Changing network interactions in models of medically refractory epilepsy

Epilepsy is among the most common neurological conditions, affecting about 3 million people in Europe alone. Remarkably, in up to 30% of these cases, the epilepsy is resistant to any of the currently available treatments. A growing body of evidence suggests that chloride may provide the key to many types of epilepsy, but critically no clinical therapies target this system. It therefore represents a major opportunity for therapeutic development.

Chloride is important because of its role in neural inhibition. It is a negatively charged ion, and for the most important type of synaptic inhibition, it provides the negative signal to neurons. For this to work, however, chloride needs to be kept very low inside neurons. In some situations however, for reasons that remain obscure, chloride can rise, thus compromising synaptic inhibition, and seizures may ensue. We therefore designed a way to correct this chloride imbalance - the first time this has been possible. Our proposal describes how we will develop this new technique both to investigate epileptic pathological mechanisms, and also to treat them.

We will explore this "chloride hypothesis" by examining the contribution of pathologically raised neuronal chloride in a widely used in vitro model of epileptiform activation. Importantly, the pathological activity in this model evolves from an early pattern of pharmaco-sensitive discharges, to a late-stage pharmaco-resistant pattern. We hypothesise that a failure in neuronal chloride regulation may be a major contributory factor in this critical transition.

In this proposal we outline a set of studies that will, firstly, characterise the changes in neuronal excitability and synaptic function that are associated with the evolution of epileptiform activity in brain slices that are deprived of Mg2+ ions. This is a widely used in vitro preparation for studying such pathological discharges. We will focus particularly on two key transitions: the initial development of pathological discharges from the pre-ictal, baseline state, and second, the aforementioned transition from pharmaco-sensitive to pharmaco-resistant activity. We will use a range of patch clamp and optogenetic strategies to assess functional changes (membrane potential, excitability, synaptic function etc) in 3 key populations of cortical neurons (pyramidal cells and interneurons expressing parvalbumin or somatostatin). There is good prior evidence that this is associated with chloride loading of neurons, but we will put this in the context of multiple other changes in cellular attributes.

The next stage will be to manipulate the timing of the network transitions, to test whether the cellular changes are also shifted in time. This will indicate causative relations between the two sets of phenomena.

Finally, we will use novel optogenetic approaches for rapid manipulation of chloride in neurons, to test whether the pharmaco-resistant state can be reversed, by reducing the chloride level.

In summary, this proposal will advance our understanding of a widely used model of pharmaco-resistant epileptiform activity, and establish it as an assay for future drug development. It will further test a key hypothesis that this clinically important state reflects pathology in how chloride is regulated in neurons.

Drug-resistant epilepsy is a major clinical problem, affecting approximately a million people across Europe today. We propose to address this issue by examining a notable feature of a widely used in vitro model of pathological epileptiform discharges, induced by washing Mg ions out of the bathing medium. Previous work has shown that this model displays a striking transition from early pharmaco-sensitive, to late pharmaco-resistant patterns of activity. This model therefore represents a highly tractable system for understanding the cellular basis of one example of drug-resistant epileptiform discharges. We will test a key hypothesis that this transition arises from dysregulation of neuronal chloride.

Initial experiments will examine changes in neuronal function and synaptic connectivity associated with the evolution of epileptiform activity in this model. We will use a combined patch-clamp / optogenetic approach, recording from brain slices prepared from young adult mice, in which various optogenetic proteins are expressed under cell-specific promoters. We will focus on the three most abundant cell types: pyramidal cells and interneurons expressing parvalbumin or somatostatin), exploring how cellular excitability and synaptic function changes as the epileptiform activity evolves.

The epileptic network transitions occur relatively abruptly, so a particular focus will be to relate these to the kinetics of change of the various cellular changes. These temporal associations will then be further tested, using several experimental manipulations, which either speed up or delay the network transitions. Finally, we will use a novel optogenetic strategy that we have recently developed, called Cl-out (Alfonsa et al, 2016), which allows a light-activated rapide extrusion of chloride from neurons. We will test whether this optogenetic chloride extrusion can reverse the late stage pharmaco-resistant state, thereby reintroducing sensitivity to diazepam.

The immediate beneficiaries will be amongst the academic community. Prime among these will be epilepsy researchers, but such is the widespread importance of chloride-dependent inhibitory mechanisms across the brain, that we predict our research to have relevance to laboratories working in all areas of neuroscience. It will be of particular interest to the large and growing optogenetic community, as the first demonstration of combinatorial opsin function, whereby emergent properties arise only from co-activation of multiple opsins. And by extension, also for the field of brain-machine interfaces (BMIs), which will feed heavily off the continued developments in optogenetics; our new constructs, or derivatives of these, will, we believe, be among the principle optogenetic tools used in such BMIs. Dissemination to this interest group will be helped by the major optogenetic research initiatives in Newcastle ("CANDO").

Novel research findings will be incorporated into the various teaching and outreach activities of the team. There will be obvious benefits for the postgraduate and early career researchers in the Institute, where there is growing interest in implementing optogenetics into research programmes. We will engage with the wider academic and non-academic communities through various routes including the University of Newcastle, local schools, the annual North East Epilepsy Research Meetings, Epilepsy Interest Group, which is part of the Northern England Stategic Clinical Network, Epilepsy Research UK and International League Against Epilepsy meetings, local and national patient groups, including through Epilepsy Action (contributions to Epilepsy Professional magazine), and other scientific and public conferences including the British Science Festival.

The Biopharma Industry can also benefit from the utilising our new techniques for manipulating chloride levels in neurons, to investigate pharmacological means of controlling chloride. The successful completion and publication of our research will be the basis for engaging with the Biopharma Industry through future collaborative funded projects including CASE studentships and industry placements.

Finally, the project ultimately is aimed at developing new therapies for treating epilepsy; currently 600,000 people in the UK alone are diagnosed with epilepsy, and of these, only around 70% are helped with currently available therapies (medication and surgical interventions). Since epilepsy is often a lifelong condition, it presents a huge economic burden, both directly through health care costs, and indirectly through its impact on the lives of people with the condition. For these reasons, serious chronic conditions like epilepsy tend to have very wide and pervasive negative impact on society. Our experience in our interactions with patient groups is that these people are empowered by recognising the research efforts that are being pursued on their behalf. Our strategy will target a facet of epileptic pathology that has not been utilised previously for therapeutic intervention, and so represents a good candidate for development for the many people who are not helped. The team has close links with clinical medical groups both through epilepsy and the mitochondrial research groups, and have extremely strong track records in translating their basic research into clinically useful applications.

Through these various pathways, these research efforts will help raise the profile of the North East and the UK generally, as leaders of biomedical and health care innovation.