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Dopamine trasporter reversal as a pathogenic mechanism for L-dopa induced dyskinesia Cheng, Christina 2005

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D O P A M I N E T R A N S P O R T E R R E V E R S A L A S A P A T H O G E N I C M E C H A N I S M F O R L - D O P A I N D U C E D D Y S K I N E S I A by CHRISTINA CHENG B.A., The University of British Columbia, 2001 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE FACULTY OF GRADUATE STUDIES (Neuroscience) THE UNIVERSITY OF BRITISH COLUMBIA August 2005 © Christina Cheng, 2005 Abstract L-dopa remains the most effective drug for improving motor symptoms of Parkinson's disease (PD). However, following long-term chronic treatment, the therapeutic effects of L-dopa are often accompanied by debilitating peak-dose dyskinesia. The mechanisms underlying L-dopa induced dyskinesia remain unknown. Rapid increases of dopamine (DA) in the severely DA denervated striatum are associated with L-dopa induced dyskinesia (Miller and Abercrombie, 1999). This D A efflux is believed to cause many post-synaptic changes that are associated with L-dopa induced dyskinesia (Olanow et al., 2000). Therefore, it is of interest to examine the underlying mechanisms of the L-dopa induced DA release. The objectives of the present experiments were to examine the role for the DA transporter (DAT) in mediating L-dopa-induced DA release. Firstly, systemic injection of a DAT antagonist, methylphenidate (MP) was used to assess the role of the DAT in L-dopa induced dyskinesia in chronically L-dopa treated animals. Results showed a dose-dependent effect of MP in the attenuation of L-dopa induced dyskinesia. Secondly, we investigated the functional mode of the D A T by examining the effects of MP pre-treatment on the L-dopa induced DA efflux and dyskinetic responses in three groups of rats 1) L-dopa-naive, 2) 1-week L-dopa treated, and 3) 3-week L-dopa treated, rats. MP pretreatment had no effect on L-dopa induced DA efflux in L-dopa naive, or 1 -week L-dopa treated animals. In contrast, systemic pre-treatment of MP significantly attenuated the L-dopa induced D A response in 3-week treated rats, which was correlated with a similar decrease in L-dopa induced dyskinesia. The results from these experiments lend support to our hypothesis that reversal of the DAT through chronic L-dopa treatment contributes to the pathogenesis of L-dopa induced iii dyskinesia. Therefore, these findings suggest that the DAT is an important pharmacological target in the study and treatment of L-dopa induced dyskinesia. Table of Contents Abstract ii Table of Contents iv List of Tables vi List of Figures vii List of Abbreviations ix Acknowledgements xi INTRODUCTION Overview 1 Parkinson's Disease a. Epidemiology and Etiology 2 b. Treatment for PD 4 c. Animal Models 6 Neural Substrates of Parkinson's Disease a. A Functional Model of the Basal Ganglia: Normal and PD states 7 b. Neurochemistry of DA 11 c. Compensatory Mechanisms Due to DA Denervation 16 Neural Mechanisms of L-dopa-induced Dyskinesia a. Pharmacology of L-dopa 18 b. Risk Factors for L-dopa-induced Dyskinesia 19 c. Current Views on Mechanisms of L-dopa-induced Dyskinesia 20 d. Treatment for L-dopa Induced Dyskinesia 27 A Pathogenic Role for the Dopamine Transporter in L-dopa-induced Dyskinesia a. Overview 27 b. The Dopamine Transporter 29 c. Evidence for DAT mediated DA release in L-dopa-induced Dyskinesia.. 30 d. Experimental Rationale 31 e. Experimental Considerations 31 G E N E R A L M E T H O D S 6-OHDA Lesion Rat Model of PD 34 Drugs and Preparation 35 Behavioural Analysis: Abnormal Involuntary Movement (AIM) Scale 35 Microdialysis 35 High Pressure Liquid Chromatography 36 Data Analysis and Statistics 37 Histology 37 EXPERIMENT 1 Introduction 38 Methods 38 Results 39 Discussion 42 EXPERIMENT 2 Introduction 45 Methods 45 Results 46 Discussion 60 GENERAL DISCUSSION 68 FUTURE DIRECTIONS AND CONCLUSION 74 REFERENCES 76 vi List of Tables Table 1. Summary of all animals used in Experiment #1 39 Table 2. Summary of all animals used in Experiment #2 45 List of Figures Figure 1. Schematic drawing representing basal ganglia function in the normal brain 9 Figure 2. Schematic drawing representing basal ganglia function in the Parkinsonian brain 13 Figure 3. Neurochemistry of dopamine 14 Figure 4. Schematic drawing representing basal ganglia function in the on-state of L-dopa -induced dyskinesia 22 Figure 5. Dose effect of methylphenidate on L-dopa-induced AIM in chronically L-dopa treated rats 41 Figure 6. Effect of fluoxetine and desipramine on L-dopa-induced AIM 44 Figure 7. Effect of L-dopa treatment on basal levels 47 Figure 8. Effect of methylphenidate pretreatment on L-dopa-induced responses in the drug-naive, unlesioned striatum 48 Figure 9. Effect of methylphenidate pretreatment on L-dopa-induced responses in the drug-naive, lesioned striatum 50 Figure 10 Effect of methylphenidate pretreatment on L-dopa-induced peak values in the drug-naive, lesioned striatum and behaviour 52 Figure 11. Effect of methylphenidate pretreatment on L-dopa-induced responses in the 1 week L-dopa treated, unlesioned striatum 53 Figure 12. Effect of methylphenidate pretreatment on L-dopa-induced responses in the 1- week L-dopa treated, lesioned striatum 55 Figure 13. Effect of methylphenidate pretreatment on L-dopa-induced peak values in the 1- week L-dopa treated, lesioned striatum and behaviour 56 Figure 14. Effect of methylphenidate pretreatment on L-dopa-induced responses in the 3- week L-dopa treated, unlesioned striatum 58 Figure 15. Effect of methylphenidate pretreatment on L-dopa-induced responses in the 3- week L-dopa treated, lesioned striatum 59 Figure 16. Effect of methylphenidate pretreatment on L-dopa-induced peak values in the 3- week L-dopa treated, lesioned striatum and behaviour 61 Vll l Figure 17. Verification of probe placement 62 Figure 18. D A T reversal in the pathogenesis of L-dopa-induced dyskinesia 73 Abbreviations 6-OHDA 6-hydroxydopamine A A D C Aromatic amino acid decarboxylase AIM Abnormal involuntary movements Axial Axial dystonia A N O V A Analysis of Variance DA Dopamine DAT Dopamine transporter DOPA Dihydroxyphenylalanine DOPAC 3,4 dihydroxyphenylacetate CREB cAMP response element binding protein COMT Catechol-O-methyl transferase G A B A y-aminobutyric-acid GPi Globus pallidus, internal segment GPe Globus pallidus, external segment H V A Homovanillic acid i.p. Intraperitoneal L-dopa Levodopa Loco Locomotive rotation Limb Limb dyskinesia LTP Long-term potentiation MAO-B Monoamine oxidase-B MP Methylphenidate MPTP 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine NET Noradrenalin transporter PD Parkinson's disease PDyn Prodynorphin PPE Pre-proenkephalin PKA Protein-kinase-A SERT Serotonin transporter SNc Substantia nigra pars compacta SNr Substantia nigra pars reticulate STN Subthalamic nucleus TH Tyrosine hydroxylase UCH-L1 Ubiquitin C-terminal hydroxylase L l UPS Ubiquitin Proteasome System Acknowledgements First and foremost, I would like to express my sincere gratitude to my supervisors, Dr. Tony Phillips and Dr . Chong Lee, for this invaluable experience. Tony: A little over five years ago, I walked into your lab as a curious little undergrad. Since then I've learned so much about science, teamwork, and diligence. Thanks for this great opportunity! Dr. Lee: I am so grateful for all that you have taught me about clinical science and Parkinson's research. Your patience and constant encouragement really helped me in the completion of this thesis. Thank you. I would also like to thank the members of my committee: Dr . Br ian Christie, Dr. Doris Doudet, and Dr . Br ian M a c Vicar , for their helpful comments and suggestions, and their careful editing of this thesis. I would also like to acknowledge Dr . T o m Ruth, who kindly provided us with the methylphenidate used in these experiments. I would like to say a big 'thank you!' to all my friends, those in, and outside, of the lab. Thanks for putting up with me these last few years, and for keeping me sane as I tackled those Neuroscience 500/501 critiques and grant assignments. In particular, I would like to thank Dr . Soyon A h n , both a mentor and a friend, and as well, Kitty So, my fellow "little-fry" in the lab from the very beginning. We make a great team, and without your friendship, support, and encouragement over these years, this thesis would have not been possible. Also, I would like to thank Josh Levinson, for providing me with continuous confidence and support, but most importantly, for allowing me to convince him (finally) that behavioural neuroscience is cool. Lastly, I would like to thank my family for everything they have done for me. It is to them that I dedicate this thesis. 1 INTRODUCTION Overview Parkinson's disease (PD) is a progressive neurodegenerative disorder that primarily affects the elderly population. The pathological hallmarks of PD are a relatively selective degeneration of the nigrostriatal dopamine (DA) neurons in the substantial nigra pars compacta (SNc), and cytoplasmic inclusions of ubiquitin-rich aggregates termed Lewy bodies. The loss of dopaminergic neurons subsequently leads to DA depletion in the striatum, producing a constellation of motor deficits, such as bradykinesia, rigidity, and resting tremor, which are the cardinal features of PD. Additional symptoms such as depression, anxiety, sleep disturbances and cognitive impairment often compound the motor deficits in PD. Together, these symptoms lead to a significantly compromised quality of life in the majority of PD patients. While genetic predisposition is a known contributor to PD, the majority of cases are idiopathic. The exact mechanisms underlying spontaneous neurodegeneration are still unclear, although recent advances in genetic research of PD, combined with improved experimental models, have provided new insight into the cellular and molecular mechanisms that may be involved in the pathogenesis of both familial and sporadic forms of PD. Without a clear understanding the etiology of PD, development of neuroprotective or neurodegenerative treatments for this disorder remains a challenge. Therefore, most patients currently rely on symptomatic treatments alone. These treatments typically involve the use of DA agonists, which act to supplement the diminishing dopaminergic stimulation in the striatum. Developed in the 1960s, levodopa (L-dopa) is one such drug that provides efficacy in the treatment of parkinsonism. Unfortunately, while most patients respond well in the first few years of L-dopa therapy, benefits may be overshadowed by the emergence of response 2 fluctuations or dyskinesia that are associated with prolonged L-dopa treatment. Despite these setbacks however, few other treatments have shown such consistent success in improving parkinsonian motor symptoms. As such, L-dopa remains the most commonly used treatment for PD. It is thus critical to elucidate the underlying mechanisms involved in L-dopa-induced motor fluctuations and dyskinesia. The key focus of the studies described in this thesis is to investigate a novel mechanism by which chronic L-dopa therapy can lead to the induction of dyskinesia. Specifically, the dynamic role of the dopamine transporter (DAT) in the development of L-dopa-induced dyskinesia is assessed using a unilateral 6-hydroxydopamine (6-OHDA) lesioned rat model of L-dopa-induced dyskinesia (Lee, 2000). The remainder of this chapter will present, 1) a more in-depth description of PD, 2) a review of the neuropathophysiology of PD, 3) the current hypotheses which have been put forward to explain L-dopa-induced dyskinesia, and finally, 4) the experimental rationale for the present experiments presented in this thesis. Parkinson's Disease Epidemiology and Etiology PD affects approximately 0.3% of the general population, and 1% of people over the age of 60. It has a slightly higher incidence in men, and is evident across all ethnic groups. The average age of onset is in the mid-50s to 60s for most patients, but can be as early as the mid 20s for patients with young-onset PD. Familial PD accounts for only a small proportion of all PD cases. This suggests that, although genetic inheritance may play a role in the etiology of PD, environmental factors, if not primarily involved, are strongly implicated as well. Recent data from a large-scale study examining the concordance of PD in monozygotic twins suggest that heredity is not a major etiological factor in PD, especially in cases involving onset after 50 yrs of 3 age. However, further analysis shows that genetic components of PD are much more prevalent in early-onset cases (Tanner et al., 1999). Genetic predisposition Although the exact causes of PD are unknown, the discovery of several genes and gene loci associated with familial PD has enhanced our understanding of the molecular pathogenesis of the disease. Mounting evidence suggest that neurodegeneration may be a consequence of dysfunction in the mitochondrial I complex, resulting in oxidative stress and production of misfolded proteins. There is further evidence that failure in the ubiquitin proteasome system may contribute to aggregation of mishandled proteins and hence, to neurodegeneration in PD. Parkin and ubiquitin C-terminal hydroxylase L l (UCH-L1) are two proteins that are directly involved in the ubiquitin proteasome system. Genetic mutations on genes (PARK 2 and PARK 5, respectively) that encode for both proteins account for some of the familial cases of PD. Mutations in PARK 1, the gene that codes for a-synuclein, have also been linked to PD. Incidentally, a-synuclein form a major component of the Lewy bodies associated with PD. Overexpression of a-synuclein in flies and rodents reiterate some of the behavioural and pathological aspects of PD, including selective, age-dependent degeneration of dopaminergic neurons and formation of Lewy body-like, a-synuclein positive inclusions (Feany and Bender, 2000; Kirik et al., 2002). Other genetic mutations that have been implicated in PD impair mitochondrial function and response to oxidative stress. The PINK1 gene encodes a protein that responds protectively to oxidative stress. Overexpression of the mutated gene has been shown to increase cell death in vitro (Valente et al., 2004). Similarly, the protein DJ-1, which has also been shown to modify mitochondrial function in response to oxidative stress, also has a critical role in neuroprotection. 4 Mutations in both PINK1 and DJ-1 genes have been documented in autosomal recessive PD (Tanner etal, 1999). Environmental factors In the early 1980's, Langston and colleagues (Langston and Ballard, 1984) reported on a series of patients who developed acute Parkinsonism after being exposed to MPTP, a small molecule which readily crosses the blood brain barrier and subsequently converted into MPP+ by astrocytes. MPP+ enters dopaminergic neurons via the DAT and causes neurotoxicity through inhibition of the mitochondrial complex I. Since MPTP is one of few exogenous substances directly linked to the development of Parkinsonism, the discovery of MPP+ induced neurotoxicity has lent support to the hypothesis that environmental exposure to chemically similar substances may contribute to the development of the disorder. Exposure to pesticides, industrial chemicals, and contaminated drinking water have all been associated with PD. At the same time, while exposure to certain substances increases the risk of PD, some appear to reduce it. For instance, cigarette smoking and caffeine intake have both been associated with neuroprotection and lowered incidence of PD (Morens et al., 1995; Ross et al., 2000). In summary, it is likely that PD is a heterogeneous disorder with a multifactorial etiology. Nigral degeneration may be a consequence of exposure to environmental toxins, particularly in individuals who are genetically predisposed. Although familial PD is rare, the genetic advances made by studying genetic mutations in these cases have increased our understanding of the pathogenesis underlying both inherited and sporadic forms. Treatment for PD Treatment for PD can be divided into two categories: one to prevent or retard neurodegeneration, and the other to alleviate the symptoms of the disorder. Since the exact 5 mechanisms for nigral cell death are not fully understood, effective neuroprotective therapies for PD are currently unavailable. However, several neuroprotective agents, including an anti-inflammatory drug, minocycline, have shown promising preliminary results, and now await clinical testing (Blum et al., 2004; Ravina et al., 2003). Although neuroprotection at any stage of the disorder should prevent further degeneration, the benefits of neuroprotective therapies would be maximized if initiated in the early stages of the disorder. Unfortunately, due to the efficient compensatory mechanisms for striatal DA depletion, Parkinsonian symptoms appear only after approximately 50% of D A cells in the SNc are lost (Bernheimer et al., 1973). Therefore, development of diagnostic measures sensitive to neuronal loss in the presymptomatic stages of the disease is greatly needed to enhance the value of neuroprotective therapies Another approach that is receiving increasing attention is cell replacement therapy. To date, several cell replacement strategies have been attempted in animal models with varying success, including intrastriatal transplantation of various stem cells, embryonic mesencephalic tissue, and retinal pigmentary epithelial cells on gelatin beads. Some studies have demonstrated significant repair of DA cells, which was accompanied by behavioural improvement (Storch et al., 2004). However, development of tumors associated with cell transplant is an important concern with this technique. Furthermore, some patients developed dyskinesia, for which the cause is unknown. While these aforementioned approaches to PD treatment have demonstrated a great deal of promise, many are still at the experimental stage. It may be many years before these treatments can be proven practical and accessible to patients. The most common and effective treatments to date, are symptomatic therapies, the majority of which restores defective dopaminergic neurotransmission. This is accomplished either by enhancing the synthesis of DA 6 via administration of an exogenous DA precursor (L-dopa), or by employing direct agonists which act on mainly the D2 class of DA receptors (pramipexole, ropinirole) (Hallett and Standaert, 2004). Unfortunately, a vast majority of patients treated with dopaminergic drugs develop unstable responses and side effects after prolonged treatment. Direct DA agonists are less likely to cause dyskinesia than L-dopa, but, they have a weaker anti-parkinsonian efficacy (Brotchie et al., 2005). Therefore, while monotherapy with a D A agonist is usually sufficient for the first few years of treatment, patients eventually turn to L-dopa treatment. The mechanisms of L-dopa treatment and consequences of chronic L-dopa use will be described in greater detail later on in this chapter. In conclusion, while traditional therapeutic approaches with L-dopa and D A agonists are usually effective for improving the symptoms of PD, shortcomings of these drug treatments related to their chronic use have incited a need for better understanding of pharmacological actions of these drugs at the molecular level, and other alternative treatments aimed towards neuroprotection or replacement of diminishing cells. Animal Models of PD The ideal animal model would capture most of the pathological features and behavioural deficits of sporadic PD. Currently, common animal models of PD use 6-OHDA lesion of nigrostriatal fibers in a variety of species, and MPP+ induced toxicity primarily in primates to produce these motor deficits. Both bilateral and unilateral 6-OHDA lesioned animal (mainly rats) are effective in producing DA depletion. As motor deficits are greater in bilaterally lesioned animals, intensive nursing is often required. Therefore, unilaterally lesioned animals are a more practical alternative, and prove to be useful for when a within-subject control design is preferred. All of these models usually entail acute and severe (>95%) depletion of DA cells in 7 the lesioned striatum. Therefore, these "end stage" models (i.e., modeling the advanced PD with severe DA denervation) are useful for evaluation of new therapies. In recent years, lesion techniques have been refined to allow experimenters better control of lesion size and severity, as well as to obtain a gradual and progressive lesion that is more characteristic to that of PD patients (Bezard et al., 1997; Lee et al., 2000; McNaught et al., 2004). Although animal models do not replicate all the features of PD, they reliably cause DA depletion and Parkinsonism and furthermore, are practical and invaluable tools for evaluation of symptomatic treatments and new emerging therapies such as cell replacement and restoration therapies. The Neural Substrates of PD A functional model of the basal ganglia: normal and PD states Situated in the basal forebrain, the basal ganglia form a complex network of parallel connections involving the cortex, the basal ganglia, and the thalamus (Figure 1). Cortical motor information is conveyed to basal ganglia via entry of the striatum. Information then passes through several basal ganglia nuclei, before arriving at the output nuclei, namely, the substantia nigra pars reticulata (SNr) and the internal segment of the globus pallidus (GPi). The GPi/SNr then communicates with the brainstem and the thalamo-cortical neurons, both of which are crucially involved with the execution of motor events. Accordingly, the basal ganglia are perfectly situated to act as an integrator and relay station for motor information. Dysfunction of this "motor circuit" commonly underlies the pathophysiology of many movement disorders. The following sections describe the classical model of basal ganglia function, and the proposed functional changes occurring in the PD state. The normal state Figure 1. Schematic drawing representing basal ganglia function in the normal brain. The red lines with round ends depict excitatory connections. The blue lines with blunt ends depict inhibitory connections. Abbreviations: Dl = dopamine DI receptor; D2 = dopamine D2 receptor; GPe = external segment of globus pallidus; GPi = internal segment of the globus pallidus; SNc = substantia nigra pars compacta; SNr = substantia nigra pars reticulata; STN subthalamic nucleus. CORTEX I STRIATUM D2 D1 i \ / i SNc GPe IT STN GPi/SNi w THALAMUS Figure 1 10 The G A B A containing medium spiny neurons in the striatum are activated by descending glutamatergic projection originating from various cortical motor areas (Figure 1). In turn, these neurons communicate with the GPi/SNr via two pathways: 1) direct and 2) indirect. Neurons that constitute the 'direct' pathway send direct GABAergic projections to the GPi/SNr. These neurons contain dopamine D l receptors, and in addition to GABA, co-express substance P and dynorphin. Activation of striatal projection neurons in the direct pathway results in inhibition of the GPi/SNr neurons. In contrast, the striatal projection neurons involved in the indirect pathway express dopamine D2 receptors and enkaphalin, and innervate the GAB A-containing cells of the external segment of the globus pallidus (GPe). Activation of the GPe exerts a tonic inhibition over the subthalamic nucleus (STN) and the GPi/SNr. Therefore, activation of the striatal projection neurons in the indirect pathway results in inhibition of the GPe, and consequently the disinhibition of the GPi/SNr. Therefore, these two pathways exert opposing influences over the GPi/SNr, and thus, the balance between the activation of these two processes allows for the facilitation of appropriate movements, and suppression of inappropriate movements (Brotchie et al., 2005). As the striatal projection neurons in both pathways contain D A receptors, nigrostriatal DA neurons modulate the activity of these two pathways. As D l receptors are facilitatory, activation of striatal D l receptors in the direct pathway results suppression of GPi/SNr neurons. In contrast, D2 receptors are inhibitory, and stimulation of striatal D2 receptors disinhibits GPe neurons, which in turn, suppresses GPi/SNr activity. Therefore, stimulation of both D l and D2 DA receptors in the striatum leads to suppression of the output nuclei in the basal ganglia 11 circuitry. In sum, this model illustrates the critical influence of D A over the basal ganglia output activity (DeLong, 1983). The PD state This model of basal ganglia circuitry has been a useful, albeit incomplete, tool used to explain the clinical syndrome of PD. It describes the neuromodulatory role of the DA system in motor function, and how deficient DA stimulation in the striatum changes the functional state of the basal ganglia circuitry (DeLong, 1983). In the direct pathway, decreased DI receptor stimulation leads to disinhibition of the GPi/SNr (Figure 2). In the indirect pathway, reduced D2 receptor stimulation-leads to increased activity in the GPe, and which in turn leads to increased activity of the STN neurons and the GPi/SNr. In sum, DA depletion in the striatum, as in PD, results in a greater inhibitory influence over the GPi/SNr, and their target structures, namely the brainstem and the cortex, which are associated with motor behaviour. Neurochemistry of DA DA synthesis Tyrosine molecules cross through the blood brain barrier by carrier-mediated transport, and enter D A nerve terminals (Figure 3), where they are converted into dihydroxyphenylalanine (DOPA) by the rate limiting enzyme tyrosine hydroxylase (TH). Subsequently, DOPA is decarboxylated by the abundant enzyme aromatic amino acid decarboxylase (AADC) to form DA. Since endogenous levels of DOPA are low relative to the amount of A A D C in the remaining DA terminals in the denervated striatum, administration of exogenous L-dopa can substantially increase synthesis of DA (Cooper, Bloom, & Roth 1996). 12 F i g u r e 2. Schematic drawing representing basal ganglia function in the parkinsonian brain. The red lines with round ends depict excitatory connections. The blue lines with blunt ends depict inhibitory connections. Thick lines represent overactive projections, whereas thin lines represent underactive projections. Consistently, boxes with thick outlines (the STN, GPi/SNr) represent nuclei which are overactive. Dotted Lines or outlines represent projections or nuclei that are eliminated due to D A cell death. Abbreviations: Dl= dopamine Dl receptor; D2 = dopamine D2 receptor; GPe = external segment of globus pallidus; GPi = internal segment of the globus pallidus; SNc = substantia nigra pars compacta; SNr = substantia nigra pars reticulata; STN = subthalamic nucleus. 13 CORTEX T STRIATUM 02 THALAMUS Figure 2 14 Figure 3. Neurochemistry of DA DA originates from tyrosine molecules entering DA neurons. Tyrosine is then converted into dopa molecules by the rate limiting enzyme tyrosine hydroxylase (TH). DOPA is then decarboxylated by aromatic amino acid decarboxylase (AADC), converting it to DA. DA then forms a transient, cytosolic pool, and then transported into DA vesicles. D A release occurs via two known mechanisms, namely vesicular and DAT-mediated release. Exogenous L-dopa, on the other hand, enters D A neurons, and is directly converted into D A by A A D C , bypassing the rate limiting factor, TH. 15 DA release Once synthesized, intraterminal DA forms a primary, transient, cytosolic pool from which DA is vesicularized into a secondary pool. It is generally accepted that these two distinct pools of DA that are released through different mechanisms (Leviel, 2001). While vesicular DA release is known to be dependent on the depolarization of the nerve terminal and subsequent increase in intracellular C a 2 + . Although the mechanisms mediating cytosolic DA release is less clear, it is known to be independent of neural depolarization. However, considering the established role of D A T in mediating non-vesicular release in amphetamine induced DA efflux (Leviel, 2001; Rothman and Baumann, 2003; Elliott and Beveridge, 2005), and more recently, under physiological conditions (Falkenburger et al., 2001), the DAT may be an important contributor to impulse independent release of cytosolic DA. Homeostatic mechanisms regulating extracellular DA Physiological basal striatal DA levels, as measured by in vivo sampling techniques such as microdialysis or voltammetry, are maintained at a relatively stable 4-20nM concentration range (Parsons et al., 1991; Smith and Justice, 1994; Jones et al., 1998; Smith and Weiss, 1999; Chen, 2005). This is in contrast to the uM range of DA levels found in the synaptic cleft after burst firing of DA neurons in response to behaviourally relevant stimuli (Chen, 2005). However, despite these bursts of D A neuronal discharge causing massive release of DA into the synaptic cleft, extracellular DA levels remain relatively stable (Chen, 2005), suggesting the existence of mechanisms regulating the diffusion of synaptic DA into the extrasynaptic extracellular space. Although enzymatic breakdown of DA by extraneuronal catechol-O-methyl transferase (COMT) into H V A and monoamine oxidase-B (MAO-B) into DOPAC contributes to the degradation of released DA, the primary mechanism regulating extracellular DA levels is the DAT, which is 16 located at the vicinity of the synaptic cleft, and quickly removes released synaptic DA before it diffuses into the extracellular space. These homeostatic mechanisms play a vital role in maintaining constant, tonic stimulation of the extrasynaptic D A receptors. However, with progressive DA denervation in PD, these mechanisms alone can not maintain the same pattern of activity and stimulation in the striatum. Therefore, DA depletion triggers different compensatory mechanisms in attempt to sustain the same level and pattern of D A receptor stimulation. Compensatory mechanisms in the striatum with D A terminal loss Symptoms of PD begin to appear only after about 50% of D A neuronal loss in the substantia nigra, and about 80% of DA loss in the striatum (Bernheimer et al., 1973; Fearnley and Lees, 1991). As suggested by these pathological observations, experimental studies have shown evidence that highly effective compensatory mechanisms take place both in presynaptic and postsynaptic phases of dopaminergic neurotransmission. Presynaptic mechanisms It has been well recognized that extracellular D A levels in the striatum remain within a normal range despite a substantial loss of nigrostriatal D A neurons (Abercrombie et al., 1990). Normalization of extracellular D A levels in the striatum with DA terminal loss is an outcome of presynaptic compensatory mechanisms that affect all aspects of D A metabolism in remaining DA terminals, which are working concurrently or in stages: (i) increased D A synthesis in the remaining DA neurons by increased activity of T H protein (Pasinetti et al., 1992; Blanchard et al., 1995) and increased expression of T H mRNA (Blanchard et al., 1995); (ii) increased DA release by increased activity of surviving neurons (Agid et al., 1973; Zigmond et al., 1984), and increased extracellular levels of glutamate, which have a facilitative effect on DA release (Robelet et al., 2004) and (iii) decreased DA uptake by the downregulation of DAT (Bezard et 17 al., 2000; Lee et al., 2000). These^regulatory changes in D A metabolism are expected to increase extracellular D A levels towards the normal range, representing major presynaptic compensatory mechanisms in the striatum of PD patients. Altered regulation of postsynaptic dopamine receptors While the studies described above relate to the intrinsic properties of presynaptic DA neurons, post-synaptic changes also occur in response to reduced D A stimulation. One of these changes is mediated by "supersensitivity" of DA receptors, which is characterized by enhanced responsiveness, mainly behavioural, to certain dopaminergic agents such as L-dopa or apomorphine (Kostrzewa et al., 2005). This phenomenon may appear late in the development of PD, when D A denervation is severe (Zigmond and Strieker, 1980; Chen, 2005). Receptor "supersensitivity" can be attributed to two main causes: 1) an increase in DA receptor levels, and 2) increased sensitivity of D A receptors (Bezard and Gross, 1998). DA denervation does not affect D1 and D2 receptors similarly. There is a plethora of evidence which suggests, in humans and in several animal models, that expression of D2 receptors in the untreated striatum of PD is upregulated (Qin et al., 1994; Frohna et al., 1995; Narang and Wamsley, 1995; Morissette et al., 1996; Rioux et al., 1997; Araki et al., 1998; Piggott et al., 1999; Betarbet and Greenamyre, 2004). Effect of D A denervation on DI expression is more equivocal; upregulated in some studies (Narang and Wamsley, 1995; Morissette et al., 1996), but downregulated in others (Marshall et al., 1989; Qin et al., 1994). In other studies (Gerfen et al., 1990; Qin et al., 1994; Morissette et al., 1996), downregulation of DI mRNA was also observed. These discrepancies regarding the regulation of DI receptors remain unexplained. It is possible that regulatory changes of the DA receptors are time dependent, and the time at which these experiments are conducted following DA denervation 18 may contribute to the differences observed (Narang and Wamsley, 1995). In line with this view, Araki et al (2000) demonstrated a sequential pattern of receptor regulation with D2 changes preceding D l changes (Araki et al., 2000). In addition, D l changes are more transient than those of the more sustained D2 receptor changes. In addition, there are also differing patterns of regulation in various parts of the striatum. For instance, D2 receptor increase in the ventromedial striatum is sustained much longer than the D2 increase in the dorsolateral striatum (Araki et al., 2000). Together, these data demonstrate a complex pattern of changes in DA receptor levels after D A denervation. In conclusion, multiple mechanisms are involved in maintenance of physiological levels of DA stimulation despite the progressing depletion of DA cells. Initially, increased release combined with decreased uptake may be sufficient to uphold extracellular levels. However, with further progression, post synaptic changes take effect. Therefore, at different stages of the disease and D A denervation, different compensatory mechanisms may be essential to regulate DA. Neural Mechanisms of L-dopa-induced dyskinesia Pharmacology of L-dopa Since its discovery in the early1960s, L-dopa has been the cornerstone in the treatment of PD. Exogenous L-dopa is converted to DA by A A D C in the striatum, bypassing TH, the rate limiting factor in DA synthesis. Therefore, administration of L-dopa may increase striatal DA levels in a dose-dependent way (Henry et al., 1998; Lundblad et al., 2004). It has been proposed that L-dopa is also converted to D A in cells other than DA neurons in the striatum, such as AADC-containing glia (Nakamura et al., 2000), or other monoaminergic neurons, particularly serotonergic (Arai et al., 1994), and GABAergic neurons (Melamed et al., 1980; Hefti et al, 19 1981). This view has been used to explain how L-dopa is converted to DA in the striatum with almost complete loss of DA terminals (Poewe and Wenning, 2002; Kostrzewa et al., 2005). The benefit of L-dopa treatment often erodes after the first few years of treatment due to the emergence of fluctuating response, dyskinesia or both. The anti-parkinsonian effect of L-dopa may change gradually over time: becoming shorter in duration ("wearing-off'), and greater in magnitude, eventually leading to fluctuating responses after each dose of L-dopa. Similarly, chronic treatment with L-dopa may induce dyskinesia, which is involuntary movements appearing after administration of L-dopa, particularly when anti-parkinsonian effects of L-dopa are maximal (hence, peak-dose dyskinesia). Risk Factors for L-dopa-induced Dyskinesia Although mechanisms of L-dopa-induced dyskinesia are not fully understood, it has been recognized that certain factors related to the drug or the disorder increase the risk of L-dopa-induced dyskinesia. In regards to the drug-related factors, it appears that not only the duration of treatment, but also the type, dose and preparation of the drug influence the incidence of L-dopa-induced dyskinesia: (i) PD patients treated are less likely to develop dyskinesia with DA agonists than with L-dopa; (ii) incidence tends to be higher in PD patients treated with higher doses. At moderate doses, L-dopa elicits dyskinesia in approximately 50% of treated patients, and rises to 75%-80% with a higher dose (Calon et al., 2000; Nutt, 2001). This dose dependent effect of L-dopa on dyskinesia seen in patients is observed in animals as well (Henry et al., 1998; Lundblad et al., 2004); (iii) Observations in experimental and clinical studies suggest that pulsatile drug treatment, but not continuous drug treatment, causes motor complications including L-dopa-induced dyskinesia in animal models of PD and patients with PD (Olanow et al., 2000). 20 Although the mechanisms of this phenomenon are not fully understood, it is, at least in part, associated with downstream changes induced by pulsatile stimulation of DA receptors (discussed in detail at a later section). In regards to PD related factors, cumulated evidence from clinical and experimental studies has shown that D A denervation is necessary for L-dopa treatment to induce dyskinesia. (Mones, 1971; Nutt, 1990; Langston et al., 2000). For instance, in patients with asymmetric PD, L-dopa-induced dyskinesia appears earlier and is more severe on the more affected side than the less affected side (Mones, 1971). Current Views on the Mechanisms of L-dopa-induced Dyskinesia Although the neural mechanisms of L-dopa-induced dyskinesia are not fully understood, the next section highlights several of the neural changes that have been established as key contributors to this phenomenon. Increased Activity in the Basal Ganglia Output Nuclei It has been suggested that dyskinesia results from reduced activity in the STN and GPi/SNr neurons (Obeso et al., 2000). We can again use the classical model of basal ganglia to describe the consequences of this DA-induced overstimulation of the direct and indirect pathways on the output nuclei of the basal ganglia, the GPi/SNr (Figure 4). First, c overstimulation of striatal GABAergic projections in the direct pathway reduces the activity of the GPi/SNr. Secondly, overstimulation of striatal GABAergic projections in the indirect pathway reduces the activity of GPe neurons, which in turn, disinhibits GPi/SNr activity. Furthermore, it has been proposed that this reduces the tonic inhibitory influence of the GPi/SNr on the thalamo-cortical neurons, along with excess cortical-striatal activity, drive the expression of unwanted movements (Obeso et al , 2000). Data from electrophysiological studies have 21 Figure 4. Schematic drawing representing basal ganglia function in the on-state of L-dopa-induced dyskinesia. The red lines with round ends depict excitatory connections. The blue lines with blunt ends depict inhibitory connections. Thick lines represent overactive projections, whereas thin lines represent underactive projections. Consistently, boxes with thick outlines (the GPe, thalamus) represent nuclei which are overactive. Dotted Lines or outlines represent projections or nuclei that are eliminated due to DA cell death. Abbreviations: Dl= dopamine DI receptor; D2 = dopamine D2 receptor; GPe = external segment of globus pallidus; GPi = internal segment of the globus pallidus; SNc = substantia nigra pars compacta,; SNr = substantia nigra pars reticulata; STN = subthalamic nucleus 22 CORTEX 1 J STRIATUM D2 01 $ SNc :> J - I GPe II STN GPi/SNr : THALAMUS Figure 4 23 confirmed reduced neuronal firing in the GPe and increased activity in the GPi during apomorphine induced dyskinesia in MPTP treated monkeys (Filion et al., 1991) and as well, in PD patients (Lozano et al., 2000). This model of L-dopa-induced dyskinesia provides a useful theoretical framework to explain many experimental results and to generate effective working hypotheses. However, to say that an underactive GPi/SNr is the sole mechanism underlying L-dopa-induced dyskinesia would be inaccurate. Accordingly, this model is unable to explain why reduction of GPi neuronal firing through pallidotomy, consistently improves, rather than induce, dyskinesia (Lang et al., 1997). Closer investigation of GPi/SNr activity suggested that not only is the net activity decreased, but patterns of firing and synchronization is also affected in L-dopa-induced dyskinetic animals (Matsumura et al., 1995; Vitek and Giroux, 2000; Boraud et al., 2001). Therefore, an interaction of many factors is likely involved in the production of downstream neuronal changes that lead to abnormal firing rate and pattern associated with L-dopa-induced dyskinesia. As regulation of the basal ganglia involves several neurotransmitter systems, the following section will review some evidence of how key systems may be involved in the generation of L-dopa-induced dyskinesia. Postsynaptic changes due to Dl and D2 pulsatile stimulation As mentioned earlier, pulsatile, but not continuous, D A receptor stimulation is associated with development of L-dopa-induced dyskinesia (Chase and Oh, 2000). D A receptor stimulation is relatively tonic in the striatum, except for short phasic bursts that occur with expectation of certain behaviours and reward (Schultz and Romo, 1990). Therefore, in the DA denervated striatum, DA receptors are more dependent on exogenous stimulation by D A agents such as L-dopa. Due to the short half-life of L-dopa (60-90 minutes), the peripheral or plasma levels of L-dopa derived DA fluctuates. As the number of DA terminals decreases, the storage capacity of 24 L-dopa derived D A decreases, and the ability of DA neurons to buffer fluctuating plasma levels become reduced (Chase and Oh, 2000). Therefore, DA receptors in the D A denervated striatum are exposed to an abnormal pattern of high and low stimulation. (Sealfon and Olanow, 2000). Consistent with this concept, chronic treatment of a really high dose of L-dopa in unlesioned monkeys elicit the expression of dyskinesia, suggesting that even with a normal number of DA terminals, the buffering capacity can be overwhelmed if a large enough dose is used (Pearce et al., 1999). It is likely that this abnormal pattern of DA receptor stimulation leads to downstream neuronal changes leading to L-dopa-induced dyskinesia. Data from animal studies showed that DA denervation caused upregulation of pre-proenkephalin (PPE) and downregulation of pro-dynorphin (PDyn) mRNA expression in animal striatum (Gerfen et al., 1990; Jolkkonen et al., 1995). Enkaphalin levels remain elevated in rats which develop dyskinesia, but are normalized with administration of long-acting DA agonists (Herrero et al., 1995; Jolkkonen et al., 1995). Similar observations were made by Cenci and colleagues (1998), who detected increased mRNA expression of PPE and PDyn in the lesioned striata of dyskinetic rats (Cenci et al., 1998). Furthermore, they found that PDyn mRNA levels had the strongest correlation to severity of dyskinesia. These data suggest a role for stimulus-induced, post-synaptic changes in the gene expression of PPE and PDyn in L-dopa-induced dyskinesia. Role of D3 receptors in L-dopa-induced dyskinesia Another receptor subtype that is receiving increasing attention for its role in L-dopa-induced dyskinesia is the D3 receptor. In the normal brain, D3 receptors are typically localized in the shell of the nucleus accumbens, which is associated with emotional and cognitive processes. They are virtually absent in the dorsal striatum (Sokoloff et al., 1990; Levesque et al., 1995) and the core region of the nucleus accumbens, two areas that are involved in the motor 25 disturbances. However, studies conducted by Bordet and colleagues (1997) in 6-OHDA lesioned rats demonstrated D3 receptor upregulation in the nucleus accumbens core by 680%, and in the dorsal caudate putamen by 130%, following repeated L-dopa treatment (Bordet et al., 1997). Induction of gene expression was correlated in D3 upregulation. Behavioural sensitization can be elicited by administration of D3 agonists alone, and can be blocked by pretreatment of D3 antagonists, suggesting the critical role for D3 receptors in expression of behavioural sensitization. Furthermore, it appears that D1 receptors are involved in the induction of D3 gene expression following L-DOPA, because D l agonist SKF38393 produced similar behavioural effects as L-dopa, and D l antagonist SCH23390 blocked the L-dopa-induced behavioural effect. In MPTP treated primates and in humans, D3 binding decreases following denervation, a trend that is reversed with repeated treatment of L-dopa (Morissette et al., 1998; Ryoo et al., 1998; Bezard et al., 2003). Coincidently, D3 upregulation correlates with the development of L-dopa-induced dyskinesia and motor fluctuations, and when normalized, is correlated with the attenuation of L-dopa-induced dyskinesia (Bezard et al., 2003). Role of glutamate receptors in L-dopa-induced dyskinesia As previously mentioned, the excessive drive from the motor cortices may an important contributing factor in the etiology of L-dopa-induced dyskinesia. Indeed, many studies have reported changes in glutamate regulation following DA denervation in animal models, and after chronic L-dopa treatment. Enhancement of corticostriatal glutamate transmission has been noted following DA denervation (Lindefors and Ungerstedt, 1990; Meshul et al., 1999; Calabresi et al., 2000), but is further augmented with L-dopa therapy (Robelet et al., 2004). Therefore, numerous studies have examined the effects of L-dopa treatment on glutamate receptors and their possible roles in L-dopa-induced dyskinesia. Co-administration of NMDA-receptor antagonists in MPTP 26 lesioned monkeys substantially prevented the dykinesiogenic, but not the anti-parkinsonian effects of L-dopa (Gomez-Mancilla and Bedard, 1993; Papa and Chase, 1996; Blanchet et al., 1998). Similarly, administration of AMPA receptor antagonist N B Q X produced similar effects (Marin et al., 2000). Metabotropic glutamate receptors also colocalize with the ionotropic glutamate receptors on the dendrites of the medium spiny neurons (Testa et al., 1994). However, their contribution to L-dopa-induced dyskinesia has yet to be tested. Faulty learning hypothesis It can take up to several weeks of chronic L-dopa administration in animal models before dyskinesia develops. Once the symptoms appear, however, they are difficult to extinguish. Even weeks after treatment withdrawal, the first dose of L-dopa will trigger the onset of dyskinesia, indicating that long-term treatment with LD has made lasting modifications in the response of the basal ganglia to DA (Calon et al., 2000). Recently, new experimental evidence has been used to support a new hypothesis explaining the irreversibility of L-dopa-induced dyskinesia, based on a model of synaptic memory (Picconi et al., 2003). Long-lasting synaptic changes due to non-physiological stimulation of glutamate receptors have been studied extensively in animal models of learning and memory, especially in relevance to long term potentiation (LTP). The mechanisms of LTP involve several phosphatase-kinase signaling pathways, alterations in gene expression and subsequent protein regulation. Remarkably, the development of L-dopa-induced dyskinesia is associated with similar processes in striatal neurons, including alterations in the expression of immediate early genes, phosphorylation of glutamate receptors, activation of similar signaling cascades, and expression of transcription factors. Phosphorylation of one such transcription factor, the cAMP-response-element-binding protein (CREB), is critical in the induction of LTP. PKA-dependent activation of CREB is also 27 triggered by stimulation of D l receptors (Dudman et al., 2003), providing further support that mechanisms involved in LTP may underlie the pathology of L-dopa-induced dyskinesia. Recent studies performed in vitro demonstrated LTP in striatal neurons in response to high frequency stimulation of glutamate which as DA dependent, suggesting that L-dopa treatment may be a substrate of the long term synaptic changes associated with L-dopa-induced dyskinesia (Picconi et al., 2003). Furthermore, striatal slices taken from non-dyskinetic animals, but not dyskinetic animals, demonstrated the ability to depotentiate after 10 minutes of low frequency stimulation. Taken together, these data suggest bidirectional plasticity in striatal neurons may be important for the storage, as well as deletion of appropriate and inappropriate motor information. Therefore, inability to erase nonessential motor information may form a pathological basis for L-dopa-induced dyskinesia. Treatment of L-dopa-induced Dyskinesia Although currently there is no cure for L-dopa-induced dyskinesia, several approaches have been attempted to reduce the severity of L-dopa-induced dyskinesia. The first measure is typically reduction of drug (L-dopa or DA agonists) dosage. Lower dosage is typically correlated with lessening of dyskinesia severity. Anti-dyskinetic drug (usually anti-glutamatergic) supplements can also be used conjunctively with L-dopa. However, while many of these drugs alleviate dyskinesia, they also reduce the efficacy of L-dopa (Brotchie et al., 2005). Furthermore, tolerability of these drugs is highly variable among patients (Brotchie et al., 2005). Recently, the development of surgical procedures facilitating stimulation of deep brain nuclei has also proved effective. A Pathogenic Role for the Dopamine Transporter in L-dopa-induced Dyskinesia Overview 28 Previous studies have shown that in 6-OHDA treated rats, an acute injection of high dose of L-dopa induced a surge of D A efflux in the lesioned striatum that showed a similar time course of L-dopa-induced rotational behaviour (Nakazato and Akiyama, 1989; Abercrombie et al., 1990). Increases in striatal DA have also been observed in the denervated striatum of PD patients who display response fluctuations to L-dopa and dyskinesia (de la Fuente-Fernandez et al., 2001). In contrast, D A level increases in the unlesioned striatum are either mild or absent. This L-dopa-induced efflux of extracellular DA in the lesioned striatum may be pathogenic as it exposes the D A receptors to bursts of a fluctuating pattern of stimulation, resulting in downstream, long lasting changes in post-synaptic striatal projection neurons (Calon et al., 2000; Olanow et al., 2000). Furthermore, it seems that once these changes are present, they are very difficult, if not impossible, to reverse (Brotchie et al., 2005). Therefore, in order to better understand the pharmacology of L-dopa and its effect on D A regulation, it is important understand the mechanisms mediating L-dopa-induced D A efflux in the DA denervated striatum. To study the mechanism underlying this DA efflux, Miller and Abercrombie (1999) used in vivo microdialysis to examine the effect of TTX, an inhibitor of fast Na + channels, on L-dopa (50mg/kg) induced DA efflux in the DA-denervated striatum (Miller and Abercrombie, 1999). Their results indicate that T T X significantly attenuates the DA surge response, suggesting the mechanisms for release is largely C a 2 + mediated. However, T T X administration only resulted in a partial attenuation, suggesting that part of the L-dopa-induced D A efflux may be mediated by a depolarization-dependent mechanism. Ca2+-mediated vesicular release is one mode of D A release, however, recent evidence suggests that the DAT is also involved in the release of DA under physiological conditions, in particularly under the influence of increased glutamatergic activity, as observed in the DA 29 dendrites in the SNr (Falkenburger et al., 2001). In addition to DA denervation, chronic treatment with L-dopa appears to be necessary to further enhance glutamate release in the striatum (Robelet et al., 2004). Given this evidence, it is of interest to study the role of reverse transport as a mechanism for L-dopa-induced DA release. The Dopamine Transporter The DAT is a critical player in the modulation of the expression of movement and in PD. By uptaking released DA molecules back into intracellular stores, it has a key role in regulating the intensity and duration of DA stimulation. The DAT belongs to a family of monoamine uptake transporters which also includes the NET and SERT [for noradrenalin (NA) and serotonin, respectively]. Furthermore, the monoamine transporters are established targets for pharmacological agents such as anti-depressants and psychostimulant drugs of abuse such as cocaine, amphetamines and amphetamine-like drugs. Therefore, an examination of the mechanisms by which these drugs interact with the DAT has greatly contributed to our understanding of the function and regulation of this transporter protein, as well as the interactions between the D A T and DA. The DAT is localized mainly on the pre-synaptic terminals of dopaminergic neurons (Ciliax et al., 1995), but is also found on axons and dendritic spines, (Falkenburger et al., 2001) along with extraneuronal sites such as glial cells. The abundance of presynaptic DATs on nigrostriatal neurons has led to imaging of the transporter as a common measure of the progressive DA neuronal loss in PD. Decrease in DAT binding in PD patients is widely established in clinical studies. Similarly, animals that have received nigrostriatal lesions also consistently show decreased binding to the DAT. 30 Uptake of D A molecules is dependent on the sequential binding and co-transport of Na + and CI" ions, and is driven by the electrochemical gradient generated by the Na + /K + ATPase pump. Binding of 2 Na + and 1 CI" and 1 DA molecule activates a conformational change in the DAT that allows the translocation of ions and DA into the cell, coupled with the binding and outward transport of K + . Inward transport of DA involves the net movement of positively charged ions into the cell; therefore, a negative membrane potential facilitates DA influx. Conditions that change membrane potential, ion gradients, or amount of ATP molecules can evidently change the function of the DAT, including the directional mode (Blakely, 2001). DAT blocking drugs fall into two basic categories. Drugs like cocaine and methylphenidate (MP) are non-selective competitors of DAT which blocks substrate. Amphetamines, on the other hand, are carrier substrates which themselves are transported into the cell, resulting in a block of D A uptake and additionally, simultaneous stimulation of DA efflux from intracellular stores. Therefore, it seems that while the normal function of the DAT is uptake DA, under certain circumstances, the DAT can partake in the release of DA as well via a reversal of its functional mode. Traditionally, reversed transport was considered a drug-induced phenomenon; however, more recent findings suggest that DAT mediated DA release occurs under more physiological circumstances (Falkenburger et al., 2001). Evidence for D A T mediated DA release in L-dopa-induced dyskinesia Recent findings from our lab demonstrated that in unilaterally lesioned rats chronically exposed to a low, clinical dose of L-dopa (lOmg/kg, i.p.), reverse dialysis of specific DAT blocker GBR 12909 significantly attenuated the L-dopa-induced DA efflux (Ahn et al., 2004). Although GBR 12909 also attenuated the DA response in L-dopa na'ive rats, the effect was much smaller compared to that in chronically treated animals. Additionally, perfusion of Ca free 31 artificial cerebral spinal fluid resulted in a significant decrease of the L-dopa-induced DA response in drug-naive, but not chronically treated, rats. Taken together, these data indicate that over a course of 3 weeks chronic treatment, L-dopa induces a functional reversal of the DAT, from uptake, to release mode. More importantly, the role of D A T in mediating L-dopa-induced DA release has important implications for L-dopa-induced dyskinesia. Experimental rationale The rationale for the experiments conducted for this thesis is based in part on the findings, that although reverse dialysis of GBR 12909 diminished the L-dopa-induced DA response, this DAT antagonist failed to attenuate dyskinetic behaviour. It is possible that when administered via reverse dialysis, the amount of drug that diffuses into the immediate column of tissue surrounding the microdialysis probes is insufficient to cause any behavioural effect. The objectives of the studies presented in this thesis are to 1) replicate the effects of intrastriatal GBR 12909 using systemic administration of an alternate DAT blocker, methylphenidate (MP), 2) to assess the effects of MP on L-dopa-induced DA efflux in 6-OHDA lesioned rats receiving different periods of L-dopa treatment, and 3) assess the effect of MP on L-dopa-induced dyskinesia. Experimental considerations To achieve the aims of these experiments, we selected MP as the drug of chose to investigate the function of the DAT. As well, the unilateral 6-OHDA lesioned rat model was selected for these studies. The following sections provide some background information on these two investigative tools, as well as our rationale for using them. Methylphenidate 32 Like amphetamine, MP is classified as a DAT-blocking psychostimulant. MP is the active ingredient used in the clinically approved drug Ritalin, which has been established primarily for treatment of ADHD, but has been used as well to treat depression, narcolepsy, and fatigue (Leonard et al., 2004). Although the main mechanism of action of MP is blocking of the DAT, MP shows affinity for the NET as well. Therefore, the therapeutic effects of MP most likely come from increases in both extracellular D A and noradrenalin levels (Leonard et al., 2004). Although MP has a high pre-systemic metabolism (i.e., metabolized in the mouth and the gut), only a small amount is needed for extensive diffusion in the brain (Leonard et al., 2004). PET imaging shows that the peak uptake of MP in humans was approximately 60 minutes following oral administration (Volkow et al., 2002). Plasma concentrations of MP correlate well with DAT occupancy, suggesting that measuring plasma concentrations is a good indicator of brain levels of MP. In animal studies of MP, animals are typically injected intraperitoneally, allowing for quicker availability in the brain. Microdialysis studies showed that (Huff and Davies, 2002) increases in plasma levels of MP are correlated with extracellular striatal DA levels and drug-induced behaviour, all of which peaked at approximately 20-40 minutes after administration and persisted for about 3 hours. MP was selected as the DAT blocker used for the experiments in this thesis for several reasons. First, it demonstrates a similar time course as L-dopa, displaying similar peak and duration. Second, as opposed to some other DAT blocking agents, MP is relatively easy to dissolve and inject. Finally, as MP is already a clinically approved drug, any therapeutic value for L-dopa-induced dyskinesia that may be demonstrated from theses studies can be easily tested in pre-clinical trials. 33 Animal models of L-dopa-induced dyskinesia 6-OHDA and MPTP lesioned animals display L-dopa-induced dyskinesia in a dose-dependent way (Henry et al., 1998; Lundblad et al., 2004). With chronic L-dopa treatment, MPTP-lesioned primates develop dyskinetic movements that resembles very closely to that seen in PD patients (Cenci et al., 2002). Unilaterally 6-OHDA lesioned rats also display dyskinesia in the contralateral forelimb, trunk, and orafacial musculature (Cenci et al., 2002). Both rats and primates show a time course that is similar to peak-dose dyskinesia in humans (Lee et al., 2000). Furthermore, both models show similar molecular changes that include upregulation of dynorphin and enkephalin mRNA and receptor binding (Cenci et al., 1998), and similar responses to pharmacological agents used to treat L-dopa-induced dyskinesia in patients. Despite this accumulating evidence, the suitability of the 6-OHDA lesioned rat model for L-dopa-induced dyskinesia has been questioned (Cenci et al., 2002). Critics of this model argue that the main behavioural effect elicited by L-dopa in rats is contraversive rotational locomotion, a behaviour that is absent from human dyskinesia. Furthermore, they argue that limb and trunk movements may simply be compensatory responses to the intense rotation (Langston et al., 2000). However, recent findings challenge this view by showing that limb and axial dyskinesia can be produced separate from locomotive rotation, using specific unilateral intrastriatal lesions in the lateral regions of the putamen (Lundblad et al , 2002). Similarly, lesions in the medial putamen elicit L-dopa-induced rotation without forelimb and trunk movements. Furthermore, L-dopa causes dyskinesia without rotation in bilaterally lesioned rats. Taken together, these data suggest that rotation is distinct from the abnormalities in limb, mouth, and trunk movements. 34 G E N E R A L M E T H O D S L - O H D A Lesioning and Probe Implantation Sprague-Dawley female rats weighing 275-300 grams from Charles River (St. Constant, Quebec, Canada) or University of British Columbia (Animal Care Centre Breeding Unit, South Campus) were used in all experiments. All animals were pair-housed upon arrival and individually house after surgery in plexiglass cages with free access to rat chow and water, in a colony maintained at 19-22 °C, with a 12:12 hour light-dark cycle. For induction of anaesthesia, 4% isoflurane mixed with oxygen was used, and then subsequently lowered to 1.5 - 2.5% isoflurane (AErrane, Baxter Co., Ontario, Canada) for maintenance throughout the rest of surgery. Animals were positioned in flat skull position (mouth bar at -3.2mm) in a stereotaxic apparatus that was lined with a heating pad to maintain the animals' body temperature. DA-denervating lesions were performed by unilateral injection of 6-OHDA (3ug/uL; 2ug/uL) prepared in 0.02% ascorbic acid solution into 2 locations: the SNc (from bregma, -5.2 mm AP, -2.0 mm M L ; from dura, -7.1 mm DV), and the medial forebrain bundle (MFB; -4.2mm AP, -1.7 M L , -7.9 DV); all coordinates were determined according to Paxinos and Watson (1997). Injection of 6-OHDA into the MFB and SNc, the origins of the nigrostriatal pathway, is the lesion procedure used in most studies using the 6-OHDA model (Schwarting & Huston, 1996). Injections were performed at the rate of 1 mL/min, and the injection needle was left at the lesion site for additional 3 minutes to allow for drug diffusion. Additionally, 19 gauge stainless steel guide cannulae (15mm) were implanted directly above the lesioned and unlesioned striata (+1.0 AP, +/-3.1 MP, -1.0 DV). Approximately 30 minutes before rats were taken of isoflurane anaesthesia, rats were given a subcutaneous dose of 35 analgesic (ketoprofen 0.05mL or buprenorphine 0.1 mL) before placed under a heat lamp for recovery. Drugs and Preparation 6-hydroxydopamine hydrobromide (6-OHDA), L-3.4-dihydroxyphenylalanine methyl ester (L-dopa), benserazide hydrochloride, fluoxetine hydrochloride, and desipramine hydrochloride were all obtained from Sigma Aldrich, Ontario, Canada. L(+)-ascorbic acid was obtained from E M Science, Gibbstown, NJ, USA. Methylphenidate hydrochloride was synthesized from the lab of Dr. Tom Ruth at the University of British Columbia (Vancouver, BC, Canada). 6-OHDA was dissolved in 0.02% L-ascorbic acid solution immediately prior to infusion. L-dopa was prepared fresh daily in a mixed solution with peripheral DOPA-decarboxylase inhibitor, benserazide hydrochloride (15mg/kg) in a 0.9% saline solution. MP, fluoxetine, and desipramine were all dissolved in 0.9% saline solution Behavioural Analysis: Abnormal Involuntary Movement (AIM) Scale A modified version of the of AIM scale (Lee et al., 2000) was used to classify and rate three different components of L-induced behaviour according to their topographic distribution: 1) locomotive rotation (loco), 2) axial dystonia (axial), and 3) limb extension (limb). Each component was rated on a scale of 0-4 based on the proportion of time/monitoring period: 0 being absence of behaviour, 1 being intermittence under 50% of the time, 2 being intermittence over 50% of the time, 3 being continuous dyskinesia that can be interrupted by sensory distraction, and 4 being continuous dyskinesia that is oblivious to sensory distraction. A total-AIM score was calculated summing all AIM scores during total observation period (160 minutes after L-dopa administration) Microdialysis 36 Microdialysis probes consisted of a semipermeable membrane 4.0 mm in length (340 um outer diameter; 65,000 Dalton molecular weight cutoff; Filtral 12; Hospal, Neurnberg, Germany), and were flushed continuously at 1 ul/min with artificial cerebrospinal fluid (147.0 mM NaCl, 1.2 mM CaC12, 3.0 mM KC1, 1.0 mM MgC12, and a 10.0 mM sodium phosphate buffer) using a 2.5 ml gas-tight syringe (Hamilton, Reno,NV) and a syringe pump (model 22; Harvard Apparatus, South Natick, MA) before implantation into rat striata. Rats then remained in testing chambers overnight with access to food and water. On test day, samples were collected at 10 minute intervals and assayed for DA and its metabolites 3,4 dihydroxyphenylacetate (DOPAC) and homovanillic acid (HVA) until a stable baseline was established (4 samples within 10% variation). Drugs (L-dopa or MP) were administered to rats, and samples continued to be collected every 10 minutes for 165 minutes after L-dopa injection. L-dopa-induced AIM was also monitored and rated using a modified AIM rating scale at 10 minute intervals, and videotaped for later reference. High Pressure Liquid Chromatography Two HPLC-electrical detection systems were employed for assaying DA and DA metabolite contents in striatal dialysates. The systems consisted of an ESA 582 pump (Chelmsford, MA), a Scientific Systems, Inc. (State College, PA) pulse damper, a Rheodyne manual injector (20 ul injection loop, Rhonert Park, CA), a TOSOH Biosep (Montgomeryville, PA) Super ODS TSK column (2 mm x 10 mm; 2uM particle),), an Antec Leyden (Leyden, The Netherlands) Links system and an Antec Intro detector with a VT-03 electrochemical flowcell (^applied= +0.7 V). The mobile phase consisting of 70mM sodium acetate, 40mg/L of EDTA, and 50mg/L of sodium octyl sulfate (adjustable), pH 4.0, and 12% methanol, was flowed through 37 the HPLC system at 0.18mL/min. Chromatographic data was analyzed using EZChrom Elite software. Data Analysis and Statistics Neurochemistry All neurochemical data are expressed as difference from baseline (average of the last four baselines). Statistical significance was based on analysis of variance (ANOVA) and followed when appropriate by Dunnett's post hoc test for comparisons (for within-groups comparisons, i.e. time). For between-groups analysis, a 2-way A N O V A was used to compare the effects of MP pretreatment vs. L-dopa only on L-dopa induced neurochemical changes. In addition, a T -test was used to compare L-dopa induced peak neurochemical changes in the lesioned striatum. Peak response was calculated as the average of the three highest consecutive data points following L-dopa injection. Using a T-test, behavioural data (total AIM score) were compared statistically between MP-pretreated vs. L-dopa only groups. In this thesis, only p values were presented for those results that did not reach statistical significance. Histology After testing, rats were given a minimum of 1-week washout period, before being deeply anaesthetized and decapitated. Brains were promptly removed and flash frozen at 2-methylbutane (Fisher Scientific, New Jersey, USA) cooled in dry ice, and stored at -80°C until being sectioned at 50um for verification of probe placement according to Paxinos and Watson's rat brain atlas (Paxinos and Watson, 1997). 38 E X P E R I M E N T 1 -Dose dependent effect of M P on L-dopa-induced dyskinesia Introduction In the first experiment of this study, we investigated whether a systemic dose of MP would attenuate L-dopa-induced AIM, and in addition, whether a dose effect of MP would be observed. In chronically L-dopa treated animals, we hypothesized that MP would result in a dose-dependent attenuation of the L-dopa-induced AIM. In the second experiment, we examined the possible role for other monoaminergic transporter systems in L-dopa-induced dyskinesia, by using pretreatment of fluoxetine and desipramine to inhibit serotonin and noradrenalin transporters, respectively. Methods Following a 4 week recovery period after surgery, rats with unilateral 6-OHDA lesions received 3 weeks of daily L-dopa injections (lOmg/kg i.p., co-administered with 15mg/kg i.p. of benserazide). Development of dyskinesia on the side contralateral to the lesion was monitored daily using the modified AIM rating scale. L-dopa-induced AIM severity progressed steadily over the first week of treatment, and stabilized after approximately 2 weeks. Using the modified AIM scale, we obtained a pretest baseline measure of L-dopa-induced AIM on each of the two days leading to test day (Days 19 and 20). Scoring began 5 minutes after L-dopa administration, and subsequently every 10 minutes for 160 minutes. Only rats that displayed dyskinetic behaviour on their pre-test baseline were selected for testing. On test day, animals were divided into 5 treatment groups, and were pretreated with MP, fluoxetine, or desipramine 20 minutes between receiving an L-dopa challenge. The animals used in each of the treatment groups are summarized in Table 1. 39 Table 1. Summary of animals and treatment groups used in Experiment 1. Treatment Group Number of Subjects Used Experiment #1 MP 7 mg/kg n=15 MP 14 mg/kg n=17 MP 21 mg/kg n=10 Experiment #2 Fluoxetine 25 mg/kg n=6 Desipramine 25 mg/kg n=8 Results Dose Effect of Methylphenidate With daily repeated L-dopa treatment, the severity of dyskinesia severity increased during the first week, and reached a stable plateau within 2 weeks (Figure 5). Rats in all three groups demonstrated moderate to severe dyskinesia as observed in the pretest baseline measures. After MP administration, rats in all three groups displayed moderate turning behaviour ipsiversive to their lesion due to a greater relative increase of DA in the unlesioned striatum. Direction of turning was reversed soon after L-dopa was administered. This pattern of directional change was observed in all animals that received MP pretreatment in the experiments described in the later sections. MP at all three doses attenuated L-dopa-induced dyskinesia in all rats in comparison to their pre-test baseline (Figure 5). 7mg/kg of MP, resulted in a significant decrease of L-dopa-induced dyskinesia (L-dopa pre-test baseline vs. L-dopa with MP pretreatment: 87.63 ± 3.75 vs. 48.00 ± 6.83, T = 5.266,p < 0.001), as did 14mg/kg (76.20 ± 4.28 vs. 22.94 ± 8.67, T = 5.680,/? < 0.001), and 21mg/kg (67.85 ± 7 . 1 5 vs. 23.60 ± 5.32, T = 5.231, p < 0.001). While the attenuation produced by 14mg/kg and 21 mg/kg were significantly greater than that by 7mg/kg (T = 2.300, p = 0.029; T = 2.681,/? = 0.013, respectively), there were 40 Figure 5. Dose Effect of Methylphenidate on L-dopa-induced AIM in chronically L-dopa treated rats. Three groups of animals were each tested with a different dose of MP pretreatment (Panel A = 7mg/kg MP, i.p., Panel 5=14 mg/kg MP, i.p., Panel C = 21mg/kg MP, i.p.). The top row of graphs represents L-dopa-induced AIM scores on the day prior to test day. The middle three graphs represent the effect of MP pretreatment on L-dopa-induced AIM scores. The graphs on the bottom row represent a between groups comparison of the total AIM scores on pre-test and test day. The blue bars indicate locomotive turning, either ipsiversive or contraversive to the lesioned hemisphere. The orange bars indicate axial dystonia, and the white bars indicate limb dyskinesia. Significance for experimental group relative to their own control * p< 0.05; ** p< 0.01; *** p< 0.001. Significance effect of treatment between groups #p < 0.05. 41 A Pre- Test AIM 8 < 4 0 4 8 12 Time (xlOmins) Effect of MP on AIM 7mg/kg < Qj 0 4 8 12 Time(xlOmins) Effect of MP on Total AIM B Pre- Test AIM 0 4 8 12 Time (xlOmins) Effect of MP on AIM 14mg/kg 4 8 12 Time (xlOmins) #1 Effect of MP on Total AIM # 100 S 80 < 60 £ 40 *- 20 *** L D + M P L D L D + M P c Pre- Test AIM 8 12 Time (xlOmins) Effect of MP on AIM 21 mg/kg ... 4 8 12 Time (xlOmins) Effect of MP on Total AIM 100 1 80 < 60 £ 40 20 L D L D + M P • Limb • Axial • L o c o Figure 5 42 no significant differences between 14mg/kg and 21mg/kg (p = 0.956). Hence, 14mg/kg MP was the chosen dose for the subsequent experiments for this thesis. Effect of Fluoxetine and Desipramine After 3 weeks of daily L-dopa treatment, all animals developed a stable dyskinetic response to L-dopa. On test day, pretreatment of either fluoxetine (Figure 6A) or desipramine (Figure 6B) failed to significantly attenuated AIM scores, (fluoxetine: p = 0.220; desipramine: p = 0.415). While there is some data that suggest fluoxetine may improve apomorphine-induced dyskinesia (Durif et al., 1995), our data lend support to the present hypothesis that L-dopa-induced dyskinesia is dependent on the DAT system, but not other monoaminergic transporters such as the SERT or the NET. Discussion The findings from this experiment demonstrated the dose-dependent attenuation of systemic MP on L-dopa-induced dyskinesia in 3-week L-dopa treated rats. These data support our hypothesis that the D A T may be involved in L-dopa-induced dyskinesia in chronically treated rats, possibly by releasing DA by reversed transport. Furthermore, blockade of the SERT and NET did not give the same results, indicating that the DAT is the key player in this effect. However, the lack of effect from SERT and NET contradict previous reports in the literature that showed the anti-dyskinetic effects of fluoxetine (Durif et al., 1995). However, these tests were conducted in patients, and much of the improvements shown were for onset- and end-of-dose dyskinesia, which has a different behavioural profile than peak-dose dyskinesia. These differences may help explain the lack of effect of fluoxetine seen in the present study. 43 Figure 6. Effect of Fluoxetine and Desipramine on L-dopa-induced AIM. Two groups of animals were each tested with either pretreatment with 25mg/kg i.p of fluoxetine (Panel A) or 25mg/kg i.p. of desipramine (Panel B). The top row of graphs represents L-dopa-induced AIM scores on the day prior to test day. The middle graphs represent the effect of fluoxetine or desipramine pretreatment on L-dopa (lOmg/kg) induced AIM scores. The graphs on the bottom row represent a between groups comparison of the total AIM scores on pre-test and test day. The blue bars indicate locomotive turning, either ipsiversive or contraversive to the lesioned hemisphere. The orange bars indicate axial dystonia, and the white bars indicate limb dyskinesia. Significance for experimental group relative to their own control * p< 0.05; ** p< 0.01; ***p< 0.001. A Pre-Test AIM 0 4 8 12 Time (xlOmins) Effect of 25mg/kg Fluoxetine 4 8 12 16 Time (xlOmins) Effect of 25mg/kg Fluoxetine on Total AIM 100 S < i 50 O LD Fluoxetine + LD B • Limb • Axial B L o c o Pre-Test AIM l l . . l i f t . 4 8 12 16 Time (xlOmins) Effect of 25mg/kg Desipramine 4 8 12 Time (xlOmins) 16 Effect of 25mg/kg Desipramine on Total AIM 100 < . n Desipramine L U +LD Figure 6 45 E X P E R I M E N T 2- Effects of Methylphenidate on L-dopa-induced D A Efflux and Behaviour: a Microdialysis Study Introduction After establishing dosage effect with MP, 14mg/kg of MP was chosen for this study. To examine L-dopa-induced functional changes in the DAT, we examined the effects of MP on three groups of rats receiving different durations of L-dopa treatment. Methods Following surgery and at least 4 weeks of recovery period, rats were divided into 3 L-dopa treatment groups: 1) no treatment of L-dopa, 2) 6 days of daily L-dopa treatment, or 3) 20 days of daily L-dopa treatment (lOmg/kg i.p. L-dopa, coadministered with 15mg/kg i.p. benserazide). On test days, which was the day following the last day of LD treatment period, rats in each group were administered either with L-dopa only (lOmg/kg i.p. coadministered with benserazide, 15mg/kg i.p.) or LD with a 20 minute pretreatment of MP (14mg/kg, i.p.). Microdialysis and AIM rating were used to assess neurochemical and behavioural responses to test challenge. Only rats that had developed dyskinesia and had complete unilateral lesions (if basal D A levels in the lesion striatum <10% of basal DA levels in the unlesioned striatum) were used for analysis in this study. The animals used in each of the treatment groups are summarized in Table 2. Table 2. A summary of the animals and treatment groups in Experiment 2 Treatment Group Challenge on Test day Number of Animals L-dopa-naiVe LD alone N=7 L-dopa-naiVe LD + MP N=6 L-dopa treatment (1-week) LD alone N=7 L-dopa treatment (1-week) LD + MP N=7 L-dopa treatment (3-week) LD alone N=9 L-dopa treatment (3-week) LD + MP N=6 46 Results Effect of L-dopa treatment on basal neurochemical levels Basal D A levels in the lesioned striatum of were 5% of that in the unlesioned striatum in L-dopa naive animals [lesioned striatum vs. unlesioned striatum, respectively; (mean ± SEM) 0.44 ± 0.25 vs. 9.63 ± 2.92 nM, T= 2.92;p = 0.008]; 5.5% in 1-week L-dopa treated animals (0.81 ± 0.22 vs. 14.64 ± 4.25 nM, T = 3.38,/? = 0.002), and 5% in 3-week L-dopa treated animals (0.50 ± 0.25 vs. 10.28 ± 3.44 nM, T = 3.29,;? = 0.003) (Figure 7, top graph). Basal DOPAC levels in the lesioned striatum of were 3% of that in the unlesioned striatum in L-dopa naive animals [lesioned striatum vs. unlesioned striatum, respectively; (mean ± SEM) 1066.92 ± 138.93 vs. 12.46 ± 6.02 nM, T= 7.83;p < 0.001]; 1.2% in 1-week L-dopa treated animals (1526.85 ± 181.46 vs. 18.87 ± 6 . 3 1 nM, T = 8.61,p< 0.001), and 1% in 3-week L-dopa treated animals (1342.02 ± 141.93 vs. 15.54 ± 7.33 nM, T = 11.20,p < 0.001) (Figure 7, middle graph). Furthermore, a comparison between the unlesioned and lesioned striata showed no effect of 6-OHDA on basal 5-HIAA levels (Figure 7, bottom graph). This is consistent with the view that of 6-OHDA targets mainly dopaminergic, but not serontonergic, neurons. An A N O V A of basal D A and DOPAC levels in the L-dopa-naive, 1-week, and 3-week treated groups failed to show a significant effect of L-dopa treatment duration in the lesioned (DA: p = 0.699, DOPAC: p = 0.619) and unlesioned (DA: p = 0.499, DOPAC: p = 0.082) striata. While there is some controversy relating to the neurotoxic effects of L-dopa, the present data lend support to the emerging view that it does not (Olanow et al., 2004). Effect of DAT blockade on L-dopa-naive rats In the unlesioned striatum (Figure 8A), A 1-way A N O V A revealed that L-dopa injection alone had no significant effect on extracellular D A levels (p = 0.119), but showed a significant 47 < L-dopa Naive 1 week L-dopa treated 1 6 0 0 _ 1 2 0 0 O Q 8 0 0 O 4 0 0 L-dopa Naive 1 week L-dopa treated 3 week L-dopa treated 3 week L-dopa treated 400 zn 200 JO D 100 L-dopa Naive l week L-dopa treated 3 week L-dopa treated Figure 7. Effect of L-dopa treatment on basal levels. The top graph: effect of L-dopa treatment (lOmg/kg) duration (0,1-,3- weeks) on basal levels of DA. The middle graph: effect of treatment of L-dopa (lOmg/kg) treatment duration (0,l-,3- weeks) on basal levels of DOPAC. The bottom graph: effect of L-dopa (lOmg/kg) treatment duration (0,1-,3- weeks) on basal levels of 5-HIAA. The blue bars indicate levels in the unlesioned striatum, and the purple bars indicate levels in the lesioned striatum. Significance of lesioned side relative to unlesioned side; * =p < 0.01; and * * = p< 0.001 48 Unlesioned Striatum (L-dopa Naive Group) A LD only 30 -= 20 < Q < 10 •10 S 750 £ 500 O < 250 CL § 0 < -250 „2000 2 £.1000 % < -1000 0 4 8 12 Time (xlOmins) i mi 0 4 8 12 Time (xlOmins) [Lb] 5 750 o 5 0 0 6 250 < -250 2000 LD + MP -1000 0 4 8 12 Time (xlOmins) 0 4 8 12 Time (xlOmins) M P i L D Figure 8. Effect of MP pretreatment on L-dopa-induced responses in the drug-naive, unlesioned striatum. Panel A illustrates the L-dopa-induced changes in DA, DOPAC, and HVA levels. Panel B illustrates L-dopa induced changes in DA, DOPAC, and HVA levels 20 minutes after treatment with MP. 8A: Following four baseline samples, injection of L-dopa (10 mg/kg, i.p.) was given. Collection of dialysate samples continued for 160 minutes after L-dopa injection. Each diamond represents a dialysate sample collected over a 10-minute bin. 8B: Following four baseline samples, injection of MP (14mg/kg, i.p.) was given. Twenty minutes later, injection of L-dopa (10 mg/kg, i.p.) was administered. Collection of dialysate samples continued for 160 minutes after L-dopa injection, f denotes significance at p < 0.05 relative to the last prc-drug baseline; and I denotes significance at p < 0.001 relative to the last prc-drug baseline. 49 increased above baseline for DOPAC and H V A levels (DOPAC: Fi6,96 = 6.89,p = 0.014; HVA: F - 6 , 1 9 2 = 7.41, p= 0.008, Dunnett's test, p < 0.01). In the MP pretreated group (Figure 8B), MP significantly increased extracellular DA from baseline, with no further increase following administration of L-dopa (Fig,9o = 10.96; p = 0.003). This MP-induced DA increase peaked approximately 40 minutes after MP injection, and gradually returned to baseline levels after 2 hours. Although MP pretreatment alone did not increase basal DOPAC or H V A levels, a significant delayed rise in H V A levels was observed (HVA: F 18,90-= 9A\;p = 0.002, Dunnett's test, p< 0.01). No changes from baseline were observed in DOPAC levels, although a slight, but insignificant decrease was observed in the first 60 minutes after L-dopa. A 2-way A N O V A test showed that D A levels in L-dopa with MP pretreatment group was significantly different compared to that in L-dopa only control group (DA: F i 6 , i 7 6 = 9.38,p < 0.001). In contrast, no effect of MP pretreatment was observed in L-dopa-induced DOPAC, or H V A response (DOPAC: p = 0.139; H V A : p = 0.380). This pattern of response in the unlesioned striatum was similar in all three groups (drug naive, 1-week treated, 3-week treated), with only some minor differences. In the lesioned striatum (Figure 9A), injection of L-dopa caused a significant rise in extracellular D A concentration (Fi 6 ,96 = 9.00, p = 0.001, Dunnett's test, p < 0.01), which was accompanied by a similar pattern of L-dopa-induced AIM. The D A efflux was followed by a significant delayed increase in DOPAC ( F 1 6 , 96 = 9.54, p = 0.006, Dunnett's test, p< 0.01), and H V A levels (F\6i96 = 16.70, p< 0.001, Dunnett's test, p< 0.01). MP pretreatment (Figure 9B), had no effect on basal DA, DOPAC, and H V A levels. L-dopa given 20 minutes following pretreatment of MP resulted in an increase of DA levels (DA: F i6 ,80 = \ \.%5,p = 0.009, Dunnett's test, p< 0.01), which was accompanied by a similar pattern of AIM. DA efflux was 50 Lesioned Striatum (L-dopa-naive Group) immmiii,, 0 4 8 12 Time (xlOmins) f 4 8 12 Time (xlOmins) 4 8 12 Time (xlOmins) > 2 LD MP LD 0 4 8 12 Time (xlOmins) _,. „ • L i m b • Ax ia l B L o c o Figure 9. Effect of MP pretreatment on L-dopa-induced responses in the drug-naive, lesioned striatum. Panel A illustrates the L-dopa-induced changes in DA, DOPAC, and HVA levels. Panel B illustrates L-dopa-induced changes in DA, DOPAC, and HVA levels 20 minutes after treatment with MP. 9A: Following four baseline samples, injection of L-dopa (10 mg/kg, i.p.) was given, after which dialysate sample collection was continued for 160 minutes. 9B: Following four baseline samples, injection of MP (14mg/kg, i.p.) was given. Twenty minutes later, injection of L-dopa (10 mg/kg, i.p.) was administered. Collection of dialysate samples continued for 160 minutes after L-dopa injection. Each diamond represents a dialysate sample collected, and is drawn over a corresponding measure of AIM (colored bars) observed in a-10 minute bin. The blue bars indicate locomotive turning, either ipsiversivc or contraversive to the lesioned hemisphere. The orange bars indicate axial dystonia, and the white bars indicate limb dyskinesia, t denotes significance at p < 0.05 relative to the last pre-drug baseline; and I denotes significance at p < 0.001 relative to the last prc-drug baseline. 51 followed by a delayed rise in DOPAC and H V A levels (DOPAC, F i 6 , 8 0 = 42.60,/? < 0.001; HVA: Fie.go = 16.10,/? = 0.001; Dunnett's test, p< 0.01). A 2-way A N O V A test (Figure 9) showed that no significant effect of MP pretreatment on L-dopa-induced DA, DOPAC, or H V A response (DA: p = 0.857; DOPAC: p = 0.547; HVA: p = 0.298). A T-test used to compare L-dopa-induced peak neurochemical responses of DA, DOPAC, or H V A levels showed no significant effect of MP pretreatment (Figure 10A). (DA: p = 0.971; DOPAC: p = 0.874; HVA: p = 0.249). In comparison to rats which only received L-dopa, a T-test showed that MP pretreatment resulted in a significant reduction in the axial component of L-dopa-induced dyskinesia (FigurelOB; control vs. treatment; Axial: 13.71 ± 5.25 vs. 0.33 ± 0.37; T = 2.53;p = 0.028), but not in locomotor and limb components (Loco: p = 0.095; Limb: p = 0.665). Effect of DAT blockade on 1-week L-dopa treated rats In the unlesioned striatum (Figure 1 IA), a 1-way A N O V A revealed that L-dopa injection alone had no significant effect on extracellular DA levels (p = 0.201), but showed an increased for both DOPAC and H V A from baseline levels; however, this was only statistically significant for H V A (HVA: F i 6 , 9 6 = 16.96,/? < 0.001, Dunnett's test, p < 0.01; DOPAC: p = 0.271). In the MP pretreated group (Figure 1 IB), MP significantly increased extracellular DA relative to baseline, with no further increase following administration of L-dopa (Fig.ios= 7.95; p < 0.001). This MP-induced DA increase peaked approximately 40 minutes after MP injection, and gradually returned to baseline levels after 2 hours. Although MP pretreatment alone did not increase basal DOPAC or H V A levels, a delayed rise in H V A levels was observed after L-dopa injection (HVA: Fi 8 , io8 = 4.70,/? = 0.004, Dunnett's test, p < 0.01). Further analysis showed a significant decrease in the first 60 minutes after L-dopa ( F 6 j 3 6 = 7.26,/? < 0.001, Dunnett's test, p L-dopa-na'i've A < Q < m > 0 10 OL Neurochemistry LD LD+MP B Behaviour 40 | 30 *. 20 o W 10 LD LD+MP £ . 2 0 0 O | 150 a < •g 100 "5 m 50 © a. 5 250 £ 200 X 2 150 o I 100 2C CL 50 0 50 40 a | 30 | 20 w 10 LD LD+MP LD LD+MP LD LD+MP Figure 10. WA. Effect of MP pretreatment on L-dopa-induced peak values in the drug-naive, lesioned striatum (14mg/kg, i.p. MP; lOmg/kg, i.p. L-dopa). These graphs represent a between-groups comparison of the L-dopa-induced peak response of DA, DOPAC, and HVA (mean ± SEM) without MP pretreatment (green bar), and with MP pretreatment (white bar). 10B. Effect of MP pretreatment on L-dopa-induced behaviour (14mg/kg, i.p. MP; lOmg/kg, i.p. L-dopa). These graphs represent a betwecn-groups comparison of the L-dopa-induced total AIM scores of each subtype (mean ± SEM). Top graph: Limb dyskinesia; Middle graph: Axial dystonia; Bottom graph: Locomotion. In each graph, the left bar represents L -dopa-induced AIM without MP pretreatment, and right bar represents L-dopa-induced AIM with MP pretreatment. Significance relative to L-dopa only control; * p < 0.05 Unlesioned Striatum (1-week L-dopa Treated Group) Figure 11. Effect of MP pretreatment on L-dopa-induccd responses in the 1-week L-dopa treated, unlesioned striatum. Panel A illustrates the L-dopa-induced changes in DA, DOPAC, and HVA levels. Panel B illustrates L-dopa-induccd changes in DA, DOPAC, and HVA levels 20 minutes after treatment with MP. 11 A: Following four baseline samples, injection of L-dopa (10 mg/kg, i.p.) was given. Collection of dialysate samples continued for 160 minutes after L-dopa injection. Each diamond represents a dialysate sample collected over a 10-minute bin. 11B: Following four baseline samples, injection of MP (14mg/kg, i.p.) was given. Twenty minutes later, injection of L-dopa (10 mg/kg, i.p.) was administered. Collection of dialysate samples continued for 160 minutes after L-dopa injection, t denotes significance at p < 0.05 relative to the last pre-drug baseline; and + denotes significance at p < 0.001 relative to the last pre-drug baseline. 54 < 0.01). A 2-way A N O V A test showed that DA levels in the MP pretreated group showed that L-dopa induced DA levels were significantly different from that of the L-dopa only control group (DA: F i 6 , i 9 2 = 10.86, p < 0.001). In contrast, no effect of MP pretreatment was observed in L-dopa-induced DOPAC, or H V A response (DOPAC: p = 0.482; HVA: p = 0.522). In the lesioned striatum (Figure 12A), injection of L-dopa resulted in a significant rise in extracellular DA concentration (Fi6 ,96 = 17.85,p < 0.001, Dunnett's test, p < 0.01), which was accompanied by a similar pattern of L-dopa-induced AIM. The DA increase was followed by a significant delayed increase in DOPAC (Fi 6 > 9 6 = 26.65,/? = 0.001, Dunnett's test, p < 0.01), and H V A levels (Fi 6 > 96 = 42.23, p < 0.001, Dunnett's test, p < 0.01). MP pretreatment (Figure 12B), had no effect on basal DA, DOPAC nor H V A levels. L-dopa given 20 minutes following pretreatment of MP resulted in an increase of D A levels (DA: F i6 ,96 7.65,/? = 0.013), which was accompanied by a similar pattern of L-dopa-induced AIM. DA efflux was followed by a delayed rise in DOPAC and H V A levels (DOPAC, F 1 6 ; 9 6 = 59.175,/? < 0.001; HVA: F 1 6 ; 9 6 = 22.45,/? < 0.001, Dunnett's test, p< 0.01). A 2-way A N O V A (Figure 12) test showed no significant effect of MP pretreatment on L-dopa-induced DA nor H V A , but significant effect of MP pretreatment on L-dopa-induced DOPAC response (DA: p = 0.397; HVA: p = 0.184; for DOPAC : F i 6 , i 9 2 = 5.46,/? = 0.005, Dunnett's test, p < 0.01). A T-test used to compare L-dopa-induced peak neurochemical responses of DA, DOPAC, and H V A showed insignificant effect of MP on DA or H V A (Figure 13A), but significantly decreased the peak L-dopa-induced response for DOPAC (DA: p = 0.572; HVA: p = 0.416; DOPAC: 181.99 ± 25.11 vs. 96.52 ± 8.41 nM; T = 3.49;p = 0.004). In comparison to rats which only received L-dopa, a T-test showed that MP pretreatment showed significant reduction in the axial and limb components of L-dopa-induced dyskinesia, 55 Lesioned Striatum (1-week L-dopa Treated Group) Figure 12. DLimb • Axial BLoco Effect of MP pretreatment on L-dopa-induced responses in the 1-week L-dopa treated, lesioned striatum. Panel A illustrates the L-dopa-induced changes in DA, DOPAC, and HVA levels. Panel B illustrates L-dopa-induced changes in DA, DOPAC, and HVA levels 20 minutes after treatment with MP. 12A: Following four baseline samples, injection of L-dopa (10 mg/kg, i.p.) was given, after which dialysate sample collection was continued for 160 minutes. 12B: Following four baseline samples, injection of MP (14mg/kg, i.p.) was given. Twenty minutes later, injection of L-dopa (10 mg/kg, i.p.) was administered. Collection of dialysate samples continued for 160 minutes after L-dopa injection. Each diamond represents a dialysate sample collected, and is drawn over a corresponding measure of AIM (colored bars) observed in a 10-minute bin. The blue bars indicate locomotive turning, either ipsiversive or contraversive to the lesioned hemisphere. The orange bars indicate axial dystonia, and the white bars indicate limb dyskinesia, t denotes significance at p < 0.05 relative to the last pre-drug baseline; and } denotes significance at p < 0.001 relative to the last pre-drug baseline. 1 -week L-dopa Treated Figure 13. 13A. Effect of MP pretreatment on L-dopa-induced peak values in the 1-week L-dopa treated, lesioned striatum (14mg/kg, i.p. MP; lOmg/kg, i.p. L-dopa). These graphs represent a between-groups comparison of the L-dopa-induced peak response of DA, DOPAC, and HVA (mean ± SEM) without MP pretreatment (green bar), and with MP pretreatment (white bar). 13B. Effect of MP pretreatment on L-dopa-induced behaviour (14mg/kg, i.p. MP; lOmg/kg, i.p. L-dopa). These graphs represent a between-groups comparison of the L-dopa-induced total AIM scores of each subtype (mean ± SEM). Top graph: Limb dyskinesia; Middle graph: Axial dystonia; Bottom graph: Locomotion. In each graph, the left bar represents L-dopa-induced AIM without MP pretreatment, and right bar represents L-dopa-induced AIM with MP pretreatment. Significance relative to L-dopa only control; *p<0.05; **p<0.01 57 but not in locomotor rotation (Figure 13B) (Loco: p = 0.653; Axial: 24.14 ± 7.07 vs. 5.43 ± 3.85, /? = 0.027; Limb: 34.14 ± 4 . 3 1 vs. 18.86 ± 3.24,/? = 0.010) Effect of DAT blockade in 3-week L-dopa treated rats In the unlesioned striatum (Figure 14A), a 1-way A N O V A revealed that L-dopa injection alone had no significant effect on extracellular DA levels (p = 0.764). Both DOPAC and H V A increased from basal levels (DOPAC: F i 6 , i i 2 = 6.79, p = 0.007; HVA: F , 6 , n 2 = 5.19,/? = 0.037, Dunnett's test, p < 0.01). In the MP pretreated group (Figure 14B), MP significantly increased extracellular D A from baseline, with no further increase following administration of L-dopa (F 18,90 = 12.81; p = 0.001, Dunnett's test, p < 0.01). This MP-induced DA increase peaked approximately 40 minutes after MP injection, and gradually returned to baseline levels after 2 hours. Although MP pretreatment alone did not increase basal DOPAC or H V A levels, a significant delayed rise in H V A levels was observed about 80 minutes after L-dopa injection (for HVA: F 18,90= 17.40,/? < 0.001, Dunnett's test, p < 0.01). In contrast, L-dopa administration following MP pretreatment induced a significant decrease in DOPAC levels in the first 60 minutes after L-dopa (F 6 > 3 6 = 7.42,/? = 0.001, Dunnett's test, p < 0.01). A 2-way A N O V A test showed that L-dopa induced DA levels with MP pretreatment were significantly different from that in L-dopa only control group (DA: F i 6 , i76= 5.18,/? = 0.011). In contrast, no effect of MP pretreatment were observed in L-dopa-induced DOPAC, or H V A response (DOPAC: p = 0.164; HVA: p = 0.477). In the lesioned striatum (Figure 15 A), injection of L-dopa caused in a significant rise in extracellular DA concentration (Fi6,i28 = 24.17, p< 0.001, Dunnett's test, p < 0.01), which was accompanied by a similar pattern of L-dopa-induced AIM. The DA efflux was followed by a delayed increase in DOPAC (Fi6,i28 = 33.71,/? < 0.001, Dunnett's test, p < 0.01), and H V A Unlesioned Striatum (3-week L-dopa Treated Group) A 30 | 20 Q < 0 -10 S 750 £ 500 O < 250 o. § 0 < -250 „2000 s £.1000 1 • < -1000 LD only 4 8 12 Time (xlOmins) I T 4 8 12 Time (xlOmins) s 750 c 500 u 1 250 o 0 Q < -250 2 2000 c 1000 < > I 0 < -1000 B P 0 4 8 12 Time (xlOmins) LD MP LD I 4 8 12 Time (xlOmins) Figure 14. Effect of MP pretreatment on L-dopa-induced responses in the 3-week L-dopa treated, unlesioned striatum. Panel A illustrates the L-dopa-induced changes in DA, DOPAC, and HVA levels. Panel B illustrates L-dopa-induced changes in DA, DOPAC, and HVA levels 20 minutes after treatment with MP. 14A: Following four baseline samples, injection of L-dopa (10 mg/kg, i.p.) was given. Collection of dialysate samples continued for 160 minutes after L-dopa injection. Each diamond represents a dialysate sample collected over a 10-minute bin. 14B: Following four baseline samples, injection of MP (14mg/kg, i.p.) was given. Twenty minutes later, injection of L-dopa (10 mg/kg) was administered. Collection of dialysate samples continued for 160 minutes after L-dopa injection, f denotes significance at p < 0.05 relative to the last pre-drug baseline; and % denotes significance at p < 0.001 relative to the last pre-drug baseline. 59 Lesioned Striatum (3-week L-dopa Treated Group) A LD only B LD + MP s £ o o Q < S c 200 100 200 100 X < o Time (xlOmins) I i 4 0 4 8 12 Time (xlOmins) (Lb] j W 4 8 12 Time (xlOmins) .8 4 2 0 SI 2 > i 0 4 8 12 Time (xlOmins) MP LD • Limb • Axial BLoco Figure 15. Effect of MP pretreatment on L-dopa induced responses in the 3-weck L-dopa treated, lesioned striatum. Panel A illustrates the L-dopa-induced changes in DA, DOPAC, and HVA levels. Panel B illustrates L-dopa-induccd changes in DA, DOPAC, and HVA levels 20 minutes after treatment with MP. 15A: Following four baseline samples, injection of L-dopa (10 mg/kg, i.p.) was given, after which dialysate sample collection was continued for 160 minutes. 15B: Following four baseline samples, injection of MP (14mg/kg, i.p.) was given. Twenty minutes later, injection of L-dopa (10 mg/kg, i.p.) was administered. Collection of dialysate samples continued for 160 minutes after L-dopa injection. Each diamond represents a dialysate sample collected, and is drawn over a corresponding measure of AIM (colored bars) observed in a 10-minute bin. The blue bars indicate locomotive turning, either ipsiversive or contraversive to the lesioned hemisphere. The orange bars indicate axial dystonia, and the white bars indicate limb dyskinesia, f denotes significance at p < 0.05 relative to the last pre-drug baseline; and + denotes significance at p < 0.001 relative to the last pre-drug baseline. 60 levels (Fi6ji2g = 71.25,/? < 0.001, Dunnett's test, p < 0.01). MP pretreatment (Figure 15B) had no effect on basal DA, DOPAC, and H V A levels. L-dopa given 20 minutes following pretreatment of MP resulted in an increase of DA levels (DA: Fi6,8o 9.42,/? = 0.009, Dunnett's test, p < 0.01), accompanied by a similar pattern of L-dopa-indueed AIM. DA efflux was followed by a delayed rise in DOPAC and H V A levels (DOPAC, F i 6 j 8 0 = 24.70,/? < 0.001; HVA, Dunnett's test, p < 0.01: Fi 6 , 8 o = 13.47,/? = 0.004, Dunnett's test, p < 0.01). A 2-way A N O V A (Figure 15) test showed significant effect of MP pretreatment on L-dopa-induced DA, DOPAC and H V A responses (DA: F , 6 > 2 08 = 3.52, p = 0.014, DOPAC : Fi 6, 208 = 3.964,/? = 0.033; HVA: F i 6 > 2 0 8 = 19.04,/? < 0.001). A T-test used to compare L-dopa-induced peak neurochemical responses of DA, DOPAC, and H V A response showed significant effect of MP pretreatment (Figure 16A) (control vs. treatment; DA: 3.00 ± 0.33 vs. 1.66 ± 0.31 nM; T = 3.009; p= 0.010; DOPAC: 124.79 ± 14.82 vs. 64.58 ± 10.12nM; T = 3.196;p = 0.007; HVA: 150.17 ± 9.38 vs. 48.80 ± 5.29nM; T = 8.002;p < 0.001) In comparison to rats which only received L-dopa, a T-test showed that MP pretreatment resulted in a significant reduction of all components of L-dopa-induced dyskinesia (Figure 16B) (control vs. treatment: Loco: 27.89 ± 3.31 vs. 13.67 ± 3.67,/? = 0.010; Axial: 29.00 ± 4.06 vs. 0.17 ± 0.18,/? < 0.001; Limb: 32.22 ± 4.22 vs. 8.67 ± 3.83,/? = 0.001). Histology Bilateral probe tracts were verified in fixed 50|im coronal sections (Figure 17). 15 animals were randomly selected for presentation in this figure. Discussion The aim of this experiment was to assess the functional mode in the DAT associated with DA denervation, and with chronic L-dopa administration, in the severely lesioned striatum. The 3-week L-dopa Treated 61 A s < a < > 0 _ J re 0 CL Neurochemistry ** T B 40 | 30 o E 3 CO 20 10 0 Behaviour kkk LD LD+MP LD LD+MP £ . 2 0 0 o <c § 1 5 0 a < • s i o o 0 0 50 re 0 0 CL LD LD+MP LD+MP 5 250 £ 200 X ^ 1 5 0 o • 1 0 0 j< 50 re 0 a. o LD LD+MP Figure 16. /d/4. Effect of M P pretreatment on L-dopa-induced peak values in the 3-week L-dopa treated, lesioned striatum (14mg/kg, i.p. M P ; lOmg/kg, i.p. L-dopa). These graphs represent a between-groups comparison of the L-dopa-induccd peak response of D A , D O P A C , and H V A (mean ± S E M ) without M P pretreatment (green bar), and with M P pretreatment (white bar). 16B. Effect of M P pretreatment on L-dopa-induced behaviour (14mg/kg, i.p. M P ; lOmg/kg, i.p. L-dopa). These graphs represent a between groups comparison of the L-dopa-induced total A I M scores of each subtype (mean ± S E M ) . Top graph: L imb dyskinesia; Middle graph: A x i a l dystonia; Bottom graph: Locomotion. In each graph, the left bar represents L-dopa-induced A I M without M P pretreatment, and right bar represents L-dopa-induced A I M with M P pretreatment. Significance relative to L-dopa only control; * * p < 0.01; kkk p < 0.001 62 Left Hemisphere Right Hemisphere Unlesioned Lesioned Figure 17. Verification of probe placement. The black lines represent the 4mm of exposed membrane area of the probes used for microdialysis. 63 results of this experiment showed that in the unlesioned striatum, L-dopa alone failed to increase extracellular basal D A levels (Figures 8A, 11 A, and 14A). In rats that received MP pretreatment, basal DA levels increased with MP administration, but did not with a subsequent L-dopa injection (Figures 8B, 1 IB, and 14B). This effect was seen in all three groups (L-dopa naive, 1-week L-dopa treated, and 3-week L-dopa treated). In contrast, in the lesioned striatum, L-dopa administration resulted in a surge of extracellular DA levels (Figures 9A, 12A, and 15 A). In rats that received MP pretreatment, MP alone failed to increase DA basal levels, or attenuate L-dopa induced DA efflux in the lesioned striatum of L-dopa-naive, or 1-week L-dopa treated animals. Functional Mode of the D A T in the Striatum: role of D A terminal loss As previous data have suggested that DAT function may be reversed in the severely DA denervated striatum (Miller and Abercrombie, 1999; Ahn et al., 2004), we examine the role of DA terminal loss by comparing the effects of MP pretreatment on L-dopa induced DA efflux in the unlesioned and lesioned striatum of L-dopa-nai've rats. DAT uptake in the unlesioned striatum The absence of D A efflux in the unlesioned striatum following exogenous L-dopa administration may reflect the efficiency of the DAT system by clearing excess DA by reuptake. Consistent with this view, blockade of the DAT with systemic MP caused a significant increase in DA and H V A levels, but a decrease in DOPAC levels (Figure 8). As D A is catabolized into H V A within the extracellular space, the increase in H V A levels is dependent on increased extracellular DA levels. On the other hand, intracellular conversion of DA to DOPAC requires DAT-dependent uptake of D A back into the cytosol, and therefore, following MP pretreatment, further DOPAC synthesis is inhibited and a decrease was observed following L-dopa injection. 64 Therefore, these observed changes in DA, DOPAC, and H V A indicate that the DAT functions in the uptake mode in the unlesioned striatum. DAT function in the lesioned striatum In the lesioned striatum, MP administration alone failed to increase basal levels of DA, DOPAC, or H V A . Furthermore, MP pretreatment did not significantly attenuate L-dopa-induced DA efflux (Figures 9, 10A). These data are inconsistent with previous reports (Miller and Abercrombie, 1999; Ahn et al., 2004) which demonstrated, in L-dopa-nai've rats, that (i), the L-dopa-induced DA efflux was partly mediated by a depolarization-independent mechanism (Miller and Abercrombie, 1999); and (ii) continuous reverse-dialysis of GBR 12909 in the denervated striatum attenuated the L-dopa induced DA efflux (Ahn et al., 2004). One explanation for the lack of effect of MP on basal D A levels and L-dopa-induced DA efflux in L-dopa-nai've rats may be due to the dose and method of administration of MP used in the present study. Reverse-dialysis of GBR 12909 may allow for a more continuous and concentrated delivery of the drug into a small amount of tissue, whereas systemic injection of the drug may be subject to peripheral metabolism. Therefore, a higher dose of MP may be required to detect changes in very low basal levels. Similarly, systemic injection of GBR 12909 also failed to affect the L-dopa-induced DA efflux in the DA denervated striatum (Miller and Abercrombie, 1999). Therefore, the role of DA denervation on DAT function is inconclusive in the present results. Together, these data suggest that in L-dopa-naive animals, at least two mechanisms maybe in involved in L-dopa-induced DA release: first, a predominant impulse-dependent mechanism, possibly through conversion of exogenous L-dopa and release of DA by other AADC-containing neuronal cells, such as the striatal GABAergic interneurons (Melamed et al., 1980; Hefti et al., 1981)and serotonergic afferents (Arai et al., 1994; Mura et al., 1995; Tanaka et 65 al., 1999); second, a partial impulse-independent mechanism, involving reverse transport of DA via the DAT (Miller and Abercrombie, 1999. Ahn et al., 2004). Functional Changes in the D A T Following Chronic L-dopa Treatment To investigate how chronic L-dopa treatment changes the mechanism underlying L-dopa-induced D A release in the striatum, the effect MP pretreatment was examined in 3-week L-dopa treated rats. In these rats, systemic MP substantially reduced L-dopa-induced DA, DOPAC and H V A efflux in the lesioned striatum (Figure 15), suggesting that, in contrast to L-dopa naive, denervated striatum, these responses are predominantly DAT-dependent. Other results from our lab also support this, demonstrating that intrastriatal reverse dialysis of GBR-12909 almost abolishes completely the L-dopa-induced DA, DOPAC, and H V A responses in the lesioned striatum of 3-week L-dopa treated rats (Ahn et al., 2004). Therefore, these data suggest that 3 weeks of chronic L-dopa treatment is sufficient to alter the mode of L-dopa-induced DA release from one that is predominately impulse-dependent, to one that is primarily DAT-dependent. Although it is unknown when these changes occur during the 3-week treatment period, the present data show that with one week of L-dopa treatment, changes in the functional mode of the DAT have begun to occur. Although the effect of MP on L-dopa-induced DA efflux was not significant in L-dopa naive or 1-week treated rats, there was a consistent trend for MP-induced attenuation in each of the 1-week treated rats (Figures 12, 13 A). These observations may reflect a progressive change in the ratio of DATs in uptake vs. release mode, suggesting that while 1-week treated animals may have a higher proportion of DATs functioning in the release mode than L-dopa naive animals, longer treatment duration (at least 3 weeks) is required for significant attenuation of DA release by MP blockade of the DAT. The Role of Striatal D A in L-dopa-induced Dyskinesia 66 One of the principal goals of the present study was to examine the relationship between L-dopa-induced DA levels in the lesioned striatum and the expression of L-dopa-induced dyskinesia. While the L-dopa-induced DA increase was not significantly different between L-dopa naive, 1-week, and 3-week treated rats, AIM scores increased progressively with longer duration of treatment (Figures 9A, 12A, 15 A). These observations suggest that a threshold for triggering dyskinesia was reduced, most likely attributable to sensitization of DA receptors with repetitive exposure to L-dopa. These data are in line with the current view that L-dopa-induced dyskinesia is mediated through downstream gene changes (Calon et al., 2000; Olanow et al., 2000). It is still unclear exactly how chronic L-dopa treatment alters the relationship between striatal DA levels and expression of L-dopa-induced dyskinesia (kinetic-dynamic association). It could be that D A acts only as a trigger for dyskinesia, or D A may also mediate the magnitude of behaviour. Our data suggest that DA's role in mediating behaviour may be altered by L-dopa treatment. In animals which were treated for 1 or 3 weeks, changes in DA levels were well correlated with changes in AIM. Consistently, pretreatment with MP in L-dopa treated animals (1- or 3-weeks treated) resulted in a blunted DA response, which was correlated with similar attenuation in AIM scores. In contrast, this correlation between DA and AIM was not the matched in L-dopa naive rats. While the rise of D A levels correlated with the appearance of AIM, peak DA levels were earlier compared to the peak of the AIM response. The peak D A response was almost 100 minutes earlier than that of the AIM response. Furthermore, pretreatment with MP did not change the DA response, but selectively attenuated the axial component of AIM. These data suggest that in L-dopa-naive animals, DA may act simply as a trigger for AIM, which will express as long as a certain threshold of DA level is reached. However, other non-dopaminergic mechanisms may be involved in determining the intensity of AIM (Cenci et al., 1998; Brotchie et al., 2005). Repeated L-dopa exposure, on the other hand, elicits downstream changes such as increased DA receptor sensitivity and long lasting alterations in gene expression (Olanow et al., 2000), which could change the relationship between DA and AIM. More precisely, in treated animals, DA levels influence the intensity and duration of L-dopa-induced behaviour. 68 G E N E R A L DISCUSSION The aim of the experiments presented in this thesis was to assess functional changes in the DAT associated with chronic L-dopa administration in the severely lesioned striatum, and how these changes contribute to L-dopa-induced striatal efflux of DA and dyskinetic behaviour. These findings support our hypothesis that DAT reversal is a contributing factor in this L-dopa derived DA efflux. While systemic administration of MP failed to attenuate L-dopa-induced DA efflux in the DA-denervated striatum of L-dopa naive rats, it significantly reduced both L-dopa-induced DA efflux and the corresponding AIM scores in rats treated for 3 weeks. These findings emphasize the importance of chronic L-dopa treatment, as well as D A denervation, as contributing factors for DAT reversal. DAT Reversal as a Compensatory Mechanism Extracellular D A levels, and hence motor behaviour, remain normal with up to 80% of DA terminal loss (Abercrombie et al., 1990; Benazzouz et al., 1992; Bezard et al., 1997), indicating that D A denervation causes a sequence of compensatory mechanisms which serve to maintain normal extracellular D A levels. In the initial stages of progressive DA denervation in PD, normal DA levels are maintained by increased DA synthesis by increased T H expression (Pasinetti et al., 1992; Blanchard et al , 1995), increased D A release (Agid et al., 1973; Zigmond et al., 1984), and reduced D A uptake (Bezard et al., 2000; Lee et al., 2000). However, in the severely lesioned striatum, these compensatory mechanism are no longer sufficient in upholding normal levels of D A stimulation. As previously suggested, D A denervation alone may induce reversal of transport in a proportion of the remaining DATs. Therefore, D A T reversal may be an additional compensatory mechanism which appears only with severe DA denervation. As the present data indicate, chronic treatment with L-dopa exaggerates this phenomenon, possibly by 69 increasing the ratio of striatal D A T in the reverse mode, or by enhancing the reversibility of the DAT (i.e. transient reversal vs. sustained reversal). However, the mechanisms by which L-dopa induces flux reversal of the DAT remain unknown. Mechanisms for D A T Reversal The concept of reverse transport was first proposed following by the discovery of pharmacological agents, such as amphetamine, that were capable of releasing monoamines in an impulse, Ca independent fashion. During the last two decades, a great deal of research has identified some of the key mechanisms by which these monoamine releasing agents cause the reversal of monoaminergic transporters. Lessons taken from these studies have been useful in determining the causes of reverse transport in pathophysiological conditions. 1) DAT reversal is facilitated by L-dopa-induced DA synthesis and accumulation of the cytosolic DA pool: D A is synthesized in the terminal, and is stored in two separate releasable compartments: a primary cytosolic pool, and then vesicularized into a secondary pool. DATs are located preferentially on extrasynaptic sites, therefore it is most likely that DA released by reverse transport originates from the cytosolic pool of the releasable compartment of DA terminals (Leviel, 2001). Thus, increasing the cytosolic DA levels may be an important step in DAT reversal. Support for this suggestion comes from the observation that sustained DA efflux by amphetamine is contingent on amphetamine-induced DA synthesis. However, increasing cytosolic DA levels alone by treatment with reserpine does not induce reverse transport (Jones et al., 1998). These data suggest that while increased cytosolic D A may not be an essential factor in DAT reversal, it may be a facilitative step (Leviel, 2001). Therefore, increasing the concentration of cytosolic DA may be one mechanism through which L-dopa may promote DAT reversal in the lesioned striatum. 70 2) The directional transport of the DAT is driven by the electrochemical gradient ofNa+: the role of glutamate: D A is co-transported with Na + , which moves along its concentration gradient, generated by plasma membrane Na + -K ATPases. Thus, the relative intracellular/extracellular concentration of Na + is a critical determinant of the direction of DA flux. Inversion of the Na + gradient by decreasing extracellular Na + levels during microdialysis significantly increases D A efflux in the striatum. This increase is attenuated by pretreatment with the DAT blocker nomifensine (Hurd and Ungerstedt, 1989). These data suggest that molecular agents which increase intracellular Na + may facilitate the reversed transport of the DAT. Particularly relevant to DAT mediated efflux in the striatum are the corticostriatal glutamatergic afferents, activation of which leads to increased intracellular Na + in DA terminals. Recent experiments have demonstrated that reverse dialysis of N M D A increased DA efflux in the striatum (Andres et al., 1998). Furthermore, under more physiological conditions, both nigral DA neurons and D A terminals in the striatum display DAT mediated DA release, both of which are regulated by glutamate (Lonart and Zigmond, 1991; Falkenburger et al., 2001). Enhanced corticostriatal glutamatergic activity has been long associated with PD (Leviel, 2001). Furthermore, chronic L-dopa treatment in rats has been shown to elevate extracellular glutamate levels (Robelet et al., 2004). It seems very likely, then, that the L-dopa-induced glutamate increase is a mechanism through which chronic L-dopa treatment facilitates DAT reversal. D A T Reversal as a Pathogenic Mechanism of L-dopa-induced Dyskinesia As mentioned previously, low doses of L-dopa provide therapeutic effects for the motor deficits in PD patients in the first few years of treatment. With extended treatment, neural changes occur that alter the response to L-dopa, including the emergence of L-dopa-induced dyskinesia. While pathogenic mechanisms of dyskinesia are not yet fully understood, many 71 post-synaptic factors associated with L-dopa-induced dyskinesia have been identified, including alterations in the expression of peptides (PPE, PDyn) and receptors (D3, glutamate). These changes are most likely due to abnormal activation of receptors in the post-synaptic neurons, due to the cycling pattern of high stimulation (when L-dopa is given) and low stimulation (periods without L-dopa), attributable to pulsatile administration of exogenous L-dopa. Therefore, determination of mediating L-dopa-induced DA efflux is critical for understanding the initial events triggering L-dopa-induced dyskinesia. The findings from the present study support a novel hypothesis involving the reversal of the DAT as a mechanism for D A release in the severely denervated striatum, following chronic L-dopa administration. Therefore, DAT reversal may have important implications in the pathogenesis of L-dopa-induced dyskinesia. In the initial stages of treatment, stable response to L-dopa administration may be explained by the regulated release of DA due to vesicular storage in the remaining DA terminals (Figure 18). Over the course of the disease, the storage capacity decreases due to loss of DA terminals (Chase and Oh, 2000); therefore, non-dopaminergic AADC-containing cells become more involved in the conversion and vesicular release of exogenous L-dopa to DA in the severely D A denervated striatum (Miller and Abercrombie, 1999). However, assuming that non-dopaminergic cells participate in vesicular, and therefore, regulated, release of L-dopa derived DA, this theory does not fully explain why over time, a stable L-dopa response changes to a fluctuating pattern, with chronic L-dopa treatment. Our results, which indicate that DAT reversal is treatment dependent, may offer new insights into how L-dopa-induced DA response becomes unstable over long term administration. As suggested in Figure 18, chronic treatment results in a DAT reversal, which, combined with reduced storage capacity, leads to an 72 Figure 18. DAT reversal as a pathogenic mechanism of L-dopa-induced dyskinesia Chronic treatment with L-dopa changes the functional mode of D A T from uptake to release. This mechanism underlies the change from "steady" to "surged" L-dopa induced DA release. This DA surge forms an abnormal and pathogenic pattern of DA receptor stimulation which leads to downstream gene alternations associated with response fluctuations and dyskinesia. 73 Figure 18 74 "surged" DA response. Repeated exposure to this short lasting surge of D A stimulation could lead to altered gene expression and ultimately, dyskinesia. Clinical Studies The effect of MP pretreatment on dyskinesia has also been studied in PD patients. Preliminary clinical trials by Camicioli and colleagues (2001) demonstrated, in contrast to our data, that MP increased L-dopa-induced dyskinesia in three PD patients (Camicioli et al., 2001). In these patients, MP was administered orally, followed 30 minutes later by an infusion of L-dopa. One possible explanation for these findings could be differences in the degree of DA denervation, as well as the treatment history, in these patients and the animals used in our studies. The patients were all described as "functionally independent" which, in clinical terms, refers to patients who have mild to moderate Parkinsonism. As our data indicate, severe DA denervation and treatment history are both critical factors that can result in differential effects of MP on L-dopa-induced dyskinesia. While our animals received only L-dopa treatment, patients may have received a combination of drugs that could alter DAT regulation/function, and consequently, the effects of MP. F U T U R E DIRECTIONS AND C O N C L U S I O N In light of the finding of a role for DAT reversal in dyskinesia, it will be of interest to examine further the factors associated with DA denervation and chronic, pulsatile L-dopa treatment that facilitate the reversal of DAT. As mentioned before, one of the possible mechanisms through which the DAT becomes reversed may be elevated glutamate levels in the striatum. In this view, co-administration of anti-glutamatergic and DAT-blocking drugs may offer new perspective regarding the interactions of the glutamate and the DAT in the DA-denervated striatum. 75 In conclusion, the findings from the experiments in this thesis provide support for our hypothesis that reversal of the DAT may be a pathogenic mechanism for L-dopa-induced dyskinesia. As mentioned before, once dyskinesia appears, it is difficult to distinguish (Brotchie et al., 2005), hence methods of treatment which prevent the emergence of dyskinesia is highly important. Therefore, as our data propose DAT reversal as a novel, presynaptic mechanism for L-dopa induced D A efflux and hence, dyskinesia, they highlight the importance of the DAT as a target for the prevention. 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