Elsevier

Brain Stimulation

Volume 7, Issue 6, November–December 2014, Pages 890-899
Brain Stimulation

Other
Original Article
Seizure Suppression by High Frequency Optogenetic Stimulation Using In Vitro and In Vivo Animal Models of Epilepsy

https://doi.org/10.1016/j.brs.2014.07.034Get rights and content

Highlights

  • High frequency optogenetic stimulation induces acute seizure suppression.

  • Optical stimulation suppresses focal and distal epileptiform activity.

  • Seizure suppression decreases over time but can be reinstated by intermittent stimulation.

  • GABA transmission activated by optical stimulation is implicated in seizure suppression.

Abstract

Background

Electrical high frequency stimulation (HFS) has been shown to suppress seizures. However, the mechanisms of seizure suppression remain unclear and techniques for blocking specific neuronal populations are required.

Objective

The goal is to study the optical HFS protocol on seizures as well as the underlying mechanisms relevant to the HFS-mediated seizure suppression by using optogenetic methodology.

Methods

Thy1-ChR2 transgenic mice were used in both vivo and in vitro experiments. Optical stimulation with pulse trains at 20 and 50 Hz was applied on the focus to determine its effects on in vivo seizure activity induced by 4-AP and recorded in the bilateral and ipsilateral-temporal hippocampal CA3 regions. In vitro methodology was then used to study the mechanisms of the in vivo suppression.

Results

Optical HFS was able to generate 82.4% seizure suppression at 50 Hz with light power of 6.1 mW and 80.2% seizure suppression at 20 Hz with light power of 2.0 mW. The suppression percentage increased by increasing the light power and saturated when the power reached above-mentioned values. In vitro experimental results indicate that seizure suppression was mediated by activation of GABA receptors. Seizure suppression effect decreased with continued application but the suppression effect could be restored by intermittent stimulation.

Conclusions

This study shows that optical stimulation at high frequency targeting an excitatory opsin has potential therapeutic application for fast control of an epileptic focus. Furthermore, electrophysiological observations of extracellular and intracellular signals reveled that GABAergic neurotransmission activated by optical stimulation was responsible for the suppression.

Introduction

Approximately 60 percent of epilepsy disorders are classified as partial epilepsy, of which temporal lobe epilepsy involving the hippocampus is the most common [1], [2]. Temporal lobe epilepsy is also among the most difficult to treat medically, often necessitating surgical resection of an epileptic focus. Electrical high frequency stimulation (HFS) is an alternative treatment for seizure disorders. Previous studies have shown that high frequency electrical stimulation can suppress seizures in animal models of epilepsy [3], [4], [5] and reduce seizure frequency in patients [6], [7], [8]. However, the mechanisms of seizure suppression remain unclear, and multiple mechanisms are most likely involved [9], [10], [11], [12].

Optogenetics provides a possible alternative treatment for epilepsy by allowing for the reversible excitation and inhibition of neurons with millisecond time resolution using light-activated ion channels and pumps expressed in target cell populations. Cell activation can be achieved using channelrhodopsin-2 (ChR2), a light-gated cation channel isolated from the algae Chlamydomonas reinhardtii that has been successfully expressed in mammalian neurons [13]. After illumination with blue light, ChR2 opens to allow the passive movement of Na+, H+, Ca2+ and K+ ions, causing depolarization of the cell membrane [14]. The light-activated chloride pump, halorhodopsin (NpHR), that is naturally expressed by the halobacterium Natronomonas pharaonis [15], can cause membrane hyperpolarization and inhibition of action potential firing in neurons after exposure to yellow light [16]. Given that seizure disorders result from excessive neuronal activity, common optogenetic strategies currently being investigated for the treatment of epilepsy are to inhibit excitatory neurons using NpHR or to excite inhibitory neurons using ChR2 that is selectively expressed in these cells [17]. Previously, NpHR expression in the hippocampal formation was shown to provide sufficient inhibition to curtail excessive hyper-excitability induced by an electrical stimulus burst in organotypic slice cultures [18]. Similarly, optical activation of NpHR in neurons at the site of an epileptic focus transduced using lentiviral gene delivery can attenuate electrographic seizures in a rodent model of focal neocortical epilepsy using open-loop optical stimulation paradigms [19]. Closed-loop control using seizure detection algorithms to apply optical stimulation only at seizure onset has also been shown to be effective to suppress seizures either by temporarily inhibiting pyramidal neurons [20] or through the activation of a sub-population of GABAergic inter-neurons [21]. These studies indicate that seizures can be aborted and hyper-excitability suppressed by optical stimulation that could induce either neural activation or inhibition.

In the present study, the effects of optical HFS protocol on seizures as well as the underlying mechanisms relevant to the HFS-mediated seizure suppression were evaluated. ChR2 expression driven by the Thy-1 promoter is present both in excitatory and inhibitory neurons [22] and is well suited for studying the mechanism of seizure suppression as compared with electrical HFS that activates both excitatory and inhibitory neurons.

Section snippets

Animals

Thy1-ChR2-YFP transgenic mice [22] were used in this study. In the in vitro experiments, mice were used at the age of approximately postnatal day 14 (P14, range P11–P16). In the in vivo experiments, adult mice were used at age P90–P110. All experimental protocols were reviewed and approved by the Institution Animal Care and Use Committee at Case Western Reserve University.

In vitro preparation for hippocampal recordings

Transgenic and wildtype mice were anesthetized by isoflurane inhalation and decapitated. 350 μm transverse hippocampal

Optically-evoked potentials in the hippocampus

To understand the optical response in the normal condition, the laser light was first applied on wild type and Thy1-ChR2 mice without seizure induction. Optical stimulation in-vivo in wild type mice did not induce any activity (Fig. 1A). However, stimulation in Thy1-ChR2 mice could elicit a hippocampal evoked potentials in the in vivo preparation for stimulation for 1, 20, and 50 Hz trains (Fig. 1B–D). The field potential responses to optical stimulation were biphasic, with a steep negative

Discussion

Epilepsy has been thought to result from hyper-excitability spreading to an increasing number of neurons in the neural network [23]. Therefore, optogenetic strategies are commonly aimed at inhibiting the excitatory neurons using a NpHR construct or at activating inhibitory neurons by means of ChR2 expression. Previous studies have supported these strategies and showed effective seizure suppression in different parts of the brain [18], [19], [20], [21]. However, a different optogenetic strategy

Conclusion

In this study, HFS was applied through an optogenic construct to suppress epileptiform activity in vitro and in vivo reaching maximum suppression ratios of 70 and 82.4%, respectively. The local suppressive effect in CA3 spreads contra-laterally and longitudinally across the whole hippocampus from a single site of excitation. Histological and electrophysiological experiments show that this suppression was mediated through GABAA receptors. Furthermore, we show that on/off intermittent stimulation

Acknowledgment

This research was supported by the National Institutes of Health (NINDS grant #: 2R01NS060757-05A1).

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