Anatomy and Neuroscience - Theses
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ItemAnatomical changes at the axon initial segment in neuronal hyperexcitability( 2013)The axon initial segment (AIS) is an important sub-cellular region in neurons, playing diverse and critical roles in neuronal excitability, the maintenance of neuronal polarity, and the regulation of cytoplasmic trafficking. Previously thought to be a uniform, static structure, it is now apparent that the AIS exhibits greater levels of complexity and plasticity than previously predicted, and is an increasingly interesting and relevant focus of research in neuroscience. A range of proteins are expressed at high densities at the AIS, some exclusively, including structural molecules, ion channels and cell adhesion molecules. The molecular composition and structural characteristics of the AIS vary by neuronal subtype, brain region and developmental stage, resulting in differences in functional phenotypes of these neurons, although the more subtle aspects of this are yet to be elucidated. The important roles played by AIS-localised proteins, along with the potential consequences of disruption to AIS integrity, composition or structure, make this an incredibly important neuronal region to consider in a variety of pathophysiological pathways in the brain. Many AIS proteins have been implicated in CNS disease; in particular a large number of AIS ion channels are implicated in epilepsy. Additionally, the emerging phenomenon of AIS plasticity, by which neuronal excitability is altered as a result of changes in the gross structural anatomy of the AIS, could potentially play a role in epilepsy. In this thesis I explore two aspects of AIS involvement in disorders of neuronal hyperexcitability using immunohistochemistry and high-resolution confocal microscopy. The first study analyses the effects of seizures on AIS structure in two animal models of neuronal hyperexcitability, in which I have identified structural changes in the position of the AIS relative to the soma in animals experiencing seizures. This is the first study to demonstrate plasticity of the AIS in epilepsy, and the results suggest differing roles of this phenomenon in established genetic epilepsy and in the pathogenesis of acquired seizure disorders. The second study describes the AIS localisation of an ion channel subtype – the β1 subunit of the voltage-gated sodium channel – in both health and disease states, using a genetic mouse model of a human epilepsy syndrome. I have demonstrated the endogenous localisation of this subunit to the AIS and revealed its disruption in genetic epilepsy, an important finding complementing functional studies in elucidating the pathogenic mechanisms in this type of epilepsy. These studies reveal the novel involvement of AIS structural plasticity in neuronal hyperexcitability as well as a mechanism of AIS dysfunction in genetic epilepsy, together highlighting the ubiquitous influence of AIS function on neurological health. The linking of genetic mutations, environmental conditions and anatomical AIS phenotypes will further enhance our understanding of the pathophysiological basis of disorders of neuronal hyperexcitability and aid identification of novel therapeutic targets for neurological disease.
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ItemFunctional analysis of sodium channel gene variation in epilepsy( 2013)A genetic etiology of epilepsy is widely accepted in 50-70% of all epilepsy syndromes. With genome sequencing now increasingly efficient and affordable, more and more novel genes and mutations are being discovered that are associated with epilepsy. However, most of the mutations have been discovered in genes that code for ion channels which has led to the theory that the genetic epilepsies are a family of channelopathies. The voltage-gated sodium channel family have been particularly implicated with over 800 variants discovered in this gene family. Given their critical role in regulating neuronal excitability it is not surprising that genetic variations in sodium channels can have functional and potentially devastating consequences. With a focus on the voltage-gated sodium channels, the three chapters in this thesis used high-throughput automated planar patch-clamp technology to try and develop a deeper understanding of genetic risk in epilepsy. Chapter two examines a novel cause in a mouse model of absence epilepsy that harbours a mutation in the Scn8a gene. The phenotype of this mouse is enhanced on the C3H background, as opposed to C57, where the C3H animal also has a mutation in the Scn2a gene. The individual biophysical profiles of these two mutations were examined on the Nanion patchliner, and their potential genetic interaction was investigated in a computer model of a layer 5 pyramidal neuron, to see if this could be explained by a biological interaction at the axon initial segment. The results revealed an overall loss of function of the NaV1.6V752F mutant, and an overall gain of function in the NaV1.2V929F mutant. When these changes were implemented in the computer model, it revealed that the output was dominated by the NaV1.2V929F mutant, which suggests there is not a biological interaction of these two genes at the axon initial segment. Alternative scenarios where there may be an alternative site for biological epistasis will be revealed with future studies using immunohistochemistry and brain slice patch clamp recording in the mice. It may also be the case that the NaV1.2V929F mutant is not a modifier of the NaV1.6V752F mutant, which will be revealed by genetic studies to identify the modifier genes. The third chapter examined the modulation of NaV1.2 and NaV1.1 by the β1 auxiliary subunit. As mutations in the β1-subunit have been detected in patients with epilepsy, understanding their impact on subunits from excitatory and inhibitory neurons is critical for understanding how this variation impacts on risk for epilepsy. There was a differential modulation revealed where β1 had a greater functional effect on the NaV1.2 channel but a greater effect on current density on the NaV1.1 channel. Therefore if a variant in β1 experiences a functional change this suggests differentially altered levels of excitation and inhibition in the brain, which could feasibly result in an epileptic phenotype. The fourth chapter looked at exploiting the high-throughput capabilities of the Nanion patchliner, and examined eight mutations in the β1-subunit co-expressed with NaV1.1 and NaV1.2 that have been associated with epilepsy. With this influx of data we needed to devise a new way to represent this data, and converted all raw measurements to effect size values, and represented them on tornado diagrams. With this measurement we could then more easily directly compare parameters from the individual protocols and calculate averages both across mutations, and across parameters. From this data set it is quite apparent that the β1 mutants modulate the α-subunits quite differently, both comparing α-subunits, and comparing mutations. More importantly however this chapter highlighted a new way of thinking about analysis of high-throughput electrophysiology data. As people continue to look into the genetics of epilepsy and reveal novel genes and novel mutations implicated in the disease, we need to look for new ways to tame the genetic complexity, and look for points of convergence. High-throughput technology allows us to decrease the time lapse between the discovery of the genetic variants and the corresponding functional analysis. And the type of analysis as suggested in chapter four, enables us to start to look for points of convergence in the functional data. This data can then be used to train clustering algorithms to group the variants based on their ‘channelomic’ profile. To do this we need a large volume of functional data obtained from variants that have strong corresponding phenotypic data, and future studies should endeavour to accomplish this.
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ItemGenetic, metabolic and pharmacological modulation of seizure susceptibility in mouse models of genetic epilepsy( 2013)Epilepsy is a common neurological disorder that is poorly understood. A large proportion of epilepsies have a strong familial component. The GABAA γ2 (R43Q) mutation was discovered in an Australian family with genetic epilepsy with febrile seizures + (GEFS+) that predominantly have febrile seizures (FS) and childhood absence epilepsy (CAE). A mouse model based on the mutation recapitulates these seizure types and is sensitive to first-line antiepileptic drugs. The model therefore provides an opportunity to study aspects of the genesis of epilepsy with relevance to the human condition. The work performed in this thesis describes the use of this syndrome specific mouse model to investigate aspects of seizure genesis and modulation. Three research questions are addressed; the genetic mechanisms underlying seizure genesis, metabolic and dietary modulation of seizure activity and pharmacological sensitivity to new anti-epileptic drugs in the GABAA γ2 (R43Q) mouse. Clinical heterogeneity in genetic epilepsy is common and is typically characterized by multiple seizure types and incomplete penetrance for a given protein mutation. However, the molecular and genetic basis of clinical heterogeneity is not well understood. Here, two models, GABAA γ2 (R43Q) knock-in and GABAA γ2 knock-out were used to determine the fundamental molecular mechanisms of the GABAA γ2 (R43Q) mutation underlying individual seizure phenotype. Spike-wave discharges (SWD) recorded on electroencephalogram from the GABAA γ2 (R43Q) mouse are associated with behavioural arrest and model absence epilepsy. A reduced latency to first heat-induced tonic-clonic seizure is consistent with a FS phenotype. Both the knock-in and knock-out models expressed SWDs while only the knock-in had a reduced latency to thermogenic seizures. This comparison demonstrates that two fundamental molecular mechanisms independently cause the two major seizure types in the mouse model. Haploinsufficiency could account for the SWD phenotype while a dominant impact of the mutation must be required for the FS phenotype. Subsequent investigation using mice of varying genetic background showed that the SWD phenotype required additional genetic susceptibility. In contrast, FS phenotype occurred independently of background genetics consistent with its higher penetrance compared to absence epilepsy in the GABAA γ2 (R43Q) family. Environmental modulation of neuronal excitability has been long known to alter seizure susceptibility. Altered metabolism using dietary intervention, such as the ketogenic diet, is a well recognized epilepsy therapy. The ketogenic diet conveys its anticonvulsant effects presumably through the stabilization of blood glucose and/or providing an alternative energy substrate. Here, the impact of a number of metabolic manipulations was investigated in the GABAA γ2 (R43Q) mouse model. Overnight fasting lowered blood glucose levels and increased SWD occurrence suggesting it as a potential seizure precipitant. Low-GI and triheptanoin diets on the other hand reduced SWD activities suggesting that both stabilization of blood glucose levels and provision of additional energy substrates may independently offer anticonvulsant effects. Importantly, these diets have less tolerability issues making them a potential alternative to the poorly tolerated ketogenic diet. In-vivo drug testing is a critical step for drug discovery. Oxcarbazepine (OXC) is a second-generation drug that is typically used to control partial seizures. Like its older generation carbamazepine, OXC is contraindicated in patients with generalized epilepsy. OXC is metabolized to monohydroxy derivatives (MHD) in two enantiomeric-forms, S-(+)-licarbazepine and R-(+)-licarbazepine. The effects of individual metabolites have not been adequately characterized. In this study, OXC increased the frequency of SWDs in the GABAA γ2 (R43Q) model, consistent with clinical observation. Similarly, both MHDs also caused seizure aggravation. However, OXC and MHDs were ineffective at altering the sensitivity of mice to thermogenic seizures. The findings indicate that like OXC, its derivatives may be contraindicated in certain forms of generalized epilepsy.