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University of Macau / Institute of Chinese Medical Sciences

University of Macau / Institute of Chinese Medical Sciences

Parkinson’s disease (PD) is a multi-organ disorder. It is diagnosed from motor impairments that arise from neurodegeneration in the midbrain. However, the disease begins decades earlier in the gut prior to involvement of the brain. PD is characterized by persistent inflammation, both in the brain and in the periphery in addition to neurodegeneration. Here, I investigate the role of the adaptive immune system in disease pathogenesis and as a driver of prodromal symptoms of PD in both humans and mice. In Chapter 1, I introduce Parkinson’s disease, its pathological hallmarks and the progression of the symptoms, and discuss genetic and environmental influences. Then, I elaborate on the inflammatory phenotypes observed in the disease and recent work describing the role of inflammation in animal models for PD. In Chapter 2, I examine the autoimmune features of Parkinson’s disease from analysis of patients’ blood. I found that approximately 40% of PD patients possess aspects of autoimmunity against α-synuclein. By screening peripheral blood mononuclear cells of patients and healthy controls for potential neoantigens derived from α-synuclein protein, I identified two antigenic regions of the protein that elicit an immune response. The immune responses to a specific α-synuclein neo-antigens were linked to unique HLAs that are over-represented in our PD cohort and are associated with PD in genome wide association studies (GWAS). In Chapters 3 and 4, I describe the effects of recapitulating α-synuclein autoimmunity in a humanized mouse strain expressing the HLA allele risk for PD. In Chapter 3, I show that the humoral and cellular immunity is mounted against α-synuclein in the humanized mice, similar to what is observed in PD patients; however, there is no inflammation or immune response toward the brain. In Chapter 4, I show how the autoimmune response to α-synuclein induces inflammation and neurodegeneration in the gut leading to constipation in mice, recapitulating the prodromal aspects of the human disease. Finally, in Chapter 5, I discuss the implications of these findings for α-synuclein autoimmunity in the periphery, gut and brain in Parkinson’s disease. I also elaborate on the implications of these findings for potential future diagnostic screening and treatments for Parkinson’s disease.

published_or_final_version / Medicine / Master / Master of Philosophy

Parkinson’s disease (PD) is the second most common neurodegenerative disorder characterized by the accumulation of protein aggregates (namely Lewy bodies) in dopaminergic neurons in the substantia nigra region of the brain. Alpha-synuclein (α- syn) is the major component of Lewy bodies (LBs) in PD, and impairment of the autophagy-lysosomal pathway has been linked to its accumulation. In our previous study, we identified corynoxine B (Cory B), an oxindole alkaloid isolated from Uncaria rhynchophylla (Miq.) Jacks (Gouteng in Chinese), as a Beclin-1-dependent autophagy enhancer. In this work, we continued to screen autophagy enhancers from Gouteng alkaloids, and found corynoxine (Cory), an isomer of Cory B, also induces autophagy in different neuronal cell lines and primary neurons. Meanwhile, Cory promotes the formation of autophagosomes in the fat bodies of Drosophila. By inducing autophagy, Cory promotes the clearance of wild-type and A53T α-syn in inducible PC12 cells. Interestingly, different from its enantiomer Cory B, Cory induces autophagy through the Akt/mTOR pathway as evidenced by the reduced levels of phospho-TSC2, phospho-Akt, phospho-mTOR and phospho-p70 S6 Kinase. To identify the different pathway between Cory and Cory B, we performed phosphoproteomic study on N2a cells. With the help of iGPS (In vivo Group-based Prediction System), protein kinases which were significantly regulated by Cory or Cory B were predicted. Based on these kinases, we drew the detailed kinasesubstrates network regulated by Cory or Cory B. The structures of Cory and Cory B differ only in the stereochemistry at the spiro carbon; however, Cory has more effect on the CAMK, Trb and TSSK families, while CDK and CDKL families are more sensitive to Cory B. Furthermore, we established a rotenone rat model of PD via injecting rotenone into the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA), and evaluated the neuroprotection of Cory and Cory B on this rat model. Motor dysfunction, decreased TH level, impairment of autophagy, aggregation of α-syn and activation of microglia were all found on this PD model, which were consistent with previous reports. After the treatment of Cory or Cory B, we found that both Cory and Cory B improve motor dysfunction, increase the TH level, and inhibit microglial activation. Both Cory and Cory B decrease the puncta number of aggregated α-syn, likely due to the induction of autophagy. All these results indicate the neuroprotection of Cory and Cory B against PD. Collectively, our findings (1) provide the original finding of Coy to be an autophagy enhancer with experimental evidences that Cory inhibited the pathway of Akt/mTOR; (2) provide cellular and animal experimental evidences for developing Cory or Cory B as anti-PD agent, by inducing autophagy in neurons; and (3) provide candidate pathways to identify the primary molecular target of Cory or Cory B, which may turn out to be potential therapeutic targets for treating PD. Keywords: Parkinson’s disease, Cory, Cory B, autophagy, phosphoproteomic, neuroprotection.

Previous controlled studies have shown that severe daytime restriction of dietary protein improves the efficacy of L-dopa and reduces response fluctuations in some Parkinson's disease in-patients. The main purpose of the present study was to investigate the nutritional adequacy of the restricted protein diet. Other objectives were to assess the patients' acceptance of the diet and to identify the practical difficulties encountered in following the diet at home. The effect of the restricted protein diet on the subjects' response to levodopa was also examined. Subjects were 11 free-living, otherwise healthy Parkinson's disease patients who suffered from unpredictable response fluctuations to Sinemet (L-dopa containing medicine). This condition is also described as the "on-off" phenomenon. They were counselled to consume a daytime restricted protein diet for 6 weeks (mean ± s.d. protein intake before evening meal 12 ± 2 g for females and 14 ± 3 g for males). Food intake was unrestricted from dinner until bedtime, and subjects were encouraged to consume nutrient-dense foods during this period. The subjects were required to complete a series of three 6-day food records and "on-off" charts (pre-diet, diet week 2, diet week 6). The "on-off" charts indicated the daily number of hours spent in the "on" state (when medication is effective and parkinsonian symptoms are controlled) and in the "off" state (when medication is not effective and symptoms are not well controlled). Hemoglobin, plasma albumin, prealbumin and ferritin were measured before and after the 6-week diet period. Subjective evaluation questionnaires were completed by all participants and their spouses or caregivers. Results from dietary record analysis showed that the restricted protein diet was associated with significant decreases in total intakes of protein, calcium, iron, magnesium, phosphorus, niacin, riboflavin, vitamin B6 and pantothenate, in comparison to "usual" intakes. Intake of energy, carbohydrates, lipids, potassium, thiamin, folacin, and vitamins A, C and B12 did not change significantly. While on the restricted protein diet, only calcium intake was substantially less than the RNI. Biochemical measures of nutritional status were not significantly reduced. Mean body weight tended to decrease (p=.054) over the first 2 weeks and then stabilized until the end of the study period. The results of the "on-off" charts showed that three of the eleven subjects significantly increased their daily time "on" while on the restricted protein diet. Subjectively, six individuals noted an improvement in daytime mobility and indicated that they would maintain the diet for an indefinite period of time. Problems identified by the subjects included hunger prior-'to the evening meal and a lack of variety in food choices. These results show that otherwise healthy and motivated patients with Parkinson's disease can maintain an adequate intake of energy, protein, and most nutrients while on the daytime restricted protein diet. The diet appeared to be relatively well tolerated by patients who obtained a subjective benefit. It is suggested that in patients whose regular diets are marginally adequate, the restricted protein diet might compromise nutrient intakes. Counselling by a registered dietitian is recommended for all patients who undertake this type of diet. / Land and Food Systems, Faculty of / Graduate

Progressive disorders of airway protection, namely cough (dystussia) and swallowing (dysphagia) dysfunction, are highly prevalent in Parkinson’s disease (PD), impacting quality of life and contributing to the development of aspiration pneumonia – a leading cause of death in this population. To date, dysphagia rehabilitation has remained the primary (and often only) treatment target of choice by clinicians managing dysphagia in patients with PD and other neurodegenerative disease. This is a major concern since the progressive nature of PD makes it somewhat unreasonable to expect that treatments can fully rehabilitate swallowing dysfunction and eliminate chronic aspiration. Instead, rehabilitating cough dysfunction can serve as an adjunctive approach to promote pulmonary health. Considering that impairments in motor control and organization are primary features of PD, skill training may have a necessary role in cough rehabilitation. Despite a growing body of research supporting the feasibility and effectiveness of cough skill training, a significant gap remains in our understanding of optimal skill training parameters that maximize treatment outcomes through motor learning. This document addresses this gap in the literature in a series of three research studies. Chapter 1 will begin by reviewing the current body of literature related to normal and disordered mechanisms of airway protection dysfunction in PD, skill training as an efficacious approach to rehabilitate cough dysfunction, and motor learning considerations to maximize treatment outcomes. Chapter 2 will characterize motor performance and learning during a voluntary cough skill training paradigm, and evaluate the contributions of physiologic (i.e., lung volume) and treatment-specific (i.e., biofeedback) factors to treatment response in PD. Chapter 3 will then characterize trajectories of motor performance during multiple sessions of sensorimotor cough skill training and explore the role of task-specific predictors (i.e., variability, motor learning) on motor performance. Chapter 4 will examine the effects of cough skill training with variable practice on motor performance and motor learning and characterize contributions of laryngeal and respiratory subsystems to cough strength. This document will then conclude (Chapter 5) by synthesizing results from these studies and discussing clinical implications, limitations, and potential directions for future research.

Thesis (PhD (Biomedical Sciences. Medical Physiology))--Stellenbosch University, 2008. / The overall objective of this thesis is to provide additional data to assist clinicians and experimental neurologists alike in the quest for better understanding, more accurately diagnosing and more successfully treating patients suffering from Parkinson’s disease (PD). The general theme of the thesis is the interaction between certain environmental stimuli, including the exposure to adverse events during early central nervous system (CNS) development and the manifestation of elements of neurodegeneration, whether by means of neurochemical changes or expressed as a dysfunctional voluntary motor system. The first chapter provides a general introduction to the research theme of the thesis. This includes, in particular, a discussion on current understanding concerning the etiology and clinical profile of PD, the relative contribution made by genetic factors compared to environmental ones, and current treatment strategies for treating the disease. Mention is also made of the failure of these therapeutic applications for reversing or protecting against the disease, due to the side-effects associated with them. The material covered in chapter 1 provides the basis for the more complete discussion concerning these various aspects, contained in the chapters to follow. The overall aim was also to characterise the effects of commonly used toxin-induced animal models of PD, and the extent of vulnerability that the CNS displays towards them. The destruction of dopaminergic neurons following the administration of 6-OHDA at targeted points along the nigrostriatal tract is used extensively to model PD pathology in rats and is an established animal model of the disease. However, mature or even aged animals are mainly used in these studies, while the effects that the toxin might have on the developing CNS remain unclear. The study reported in chapter 4 aimed to elucidate some of 6-OHDA’s actions on the young adolescent (35 days-old) CNS by comparing the motor and biochemical effects of a unilateral infusion of the toxin into two anatomically distinct basal ganglia loci: The medial forebrain bundle (MFB) and the striatum. Animals were randomly assigned to receive either a direct delivery of 6-OHDA (12μg/4μl) into the MFB or an indirect injection, into the striatum. Although both lesion types were used, the MFB model is considered a more accurate portrayal of end-stage PD, while the striatum-model better reflects the long-term progressive pathology of the disease. The different lesions’ effects on motor function were determined by observing animal’s asymmetrical forelimb use to correct for weigh shifting during the vertical exploration of a cylindrical enclosure. Following the final behavioral assessment, the concentration of dopamine (DA) and DA metabolites remaining in the post-mortem brains were determined using 4 HPLC electrochemistry (HPLC-EC) and the levels compared between the two groups. The HPLC-EC results revealed a compensatory effect for DA production and DA turnover on the lesioned hemisphere side of the toxin-infused animal group. Thus, following 6-OHDA treatment, there appears to be extensive adaptive mechanisms in place within the remaining dopaminergic terminals that may be sufficient for maintaining relatively high extracellular and synaptic concentrations of DA. However, since substantial changes in motor-function were observed, it is suggested that the capacity of the remaining dopaminergic neurons to respond to increased functional demands may be limited. In addition, the behavioral results indicate that the distinct indices relating to different functional deficits depend on the lesioning of anatomically distinct structures along the nigrostrial tract. It has long been known that far fewer women are diagnosed with PD than men are. This seeming protection offered to females against degenerative disease of the CNS may relate to estrogen, although the hormone’s mechanism of action on the dopaminergic system is poorly defined. With an estimated 10-15 million women using oral contraceptives (OCs) in the United States alone, the aim of chapter 2 was to examine the evidence for a possible relationship between PD and the female reproductive hormone estrogen. A review of the current literature available on the topic was performed by consulting Medline, and by performing a search of the case-reports contained within the World Health Organization’s (WHO) International Drug Monitoring database, for possible PD-related symptoms that may arise from estrogen replacement therapy (ERT). The results, whilst conflicting, seem to suggest that estrogen protects women from obtaining the disease, or at least some features of it. Intensive research efforts are called for, with sufficient power to establish the relationship between ERT and the onset and development of parkinsonism. Chapter 3 reports on the results obtained from an experiment that subjected young Sprague-Dawley rats, 35 days of age, to a lower and a higher dose of 6-OHDA delivered to the MFB. Control rats received equivalent saline infusions. At 14 days post-surgery, the rats were evaluated for forelimb akinesia. For the higher dose of 6- OHDA the female rats were less impaired than males in making adjustment steps in response to a weight shift and in the vibrissae-evoked forelimb placing test. In addition, Tyrosine hydroxylase (TH) immunoreactivity was significantly higher for the female rats. Early gender differences in cell survival factors and/or other promoters of neuroplasticity may have contributed to the beneficial outcome seen in the females. For example, nerve growth factor (NGF) was found to be higher in the female rats following administration of the DA neurotoxin. It is unclear whether gonadal steroids are involved, and, if so, whether female hormones are protective or whether male hormones are prodegenerative. Determining the mechanisms for the improved outcome seen in the young female rats may lead to potential treatment strategies against PD. 5 Many studies have shown that early life stress may lead to impaired brain development, and may be a risk factor for developing psychiatric diseases, including clinical depression. However, few studies have investigated the impact that early stress may have on the onset and development of neurodegenerative disorders such as PD. The study reported on in chapter 5 conjointly subjected rat pups to a maternal separation (MS) paradigm that is a well characterised model of adverse early life events, and a unilateral, intrastriatal injection of 6- OHDA. The combined effects of these models on motor deficits and brain protein levels were investigated. Specifically, the animals were assessed for behavioral changes at 28 days postlesion with a battery of tests that are sensitive to the degree of DA loss sustained. The results show that animals that had been subjected to MS display poorer performance in the vibrissae and single-limb akinesia test compared to non-MS control animals (that had also been subjected to the toxin exposure). In addition, there was a significant increase in the loss of TH staining in MS rats compared to non-MS ones. The results from this study therefore suggest that exposure to adverse experiences during the early stages of life may contribute towards making dopaminergic neurons more susceptible to subsequent insults to the CNS occurring during mature stages of life. Therefore, taken together, early exposure to stress may predispose an individual towards the onset and development of neurodegenerative disease, which especially becomes a threat during the later stages of adult life. Moreover, within the framework of these characteristics, the capacity of a widely-used pharmacological agent (statins) was tested for possible future therapeutic application in PD (chapter 7). Although the precise cause of sporadic PD remains an enigma, evidence suggests that it may associate with defective activity of complex I of the mitochondrial electron transport chain. Mitochondrial DNA transmit and express this defect in host cells, resulting in increased oxygen free radical production, depressed antioxidant enzyme activities, and greater susceptibility to apoptotic cell death. Simvastatin is a member of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) group of drugs that are widely used for lowering cholesterol levels in patients who display elevated concentrations of low-density lipoprotein cholesterol. The study aimed to investigate the effects that statin-treatment have on motor-function and at the mitochondrial-protein level, using rotenone, a mitochondrial complex I inhibitor, as a rat-model of PD. Adult male Sprague-Dawley rats were treated either with simvastatin (6mg/day for 14 days) or with a placebo. Two different tests to assess motor function were used: The apomorphine-rotation test, and the vibrissae-elicited forelimb placement test. Following the drug administration protocol, the nigrostriatal tract was unilaterally lesioned with either rotenone (3 μg/4 μl) or, for the controls, were sham-operated by infusing the vehicle (DMSO:PEG) only. Five days later the rats were killed and a highly purified concentration of isolated mitochondria was prepared from the substantia nigra (SN) sections. 2- 6 Dimensional electrophoresis (2-DE) with subsequent identification of the spots using electronspray ionization quadruple time-of-flight mass spectrometrical (ESI-Q-TOF MS) was performed and the results BLAST-searched using bio-informatics tools for naming the identified peptides. The motor test results indicate that while unilateral rotenone causes behavioral asymmetries, treatment with simvastatin improved motor function relative to the rotenoneinduced ones. Mass Spectroscopy identified 23 mitochondrial proteins that differ significantly in protein expression (p < 0.05) following simvastatin treatment. The altered proteins were broadly classified according to their cellular function into 6 categories, with the majority involved in energy metabolism. This study effectively illustrated how neuroproteomics, with its sophisticated techniques and non-biased ability to quantify proteins, provides a methodology with which to study the changes in neurons associated with neurodegeneration. As an emerging tool for establishing disease-associated protein profiles, it also generates a greater understanding as to how these proteins interact and undergo post-translational modifications. Furthermore, due to the advances made in bioInformatics, insight is created concerning their functional characteristics. Chapter 4 summarises the most prominent proteomics techniques and discuss major advances made in the fast-growing field of neuroproteomics in PD. Ultimately, it is hoped that the application of this technology will lead towards a presymptomatic diagnosis of PD, and the identification of risk factors and new therapeutic targets at which pharmacological intervention can be aimed. The final chapter (chapter 8) provides a retrospective look at the academic work that had been performed for the purpose of this thesis, recaps on the main findings, and also highlights certain aspects of the project and provides relevant suggestions for future research. Lastly, the appendix provides a detailed overview of the methods followed for the experiments described in this thesis. It provides not only a comprehensive description of the techniques that had been followed, but provides information concerning the care taken with the animals (i.e. post-surgery) in order to control for the potential influence of experimental variables on the results.

初級運動皮質直接負責運動控制。大量關於帕金森式癥(PD)的有效治療手段的研究已經證明，初級運動皮質在病理情況下的功能改變，直接與患者運動障礙相關。本論文的研究重點在於探索初級運動皮質在深部腦刺激治療帕金森氏症的運動障礙的過程中發揮的作用及其與運動學習功能障礙的聯繫。 / 丘腦底核深部腦刺激(STN-DBS) 已被廣泛應用於治療帕金森式症。雖然該項治療手段能顯著地改善患者的運動功能障礙，但其確切的治療機制仍未明確。理論上來說，丘腦底核深部腦刺激能夠直接啟動丘腦底核內部和其周圍很大範圍的神經組織，包括丘腦底核內部本身的神經元胞體，以及與其相連接的輸入輸出核團的神經元軸突。在丘腦底核眾多輸入核團之中，一個重要的神經輸入來自於初級運動皮質(MI)第五層的離皮質神經元(CxFn)，電刺激引起的逆行皮質啟動作用被提出，用於解釋丘腦底核深部腦刺激的治療機制。 / 為了研究逆行皮質啟動效應究竟如何在丘腦底核深部腦刺激的過程之中帶來治療效果，我們採用多通道神經電生理信號記錄系統在自由活動的單側帕金森大鼠的初級運動皮質進行鋒電位元和局部場電位元信號的記錄。實驗結果證明，當對丘腦底核進行高頻電刺激，在運動皮質第五層的離皮質神經元能成功記錄到保持固定延時的逆行鋒電位。由於增加刺激頻率會引起逆行鋒電位被成功記錄到的百分比下降，因此當深部腦刺激的頻率選擇在125Hz時，逆行鋒電位的放電頻率達到最高，而此刺激頻率正好與行為學實驗中帶來最佳治療效果的刺激頻率一致。於此同時，逆行皮質啟動作用還伴隨著初級運動皮質離皮質神經元的自發放電頻率增加、同步性爆發式放電減少等電生理信號特點。場電位分析的結果進一步表明，丘腦底核深部腦刺激減弱了病理情況下出現的beta波頻譜能量增高以及鋒電位-場電位相干性增強。更重要的是，我們發現只有逆行鋒電位被成功誘發，離皮質神經元的發放電機率才能被調節。這點有力地表明由電刺激隨機誘發的逆行鋒電位傳導至初級運動皮質，直接幹預並抑制了離皮質神經元在病理情況下的同步性爆發式放電活動，從而緩解了帕金森氏症的運動障礙。 / 另外，初級運動皮質並不僅僅是一個靜態的運動控制中樞，更為重要的功能在於它參與著與運動學習和運動記憶相關的動態資訊編碼。帕金森氏症患者普遍存在皮質可塑性減弱以及運動技能學習障礙。由於初級運動皮質分層結構的存在，層內神經元之間的突觸連接為神經可塑性提供了很好的結構基礎。因此，我們在初級運動皮質誘發在體長時程增強(LTP)，旨在研究與運動技能學習相關的皮質神經可塑性的動態變化過程，以及探索中腦多巴胺能投射系統對皮質神經可塑性的影響。 / 一方面，我們採用間斷性高頻刺激誘發在體長時程增強，證實六羥多巴損毀後皮質的長時程增強水準顯著下降。另一方面，我們設計前肢抓食的行為學範式用來評價動物在運動技能學習的不同階段皮質可塑性發生的動態變化。實驗結果表明，直接損毀皮質的多巴胺能輸入，模型組大鼠與假實驗組大鼠的行為表現在初期的技能獲取階段並無明顯差異，而只在後期的技能鞏固階段模型組大鼠表現出技能鞏固障礙。更為有趣的是，兩組行為學變化趨勢與各自的在體長時程增強的變化趨勢有很高的一致性。本研究表明多巴胺對初級運動皮質的支配在運動記憶的鞏固過程中起著關鍵作用。在帕金森氏症的病理情況下，多巴胺耗竭將影響皮質的突觸可塑性，從而造成帕金森患者在運動技能的鞏固階段表現出障礙。 / The primary motor cortex (MI) controls movement directly, but is an under-investigated brain region in the pathogenesis and treatment of Parkinsonian motor disability, when compared with the basal ganglia circuitry. In this study, the roles of MI in underlying the therapeutic action of surgical deep brain stimulation and motor learning impairment were investigated. / Deep brain stimulation of the subthalamic nucleus (STN-DBS) is now a recognized therapeutic option for Parkinson’s disease (PD). Although this surgical strategy provides behavioral benefits remarkably, its exact mechanism is still a matter of controversy. In principle, STN-DBS can directly activate a wide range of neuronal elements within the STN and surrounding areas. As the corticofugal neurons (CxFn) in the layer V motor cortex provide a major input to the STN, we hypothesized that the stimulation evoked antidromic cortical activation is involved in the therapeutic mechanism of STN-DBS. In the first series of experiments, we performed simultaneous recordings of multi-unit neuronal activities and local field potentials (LFPs) in MI in freely moving hemi-parkinsonian rats. By identifying stimulation evoked antidromic spike, which occurred at a fixed, short latency, CxFn located in the layer V MI were identified. Increasing stimulation frequency also increased failure rate of activation, resulting in a peak frequency of stochastic antidromic spikes at 125Hz STN-DBS, which was correlated with the optimal therapeutic efficacy observed in behavioral tests. Meanwhile, this antidromic effect was accompanied by the rectification of pathological neuronal activities including increased spontaneous firing rate, reduced burst discharge and synchrony among the CxFn. Field potential analysis revealed that STN-DBS alleviated the dominance of pathological beta band oscillation and spike-field coherence in the MI. More importantly, it was found that the firing probability of CxFn could only be modified following the occurrence of antidromic spikes, suggesting that direct interference of stochastic antidromic spikes with pathological neuronal activities underlies the beneficial effect of STN-DBS. / The MI is not simply a static motor control structure. It also contains a dynamic substrate that participates in motor learning or stores motor memory. In PD patients, loss of cortical plasticity and impaired motor learning is a common feature. As the intrinsic horizontal neuronal connections in MI are a strong candidate of cellular correlate for activity-dependent plasticity, in the second series of experiments, we developed in vivo long-term potentiation (LTP) technique in the MI to investigate the dynamics of cortical plasticity during motor skill learning and the role of the innervation by mesocortical dopamine input. Local depletion of dopamine in the primary motor cortex resulted in reduced performance in the forelimb reaching for food learning task. Although the performance of the PD rats in the initial learning phase was comparable to that of the sham-operated group, as training continued, these animals exhibited deficit in consolidating the motor skill. These deficits closely paralleled the impairment in training-enhanced synaptic connections in layer V neurons, and the in vivo LTP of evoked field excitatory postsynaptic potentials induced by intermittent high frequency stimulation. In addition, progressive recruitment of task-specific neurons was suppressed. Our study therefore revealed that dopamine depletion confined to the MI could lead to impairment in cortical synaptic plasticity which may preferentially affect the consolidation, but not the acquisition, of motor skills. These findings shed light on the cellular mechanisms of motor skill learning and could explain the decreased ability of PD patients in learning new motor skills. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Qian. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 168-190). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / CHAPTER 1 --- p.1 / General Introduction --- p.1 / Chapter 1.1 --- Anatomical organization of the basal ganglia --- p.1 / Chapter 1.1.1 --- Overview of the basal ganglia circuit --- p.1 / Chapter 1.1.2 --- Cortico-basal ganglia-cortical circuit --- p.1 / Chapter 1.1.2.1 --- Direct and indirect pathway --- p.2 / Chapter 1.1.2.2 --- Hyperdirect pathway --- p.2 / Chapter 1.1.2.3 --- The midbrain dopamine system --- p.2 / Chapter 1.2 --- Striatum --- p.3 / Chapter 1.2.1 --- Cell types in the striatum. --- p.3 / Chapter 1.2.2 --- The Cortico-striatal system --- p.4 / Chapter 1.3 --- Subthalamic Nucleus --- p.5 / Chapter 1.3.1 --- Neuronal property of the STN. --- p.5 / Chapter 1.3.2 --- Electrophysiological property of the STN --- p.6 / Chapter 1.3.3 --- Cortico-subthalamic system --- p.7 / Chapter 1.3.4 --- Functional significance of the cortico-subthalamic and corticostriatal system. --- p.8 / Chapter 1.4 --- Parkinson’s disease --- p.9 / Chapter 1.4.1 --- Pathogenesis of PD --- p.9 / Chapter 1.4.2 --- Genetic risk factors of PD --- p.10 / Chapter 1.4.3 --- Progressive motor symptoms of PD --- p.11 / Chapter 1.4.4 --- Non-motor symptoms of PD --- p.13 / Chapter 1.4.5 --- Pathological neuronal rhythms in the basal ganglia of PD. --- p.16 / Chapter 1.5 --- Experimental studies of PD. --- p.18 / Chapter 1.5.1 --- Animal modeling of PD. --- p.18 / Chapter 1.5.2 --- Motor deficits evaluation in rodent models of PD --- p.21 / Chapter 1.5.3 --- Non-motor symptoms evaluation in experimental models of PD --- p.24 / Chapter 1.6 --- Deep Brain Stimulation --- p.27 / Chapter 1.6.1 --- DBS in alleviating Parkinsonian motor symptoms --- p.28 / Chapter 1.6.2 --- DBS in alleviating Parkinsonian non-motor symptoms --- p.29 / Chapter 1.6.3 --- Investigation of the STN-DBS mechanism. --- p.31 / Chapter 1.6.3.1 --- Local inhibitory effect within the STN --- p.32 / Chapter 1.6.3.2 --- Excitatory effect at output nuclei --- p.33 / Chapter 1.6.3.3 --- The de-coupling of soma and axons at system level --- p.34 / Chapter 1.6.3.4 --- Effects of DBS on abnormal rate or pattern --- p.35 / Chapter 1.6.3.5 --- Antidromic propagation of DBS effect towards cortex --- p.37 / Chapter 1.7 --- Objective --- p.38 / Chapter 1.8 --- Figures --- p.41 / CHAPTER 2 --- p.47 / General Methods --- p.47 / Chapter 2.1 --- Animals --- p.47 / Chapter 2.2 --- Stereotaxic surgery --- p.47 / Chapter 2.2.1 --- Preoperative preparation --- p.47 / Chapter 2.2.2 --- Anesthesia and craniotomy --- p.48 / Chapter 2.2.3 --- Induction of hemi-Parkinsonian rat model --- p.48 / Chapter 2.2.4 --- Electrode implantation techniques. --- p.49 / Chapter 2.3 --- Behavioral assessment. --- p.50 / Chapter 2.3.1 --- Apomorphine-induced contralateral rotation. --- p.50 / Chapter 2.3.2 --- Open field test --- p.50 / Chapter 2.4 --- STN-DBS protocol --- p.50 / Chapter 2.5 --- Electrophysiological data acquisition --- p.51 / Chapter 2.6 --- Data analysis --- p.52 / Chapter 2.6.1 --- Statistical analysis of behavioral data --- p.52 / Chapter 2.6.2 --- Electrophysiological data --- p.52 / Chapter 2.6.2.1 --- Stimulation artifact removal --- p.52 / Chapter 2.6.2.2 --- Multi-unit spike sorting --- p.53 / Chapter 2.6.2.3 --- Electrophysiological identification of pyramidal neuron and interneuron. --- p.54 / Chapter 2.6.2.4 --- Identification of antidromic cortical activation --- p.54 / Chapter 2.6.2.5 --- Discharge pattern classification --- p.54 / Chapter 2.6.2.6 --- Synchrony level evaluation --- p.55 / Chapter 2.6.2.7 --- Oscillatory rhythm characterization --- p.55 / Chapter 2.6.2.8 --- Coherence Level Measurement --- p.56 / Chapter 2.7 --- Histological verification --- p.56 / Chapter 2.8 --- Figures --- p.58 / CHAPTER 3 --- p.60 / Alleviation of Parkinsonian Motor Symptoms during Deep Brain Stimulation in Hemi-Parkinsonian Rats --- p.60 / Chapter 3.1 --- Introduction --- p.60 / Chapter 3.2 --- Materials & Methods --- p.61 / Chapter 3.2.1 --- Animals --- p.61 / Chapter 3.2.2 --- Chemicals --- p.61 / Chapter 3.2.3 --- Equipment --- p.61 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- Time course of the Apomorphine induced rotation behavior --- p.62 / Chapter 3.3.2 --- Dose-dependence of the Apomorphine induced rotation --- p.62 / Chapter 3.3.3 --- Acute behavioral response to STN-DBS. --- p.63 / Chapter 3.3.4 --- The dependence of STN-DBS effect on stimulation paradigm. --- p.64 / Chapter 3.3.5 --- Acute effects of STN-DBS on APO induced rotation. --- p.64 / Chapter 3.3.6 --- Long-term effects of STN-DBS on APO induced rotation --- p.64 / Chapter 3.3.7 --- Histological confirmation of the stimulation electrodes localization --- p.65 / Chapter 3.3.8 --- Loss of DA neurons in the SNc --- p.65 / Chapter 3.3.9 --- Reductions of the DA axon terminals in the striatum --- p.65 / Chapter 3.3.10 --- Chronic STN-DBS failed to rescue nigrostsriatal and striatal DA --- p.66 / Chapter 3.4 --- Discussion --- p.66 / Chapter 3.4.1 --- Neurotoxic mechanism of 6-OHDA --- p.66 / Chapter 3.4.2 --- Time course of dopamine degeneration induced by 6-OHDA --- p.66 / Chapter 3.4.3 --- Failure in observing worsened motor symptoms during low frequency STN-DBS. --- p.67 / Chapter 3.4.4 --- Experimental DBS based on rat model: does it mimic human case? --- p.67 / Chapter 3.4.5 --- Technical issues about STN-DBS --- p.69 / Chapter 3.5 --- Figures --- p.72 / CHAPTER 4 --- p.82 / Direct involvement of the Corticofugal Neurons in Motor Cortex during Therapeutic Deep Brain Stimulation --- p.82 / Chapter 4.1 --- Introduction --- p.82 / Chapter 4.2 --- Materials --- p.83 / Chapter 4.2.1 --- Animals --- p.83 / Chapter 4.2.2 --- Chemicals --- p.83 / Chapter 4.2.3 --- Equipment --- p.83 / Chapter 4.3 --- Results --- p.84 / Chapter 4.3.1 --- Identification of CxFn based on antidromic effect --- p.84 / Chapter 4.3.2 --- Antidromic spikes frequency correlates with therapeutic effect of STN-DBS. --- p.84 / Chapter 4.3.3 --- Pathological changes of neuronal firing rate in MI --- p.85 / Chapter 4.3.4 --- Only high frequency STN-DBS normalizes neuronal firing rate in MI --- p.86 / Chapter 4.3.5 --- Pathological changes of neuronal discharge pattern in MI --- p.88 / Chapter 4.3.6 --- Pathological synchrony of MI neuronal population, especially during burst discharge --- p.89 / Chapter 4.3.7 --- High frequency STN-DBS successfully suppresses synchronized burst discharge in MI --- p.89 / Chapter 4.3.8 --- Pathological β-band oscillatory activity in MI-LFPs induced by 6-OHDA lesion --- p.90 / Chapter 4.3.9 --- High frequency STN-DBS alleviates the β-band oscillation in MI-LFPs --- p.90 / Chapter 4.3.10 --- Synchronized bursting discharge correlates with oscillatory activity --- p.91 / Chapter 4.3.11 --- Pathological increased spike-LFP coherence level induced by 6-OHDA lesion --- p.92 / Chapter 4.3.12 --- High frequency STN-DBS modulated the spike-LFP coherence properties --- p.92 / Chapter 4.3.13 --- Antidromic spikes directly modulate the firing probability of CxFn --- p.93 / Chapter 4.3.14 --- Antidromic spikes modulate the firing probability of INs and non-CxFn nearby. --- p.94 / Chapter 4.3.15 --- The efficiency of antidromic cortical modulation depends on DBS frequency --- p.94 / Chapter 4.3.16 --- Orthodromic vs. antidromic effect: which one is responsible for the beneficial effect of DBS? --- p.95 / Chapter 4.3.17 --- Histology --- p.96 / Chapter 4.4 --- Discussion --- p.96 / Chapter 4.4.1 --- Origin of pathogenic rhythm in basal ganglia circuit --- p.96 / Chapter 4.4.2 --- Suppression of oscillatory synchronization equals to therapeutic effects of DBS? --- p.97 / Chapter 4.4.3 --- Beneficial effect of DBS corresponds to the topographic distribution of cortico-subthalamic projection. --- p.98 / Chapter 4.4.4 --- What is the reason for a stochastic pattern of antidromic activation effect? --- p.99 / Chapter 4.4.5 --- Desynchronization of pathological oscillatory rhythm by antidromic activation --- p.100 / Chapter 4.4.6 --- Antidromic vs. orthodromic: which is the cause of the beneficial effects of DBS? --- p.101 / Chapter 4.4.7 --- Wide propagation of antidromic effect by cortical horizontal circuits --- p.102 / Chapter 4.4.8 --- Significance of antidromic cortical activation in during STN-DBS --- p.102 / Chapter 4.4.9 --- Implication of antidromic activation effect on pathogenesis and treatment of PD --- p.104 / Chapter 4.5 --- Figures --- p.105 / CHAPTER 5 --- p.132 / Impaired Synaptic Plasticity in the Primary Motor Cortex after Dopamine Depletion: Potential Role in Motor Memory Consolidation --- p.132 / Chapter 5.1 --- Introduction --- p.132 / Chapter 5.1.1 --- Characteristics of motor learning --- p.132 / Chapter 5.1.2 --- Motor learning related cortical plasticity. --- p.133 / Chapter 5.1.3 --- Dopaminergic signals in the primary motor cortex --- p.134 / Chapter 5.1.4 --- Impaired cortical plasticity in PD --- p.135 / Chapter 5.1.5 --- Objective --- p.136 / Chapter 5.2 --- Materials --- p.136 / Chapter 5.2.1 --- Animals --- p.136 / Chapter 5.2.2 --- Chemicals --- p.136 / Chapter 5.2.3 --- Equipment --- p.136 / Chapter 5.3 --- Methods --- p.136 / Chapter 5.3.1 --- Functional mapping of the forelimb territory in MI --- p.136 / Chapter 5.3.2 --- Stereotaxic surgery --- p.137 / Chapter 5.3.3 --- Forelimb-reaching Task. --- p.137 / Chapter 5.3.4 --- In-vivo LTP Induction. --- p.138 / Chapter 5.4 --- Results --- p.139 / Chapter 5.4.1 --- Functional mapping of rat forelimb territory. --- p.139 / Chapter 5.4.2 --- Morphologies of evoked field potential response --- p.139 / Chapter 5.4.3 --- LTP of the early, monosynaptic plasticity within horizontal layer V MI --- p.140 / Chapter 5.4.4 --- LTP of the late, polysynaptic plasticity within horizontal layer V MI --- p.140 / Chapter 5.4.5 --- Impaired synaptic plasticity in MI after dopamine depletion --- p.140 / Chapter 5.4.6 --- Learning curve of forelimb-reaching task --- p.140 / Chapter 5.4.7 --- Physiologically enhanced cortical plasticity during motor learning --- p.141 / Chapter 5.4.8 --- Dynamic modulation of cortical neuronal activities during motor skill learning. --- p.142 / Chapter 5.4.9 --- Statistical analysis of ‘task related’ neuron’s modulation pattern. --- p.143 / Chapter 5.4.10 --- Loss of dopamine modulation in the MI --- p.144 / Chapter 5.5 --- Discussion --- p.144 / Chapter 5.5.1 --- Distinguishing between monosynaptic and polysynaptic transmission --- p.144 / Chapter 5.5.2 --- Artificially vs physiologically induced cortical plasticity. --- p.145 / Chapter 5.5.3 --- Cortical synaptic plasticity interprets motor learning dynamics --- p.146 / Chapter 5.5.4 --- Balance between neuronal recruitment and withdrawal in the consolidation stage --- p.147 / Chapter 5.5.5 --- Dopamine’s involvement in mediating the cortical synaptic plasticity. --- p.148 / Chapter 5.6 --- Figures --- p.150 / Conclusion --- p.162 / Abbreviations --- p.165 / References --- p.168