) Anxiolytic- and antidepressant-like activities of H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512), a novel selective delta

opioid receptor agonist. Peptides 29:93–103.

(Accepted 25 August 2009)(Available online 1 September 2009)