Optogenetic inhibition of chemically induced hypersynchronized bursting in mice☆,☆☆,★
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.
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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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2020, World NeurosurgeryCitation Excerpt :First, in vitro studies showed that kainate-injected transgenic animals in which hippocampal slices were bathed in Mg-free artificial cerebrospinal fluid containing picrotoxin and 4-aminopyridine had an 80% reduction in epileptic bursting, compared with controls. However, in vivo studies were less impressive, showing a reduction in bicuculline-induced spikes by only 20%.57 Rather than directly modulating the seizure focus itself, which may comprise a large and diffuse network, optogenetics has been used to modulate seizures by targeting other areas of the brain that may interact with the seizure network.
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2019, Pharmacology and TherapeuticsCitation Excerpt :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.
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The funding agencies had no role in any element of planning, data collection, analysis or writing of this research paper.
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The authors declare no competing financial interests.
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Present address: Institute of Experimental Medicine, Hungarian Academy of Sciences, 1083 Budapest, Hungary.
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Present address: McGovern Institute for Brain Research, Massachusetts Institute of Technology, 02139 Cambridge, MA, USA.
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Present address: Department of Neuroscience “B.B. Brodie”, University of Cagliari, Cittadella di Monserrat, SS554 bivio per Sestu, Monserrato, Italy.
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Present address: Swammerdam Institute for Life Sciences, Amsterdam University, 1098 XH Amsterdam, The Netherlands.