An investigation into dysfunctional feed-forward inhibition within the cortico-thalamocortical network on absence seizure generation using DREADD technology
Childhood absence epilepsy (CAE) is one of the most prevalent paediatric epilepsies, accounting for between 10-17% of all diagnosed cases of epilepsies seen in school-aged children. Absence seizures are characterized by behavioural arrest/loss of awareness and electrographic signature of spike-wave discharges (SWDs) measuring 2.5-4 Hz on an electroencephalogram (EEG). These brief episodes of impaired consciousness can occur hundreds of times a day and might increase the chance of physical injury when undertaking activities like swimming and cycling. Current treatment options are not sufficient and up to 30% of patients are pharmaco-resistant. ~60% of children with CAE have severe neuropsychiatric comorbid conditions including attention deficits, mood disorders, impairments in memory and cognition. Ethosuximide (ETX), an anti-absence epileptic drug which was first introduced almost six decades ago remains the first choice for initial monotherapy for the treatment of CAE. Large-scale clinical trials suggested that efficacy of ethosuximide is considerably lower than previous findings. Thus, safe, effective and patient specific treatment approach is imperative. For this, it is crucial first to understand the precise cellular and molecular mechanisms of absence seizures which may enable the development of novel therapeutic targets and discovery of new anti-epileptic drugs (AEDs). EEG and functional imaging evidence suggest that absence seizures are likely due to aberrant activity within the cortico-thalamocortical (CTC) network. Studies involving the genetic rodent models have shown that the cortex is the driving source for the origin of SWDs but is not capable of maintaining discharges on its own, nor is the thalamus. General consensus is that, within the CTC network, a cortical focus initiates rhythmic epileptic discharges, however, once the rhythmic oscillations are established, both the cortex and thalamus form an integrated network. Rhythmic absence-SWDs are sustained via the cortex and thalamus driving each other. Within the CTC network, feed-forward inhibition (FFI) is essential to prevent runaway excitation. FFI is mediated by fast spiking parvalbumin expressing (PV+) inhibitory interneurons in the somatosensory cortex (SScortex) and the reticular thalamic nucleus (RTN). Studies conducted in well-established stargazer mouse model of absence epilepsy with a genetic deficit in stargazin i.e. TARP [a transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor regulatory protein] have shown reduced expression of GluA4-AMPARs at excitatory synapses in feed-forward inhibitory (PV+) interneurons in the SScortex and RTN thalamus of the CTC network. However, the extent of this deficit in AMPARs expression impacting FFI and possibly contributing towards generation of absence-SWDs is not established via functional studies. Hence, this thesis was aimed at investigating the impact of dysfunctional feed-forward inhibitory PV+ interneurons within CTC network on absence seizure generation and behaviour. For this purpose, inhibitory and excitatory Designer Receptors Exclusively Activated by Designer Drug (DREADD) approach was utilized to silence/excite feed-forward inhibitory PV+ interneurons within the CTC network. DREADD mediated regional silencing of PV+ interneurons within the CTC network generated ETX-sensitive absence-like SWDs. Activating PV+ interneurons either prevented or suppressed pentylenetetrazole (PTZ)-induced absence-SWDs. Finally, impact of impaired FFI in γ-aminobutyric acid (GABA) levels by affecting its synthesizing enzymes (GADs) and transporter proteins (GATs) in stargazer animal model of absence epilepsy and CNO treated inhibitory Gi-DREADD animals was determined. Results indicate that upregulation of GAD65 in the SScortex of epileptic stargazers may be a consequence of absence seizures or this may have contribution in absence seizure generation. The work presented in this thesis provide an electrophysiological insight into the possible mechanism underlying the absence seizure generation. This work provides convincing evidence that dysfunctional feed-forward inhibitory PV+ interneurons within the CTC network is likely to be involved in altered excitation/inhibition balance resulting SWDs as activating these interneurons dramatically protected animals from PTZ induced absence seizures. The clinical relevance of this study is that it potentially uncovers the possibility of focally targeting PV+ interneurons within the CTC network to control absence seizures in human patients.
Advisor: Leitch, Beulah
Degree Name: Doctor of Philosophy
Degree Discipline: Anatomy
Publisher: University of Otago
Keywords: Parvalbumin; Feedforward inhibition; GABAergic inhibition; DREADDs; Absence Seizures; Cortico-thalamocortical; SScortex; RTNthalamus; CAE
Research Type: Thesis