Florey Department of Neuroscience and Mental Health - Theses

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    Exploring how the indoor hospital physical environment may impact physical and social behaviour, and affective responses of people with brain injury
    Shannon, Michelle Maura ( 2022)
    The physical environment of the interior hospital environment is an aspect of the environment of care of the person with brain injury that has received little attention in studies. Although several physical design attributes (e.g. daylight, patient room) have been studied in diverse healthcare settings (neonatal, cancer, ICU) few studies have involved people with brain injury. In addition, this field of research in the design of the physical environment is complex, requiring a multi-pronged approach to examine how and to what extent the physical environment might impact on people with brain injury in hospital. The overall objective of my thesis research was to explore how the physical environment of hospitals, specifically the indoor physical environment, might impact the physical and social behaviour, and affective responses of people with brain injury. Methods A series of sequentially-conducted studies were done over the course of this thesis. Firstly, I utilised a behavioural mapping approach and an adapted environmental checklist to compare the physical, and social activity of people in hospital for neurological conditions (including stroke) during a ‘before and after’ hospital re-location study. Then I conducted a systematic review where I aimed to differentiate the physical design attributes in a ‘single patient room’ from a ‘multi-patient room’, and to establish the strength of empirical evidence supporting one patient room type over the other. The systematic review motivated me to embark on a scoping review of the theories utilised by others in the field to attempt to explain or to test how the physical environment impacts on adults in health facilities. With the findings of studies 1-3, I was positioned to be able to design and conduct a factorial experimental design, using a virtual reality technology, to explore the impact of the physical design attributes on people with stroke, using numerical, categorical, and interviewing data collection. Results Firstly, the observational study showed that people with neurological conditions (>50% with stroke) spend most of the daytime in the patient room (social activity, physical activity), in both the old and new hospital ward environments. Further, these people spent time in close proximity to the window outlook and window seat (new ward), and not in communal areas. The systematic review revealed the paucity of diverse physical design attributes, beyond occupancy, used to examine the content of patient rooms, and highlighted the heterogeneity of outcomes (mostly hospital-acquired infection and falls rates) with which to compare the room types. Although a variety of theories were identified through the scoping review, few of them had been utilised consistently during a health research design study. One theory (the Theory of Supportive Design) was found to be consistently applied through a research study of the hospital ward environment. Finally, putative physical design characteristics (including ‘single’ versus ‘multi-patient’ room), led to the final thesis study (RiSE-VR). Identification of the magnitude and direction of stroke participant choice-preference and affective responses during exposures to a number of physical design characteristics were primarily sought. Quantitative findings were elaborated using interviews in the qualitative study. Feasibility of use of Virtual Reality in stroke was shown. Conclusions/ Implications New insights have been provided about how to study the impact of the interior hospital physical environment and people with brain injury. A novel study approach using virtual reality was developed and utilised to evaluate selected typical hospital physical design attributes in people after stroke. Feasibility and safety of this approach was shown. Regression analyses revealed the impact of different physical design attributes when co-existing with other physical attributes, in daytime and night-time. Thematic analysis provided triangulation and explanation of the quantitative data findings
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    α-synuclein, iron and Multiple System Atrophy
    Shukla, Jay Jayeshbhai ( 2019)
    Multiple System Atrophy (MSA) is an atypical parkinsonian disorder characterised by progressive neurodegeneration in substantia nigra, striatum, cerebellum, pons, inferior olives and spinal cord. The presence of protein aggregates primarily composed of misfolded α-synuclein in oligodendrocytes is the pathological hallmark of MSA, classifying it as a synucleinopathy. However, the aetiology of MSA remains poorly understood and due to the lack of identification of potential targets for drug therapy, no disease modifying therapies are available. Brain region-specific changes in the metabolism of biological trace metals – especially iron and copper – have been reported in α-synucleinopathies like Parkinson’s disease but, their contribution in MSA pathogenesis requires further investigation. Hence, in this thesis, I studied the role of iron and copper in the pathogenesis of MSA using post mortem human MSA brains and a mouse model of MSA. Quantification of metal levels using inductively coupled plasma-mass spectrometry (ICP-MS) revealed an increase in cytosolic iron content in putamen and occipital cortex from MSA brains. Since ferritin is a major iron storage protein, the amount of iron bound to ferritin was investigated using size exclusion chromatography-ICP-MS and it was found that ferritin-bound iron remained unchanged in MSA brain. Furthermore, ferritin protein levels were also unchanged in MSA putamen and occipital cortex. In order to better understand how iron and copper levels change through the course of disease progression in MSA, I used a transgenic mouse model of MSA and studied age-dependent changes in these metals. I found increased iron in substantia nigra, putamen and cerebellum in aged MSA mice compared with non-transgenic littermates, and a copper-binding protein with a molecular weight consistent with ceruloplasmin had a significantly decreased copper content. Ceruloplasmin is a copper-dependent protein that is involved in iron export from cells. In addition, the levels of ferritin were found to be decreased. These results indicate that elevated iron in MSA mice may result from ceruloplasmin dysfunction. Decreased copper binding to ceruloplasmin may result into loss of activity and hence, impaired iron export from the cell leading to iron accumulation that could contribute to the ongoing neurodegeneration in MSA. I further investigated if administration of ceruloplasmin or deferiprone alleviated neuronal pathology and motor impairment in MSA mice. Deferiprone is a clinically approved iron chelator and exogenous ceruloplasmin administration has been shown to be therapeutic in animal models. Compared to vehicle treated mice, deferiprone and ceruloplasmin treatments prevented the decline in motor performance, prevented loss of substantia nigra neurons and reduced the number of α-synuclein aggregates in substantia nigra. The results from this proof of concept pre-clinical trial provide evidence that targeting iron in MSA could be a viable therapeutic option.
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    Spatial and temporal surveillance of the mechanisms controlling proteome foldedness via a FRET-based biosensor
    Raeburn, Candice ( 2018)
    Proteostasis (protein homeostasis) is essential for keeping the proteome functional. This process controls protein synthesis, folding and degradation and involves hundreds of genes, including those encoding chaperones, to form extensive quality control (QC) networks (Kim et al., 2013). Imbalances in proteostasis are implicated in a range of aggregation-based neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS), Huntington’s and Alzheimer’s diseases (Morimoto et al., 2014; Vilchez et al., 2014). Currently there is a lack of capacity to quantitatively measure proteostasis imbalance and therefore we are limited in understanding how proteostasis imbalance manifests during disease. A new biosensor system has been developed by our lab to address this shortfall. The biosensor is a genetically encoded unfolded “bait” flanked by two fluorescent proteins to assay foldedness by fluorescence resonance energy transfer (FRET). Proteostasis efficiency is reported by measurement of the efficiency to which the bait interacts with the QC network. In this master’s project, the biosensor was further targeted to organelles to allow for a higher degree of spatiotemporal control. Signalling peptides were used to target the biosensor to specialised microenvironments, and successful targeting was achieved in the Golgi apparatus and nucleus. Investigations into nuclear proteostasis revealed the biosensor behaved predictably to chaperone overexpression (Hsp40 and Hsp70 co-expression) or inhibition (Hsp70 or Hsp90 inhibition). Polyglutamine (PolyQ) expansions of non-pathogenic (Q25) to pathogenic (Q72) lengths reduced the biosensor foldedness and decreased aggregation, which is consistent with an increase in chaperone supply. The biosensor was also adapted to express in the body wall muscles of Caenorhabditis elegans to examine change in proteostasis across age and in an organismal context. The biosensor was successfully expressed in the model organism, with potential sub-microscopic and variant biosensor expression level confounding data analysis. The C. elegans reporter lines were successfully crossed with lines expressing Aβ (1-42) demonstrating the ability of the biosensor to report on disease states. Moving forward, the generation of low-expression, single-copy C. elegans biosensor lines would allow for steady, matched expression and enhanced capacity for comparison between worm lines.
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    Regional brain responses associated with the neural control of drinking in humans
    Saker, Pascal Dorian ( 2017)
    Drinking in response to dehydration is a tightly regulated behaviour that accurately corrects the fluid deficit present within the body. The critical importance of this behaviour is underlined by its conservation across evolution along with the adverse health effects produced by its dysregulation. One of the most notable of these effects is water intoxication caused by excess water intake, exemplified by the polydipsia associated with schizophrenia. Given the everyday occurrence of drinking, our critical dependence upon it for survival, and the range of detrimental health effects associated with its dysfunction, understanding how drinking is regulated in response to hydration status represents a topic of considerable scientific and medical importance. Remarkably, despite its importance, this regulatory process has received little investigation in humans. Furthermore, as both the motivation to drink and the cessation of drinking are associated with the subjective states of thirst and satiation respectively, the fundamental relationship between these states, hydration status, and drinking behaviour remains unknown. This relationship can be investigated using functional magnetic resonance imaging (fMRI), an imaging modality capable of interrogating the fully integrated system of brain, body and behaviour. fMRI was therefore used in this PhD project to investigate the neural control of drinking in humans, with the aim of gaining novel insight into how dehydration status influences the regulation of fluid intake and its associated subjective states. Twenty healthy participants were recruited for the first study in this thesis. The experimental protocol consisted of inducing two opposing conditions of hydration in each participant: a state of thirst produced by exercise-related dehydration and a ‘post-satiation’ (over-sated) state that followed excess fluid intake. Participants were scanned during both conditions while they drank 5mL volumes of liquid and the pleasantness of this drinking was subsequently rated. The principle finding of the first study was a bilateral increase in primary motor cortex activation when water was swallowed during the over-sated condition relative to the thirsty condition. A difference in mean pleasantness ratings was also found between the thirsty and over-sated conditions, with drinking found to be pleasant during the thirsty condition and unpleasant during the over-sated condition. Finally, between-participant correlations of subjective ratings and brain activity revealed a positive association during the thirsty condition for pleasantness ratings and activity in the orbital frontal cortex (OFC) and the anterior cingulate cortex (ACC), while for the over-sated condition activity in the amygdala (amongst other regions) showed a negative association with unpleasantness ratings. For the second study, twenty healthy participants were again recruited. In this study subjective ratings of “swallowing effort” were added to the existing experimental protocol and brain activity prior to swallowing was examined. A threefold increase in swallowing difficulty was revealed psychometrically for the over-sated condition compared to the thirsty condition, while for the same contrast increased activity was observed in parietal, prefrontal, and motor cortices along with the striatum and thalamus. Between-participant correlations of subjective ratings and brain activity revealed a negative association during the over-sated condition between ratings of swallowing effort and activity in the prefrontal cortex and several nuclei in the pontine region of the brainstem. For the final study, data from the second experiment was reanalyzed and regional brain responses were investigated during a ten second period designated ‘drinking behaviour’, which consisted of a conjunction of pre-swallow and swallow events. Psychophysical interaction (PPI) analyses were also used to investigate the functional connectivity between an anterior mid cingulate cortex (aMCC) ‘seed’ and other brain regions. For the thirsty condition relative to the over-sated condition, greater activation was observed in the aMCC along with several swallowing-related regions. An increase in functional connectivity, relative to a non-drinking baseline, was also observed in the thirsty condition between the aMCC seed and these swallowing-related regions. When the thirsty and over-sated conditions were contrasted during drinking behaviour, a greater increase in functional connectivity compared to the non-drinking baseline was observed in the thirsty condition between the aMCC and two subcortical regions, the cerebellum and the rostroventral medulla, the latter containing nuclei responsible for the swallowing reflex. Finally, during drinking behaviour in the over-sated condition, ratings of swallowing effort showed a negative association with functional connectivity between the aMCC and two cortical regions, the sensorimotor cortex and the supramarginal gyrus. The three empirical chapters in this thesis present several important findings with the potential to inform knowledge of the neural control of fluid intake: the identification of swallowing inhibition following satiation; a disinhibitory counterresponse provided by the prefrontal cortex; and the identification of a brain region (the aMCC) likely to play an important role in regulating fluid intake in response to changes in hydration status. As these outcomes show considerable promise for improving our understanding of pathologicial and non-pathological cases of excess fluid intake, the work presented in this PhD thesis is likely to provide an important empirical foundation for future research in this emerging field.
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    Functional tracing of brain pathways with optogenetics: insights into the hierarchy of sympathetic control
    Pracejus, Natasha Heide ( 2017)
    Brainstem premotor neurons can selectively drive sympathetic neurons of one functional type, but methodological constraints have prevented previous studies from identifying premotor neurons that drive more than one functional type. This project tested whether individual premotor neurons might drive two or more functional outputs using optogenetics, which genetically renders selected neurons light-sensitive. This study found that there are indeed premotor neurons with branched projections capable of exciting more than one functional type of sympathetic neuron.
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    Stem cells for the treatment of neurodegenerative disorders
    Turner, Christopher ( 2016)
    Neurological disorders present a special challenge to medical science, because of their increasing prevalence, and the inability of the brain and spinal cord to repair itself. While the prevention of these conditions is preferable, the reality is that there will most likely always be a heavy dependence on therapies that treat established disease. Cell therapy holds significant promise for the treatment of these disorders, although substantial challenges remain before they can be progressed into mainstream therapies. This thesis explores some of the factors that affect the successful integration of stem cell-derived neural cells into the brain, in a series of experiments presented across four research chapters. Notably, the research has demonstrated that therapeutic neural cells derived from human iPS cells require a period of 12 months after implantation to mature, in a similar manner as what is observed in normal human development. This has implications for preclinical investigations utilising human cells to repair the damaged brain, which may require studies to run for periods of up to 1 year. These studies rely heavily on the transplantation of human therapeutic cells into discordant species, such as rodents and non-human primates. Accordingly, we conducted a systematic evaluation of the ability range of immune-modulating approaches to sustain human xenograft survival in the rat. While robust survival of hESC-derived neural cells was observed in athymic animals, survival of the same cells in immunocompetent adult or neonatal-grafted animals did not exceed 14 weeks. Immunosuppression by pharmaceutical agents resulted in cell-survival beyond 20 weeks, which was associated with a reduction in blood and brain T-cell quantities. These results demonstrate the utility of the athymic rat in xenografting studies, and provide practical information for the design of preclinical studies. When we attempted to utilise pharmacological immunosuppression to bolster grafted-cell survival in the SOD1G93A rat model of motor neuron disease, we observed a significantly detrimental impact associated with this agent on the motor performance of these animals. This highlighted the problem of immunosuppression in models of neurodegeneration, where the disease pathology is influenced by immune-effects. This presents a particular challenge to cell therapies aimed at treating these conditions, and we therefore evaluated the impact of treatment with three commonly used immunosuppressants, cyclosporin A, FK506 and rapamycin, on core disease characteristics in the SOD1G93A rat. Rotarod performance was disrupted by treatment with rapamycin and cyclosporin A, but not by treatment with FK506. The observed impairment occurred despite rapamycin reducing local expansion and recruitment of microglia, and cyclosporin A reducing levels of misfolded SOD1 protein within the spinal cord. In contrast, FK506 appeared to increase astrocyte activation, whilst not impairing behavioural outcomes. This study highlights that immunosuppression to support xenograft survival may directly affect important disease traits in models of neurodegeneration. Thus, the use of immunosuppression in such paradigms should be carefully considered within study design. While the long-term survival of implanted cells is a critical feature of successful stem cell-based therapies for neurodegenerative disorders, understanding the factors that guide the growth and integration of implanted neural cells is equally important. We performed a study that compared the fibre pathways of orthotopically and heterotopically transplanted fetal tissue-, as well as mouse embryonic stem cell- derived neural progenitors, with endogenous fibres of the intact adult murine cortex. Fetal tissue transplanted into the visual and motor cortices projected fibres across the entire dorsoventral axis of the adult brain. Quantification of the innervation of these fibres in specific targets of the cortex did not reveal an overwhelming tendency for grafted cells to target nuclei relevant to their intrinsic identity. While mouse embryonic stem cell-derived neural progenitors survived and expressed markers of mature cortical cells types in vivo, these cells did not demonstrate a high degree of axonal outgrowth. When quantified, there was no substantial difference in the innervation of specific cortical areas when mouse embryonic stem cell-derived neural progenitors were placed in either the motor or visual cortices. This suggests that the innervation patterns of implanted cells cannot be used to assign a particular areal identity to donor cells, as the passive outgrowth of fibres along host white matter tracts cannot be excluded as a possibility. Instead, immunohistochemical analysis of the expression of markers for specific areal identity may assist future studies in this purpose. Taken together, this work enhances our understanding of how certain factors must be managed to ensure the integration of donor cells in preclinical investigations, so that one day these studies can be progressed into therapies that support full patient rehabilitation.
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    ‘Somnivore’ a user-friendly platform for automated scoring and analysis of polysomnography data
    Allocca, Giancarlo ( 2016)
    The low-throughput nature of manual scoring of polysomnography (sleep) data, both in terms of speed and consistency, is a major factor preventing sleep research from reaching its full efficiency and potential. Automated approaches developed previously have generally failed to provide sufficient accuracy or 'usability' for sleep scientists lacking specialist-engineering expertise. Moreover, all earlier approaches have only been validated using baseline data, suggesting a failure to embed in the algorithm the robustness to remain effective when used to analyse the effect on sleep of treatment or disease. Finally, no single approach has been validated for mouse, rat and human data. Therefore, the aim of my research was to develop a user-friendly platform for real-time automated scoring and analysis of polysomnography data. The program is known as ‘Somnivore’ (from Latin somnus, ‘sleep’, and vorare, ‘to devour’), and was developed using state of the art supervised machine learning technology, with support vector machine (SVM) at its core, and coded as a graphical user interface (GUI)-based solution in the Matlab™ ambient. Somnivore learns, in parallel, by surveying features from a variety of different inputs (including EEG, EMG, EOG and ECG) and outputs data into the various sleep stages (wake, NREM, N1, N2, N3, REM). The classifier is trained for each subject via a brief session of manual scoring. Design and development strategies were built around both theoretical and heuristic approaches. This led to a multi-layered system capable of learning from extremely limited training sets, using large input space dimensionalities from a rich variety of polysomnography inputs, and with rapid computational times. Validation was pursued to approach the numerous contentious dynamics that have led to the demise of previous solutions. Somnivore generalisation was evaluated at the level of canonical classifier evaluation metrics such as F-measure, as well as experimental end-measures more germane to traditional biological sleep research. Somnivore, generated superior generalisation, with high power, on both murine (n = 54) and human (n = 52) recordings. These included multiple rat strains (Sprague-Dawley, Wistar) and mouse phenotypes (wild type, orexin neuron-ablated transgenic), various pharmacological interventions (placebo, alcohol, muscimol, caffeine, zolpidem, almorexant), and in humans, both genders, younger and older subjects, and subjects with mild cognitive impairment (MCI). Somnivore’s generalisation was also evaluated in conditions of signal challenged data, and provided excellent performance in all conditions using only one EEG channel for learning. Remarkable results were also reported for learning undertaken using only one EMG channel or two EOG channels. Furthermore, validation studies highlighted that a substantial part of the disagreement between manual and automated hypnograms was located within transition epochs. As Somnivore has several features geared towards the management of transition epochs, further control over generalisation is also possible. Comprehensive inter-scorer agreement analysis was conducted on human data, showcasing how inter-scorer agreement between manual hypnograms and their automated counterparts provided by Somnivore is comparable to the gold-standard of the inter-scorer agreement between two experts trained in the same laboratory. Results also highlighted critical problems within the scoring of stage N1. However, inter-scorer agreement validation studies also confirmed what has already been reported in the literature, that N1 is a volatile stage that systematically produces inadequate agreement even between trained experts, both within or outside the same laboratory. Accordingly, Somnivore performed as well on N1 as reported in the literature for manually scored data. Due to the high-throughput nature of Somnivore’s analyses of experimental end-measures, several novel, cautionary findings were extracted from the recordings provided by external laboratories for this research evaluations. Additionally, as Somnivore is also capable of scoring real-time during polysomnography recordings, it will facilitate the development of more advanced protocols such as biofeedback sleep-deprivation protocols and integrated optogenetics. In conclusion, Somnivore, has been comprehensively validated as an accurate, reliable, high-throughput solution for scoring and analysis of polysomnography data, in a range of experimental situations including studies of normal physiology and tests related to drug discovery for the improved treatment of sleep disorders and psychiatric diseases.
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    Prefrontal dopaminergic mechanisms of adolescent cue extinction learning
    Zbukvic, Isabel ( 2016)
    Addiction and anxiety disorders represent the most prevalent mental illnesses in young people worldwide. Unfortunately, adolescents attain poorer outcomes following extinction-based treatment for these disorders compared to adults. Cue extinction learning involves dopamine signaling via the dopamine 1 receptor (D1R) and dopamine 2 receptor (D2R) in the medial prefrontal cortex. In particular, the infralimbic cortex, a subregion of the medial prefrontal cortex, has been implicated in extinction learning in both adolescent and adult rodents. The prefrontal dopamine system changes dramatically during adolescence. However, the role of prefrontal dopamine in adolescent cue extinction learning is poorly understood. Therefore, this thesis aimed to elucidate the role of prefrontal dopamine in adolescent cue extinction, using cocaine self-administration and fear conditioning in rats. My first study examined cocaine self-administration and cocaine-associated cue extinction in adolescent versus adult rats. Adolescents displayed a deficit in cocaine-cue extinction learning compared to adults (postnatal day [P]53 and P88 on cue extinction day, respectively). A single infusion of the full D2R agonist quinpirole into the infralimbic cortex prior to extinction enhanced adolescent cue extinction to reduce relapse-like behavior the next day. This effect was recapitulated by a systemic injection of the partial D2R agonist aripiprazole, an FDA-approved drug for the treatment of psychosis with strong translational potential. My second study examined fear conditioning and extinction in adolescent and adult rats. I first aimed to optimize behavior in late adolescent (P53) and adult (P88) rats during the dark phase of their 12-hour light-dark cycle, to remain consistent with conditions of the previous chapter. However, this produced unreliable behavioral results. In contrast, adolescent rats (P35) consistently display a deficit in long-term fear extinction compared to adults (P88) during the light phase. Infusion of the D1R agonist SKF-81297 into the infralimbic cortex prior to fear extinction had no effect for either age group. However, infusion of quinpirole into the infralimbic cortex significantly enhanced long-term fear extinction in adolescents, whereas it delayed within-session extinction in adults. Interestingly, an acute systemic injection of aripiprazole improved long-term fear extinction in adults. My final experiments measured prefrontal gene expression for D1R, D2R, and D1R relative to D2R (D1R/D2R ratio) in naïve rats across adolescent development, or following cocaine-cue, or fear extinction. There were no significant differences in prefrontal dopamine receptor gene expression across naïve rats age P35, P53, and P88. Following cocaine-cue extinction, prefrontal D1R gene expression was upregulated in adults but not adolescents. By comparison, following fear conditioning, adolescents showed higher D1R and D1R/D2R ratio gene expression compared to adults. D1R/D2R ratio was modulated in opposite directions following fear extinction learning during adolescence versus adulthood. These findings show that adolescents are impaired in extinction of emotionally salient cues across both appetitive (drug) and aversive (fear) learning domains. Functional and molecular data provide novel evidence for divergent involvement of prefrontal dopamine in cue extinction learning across adolescent development. Results not only extend understandings of extinction learning in general, but represent an exciting step towards finding new therapeutic targets to facilitate exposure-based therapy in the clinic.
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    The therapeutic effects of N-acetylcysteine in a mouse model of Huntington’s disease
    Wright, Dean ( 2016)
    Huntington's disease (HD) is a neurodegenerative disorder, involving psychiatric, cognitive and motor symptoms, caused by a CAG-repeat expansion encoding an extended polyglutamine tract in the huntingtin protein. Oxidative stress and glutamate pathology have been implicated as key factors in the pathogenesis of HD. The cysteine pro-drug, N-acetylcysteine (NAC) has the potential to alleviate both oxidative stress and glutamate pathology. NAC aids in the production of the primary central nervous system antioxidant, glutathione (GSH), and also provides cystine, a substrate for the glutamate transporters, system xc- and GLT-1. The aim of this thesis was to establish whether cysteine supplementation, using NAC, could ameliorate oxidative stress and glutamate pathology, and subsequently improve motor and behavioural outcomes in HD. The R6/1 transgenic mouse model of HD was used to investigate the effects of NAC on HD pathology. Two pilot cohorts were conducted on both male and female HD mice, assessing a variety of tests looking at motor, cognitive, affective and ethologically relevant phenotypes. NAC appeared to have it strongest effects on motor and depressive-like behaviours in male HD mice. These behaviours were then assayed in well-powered cohorts. It was found that chronic NAC administration delayed the onset and progression of motor deficits in male HD mice as well as reducing forced swim immobility in both WT and HD mice. These behavioural changes were associated with reduced oxidative stress and improved function in mitochondria isolated from the HD striatum. Reductions in GLT-1 protein were found in the HD brain, but these were not affected by NAC treatment. In female HD mice, both acute and chronic NAC treatment was able to reduce depressive-like behaviour in the forced swim-test. These antidepressant-like effects were blocked by co-administration of inhibitors of system xc- and GLT-1 (CPG and DHK). Using in vivo microdialysis, female HD mice were found to have a baseline decrease in extracellular cystine. By donating cysteine with NAC, extracellular glutamate was transiently increased. CPG and DHK were able to block the NAC-induced spike in glutamate, indicating that NAC is working by ameliorating deficits in cystine, restoring glutamate homeostasis through system xc- and GLT-1. These in vivo changes reflect changes in glutamate transporter protein in HD mice and human HD post-mortem tissue. Furthermore, NAC was able to rescue changes in key glutamate receptor proteins related to excitotoxicity in HD, including NMDAR2B. Thus, we have shown that baseline reductions in cysteine underlie glutamatergic dysfunction and depressive-like behaviour in HD and these changes can be rescued by treatment with NAC. Furthermore, chronic NAC administration was able to ameliorate downstream effects on mitochondrial function and subsequent oxidative stress, and also delay the onset of core motor symptoms. These findings have elucidated a novel mechanism by which glutamate-pathologies may be acting in HD, providing rationale for the trial of treatments supplementing cysteine, including the FDA-approved drug, NAC.
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    Relaxin-3/RXFP3 system in alcohol self-administration and relapse
    Kastman, Hanna Erika ( 2016)
    Alcoholism is a chronic relapsing disorder, accounting for 10% of disability-adjusted life years lost in industrialized countries. Our understanding of the neurobiology of addiction is far from complete and due to high relapse rates, there is a need to identify new therapeutic targets to assist the development of better treatments. In early studies, the neuropeptide relaxin-3 was implicated in the regulation of stress responses and arousal/motivational behaviours such as feeding, as well as spatial memory. Relaxin-3 is primarily expressed in large GABAergic neurons of the nucleus incertus (NI) in the hindbrain that project topographically to forebrain regions containing neurons expressing the native relaxin-3 G-protein-coupled receptor, RXFP3, several of which are implicated in the control of drug-seeking behaviour. Therefore, the present study directly investigated the role of the central relaxin-3/RXFP3 signalling system in alcohol- and sucrose-seeking in alcohol-preferring (iP) and Wistar rats. Firstly, the effect of central antagonism of RXFP3 using a receptor-selective relaxin-3 analogue peptide on self-administration of alcohol and sucrose was investigated; and then the effect of the same antagonist treatment on cue- and stress-induced reinstatement of both sucrose- and alcohol-seeking was studied. Thereafter, the impact of RXFP3 antagonism within the stress-related bed nucleus of stria terminalis (BNST) on stress-induced reinstatement of alcohol-seeking was also examined. Central antagonism of RXFP3 reduced cue- and stress-induced reinstatement of alcohol-seeking, but did not markedly alter sucrose-seeking in either paradigm, suggesting a specific effect on alcohol-related behaviour(s). Alcohol consumption and stress-induced reinstatement were also both attenuated by local RXFP3 antagonism within the BNST. These data are the first identifying a role for relaxin-3/RXFP3 signalling in alcohol use and seeking. In light of these initial findings and the enrichment of RXFP3 in other stress-related brain areas, the ability of RXFP3 antagonism within the central amygdala (CeA) to modulate alcohol self-administration and stress-induced reinstatement of alcohol-seeking was also examined. Indeed, RXFP3 antagonism in CeA also reduced alcohol self-administration and stress-induced reinstatement of alcohol-seeking, further indicating an involvement of native relaxin-3/RXFP3 signalling in stress-induced reinstatement and potential interactions with other major peptide transmitter systems implicated in these behaviours, such as corticotropin-releasing factor (CRF) and orexin. NI relaxin-3 neurons express the CRF type 1 receptor (CRF1) and are activated by CRF; likewise there is anatomical evidence for the expression of orexin-1 (OX1) and orexin-2 (OX2) receptors in the NI. Accordingly, the effect of bilateral NI infusions of the CRF1receptor antagonist (CP376395), the OX1 receptor antagonist (SB-334867) and the OX2 receptor antagonist (TCS-OX2-29) on stress-induced reinstatement of alcohol-seeking was examined. CP376395 and TCS-OX2-29 reduced stress-induced reinstatement of alcohol-seeking, whereas SB-334867 was ineffective. These data suggest that CRF- and orexin-mediated activation of NI neurons occurs during stress-induced reinstatement, via CRF1 and OX2 receptor signalling events. Together, these data demonstrate that the relaxin-3/RXFP3 system can modulate stress-induced reinstatement of alcohol-seeking via both forebrain sites and a potential CRF- and orexin-primed activation of the NI during stress exposure. These findings have added significantly to our knowledge of the neurocircuitry that underpins stress-induced relapse-like behaviour and to our general understanding of addictive behaviours, with implications for the development of better treatment strategies and the identification of potential drug targets for treating alcoholism and other stress-related addictions.