OtherOriginal ArticleSeizure Suppression by High Frequency Optogenetic Stimulation Using In Vitro and In Vivo Animal Models of Epilepsy
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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The first two authors have equal contribution.