Anatomy and Neuroscience - Theses
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ItemSodium channels and epilepsy: neuronal dysfunction in genetic mouse models( 2014)Mutations in sodium channels have long been linked to inherited epilepsies. Recent clinical findings identified patients with Dravet syndrome that were homozygous for a mutation in SCN1B which encodes the β1 auxiliary subunit of sodium channels. Dravet syndrome is a severe childhood epileptic encephalopathy, and patients commonly present with frequent seizures, developmental regression, ataxia with associated gait abnormalities, and shorter lifespans. We have engineered a mouse model based on the human C121W epilepsy mutation (β1-C121W). Mice homozygous for this C121W mutation displayed similar deficits in health and motor skills to Dravet syndrome. Our experiments showed that β1-C121W homozygous neurons fired more action potentials per current injection, had significantly higher membrane resistance, and were more prone to demonstrate a bursting subtype. These hallmarks of neuronal excitability may contribute to the increased sensitivity to thermal seizures in the homozygous mice. Neuron morphology analysis also revealed that neurons within the subiculum of these animals were significantly smaller in size, consistent with the observed increased input resistance. Application of a new anti-epileptic drug, retigabine, successfully reversed the input resistance in homozygous animals down to wildtype levels, and dampened neuronal excitability. Retigabine injected intraperitoneally into homozygous mice was extremely efficient at reducing thermal seizure susceptibility. These findings highlight the potential utility of applying disease-mechanism based strategies to aid anti-epileptic therapy. In order to examine network excitability in another genetic model of epilepsy, the function of the Nav1.2 sodium channel alpha subunit during development was studied. The NaV1.2 gene has two developmentally regulated splice variants; the ‘neonatal’ and ‘adult’ isoforms. A mutation discovered in patients with benign familial neonatal-infantile epilepsy (BFNIE) increases the excitability of the ‘neonatal’ isoform such that it resembles the adult isoform. Moreover, previous work from the current laboratory using human NaV1.2 expressed in HEK293 cells showed that the ‘neonatal’ form is less excitable than the ‘adult’ form. Based on these data and because the proportion of the neonatal Nav1.2 mRNAs gradually decreases with age during development we hypothesize that the ‘neonatal’ NaV1.2 isoform reduces neuronal excitability in infant brain and therefore plays a protective physiological role. To test this the current laboratory engineered a mouse line which continuously expresses the adult form of Nav1.2 from birth (NaV1.2adult) and investigated seizure susceptibility and neuronal phenotypes. Homozygous NaV1.2adult mice were of normal size and had no obvious seizures under observation during routine video analysis. However, NaV1.2adult mice had increased susceptibility to PTZ-induced seizures, suggesting that the neonatal isoform of NaV1.2 may confer an a novel form of seizure protection. Pyramidal neurons recorded from cortical layers 2/3 of postnatal day 3 (P3) Nav1.2adult neonates show heightened excitability reflected by the presence of a fast-firing neuronal population, which was not seen in the wild-type. At P15, the differences between Nav1.2adult and wildtype at a single neuron level were no longer evident. Interestingly, we also identified an increase in the amplitude of miniature inhibitory post synaptic currents in Nav1.2adult mice compared to the wildtype mice. These results suggest that inherent changes in the neuronal networks occur as a consequence of continuous expression of the adult isoform of NaV1.2 during development. Although further investigation is required to fully understand the biological roles of the two NaV1.2 isoforms, it is predicted that the neonatal isoform of NaV1.2 confers seizure protection in the NaV1.2 mouse model of BFNIE.
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ItemThe effects of stress on the onset and progression of Huntington's disease in a transgenic mouse model( 2014)Huntington’s disease (HD) is a neurodegenerative disorder largely governed by genetics. The cause of the disease is a fully penetrant gene mutation, inherited by autosomal dominant transmission. The length of this mutation also predicts the age of disease onset, which can range from childhood to late adulthood. Work from our lab on the R6/1 transgenic mouse model of HD was the first to show that environmental factors can alter symptom progression. Environmental enrichment and voluntary wheel running delayed or ameliorated the triad of motor, affective and cognitive dysfunctions in HD mice. Recent clinical studies also suggest that lifestyle factors can affect the age of onset. Currently, there are no treatments to slow or change the course of HD so environmental interventions may offer a feasible approach to extend the symptom-free years in HD gene-positive individuals. There is evidence to suggest that the stress response is abnormal in HD mice and patients. The present study is the first to investigate the impact of stressors on the onset and progression in an animal model of HD. We used an acute (Chapter 3) and two chronic stress paradigms (Chapters 4 and 6) to assess the impact on characteristic symptoms of HD. We also extended the phenotyping of R6/1 HD mice to include behaviours of ethological relevance (Chapter 5). All 3 stress protocols were able modify various functions in R6/1 HD mice, notably accelerating cognitive decline and further impairing olfactory deficits. This work contributes data for sex differences in the HD phenotype and to the general stress literature. Importantly, we show that stress is not only able to modulate specific behaviours in HD mice, but that the gene mutation may confer a susceptibility to the negative effects of stress. Therefore, behavioural management therapy in combination with other lifestyle changes may help manage the course of the disease in gene positive individuals.