Effects of rimonabant, a selective cannabinoid CB1 receptor antagonist, in a rat model of Parkinson's disease

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Effects of rimonabant, a selective cannabinoid CB1 receptor antagonist, in a rat model of Parkinson's disease Sara González 1 , Camila Scorticati 1 , Moisés García-Arencibia, Rosario de Miguel, José A. Ramos, Javier Fernández-Ruiz⁎ Department of Biochemistry and Molecular Biology, Faculty of Medicine, Complutense University, 28040-Madrid, Spain

A R T I C LE I N FO

AB S T R A C T

Article history:

Recent evidence suggest that the blockade of cannabinoid CB1 receptors might be

Accepted 6 December 2005

beneficial to alleviate motor inhibition typical of Parkinson's disease (PD). In the present

Available online 17 January 2006

study, we have explored the motor effects of rimonabant, a selective antagonist of CB1 receptors, in a rat model of PD generated by an intracerebroventricular injection of 6-

Keywords:

hydroxydopamine. Compared with rats subjected to unilateral injection of this toxin in the

Cannabinoid

medial forebrain bundle, this model allows nigral dopaminergic neurons be symmetrically

CB1 receptor

affected. Dose-response studies with 6-hydroxydopamine revealed that the application of

Rimonabant

200 μg per animal caused hypokinetic signs (decreased ambulatory activity, increased

Parkinson's disease

inactivity, and reduced motor coordination), which paralleled several signs of degeneration

Hypokinesia

of nigrostriatal dopaminergic neurons (dopamine depletion in the caudate-putamen, and

Basal ganglia

decreased mRNA levels for tyrosine hydroxylase and superoxide dismutase-1 and -2 in the

Dopamine

substantia nigra). In these conditions, the degree of hypokinesia and dopaminergic

GABA

degeneration may be considered moderate, comparable to the disturbances occurring in

Glutamate

early and middle stages of PD in humans, a period that might be appropriate to test the effects of rimonabant. There is also degeneration of other dopaminergic pathways out of the basal ganglia, but this does not appear to interfere significantly with the hypokinetic profile of these rats. Higher doses of 6-hydroxydopamine elevated significantly animal mortality and lower doses failed in general to reproduce motor inhibition. Like other animal models of PD, these rats exhibited an increase in the density of CB1 receptors in the substantia nigra, which is indicative of the expected overactivity of the cannabinoid transmission in this disease and supports the potential of CB1 receptor blockade to attenuate hypokinesia associated with nigral cell death. Thus, the injection of 0.1 mg/kg of rimonabant partially attenuated the hypokinesia shown by these animals with no effects in control rats, whereas higher doses (0.5–1.0 mg/kg) were not effective. We also found that the antihypokinetic effects of low doses of rimonabant did not influence the dopamine deficits of these animals, as well as it did not modify GABA or glutamate transmission in the caudate-putamen. In summary, rimonabant may have potential antihypokinetic activity in moderate parkinsonism at low doses, but this effect is not related to changes in dopaminergic, GABAergic, or glutamatergic transmission in the striatum. Therefore, the

⁎ Corresponding author. Fax: +34 91 3941691. E-mail address: [email protected] (J. Fernández-Ruiz). 1 Both authors contributed equally to this article. 0006-8993/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.12.014

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elucidation of the neurochemical substrate involved in this effect remains a major challenge for the future. © 2005 Elsevier B.V. All rights reserved.

1.

Introduction

Parkinson's disease (PD) is a progressive neurodegenerative disorder, in which the capacity for executing voluntary movements is gradually lost. The major clinical symptomatology in PD includes tremor, rigidity, and bradykinesia (slowness of movement). The main pathological hallmark of this disease is the degeneration of melanine-containing dopaminergic neurons of the substantia nigra pars compacta that leads to severe dopaminergic denervation of the striatum (for a recent review, see Blandini et al., 2000). Recently, we have found that cannabinoid CB1 receptor binding and the activation of G proteins by cannabinoid agonists were significantly increased in the postmortem basal ganglia of humans affected by PD (Lastres-Becker et al., 2001). The same stimulatory effect has been observed by Pisani et al. (2005) measuring the levels of endocannabinoid ligands in cerebrospinal fluids of PD patients. Interestingly, these patients were untreated (Pisani et al., 2005), so the effects found were not related to dopaminergic replacement therapy with levodopa frequently used in this disease, as we also concluded in our study with human tissues (Lastres-Becker et al., 2001). The increase in CB1 receptors was also seen in MPTP-treated marmosets, a primate PD model, and this disappeared after chronic levodopa administration to these animals (Lastres-Becker et al., 2001). The same pattern was observed by Maccarrone et al. (2003) for the increase in endocannabinoid levels reported by these authors in a rat model of PD (Gubellini et al., 2002), which was also reversed by levodopa. This suggests the existence of an unbalance between dopamine and endocannabinoids at the basal ganglia in PD (see Fernández-Ruiz and González, 2005, for review). Overactivity of the endocannabinoid transmission (recording CB1 receptors or endocannabinoid levels) has been also found in the basal ganglia in different rat models of PD (Mailleux and Vanderhaeghen, 1993; Romero et al., 2000; Di Marzo et al., 2000; Gubellini et al., 2002). However, the issue is controversial since other authors reported no changes (Herkenham et al., 1991), reductions (Silverdale et al., 2001), or dependency on a chronic levodopa cotreatment (Zeng et al., 1999). Despite these conflicts, most of data indicate that endocannabinoid transmission becomes overactive in the basal ganglia in PD. This occurred in the case of administration of reserpine (Di Marzo et al., 2000) or dopaminergic antagonists (Mailleux and Vanderhaeghen, 1993) or during the degeneration of these neurons caused by the local application of 6-hydroxydopamine (Mailleux and Vanderhaeghen, 1993; Romero et al., 2000; Gubellini et al., 2002) or MPTP (Lastres-Becker et al., 2001), and it is compatible with the hypokinesia that characterizes this disease. This would also support the suggestion that CB1 receptor antagonists might be useful to alleviate bradykinesia in PD, as well as to reduce the development of dyskinesia caused by prolonged replacement therapy with levodopa (Brotchie, 2000, 2003; Romero et al., 2000; Di Marzo et al., 2000; Lastres-Becker et al., 2001; Fox et al., 2002; see Fernández-Ruiz and González, 2005, for review). In theory,

CB1 receptor blockade would avoid the excessive inhibition of GABA uptake produced by the increased activation of CB1 receptors in striatal projection neurons (Maneuf et al., 1996; Romero et al., 1998), thus allowing a faster removal of this inhibitory neurotransmitter from the synaptic cleft, which would reduce hypokinesia. Several studies have recently addressed the capability of rimonabant, a selective antagonist of CB1 receptors, to improve hypokinesia in animal models of PD, but they have shown opposite results (Di Marzo et al., 2000; Meschler et al., 2001; El-Banoua et al., 2004). In addition, no effects were found in the only clinical trial developed so far (Mesnage et al., 2004). It is possible that the blockade of CB1 receptors might be effective only at specific phases of the disease. In this sense, Fernández-Espejo et al. (2005) have demonstrated that rimonabant improved motor inhibition in 6hydroxydopamine-lesioned rats with extremely high degeneration of dopaminergic neurons (N95% of neuronal loss) but not in rats with high dopaminergic degeneration (85–95%), which presents an additional advantage since it would allow for an antiparkinsonism compound in a stage of the disease when the classic dopaminergic therapy is generally failed. However, these authors did not examine whether rimonabant might improve motor inhibition in rats with more moderate lesions. On the other hand, rimonabant might provide alleviation of symptoms through mechanisms independent of recovering dopamine transmission, which might be particularly interesting for those patients that do not respond to classic therapy of dopaminergic replacement. In this context, the present study was designed to explore the motor effects of rimonabant in rats subjected to intracerebroventricular (icv) injection of 6-hydroxydopamine. This is a rat model of PD (Rodriguez Diaz et al., 2001) which, compared with the classic model of unilateral injection of this toxin in the medial forebrain bundle, allows that 6-hydroxydopamine affects nigral neurons in both brain sides, improving the homogeneity of experimental lesions and reaching a moderate degree of degeneration of dopaminergic neurons (see details in Rodriguez Diaz et al., 2001), that looks appropriate to test the effects of rimonabant in a condition different to those reported by Fernández-Espejo et al. (2005). This model seems also more appropriate for the analysis of bradykinesia than the classic model of unilateral injections which only allows the analysis of drug-induced rotational behavior (see Deumens et al., 2002, for review). In addition, by using controlled conditions (i.e., specific animal body weights, protection of noradrenergic neurons, and specific anesthesia; see details in Rodriguez Diaz et al., 2001), icv injections of 6-hydroxydopamine result in less mortality and side-effects (aphagia and adipsia) than other approaches (Berridge et al., 1989). The major problem of icv injection of 6hydroxydopamine is that it also produces the degeneration of other dopaminergic pathways out of the basal ganglia. However, the previous evidence (Rodriguez Diaz et al., 2001) and the preliminary analysis performed here have revealed that dopaminergic neurons in limbic and hypothalamic structures are less responsive to 6-hydroxydopamine than nigral dopaminergic neurons. In addition, the existence of degeneration in

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mesolimbic and hypothalamic dopaminergic neurons does not appear to interfere significantly with the hypokinetic profile of these rats (Rodriguez Diaz et al., 2001). Therefore, using this model, (i) we evaluated the existence of hypokinesia and degeneration of nigral tyrosine hydroxylase (TH)-positive neurons (measured by dopamine depletion in the caudate-putamen and TH-mRNA levels in the substantia nigra), (ii) we controlled the degree of impairment of dopamine transmission in limbic and hypothalamic structures, (iii) we assessed the occurrence of overactivity of the endocannabinoid transmission (up-regulation of CB1 receptors), as reported in previous PD models (Mailleux and Vanderhaeghen, 1993; Di Marzo et al., 2000; Romero et al., 2000; Lastres-Becker et al., 2001; Gubellini et al., 2002; Pisani et al., 2005), and (iv) we examined the effects of rimonabant on the degree of hypokinesia in this model and their relation with the correction of dopamine deficits and/or with the influence on other key transmitters in the basal ganglia, such as GABA or glutamate.

2.

Results

2.1. Characterization of rats injected i.c.v. with 6-hydroxydopamine as a model of PD First of all, we carried out dose-response studies, using a range of doses for 6-hydroxydopamine between 50 μg and 300 μg, to search a dose able to reproduce a parkinsonism phenotype characterized by moderate hypokinesia and dopamine deple-

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tion of the caudate-putamen. We also controlled that the impairment of other dopaminergic pathways was in a level compatible with the hypokinetic profile of these animals. The application of a dose of 200 μg per animal resulted in a set of behavioral and neurochemical modifications comparable to those occurring in early and middle phases of PD (Deumens et al., 2002) and similar to those reported by Rodriguez Diaz et al. (2001) who used the same experimental model. Higher doses (300 μg) elevated significantly the mortality (data not shown), and lower doses failed in general to reproduce motor inhibition (see Fig. 1). Thus, animals injected with 6-hydroxydopamine (200 μg) exhibited 14 days after the injection a reduction of movement of approximately 50% according to data obtained in a computer actimeter. The parameter most affected was the ambulatory activity (distance traveled: F (5,29) = 2.826, P b 0.05; Fig. 1). We also found trends towards a decrease in the mean velocity developed by the animals (F (5,29) = 1.828, P = 0.145; Fig. 1) and in the frequency of fast movements (F(5,28) = 2.063, P = 0.107; Fig. 1). We also recorded an equivalent trend towards an increase in the time spent by animals in inactivity (F(5,27) = 2.024, P = 0.115; Fig. 1). The motor inhibition developed by animals injected with 6hydroxydopamine (200 μg) was particularly evident when animals were analyzed in the rotarod. In this test, animals exhibited a high total number of falls and a low maximal working time (time on the rod) (Fig. 2), which indicate a notable impairment in motor coordination. It is also important to mention that we also recorded the responses of these rats and their controls in both tests (actimeter and rotarod) at 7

Fig. 1 – Motor activity recorded in a computerized actimeter in rats i.c.v. injected with 6-hydroxydopamine at different doses (50–200 μg) or saline (0 μg). Data corresponded to 14 days postinjection. Details in the text. Values are means ± SEM of 5–6 animals per group (n = 5 for vehicle and n = 6 for the different doses of 6-hydroxydopamine). Data were assessed by one-way analysis of variance followed by the Student–Newman–Keuls test (*P b 0.05).

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Fig. 2 – Motor response recorded in a rotarod in rats i.c.v. injected with 6-hydroxydopamine (200 μg) or saline. Data corresponded to 14 days postinjection. Details in the text. Values are means ± SEM of 5–6 animals per group (n = 5 for controls and n = 6 for 6-hydroxydopamine-lesioned animals). Data were assessed by Student's t test (*P b 0.05, **P b 0.01).

days post-injection and found that the animals exhibited the same trends as those observed at 14 days, although the magnitude of these changes was always lesser at this early time post-injection (data not shown). Based on this observation, we decided to carry out the neurochemical analyses and, in particular, to conduct the experiments with rimonabant at 14 days after the application of 6-hydroxydopamine (200 μg). 6-Hydroxydopamine-injected rats, that showed a decrease in the motor responses in the two tests, also showed a marked reduction in dopamine contents, accompanied by a reduction of its intraneuronal metabolite DOPAC, in the caudate-putamen (Table 1) and a reduction in mRNA levels for tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, in the substantia nigra (Fig. 3). Reductions were mostly in a range of 65–85%, which is concordant with our hypothesis to use a model of moderate dopaminergic injury that can be compared with the data published by Fernández-Espejo et al. (2005) using animals with more severe lesions (N85%) The data presented in Table 1 corresponded to the mean of amounts of dopamine or DOPAC in the right and the left caudate-putamen. However,

we processed both sides separately to establish whether there might be a possible asymmetry in the response to 6hydroxydopamine, but we found no differences (data not shown) in concordance with the previous results published by Rodriguez Diaz et al. (2001). We also recorded significant reductions in mRNA levels for superoxide dismutase-1 and -2 (Fig. 3), which are a part of the endogenous defenses against oxidative stress, a key event in the pathogenesis of PD. This was observed in the substantia nigra but not in the caudate-putamen. These last data strongly indicate the occurrence of degeneration of nigrostriatal dopaminergic neurons. Degeneration was also evident in other dopaminergic pathways, such as those reaching the nucleus accumbens or the medial basal hypothalamus (Table 1), but they did not reach the magnitude found in the caudateputamen, in particular in the case of the hypothalamus. In any case, these effects did not appear to influence hypokinetic profile of these animals. It is important to mention that this was also observed by Rodriguez Diaz et al. (2001), and it is concordant with the generally accepted notion that mesolimbic dopaminergic neurons also show some degree of degeneration during the progression of PD (Damier et al., 1999). We also addressed the changes in the status of CB1 receptors in the two regions of the basal ganglia, caudateputamen, and substantia nigra, affected by dopamine degeneration. Like other animal models of PD, these rats exhibited an increase in the density of CB1 receptors in the substantia nigra (Table 2) which is indicative of the expected overactivity of the cannabinoid transmission in this disease. However, a reduction of mRNA levels for this receptor subtype was also evident in the lateral caudate-putamen (Table 2), which might be indicative of a certain compensatory response in receptor

Table 1 – Concentrations of dopamine and DOPAC in several brain regions of rats i.c.v. injected with 6hydroxydopamine (200 μg) or saline Parameters Caudate-putamen DA contents (ng/mg protein) DOPAC contents (ng/ mg protein) Nucleus accumbens DA contents (ng/mg protein) DOPAC contents (ng/ mg protein)

Control rats

6-Hydroxydopaminelesioned rats

99.9 ± 14.1 (5)

35.9 ± 6.3 (6)**

16.5 ± 3.7 (5)

2.7 ± 0.7 (6)**

65.0 ± 2.7 (5)

27.7 ± 5.4 (6)*

21.8 ± 1.5 (5)

8.8 ± 2.8 (6)*

Medial–basal hypothalamus DA contents (ng/mg 3.32 ± 0.56 (5) protein) DOPAC contents (ng/ 0.39 ± 0.08 (5) mg protein)

2.48 ± 0.76 (6) 0.48 ± 0.12 (6)

Data corresponded to 14 days post-injection. Details in the text. Values are means ± SEM with the number of animals per group in parentheses. Data were assessed by Student's t test (*P b 0.005, **P b 0.0005).

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2.2. Effects of rimonabant on motor activity and neurochemical parameters in rats i.c.v. injected with 6-hydroxydopamine The injection of rimonabant at a dose of 0.1 mg/kg partially attenuated the hypokinesia shown by these animals (rimonabant treatment: F(3,41) = 3.68, P b 0.05) with no effects in control rats, although highest doses (0.5–1.0 mg/kg) exhibited a trend towards an enhancement of motor inhibition in both 6-hydroxydopamine-lesioned and control rats (Fig. 4). This last effect remained only as a trend because the two groups showed high values of SEM generally indicative of a greater variation in the individual animal response (see Fig. 4). We also found that the antihypokinetic effects of low doses of rimonabant did not influence the dopamine deficits of these animals. Thus, the low values of dopamine (6-hydroxydopamine lesion: F(1,54) = 92.45, P b 0.0001) and DOPAC (6hydroxydopamine lesion: F(1,54) = 40.59, P b 0.0001) contents and TH activity (6-hydroxydopamine lesion: F(1,45) = 9.90, P b 0.005) measured in the caudate-putamen of 6-hydroxydopamine remained low after the administration of the three

Fig. 3 – mRNA levels for superoxide dismutase-1 (SOD-1), superoxide dismutase-2 (SOD-2), and tyrosine hydroxylase (TH) in the basal ganglia of rats i.c.v. injected with 6-hydroxydopamine (200 μg) or saline. Data corresponded to 14 days postinjection. Details in the text. Values are means ± SEM of 6–8 animals per group (n = 6 for controls and n = 8 for 6-hydroxydopamine-lesioned animals). Data were assessed by Student's t test (*P b 0.01, **P b 0.005, ***P b 0.0005).

synthesis to the increase in receptor density. No changes were found for other cortical or subcortical (limbic) regions for both parameters (data not shown).

Table 2 – Cannabinoid CB1 receptor binding and mRNA levels in the basal ganglia of rats i.c.v. injected with 6-hydroxydopamine (200 μg) or saline Parameters

Control rats

Receptor binding (fmol/mg protein) Lateral caudate148.1 ± 7.7 (8) putamen Medial caudate109.2 ± 4.6 (6) putamen Substantia nigra 264.4 ± 16.2 (6) Receptor mRNA levels (optical density) Lateral caudate0.444 ± 0.017 (7) putamen Medial caudate0.205 ± 0.007 (6) putamen

6-Hydroxydopaminelesioned rats 137.3 ± 6.6 (6) 99.9 ± 4.9 (6) 324.3 ± 12.7 (6)*

0.369 ± 0.020 (7)* 0.187 ± 0.013 (8)

Data corresponded to 14 days post-injection. Details in the text. Values are means ± SEM with the number of animals per group in parentheses. Data were assessed by Student's t test (*P b 0.05).

Fig. 4 – Effects of an acute injection of rimonabant (0.1–1.0 mg/kg) or vehicle on motor activity recorded in a computerized actimeter and on the ratio between dopamine and its intraneuronal metabolite DOPAC in the striatum of rats i.c.v. injected with 6-hydroxydopamine (200 μg) or saline. Data corresponded to 14 days postinjection. Details in the text. Values are means ± SEM of 6–8 animals per group (n = 6 for vehicle and n = 8 for the different doses of rimonabant). Data were assessed by two-way analysis of variance followed by the Student–Newman–Keuls test (*P b 0.005 versus controls, #P b 0.05 versus vehicle).

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Table 3 – Effects of an acute injection of rimonabant (0.1–1.0 mg/kg) or vehicle on dopamine contents and tyrosine hydroxylase activity in the caudate-putamen of rats i.c.v. injected with 6-hydroxydopamine (200 μg) or saline Parameters DA contents (ng/mg protein)

DOPAC contents (ng/mg protein)

TH activity (ng/mg protein h)

GABA contents (μg/mg protein)

Glutamate contents (μg/mg protein)

Rimonabant (mg/kg)

Control rats

0 0.1 0.5 1.0 0 0.1 0.5 1.0 0 0.1 0.5 1.0 0 0.1 0.5 1.0 0 0.1 0.5 1.0

90.4 90.1 96.0 151.4 18.7 13.5 21.3 22.6 172.3 200.8 196.2 221.6 3.45 2.61 3.07 3.26 12.2 13.2 11.8 14.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

15.1 (9) 12.4 (9) 15.5 (8) 34.5 (6) 4.7 (9) 2.3 (9) 4.6 (8) 7.9 (6) 43.5 (7) 43.7 (8) 29.3 (6) 20.6 (6) 0.56 (8) 0.39 (8) 0.35 (8) 0.78 (6) 0.2 (6) 0.9 (6) 0.9 (6) 1.5 (5)

6-Hydroxydopamine-lesioned rats 10.1 13.4 12.3 11.3 2.4 2.8 2.7 2.4 109.6 108.4 112.9 138.7 2.53 2.41 3.87 4.36 13.4 11.3 13.0 10.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.7 (8)*** 3.9 (8)*** 2.9 (8)*** 7.7 (6)*** 1.0 (8)** 1.0 (8)*** 0.4 (8)*** 1.5 (6)** 29.4 (7) 26.4 (7)* 26.2 (6)* 29.8 (6)* 0.23 (7) 0.27 (6) 0.40 (7) 0.58 (6) 1.1 (6) 1.5 (6) 0.4 (6) 1.6 (5)*

Data corresponded to 14 days post-injection. Details in the text. Values are means ± SEM with the number of animals per group in parentheses. Data were assessed by two-way analysis of variance (lesion × rimonabant treatment) followed by the Student–Newman–Keuls test (*P b 0.05, **P b 0.005; ***P b 0.0005 versus the corresponding control group).

doses of rimonabant (Table 3). In addition, no changes were found for the ratio between the contents of dopamine and its metabolite, which is an index of activity of dopaminergic neurons (Fig. 4), thus indicating that the recovery of movement elicited by rimonabant in 6-hydroxydopamine-injected rats is a dopamine-independent phenomenon. This drove us to explore the possible involvement of other transmitters in the basal ganglia such as GABA or glutamate. However, we found that GABA contents in the caudate-putamen were not affected in a statistically significant manner neither by 6hydroxydopamine lesion nor by the treatment with rimonabant (Table 3), at least at the lowest dose, the one that was effective against hypokinesia, and the same happened for glutamate (Table 3). In contrast with the lack of dopamine mediation in the antihypokinetic effect of low doses of rimonabant in 6hydroxydopamine-injected rats, the trend toward a further motor depression caused by higher doses of this antagonist in lesioned and control rats (Fig. 4) might be mediated through a reduction in dopamine activity in the caudate-putamen, as revealed by the trends towards a reduction recorded in DOPAC/ DA ratio, in particular in control rats (Fig. 4). In addition, the analysis of GABA contents in this situation also revealed that the highest doses of rimonabant tended to increase the low GABA contents recorded in the caudate-putamen of 6-hydroxydopamine-lesioned rats (F(3,48) = 2.53, P = 0.0639; see Table 3), a fact that did not occur in controls (Table 3). The opposite happened for glutamate since the contents of this neurotransmitter after the administration of the highest dose of rimonabant to 6-hydroxydopamine-lesioned rats were significantly lower than when this dose was administered to controls (two-way interaction: F(3,37) = 3.67, P b 0.05; see Table 3). These observations are all compatible with the trend

towards a decrease in the ambulatory activity caused by rimonabant at the dose of 1 mg/kg (Fig. 4).

3.

Discussion

The hypokinetic profile of PD (Blandini et al., 2000) and the evidence of an increase in the endocannabinoid transmission in the basal ganglia in patients and animal models of this disease (Mailleux and Vanderhaeghen, 1993; Romero et al., 2000; Di Marzo et al., 2000; Lastres-Becker et al., 2001; Gubellini et al., 2002; Pisani et al., 2005) support the potential of rimonabant or other CB1 receptor antagonists to alleviate bradykinesia in this disorder. The same conclusion derives from the data of CB1 receptors found in the animal model of PD used in the present study, since we recorded an increase in the density of these receptors in the substantia nigra of these parkinsonian rats. This was accompanied by a small reduction in the levels of transcripts for this receptor subtype in the striatum, the area where cell bodies of striatal projection neurons containing CB1 receptors are located (Mailleux and Vanderhaeghen, 1992), which might be indicative that a compensation in receptor synthesis seems to take place. However, an alternative possibility is that the increase of CB1 receptors recorded in the substantia nigra occurs in glutamatergic terminals coming from the subthalamic nucleus, which also contain CB1 receptors (Mailleux and Vanderhaeghen, 1992), rather than in striatal GABA projection neurons. If this was the case, the possible reduction of CB1 receptor binding in striatonigral GABAergic neurons (that the decrease in mRNA levels for this receptor subtype seems to indicate) would be completely surpassed by the marked increase in CB1 receptor binding in subthalamonigral glutamatergic terminals. This

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would result in a significant increase in CB1 receptor binding in the substantia nigra, the key region for the degeneration typical of PD, in concordance with our previous studies in humans and MPTP-treated primates (Lastres-Becker et al., 2001). Despite the strong evidence indicating a beneficial effect of CB1 receptor antagonists in PD, the data obtained so far do not completely support this assumption since, while some authors have demonstrated that rimonabant might be effective to increase movement in animal models of PD (Di Marzo et al., 2000; El-Banoua et al., 2004), other studies have documented conflicting results (Meschler et al., 2001). In addition, the only clinical study available to date has not demonstrated an efficacy of rimonabant in PD (Mesnage et al., 2004). It is possible that rimonabant may be effective in PD only in specific groups of patients and/or in specific phases of the disease. Thus, rimonabant might work only in those groups of patients more refractory to classic dopaminergic replacement therapy. If this was the case, it would explain the lack of positive results in the clinical trial conducted with rimonabant by Mesnage et al. (2004) because the patients included in this study were all clinically homogeneous and with an excellent response to levodopa treatment. The question is whether rimonabant would have been effective if the authors would have included in their study patients with lesser response to levodopa. With our present data, we can not demonstrate this hypothesis, but we can provide some indirect evidence. We have observed that rimonabant, at a dose of 0.1 mg/kg, increased the low ambulatory activity exhibited by rats lesioned with 6-hydroxydopamine, but these positive effects were not produced through a transient recovery of the dopaminergic deficit typical of these rats, so they were not dopamine-dependent. The second of the two options (that rimonabant works only at specific phases of the disease) is supported by recent data published by Fernández-Espejo et al. (2005) who described a positive effect in rats unilaterally lesioned with 6-hydroxydopamine but only in conditions of very severe nigral degeneration (N95% neuronal loss), which would be equivalent to very advanced stages in the human pathology and not in rats with 85–95% of neuronal loss (see details in the Introduction). Our present study was addressed to complete the findings derived from the study conducted by Fernández-Espejo et al. (2005), using a model of PD with a more moderate degree of hypokinesia and dopaminergic injury (approximately 65–85% of dopamine impairment) and with the additional advantage to produce a bilateral damage more appropriate to test compounds potentially beneficial on motor inhibition than unilateral models (see Deumens et al., 2002, for details on the advantages of bilateral models). Our results indicate that the blockade of CB1 receptors might be effective also in conditions of more moderate dopaminergic lesions although the key condition here would be the use of low doses of rimonabant (around 0.1 mg/kg), mostly the same doses that were used by Fernández-Espejo et al. (2005). In our model, higher doses of rimonabant, rather than improving motor deficits, tended to aggravate bradykinesia, possibly through an effect involving a reduction in dopamine and glutamate transmission and an enhancement in GABA activity in the caudate-putamen (see Fernández-Ruiz and González, 2005, for a recent review). Therefore, the use of low doses seems critical.

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Another important aspect is that rimonabant would act here to reduce bradykinesia through neurobiological substrates, whose description remains to be determined. To explore this issue, we first examined the two transmitters that are more affected in PD, GABA, and, in particular, dopamine. We observed that rimonabant did not reverse the dopamine deficit produced by 6-hydroxydopamine in the caudate-putamen, as well as it did not affect GABA transmission in this structure. An additional possibility was that rimonabant might be able to influence glutamate transmission at the level of cortical efferent projections to the striatum, a target that has been proposed of relevance for improving parkinsonian signs in PD patients (see Ossowska et al., 2002, for details). This option was also supported by the fact that the activation of CB1 receptors has been reported to inhibit glutamate release (see Fernández-Ruiz and González, 2005, for review), so the blockade of these receptors with rimonabant, an antagonist with inverse agonist properties (Pertwee, 1997), would be expected to increase glutamate release. However, our data demonstrated that glutamate contents in the caudate-putamen were not altered by the application of 6hydroxydopamine and by the treatment with rimonabant. Therefore, the elucidation of the neurochemical substrate(s) involved in the beneficial effects of rimonabant in these rats remains the major challenge for the future. In this respect, an attractive possibility, in view of the lack of effects of rimonabant on presynaptic activities of the three major transmitters in the basal ganglia, is that the blockade of CB1 receptors might facilitate dopaminergic receptor function by interactions at the postsynaptic level, in absence of an increase in dopamine synthesis and release from presynaptic terminals. In support of this hypothesis, there is evidence that (i) CB1 receptors colocalize with D1 and D2 receptors in striatal projection neurons (Herkenham et al., 1991); (ii) the activation of D2 but not D1 receptors is associated with an increase in the production of endocannabinoids in control rats (Giuffrida et al., 1999); (iii) these endocannabinoids, acting through CB1 receptors located in striatal projection neurons, are able to reduce the efficacy of selective agonists that activate D1 (Meschler and Howlett, 2001) or D2 (Beltramo et al., 2000) receptors in control rats; and (iv) rimonabant enhanced the stimulation of motor activity induced by D2 receptor agonists in control rats (Giuffrida et al., 1999). Therefore, it is possible that rimonabant, by closing CB1 receptors, might release endocannabinoid-induced inhibition of dopaminergic receptors, thus producing a higher activation of these receptors, even in conditions of low dopamine levels, as those found in PD. A similar hypothesis has been also proposed by Fernández-Espejo et al. (2005) to explain the potential of rimonabant to improve parkinsonian signs in animals with very severe dopaminergic lesions. Therefore, taking the previous reports (Di Marzo et al., 2000; Mesnage et al., 2004; Fernández-Espejo et al., 2005) and the data obtained here, we can conclude that rimonabant may have potential antihypokinetic activity in PD. However, the dosage of rimonabant (it must be used at low doses, since higher doses might increase motor disability) and the severity of dopaminergic injury (rimonabant appears to work only in conditions of moderate or very severe injury) may be critical factors for determining the efficacy of this CB1 receptor antagonist to

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improve motor inhibition in PD. The neurochemical mechanism(s) underlying this antihypokinetic effects in the conditions of moderate damage used here does not appear to be related to a transient recovery of dopamine transmission or the normalization of GABA or glutamate activities, so its elucidation remains a major challenge for the future. Another critical factor for rimonabant efficacy in PD might be a low response to levodopa, but this has not been directly studied here, although the observation that the antihypokinetic activity of rimonabant was not produced through a recovery in the activity in those dopaminergic terminals that did not degenerate by the application of 6-hydroxydopamine might indirectly support the dopamine-independent characteristics of rimonabant effect in this disease.

of motor inhibition) or small dopamine impairment (b50% of dopamine depletion)] were not included in the study. 4.1.3. Treatment with rimonabant In those experiments addressed to establish the potential of the cannabinoid CB1 receptor blockade in the treatment of hypokinesia in PD, 6-hydroxydopamine (200 μg)-lesioned and control rats were subjected at 2 weeks postinjection to an acute i.p. administration of rimonabant (kindly supplied by Sanofi-Synthelabo, Montpellier, France), at three doses: 0.1, 0.5, and 1.0 mg/kg (n = 8 animals per dose), or vehicle (Tween 80-saline, 1:16; n = 6 animals). Ten minutes later, animals were assessed in a computerized actimeter for a period of 10 min, at the end of which the animals were killed by rapid and careful decapitation. Their brains were rapidly removed and frozen in 2-methylbutane cooled in dry ice and stored at −80 °C for neurochemical evaluation. 4.2.

4. 4.1.

Evaluation of motor activity: actimeter and rotarod

Experimental procedure Animals, surgical procedures, treatments, and sampling

4.1.1. Animals Male Sprague–Dawley rats (N2 months; approximately 350 g weight at the start of the experiments) were housed in a room with controlled photoperiod (08:00–20:00 light) and temperature (23 ± 1 °C). They had free access to standard food and water. All experiments were conducted according to European rules (directive 86/609/EEC). 4.1.2. Intracerebroventricular injection of 6-hydroxydopamine The procedure used to induce moderate parkinsonism in rats was based in the model previously described by Rodriguez Diaz et al. (2001) using intracerebroventricular application of 6hydroxydopamine. After pretreatment (30 min before) with desipramine (25 mg/kg, i.p.), rats were anesthetized (ketamine 40 mg/kg + xylazine 4 mg/kg, i.p.) and subjected to stereotaxic injection into the third ventricle (coordinates: midline, 2 mm posterior to bregma and 8 mm below the dura, according to Paxinos and Watson, 1998; see Rodriguez Diaz et al., 2001, for details) of 6-hydroxydopamine free base (50, 100, 125, 150, 200, or 300 μg in a volume of 5 μl of saline containing 0.05% ascorbate to avoid oxidation; n = 6 animals per each dose) or saline (for control rats; n = 5 animals). These coordinates are the same than those used by Rodriguez Diaz et al. (2001) and were previously validated in a series of preliminary injections using black ink in a few animals and visual inspection of the injection site, as well as of the damage produced by the needle, after slicing the brains at different levels close to the injection place. The injection was done slowly (0.5 μl/30 s), and the needle was left in place for 5 min before being slowly withdrawn. This avoids the occurrence of reflux and of a rapid increase in intracranial pressure. To avoid a dose effect, the experiment was conducted in different series each containing all doses and the corresponding controls. After the application of 6-hydroxydopamine or saline, animals were behaviorally examined at 1 and 2 weeks postinjection in a computerized actimeter or in a rotarod, and they were killed immediately after their last behavioral assessment. Sacrifice was done by rapid and careful decapitation, and their brains were rapidly removed and frozen in 2methylbutane cooled in dry ice and stored at −80 °C for evaluation of the degree of dopaminergic injury and basal levels of CB1 receptors and other phenotypical markers in the basal ganglia. We always did a verification of the correct injection site at the time that these brains were sliced for the different neurochemical analyses. Animals showing an incorrect injection site or any other potential problems [lack of hypokinesia (b30%

Motor activity was analyzed in a computerized actimeter (Actitrack, Panlab, Barcelona, Spain). This consisted of a 45 × 45-cm arena, with infrared beams all around, spaced 2.5 cm, coupled to a computerized control unit, which allows the analysis of the following parameters: (i) distance run in the actimeter (ambulation); (ii) mean velocity to run all that distance; (iii) time spent in fast movements (speed N5 cm/s); and (iv) resting time (inactivity). The analysis of motor activity was done for a period of 10 min, a time that we have previously demonstrated that is adequate for testing the effects of cannabinoid agonists or antagonists (see Fernández-Ruiz and González, 2005, for review). In a separate group of animals, motor coordination was measured in a rotarod apparatus following the procedure described by Rozas et al. (1997). This consisted of a rotating spindle (diameter: 7.3 cm for rats) with individual compartment for each animal. The system software allows preprogramming of session protocols with varying rotational speeds (0–80 rpm). Infrared beams are used to detect when a rat has fallen onto the grids beneath the rotarod. The system logs the fall as the end of the experiments for that subject and the total time running on the rotarod (maximal working time), as well as the time of the fall and all experimental set-up parameters are recorded. Analyses presented in Fig. 2 were carried out at 18 rpm. 4.3.

Autoradiographic analyses

4.3.1. Brain slicing Coronal sections 20 μm thick were cut in a cryostat, according to the Paxinos and Watson (1998) atlas. Sections were thaw-mounted onto RNAse-free gelatin/chrome alum coated slides and dried briefly at 30 °C and stored at −80 °C until used. Adjacent sections to those used for autoradiographic analysis were stained with cresylviolet for the identification of the different brain nuclei. 4.3.2. Autoradiography of cannabinoid receptor binding The protocol used is basically the method described by Herkenham et al. (1991). Briefly, slide-mounted brain sections were incubated for 2.5 h, at 37 °C, in a buffer containing 50 mM TRIS with 5% bovine serum albumin (fatty acid-free), pH 7.4, and 10 nM [3H]-CP-55,940 (Du Pont NEN) prepared in the same buffer, in the absence or the presence of 10 μM non-labeled CP-55,940 (kindly supplied by Pfizer) to determine the total and the nonspecific binding, respectively. Following this incubation, slides were washed in 50 mM TRIS buffer with 1% bovine serum albumin (fatty acid-free), pH 7.4, for 4 h (2 × 2 h) at 0 °C, dipped in ice-cold distilled water, and then dried under a stream of cool dried air. Autoradiograms were generated by apposing the labeled tissues, together with autoradiographic standards ([3H]

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micro-scales, Amersham), to tritium-sensitive film ([3H]-Hyperfilm MP, Amersham) for a period of 2 weeks. Autoradiograms were developed (D-19, Kodak) for 4 min at 20 °C, and the films were analyzed and quantitated in a computer-assisted videodensitometer using the curve generated from [3H]-standards. 4.3.3. Analysis of mRNA levels for CB1 receptor, TH, SOD-1, and SOD-2 by in situ hybridization The analysis of CB1 receptor mRNA levels was carried out according to Rubino et al. (1994). Briefly, sections were fixed in 4% paraformaldehyde for 5 min and, after rinsing twice in phosphate buffer saline, were acetylated by incubation in 0.25% acetic anhydride, prepared in 0.1 M triethanolamine/0.15 M sodium chloride (pH 8.0), for 10 min. Sections were rinsed in 0.3 M sodium chloride/0.03 M sodium citrate, pH 7.0, dehydrated, and delipidated by ethanol/chloroform series. A mixture (1:1:1) of the three 48-mer oligonucleotide probes complementary to bases 4– 51, 349–396, and 952–999 of the rat CB1 receptor cDNA (Du Pont; the specificity of the probes used was assessed by Northern Blot analysis) was 3′-end labeled with [35S]-dATP using terminal deoxynucleotidyl-transferase. Sections were, then, hybridized with [35S]-labeled oligonucleotide probes (7.5 × 105 dpm per section), washed, and exposed to X-ray film (βmax, Amersham) for 1 week, and developed (D-19, Kodak) for 6 min at 20 °C. The intensity of the hybridization signal was assessed by measuring the grey levels in the autoradiographic films with a computerassisted videodensitometer. Adjacent brain sections were cohybridized with a 100-fold excess of cold probe or with RNAse to assert the specificity of the signal (data not shown). Similar procedures were used for the analysis of mRNA levels of TH, SOD1, and SOD-2. We used commercial probes (NEN-Du Pont, Itisa, Madrid, Spain) for TH (García-Gil et al., 1998), and synthetic 40–45 base probes, selected from the previously published sequence, for SOD-1 (5′-TCCAGTCTTTGTACTTTCTTCATTTCCACCTTTGCCCAAGTCATC-3′; Kunikowska and Jenner, 2001) and SOD-2 (5′TGATCTGCGCGTTAATGTGCGGCTCCAGCGCGCCATAGT-3′; Kunikowska and Jenner, 2001). 4.4. Determinations of GABA, glutamate and dopamine contents, and TH activity by HPLC with electrochemical detection 4.4.1. Brain dissection Brains were used to manually obtain coronal slices (around 500 μm thick) at the level containing the caudate-putamen. Subsequently, this structure was dissected and homogenized in 20–40 vol of cold 150 mM potassium phosphate buffer, pH 6.8. Each homogenate was distributed for the analysis, using HPLC coupled to electrochemical detection, of (i) GABA and glutamate contents, following the procedure described by Smith and Sharp (1994), (ii) dopamine and L-3,4-dihydroxyphenylacetic acid (DOPAC) contents, following the method described by Romero et al. (1995), and (iii) TH activity, following the procedure described by Nagatsu et al. (1979). An aliquot of each homogenate was used to analyze protein concentration (Lowry et al., 1951). In those experiments addressed to characterize the deficits caused by icv administration of 6hydroxydopamine, dopamine and DOPAC contents were also analyzed in the nucleus accumbens and the medial basal hypothalamus.

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the samples for determination of GABA and glutamate contents that were subjected to a previous derivatization process with ophthaldehide (OPA)-sulfite solution (14.9 mM OPA, 45.4 mM sodium sulfite and 4.5% ethanol in 327 mM borate buffer, pH 10.4) (see details in González et al., 1999; Cabranes et al., 2005). HPLC system consisted of the following elements. The pump was an isocratic Spectra-Physics 8810. The column was a RP-18 (Spherisorb ODS-2; 150 mm, 4.6 mm, 5 μm particle size; Waters, Massachusetts, USA). The mobile phase, previously filtered and degassed, consisted of (i) 0.06 M sodium dihydrogen phosphate, 0.06 mM EDTA, and 20–30% methanol (pH 4.4), for determination of GABA and glutamate, and (ii) 100 mM citric acid, 100 mM sodium acetate, 1.2 mM heptane sulphonate, 1 mM EDTA, and 7% methanol (pH 3.9), for the determination of dopamine and DOPAC. The flow rate was 0.8 ml/min. The effluent was monitored with a Metrohm bioanalytical system amperometric detector using a glassy carbon electrode. The potential was 0.85 V (GABA and glutamate) relative to an Ag/AgCl reference electrode with a sensitivity of 50 nA (approximately 2 ng per sample). In the case of the determination of dopamine and DOPAC, the effluent was monitored with a coulochemical detector (Coulochem II, ESA) using a procedure of oxidation/reduction (conditioning cell: +360 mV; analytical cell #1: +50 mV; analytical cell #2: −340 mV), procedure that reaches a sensitivity of 50 nA (10 pg per sample). In both cases, the signal was recorded on a Spectra-Physics 4290 integrator. The results were obtained from the peaks and calculated by comparison with the area under the corresponding internal standard peak. Values were expressed as ng or μg/mg of protein. 4.4.3. Analysis of TH activity The aliquots used for the measurement of TH activity were incubated at 37 °C in the presence of 0.1 M sodium acetate, 1 mM 6methyl-5,6,7,8-tetrahydropterine (prepared in 1 M mercaptoethanol solution), 0.1 mg/ml catalase, and 0.2 mM L-tyrosine. For the blank incubation, L-tyrosine was replaced by D-tyrosine. Blank tubes containing 1 μM L-3,4-dihydroxyphenylalanine (L-DOPA) were also used as an internal standard for each tissue. After 30 min of incubation, the reaction was stopped by the addition of 0.2 N perchloric acid containing 0.2 mM sodium disulfite and 0.45 mM EDTA. Dihydroxybenzylamine was also added as an internal standard for HPLC determination. The amounts of L-DOPA formed were evaluated by HPLC following the same procedure as for the direct analysis of dopamine and DOPAC contents, with the only difference of a previous extraction with alumina. Values were expressed as ng/mg of protein per hour. 4.5.

Statistics

Data were assessed by Student's t test [rotarod data, analyses of neurotransmitters, CB1 receptors and mRNA levels in rats injected with 6-hydroxydopamine (200 μg) or saline] or one (dose-response studies with 6-hydroxydopamine and evaluation of motor activity)- or two-way (neurochemical and behavioral data after the administration of rimonabant to 6-hydroxydopamine-injected rats) analysis of variance followed by the Student–Newman– Keuls test, as required.

Acknowledgments 4.4.2. Analysis of GABA, glutamate, and dopamine contents The aliquots used for the measurement of neurotransmitter contents were diluted (1/2) with 0.4 N perchloric acid containing 0.4 mM sodium disulfite, 0.90 mM EDTA, and the corresponding internal standard (β-aminobutyric acid for GABA and glutamate, and dihydroxybenzylamine for catecholamines). Afterwards, samples were centrifuged for 3 min (15000 g), and the supernatants were directly injected into the HPLC system, expect in the case of

This work has been supported by grants from MCYT (SAF200308269) and “Red CIEN” (C03/06). Sara González is a postdoctoral fellow supported by the “Red CIEN”, whereas Camila Scorticati and Moisés García-Arencibia are predoctoral fellows supported by “Fundación Carolina” and “MCYT (Plan FPI)”. Authors are indebted to Sanofi-Synthelabo for the gift of SR141716.

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