Abstract
The aim of this thesis was to investigate cortical changes in GABAergic neurotransmission that could underlie absence seizures generation, using the well-established genetic stargazer mouse model of absence epilepsy. Absence seizures are non-convulsive, generalized seizures that involve sudden, brief loss of awareness with behavioural arrest. These seizures are more common in children than in adults, being the principal clinical presentation of childhood absence epilepsy, which accounts for 10-17% all paediatric epilepsies. Absence seizures attacks in affected children are brief but can occur hundreds of times a day which are highly disruptive to their daily lives and carry adverse sequelae such as attention deficits, delay in learning and behavioural development, cognitive decline, and the risk of developing into other forms of epilepsies. Therefore, treatment of these seizures is imperative which is currently available in the form of anti-absence drugs. Although effective to a certain extent, these drugs carry a significant failure rate of nearly 30% and are associated with debilitating side-effects which necessitate switching drugs and can force some to withdraw from treatment altogether. The variable response of patients to anti-absence drugs indicates complex, multifactorial mechanisms that underlie absence seizures generation, understanding of which is crucial for the development of highly efficient novel treatment strategies.
The distinctive feature of absence seizures is the appearance of bilaterally hypersynchronous 2.5 – 4 Hz spike-wave discharges (SWDs) on electroencephalography during the event. It is known that these SWDs mainly arise in the cortico-thalamocortical network due to excitatory/inhibitory (E/I) imbalance, switching normal physiological oscillations into pathological SWDs. Given that seizures are essentially hyperexcitable neuronal circuitries, several potential mechanisms are implicated in absence seizures, such as altered glutamatergic excitation, altered channel activity and failure of inhibitory GABAergic neurotransmission. Since GABAergic neurotransmission is critical not only for the development of the nervous system but gating the flow of excitations as well as neuronal plasticity, its failure is bound to disrupt the normal E/I homeostasis. Several human patients have been reported with point-mutations in GABAA receptor (GABAAR) subunits, indicating a definitive link to absence seizures, since similar mutations in animal models generate absence seizures as well. Moreover, the vast range of genetic rodent models used in research, with genetic defects that do not have a direct effect on GABAergic neurotransmission, show downstream changes in GABAergic neurotransmission that are potentially important for the generation of absence seizures.
The well-established stargazer mouse model with human-like absence seizures presents with a genetic defect that results in a deficit in the stargazin protein. This protein is essential for the modulation and synaptic trafficking of AMPA receptors, loss of which results in the loss of these receptors in various regions of the brain. While this might be considered a loss of excitatory signalling, loss of AMPA receptors at inputs onto the predominantly fast-spiking parvalbumin-positive (PV+) GABAergic interneurons of the cortico-thalamocortical network, indicates dysfunctional feed-forward inhibitory gating of excitations. In addition to this, the stargazer also presents with downstream changes in GABAergic neurotransmission which could be critical in the mechanisms that result in the stargazer’s phenotype including absence seizures. Absence seizures likely originate in the cortex, based on evidence from both animal models and human patients, with the site of initiation in rodent models localized to the primary somatosensory cortex. Hence, the current thesis investigated changes in GABAergic neurotransmission in the primary somatosensory cortex, that could be critical to the pathogenesis of absence seizures, using the stargazer mouse. Although the stargazer mutation has not been reported in human patients, the monogenic nature of this model provides the benefit of a controlled framework for investigating underlying mechanisms that causes the primary genetic defect to eventually develop into absence seizures. Such mechanisms could represent at least a subset of human patients.
GABAergic neurotransmission is chiefly mediated as phasic and tonic inhibition through activation of synaptic and extra-synaptic GABAARs, respectively. Changes in these receptors have been reported in absence seizures. Thus, the first part of this thesis investigated changes in GABAAR subunit expression in the stargazer primary somatosensory cortex. I first examined the expression levels of GABAAR subunits in the adult stargazer primary somatosensory cortex using western blotting, with particular focus on GABAAR α1 as a representative subunit given its ubiquitous expression in synaptic GABAARs. Synaptic and extra-synaptic changes in relevant subunits were then evaluated after biochemically isolating the subcellular fractions from the adult stargazer primary somatosensory cortex. A significant reduction in global (~18%) and synaptic (~12%) expression of GABAAR α1 only was revealed. No change was found in other GABAAR subunits (α3, α4, α5, β2, β3, γ2 and δ) investigated. Synaptic density (gold particles/μm) of GABAAR α1 was then assessed using post-embedding immunogold electron microscopy (ICC-EM). However, no apparent change in GABAAR α1 density within individual synapses from PV+ interneurons was observed. Dysfunctional inhibitory gating of excitations and reduced expression of GABAAR α1 in the primary somatosensory cortex indicate increased cortical excitability which could be reflected in altered neurotransmitter dynamics (synthesis, release, and re-uptake). Hence, the second part of the study investigated changes in the major brain neurotransmitters, GABA and glutamate. Here, the changes in total primary somatosensory content of glutamate and GABA were first examined using high-performance liquid chromatography. Significantly enhanced global GABA levels (~19%) but not glutamate in the adult stargazer primary somatosensory cortex was revealed. This was followed by intracellular synaptic assessment of GABA levels in the PV+ and GABAergic terminals of the stargazer primary somatosensory cortex using immunogold ICC-EM, where GABA levels showed a reduction in both PV+ (~19%) and GABAergic (~27%) terminals. Results were further substantiated with investigating changes in PV+ and GABAergic neuron populations and immunoreactive profiles (cellular branches) using immunoperoxidase immunohistochemistry. However, no difference in the actual number of GABAergic and PV+ cells or their immunoreactive profiles was found. Finally, the third part of the study investigated developmental changes in the expression of synaptic GABAAR α1 and GAD65 to identify whether changes found in the adult stargazer primary somatosensory cortex are a potential cause or downstream effect of seizures as the stargazer shows evidence of seizures at postnatal days 17-18. GAD65 was selected because of its correlation with GABA release from inhibitory terminals during intense activity and a previous study indicating its increase in the adult stargazer primary somatosensory cortex. Both synaptic GABAAR α1 and GAD65 showed no difference between stargazers and non-epileptic control before or at the onset of seizures and may not initially contribute to the pathogenesis of absence seizures.
Overall, the results indicate a downstream impairment in GABAergic neurotransmission in the adult stargazer primary somatosensory cortex that appears after seizures initiate at postnatal days 17-18 due to the dysfunctional engagement of PV+ feed-forward inhibitory interneurons. Such impairment may be a compensatory change that attempts to correct the original defect but given the chronic genetic nature of the defect, the compensatory mechanisms not only fail to suppress seizures but may even potentially exacerbate them. Such impairment may also explain why certain GABA neurotransmission-enhancing anti-epileptic drugs actually exacerbate absence seizures. The findings in the current study are crucial because they indicate that the primary genetic defect that causes absence seizures may cause downstream changes that may further contribute to the pathology and affect the outcome of treatment strategies. Such changes need to be considered in the development of novel future treatment designs.