Changes in action potential features during focal seizure discharges in the entorhinal cortex of the in vitro isolated guinea pig brain Federica Trombin, Vadym Gnatkovsky, and Marco de Curtis Unit of Experimental Neurophysiology and Epileptology, Fondazione Istituto Neurologico Carlo Besta, Milan, Italy Submitted 14 March 2011; accepted in final form 9 June 2011 Trombin F, Gnatkovsky V, de Curtis M. Changes in action poten- tial features during focal seizure discharges in the entorhinal cortex of the in vitro isolated guinea pig brain. J Neurophysiol 106: 1411–1423, 2011. First published June 15, 2011; doi:10.1152/jn.00207.2011.—Temporal lobe seizures in humans correlate with stereotyped electrophysiolog- ical patterns that can be reproduced in animal models to study the cellular and network changes responsible for ictogenesis. Seizure-like discharges that mimic seizure patterns in humans were induced in the entorhinal cortex of the in vitro isolated guinea pig brain by 3-min arterial applications of the GABA A receptor antagonist bicuculline. The onset of seizure is characterized by a paradoxical interruption of firing for several seconds in principal neurons coupled with both enhanced interneuronal firing and increased extracellular potassium (Gnatkovsky et al. 2008). The evolution of action potential features from firing break to excessive and synchronous activity associated with the progression of seizure itself is analyzed here. We utilized phase plot analysis to characterize action potential features of ento- rhinal cortex neurons in different phases of a seizure. Compared with preictal action potentials, resumed spikes in layer II–III neurons (n = 17) during the early phase of the seizure-like discharge displayed 1) depolarized threshold, 2) lower peak amplitude, 3) depolarized voltage of repolarization and 4) decelerated depolarizing phase, and 5) spike doublettes. Action potentials in deep-layer principal cells (n = 8) during seizure did not show the marked feature changes observed in superficial layer neurons. Action potential reappearance correlated with an increase in extracellular potassium. High-threshold, slow-action potentials similar to those observed in the irregular firing phase of a seizure were reproduced in layer II–III neurons by direct cortical application of a highly concentrated potassium solution (12–24 mM). We propose that the generation of possibly nonsomatic action potentials by increased extracellular potassium represents a crucial step toward reestablish firing after an initial depression in an acute model of temporal lobe seizures. Resumed firing reengages principal neurons into seizure discharge and promotes the transition toward the synchronized burst firing that characterizes the late phase of a seizure. phase plot analysis; intracellular recordings THE STUDY OF SEIZURE GENERATION (ictogenesis) is one of re- search priorities recognized by the international epilepsy re- search community (Baulac and Pitkanen 2009). A better un- derstanding of seizure initiation and progression will possibly lead to new strategies to cure seizures resistant to available treatments. Focal seizure patterns recorded with intracranial electrodes (Engel 1993) during presurgical studies aimed at defining the boundaries of the epileptogenic region in pharma- coresistant patients demonstrated that seizure onset is often characterized by an abrupt waning of the background activity coupled with the emergence of fast rhythms in the / range (for review, see de Curtis and Gnatkovsky 2009). This pattern is followed by a period of irregular activity that becomes progressively larger in amplitude and more synchronous. Within seconds, the discharges organize in wide bursts regu- larly spaced that precede seizure ending and postictal depres- sion. A similar progression of events was observed in chronic animal models of focal epilepsy (Bragin et al. 1999; Kharat- ishvili et al. 2006; Williams et al. 2009; Kadam et al. 2010) and in models of seizures developed in vitro (Lopantsev and Avoli 1998; Avoli et al. 2006; for review, see de Curtis and Gnatk- ovsky 2009). We utilized an acute model of limbic lobe seizures developed on the in vitro-isolated brain of adult guinea pigs to reproduce the focal electrographic seizure pattern ob- served in humans (Uva et al. 2005). Transient and partial (30 – 40%) disinhibition induced in this preparation by a 3-min arterial infusion of the GABA A receptor antagonist bicuculline methiodide promoted focal seizures in the hippocampal-para- hippocampal region. These events were characterized at onset by fast activity at 20 –30 Hz, sequentially followed by irregular firing and rhythmic bursting (Gnatkovsky et al. 2008). In the same study, we showed that in the medial entorhinal cortex (EC) the fast activity observed at seizure onset was generated by enhanced synchronization of inhibitory networks, mediated by intense firing of putative interneurons, and correlated with the complete disruption of neuronal firing in principal neurons (Table 1). Similar enhancement in interneuronal firing and reduction of excitation ahead of seizures was also reported in other in vitro studies performed on either hippocampal slices (Dzhala and Staley 2003; Ziburkus et al. 2006; Fujiwara- Tsukamoto et al. 2007) or in toto hippocampal/EC preparations from immature rats (Derchansky et al. 2008). How this paradoxical blockade of firing in principal cells associated with strong interneuron firing at seizure onset can develop into the hypersynchronous activation of the bursting phase is an open question. On the basis of intracellular record- ings coupled with extracellular potassium ([K + ] o ) measure- ments, we hypothesized that the reappearance of neuronal firing in principal neurons could be accounted for by two synergic effects produced by the [K + ] o elevation: 1) a reduc- tion of the efficacy of GABAergic inhibition due to a depolar- ization of the GABA A receptor-mediated reversal potential (Thompson and Gahwiler 1989) and 2) the induction of regen- erative action potentials (APs) in principal cells. The latter hypothesis was verified in the present study by performing phase plot analysis of AP features in different populations of neurons during seizure progression. Address for reprint requests and other correspondence: M. de Curtis, Unit of Experimental Neurophysiology and Epileptology, Fondazione Istituto Neuro- logico Carlo Besta, via Celoria 11, 20133 Milan, Italy (e-mail: decurtis @istituto-besta.it). J Neurophysiol 106: 1411–1423, 2011. First published June 15, 2011; doi:10.1152/jn.00207.2011. 1411 0022-3077/11 Copyright © 2011 the American Physiological Society www.jn.org Downloaded from journals.physiology.org/journal/jn (034.238.191.092) on November 24, 2021.