Florey Department of Neuroscience and Mental Health - Theses
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The modulation of cortical dendrites during sensory perception and learning
Sensory perception arises in the cortex by integrating external information from the environment with internal representations and the current brain state. This process is supported by the structure of the neocortex and the organisation of excitatory inputs onto its core computational element: the pyramidal neuron. In layer 2/3 pyramidal neurons, external (feedforward) information mainly target the somatic region, while internal (feedback) information runs through layer 1 and lands onto distal tuft dendrites. Cortical dendrites have active properties that could be important for sensory processing and could provide a cellular mechanism to support the flexibility of sensory representations during learning. In this thesis, I addressed the role of L2/3 pyramidal neuron dendrites during sensory perception and learning in three parallel studies that investigated: 1) how inputs from another sensory area affect early stages of sensory processing; 2) the modulation of sensory processing in dendrites of the auditory cortex following fear conditioning; 3) changes in dendritic activity during perceptual learning of an auditory discrimination task. To tackle these questions, a combination of two photon Ca2+ imaging and whole cell patch clamp electrophysiology in vivo, together with behavioral testing and optogenetics manipulation, was used. The results presented here confirm that dendrites encode sensory information and show that they can undergo plastic changes during learning. The findings also illustrate the existence of compartmentalised activity in L2/3 pyramidal neurons and of a cellular mechanism for the control of action potential generation that involve dendritic integration. The results presented in this thesis highlight the importance of the upper layers of the cortex for flexible sensory representation through the integration of feedback and feedforward information.
Remyelination in the aging brain and the effects of growth factor supplementation on oligodendrogenesis
In multiple sclerosis (MS), chronic demyelination initiated by immune-mediated destruction of myelin, leads to axonal damage and neuronal cell death, resulting in a progressive decline in neurological function. The development of interventions that potentiate remyelination could hold promise as a novel treatment strategy for MS. Seminal work in the Merson group has demonstrated that neural precursor cells (NPCs) residing in the subventricular zone (SVZ) of the adult mouse brain contribute significantly to remyelination in response to CNS demyelination and can regenerate myelin of normal thickness. However, aging takes its toll on the regenerative potential of NPCs and reduces their contribution to remyelination. In this study, I investigated how aging could affect the NPCs contribution to oligodendrogenesis during the remyelination process and whether the delivery of growth factors into the brains of aged mice could potentiate the oligodendrogenic potential of NPCs. To map the fate of NPCs in response to demyelination induced at different postnatal ages, Nestin-CreERT2; Rosa26-LSL-eYFP mice were gavaged with tamoxifen at either 8 weeks, 30 weeks or one year of age before being challenged with cuprizone for a period of six weeks. Using osmotic minipumps, I infused epidermal growth factor (EGF) and/or heparin-binding EGF-like growth factor (HB-EGF) or artificial cerebrospinal fluid (vehicle) into the cisterna magna for a period of two weeks beginning at the peak of cuprizone-induced demyelination (n=6-8 mice per group). Mice were perfused six weeks after cuprizone withdrawal and the contribution of NPCs to oligodendrocyte regeneration in the corpus callosum was assessed. Our data reveal that although NPC-derived oligodendrocyte generation declined dramatically with aging, this decline was at least partially reversed by growth factor infusion. Notably, co-infusion of EGF and HB-EGF increased oligodendrocyte regeneration by two-fold in some regions of the corpus callosum. The results of this study demonstrate the beneficial effects of EGF and HB-EGF for increasing the contribution of NPCs to oligodendrogenesis during the remyelination process and indicate that modulation of progenitor responses could hold therapeutic potential for combating the negative effects of aging upon remyelination efficacy.
Age related dopamine 1 and dopamine 2 receptor expression in addiction-related behaviours
Methamphetamine (meth) is a significant social and public health concern worldwide, and a growing problem in Australia. One factor contributing to meth use disorder is the lasting memory of its rewarding experience, which can lead to persistent use in vulnerable individuals. Epidemiological data show that adolescence is a period of heightened vulnerability for developing meth use disorder. Furthermore, sex differences exist in numerous aspects of meth use motivations, behaviours, and consequences. Despite this, few studies have investigated age and sex effects of meth on brain and behaviour. A unified neural mechanism by which substances of abuse, including meth, produce their addictive properties is by increasing dopaminergic transmission throughout the mesocorticolimbic dopamine system. Although dopamine binds to and activates several subclasses of receptor in brain regions implicated in reward processing, the dopamine receptors 1 (D1) and 2 (D2) have been reported to be particularly important for drug-affected behaviour. Importantly, levels of D1 and D2 have been shown to fluctuate throughout development. D1 and D2 signalling may therefore be important mediators of adolescent vulnerability to substance use. However, there are numerous inconsistencies in the literature that describe developmental changes in D1 and D2 expression. Systematic characterisation of these changes is therefore critical for a full understanding of how changes to the dopamine system may affect susceptibility to meth (and other substance) use disorder. As such, the first aim of my thesis was to investigate the postnatal developmental trajectory of D1 and D2 in addiction-related brain regions. This was achieved using D1- and D2-green fluorescent protein (GFP) transgenic mice, starting from the juvenile period through to adulthood. The results showed region-specific changes in D1 and D2 expression occur across development, with the insular cortex (insula) showing the most dramatic changes. In particular, the density of D1 compared to D2 expressing neurons (D1:D2 ratio) in the insula substantially increased from adolescence to adulthood in males. Since substance use disorders are male dominant disorders which often have an adolescent onset, this reduced D1:D2 ratio may be relevant in understanding the neurobiological basis of substance use disorders. The second aim of my thesis was to investigate the role of insula D1 and D2 in potential age and sex differences in meth conditioned place preference (CPP) and aversion (CPA). Although no age or sex differences were observed in CPP to 0.1 or 3 mg/kg meth at a group level, analysis of individual data revealed females were less likely to form a place aversion compared to males, and adolescents formed more of a place preference to 0.1 mg/kg meth compared to adults. Conditioning with 3 mg/kg meth led to age differences in insula D1:D2 ratio in males, reduced age differences in insula D1:D2 ratio in females, and increased the activation of insula D2 expressing cells in adults compared to adolescents. Insula activity and expression of D1 and D2 did not correlate with place preference behaviour. Taken together, these findings suggest distinct sex differences in D1 and D2 expression across development in addiction-related cortical and striatal brain regions may underly age- and sex-associated vulnerability to meth-related behaviours.
Space for recovery after stroke: Exploring the role of the physical environment in inpatient rehabilitation facilities
The aim of this thesis was to explore the role of the physical environment of inpatient rehabilitation facilities in stroke recovery. The purpose of rehabilitation is to help stroke survivors to re-learn skills and abilities lost as a result of stroke, or to learn new skills to adapt to their changed condition. Research in other healthcare environments suggests that hospital design can impact patient outcomes, but there is little evidence specific to rehabilitation. This thesis embraces the complexity of rehabilitation environments and explores the physical environment as an essential and integrated component of this complex system. This exploration began with a scoping survey to identify and describe all inpatient rehabilitation facilities in Victoria, Australia. This survey revealed 64 facilities, most of which had not been purpose-built for rehabilitation. Rehabilitation facility design appears to be influenced by evidence from acute medical settings and current design trends, rather than reflecting the unique purpose of rehabilitation. A series of expert elicitation workshops were then conducted to define – for the first time – what is important in the physical environment of inpatient stroke rehabilitation facilities. Thirty experts participated, including policy makers, researchers and designers in learning and healthcare environments, clinical staff, and patients. A Value-Focused Thinking methodology was used to facilitate the workshops. The experts defined 16 criteria thought to be fundamentally important (including efficiency, patient practice, activity and rest, emotional well-being, and safety), and 14 criteria that could be a means to achieving these fundamentally important things. Together, these criteria comprise a framework which can be used to guide research and design in this complex area. This framework informed a multiple-case study in two stroke inpatient rehabilitation facilities. Convergent mixed-methods were used to produce a rich and thorough exploration of the cases. Twenty inpatients from Case 1 participated, and 16 from Case 2. The physical environment was described using field notes, photographs, floor plans, and checklists. Walk-through semi-structured interviews were used to explore patients’ experience of the physical environment. Systematic observation (behavioural mapping) and questionnaires were used to investigate patients’ behaviour and emotional well-being in the environment, and a retrospective audit of patient falls was conducted to investigate patient safety. Four interrelated themes described the patient experience: 1) entrapment and escape; 2) power, dependency, and identity in an institutional environment; 3) the rehabilitation facility is a shared space; and 4) the environment should be legible and patient-centred. Quantitative data revealed that patients spent over 75% of their time in their bedrooms. Comparison between cases suggested that the physical environment played a role in patients’ behaviour, emotional well-being, and safety. Qualitative and quantitative findings were then merged using joint display tables and narrative integration. This robust analytic process produced a new conceptual model of the role of the physical environment in stroke patients’ behaviour, emotional well-being, and safety in rehabilitation, emphasising the importance of variety and interest in the environment, privacy without isolation, and patient-centred design. The findings from this study provide meaningful direction for rethinking rehabilitation facilities and guiding real-world health design practice.
Sex differences in fear learning in juvenile rats
The present thesis examined the sex differences in fear learning in the developing rat. Traditionally, it has been widely assumed that pre-pubertal sex differences are negligible in developmental studies. However, epidemiological studies and sexual dimorphism in the brain prior to puberty, indicate that sex differences in fear learning may emerge early in life. In chapter 2, I assessed renewal, reinstatement and spontaneous recovery of extinguished fear (i.e. relapse of extinguished fear) in juvenile male and female rats. I found that P18 females showed all three fear relapse behaviors while P18 males did not. This finding implied that P18 female rats were able to form the contextual memories while P18 males could not. However, whether the ability to form a context-shock association contributes to fear extinction in juvenile rats, has not been explicitly tested. Therefore in chapter 3a and 3b, I directly compared renewal with context fear, using identical fear conditioning parameters in the developing rats. In chapter 3a, I found that P18 male rats did not show renewal while P25 male rats did. P18 and P25 male rats displayed comparable contextual freezing immediately after conditioning and after 24 hours delay. In chapter 3b, both P18 and P25 female rats displayed renewal. I also found that P18 female rats displayed a developmental deficit in context fear learning compared to P25 female rats. Together, findings from chapter 2, 3a and 3b strongly suggested that the emergence of context memory may be sex-dependent, where context-specific extinction emerges earlier in females while context fear learning emerges first in males. Given the critical role of the hippocampus in context-dependent learning, I thought the sex differences in the hippocampal function may underlie the observed sex differences in fear learning. In chapter 4, I examined the role of dHPC and vHPC in fear extinction in P18 male and female rats. I showed that temporary inactivation of the dHPC prior to extinction accelerated extinction acquisition in both male and female rats. I also showed that pre extinction inactivation of the vHPC reduced freezing during extinction and impaired extinction recall, regardless of sex. Collectively, these findings strongly suggest sexually dimorphism in fear learning emerges early in life and emphasize the importance of sex as a factor in the field of developmental learning and memory.
Improving the safety and function of pluripotent stem cell-derived neural transplants for the treatment of Parkinson's disease
Clinical trials using fetal tissue have provided the necessary proof-of-principle evidence that transplanted dopamine neurons can appropriately integrate into the brain and alleviate motor symptoms in Parkinson’s disease patients for > 20 years. Pluripotent stem cells (PSCs), are now being pursued as an alternative cell source, circumventing ethical and availability issues, given their competent self-renewal and differentiation capabilities. Despite rapid progress in the field over the past decade, and recent advancement of these cells into clinical trials (Japan October 2018), we recognise a number of short-comings that require further attention. Despite the existence of efficient protocols for the directed differentiation of human PSC into ventral midbrain (VM) progenitors (i.e. cells capable of maturing into dopamine neurons and amenable to transplantation), a small proportion (10%) of cells in culture remain incorrectly specified. These cells are capable of significant expansion after transplantation, such that grafts commonly contain <5% dopamine neurons when examined after many months. These highly proliferative cells not only present an evident risk of neural overgrowth, but there also remains little knowledge of the identity these cells become and the impact they may have on the graft and patient. For these reasons, strategies have been pursued to improve the purity of donor tissue for grafting. However, to date, cell sorting approaches, to select for correctly patterned human PSC-derived cells prior to implantation have been suboptimal. In chapter 3 of the thesis we employ two human PSC reporter lines to demonstrate that the isolation of vm progenitors, but not vm precursors, results in viable cell grafts that are more predictable in their composition, retain integration and functional capacity to restore motor deficits in Parkinsonian rats, and importantly eliminate highly proliferative cells and populations known to contribute to graft-induced dyskinesias. An alternative safeguarding approach for transplantation studies is to employ suicide gene therapy – targeted at eliminating unwanted cells after transplantation. In Chapter 4, we employ a human PSC line carrying a suicide gene (thymidine kinase) that can be activated by administration of a prodrug (ganciclovir), to enable remote killing of proliferating cells within the transplant. We demonstrate that the timely activation of this ‘suicide switch’ can prevent excessive expansion of undesirable cells in the graft whilst preserving DA integrity - yet surprisingly, a small proportion of persistent dividing cells remained. We speculate that this was a result of suboptimal delivery of ganciclovir caused by insufficient vascularisation of dopamine grafts. In a third approach to improve graft outcomes, we address the impact of human PSC-derived VM progenitor donor age (Chapter 5). Despite historical efforts to understand the impact of different aged VM fetal tissue on graft survival, dopamine contribution and functional integration, there remains no comparable assessment of human PSC-derived vm progenitor age. While the current clinical trial in Japan has elected to use late stage human PSC-derived VM progenitors, in Chapter 5 we surprisingly show that VM-specified early-stage progenitors generated the most homogeneous DA grafts, containing a considerably lower component of unwanted, off-target cells than grafts derived from older progenitors. Importantly we demonstrated that while this effect was extremely consistent within human PSC lines, considerable inter-line variability was evident, highlighting the need for rigorous assessment on a line to line basis, prior to translation. In summary, the work provided within this thesis provides significant new insight into strategies to standardize human PSC-derived transplantation for Parkinson’s disease – importantly addressing the need to consider graft survival, proportion of dopamine neurons and their function, as well as the critical requirement to eliminate unwanted cell types to ensure maximal safety.
Studies on the mechanism of binding and activation of relaxin family peptide receptors
The peptide hormone relaxin is involved in reproductive processes but has also been investigated for several decades as a treatment for a range of disease states such as scleroderma, acute heart failure, and fibrotic conditions. The receptor for relaxin, RXFP1, is an integral membrane protein belonging to the G protein coupled receptor (GPCR) family. RXFP1 is therefore a therapeutically tractable target for which a thorough understanding of its mechanism of binding and activation is required to develop better relaxin-like drugs. The aims of these studies are to investigate the mechanism by which relaxin binds and activates RXFP1 using a variety of molecular pharmacology approaches in a HEK293T cell model system recombinantly expressing RXFP1 in various forms. Specifically, a hypothesis was tested that a homodimer of RXFP1 might be the minimal functional unit required for receptor activation. GPCR dimers are postulated to interact via their transmembrane helices, so initial investigations aimed to disrupt RXFP1 homodimerisation by incorporation of peptides representing single transmembrane segments of RXFP1 as well as recombinant expression of RXFP1 transmembrane domains. There was no evidence that RXFP1 homodimerisation is required for receptor activation. Following this, the evidence for RXFP1 homodimerisation was re-evaluated in the development of two methods which utilise principles of Bioluminescence Resonance Energy Transfer (BRET). Firstly, split Nanoluciferase was used to tag cell surface localised RXFP1 receptors in combination with mCitrine-tagged RXFP1 and BRET was measured to assess relative receptor proximity. This indicated that RXFP1 is unlikely to be a stable homodimer, intracellularly localised receptors predominate, and there is no change in receptor:receptor proximity upon relaxin stimulation. Secondly, Nanoluciferase-tagged RXFP1 receptors were used in combination with fluorescently labelled relaxin and BRET was measured to track relaxin:RXFP1 binding interactions. This allowed sensitive, real time measurements of the relaxin:RXFP1 binding interactions, demonstrating a multi-step mechanism of relaxin binding in which the linker domain of RXFP1 is critical for high-affinity interactions. Furthermore, there was no evidence of negative co-operativity of relaxin binding, contrary to previous reports which were used as evidence of RXFP1 homodimerisation. Overall, these studies indicate that relaxin does not activate RXFP1 via a mechanism involving a receptor homodimer. Several molecular tools were developed which will be useful for future investigations into RXFP1 pharmacology. This work adds incremental detail to the understanding of how relaxin activates RXFP1, hopefully leading to the development of novel therapeutically useful relaxin-like molecules in future.
Early gene expression profiling of motor neurons in the SOD1G93A mouse model of amyotrophic lateral sclerosis: insights into earliest pathogenesis
Although amyotrophic lateral sclerosis (ALS) typically presents in mid to late life, there is increasing evidence in patients and mouse models for a protracted preclinical period of motor neuron vulnerability and damage before clinical onset. We hypothesized that the seeds for the development of ALS may be sown shortly after conception and motor neuron vulnerability is induced in the perinatal period of life, leading to subsequent neuronal dysfunction and degeneration. The goal of this study was to identify the earliest gene expression patterns in the vulnerable lower motor neurons at very early and key developmental ages in the superoxide dismutase 1 (SOD1)G93A mouse model of ALS. We have implemented and fully characterised HB9:GFP reporter mouse to unambiguously identify and isolate the spinal alpha motor neurons for transcriptomic profiling using RNA sequencing, pathway analysis and target validation. Isolation of HB9:GFP+ spinal motor neurons using FACS was employed at embryonic day 12.5 (E12.5), E17.5, postnatal day 3 (P3) and P8 in SOD1G93A mice and control littermates. Purification of enriched motor neurons was only successful from E12.5 mice. At E17.5, P3 and P8 samples were enriched in glial cells. Gene expression profile of HB9:GFP+ spinal motor neurons from E12.5 SOD1G93A mice revealed significant dysregulation of RNA processing genes, consistent with key pathological pathways in ALS. Dysregulation of Gria2, an AMPA receptor subunit gene, implicated in Ca2+ mediated excitotoxicity was confirmed by real-time qPCR and immunohistochemical analyses in motor neurons from embryonic SOD1G93A mice and induced pluripotent stem cell lines of ALS patient carrying SOD1 mutations. We propose that dysregulation of the AMPA receptor may lead to mitochondrial pathology induced by increased Ca2+ influx and the intracellular increase of free radicals which confers an early susceptibility of spinal motor neurons to excitotoxicity in ALS. In summary, this study provides the first insights into gene expression profiles of motor neurons from SOD1G93A mice in utero. Importantly, our data identified dysregulation of RNA processing and the AMPA-mediated excitotoxicity receptor subunit as early as E12.5 which may account for an early, and likely a causal event in triggering selective motor neuron vulnerability in ALS, arguing for administration of early interventions using anti-excitotoxic therapeutic approaches in ALS.
Understanding and promoting the functional integration of neural transplants in Parkinson’s disease
Clinical trials have provided evidence that the transplantation of new dopamine neurons (DAn) into the striatum of Parkinson’s disease (PD) patients can survive, integrate and restore motor function. Demonstrated first using fetal donor tissue isolated from the developing ventral midbrain (VM), the procedure was hindered by challenges of poor tissue standardisation, limited availability and ethical considerations. Recently, studies have seen a rapid advancement in the use of human pluripotent stem cells (hPSC), differentiated into dopamine progenitors, as a viable alternative and standardized donor source. Despite recently advancing to clinical trials, several questions remain regarding the safety, predictability and efficacy of these cells. While fetal tissue has an estimated 20% cell survival and just 10% of cells within the graft are DA neurons, hPSC-derived neural transplants are notably poorer – remaining highly heterogeneous with a high proportion of non-dopaminergic cells and display inferior reinnervation of target tissues compared to their fetal counterpart. Such observations suggest that a more conducive environment, reminiscent of the developing brain, to support newly integrate fetal and hPSC-DA progenitors could improve the differentiation and functional capacity of neural transplants for PD. The present thesis had focused on improving the host environment into which cells are delivered by using a tissue-specific hydrogel/biomaterial to mimic the extracellular matrix (ECM). The employed biomaterial was capable of signalling to the cells by presenting a high density of a laminin epitope (the main brain ECM component) capable of influencing not only cell adhesion but also survival, differentiation and plasticity. In addition, the biomaterial was utilised to sustain the delivery of trophic proteins, absent in adult brain. This thesis demonstrated the synergistic capacity of the tissue-specific hydrogel, sustaining the delivery of stromal derived factor1 (SDF1) or glial cell-derived neurotrophic factor (GDNF) to enhance grafting outcome for rodent fetal tissue grafts and human pluripotent stem cell derived grafts, respectively, in rodent models of PD. Finding demonstrated these functionalised scaffolds promoted graft survival, improved the specification of A9 dopaminergic neurons (the subpopulation of DA neurons responsible influencing motor function) and enhance plasticity – resulting in restoration of motor deficits. Recognising the heterogeneity of hPSC-derived VM grafts to date, yet with little knowledge of the cellular composition, the work within this thesis also developed a new method to rapidly and efficiently transcriptionally profile xenografts. The method employs bulk tissue dissection, inclusive of the graft and surrounding host tissue, and utilises differences in the RNA sequences between the species to discriminate the xenograft from host gene expression -using either real-time qPCR (qPCR) or whole RNA sequencing (RNAseq). The technique is validated and demonstrated by assessing and comparing the composition of undifferentiated hPSC grafts/teratomas to hPSC-derived VM progenitor grafts in the rodent brain. The approaches enable identification of known and novel genes and provides the first complete characterisation of human VM grafts to date. Collectively, this thesis provides new insight into improving the functional outcomes of VM progenitors’ grafts, as well as a means to screen grafts for both safety and predictable efficacy. Such knowledge will be of critical important as these cells continue to move into clinical trials.
A platform for analysis of in vitro neuronal networks for the development of precision therapeutics in SCN2A disease
Background and Purpose Developmental and epileptic encephalopathies are a group of devastating neurological disorders in which the patients have developmental impairment as well as refractory seizures. Comorbid states are common and include cognitive and movement disorders. SCN2A, which encodes the brain sodium channel Nav1.2, has emerged as one of the most prominent developmental and epileptic encephalopathy genes. Based on the onset of disease, patients with SCN2A epilepsy variants can be divided into two major groups. In the early onset group, seizures start within the first three months of life, whereas in the second group, the onset is after three months of age. Sodium channel blockers such as phenytoin are effective in some of the early onset patients. In contrast phenytoin is ineffective and may in fact worsen seizure outcomes in late onset disease. This suggests different molecular pathomechanisms. The lack of efficacious therapies underscores an urgent need for novel treatment strategies. Experimental Approach Two knock-in mouse lines were generated carrying Scn2a p.R1883Q and p.R854Q variants corresponding to human SCN2A p.R1882Q and p.R853Q variants. These are the most common recurrent variants found in the early and the late onset group of SCN2A developmental and epileptic encephalopathies, respectively. In vitro signatures of neuronal network behaviour were assessed using multi-electrode array analysis of the primary cortical cultures obtained from postnatal day 0-1 animals carrying the respective variants up to 28 days in vitro. Acute pharmacological effects were evaluated around 22 days in vitro. Key Results After 2-4 weeks network analysis in culture showed increased activity for neurons harbouring the heterozygous p.R1882Q variant associated with early onset disease. Conversely, a decreased firing rate was observed in cultures in which neurons carried the heterozygous p.R853Q variant associated with late-onset disease. The excitability in both cultures was reduced by phenytoin, which resulted in shifting the p.R1882Q in vitro phenotype towards the wild types and p.R853Q away from the wild types, consistent with clinical observations. Interestingly, one of the tested antiepileptic drugs changed the activity of both cultures was towards the wild type phenotype indicating potential benefits of this drug for both early and late onset SCN2A developmental and epileptic encephalopathies. Conclusion and Implications The assumption that early onset SCN2A variants are more likely to cause gain-of-function and the late onset SCN2A variants to a loss-of-function of the Nav1.2 channel was confirmed for the two studied variants using in vitro neuronal cultures. Moreover, the clinical observations regarding the effectiveness of the sodium channel blocker phenytoin in patients with early and late onset seizures was corroborated by our in vitro models. Lastly, an antiepileptic drug was identified as a potential treatment for both early and late onset SCN2A developmental and epileptic encephalopathies.
The role of the higher order thalamus during goal-directed behavior and sensory processing
The integration and interpretation of sensorial information is a fundamental requirement for cognition. The thalamus is the sensory gate to the cortex. With its widespread cortical and subcortical connectivity, the thalamus has the network capabilities to function as an integrator of sensory relevant information necessary to drive behavior. Higher order regions of the thalamus send extensive projections to the cortex which exert a powerful influence on cortical encoding of sensory information. However, despite both anatomical and functional evidence, this aspect of thalamic functions has remained largely overlooked likely due to the complexity of investigating thalamocortical circuits in behaving animals. The posteromedial nucleus of the thalamus (POm) is the higher order thalamic nucleus mediating somatosensation. Studies have shown that the POm can modulate sensory processing of cortical pyramidal neurons in the primary somatosensory cortex and drive the cortical plasticity thought to underlie perceptual learning. Despite these reports however, the extent to which POm can influence sensory processing remains unclear and little is known about the role of the POm during sensory-based behavior, let alone goal- directed behavior. In this thesis, I aimed to investigate the effect of POm modulatory input on the primary somatosensory cortex during sensory encoding and goal-directed behavior and determine how this thalamic influence contributes to correct behavioral performance. To achieve this, I used a combination of patch clamp electrophysiology, optogenetics and two-photon Ca2+ imaging of POm axonal projections in the forepaw area of the primary somatosensory cortex (forepaw S1) in anaesthetized and behaving mice trained to perform a sensory based goal-directed task. The work presented in this thesis demonstrates that the activity of POm axonal projections within forepaw S1 encodes correct goal-directed active behavior and dynamically shifts according to task requirements and brain state. In addition, by activating cortical pyramidal neurons either directly or indirectly (through GABAergic interneurons), we show that POm input can both enhance or suppress sensory responses, exerting a powerful modulatory influence over cortical sensory processing. These findings expand the known roles of the higher-order-somatosensory thalamus, from sensory encoding to action selection and flexible switching. Overall, the thalamus is not a simple relay system but mediates complex cognitive functions which are crucial to guide behavior.
Exploring potential mechanisms of environmental factors in mediating risks for schizophrenia: immune and EGF system interactions
Schizophrenia is a severe neuropsychiatric disorder with unknown aetiology, however, environmental stressors that operate early in life are proposed to increase the vulnerability to schizophrenia, but how they act is unknown. One mechanism may be that the diverse environmental risk factors converge on stress-immune pathways and the resultant immune activation perturbs growth factor systems, with consequential neurodevelopmental changes that increase vulnerability to schizophrenia. This research examined the epidermal growth factor (EGF) system, a key growth factor system, the immune system and potential interactions between them in schizophrenia to determine a plausible pathological mechanism. In a targeted candidate gene and pathway approach, expression of >200 selected EGF and immune system genes and the relevant signalling pathways were studied in microarray data derived from BA9 and BA46 of dorsolateral prefrontal cortex from schizophrenia, mood disorder and healthy control subjects. In BA46 and BA11 (orbitofrontal cortex), mRNA of >100 relevant genes, and protein levels of eight molecules were quantified. To determine EGF and immune system changes in association with environmental insult, six markers were protein quantified in brain of spiny mice offspring exposed to maternal immune activation (MIA). In BA9, IFNA1 emerged as the only differentially expressed gene, which was upregulated in both schizophrenia and mood disorder groups. Microarray gene expression profiles from BA46 revealed more transcriptomic changes in schizophrenia than in bipolar affective disorder and major depressive disorder patients across the whole genome and in the cohort of selected EGF and immune genes. Pathway analysis showed dampened EGF system signalling as indicated by the downregulation of the MAPK-ERK and PI3K-AKT-mTOR pathways, in association with upregulated immune pathways TLR-NFkB, TNF, JAK-STAT and the complement cascade in schizophrenia. In BA46, 68 genes showed differential mRNA expression, predominantly in schizophrenia, where decreased EGF system signalling, as indicated by attenuated expression of the MAPK-ERK, NRG1-PI3K-AKT and mTOR cascades, and similarly diminished immune molecular expression, notably in TLR, TNF and complement pathways, along with low NF-kB1 and elevated IL12RB2 protein levels were noted. Additionally, nominal evidence for altered convergence between ErbB-PI3K and TLR pathways was found in BA46 in schizophrenia. Comparatively minimal changes were noted in BA11. Spiny mouse MIA offspring had elevated NF-kB1 protein in nucleus accumbens but only trend EGF system changes in the brain. This research revealed that the EGF and immune systems play a significant role in the pathology of schizophrenia, potentially aberrantly converging, and that the PI3K-AKT-mTOR, TLR and complement pathways may be more closely implicated in the illness pathology. Distinct pathway changes between schizophrenia and mood disorder may reflect variant pathological processes between them. The DLPFC, specifically BA46, appeared a critical regional substrate in schizophrenia in relation to EGF and immune system dysregulation. Further, exposure to environmental insult during gestation can lead to immune dysregulation in the postnatal brain not necessarily associated with marked EGF system perturbations. These findings could inform on a plausible pathological mechanism in schizophrenia linked with environmental risk factors, and lay a foundation for the development of a biological risk profile to allow early recognition and intervention.