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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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.