pontaneous, and recurrent seizures or ictal events. Typically, inter-ictal events or large bouts of population level activity can be measured between seizures and are generally asymptomatic. Decades of research have focused on understanding the mechanisms leading to the development of seizure-like activity using various pro-convulsive pharmacological agents, including 4-aimnopyridine (4AP). However, the lack of consistency in the concentrations used for studying 4AP-induced epileptiform activity in animal models may give rise to differences in results and interpretation thereof. Indeed, the range of 4AP concentration in both in vivo and in vitro studies varies from 3 µM to 40 mM. Here, we explored the effects of various 4AP concentrations on the development and characteristics of hippocampal epileptiform activity in acute mouse brain slices of either sex. Using multi-electrode array recordings, we show that 4AP induces hippocampal epileptiform activity for a broad range of concentrations. The frequency component and the spatiotemporal patterns of the epileptiform activity revealed a dosedependent response. Finally, in the presence of 4AP, reduction of KCC2 co-transporter activity by KCC2 antagonist VU0240551 prevented the manifestation of the frequency component differences between different concentrations of 4AP. Overall, the study predicts that different concentrations of 4AP can result in the different mechanisms behind hippocampal epileptiform activity, of which some are dependent on the KCC2 co-transporter function.

K+-channels regulate the intrinsic excitability of neurons and maintain the repolarization phase of action potentials. Specifically, the IA and ID seem to account for nearly all of the K+-current during this repolarization phase in principal neurons. Due to the roles of these two currents in the regulation of neuronal excitability, they have been the focus of decades of intense study with the hope of gaining a better understanding of the mechanisms leading to the development of epilepsy. In this new study, we tested the hypothesis that the effect of IA and ID channel antagonist 4-aminopyridine (4AP) is strongly concentration-dependent. Different 4AP concentrations are characterized by qualitatively different spatiotemporal patterns of epileptiform activity in acute mouse hippocampal brain slices. Our in vitro data suggests that the spatiotemporal propagation, underlying frequency components, and inter-event intervals of 4AP-induced epileptiform activity vary with concentration. Furthermore, high concentrations of 4AP lead to the appearance of the high frequency component in epileptiform bursts, which may be dependent on the activation of the KCC2 co-transporter. We found that for all concentrations used in our study, epileptiform activity originated in the CA3 hippocampal subfield before propagating to CA1 and DG. This result is similar to previously reported findings that the CA3 region is the initiator of epileptiform activity induced by elevated K+. The local architecture of the CA3 makes it a prime candidate for seizure generation. Indeed, the pyramidal neurons within the CA3 exhibit a high degree of recurrent connectivity. The differences in the spatiotemporal patterns of epileptiform activity arise during the application of “transition” 4AP concentrations (75- 100 µM) and more so at high concentrations (125-200 µM). Low 4AP concentrations (25-50 µM) generate longer lasting epileptiform bursts as compared to those induced by transitional and high concentrations. This may be due to altered excitability of hippocampal pyramidal neurons in response to reduced IA and ID. Previous studies in cultured pyramidal neurons showed that the elimination of IA, by expression of Kv-4.2 dominant negative mutant, resulted in increased neuronal excitability to low-amplitude current stimulation. Interestingly, large-amplitude current injections generated transient spiking before the neuron ceased spiking activity. This reduction of prolonged spiking as a result of IA reduction may explain the reduced durations of epileptiform bursts seen in at higher 4AP concentrations.

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