Anatomy and Neuroscience - Theses

Permanent URI for this collection

Search Results

Now showing 1 - 2 of 2
  • Item
    Thumbnail Image
    Sodium channels and epilepsy: neuronal dysfunction in genetic mouse models
    LEAW, BRYAN ( 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.
  • Item
    Thumbnail Image
    Anatomical changes at the axon initial segment in neuronal hyperexcitability
    Harty, Rosemary Colette ( 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.