Elsevier

Neurobiology of Disease

Volume 65, May 2014, Pages 133-141
Neurobiology of Disease

Optogenetic inhibition of chemically induced hypersynchronized bursting in mice,☆☆,

https://doi.org/10.1016/j.nbd.2014.01.015Get rights and content

Highlights

  • Seizures caused by PTX in vitro are blocked by principal cell optogenetic silencing.

  • Bicuculline-induced seizures in vivo are reduced by principal cell optogenetic silencing.

  • Optogenetic silencing of principal cells arrests seizures caused by reduced inhibition.

Abstract

Synchronized activity is common during various physiological operations but can culminate in seizures and consequently in epilepsy in pathological hyperexcitable conditions in the brain. Many types of seizures are not possible to control and impose significant disability for patients with epilepsy. Such intractable epilepsy cases are often associated with degeneration of inhibitory interneurons in the cortical areas resulting in impaired inhibitory drive onto the principal neurons. Recently emerging optogenetic technique has been proposed as an alternative approach to control such seizures but whether it may be effective in situations where inhibitory processes in the brain are compromised has not been addressed. Here we used pharmacological and optogenetic techniques to block inhibitory neurotransmission and induce epileptiform activity in vitro and in vivo. We demonstrate that NpHR-based optogenetic hyperpolarization and thereby inactivation of a principal neuronal population in the hippocampus is effectively attenuating seizure activity caused by disconnected network inhibition both in vitro and in vivo. Our data suggest that epileptiform activity in the hippocampus caused by impaired inhibition may be controlled by optogenetic silencing of principal neurons and potentially can be developed as an alternative treatment for epilepsy.

Introduction

Epilepsy is a heterogeneous chronic neurological disorder with a prevalence of up to 1% in the general population (Duncan et al., 2006, Sander, 2003). While drug treatment is successful in a majority of cases, certain types of epilepsies are not as easily controlled. About 30–40% of patients with one of the most common forms of epilepsy, temporal lobe epilepsy (TLE), are drug resistant (Duncan et al., 2006, Engel, 2001). This puts demand on developing alternative treatment strategies.

It has been proposed that epileptic seizures could be the result of synchronization of principal cells by interneurons (Avoli and de Curtis, 2011, Isomura et al., 2008) through activation of GABAA receptors. However, both experimental models and human temporal epilepsies are often associated with degeneration or dysfunction of various inhibitory interneuron populations (de Lanerolle et al., 1989, Malmgren and Thom, 2012, Morimoto and Goddard, 1986, Zhang and Buckmaster, 2009). This is believed to significantly reduce inhibitory drive on pyramidal neurons and thereby promote hyperexcitability and seizures (Kumar and Buckmaster, 2006, Morimoto, 1989, Zhang and Buckmaster, 2009). Additional support for this assumption comes from experimental data showing induction of epileptiform activity, both in vitro and in vivo, by application of GABAA receptor antagonists (Hwa et al., 1991, Miles and Wong, 1983, Piredda et al., 1985, Strombom et al., 1979) indicating that one of the main consequences of inhibitory interneuron degeneration leading to seizures is decreased GABAA receptor-mediated inhibition in the epileptic network. We have previously demonstrated that optogenetic hyperpolarization (Zhang et al., 2007) of principal neurons by light-induced activation of NpHR (Halorhodopsin) can counteract synchronized epileptiform activity generated by electrical stimulation when GABAergic transmission in the hippocampus is not compromised (Tonnesen et al., 2009). We now hypothesized that in a scenario where interneuron inhibitory function is compromised by blockade of GABAA receptors, resulting in epileptiform activity presumably driven by principal cells, hyperpolarization of these cells by optogenetic approach would be strong enough to stop epileptiform activity. Indeed, we demonstrate that epileptiform bursting in the hippocampus induced by GABAA receptor antagonists is attenuated by hyperpolarization of principal neurons using an optogenetic strategy both in vitro and in vivo.

Section snippets

Animals and surgery

Female FVB mice (Charles River) were housed according to local animal house standards with 12-hour light/dark cycles and food & water ad libitum. All procedures followed ethical permits approved by the Malmö/Lund Animal Research Ethics Board. Animals were anesthetized with 1.5–2.5% isoflurane (4% at induction) (Baxter), 0.5 mL bupivacaine (Marcain, AstraZeneca) was used as local anesthetic and Chlorhexidine (Fresenius Kabi) was used for wound cleaning. During and after surgery, physiological

NpHR expression in the hippocampus

The AAV vector construct with the NpHR gene (enhanced version 3.0 (Gradinaru et al., 2010)) and human Synapsin (hSyn) promoter, driving expression in both excitatory and inhibitory neurons (Bogen et al., 2009), was injected into the ventral hippocampus several weeks before in vitro or in vivo experiments to allow for optimal and widespread transgene expression. The hSyn promoter was chosen to minimize GABA release from interneurons during the light illumination. The expression of NpHR3.0 in the

Discussion

Here we show for the first time that when hyperexcitability in the hippocampus is induced by compromised inhibitory drive onto the principal neurons, optogenetic hyperpolarization of principal cells attenuates epileptiform activity both in vitro and in vivo.

Optogenetic silencing of principal neurons in organotypic hippocampal cultures has been shown to suppress stimulation-induced bursting (Tonnesen et al., 2009), with silencing achieved by yellow light illumination of slices selectively

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      Optogenetic hyperpolarization of these cells prevented generation of action potentials and epileptiform discharge in an in vitro seizure model. Later, studies from different groups verified that optogenetic inhibition of excitatory principal cells in the hippocampus significantly delayed the initiation of status epilepticus in a pilocarpine-induced seizure model (Sukhotinsky et al., 2013) and greatly control seizure activities in an in vivo mouse model of TLE (Berglind et al., 2014; Sukhotinsky et al., 2013). In addition, Osawa et al. (2013) found that optogenetic activation of hippocampal excitatory neurons directly induced focal seizure-like afterdischarges in rat hippocampus (Osawa et al., 2013), providing additional evidence of the potential role of hippocampal excitatory neurons in seizure genesis.

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    Funding for the research was provided by: the Swedish Research Council, the Swedish Brain Foundation, the Nanometer Structure Consortium at Lund University, the Thorsten and Elsa Segerfalk Foundation, the Johan and Greta Kock Foundation and the Royal Physiographic Society in Lund.

    ☆☆

    The funding agencies had no role in any element of planning, data collection, analysis or writing of this research paper.

    The authors declare no competing financial interests.

    1

    Present address: Institute of Experimental Medicine, Hungarian Academy of Sciences, 1083 Budapest, Hungary.

    2

    Present address: McGovern Institute for Brain Research, Massachusetts Institute of Technology, 02139 Cambridge, MA, USA.

    3

    Present address: Department of Neuroscience “B.B. Brodie”, University of Cagliari, Cittadella di Monserrat, SS554 bivio per Sestu, Monserrato, Italy.

    4

    Present address: Swammerdam Institute for Life Sciences, Amsterdam University, 1098 XH Amsterdam, The Netherlands.

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