Repetitive low-frequency stimulation reduces epileptiform synchronization in limbic neuronal networks

Abstract

Deep-brain electrical or transcranial magnetic stimulation may represent a therapeutic tool for controlling seizures in patients presenting with epileptic disorders resistant to antiepileptic drugs. In keeping with this clinical evidence, we have reported that repetitive electrical stimuli delivered at approximately 1 Hz in mouse hippocampus-entorhinal cortex (EC) slices depress the EC ability to generate ictal activity induced by the application of 4-aminopyridine (4AP) or Mg²⁺-free medium (Barbarosie, M., Avoli, M., 1997. CA3-driven hippocampal–entorhinal loop controls rather than sustains in vitro limbic seizures. J. Neurosci. 17, 9308–9314.). Here, we confirmed a similar control mechanism in rat brain slices analyzed with field potential recordings during 4AP (50 μM) treatment. In addition, we used intrinsic optical signal (IOS) recordings to quantify the intensity and spatial characteristics of this inhibitory influence. IOSs reflect the changes in light transmittance throughout the entire extent of the slice, and are thus reliable markers of limbic network epileptiform synchronization. First, we found that in the presence of 4AP, the IOS increases, induced by a train of electrical stimuli (10 Hz for 1 s) or by recurrent, single-shock stimulation delivered at 0.05 Hz in the deep EC layers, are reduced in intensity and area size by low-frequency (1 Hz), repetitive stimulation of the subiculum; these effects were observed in all limbic areas contained in the slice. Second, by testing the effects induced by repetitive subicular stimulation at 0.2–10 Hz, we identified maximal efficacy when repetitive stimuli are delivered at 1 Hz. Finally, we discovered that similar, but slightly less pronounced, inhibitory effects occur when repetitive stimuli at 1 Hz are delivered in the EC, suggesting that the reduction of IOSs seen during repetitive stimulation is pathway dependent as well as activity dependent. Thus, the activation of limbic networks at low frequency reduces the intensity and spatial extent of the IOS changes that accompany ictal synchronization in an in vitro slice preparation. This conclusion supports the view that repetitive stimulation may represent a potential therapeutic tool for controlling seizures in patients with pharmacoresistant epileptic disorders.

Introduction

Over the last few years, several attempts have been made to use deep-brain electrical (Velasco et al., 2000a, Velasco et al., 2000b, Velasco et al., 2001, Vonck et al., 2002, Yamamoto et al., 2002) or transcranial magnetic stimulation (Menkes and Gruenthal, 2000, Tergau et al., 1999) to abate seizures in patients presenting with epileptic disorders resistant to antiepileptic drugs, including mesial temporal lobe epilepsy (MTLE). These stimulating procedures have been often proved to be effective in reducing and/or abolishing both interictal and ictal discharges. However, these experiments included a limited number of patients. Moreover, two fundamental points for establishing a solid scientific rationale are as yet missing: first, which brain structure should be stimulated in a specific epileptic disorder; second, what patterns of stimulation (e.g., frequency or intensity) have to be used.

By analyzing the mechanisms of ictogenesis in combined mouse entorhinal cortex (EC)-hippocampus slices superfused with 4-aminopyridine (4AP) or Mg²⁺-free medium, we have found that CA3-driven interictal activity inhibits the EC propensity to generate ictal discharges resembling the electrographic limbic seizures seen in MTLE patients (Barbarosie and Avoli, 1997, Barbarosie et al., 2002). Moreover, we discovered that when this inhibitory control is removed by cutting the Schaffer collaterals (which represent the main output pathway for CA3 pyramidal cells), electrical stimuli delivered in the subiculum at frequencies similar to those of CA3-driven interictal discharges are capable of depressing EC ictogenesis. Similar evidence has recently been obtained in the EC of rat brain slices in which repetitive stimuli were delivered in the lateral–basolateral nucleus of the amygdala (Benini et al., 2003). Overall, these data indicate that repetitive electrical stimuli of parahippocampal networks (which were surgically separated from the CA3 outputs) can mimic the inhibitory control exerted on EC ictogenesis by CA3-driven output activity. In addition, they suggest that this in vitro experimental model may be used for analyzing the mechanisms by which repetitive stimulation controls the generation and spread of ictal activity within limbic neuronal networks.

Here, we have further characterized the effects induced by repetitive electrical stimulation on ictal synchronization in vitro by using standard field potential recordings along with intrinsic optical signals (IOSs) in rat hippocampus-EC slices superfused with 4AP-containing medium. IOSs recorded in vitro reflect the changes in light-scattering properties and absorption that are secondary to cell swelling leading to alterations in the extracellular space volume (Andrew and MacVicar, 1994, Buchheim et al., 1999, Buchheim et al., 2000, Holthoff and Witte, 1996, Hochman et al., 1995, MacVicar and Hochman, 1991, Meierkord et al., 1997; see, for review, Andrew et al., 1999). Moreover, excitatory synaptic transmission represents a prerequisite for IOS generation (D'Arcangelo et al., 2001, Dodt et al., 1996, MacVicar and Hochman, 1991). Although it is known that the time course of IOSs is much slower than what obtained with field potential recordings (cf. Dodt et al., 1996, Buchheim et al., 2000, Holthoff and Witte, 1996, Hochman et al., 1995, Meierkord et al., 1997), this method can provide valuable quantitative information on the intensity and spatial characteristics of the inhibitory influence that is induced by repetitive electrical stimuli over the entire extent of the slice.

Since spontaneous epileptiform activity is uncommon in the submerged slice preparation (D'Arcangelo et al., 2001, Meierkord et al., 1997), discharges resembling ictal epileptiform events in field potential recordings were elicited during 4AP application by focal extracellular stimuli. In particular, we were interested to answer the following questions: (i) which is the extent of the inhibitory influence exerted by repetitive stimulation on ictogenesis in different limbic areas that are comprised in the brain slice? (ii) which are the repetitive stimulation frequencies that are most effective in reducing ictogenesis? and (iii) are these inhibitory effects input and/or activity dependent?