The Enlightened Brain: Novel Imaging Methods Focus on Epileptic Networks at Multiple Scales

Epilepsy research is rapidly adopting novel fluorescence optical imaging methods to tackle unresolved questions on the cellular and circuit mechanisms of seizure generation and evolution. State of the art two-photon microscopy and wide-field fluorescence imaging can record the activity in epileptic networks at multiple scales, from neuronal microcircuits to brain-wide networks. These approaches exploit transgenic and viral technologies to target genetically encoded calcium and voltage sensitive indicators to subclasses of neurons, and achieve genetic specificity, spatial resolution and scalability that can complement electrophysiological recordings from awake animal models of epilepsy. Two-photon microscopy is well suited to study single neuron dynamics during interictal and ictal events, and highlight the differences between the activity of excitatory and inhibitory neuronal classes in the focus and propagation zone. In contrast, wide-field fluorescence imaging provides mesoscopic recordings from the entire cortical surface, necessary to investigate seizure propagation pathways, and how the unfolding of epileptic events depends on the topology of brain-wide functional connectivity. Answering these questions will inform pre-clinical studies attempting to suppress seizures with gene therapy, optogenetic or chemogenetic strategies. Dissecting which network nodes outside the seizure onset zone are important for seizure generation, propagation and termination can be used to optimize current and future evaluation methods to identify an optimal surgical strategy

Involvement of fast-spiking cells in ictal sequences during spontaneous seizures in rats with chronic temporal lobe epilepsy

Epileptic seizures represent altered neuronal network dynamics, but the temporal evolution and cellular substrates of the neuronal activity patterns associated with spontaneous seizures are not fully understood. We used simultaneous recordings from multiple neurons in the hippocampus and neocortex of rats with chronic temporal lobe epilepsy to demonstrate that subsets of cells discharge in a highly stereotypical sequential pattern during ictal events, and that these stereotypical patterns were reproducible across consecutive seizures. In contrast to the canonical view that principal cell discharges dominate ictal events, the ictal sequences were predominantly composed of fast-spiking, putative inhibitory neurons, which displayed unusually strong coupling to local field potential even before seizures. The temporal evolution of activity was characterized by unique dynamics where the most correlated neuronal pairs before seizure onset displayed the largest increases in correlation strength during the seizures. These results demonstrate the selective involvement of fast spiking interneurons in structured temporal sequences during spontaneous ictal events in hippocampal and neocortical circuits in experimental models of chronic temporal lobe epilepsy

GABAergic Interneurons in Seizures: Investigating Causality With Optogenetics

Seizures are complex pathological network events characterized by excessive and hypersynchronized activity of neurons, including a highly diverse population of GABAergic interneurons. Although the primary function of inhibitory interneurons under normal conditions is to restrain excitation in the brain, this system appears to fail intermittently, allowing runaway excitation. Recent developments in optogenetics, combined with genetic tools and advanced electrophysiological and imaging techniques, allow us for the first time to assess the causal roles of identified cell-types in network dynamics. While these methods have greatly increased our understanding of cortical microcircuits in epilepsy, the roles played by individual GABAergic cell-types in controlling ictogenesis remain incompletely resolved. Indeed, the ability of interneurons to suppress epileptic discharges varies across different subtypes, and an accumulating body of evidence paradoxically implicates some interneuron subtypes in the initiation and maintenance of epileptiform activity. Here, we bring together findings from this growing field and discuss what can be inferred regarding the causal role of different GABAergic cell-types in seizures

Initiation and slow propagation of epileptiform activity from ventral to dorsal medial entorhinal cortex is constrained by an inhibitory gradient.

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this record.The medial entorhinal cortex (mEC) has an important role in the generation and propagation of seizure activity. The organisation of the mEC is such that a number of dorso-ventral relationships exist in neurophysiological properties of neurons. These range from intrinsic and synaptic properties to density of inhibitory connectivity. We examined the influence of these gradients on generation and propagation of epileptiform activity in the mEC. Using a 16-shank silicon probe array to record along the dorso-ventral axis of the mEC in vitro, we found 4-aminopyridine (4-AP) application produces ictal-like activity originating predominantly in ventral areas. This activity spreads to dorsal mEC at a surprisingly slow velocity (138 μm.s-1), while cross-site interictal-like activity appeared relatively synchronous. We propose that ictal propagation is constrained by differential levels of GABAergic control since increasing (diazepam) or decreasing (Ro19-4603) GABAAreceptor activation, respectively, reduced or increased the slope of ictal initiation. The observation that ictal activity is predominately generated in ventral mEC was replicated using a separate 0-Mg2+model of epileptiform activity in vitro. By using a distinct disinhibition model (co-application of kainate and picrotoxin) we show that additional physiological features (for example intrinsic properties of mEC neurons) still produce a prevalence for interictal-like initiation in ventral mEC. These findings suggest that the ventral mEC is more likely to initiate hyperexcitable discharges than dorsal, and that seizure propagation is highly dependent on levels of GABAergic expression across the mEC. This article is protected by copyright. All rights reserved.This work was supported by a University of Exeter and Eli Lilly studentship (T.R). P.M 513 was supported by an MRC Proximity to Discovery award in partnership with 514 AstraZeneca. K.G.P was an employee of Eli Lilly. A.D.R was part funded by a Royal 515 Society Industrial Fellowship. J.T.B was an Alzheimer’s Research UK Senior Research 516 Fellow (ARUK-SRF2012-6)

KCC2 overexpression prevents the paradoxical seizure-promoting action of somatic inhibition

Although cortical interneurons are apparently well-placed to suppress seizures, several recent reports have highlighted a paradoxical role of perisomatic-targeting parvalbumin-positive (PV+) interneurons in ictogenesis. Here, we use an acute in vivo model of focal cortical seizures in awake behaving mice, together with closed-loop optogenetic manipulation of PV+ interneurons, to investigate their function during seizures. We show that photo-depolarization of PV+ interneurons rapidly switches from an anti-ictal to a pro-ictal effect within a few seconds of seizure initiation. The pro-ictal effect of delayed photostimulation of PV+ interneurons was not shared with dendrite-targeting somatostatin-positive (SOM+) interneurons. We also show that this switch can be prevented by overexpression of the neuronal potassium-chloride co-transporter KCC2 in principal cortical neurons. These results suggest that strategies aimed at improving the ability of principal neurons to maintain a trans-membrane chloride gradient in the face of excessive network activity can prevent interneurons from contributing to seizure perpetuation

SCN1A channelopathies: Navigating from genotype to neural circuit dysfunction

The SCN1A gene is strongly associated with epilepsy and plays a central role for supporting cortical excitation-inhibition balance through the expression of NaV1.1 within inhibitory interneurons. The phenotype of SCN1A disorders has been conceptualized as driven primarily by impaired interneuron function that predisposes to disinhibition and cortical hyperexcitability. However, recent studies have identified SCN1A gain-of-function variants associated with epilepsy, and the presence of cellular and synaptic changes in mouse models that point toward homeostatic adaptations and complex network remodeling. These findings highlight the need to understand microcircuit-scale dysfunction in SCN1A disorders to contextualize genetic and cellular disease mechanisms. Targeting the restoration of microcircuit properties may be a fruitful strategy for the development of novel therapies

Volume-transmitted GABA waves pace epileptiform rhythms in the hippocampal network

Mechanisms that entrain and pace rhythmic epileptiform discharges remain debated. Traditionally, the quest to understand them has focused on interneuronal networks driven by synaptic GABAergic connections. However, synchronized interneuronal discharges could also trigger the transient elevations of extracellular GABA across the tissue volume, thus raising tonic conductance (Gtonic) of synaptic and extrasynaptic GABA receptors in multiple cells. Here, we monitor extracellular GABA in hippocampal slices using patch-clamp GABA "sniffer" and a novel optical GABA sensor, showing that periodic epileptiform discharges are preceded by transient, region-wide waves of extracellular GABA. Neural network simulations that incorporate volume-transmitted GABA signals point to a cycle of GABA-driven network inhibition and disinhibition underpinning this relationship. We test and validate this hypothesis using simultaneous patch-clamp recordings from multiple neurons and selective optogenetic stimulation of fast-spiking interneurons. Critically, reducing GABA uptake in order to decelerate extracellular GABA fluctuations-without affecting synaptic GABAergic transmission or resting GABA levels-slows down rhythmic activity. Our findings thus unveil a key role of extrasynaptic, volume-transmitted GABA in pacing regenerative rhythmic activity in brain networks

The spatiotemporal dynamics of human focal seizures

Spontaneous human focal seizures can present with a plethora of behavioral manifestations that vary according to the affected cortical regions; however, several key features have been consistently observed. During my doctoral studies, I applied both theoretical and experimental methods to study mechanisms underpinning these consistently seen dynamics. I first analyzed human intracranial EEG recordings, describing statistical methods for measuring their electrophysiological signatures. I next proposed several neurophysiological hypotheses that could explain seizure dynamics and verified them in rodent seizure models. Finally, a computational model was developed, successfully explaining how the complex spatiotemporal evolution of focal seizures emerges from simple neurophysiological principles. In Chapter 1, the long-standing behavioral manifestations and the most up-to-date electrophysiology findings are reviewed. This section details the inspiration for the studies reported in the subsequent chapters. In Chapter 2, I describe several statistical methods for estimating traveling wave velocities. I show most ictal discharges can be described as traveling waves whose velocities contain rich information about the stages of seizure evolution. I compare performance of various statistical methods and propose a robust approach to boost the quality of each method’s estimation results. In Chapter 3, I show how inhibition modulates seizure propagation patterns. Surround inhibition spatially restrains focal seizures and masks excitatory projections of ictal activities. When compromised, two patterns of seizure propagation emerge according to the position of inhibition defects relative to the ictal focus. I show that two distant ictal foci can communicate via physiological connectivity without any chronic rewiring processes – confirming the existence of long-range propagation pathways that could lead to epileptic network formation. In Chapter 4, I show that thalamic inputs might be necessary for interictal epileptiform discharges (IEDs). The relative positions between IEDs and ictal foci indicate that surround inhibition, shown in the previous chapter, can be exhausted by repetitive exposure to ictal projections. In Chapter 5, I propose a neural network model that can explain both long-standing behavioral observations of seizures and account for the most up-to-date electrophysiological recordings of spontaneous human focal seizures. The model relies on few assumptions, all of which are proved or supported in earlier chapters of this thesis. The model explains phasic evolution of seizure dynamics – how the commonly observed patterns arise from simple neurophysiological principles, as well as seizure onset subtypes, traveling wave directions and speeds. The model also predicts how spontaneous seizures might arise from synaptic plasticity. The chapter ends with a discussion of the model’s implications and future work. The thesis is organized in a way that each chapter can be read independently, with Chapter 5 summarizing the central theory spanning the whole study. Each chapter is also tightly linked to a clinically relevant question. In sum, the dissertation’s goal is to provide an in-principle understanding of focal seizure dynamics. With rapid advancement of clinical and experimental tools, I believe this work provides a roadmap for future therapies for epilepsy patients

In vitro characterisation and modulation of evolving epileptiform activity

PhD ThesisUnderstanding the role of different neuronal populations in the evolution of epileptic activity remains a major goal for epilepsy research. Physiological neuronal networks may become hyperexcitable if they tip over some apparent threshold level of excitation, or below some threshold level of inhibition, although this process, termed ictogenesis is not understood. This hyperexcitable state of the network underlies the pathological condition of epilepsy. Clinical evidence suggests strongly that different regions in the brain have different epileptic-activity patterns and seizure susceptibility. The reasons for this differential susceptibility, however, are also not known. In this thesis, two widely used in vitro models of epilepsy were used – zero-magnesium, and 4-aminopyridine (4AP) models – to characterise the evolution of epileptiform activity in naïve cortical networks in different regions of brain slices taken from wild-type mice. Various metrics were then used to develop assays for measuring (1) the action of disease-modifying drugs and (2) the effects of genetic mutations on seizure susceptibility. Lastly, the firing properties of neocortical parvalbumin-positive (PV+) interneurons in 4AP were characterised. Different cortical areas showed notable differences in seizure susceptibility and activity patterns in the two models. In zero-magnesium, development of epileptiform activity in hippocampal regions facilitated transformation of early-stage epileptiform activity to late-stage in the neocortex. Furthermore, activity in the hippocampus entrained neocortical events, and this phenomenon was mediated, at least in part, by non-synaptic mechanisms, providing strong evidence for propagation through non-synaptic pathways. The effects of diazepam and baclofen were also examined. They showed distinct effects on different cortical areas. Pharmacological suppression of glial functions induced spontaneous activity patterns, and also affected the development of epileptiform activity in the neocortex. Lastly, 4AP was found to alter the firing capability of PV+ interneurons in an input intensity-dependent manner, and induced spontaneous membrane potential oscillations