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Priming for L-DOPA-induced abnormal involuntary movements increases the severity of amphetamine-induced dyskinesia in grafted rats
Experimental Neurology 219 (2009) 355–358
Contents lists available at ScienceDirect
Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r
Short Communication
Priming for L-DOPA-induced abnormal involuntary movements increases the severity of amphetamine-induced dyskinesia in grafted rats E.L. Lane a,b,⁎, L. Vercammen b, M.A. Cenci b, P. Brundin a a b
Neuronal Survival Unit, Department of Experimental Medical Science, Lund, Sweden Basal Ganglia Pathophysiology Unit, Department of Experimental Medical Science, Lund, Sweden
a r t i c l e
i n f o
Article history: Received 19 February 2009 Revised 8 April 2009 Accepted 14 April 2009 Available online 22 April 2009
a b s t r a c t In some patients, graft-induced dyskinesia develops following intrastriatal transplantation of embryonic neural tissue for the treatment of Parkinson's disease. The mechanisms underlying these involuntary movements need to be clarified before this approach to clinical cell therapy can be developed further. We previously found that rats with 6-OHDA lesions, primed with L-DOPA treatment and that have subsequently undergone intrastriatal graft surgery exhibit involuntary movements when subjected to amphetamine. This model of amphetamine-induced AIMs reflects a pattern of post-graft behaviours that in the absence of robust spontaneous GID in the rat is the closest approximation that we currently have available. We now show that they are associated with the chronic administration of L-DOPA prior to the transplantation surgery. We also demonstrate that neither changes in c-fos nor FosB/ΔFosB expression in the lateral striatum are associated with the expression of these behaviours. Taken together, these data reveal that the severity of abnormal movements elicited by amphetamine in grafted animals may relate to previous L-DOPA exposure and dyskinesia development, but they develop through mechanisms that are independent of FosB/ΔFosB upregulation. © 2009 Elsevier Inc. All rights reserved.
Intrastriatal transplantation of dopamine-rich, embryonic midbrain tissue can restore motor function in some Parkinson's disease patients. However, three independent clinical trials reported that a subset of patients displayed abnormal movements that were seemingly unrelated to ongoing drug treatment (Freed et al., 2001, Hagell et al., 2002, Olanow et al., 2003). The cause/s of these graftinduced dyskinesias (GID) are unclear. The transplantation protocols used by the three different clinical trials vary in key parameters, including cell preparation and immunosuppressive regimes, making it difficult to identify which factors directly contribute to the risk of developing GID. On the other hand, all patients, including those displaying GID, had received L-DOPA prior to surgery (Winkler et al., 2005). To help determine which patients have the least risk of developing GIDs we have used an animal model to examine the role of prior L-DOPA-induced dyskinesia as a risk factor for GIDs. In rats, chronic L-DOPA administration is associated with sustained biochemical alterations indicating a long-term sensitisation of the denervated striatum (Westin et al., 2001). Upregulation of the immediate early gene FosB/ΔFosB in striatal medium spiny neurons is one such biochemical change, the increase in FosB/ΔFosB expression correlating with the severity of L-DOPA-induced dyskinesia, measured as abnormal involuntary movements (AIMs) (Andersson ⁎ Corresponding author. Brain Repair Centre, Department of Biosciences, Cardiff University, Museum Avenue, Cardiff, CF10 3AX, UK. Fax: +44 2920876749. E-mail address: [email protected] (E.L. Lane). 0014-4886/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2009.04.010
et al., 1999, Tekumalla et al., 2001). These behavioural and molecular changes can be ameliorated by the intrastriatal transplantation of embryonic midbrain tissue. Conversely the same grafts can make striatal neurons hyper-responsive to amphetamine such that they express increased levels of c-fos, another immediate early gene that reflects neuronal activity (Abrous et al., 1992, Cenci et al., 1992). The spontaneous occurrence of AIMs in rodent models of transplantation has only been reported in the early post-grafting interval, and they were considered unreliable and difficult to quantify (Lane et al., 2006, Vinuela et al., 2008). A more consistent behavioural model is one in which rats with unilateral 6-OHDA lesions, grafted with embryonic dopaminergic neurons, develop AIMs in response to amphetamine administration (Carlsson et al., 2006, Lane et al., 2006). The mechanisms underlying these amphetamine-induced AIMs in grafted rats are not well understood however it is possible that the abnormal amphetamine-induced c-Fos expression seen in grafted rats, might be coupled to the behaviour. In this study, we first determine the influence of L-DOPA administration on the development of amphetamine-induced AIMs. Second, we examine the responses of FosB/ΔFosB and c-fos in the striatum of grafted rats and how they are influenced by prior L-DOPA treatment. Female Sprague-Dawley rats (Harlan Scandinavia, Allerod, Denmark; 220 g) with unilateral 6-hydroxydopamine lesions of the median forebrain bundle (Lane et al., 2008) were split into 4 groups, balanced on their ipsilateral rotational response to amphetamine (2.5 mg/kg i.p.); two groups were given L-DOPA daily (10 mg/kg/day
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with 15 mg/kg benserazide in 0.9% saline, i.p.) for 4 weeks to prime for L-DOPA-induced AIMs (based on Winkler et al 2002 the scoring criteria consists of a 0–4 scale for frequency and 0–4 scale for amplitude for forelimb and axial movements, 0–2 in amplitude for orolingual movements) whilst the other two groups received vehicle (0.9% saline, i.p.). Within each treatment arm (L-DOPA or saline), some rats were grafted with embryonic ventral mesencephalon whilst others had sham surgery, resulting in the following experimental groups, 1) Non-L-DOPA treated sham; 2) non-L-DOPA treated graft; 3) L-DOPA treated sham; 4) L-DOPA-treated graft (n = 6–9 per group). All the L-DOPA treated rats used in the experiment displayed AIMs in response to L-DOPA. The mean AIMs scores were equivalent in the two L-DOPA-treated groups prior to grafting (sum of axial, limb and orolingual, ALO, scores in the final preoperative testing session: LDOPA-treated sham = 37.2 ± 5.2, L-DOPA-treated graft = 41.1 ± 5.0, max possible = 108). We dissected ventral midbrain tissue from 14day old Sprague-Dawley embryos and prepared it into a suspension as described previously (Lane et al., 2006). We injected 2 μl of the tissue suspension into the striatum in two 1 μl deposits in one tract, at a rate of 0.5 μl per minute, at the following co-ordinates (in mm): AP + 0.6; ML − 3.5; DV −5.0/−4.5; tooth bar at − 2.3 (equivalent to 2/3 of a vm per transplantation). Following surgery, we gave the rats analgesia (Temgesic®, 0.03 mg/kg i.p.) and anti-sedatives (Antisedan®, 1 mg/kg i.p.). L-DOPA treated rats continued to receive L-DOPA twice per week following transplantation, until one week prior to the amphetamine administration to ensure a response purely due to the amphetamine, unrelated to residual L-DOPA effects. Sixteen weeks following transplantation, we gave the rats amphetamine (2.5 mg/kg in saline, i.p.) and assessed rotational behaviour and dyskinesia according to the AIMs rating scale (modified version of the scale reported in Winkler et al., 2002). These behaviours are fully characterised in timecourse and severity in relationship to the rotational behaviour in Lane et al. (2006). Two weeks after dyskinesia assessment (with no further L-DOPA administration), the amphetamine administration was repeated and 3 h later were terminally anaesthetised and transcardially perfused with 0.9% saline (100 ml) then 4% paraformaldehyde (200 ml). The brains were removed, post-fixed and cyroprotected before being sectioned coronally into 40 μm thick sections on a freezing microtome. We performed immunohistochemistry for tyrosine hydroxylase (TH) (to identify the graft) and FosB/ΔFosB and c-fos according to standard peroxidase-based techniques. We incubated sections in hydrogen peroxide and methanol in phosphate buffered saline (PBS) for 20 min, and followed this by multiple PBS washes. We blocked non-specific staining by incubating the sections in 5% of the appropriate serum and 0.25% Triton TX-100 before incubation in primary antibodies (TH 1:2000, mouse monoclonal, Chemicon International, CA, USA; FosB/ ΔFosB, 1:1500, mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, CA, USA; c-fos, 1:2000, rabbit polyclonal) for 16 h. We then washed the sections in PBS before exposing them to the appropriate biotinylated secondary antibody (TH and Fos/ΔFosB, horse antimouse secondary antibody, c-fos goat anti-rabbit secondary antibody) for 1 h at room temperature. Antibody binding was visualised using the ABC kit (Vectastain Elite Kit, Vector laboratories, Burlingame, CA) and the chromagen 3-3′diaminobenzadine (DAB kit, Vector laboratories). We assessed the number of FosB/ΔFosB and c-fos positive cells in medial and lateral areas of the striatum and the core and shell of the nucleus accumbens using VIS image acquisition and analysis software (Visiopharm, Horsholm, Denmark). Images taken from the 1 mm2 area in each location and the number of positively stained nuclei within the grid were counted by the image analysis software (Valastro et al., 2009) using a non-stereological method. We performed statistical analysis of cell counts by 2-way ANOVA, factoring L-DOPA administration (L-DOPA vs non-L-DOPA) and grafting (graft vs sham). Post hoc analysis was by Newman Keuls test. On challenge with amphetamine, non-grafted rats rotated ipsilateral to the 6-OHDA lesion. Prior L-DOPA treatment did not affect the
intensity or direction of amphetamine-induced rotational behaviour (data not shown). Grafted animals rotated contralaterally, but the magnitude of the response was the same irrespective of priming with L-DOPA (data not shown). The L-DOPA-treated grafted rats showed significantly higher AIMs scores following amphetamine administration compared to the grafted rats who had never received L-DOPA (Fig. 1A, p = 0.048). In the L-DOPA treated groups prior to transplantation there was an equivalent expression of L-DOPA induced AIMs (LDOPA treated sham ALO score, 74.3 ± 10 vs L-DOPA treated grafted, 79.3 ± 8). All animals showed some degree of abnormal movements. Following transplantation there was a significant improvement in LDOPA induced dyskinesia in the grafted group, but not in the sham group (L-DOPA treated sham ALO score, 81.2 ± 15 vs L-DOPA treated grafted 36.5 ± 7.31, ⁎⁎p = 0.003, T-test). The histochemical analysis showed that the grafts in the L-DOPA and non-L-DOPA treated groups contained the same number of TH positive neurons (non-L-DOPA treated graft group, 1722 ± 278 vs LDOPA treated graft group, 1412 ± 294). This is in line with previous findings of a lack of influence of L-DOPA on TH neuron survival (Blunt et al., 1991) although this issue still requires clarification (SteeceCollier et al., 1990). In the core and shell of the nucleus accumbens, LDOPA treatment significantly affected FosB/ΔFosB expression (core, Fig. 1D′, F(2,28) = 0.861, p = 0.434 for graft, F(2,28) = 3.814, p = 0.035 for L-DOPA; shell, Fig. 1D″, F(2,28) = 0.573, p = 0.571 for graft, F(2,28) = 5.652, p = 0.009 for L-DOPA). In the core, FosB/ΔFosB expression was elevated in ungrafted L-DOPA-treated rats, but not in grafted L-DOPA-treated rats. In contrast, in the shell region, the graft did not influence FosB/ΔFosB expression. The levels of FosB/ΔFosB immunoreactivity were not significantly different between groups in the medial striatum, regardless of L-DOPA treatment or grafting (Fig. 1E′, F(2,28) = 2.186, p = 0.136 for graft, F(2,28) = 2.89, p = 0.065 for L-DOPA). In the lateral striatum, FosB/ΔFosB expression was influenced by L-DOPA and transplantation (Figs. 1E and E″, F(2,28) = 0.939, p = 0.404 for graft, F(2,28) = 2.27, p = 0.082 for L-DOPA, F(2,28) = 59.73, p = 0.016 for interaction between L-DOPA. L-DOPA treatment alone increases FosB/ΔFosB expression but in combination with transplantation levels approach those of non-L-DOPA treated animals (Fig. 1E″). There was no significant difference between groups in FosB/ΔFosB expression in the intact side. The increase in c-fos expression in the medial and lateral striatum in response to amphetamine was significantly affected by grafting, but not L-DOPA treatment (Figs. 1F, F′ and F″, medial striatum, F(2,28) = 7.22, p = 0.003 for graft, F(2,28) = 2.25, p =0.127 for L-DOPA;, lateral striatum F(2,28) = 10.30, pb 0.001 for graft F(2,28) = 3.54, p =0.08 for L-DOPA). Indeed, both groups of grafted animals exhibited a 3–4-fold increase in the number of c-fos-immunoreactive cells compared to the sham-grafted groups (p b 0.05), irrespective of a previous exposure to L-DOPA (figures F′–F″). There was no significant difference between groups in c-fos expression on the intact side. The level of amphetamine-induced c-fos expression was not influenced by prior LDOPA treatment and was not correlated to the amphetamine-induced AIMs scores in the grafted rats (data not shown). We show that rats have a greater tendency to develop amphetamine-induced AIMs following transplantation if they have previously been exposed to L-DOPA. Grafts of embryonic ventral midbrain tissue can normalise several biochemical and molecular changes that are caused by dopamine-denervation in the striatum (Blunt et al., 1992, Chritin et al., 1992, Nakao et al., 1998). The grafts do not, however, completely normalise the lesion-induced changes in a dopaminedenervated striatum. For example, D1 receptor changes may not be returned to levels on the contralateral intact striatum (Blunt et al., 1992) and striatal peptide mRNA dysregulation (preprotachykinin, in particular) is only partially restored, despite substantial transplantderived reinnervation (Cenci et al., 1993, Winkler et al., 2003). The persistence of lesion-induced abnormalities in the striatum might explain why amphetamine can induce AIMs in grafted rats. Moreover, intrastriatal transplants normalise changes that occur subsequent to
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Fig. 1. The development of dyskinesia in response to amphetamine administration (A) and its associated biochemistry (B–F). (A) The score of abnormal movements in L-DOPAtreated or non-L-DOPA-treated rats prior to transplantation with embryonic ventral mesencephalon. Only grafted rats showed amphetamine-induced abnormal movements. Immunohistochemical analysis post mortem for fosB/ΔfosB (B) and c-fos (C) was performed and the number of positive cells counted in the areas indicated on the graphical representations of coronal sections of the rat brain (D–F). Counted areas are indicated by black squares, grey areas indicate approximate graft presence. Cell counts for fosB/ΔfosB were performed in the core (D′) and shell (D″) of the nucleus accumbens and in medial (E′) and lateral (E″) areas of the striatum at the level of the graft. C-fos positive cells were counted in medial (F′) and lateral (F″) areas of the striatum at the level of the graft (⁎p b 0.05, sham-grafted animals vs grafted animals, +p b 0.05 non-L-DOPA treated graft vs L-DOPA treated graft, †p b 0.05 non-L-DOPA treated sham vs L-DOPA treated sham graft, #p b 0.05 intact vs lesioned striatum of any treatment group).
chronic L-DOPA administration. Thus, they reduce the upregulation of preproenkephalin-B mRNA and FosB/ΔFosB expression that result from this treatment (Elsworth et al., 1998, Lee et al., 2000, Maries et al., 2005, Rioux et al., 1993, Roy et al., 1995, Stromberg et al., 1995). In the present study, we found that the levels of FosB/ΔFosB – a key mediator of transcriptional changes upon intermittent dopaminergic stimulation, were normalised in grafted rats, reiterating previous findings (Maries et al., 2005, McClung et al., 2004, Vinuela et al., 2008). The same rats, however, displayed pronounced AIMs in response to amphetamine. This indicates that the amphetamineinduced AIMs may require some of the maladaptive plasticity induced
by L-DOPA, but that they depend on signalling pathways differing from those that cause L-DOPA induced AIMs. While it may be argued that the use of amphetamine to stimulate post-transplantation AIMs may not mimic the spontaneous behaviours observed in patients, we are attempting to specifically investigate movements that worsen post-transplantation and that are unrelated to anti-parkinsonian medication. The clinical trials of transplantation thus far have reported either no change or an improvement in L-DOPA, despite the presence of GID when ‘OFF’ medication. This model of amphetamine-induced AIMs reflects this pattern of behaviours and in the absence of spontaneous GID is the
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closest approximation we currently have to work with. In order to move the field onwards, we may be able to cautiously interpret the evidence of a relationship of L-DOPA induced dyskinesia prior to transplantation and amphetamine-induced AIMs following grafting alluded to previously (Lane et al., 2006) and confirmed in this study. It may suggest potentially significant implications for the selection of patients for transplantation to minimise the risk of GID, indicating that it might be preferable to transplant patients that have yet to develop L-DOPA-induced dyskinesia, rather than the severely afflicted patients that have thus far been entered into clinical trials. Further studies are necessary to determine the effect of L-DOPA administration without the generation of L-DOPA induced dyskinesia and dopamine agonist administration to aid in determining further the risk factors for GID. In conclusion, we show that L-DOPA administration prior to grafting influences post-grafting AIMs and makes the rats susceptible to develop GID. In this model of GID, however, the expression of AIMs after amphetamine is not related to the upregulation of FosB/ΔFosB induced by prior treatment with L-DOPA. Acknowledgments This work was supported by grants from the Michael J Fox Foundation for Parkinson's Research (PB and MAC), the Swedish Parkinsonfonden (EL) and the Swedish National Research Council (PB, MAC) and The Strong Research Environment on Neurodegeneration and Brain Repair (NeuroFortis). We would like to acknowledge the excellent assistance of Birgit Haraldsson and Britt Lindberg. EL is now at the Brain Repair Group, Cardiff University, UK, supported by a UK Parkinson's Disease Society grant. References Abrous, D.N., Torres, E.M., Annett, L.E., Reading, P.J., Dunnett, S.B., 1992. Intrastriatal dopamine-rich grafts induce a hyperexpression of Fos protein when challenged with amphetamine. Exp. Brain Res. 91, 181–190. Andersson, M., Hilbertson, A., Cenci, M.A., 1999. Striatal fosB expression is causally linked with l-DOPA-induced abnormal involuntary movements and the associated upregulation of striatal prodynorphin mRNA in a rat model of Parkinson's disease. Neurobiol. Dis. 6, 461–474. Blunt, S.B., Jenner, P., Marsden, C.D., 1992. Autoradiographic study of striatal D1 and D2 dopamine receptors in 6-OHDA-lesioned rats receiving foetal ventral mesencephalic grafts and chronic treatment with L-dopa and carbidopa. Brain Res. 582, 299–311. Blunt, S., Jenner, P., Marsden, C.D., 1991. The effect of chronic L-dopa treatment on the recovery of motor function in 6-hydroxydopamine-lesioned rats receiving ventral mesencephalic grafts. Neuroscience 40, 453–464. Carlsson, T., Winkler, C., Lundblad, M., Cenci, M.A., Bjorklund, A., Kirik, D., 2006. Graft placement and uneven pattern of reinnervation in the striatum is important for development of graft-induced dyskinesia. Neurobiol. Dis. 21, 657–668. Cenci, M.A., Kalen, P., Mandel, R.J., Wictorin, K., Bjorklund, A., 1992. Dopaminergic transplants normalize amphetamine- and apomorphine-induced Fos expression in the 6-hydroxydopamine-lesioned striatum. Neuroscience 46, 943–957. Cenci, M.A., Campbell, K., Bjorklund, A., 1993. Neuropeptide messenger RNA expression in the 6-hydroxydopamine-lesioned rat striatum reinnervated by fetal dopaminergic transplants: differential effects of the grafts on preproenkephalin, preprotachykinin and prodynorphin messenger RNA levels. Neuroscience 57, 275–296. Chritin, M., Savasta, M., Mennicken, F., Bal, A., Abrous, D.N., Le Moal, M., Feuerstein, C., Herman, J.P., 1992. Intrastriatal dopamine-rich implants reverse the increase of
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Lane, E.L., Winkler, C., Brundin, P., Cenci, M.A., 2006. The impact of graft size on the development of dyskinesia following intrastriatal grafting of embryonic dopamine neurons in the rat. Neurobiol. Dis. 22, 334–345. Lane, E.L., Soulet, D., Vercammen, L., Cenci, M.A., Brundin, P., 2008. Neuroinflammation in the generation of post-transplantation dyskinesia in Parkinson's disease. Neurobiol. Dis. Lee, C.S., Cenci, M.A., Schulzer, M., Bjorklund, A., 2000. Embryonic ventral mesencephalic grafts improve levodopa-induced dyskinesia in a rat model of Parkinson's disease. Brain 123 (Pt 7), 1365–1379. Maries, E., Kordower, J.H., Chu, Y., Collier, T.J., Sortwell, C.E., Olaru, E., Shannon, K., Steece-Collier, K., 2005. Focal not widespread grafts induce novel dyskinetic behavior in parkinsonian rats. Neurobiol. Dis. McClung, C.A., Ulery, P.G., Perrotti, L.I., Zachariou, V., Berton, O., Nestler, E.J., 2004. DeltaFosB: a molecular switch for long-term adaptation in the brain. Brain Res. Mol. 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Contents lists available at ScienceDirect
Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r
Short Communication
Priming for L-DOPA-induced abnormal involuntary movements increases the severity of amphetamine-induced dyskinesia in grafted rats E.L. Lane a,b,⁎, L. Vercammen b, M.A. Cenci b, P. Brundin a a b
Neuronal Survival Unit, Department of Experimental Medical Science, Lund, Sweden Basal Ganglia Pathophysiology Unit, Department of Experimental Medical Science, Lund, Sweden
a r t i c l e
i n f o
Article history: Received 19 February 2009 Revised 8 April 2009 Accepted 14 April 2009 Available online 22 April 2009
a b s t r a c t In some patients, graft-induced dyskinesia develops following intrastriatal transplantation of embryonic neural tissue for the treatment of Parkinson's disease. The mechanisms underlying these involuntary movements need to be clarified before this approach to clinical cell therapy can be developed further. We previously found that rats with 6-OHDA lesions, primed with L-DOPA treatment and that have subsequently undergone intrastriatal graft surgery exhibit involuntary movements when subjected to amphetamine. This model of amphetamine-induced AIMs reflects a pattern of post-graft behaviours that in the absence of robust spontaneous GID in the rat is the closest approximation that we currently have available. We now show that they are associated with the chronic administration of L-DOPA prior to the transplantation surgery. We also demonstrate that neither changes in c-fos nor FosB/ΔFosB expression in the lateral striatum are associated with the expression of these behaviours. Taken together, these data reveal that the severity of abnormal movements elicited by amphetamine in grafted animals may relate to previous L-DOPA exposure and dyskinesia development, but they develop through mechanisms that are independent of FosB/ΔFosB upregulation. © 2009 Elsevier Inc. All rights reserved.
Intrastriatal transplantation of dopamine-rich, embryonic midbrain tissue can restore motor function in some Parkinson's disease patients. However, three independent clinical trials reported that a subset of patients displayed abnormal movements that were seemingly unrelated to ongoing drug treatment (Freed et al., 2001, Hagell et al., 2002, Olanow et al., 2003). The cause/s of these graftinduced dyskinesias (GID) are unclear. The transplantation protocols used by the three different clinical trials vary in key parameters, including cell preparation and immunosuppressive regimes, making it difficult to identify which factors directly contribute to the risk of developing GID. On the other hand, all patients, including those displaying GID, had received L-DOPA prior to surgery (Winkler et al., 2005). To help determine which patients have the least risk of developing GIDs we have used an animal model to examine the role of prior L-DOPA-induced dyskinesia as a risk factor for GIDs. In rats, chronic L-DOPA administration is associated with sustained biochemical alterations indicating a long-term sensitisation of the denervated striatum (Westin et al., 2001). Upregulation of the immediate early gene FosB/ΔFosB in striatal medium spiny neurons is one such biochemical change, the increase in FosB/ΔFosB expression correlating with the severity of L-DOPA-induced dyskinesia, measured as abnormal involuntary movements (AIMs) (Andersson ⁎ Corresponding author. Brain Repair Centre, Department of Biosciences, Cardiff University, Museum Avenue, Cardiff, CF10 3AX, UK. Fax: +44 2920876749. E-mail address: [email protected] (E.L. Lane). 0014-4886/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2009.04.010
et al., 1999, Tekumalla et al., 2001). These behavioural and molecular changes can be ameliorated by the intrastriatal transplantation of embryonic midbrain tissue. Conversely the same grafts can make striatal neurons hyper-responsive to amphetamine such that they express increased levels of c-fos, another immediate early gene that reflects neuronal activity (Abrous et al., 1992, Cenci et al., 1992). The spontaneous occurrence of AIMs in rodent models of transplantation has only been reported in the early post-grafting interval, and they were considered unreliable and difficult to quantify (Lane et al., 2006, Vinuela et al., 2008). A more consistent behavioural model is one in which rats with unilateral 6-OHDA lesions, grafted with embryonic dopaminergic neurons, develop AIMs in response to amphetamine administration (Carlsson et al., 2006, Lane et al., 2006). The mechanisms underlying these amphetamine-induced AIMs in grafted rats are not well understood however it is possible that the abnormal amphetamine-induced c-Fos expression seen in grafted rats, might be coupled to the behaviour. In this study, we first determine the influence of L-DOPA administration on the development of amphetamine-induced AIMs. Second, we examine the responses of FosB/ΔFosB and c-fos in the striatum of grafted rats and how they are influenced by prior L-DOPA treatment. Female Sprague-Dawley rats (Harlan Scandinavia, Allerod, Denmark; 220 g) with unilateral 6-hydroxydopamine lesions of the median forebrain bundle (Lane et al., 2008) were split into 4 groups, balanced on their ipsilateral rotational response to amphetamine (2.5 mg/kg i.p.); two groups were given L-DOPA daily (10 mg/kg/day
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with 15 mg/kg benserazide in 0.9% saline, i.p.) for 4 weeks to prime for L-DOPA-induced AIMs (based on Winkler et al 2002 the scoring criteria consists of a 0–4 scale for frequency and 0–4 scale for amplitude for forelimb and axial movements, 0–2 in amplitude for orolingual movements) whilst the other two groups received vehicle (0.9% saline, i.p.). Within each treatment arm (L-DOPA or saline), some rats were grafted with embryonic ventral mesencephalon whilst others had sham surgery, resulting in the following experimental groups, 1) Non-L-DOPA treated sham; 2) non-L-DOPA treated graft; 3) L-DOPA treated sham; 4) L-DOPA-treated graft (n = 6–9 per group). All the L-DOPA treated rats used in the experiment displayed AIMs in response to L-DOPA. The mean AIMs scores were equivalent in the two L-DOPA-treated groups prior to grafting (sum of axial, limb and orolingual, ALO, scores in the final preoperative testing session: LDOPA-treated sham = 37.2 ± 5.2, L-DOPA-treated graft = 41.1 ± 5.0, max possible = 108). We dissected ventral midbrain tissue from 14day old Sprague-Dawley embryos and prepared it into a suspension as described previously (Lane et al., 2006). We injected 2 μl of the tissue suspension into the striatum in two 1 μl deposits in one tract, at a rate of 0.5 μl per minute, at the following co-ordinates (in mm): AP + 0.6; ML − 3.5; DV −5.0/−4.5; tooth bar at − 2.3 (equivalent to 2/3 of a vm per transplantation). Following surgery, we gave the rats analgesia (Temgesic®, 0.03 mg/kg i.p.) and anti-sedatives (Antisedan®, 1 mg/kg i.p.). L-DOPA treated rats continued to receive L-DOPA twice per week following transplantation, until one week prior to the amphetamine administration to ensure a response purely due to the amphetamine, unrelated to residual L-DOPA effects. Sixteen weeks following transplantation, we gave the rats amphetamine (2.5 mg/kg in saline, i.p.) and assessed rotational behaviour and dyskinesia according to the AIMs rating scale (modified version of the scale reported in Winkler et al., 2002). These behaviours are fully characterised in timecourse and severity in relationship to the rotational behaviour in Lane et al. (2006). Two weeks after dyskinesia assessment (with no further L-DOPA administration), the amphetamine administration was repeated and 3 h later were terminally anaesthetised and transcardially perfused with 0.9% saline (100 ml) then 4% paraformaldehyde (200 ml). The brains were removed, post-fixed and cyroprotected before being sectioned coronally into 40 μm thick sections on a freezing microtome. We performed immunohistochemistry for tyrosine hydroxylase (TH) (to identify the graft) and FosB/ΔFosB and c-fos according to standard peroxidase-based techniques. We incubated sections in hydrogen peroxide and methanol in phosphate buffered saline (PBS) for 20 min, and followed this by multiple PBS washes. We blocked non-specific staining by incubating the sections in 5% of the appropriate serum and 0.25% Triton TX-100 before incubation in primary antibodies (TH 1:2000, mouse monoclonal, Chemicon International, CA, USA; FosB/ ΔFosB, 1:1500, mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, CA, USA; c-fos, 1:2000, rabbit polyclonal) for 16 h. We then washed the sections in PBS before exposing them to the appropriate biotinylated secondary antibody (TH and Fos/ΔFosB, horse antimouse secondary antibody, c-fos goat anti-rabbit secondary antibody) for 1 h at room temperature. Antibody binding was visualised using the ABC kit (Vectastain Elite Kit, Vector laboratories, Burlingame, CA) and the chromagen 3-3′diaminobenzadine (DAB kit, Vector laboratories). We assessed the number of FosB/ΔFosB and c-fos positive cells in medial and lateral areas of the striatum and the core and shell of the nucleus accumbens using VIS image acquisition and analysis software (Visiopharm, Horsholm, Denmark). Images taken from the 1 mm2 area in each location and the number of positively stained nuclei within the grid were counted by the image analysis software (Valastro et al., 2009) using a non-stereological method. We performed statistical analysis of cell counts by 2-way ANOVA, factoring L-DOPA administration (L-DOPA vs non-L-DOPA) and grafting (graft vs sham). Post hoc analysis was by Newman Keuls test. On challenge with amphetamine, non-grafted rats rotated ipsilateral to the 6-OHDA lesion. Prior L-DOPA treatment did not affect the
intensity or direction of amphetamine-induced rotational behaviour (data not shown). Grafted animals rotated contralaterally, but the magnitude of the response was the same irrespective of priming with L-DOPA (data not shown). The L-DOPA-treated grafted rats showed significantly higher AIMs scores following amphetamine administration compared to the grafted rats who had never received L-DOPA (Fig. 1A, p = 0.048). In the L-DOPA treated groups prior to transplantation there was an equivalent expression of L-DOPA induced AIMs (LDOPA treated sham ALO score, 74.3 ± 10 vs L-DOPA treated grafted, 79.3 ± 8). All animals showed some degree of abnormal movements. Following transplantation there was a significant improvement in LDOPA induced dyskinesia in the grafted group, but not in the sham group (L-DOPA treated sham ALO score, 81.2 ± 15 vs L-DOPA treated grafted 36.5 ± 7.31, ⁎⁎p = 0.003, T-test). The histochemical analysis showed that the grafts in the L-DOPA and non-L-DOPA treated groups contained the same number of TH positive neurons (non-L-DOPA treated graft group, 1722 ± 278 vs LDOPA treated graft group, 1412 ± 294). This is in line with previous findings of a lack of influence of L-DOPA on TH neuron survival (Blunt et al., 1991) although this issue still requires clarification (SteeceCollier et al., 1990). In the core and shell of the nucleus accumbens, LDOPA treatment significantly affected FosB/ΔFosB expression (core, Fig. 1D′, F(2,28) = 0.861, p = 0.434 for graft, F(2,28) = 3.814, p = 0.035 for L-DOPA; shell, Fig. 1D″, F(2,28) = 0.573, p = 0.571 for graft, F(2,28) = 5.652, p = 0.009 for L-DOPA). In the core, FosB/ΔFosB expression was elevated in ungrafted L-DOPA-treated rats, but not in grafted L-DOPA-treated rats. In contrast, in the shell region, the graft did not influence FosB/ΔFosB expression. The levels of FosB/ΔFosB immunoreactivity were not significantly different between groups in the medial striatum, regardless of L-DOPA treatment or grafting (Fig. 1E′, F(2,28) = 2.186, p = 0.136 for graft, F(2,28) = 2.89, p = 0.065 for L-DOPA). In the lateral striatum, FosB/ΔFosB expression was influenced by L-DOPA and transplantation (Figs. 1E and E″, F(2,28) = 0.939, p = 0.404 for graft, F(2,28) = 2.27, p = 0.082 for L-DOPA, F(2,28) = 59.73, p = 0.016 for interaction between L-DOPA. L-DOPA treatment alone increases FosB/ΔFosB expression but in combination with transplantation levels approach those of non-L-DOPA treated animals (Fig. 1E″). There was no significant difference between groups in FosB/ΔFosB expression in the intact side. The increase in c-fos expression in the medial and lateral striatum in response to amphetamine was significantly affected by grafting, but not L-DOPA treatment (Figs. 1F, F′ and F″, medial striatum, F(2,28) = 7.22, p = 0.003 for graft, F(2,28) = 2.25, p =0.127 for L-DOPA;, lateral striatum F(2,28) = 10.30, pb 0.001 for graft F(2,28) = 3.54, p =0.08 for L-DOPA). Indeed, both groups of grafted animals exhibited a 3–4-fold increase in the number of c-fos-immunoreactive cells compared to the sham-grafted groups (p b 0.05), irrespective of a previous exposure to L-DOPA (figures F′–F″). There was no significant difference between groups in c-fos expression on the intact side. The level of amphetamine-induced c-fos expression was not influenced by prior LDOPA treatment and was not correlated to the amphetamine-induced AIMs scores in the grafted rats (data not shown). We show that rats have a greater tendency to develop amphetamine-induced AIMs following transplantation if they have previously been exposed to L-DOPA. Grafts of embryonic ventral midbrain tissue can normalise several biochemical and molecular changes that are caused by dopamine-denervation in the striatum (Blunt et al., 1992, Chritin et al., 1992, Nakao et al., 1998). The grafts do not, however, completely normalise the lesion-induced changes in a dopaminedenervated striatum. For example, D1 receptor changes may not be returned to levels on the contralateral intact striatum (Blunt et al., 1992) and striatal peptide mRNA dysregulation (preprotachykinin, in particular) is only partially restored, despite substantial transplantderived reinnervation (Cenci et al., 1993, Winkler et al., 2003). The persistence of lesion-induced abnormalities in the striatum might explain why amphetamine can induce AIMs in grafted rats. Moreover, intrastriatal transplants normalise changes that occur subsequent to
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Fig. 1. The development of dyskinesia in response to amphetamine administration (A) and its associated biochemistry (B–F). (A) The score of abnormal movements in L-DOPAtreated or non-L-DOPA-treated rats prior to transplantation with embryonic ventral mesencephalon. Only grafted rats showed amphetamine-induced abnormal movements. Immunohistochemical analysis post mortem for fosB/ΔfosB (B) and c-fos (C) was performed and the number of positive cells counted in the areas indicated on the graphical representations of coronal sections of the rat brain (D–F). Counted areas are indicated by black squares, grey areas indicate approximate graft presence. Cell counts for fosB/ΔfosB were performed in the core (D′) and shell (D″) of the nucleus accumbens and in medial (E′) and lateral (E″) areas of the striatum at the level of the graft. C-fos positive cells were counted in medial (F′) and lateral (F″) areas of the striatum at the level of the graft (⁎p b 0.05, sham-grafted animals vs grafted animals, +p b 0.05 non-L-DOPA treated graft vs L-DOPA treated graft, †p b 0.05 non-L-DOPA treated sham vs L-DOPA treated sham graft, #p b 0.05 intact vs lesioned striatum of any treatment group).
chronic L-DOPA administration. Thus, they reduce the upregulation of preproenkephalin-B mRNA and FosB/ΔFosB expression that result from this treatment (Elsworth et al., 1998, Lee et al., 2000, Maries et al., 2005, Rioux et al., 1993, Roy et al., 1995, Stromberg et al., 1995). In the present study, we found that the levels of FosB/ΔFosB – a key mediator of transcriptional changes upon intermittent dopaminergic stimulation, were normalised in grafted rats, reiterating previous findings (Maries et al., 2005, McClung et al., 2004, Vinuela et al., 2008). The same rats, however, displayed pronounced AIMs in response to amphetamine. This indicates that the amphetamineinduced AIMs may require some of the maladaptive plasticity induced
by L-DOPA, but that they depend on signalling pathways differing from those that cause L-DOPA induced AIMs. While it may be argued that the use of amphetamine to stimulate post-transplantation AIMs may not mimic the spontaneous behaviours observed in patients, we are attempting to specifically investigate movements that worsen post-transplantation and that are unrelated to anti-parkinsonian medication. The clinical trials of transplantation thus far have reported either no change or an improvement in L-DOPA, despite the presence of GID when ‘OFF’ medication. This model of amphetamine-induced AIMs reflects this pattern of behaviours and in the absence of spontaneous GID is the
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closest approximation we currently have to work with. In order to move the field onwards, we may be able to cautiously interpret the evidence of a relationship of L-DOPA induced dyskinesia prior to transplantation and amphetamine-induced AIMs following grafting alluded to previously (Lane et al., 2006) and confirmed in this study. It may suggest potentially significant implications for the selection of patients for transplantation to minimise the risk of GID, indicating that it might be preferable to transplant patients that have yet to develop L-DOPA-induced dyskinesia, rather than the severely afflicted patients that have thus far been entered into clinical trials. Further studies are necessary to determine the effect of L-DOPA administration without the generation of L-DOPA induced dyskinesia and dopamine agonist administration to aid in determining further the risk factors for GID. In conclusion, we show that L-DOPA administration prior to grafting influences post-grafting AIMs and makes the rats susceptible to develop GID. In this model of GID, however, the expression of AIMs after amphetamine is not related to the upregulation of FosB/ΔFosB induced by prior treatment with L-DOPA. Acknowledgments This work was supported by grants from the Michael J Fox Foundation for Parkinson's Research (PB and MAC), the Swedish Parkinsonfonden (EL) and the Swedish National Research Council (PB, MAC) and The Strong Research Environment on Neurodegeneration and Brain Repair (NeuroFortis). We would like to acknowledge the excellent assistance of Birgit Haraldsson and Britt Lindberg. EL is now at the Brain Repair Group, Cardiff University, UK, supported by a UK Parkinson's Disease Society grant. References Abrous, D.N., Torres, E.M., Annett, L.E., Reading, P.J., Dunnett, S.B., 1992. Intrastriatal dopamine-rich grafts induce a hyperexpression of Fos protein when challenged with amphetamine. Exp. Brain Res. 91, 181–190. Andersson, M., Hilbertson, A., Cenci, M.A., 1999. Striatal fosB expression is causally linked with l-DOPA-induced abnormal involuntary movements and the associated upregulation of striatal prodynorphin mRNA in a rat model of Parkinson's disease. Neurobiol. Dis. 6, 461–474. Blunt, S.B., Jenner, P., Marsden, C.D., 1992. Autoradiographic study of striatal D1 and D2 dopamine receptors in 6-OHDA-lesioned rats receiving foetal ventral mesencephalic grafts and chronic treatment with L-dopa and carbidopa. Brain Res. 582, 299–311. Blunt, S., Jenner, P., Marsden, C.D., 1991. The effect of chronic L-dopa treatment on the recovery of motor function in 6-hydroxydopamine-lesioned rats receiving ventral mesencephalic grafts. Neuroscience 40, 453–464. Carlsson, T., Winkler, C., Lundblad, M., Cenci, M.A., Bjorklund, A., Kirik, D., 2006. Graft placement and uneven pattern of reinnervation in the striatum is important for development of graft-induced dyskinesia. Neurobiol. Dis. 21, 657–668. Cenci, M.A., Kalen, P., Mandel, R.J., Wictorin, K., Bjorklund, A., 1992. Dopaminergic transplants normalize amphetamine- and apomorphine-induced Fos expression in the 6-hydroxydopamine-lesioned striatum. Neuroscience 46, 943–957. Cenci, M.A., Campbell, K., Bjorklund, A., 1993. Neuropeptide messenger RNA expression in the 6-hydroxydopamine-lesioned rat striatum reinnervated by fetal dopaminergic transplants: differential effects of the grafts on preproenkephalin, preprotachykinin and prodynorphin messenger RNA levels. Neuroscience 57, 275–296. Chritin, M., Savasta, M., Mennicken, F., Bal, A., Abrous, D.N., Le Moal, M., Feuerstein, C., Herman, J.P., 1992. Intrastriatal dopamine-rich implants reverse the increase of
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