Neurotransmitter imaging to understand seizure mechanisms

Epilepsy affects up to 1% of the population, and even with optimal medication 30% of affected people continue to have seizures. People with drug-resistant epilepsy suffer the side effects of drugs, have a substantial risk of depression and other comorbidities, and have a several-fold increased mortality over the general population through accidents, suicide and sudden unexpected death in epilepsy. The only realistic prospect of seizure freedom is surgery to remove the region where seizures arise, but this is only possible in a small subset of affected people because the seizure-onset zone is often intermingled with brain areas that are necessary for language, memory, movement, sensation or other cognitive functions. The dismal current state of affairs is, to a great extent, due to a poor understanding of the mechanisms by which seizures occur. Different types of seizures are generally described in terms of large-scale networks, and the fine details of which neurons fire, how and where, are only understood in a very superficial way, because until very recently they could only be studied very indirectly using electrical methods. This means that it is still quite unclear which subtypes of excitatory and inhibitory neurons in different regions of the brain become active, or become silent, as seizures initiate, and in which order. A major advance has been the development of optical methods whereby the activity of populations of neurons can be visualised by using molecules that fluoresce in response to changes in the levels of calcium ions. This method can be implemented in experimental rodent models of epilepsy but is quite sluggish and does not detect how neurons signal among themselves by releasing the main excitatory and inhibitory neurotransmitters (glutamate and GABA respectively). We have recently overcome this limitation by using another type of fluorescence microscopy that detects the levels of glutamate and GABA as they are released by neurons. This gives a faster read-out of how populations of excitatory and inhibitory neurons are recruited and gives a direct insight into signalling among populations of neurons. We can also combine imaging of two neurotransmitters simultaneously, or of a neurotransmitter and calcium, by recording fluorescent light of different wavelengths, and in parallel record the electrical discharges that conventionally define seizures. Our proposal builds on our methodological breakthroughs and preliminary data that indicate that an inhibitory 'halo' surrounding the site of initiation of pathological discharges gradually fails in the lead-up to a full-blown seizure that escapes to spread across the brain. We will follow this up by asking which sub-populations of inhibitory neurons fail, and test the hypothesis that they do so because they become over-excited and unable to fire. This will be achieved by manipulating their electrical properties using light-activated proteins that mimic excitation or inhibition ('optogenetics'). We will, moreover, extend from models of epilepsy where seizures are evoked by the application of chemicals to the brain to a rodent model where seizures arise spontaneously. This reproduces a frequently drug-resistant form of human epilepsy associated with focal malformations of brain development that can have catastrophic outcomes for affected children and adults.
Ultimately our research proposal will shed light on how different populations of neurons fire at the transition to seizures, and how their activity relates to excitatory and inhibitory signalling. An improved understanding is essential to refine strategies to treat drug-resistant epilepsy with advanced tools to alter the excitability of populations of neurons. Our laboratory is at the vanguard of gene therapy for epilepsy, with one programme entering clinical trials in 2021, and so we are well positioned to translate the findings of the present research proposal for patient benefit.

Pharmacoresistant focal epilepsy is often accompanied by interictal discharges (IIDs). We have recently used Ca2+ fluorescence microscopy to show, in awake head-fixed mice, that IIDs and seizures evoked by chemoconvulsants are initially indistinguishable. Why do IIDs abate whilst seizures escape from local inhibitory restraint and propagate to contiguous cortical areas? By imaging GABA and glutamate using fluorescent reporters (iGABASnFR and igluSnFR), we have detected striking spatiotemporal differences between the two neurotransmitters, consistent with a halo of inhibition surrounding the ictal 'core'. Pilot data also suggest that a decrease in GABA release with each IID precedes the occurrence of a seizure. Why is GABA release impaired at the ictal core and how does it fail to constrain pathological activity? We will combine neurotransmitter imaging with Ca2+ fluorescence imaging confined to different subsets of interneurons at different distances from the ictal core. We will also use closed loop optogenetic manipulations to test the hypothesis that interneurons are pushed into depolarization block. We will, moreover, use structured illumination to confine the optogenetic manipulations to different sites in relation to the ictal core and wavefront. In parallel, we will ask how the emerging understanding applies to spontaneous seizures in a model of focal cortical dysplasia. We have confirmed that this important cause of pharmacoresistant epilepsy can be modelled by in utero electroporation to manipulate mToRC1 signalling. We will use a miniaturized head-mounted microscope to understand if IIDs and seizures arise from the dysplastic core or surrounding area, and relate the release of neurotransmitters to activity in different populations of neurons. The outcome of will help to refine advanced therapies that we and others are developing to replace epilepsy surgery and offer treatment to many people who currently have not prospect of seizure freedom.