A model of l-DOPA-induced dyskinesia in 6-hydroxydopamine lesioned mice: relation to motor and cellular parameters of nigrostriatal function

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Abstract

l-DOPA-induced dyskinesia is a major complication of l-DOPA pharmacotherapy in Parkinson's disease, and is thought to depend on abnormal cell signaling in the basal ganglia. In this study, we have addressed the possibility to model l-DOPA-induced dyskinesia in the mouse at both the behavioral and the molecular level. C57BL/6 mice sustained unilateral injections of 6-hydroxydopamine (6-OHDA) either in the medial forebrain bundle (MFB) or in the sensorimotor part of the striatum. Both types of lesion produced a similar degree of forelimb akinesia on the contralateral side of the body. The lowest dose of l-DOPA that could significantly relieve this akinetic deficit (i.e., 6 mg/kg) did not differ between MFB and intrastriatal lesions. The l-DOPA threshold dose for the induction of dyskinesia did however differ between the two lesion types. A daily dose of 6 mg/kg l-DOPA caused MFB lesioned mice to develop abnormal movements affecting orofacial, trunk, and forelimb muscles on the side contralateral to the lesion, whereas a daily dose of 18 mg/kg was required to produce comparable dyskinetic effects in the intrastriatally lesioned animals. The development of abnormal movements was accompanied by a striatal induction of ΔFosB-like proteins and prodynorphin mRNA, that is, molecular markers that are associated with l-DOPA-induced dyskinesia in both rats and nonhuman primates. We conclude that 6-OHDA lesioned mice exhibit behavioral and cellular features of akinesia and l-DOPA-induced dyskinesia that are similar to those previously characterized in rats. The mouse model of l-DOPA-induced dyskinesia will provide a useful tool to study the molecular determinants of this movement disorder in transgenic mice strains.

Introduction

The high level of genomic homology between rodents and humans justifies the use of mice as a model of human disorder Bradley, 2002, Gainetdinov et al., 2001, Tarantino and Bucan, 2000. Moreover, the ever-increasing availability of transgenic mouse strains offers new opportunities to study the impact of specific genetic mutations on both mechanisms and symptoms of disease. Establishing experimental paradigms that can mimic specific pathogenic processes and their behavioral outcome in mice can therefore be considered a very important task for the neurobiologist.

In addition to being one of the most common neurodegenerative disorders in humans, Parkinson's disease (PD) is a prominent topic of neurobiological investigation. The typical motor features of the disease (akinesia, rigidity, tremor, and postural abnormalities) can be replicated in animals using selective neurotoxins that cause degeneration of nigral dopamine (DA) neurons. These motor features are amenable to treatment with the main antiparkinsonian medication that is used in the clinic, namely l-DOPA (for review, see Cenci et al., 2002). In both human PD and the corresponding animal models, treatment with l-DOPA does however induce motor complications that limit its utility Cenci et al., 2002, Obeso et al., 2000. Among these complications, the occurrence of abnormal involuntary movements (dyskinesia) can be particularly debilitating (Fahn, 2000). Our group has established a paradigm for the classification and rating of l-DOPA-induced dyskinesia in the rat. In our paradigm, rats with unilateral 6-hydroxydopamine (6-OHDA) lesions are treated chronically with low, therapeutic doses of l-DOPA, causing about 80% of them to develop abnormal involuntary movements (AIMs) Andersson et al., 1999, Picconi et al., 2003. These movements reproduce the time course of peak–dose dyskinesia in PD, and affect predominantly the side of the body contralateral to the lesion Lee et al., 2000, Winkler et al., 2002. The development of AIMs is associated with changes in gene and protein expression in DA-denervated striatal neurons. In particular, we have found a strong, positive correlation between the rats' AIM scores and striatal levels of ΔFosB-like proteins and prodynorphin (PDyn) mRNA Andersson et al., 2001, Cenci et al., 1998, Winkler et al., 2002. The prime molecular determinants by which l-DOPA causes changes in gene expression in dyskinetic rats are as yet unknown, and further investigation is required to dissect the contribution of specific membrane receptors and signaling proteins to these events. A number of different laboratories have generated transgenic mouse strains carrying mutations of signaling proteins and membrane receptors that are critical to the function of striatal neurons. A paradigm for the study of l-DOPA-induced dyskinesia in mice has however not been characterized. Previous studies have used measures of stereotyped grooming and contralateral rotation as a predictor of the dyskinetic effect of l-DOPA in mice (Fredduzzi et al., 2002). We have however shown that the AIMs induced by l-DOPA in the rat are conceptually and phenomenologically distinguishable from stereotypic behavior (Andersson et al., 2001), and have different pharmacological features and anatomical substrates compared to rotation Lundblad et al., 2002, Winkler et al., 2002. It is yet unknown whether similar AIMs can be observed and measured in mice undergoing chronic l-DOPA treatment. Although rodent species share a high degree of structural similarity, neurotoxic models and behavioral testing methods that have been established in the rat are not immediately translatable to the mouse with the same degree of success and reliability. Indeed, mice and rats may show different sensitivities to the effects of DA-depleting neurotoxins and DA replacement therapy. In addition, mice have a seemingly simplified behavioral repertoire compared to rats (Whishaw et al., 2001).

In the present study, we have asked whether a reliable model of parkinsonism and l-DOPA-induced dyskinesia can be obtained in C57BL/6 mice using techniques analogous to those previously established in rats. The mice were subjected to unilateral injection of 6-OHDA either in the origin or in the termination of the nigrostriatal DA pathway. The mice were then treated chronically with l-DOPA and tested for different types of motor performance. In addition to AIMs ratings, we carried out a test of forelimb akinesia (Schallert and Tillerson, 2000) and measurements of horizontal activity and turning behavior in an open field (Steiner and Kitai, 2001). At the end of the behavioral studies, different histochemical techniques were used to measure the extent of striatal DA denervation, and the levels of ΔFosB-like proteins and prodynorphin (PDyn) mRNA in striatal neurons. Our data show that the unilateral 6-OHDA lesions induced an impairment of contralateral forelimb use that was stable during the long time course of the experiment, and that was reversed by l-DOPA. By contrast, deficits in horizontal motor activity were only seen during the first month post-lesion, showing spontaneous recovery over the time. During the course of chronic l-DOPA treatment, the mice exhibited a gradual development of AIMs affecting trunk, orofacial, and forelimb muscles on the side contralateral to the lesion, which we illustrate in the Limb dyskinesia, Axial dystonia/dyskinesia, Orolingual dyskinesia, Locomotive dyskinesia linked to this article. The severity of the abnormal movements showed a significant correlation to the levels of ΔFosB-like proteins and PDyn mRNA in the DA-denervated striatum.

Section snippets

Subjects

Male C57BL/6 mice (Møllegard, DK) were used in this study. They weighed approximately 25 g when purchased and were housed under a 12-h light/dark cycle with free access to food and water. The treatment of the animals and their conditions had been approved by Malmö-Lund Ethical Committee on Animal Research.

Lesion surgery

The mice were anaesthetized with Hypnorm®-Dormicum® (Apoteksbolaget, Sweden) (1:1:2 water to a volume of 2.7 ml/kg body weight) and mounted in a stereotactic frame (Kopf Instruments; USA) with

Lesion-induced deficits in spontaneous motor behavior; reversal by l-DOPA treatment

When tested before the initiation of the chronic l-DOPA treatment, all the experimental groups with 6-OHDA lesions showed a significant deficit in the use of the forelimb contralateral to the lesion (i.e., the left one) in the cylinder test. While sham lesioned mice used indifferently the right or the left forelimb to support their body, the proportion of wall contacts performed with the left paw ranged between 28% and 32% of total in the four groups with 6-OHDA lesions (P < 0.05 vs. sham in

Discussion

Systemic administration of MPTP is commonly used to produce degeneration of DA neurons in mice, but this approach presents some drawbacks. In particular, the possibility of substantial spontaneous recovery of the damaged nigrostriatal neurons (for review, see Sedelis et al., 2001) may be a concern in experiments involving long-term pharmacological treatment and repeated behavioral testing. This concern can be overcome using alternative lesion models. The catecholamine-selective neurotoxin

Acknowledgements

This study was supported by grants from the Swedish Association of the Neurologically Disabled, the Greta and Johan Kock Foundations, the Thorsten and Elsa Segerfalk Foundation, the Swedish Medical Research Council (contract nb. K2003-33X-13480-0A), and the Michael J. Fox Foundation for Parkinson Research. During her stay at Lund University, B.P. was supported by a Marie Curie Training Fellowship from the European Community.

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    Supplementary data associated with this article can be found at doi:10.1016/j.nbd.2004.01.007.

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