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Developmental and epileptic encephalopathies (DEE) represent a set of rare but devastating and largely intractable childhood epilepsies. While mouse models have made innumerable contributions to understanding the genetic basis of neurological diseases, only a small fraction of missense, gain-of-function DEE variants has been modeled in mice. In this dissertation, we focus on one DEE gene, ARHGEF9. With fewer than fifty ARHGEF9 patients reported to date, ARHGEF9 can be considered one of the rare players in DEE. ARHGEF9 encodes a brain-specific protein also known as collybistin (CB), a guanine nucleotide exchange factor and an essential regulator of inhibitory postsynaptic density. In Chapter 3 and Chapter 4, we present results and ongoing studies from our efforts to unravel the pathological mechanism of ARHGEF9 DEE. We studied the G55A variant on the SH3 domain, which was discovered in one severe case of DEE. Using a novel Arhgef9G55A mouse model, we examined behavioral, cellular, and electrophysiological consequences of Arhgef9G55A. Results demonstrate that the Arhgef9G55A mouse model is an adequate ARHGEF9 DEE model, because it phenocopies key aspects of human ARHGEF9 DEE. We showed the interesting protein aggregation phenotype caused by the G55A variant. Specifically, in Arhgef9G55A/Y neurons, CB forms protein aggregates at the proximal AIS, leading to dramatic disruptions in inhibitory postsynaptic components at the AIS. Furthermore, electrophysiological studies revealed significant changes in intrinsic neuronal excitability and synaptic transmission in Arhgef9G55A/Y brains. The work within this dissertation shows that the G55A variant disrupts axon initial segment structure and functions. In Chapter 2, we review and summarize current understandings on AIS structure and functions. We highlight the central role of the AIS in initiating action potential and integrating synaptic inputs through axo-axonic synapses. Based on our experimental results, we propose that disruptions in AIS function are closely tied to the pathophysiology of ARHGEF9 DEE. Aside from the clinical significance of our study, we demonstrate the important role of CB at the AIS. We propose that CB is a specific stabilizer of axo-axonic synapses. The difference in the requirement of CB in inhibitory synapse formation in different neuronal compartments could be a core molecular machinery underlying the functional diversity of inhibitory inputs.

by Tracy Man-kiu Ma. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 48-61). / Abstract and appendix in English and Chinese. / ABSTRACT --- p.ii / ACKNOWLEDGEMENTS --- p.iii / TABLE OF CONTENTS --- p.iv / LIST OF TABLES --- p.vi / LIST OF FIGURES --- p.vii / LIST OF APPENDICES --- p.viii / Chapter CHAPTER ONE- --- INTRODUCTION / Mesial temporal lobe and its sequel of damages --- p.1 / Mesial temporal lobe pathologies --- p.2 / Memory assessment instruments and the Hong Kong List Learning Test --- p.4 / The receiver operating characteristics (ROC) analysis --- p.6 / Purpose of the present study --- p.7 / Chapter CHAPTER TWO- --- METHOD / Participants --- p.9 / Materials --- p.10 / Procedure --- p.13 / Statistical analysis --- p.15 / Chapter CHAPTER THREE - --- RESULTS / Memory Profiles of NPC Patients with bilateral temporal lobe lesions --- p.18 / Receiver operating characteristics (ROC) analysis for test performance --- p.26 / Validity and reliability --- p.33 / Chapter CHAPTER FOUR - --- DISCUSSION / The clinical utility of the blocked condition --- p.39 / Optimal cutoff scores for sensitivity and specificity --- p.40 / Memory profiles of NPC patients and its implications --- p.42 / Limitations --- p.45 / Conclusions --- p.46 / REFERENCES --- p.48 / APPENDICES --- p.62

published_or_final_version / Electrical and Electronic Engineering / Master / Master of Philosophy

published_or_final_version / abstract / Medical Sciences / Master / Master of Medical Sciences

It has been demonstrated that by using 1H NMR spectroscopy in combination with multivariate statistical modelling (PLS) it is possible, using urine samples obtained from rats, to distinguish between different types of CNS lesions. Against this background this thesis will explore whether the combination of 1H NMR and PLS modelling on biofluids can be used q-1eientify biomarkers in .. - different neurological diseases and in clinically relevant animal models of neurologic disease. The results in this thesis demonstrate that it is possible to separate sets of animals at different stages of disease in models of multiple sclerosis and to identify the presence of early brain metastasis. The same methodology was also applied to human biofluids. In MS patient cohorts (RR- MS, PP-MS and SP-MS) it was also possible to differentiate between RR-MS and SP-MS as well between MS and healthy controls. Therapy for these two stages of MS are very different and therefore a rapid test to determine a patient's stage of MS would be hugely beneficial in the clinic. Further investigation revealed that it is possible to separate MS patients from individuals with Alzheimer's disease. Metabolomics was then combined with other eo- variants in a study of cerebrospinal fluid obtained from patients with HIV associated dementia (HAD) to discover whether disease progression could be followed in this manner. The results show that it is possible to detect neurocognitive changes in patients with HAD. Indeed, the results demonstrate. that metabolomics is a far more sensitive tool for the following progression than other non-PLS biomarker techniques and should provide a useful method for early diagnosis of CNS disease and the evaluation of therapy in prospective studies.

The blood-brain barrier (BBB) is physiologically essential for brain homeostasis. While it protects the brain from noxious agents, it prevents almost all currently available drugs from crossing to the parenchyma. This greatly hinders drug delivery for the treatment of neurological diseases and disorders such as Parkinson’s, Alzheimer’s and Huntington’s, as well as the development of drugs for the treatment of such diseases. Current drug delivery techniques to the brain are either invasive and target specific, or non-invasive with low special specificity. Neither group of techniques are optimal for long term treatment of patients with neurological diseases or disorders. Focused ultrasound coupled with intravenous administration of microbubbles (FUS) has been proven as an effective technique to selectively and noninvasively open the BBB in multiple in vivo models including non-human primates (NHP). Although this technique has promising potential for clinical outpatient procedures, as well as a powerful tool in the lab, the safety and potential neurological effects of this technique need to be further investigated. This thesis focuses on validating the safety and efficacy of using the FUS technique to open the BBB in NHP as well as the ability of the technique to facility drug delivery. First, a longitudinal study of repeatedly applying the FUS technique targeting the basal ganglia region in four NHP was conducted to determine any potential long-term adverse side effects over a duration of 4-20 months. The safety of the technique was evaluated using both MRI as well as behavioral testing. Results demonstrated that repeated application of the FUS technique to the basal ganglia in NHP did not generate permanent side effects, nor did it induce a permanent opening of the BBB in the targeted region. The second study investigated the potential of the FUS technique as a method to deliver drugs, such as a low dose of haloperidol, to the basal ganglia in NHP and mice to elicit pharmacodynamical effects on responses to behavioral tasks. After opening the BBB in the basal ganglia of mice and NHP, a low dose of haloperidol was successfully delivered generating significant changes in their baseline motor responses to behavioral tasks. Domperidone was also successfully delivered to the caudate of NHP after opening the BBB and induced transient hemilateral neglect. In the final section of this thesis, the safety and efficacy of the FUS technique was evaluated in fully alert NHP. The FUS technique was successful in generating BBB opening volumes larger on average to that of the BBB opening volumes in anesthetized experiments. Safety results through MRI verification as well as behavioral testing during application of the technique demonstrated that the FUS technique did not generate adverse neurological effects. Conversely, the FUS technique was found to induce slight positive effects on the response of the NHP to the behavioral task. Collectively, the work presented in this thesis demonstrates the safety and effectiveness of the FUS technique to open the BBB and deliver neuroactive drugs in the NHP.

The basal ganglia are a set of subcortical nuclei in the forebrain of vertebrates that are highly conserved among mammals. Classically, dysfunction in the basal ganglia has been linked to motor abnormalities. However, it is now widely recognized that in addition to their role in motor behavior, these set of nuclei play a role in reinforcement learning and motivated behavior as well as in many diseases that present with abnormal motivation. In this dissertation, I first provide a review of the literature that describes the current state of research on the basal ganglia and the background for the original studies I later present. I describe the anatomy and physiology of the basal ganglia, including how structures are interconnected to form two parallel pathways, the direct and the indirect pathways. I further review published studies that have investigated how the basal ganglia regulate motor behavior and motivation. And finally, I also summarize findings on how disruption in basal ganglia circuitry function has been linked to a number of neuropsychiatric diseases, with special focus on the symptoms of schizophrenia. I then present original data and discuss the results of three studies investigating basal ganglia function and behavior. In the first study, I investigated the bridging collaterals, axon collaterals of direct-pathway medium spiny neurons (dMSNs) in the striatum that target the external segment of the globus (GPe), the canonical target of indirect-pathway medium spiny neurons (iMSNs). Previous work in the Kellendonk laboratory has linked these collaterals to increased dopamine D2 receptor (D2R) function and increased striatal excitability, as well as to abnormal locomotor response to stimulation of the direct pathway. I expanded on these findings by first demonstrating that bridging collaterals form synaptic contacts with GPe cells. I was also able to generate a viral vector to selectively increase excitability in specific populations of MSNs. I used this virus to show that chronically increasing excitability of the indirect pathway, but not the direct pathway, leads to a circuit-level change in connectivity by inducing the growth of bridging collaterals from dMSNs in the GPe. I also confirmed that increased density of bridging collaterals are associated with an abnormal locomotor response to stimulation of striatal dMSNs and further demonstrated that chronic pharmacologic blockade of D2Rs can rescue this abnormal locomotor phenotype. Furthermore, I found that motor training reverses the enhanced density of bridging collaterals and partially rescue the abnormal locomotor phenotype associated with increased collaterals, thereby establishing a new link between connectivity in the basal ganglia and motor learning. In the second study, I conducted a series of experiments in which I selectively increased excitability of the direct or indirect pathway in specific striatal sub-regions that have been implicated in goal-directed behavior, namely the DMS and NA core. I found that this manipulation was not sufficient to induce significant effects in different behavioral assays of locomotion and motivation, including the progressive ratio and concurrent choice tasks. These findings also suggest that increased bridging collateral density does not have a one-to-one relationship with the motivational deficit of D2R-OEdev mice, as previously hypothesized. In the third and final study, my original aim was to determine whether the motivational deficit of D2R-OEdev mice, induced by upregulation of D2Rs in the striatum, could be reversed by acutely activating Gαi-coupled signaling in the indirect pathway in these animals. I found that this manipulation increased motivation in D2R-OEdev mice but also in control littermates. This effect was due to energized behavioral performance, which, however, came at the cost of goal-directed efficiency. Moreover, selective manipulation of MSNs in either the DMS or NA core showed that both striatal regions contribute to this effect on motivation. Further investigation aimed at understanding how Gαi-coupled signaling affects striatal circuit function revealed that activating a Gαi-coupled receptor did not lead to a significant change in somatic MSN activity in vivo or to a change in neuronal excitability in vitro. In contrast, the GPe, which receives monosynaptic inhibition from the indirect pathway, showed disinhibited activity when Gαi signaling was activated in striatal iMSNs. In addition, as drug therapies for psychiatric diseases are not usually given acutely but involve long-term, continuous administrations, I also studied how chronically decreasing function of iMSNs would affect behavior. I showed that chronically activating a Gαi-coupled receptor in iMSNs does not lead to a measurable effect on locomotion or motivation, a behavioral desensitization response that can be recovered within 48 h and may be due to receptor desensitization to the drug or circuit-level compensation to a chronic decrease in iMSN function. Finally, I conclude this dissertation with a general discussion addressing how the findings from each study can be related to each other to provide a more complete understanding of how basal ganglia function regulate behavior, how disruption in the basal ganglia can underlie neuropsychiatric disease, and how strategies to target basal ganglia function should be employed to treat disorders of motivation. I conclude this dissertation by proposing new avenues of research for further exploring my findings.

Zhang Aiqun. / "March 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 95-123). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

published_or_final_version / abstract / toc / Paediatrics / Master / Master of Philosophy