Seizures are thought to originate from a failure of inhibition to quell hyperactive neural circuits, but the nature of this failure remains unknown. Here we combine high-speed two-photon imaging with electrophysiological recordings to directly evaluate the interaction between populations of interneurons and principal cells during the onset of seizure-like activity in mouse hippocampal slices. Both calcium imaging and dual patch clamp recordings reveal that in vitro seizure-like events (SLEs) are preceded by pre-ictal bursts of activity in which interneurons predominate. Corresponding changes in intracellular chloride concentration were observed in pyramidal cells using the chloride indicator Clomeleon. These changes were measurable at SLE onset and became very large during the SLE. Pharmacological manipulation of GABAergic transmission, either by blocking GABAA receptors or by hyperpolarizing the GABAA reversal potential, converted SLEs to short interictal-like bursts. Together, our results support a model in which pre-ictal GABAA receptor-mediated chloride influx shifts EGABA to produce a positive feedback loop that contributes to the initiation of seizure activity.

We describe a simple two-photon fluorescence imaging strategy, called targeted path scanning (TPS), to monitor the dynamics of spatially extended neuronal networks with high spatiotemporal resolution. Our strategy combines the advantages of mirror-based scanning, minimized dead time, ease of implementation, and compatibility with high-resolution low-magnification objectives. To demonstrate the performance of TPS, we monitor the calcium dynamics distributed across an entire juvenile rat hippocampus (>1.5mm), at scan rates of 100Hz, with single cell resolution and single action potential sensitivity. Our strategy for fast, efficient two-photon microscopy over spatially extended regions provides a particularly attractive solution for monitoring neuronal population activity in thick tissue, without sacrificing the signal to noise ratio or high spatial resolution associated with standard two-photon microscopy. Finally, we provide the code to make our technique generally available.

Synchronous activation of neural networks is an important physiological mechanism, and dysregulation of synchrony forms the basis of epilepsy. We analyzed the propagation of synchronous activity through chronically epileptic neural networks. Electrocortigraphic recordings from epileptic patients demonstrate remarkable variance in the pathways of propagation between sequential interictal spikes (IIS). Calcium imaging in chronically epileptic slice cultures demonstrates that pathway variance depends on the presence of GABAergic inhibition and that spike propagation becomes stereotyped following GABA-R blockade. Computer modeling suggests that GABAergic quenching of local network activations leaves behind regions of refractory neurons, whose late recruitment forms the anatomical basis of variability during subsequent network activation. Targeted path scanning of slice cultures confirmed local activations, while ex vivo recordings of human epileptic tissue confirmed the dependence of interspike variance on GABA-mediated inhibition. These data support the hypothesis that the paths by which synchronous activity spread through an epileptic network change with each activation, based on the recent history of localized activity that has been successfully inhibited.

In secondary epilepsy, a seizure-prone neural network evolves during the latent period between brain injury and the onset of spontaneous seizures. The nature of the evolution is largely unknown, and even its completeness at the onset of seizures has recently been challenged by measures of gradually decreasing intervals between subsequent seizures. Sequential calcium imaging of neuronal activity, in the pyramidal cell layer of mouse hippocampal in vitro preparations, during early post-traumatic epileptogenesis demonstrated rapid increases in the fraction of neurons that participate in interictal activity. This was followed by more gradual increases in the rate at which individual neurons join each developing seizure, the pairwise correlation of neuronal activities as a function of the distance separating the pair, and network-wide measures of functional connectivity. These data support the continued evolution of synaptic connectivity in epileptic networks beyond the latent period: early seizures occur when recurrent excitatory pathways are largely polysynaptic, while ongoing synaptic remodeling after the onset of epilepsy enhances intranetwork connectivity as well as the onset and spread of seizure activity.