Anatomy and Neuroscience - Theses
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ItemGenetic and environmental modulation of neuronal excitability( 2014)Genetic epilepsy is the cause of many of the epilepsies. How identified epilepsy mutations alter function at the single neuron and whole animal levels is relatively well understood, however how neuronal networks are affected is not. Considering ~30% of epilepsy patients do not sufficiently control their seizures with current anti epileptic drugs, a deeper understanding of how epilepsy mutations change network level activity may enable the development of more efficacious treatment options. Interestingly, recent work has shown that an acidic shift in brain pH induced by respiration of carbogen gas (5% CO2 – 95% O2) is a rapid and extremely effective intervention to halt seizures. Despite various ion channels and receptors being implicated in reducing neuron excitability, we do not fully understand the mechanism(s) by which an acidic pH prevents seizures. Seizures are defined by hyper excitable and hyper synchronous network activity, therefore the anti seizure effect of carbogen gas may occur by a reduction in neuron excitability and/or synchrony. Synchronous network activity is generated in part by gap junction couplings between inhibitory interneurons. While non-mammalian gap junctions are modulated by pH this has never been shown between mammalian neurons by direct electrophysiological recordings. Therefore the aims of this thesis are: I. To investigate the neuronal network dysfunction and anti epileptic drug sensitivity in a mouse model of human genetic epilepsy, II. To investigate pH modulation of neuronal excitability with regard to carbogen gas’ anti seizure effect, and III. To investigate the role of gap junction couplings between inhibitory interneurons and to determine if these mammalian gap junction are modulated by pH. A NaVβ1(C121W) mouse model of human genetic epilepsy developed by this laboratory, has enhanced neuronal excitability and temperature sensitivity attributed to a decreased threshold for action potential firing in the axon initial segment. To investigate the network consequences of this neuronal dysfunction and to establish a genetic disease state model we developed an in vitro assay to investigate CA1 network properties and anti epileptic drug sensitivity. Tetanic stimulation reliably induced local CA1 oscillations that required excitatory and inhibitory synaptic activity and pacemaker currents for their generation. Slices from NaVβ1(C121W) heterozygous mice displayed several hallmarks of increased temperature dependent network excitability. Anti epileptic drugs were more effective in reducing network excitability in slices from NaVβ1(C121W) heterozygous mice and were more effective in suppressing time to thermogenic seizures in NaVβ1(C121W) heterozygous mice compared to WT controls. Hippocampal networks of the NaVβ1(C121W) heterozygous mouse model of genetic epilepsy show enhanced excitability consistent with earlier single neuron studies bridging important scales of brain complexity relevant to seizure genesis. Altered pharmacosenstivity further suggests that genetic epilepsy models may be useful in the development of novel anti epileptics that target disease state pathology. Recent studies have shown that an acidic shift in brain pH induced by application of carbogen gas is a rapid and extremely effective treatment to halt seizures in animal models and in human patients. However, we do not fully understand the mechanism(s) by which an acidic pH induced by carbogen gas halts seizures. Thus the aim of this study was to investigate the biophysical mechanisms underpinning the seizure protective effect of an acidic pH. Using a thermogenic seizure assay, we show that respiration of carbogen gas prevents seizures and that an acidic shift in pH reduces in vitro local CA1 network activity. Furthermore, using whole-cell patch-clamp recordings we show that there is a selective reduction in action potential firing in CA1 pyramidal neurons following an acidic shift in pH. Whereas, inhibitory interneuron action potential firing is not reduced. These data indicate that rather than an enhancement of inhibitory activity, the anti seizure effect of an acidic pH is driven by a reduction in excitatory drive. Gap junction coupled inhibitory interneurons are important for creating synchronous network activity. Recent experimental data and computer modeling predicts that pH modulation of gap junction couplings, potentially via changes in gap junction conductance, can underlie seizures, however this has not been directly demonstrated in a mammalian system. Therefore the aim of these experiments was to investigate pH modulation of gap junction coupled inhibitory interneurons in mammalian brain slices. Simultaneous whole-cell recordings from up to four neurons showed that gap junction connectivity enhances neuron excitability and synchrony. Furthermore, an acidic shift in pH reduced the gap junction conductance of connected neurons and reduced synchronous neuronal activity. Demonstrating that gap junction coupling increases microcircuit synchrony and excitability and that a physiological acidification reduces gap junction conductance that is well positioned to impact network function.