Levodopa treatment reverses endocannabinoid system abnormalities in experimental parkinsonism

Journal of Neurochemistry, 2003, 85, 1018–1025

doi:10.1046/j.1471-4159.2003.01759.x

Levodopa treatment reverses endocannabinoid system abnormalities in experimental parkinsonism Mauro Maccarrone,*,à,1 Paolo Gubellini, ,§,1 Monica Bari,* Barbara Picconi, ,¶ Natalia Battista,* Diego Centonze, ,¶ Giorgio Bernardi, ,¶ Alessandro Finazzi-Agro`* and Paolo Calabresi ,à,2 *Dipartimento di Medicina Sperimentale e Scienze Biochimiche and  Dipartimento di Neuroscienze, Universita` degli Studi di Roma ‘Tor Vergata’, Roma, Italy àDipartimento di Scienze Biomediche, Universita` di Teramo, Teramo, Italy §Laboratoire de Neurobiologie Cellulaire et Fonctionnelle, CNRS, Marseille, France ¶IRCCS Fondazione ‘Santa Lucia’, Roma, Italy

Abstract Cannabinoid receptors and their endogenous ligands are potent inhibitors of neurotransmitter release in the brain. Here, we show that in a rat model of Parkinson’s disease induced by unilateral nigral lesion with 6-hydroxydopamine (6-OHDA), the striatal levels of the endocannabinoid anandamide (AEA) were increased, while the activity of its membrane transporter and hydrolase (fatty-acid amide hydrolase, FAAH) were decreased. These changes were not observed in the cerebellum of the same animals. Moreover, the frequency and amplitude of glutamate-mediated spontaneous excitatory post-synaptic currents were augmented in striatal spiny neurones recorded from parkinsonian rats. Remarkably, the anomalies in the endocannabinoid system, as well as those in glutamatergic activity, were completely reversed by chronic treatment of parkinsonian rats with levodopa, and the

pharmacological inhibition of FAAH restored a normal glutamatergic activity in 6-OHDA-lesioned animals. Thus, the increased striatal levels of AEA may reflect a compensatory mechanism trying to counteract the abnormal corticostriatal glutamatergic drive in parkinsonian rats. However, this mechanism seems to be unsuccessful, since spontaneous excitatory activity is still higher in these animals. Taken together, these data show that anomalies in the endocannabinoid system induced by experimental parkinsonism are restricted to the striatum and can be reversed by chronic levodopa treatment, and suggest that inhibition of FAAH might represent a possible target to decrease the abnormal cortical glutamatergic drive in Parkinson’s disease. Keywords: anandamide, CB1 receptor, dopamine, glutamate, levodopa, striatum. J. Neurochem. (2003) 85, 1018–1025.

Endocannabinoids, such as anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoylglycerol (2-AG), bind to brain CB1 cannabinoid receptor, mimicking several actions of the marijuana constituent D9-tetrahydrocannabinol (Howlett and Mukhopadhyay 2000). CB1 receptor is densely expressed in the striatum (Herkenham et al. 1991), a brain region involved in motor processes, cognition and motivation (Berke and Hyman 2000; Calabresi et al. 2000), and its activation reduces glutamate release in the striatum (Gerdeman and Lovinger 2001; Huang et al. 2001). While the effect of cannabimimetic drugs on motor and cognitive performances have already been described (Ameri 1999; Giuffrida and Piomelli 2000), only recently have they been linked to Parkinson’s disease (PD) and its therapies.

Received October 15, 2002; revised manuscript received February 10, 2003; accepted February 12, 2003. Address correspondence and reprint requests to either Prof. Paolo Calabresi, Dipartimento di Neuroscienze, Universita` di Roma ‘Tor Vergata’, Via Montpellier 1, 00133 – Roma, Italy. E-mail: [email protected] or Prof. Mauro Maccarrone, Dipartimento di Scienze Biomediche, Universita` di Teramo, Piazza A. Moro 45, 64100 – Teramo, Italy. E-mail: [email protected] 1 These authors contributed equally to this work. Abbreviations used: ACSF, artificial cerebrospinal fluid; AEA, arachidonoylethanolamide, endocannabinoid anandamide; 2-AG, 2-arachidonoylglycerol; AMT, anandamide membrane transporter; DA, nigral dopamine; EPSC, excitata postsynaptic current; FAAH, fatty acid amide hydrolase; L-DOPA, levodopa; MAFP, methyl-arachidonoyl fluorophosphonate; NAPE, N-acyl-phosphatidylethanolamine; 6-OHDA, 6-hydroxydopamine; PD, Parkinson’s disease; PLD, phospholipase D; sEPSC, spontaneous excitatory post-synaptic current.

1018

 2003 International Society for Neurochemistry, J. Neurochem. (2003) 85, 1018–1025

Levodopa reverses striatal cannabinoid anomalies 1019

Interestingly, a clinically available cannabinoid, nabilone, alleviates levodopa (L-DOPA)-induced dyskinesia in primate models of PD (Fox et al. 2002), as well as in humans (Brotchie 2000; Sieradzan et al. 2001). In this context, it is now well recognized that the loss of nigral dopamine (DA) neurones in PD is responsible for a hyperactivation of corticostriatal glutamatergic transmission, which is supposed to underlie the motor symptoms of this pathology, as well as L-DOPA-induced dyskinesia (Calabresi et al. 1993, 2000; Schwartig and Huston 1996; Centonze et al. 2001; Tang et al. 2001; Gubellini et al. 2002). This abnormal synaptic transmission can be reduced by acting on endocannabinoid catabolism (Gubellini et al. 2002). Thus, it seems that CB1 receptor can modulate corticostriatal glutamatergic input in physiological and pathological conditions. The wide CB1 receptor distribution in the striatum provides the opportunity for functional interactions of AEA and 2-AG, also with the dopaminergic nigrostriatal pathway. A close interaction between dopamine and endocannabinoids in motor functions has been postulated (Beltramo et al. 2000; Meschler and Howlett 2001). In fact, recent evidence in freely moving rats has shown that AEA release is enhanced by quinpirole, a D2like DA receptor agonist (Giuffrida et al. 1999). We have investigated the interactions between the endocannabinoid and the dopaminergic systems in control, in parkinsonian, and in L-DOPA-treated parkinsonian rats. In particular, the following biochemical parameters were analysed in the striatum and cerebellum of these three experimental groups: (i) the levels of endogenous AEA and 2-AG, (ii) the activity of phospholipase D (PLD), an enzyme involved in AEA synthesis, (iii) the activity of AEA membrane transporter (AMT) and hydrolase (fatty acid amide hydrolase, FAAH), two key factors regulating brain AEA levels, and (iv) the binding of AEA and of the synthetic cannabinoid CP55 940 to CB1 receptors. Moreover, we have investigated, by electrophysiological experiments, the spontaneous glutamatergic transmission in control as well as in parkinsonian and L-DOPA-treated parkinsonian rats. Finally, we have characterized the possible modulation of corticostriatal glutamatergic activity by drugs targeting the endocannabinoid system.

Materials and methods 6-hydroxydopamine (6-OHDA) lesions and L-DOPA treatment All the experiments were conducted in conformity with the European Communities Council Directive of November 1986 (86/ 609/EEC). Wistar rats were injected unilaterally with 6-OHDA (8 lg/4 lL saline containing 0.1% ascorbic acid) rostral to the substantia nigra under stereotaxic co-ordinates (Paxinos and Watson 1986): A ¼ 3.7 mm anterior to the interaural line, V ¼ 2.2 mm dorsal to the interaural line; L ¼ 2.2 mm from the midline. The rats were tested 20 days later with 0.05 mg/kg (s.c.) apomorphine and the controlateral turns were recorded with automatic rotometers

(Biological Research Apparatus, Varese, Italy) for 3 h (Ungerstet and Arbuthnott 1970). Only those rats consistently making at least 200 controlateral turns were used for our studies. After brain dissection, we confirmed that the nigrostriatal pathway was lesioned. This was established by noting a > 95% loss of DA neurones in the substantia nigra compacta and the almost complete absence of DA terminals in the striatum. This was detected by the immunoperoxidase technique using a monoclonal antibody for tyrosine hydroxylase. Rats were used 2–3 months after the 6-OHDA lesion. As control animals, we used sham-operated rats of similar ages injected with saline not containing 6-OHDA. Chronic treatment with L-DOPA was performed by i.p. injections (25 mg/kg L-DOPA plus 6.5 mg/kg benserazide) twice per day for 3 weeks. As control animals, we used sham-operated rats of similar ages injected with saline. From each animal, striatum and cerebellum were removed and were immediately subjected to biochemical analysis. GC/MS analysis The endogenous levels of AEA and 2-AG in the striatum and in the cerebellum were determined by gas chromatography-electron impact mass spectrometry (GC/MS) as previously described (Maccarrone et al. 2001a). Immediately after decapitation, rat brains were washed in phosphate-buffered saline (PBS) pre-cooled at 4C; they were dissected and frozen in liquid nitrogen, and kept at ) 70C until processed. A maximum of 8 min elapsed between rat decapitation and freezing of dissected tissues, a time insufficient to cause artefactual rises in endocannabinoid levels (Schmid et al. 1995; Maccarrone et al. 2001a). Lipids were extracted from frozen tissues and injected into a Carlo Erba model HRGC5160 gas chromatograph (Rome, Italy) equipped with a BP5 silica capillary column (30 m · 0.25 mm) from SGE (Milan, Italy) and interfaced with a VG Micromass model QUATTRO spectrometer (Manchester, UK). Analyses were performed in ‘splitless’ mode at temperatures rising from 70C to 250C at a rate of 30C/min. The identity of AEA and 2-AG was assessed by comparison of the retention times and the mass spectra recorded at 70 eV with those of authentic standards. Quantification of AEA was achieved by isotope dilution with AEAd4, whereas 2-AG was quantified by the internal standard method with AEAd4. Calibration solutions and calibration curves were obtained as described (Maccarrone et al. 2001a). Determination of anandamide metabolism and binding The uptake of [3H]AEA by the AMT was assayed in synaptosomes prepared from the striatum or cerebellum as reported (Maccarrone et al. 2001a). Tissues were resuspended in ice-cold 0.32 M sucrose, 5 mM Tris–HCl buffer (pH 7.4), and were gently disrupted by 10 up-and-down strokes in a Teflon-glass homogenizer (weight/volume ratio ¼ 1 : 20). The homogenates were centrifuged at 1000 g for 5 min at 4C, then the supernatant fluids were centrifuged again at 17 000 g for 15 min at 4C. The final pellets were resuspended in 136 mM NaCl, 5 mM KCl, 0.16 mM CaCl2, 0.1 mM EGTA, 1.3 mM MgCl2, 10 mM glucose, 10 mM Tris–HCl buffer (pH 7.4), at a protein concentration of 3 mg/mL. The activity of AMT was measured using 100 lL synaptosomes and 300 nM [3H]AEA per test. The Q10 value was calculated as the ratio of AEA uptake at 30C and 20C (Hillard and Jarrahian 2000). Incubations (15 min)

 2003 International Society for Neurochemistry, J. Neurochem. (2003) 85, 1018–1025

1020 M. Maccarrone et al.

were also carried out with different concentrations of [3H]AEA, in the range 0–800 nM, in order to determine the apparent MichaelisMenten constant (Km) and maximum velocity (Vmax) of AMT by non-linear regression analysis, performed using the PRISM 3 program (GraphPAD Software for Science, San Diego, CA, USA) (Maccarrone et al. 2001a). Anandamide hydrolase (arachidonoylethanolamide amidohydrolase, E.C.3.5.1.4; FAAH) activity was assayed in rat brain homogenates by reversed phase high performance liquid chromatography, using 5 lM [3H]AEA as substrate (Maccarrone et al. 1998). FAAH activity was expressed as picomoles of arachidonate released per minute per milligram of protein. Apparent Km and Vmax values of the hydrolysis of [3H]AEA (in the range 0–15 lM) by FAAH were calculated by non-linear regression analysis, performed using the PRISM 3 program (GraphPAD Software for Science) (Maccarrone et al. 1998). The activity of phospholipase D (phosphatidylcholine phosphatidohydrolase, E.C.3.1.4.4; PLD) was assayed in rat brain homogenates by measuring the release of [14C]ethanolamine from (10 lM) as 1,2-dioleoyl-3-phosphatidyl-[2–14C]ethanolamine previously described (Moesgaard et al. 2000). PLD activity was expressed as picomoles of ethanolamine released per minute per milligram of protein. The binding of [3H]AEA or of the synthetic cannabinoid [3H]CP55 940 to striatal or cerebellar membranes was determined using rat membrane fractions that were prepared, quickly frozen in liquid nitrogen and stored at ) 80C for no longer than 1 week as previously reported (Maccarrone et al. 2001b). These membrane fractions were used in rapid filtration assays with 400 pM agonist, as described previously (Maccarrone et al. 2001b). The apparent dissociation constant (Kd) and maximum binding (Bmax) values of [3H]CP55.940 were calculated from saturation curves (in the range 0–800 pM) through non-linear regression analysis with the PRISM 3 program (GraphPAD Sofware for Science) (Maccarrone et al. 2001b). [3H]AEA (223 Ci/mM) and [3H]CP55 940 (126 Ci/mM) were from NEN DuPont de Nemours (Ko¨ln, Germany); 1,2dioleoyl-3-phosphatidyl[2–14C]ethanolamine (55 mCi/mM) was from Amersham Pharmacia Biotech (Uppsala, Sweden). Biochemical data were expressed as the mean ± SD and statistical analysis was performed by the Student’s t-test (between two groups, reported in the Results section) and by the 2-way ANOVA test (between the three groups, reported in the figure legends) (GraphPAD PRISM 3.03). The significance level was established at p < 0.05. Electrophysiology Preparation and maintenance of rat corticostriatal slices have been previously described (Calabresi et al. 1993). Briefly, corticostriatal

coronal slices (190–200 lm) were prepared from 2–3 month-old Wistar rats (sham-operated, 6-OHDA-lesioned, and 6-OHDAlesioned chronically treated with L-DOPA). Slices were cut with a vibratome and kept in artificial cerebrospinal fluid (ACSF) comprising (in mM): NaCl 126, KCl 2.5, MgCl2 1.2, NaH2PO4 1.2, CaCl2 2.4, glucose 11 and NaHCO3 25. The temperature of the ACSF was maintained at 35C and gassed with O2/CO2 (95%/5%). For whole-cell patch-clamp recordings, electrodes (4–5 MW) were filled with a solution containing (mM): K+-gluconate 125, NaCl 10, CaCl2 1.0, MgCl2 2.0, 1,2-Bis(2-aminophenoxy)ethane-N,N,N¢,N¢tetraacetic acid (BAPTA) 0.5, HEPES 19, guanosine triphosphate (GTP) 0.3 and Mg-adenosine triphosphate 1.0, adjusted to pH 7.3 with KOH. Striatal spiny neurones were clamped at ) 80 to ) 85 mV, close to their resting membrane potential. Spontaneous glutamatergic activity was recorded from striatal spiny neurones and monitored using Axopatch 200B and 1D amplifiers, and pClamp 8.1 software (Axon Instruments, Union City, CA, USA). Afterwards, spontaneous excitatory post-synaptic currents (sEPSCs) were analysed offline by MiniAnalysis 5.4.1 software (Synaptosoft, Decatur, GA, USA). All recordings were performed in the presence of 3 lM bicuculline to avoid the contamination of sEPSCs by a GABAA-mediated component. Striatal medium spiny neurones were selected by means of infrared videomicroscopy (Zeiss Axioskop, Jena, Germany) and a digital camera (Cohu, Japan). For data presented as the mean ± SEM, statistical analysis was performed using the Student’s t-test. The significance level was established at p < 0.05. Drugs were applied by dissolving them to the desired final concentration in the ACSF perfusing the slice. Bicuculline, HU-210 and VDM11 were from Tocris-Cookson (Bristol, UK); methylarachidonoyl fluorophosphonate (MAFP) was from Cayman Chemicals (Ann Arbor, MI, USA).

Results

Biochemical analysis of the endocannabinoid system The level of endogenous AEA was threefold higher in the striatum of 6-OHDA-lesioned rats compared with shamoperated animals, whereas endogenous 2-AG was unaffected (Table 1). The activity of PLD was almost identical (approximately 330 ± 35 pM/min/mg protein) in striata from sham-operated and 6-OHDA-lesioned rats (Fig. 1a). This activity was assayed under conditions found to be optimal for the N-acyl-phosphatidylethanolamines (NAPE)-hydrolysing

Brain area

AEA (nM/mg protein)

2-AG (nM/mg protein)

Striatum (sham-operated) Striatum (6-OHDA) Striatum (6-OHDA + L-DOPA) Cerebellum (sham-operated) Cerebellum (6-OHDA) Cerebellum (6-OHDA + L-DOPA)

0.25 0.75 0.30 0.15 0.17 0.16

1.25 1.30 1.15 2.40 2.25 2.30

± ± ± ± ± ±

0.02 0.08 0.03 0.02 0.02 0.02

(100%) (300%)* (120%)# (100%) (113%) (107%)

± ± ± ± ± ±

0.12 0.12 0.12 0.22 0.23 0.23

Table 1 Endogenous levels of AEA and 2-AG in parkinsonian rats

(100%) (104%) (92%) (100%) (94%) (96%)

*p < 0.01 compared with striatum (sham-operated); #p < 0.01 compared with striatum (6-OHDA); p > 0.05 in all other cases (n ¼ 4; Student’s t-test).

 2003 International Society for Neurochemistry, J. Neurochem. (2003) 85, 1018–1025

Levodopa reverses striatal cannabinoid anomalies 1021

Table 2 Kinetic constants of AMT and FAAH in parkinsonian rats AMT Striatum

Kma

FAAH Vmaxb

Kmc

Vmaxb

Sham-operated 383 ± 45 181 ± 9 10.5 ± 1.7 1687 ± 165 6-OHDA 351 ± 49 81 ± 4* 12.4 ± 2.2 580 ± 48* 6-OHDA + L-DOPA 365 ± 46 195 ± 8# 11.7 ± 1.9 1585 ± 154# a

Expressed as nM. bExpressed as pM/min/mg protein. cExpressed as lM. *p < 0.01 compared with sham-operated; #p < 0.01 compared with 6-OHDA; p > 0.05 in all other cases (n ¼ 4; Student’s t-test.).

Fig. 1 Changes in the endocannabinoid system in the striatum and in the cerebellum of parkinsonian rats. (a) The activity of PLD is affected neither by 6-OHDA lesion nor chronic L-DOPA treatment in the striatum and the cerebellum (p ¼ 0.22; F2,18 ¼ 1.65). (b) Conversely, the activity of AMT is reduced in the striatum, but not in the cerebellum, of parkinsonian animals, and returns to the values observed in shamoperated rats upon chronic L-DOPA treatment (p < 0.01; F2,18 ¼ 19.75). (c) Similarly, the activity of FAAH is reduced in the striatum of parkinsonian rats, and L-DOPA restores it to normal values, while the cerebellum is not affected (p < 0.01; F2,18 ¼ 8.38).

PLD (Moesgaard et al. 2000). A radiolabelled phosphatidylethanolamine was used instead of radiolabelled NAPEs, which are not yet commercially available (Gubellini et al. 2002). This is noteworthy, because NAPE-hydrolysing PLD activity is considered to be the checkpoint in AEA synthesis, although the lack of specific inhibitors of this enzyme makes it difficult to extend its analysis further and to assess conclusively its contribution to AEA metabolism (Moesgaard et al. 2000; Gubellini et al. 2002). Conversely, AMT activity decreased in the striatum of parkinsonian animals (35 ± 4 vs. 90 ± 10 pM/min/mg protein; p < 0.01; Student’s

t-test), as did the activity of FAAH, which was 180 ± 20 versus 470 ± 50 pM/min/mg protein ( p < 0.01; Student’s t-test) (Figs 1b and c). Kinetic analysis of AMT and FAAH in the striatum of sham-operated and parkinsonian rats showed that apparent Km, i.e. the affinity, of AMT and FAAH for AEA did not change, whereas the apparent Vmax of both proteins was significantly lower in the latter group (Table 2). Taken together with the data on PLD activity, these results suggest that the higher level of AEA in the striatum of parkinsonian rats might be due to a decreased cleavage rather than to an increased synthesis. On the other hand, the observation that endogenous 2-AG remained unchanged (Table 1) suggests that its synthesis by phospholipases A1 and C, and/or its degradation by monoacylglycerol lipase, were not affected by 6-OHDA denervation. Remarkably, the down-regulation of striatal AMT and FAAH by 6-OHDA denervation was completely reversed by chronic treatment with L-DOPA (AMT activity ¼ 100 ± 10 pM/min/mg protein; FAAH activity ¼ 400 ± 40 pM/min/mg protein, p < 0.01 vs. 6-OHDA-lesioned rats in both cases; Student’s t-test), as were the Vmax values of both proteins (Table 2). On the other hand, the binding of [3H]AEA was not affected after 6-OHDA denervation or L-DOPA treatment, and was approximately 160 ± 15 fM/mg protein in all animals (Fig. 2a). Rat striatal membranes were also able to bind [3H]CP55 940 according to saturation curves, which yielded an apparent Kd of 358 ± 80 pM and a Bmax of 350 ± 35 fM/mg protein (Fig. 2b). These Kd values are close to those previously reported for the binding of [3H]CP55 940 to rat striatal membranes (Pertwee 1997; references therein). Treatment with 6-OHDA or L-DOPA did not change [3H]CP55 940 binding by striatal membranes (Fig. 2b), corroborating the hypothesis that CB1 receptor function was not affected by experimental parkinsonism. Finally, it is noteworthy that the cerebellum of 6-OHDAlesioned and L-DOPA-treated lesioned animals did not show any significant alteration in endogenous levels of AEA and 2-AG (Table 1), in the activity of PLD, AMT or FAAH, or in [3H]AEA or [3H]CP55 940 (not shown) binding (Figs 1 and 2), compared with sham-operated rats (for a statistical analysis with 2-way ANOVA test see the legends of Figs 1

 2003 International Society for Neurochemistry, J. Neurochem. (2003) 85, 1018–1025

1022 M. Maccarrone et al.

Fig. 2 Binding to cannabinoid receptors in the striatum and in the cerebellum of parkinsonian rats. (a) The binding of [3H]AEA is not affected by 6-OHDA lesion in both striatum and cerebellum. Chronic L-DOPA treatment of parkinsonian animals also does not affect [3H]AEA binding (p ¼ 0.44; F2,18 ¼ 0.87). (b) Saturation curves of the binding of [3H]CP55 940 to striatal membranes of sham-operated (filled circles), 6-OHDA-lesioned (open squares) or L-DOPA treated (open triangles) animals.

and 2). This observation suggests that perturbation of the endocannabinoid system in parkinsonian animals was restricted to the striatum. Electrophysiological analysis of spontaneous glutamatergic activity Spontaneous excitatory post-synaptic currents (sEPSCs) were recorded from striatal spiny neurones by means of whole-cell patch-clamp techniques. According to previous reports, the neurones recorded from the three experimental groups had similar intrinsic membrane properties (Kita et al. 1984; Jiang and North 1991; Calabresi et al. 1993; Gubellini et al. 2002; Picconi et al. 2002). As shown in Fig. 3(a–c), the frequency and amplitude of sEPSCs recorded from spiny neurones of 6-OHDA-lesioned rats was significantly higher compared with sham-operated rats (respectively 2.0 ± 0.2 vs. 6.0 ± 1.1 Hz, n ¼ 20, and 14.0 ± 1.6 vs. 22.0 ± 2.7 pA, n ¼ 20, p < 0.01 for both; Student’s t-test). Interestingly, chronic treatment of parkinsonian rats with L-DOPA restored both sEPSC frequency and amplitude to the basal levels (Fig. 3a–c) observed in shamoperated animals (2.3 ± 0.3 Hz, n ¼ 20, and 16.0 ± 2.1 pA, n ¼ 20, p > 0.05 compared with sham-operated rats; Stu-

Fig. 3 Electrophysiology of striatal spiny neurones of control, parkinsonian and L-DOPA-treated parkinsonian rats. The frequency of glutamatergic sEPSCs is increased in 6-OHDA-lesioned animals (a), as well as their amplitude (b). L-DOPA treatment in parkinsonian rats restores both sEPSC frequency (a) and amplitude (b) to sham-operated levels. Electrophysiological traces (c) show glutamatergic sEPSCs from single cells in the three different experimental conditions (all these neurones were clamped at ) 80 mV). *p < 0.01 compared with sham-operated rats, #p < 0.01 compared with 6-OHDA-lesioned rats and p > 0.05 compared with sham-operated rats (n ¼ 6 for each condition; Student’s t-test).

dent’s t-test). The sEPSCs, recorded in the presence of 3 lM bicuculline, a GABAA receptor antagonist, were suppressed by the application of 10 lM CNQX, an AMPA receptor blocker (not shown). The pharmacological treatment with HU-210, a CB1 receptor agonist (Pertwee 1997), revealed a dose-dependent inhibition of sEPSC frequency that was comparable in the three experimental groups. Figure 4a shows the normalized effect of this drug on the frequency of sEPSCs. Similarly, the blockade of AMT by VDM11 (De Petrocellis et al. 2001) caused a dose-dependent inhibition of sEPSC frequency in the three experimental groups (Fig. 4b). Conversely, the FAAH blocker, MAFP (De Petrocellis et al. 2001), had a much stronger inhibitory effect in 6-OHDA-lesioned animals

 2003 International Society for Neurochemistry, J. Neurochem. (2003) 85, 1018–1025

Levodopa reverses striatal cannabinoid anomalies 1023

Fig. 4 Different pharmacological modulation of the cannabinoid system in sham-operated, 6-OHDA-lesioned and parkinsonian rats chronically treated with L-DOPA. (a) The CB1 receptor agonist HU-210 reduces sEPSC frequency in a dose-dependent manner. This effect, when normalized, is similar in sham-operated, 6-OHDA-lesioned and 6-OHDA-lesioned rats chronically treated with L-DOPA. (b) The AMT blocker VDM11 shows effects comparable with those obtained with HU-210. (c) Conversely, inhibition of FAAH activity by MAFP is much more effective on 6-OHDA-lesioned animals compared with shamoperated and L-DOPA-treated parkinsonian rats. The amplitude of sEPSCs is not significantly affected by the these drugs in the three experimental models (d, e and f). (*p < 0.01 compared with pre-drug control; n ¼ 6 for each condition; Student’s t-test).

than in sham-operated and in L-DOPA-treated lesioned rats (Fig. 4c). These three compounds did not alter sEPSC amplitude significantly in the three experimental groups (Fig. 4d–f), supporting a pre-synaptic action of CB1 receptor activation (Gerdeman and Lovinger 2001; Huang et al. 2001; Gubellini et al. 2002). Moreover, according to previous findings (Huang et al. 2001; Gubellini et al. 2002), CB1 receptor stimulation affected neither the frequency nor the amplitude of miniature EPSPs (not shown). Discussion

In the present study, we show for the first time that the complex plastic changes of the endocannabinoid system caused by experimental parkinsonism are restricted to the striatum and are completely reversed by chronic L-DOPA treatment.

In particular, in the striatum, but not in the cerebellum, of 6-OHDA-denervated rats: (i) the increased levels of endogenous AEA are due to down-regulation of its degradation, rather than to up-regulation of its synthesis; (ii) the binding of AEA to CB1 receptors does not change; (iii) the pharmacological inhibition of FAAH, but not that of AMT, produces a much stronger depression of striatal glutamatergic activity compared with sham-operated and L-DOPAtreated lesioned rats. This latter finding, taken together with the observation that denervated striata express a dramatic overactivity of glutamatergic transmission, suggests that targeting FAAH might be beneficial in experimental parkinsonism by reducing this abnormal synaptic transmission. Accordingly, ionotropic glutamate receptor antagonists improve experimental PD symptoms (Chase and Oh 2000). To our knowledge, this is the first report showing that alterations in the endocannabinoid system, associated with a neurological disorder, are restricted to the brain area responsible for this disorder, and are reversed by a treatment which corrects the symptoms of the disease. These observations speak in favour of a genuine cause–effect relationship. It has been reported that corticostriatal glutamatergic transmission is enhanced following DA denervation (Calabresi et al. 1993, 2000; Centonze et al. 2001; Tang et al. 2001; Gubellini et al. 2002). This effect also reflects the loss of the D2 receptor-mediated control of corticostriatal transmission (Cepeda et al. 2001; Tang et al. 2001). Interestingly, D2 and CB1 receptors share the same signal transduction pathway and co-operate closely in the negative regulation of striatal excitatory transmission (Meschler and Howlett 2001). Thus, the finding that endogenous levels of AEA are higher in parkinsonian rats may reflect a compensatory mechanism that is trying to control the cortical glutamatergic drive to the striatum. However, this mechanism seems not to be sufficient in 6-OHDA-lesioned rats, since spontaneous excitatory activity is still higher in these animals (Gubellini et al. 2002). It is not clear from our data why parkinsonian rats are more sensitive to FAAH inhibition than control and L-DOPA-treated lesioned animals while the other pharmacological tools acting on the endocannabinoid system have the same effects in the three groups. We can speculate that, since FAAH is less active in 6-OHDA-lesioned rats, this enzyme is more vulnerable to inhibition by MAFP. However, AMT is also reduced in these animals and thus, we should also expect an increased sensitivity to VDM11, which is not the case. It is clear therefore that further studies are necessary to address these issues. Presumably, FAAH activity plays a major role in determining AEA levels in the striatum of 6-OHDA-lesioned animals, in accordance with a recent report showing that mice lacking FAAH have a 15-fold augmented level of AEA in the brain (Cravatt et al. 2001).

 2003 International Society for Neurochemistry, J. Neurochem. (2003) 85, 1018–1025

1024 M. Maccarrone et al.

Since FAAH seems to be the ‘check-point’ in AEA degradation (Cravatt et al. 2001), we propose that drugs modulating its activity might represent a novel pharmacological approach in the therapy of Parkinson’s disease. This seems noteworthy, because therapeutic options for managing L-DOPA-induced dyskinesia in PD are still limited (Sieradzan et al. 2001). Acknowledgements This work was supported by a Schizophrenia Finalized Project (IRCCS ‘Santa Lucia’) to PC, and by two Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica, Consiglio Nazionale delle Ricerche Biotechnology Programs (L. 95/95) to GB and AF-A. We thank Dr A. Cartoni for the analysis of endogenous endocannabinoid levels and Mr M. Tolu for his technical assistance.

References Ameri A. (1999) The effects of cannabinoids on the brain. Prog. Neurobiol. 58, 315–348. Beltramo M., Rodrı´guez de Fonseca F., Navarro M., Calignano A., Gorriti M. A., Grammatikopoulos G., Sadile A. G., Giuffrida A. and Piomelli D. (2000) Reversal of dopamine D2 receptor responses by anandamide transport inhibitor. J. Neurosci. 20, 3401–3407. Berke J. D. and Hyman S. E. (2000) Addiction, dopamine and the molecular mechanisms of memory. Neuron 25, 515–532. Brotchie J. M. (2000) The neural mechanisms underlying levodopainduced dyskinesia in Parkinson’s disease. Ann. Neurol. 47, S105– S112. Calabresi P., Mercuri N. B., Sancesario G. and Bernardi G. (1993) Electrophysiology of dopamine-denervated striatal neurons. Implications for Parkinson’s disease. Brain 116, 433–452. Calabresi P., Centonze D. and Bernardi G. (2000) Electrophysiology of dopamine in normal and denervated striatal neurons. Trends Neurosci. 23, S57–S63. Centonze D., Picconi B., Gubellini P., Bernardi G. and Calabresi P. (2001) Dopaminergic control of synaptic plasticity in the striatum. Eur. J. Neurosci. 13, 1071–1077. Cepeda C., Hurst R. S., Altemus K. L., Flores-Hernandez J., Calvert C. R., Jokel E. S., Grandy D. K., Low M. J., Rubinstein M., Ariano M. A. et al. (2001) Facilitated glutamatergic transmission in the striatum of D2 dopamine receptor-deficient mice. J. Neurophysiol. 85, 659–670. Chase T. N. and Oh J. D. (2000) Striatal dopamine- and glutamatemediated dysregulation in experimental parkinsonism. Trends Neurosci. 23, S86–S91. Cravatt B. F., Demarest K., Patricelli M. P., Bracey M. H., Giang D. K., Martin B. R. and Lichtman A. H. (2001) Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase. Proc. Natl Acad. Sci. USA 98, 9371–9376. De Petrocellis L., Bisogno T., Maccarrone M., Davis J. B., Finazzi-Agro` A. and Di Marzo V. (2001) The activity of anandamide at vanilloid VR1 receptors requires facilitated transport across the cell membrane and is limited by intracellular metabolism. J. Biol. Chem. 276, 12856–12863. Fox S. H., Henry B., Hill M., Crossman A. and Brotchie J. (2002) Stimulation of cannabinoid receptors reduces levodopa-induced

dyskinesia in the MPTP-lesioned nonhuman primate model of Parkinson’s disease. Mov. Dis. 17, 1180–1187. Gerdeman G. and Lovinger D. M. (2001) CB1 cannabinoid receptor inhibits synaptic release of glutamate in rat dorsolateral striatum. J. Neurophysiol. 85, 468–471. Giuffrida A. and Piomelli D. (2000) The endocannabinoid system: a physiological perspective on its role in psychomotor control. Chem. Phys. Lipids 108, 151–158. Giuffrida A., Parsons L. H., Kerr T. M., Rodriguez de Fonseca F., Navarro M. and Piomelli D. (1999) Dopamine activation of endogenous cannabinoid signaling in dorsal striatum. Nat. Neurosci. 2, 358–362. Gubellini P., Picconi B., Bari M., Battista N., Calabresi P., Centonze D., Bernardi G., Finazzi-Agro` A. and Maccarrone M. (2002) Experimental parkinsonism alters endocannabinoid degradation: implications for striatal glutamatergic transmission. J. Neurosci. 22, 6900–6907. Herkenham M., Lynn A. B., Johnson M. R., Melvin L. S., de Costa B. R. and Rice K. C. (1991) Characterization and localization of cannabinoid receptor in the brain: a quantitative in vitro autoradiographic study. J. Neurosci. 11, 563–583. Hillard C. J. and Jarrahian A. (2000) The movement of N-arachidonoylethanolamine (anandamide) across cellular membranes. Chem. Phys. Lipids 108, 123–134. Howlett A. C. and Mukhopadhyay S. (2000) Cellular signal transduction by anandamide and 2-arachidonoylglycerol. Chem. Phys. Lipids 108, 53–70. Huang C.-C., Lo S.-W. and Hsu K.-S. (2001) Presynaptic mechanisms underlying cannabinoid inhibition of excitatory synaptic transmission in rat striatal neurons. J. Physiol. 523, 731–748. Jiang Z. G. and North R. A. (1991) Membrane properties and synaptic responses of rat striatal neurones in vitro. J. Physiol. 443, 533–553. Kita T., Kita H. and Kitai S. T. (1984) Passive electrical membrane properties of rat neostriatal neurons in an in vitro slice preparation. Brain Res. 300, 129–139. Maccarrone M., van der Stelt M., Rossi A., Veldink G. A., Vliegenthart J. F. G. and Finazzi-Agro` A. (1998) Anandamide hydrolysis by human cells in culture and brain. J. Biol. Chem. 273, 32332– 32339. Maccarrone M., Attina` M., Bari M., Cartoni A., Ledent C. and FinazziAgro` A. (2001a) Anandamide degradation and N-acylethanolamines level in wild-type and CB1 cannabinoid receptor knockout mice of different ages. J. Neurochem. 78, 339–348. Maccarrone M., Valensise H., Bari M., Lazzarin N., Romanini C. and Finazzi-Agro` A. (2001b) Progesterone up-regulates anandamide hydrolase in human lymphocytes: role of cytokines and implications for fertility. J. Immunol. 166, 7183–7189. Meschler J. P. and Howlett C. A. (2001) Signal transduction interactions between CB1 cannabinoid and dopamine receptors in the rat and monkey striatum. Neuropharmacology 40, 918–926. Moesgaard B., Petersen G., Jaroszewski J. W. and Hansen H. S. (2000) Age dependent accumulation of N-acyl-ethanolamine phospholipids in ischemic rat brain. A 31P NMR and enzyme activity study. J. Lipid Res. 41, 985–990. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates, 2nd edn. Academic Press, London. Pertwee R. G. (1997) Pharmacology of cannabinoid CB1 and CB2 receptors. Pharmacol. Ther. 74, 129–180. Picconi B., Pisani A., Centonze D., Battaglia G., Storto M., Nicoletti F., Bernardi G. and Calabresi P. (2002) Striatal metabotropic glutamate receptor function following experimental parkinsonism and chronic levodopa-treatment. Brain 125, 1–11.

 2003 International Society for Neurochemistry, J. Neurochem. (2003) 85, 1018–1025

Levodopa reverses striatal cannabinoid anomalies 1025

Schmid P. C., Krebsbach R. J., Perry S. R., Dettmer T. M., Maasson J. L. and Schmid H. H. (1995) Occurrence and postmortem generation of anandamide and other long-chain N-acylethanolamines in mammalian brain. FEBS Lett. 375, 117–120. Schwartig R. K. W. and Huston J. P. (1996) The unilateral 6-hydroxydopamine lesion model in behavioral brain research. Prog. Neurobiol. 50, 275–331. Sieradzan K. A., Fox S. H., Hill M., Dick J. P., Crossman A. R. and Brotchie J. M. (2001) Cannabinoids reduce levodopa-induced

dyskinesia in Parkinson’s disease: a pilot study. Neurology 57, 2108–2111. Tang K.-C., Low M. J., Grandy D. K. and Lovinger D. M. (2001) Dopamine-dependent synaptic plasticity in striatum during in vivo development. Proc. Natl Acad. Sci. USA 98, 1255–1260. Ungerstet U. and Arbuthnott G. W. (1970) Quantitative recording of rotational behavior in rats after 6-hydroxy-dopamine lesions of the nigrostriatal pathway. Brain Res. 24, 485–493.

 2003 International Society for Neurochemistry, J. Neurochem. (2003) 85, 1018–1025