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Title: The regulation of postsynaptic GABAA receptor signalling in epilepsy
Author: Ilie, Andrei-Sorin
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2013
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Fast postsynaptic inhibition in the brain is mediated by ionotropic GABAA receptors (GABAARs), which are activated by the release of the neurotransmitter GABA from presynaptic interneurons. The GABAAR is primarily permeable to chloride ions (Cl-) and therefore the transmembrane gradient for Cl- sets the reversal potential of the receptor (EGABA-A). When intracellular Cl- concentrations are relatively low, EGABA-A is more negative than the membrane potential and GABAAR responses will have a hyperpolarising and inhibitory effect upon the postsynaptic cell. In contrast, when intracellular Cl- concentrations are relatively high, EGABA-A will be more positive and GABAAR activation will have a depolarising effect. How a neuron controls its intracellular Cl- concentrations is a fundamental question that has direct relevance to hyperexcitability conditions such as epilepsy. Recently, it has become clear that Cl- homeostasis is altered in epileptic tissue such that postsynaptic inhibition via the GABAAR is reduced and, under some conditions, GABAAR signalling may even be excitatory. In my thesis I explore some of the mechanisms and factors that are responsible for regulating postsynaptic GABAAR signalling in the context of epileptic seizure activity in the rat hippocampus. In the first series of experiments I combined pharmacological approaches with electrophysiological recordings from pyramidal neurons in the CA3 region of the hippocampus to trigger seizure activity. My results show that intense neuronal activity during a seizure leads to a transient accumulation of intracellular Cl-, which generates a pronounced depolarising shift in EGABA-A. Under these conditions, GABAergic synapses become excitatory and contribute to ongoing neuronal activity rather than exerting their normal inhibitory role. I found that the same seizure activity also induces the release of a neuromodulator called adenosine, which serves to limit the deleterious effects of excitatory GABAAR responses. Adenosine exerts these effects by activating downstream potassium channels, which increase the postsynaptic cell’s membrane conductance and, in doing so, ‘shunt’ incoming GABAAR responses. In the second series of experiments I examined Cl- homeostasis and EGABA-A in the context of neonatal seizures. One of the main mechanisms by which neurons maintain their intracellular Cl- levels is through the activity of ion transporter proteins that reside in the membrane and move Cl- either into, or out of, the cell. I discovered that the intracellular trafficking of an important Cl- transporter protein, NKCC1, correlates with changes in Cl- homeostasis. Using a combination of biochemical and molecular techniques, I then identified a novel molecular association between NKCC1 and a motor protein, Myosin Va, which has been implicated in the intracellular trafficking of membrane proteins. Using electrophysiological recordings I found that Myosin Va is required for NKCC1’s contribution to Cl- homeostasis, which may be important for EGABA-A changes in epilepsy. In the final series of experiments I developed methods to study the temporal dynamics in EGABA-A during a single seizure. These revealed a Cl- unloading mechanism that emerges at the end of a seizure and which depends upon hyperpolarisation of the postsynaptic membrane potential. This mechanism aids EGABA-A recovery after the seizure and moves EGABA-A to more hyperpolarised values. This mechanism could boost postsynaptic inhibition after a seizure and thereby help to protect against further seizure episodes. In conclusion, this work extends our understanding of postsynaptic GABAergic transmission in the context of epileptic seizure activity and suggests new mechanisms that could be relevant for the development of rational anti-epileptic treatments.
Supervisor: Akerman, Colin; Platt, Fran Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Neuroscience ; Epilepsy ; seizures ; GABA-A receptors ; brain inhibition ; chloride regulation