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Optogenetic Treatment of Epilepsy

Posted in Management Topics in Epilepsy on 13th Mar 2013

Author

Laura Mantoan/Dimitri Kullmann

Laura Mantoan is a Neurology Specialist Registrar at the National Hospital for Neurology and Neurosurgery and St George's Hospital. She completed a PhD with Dimitri Kullmann at UCL using optogenetic techniques to study hippocampal oscillations and inhibit epileptiform activity. Her research interests are viral and optogenetic treatment approaches for epilepsy.

Dimitri Kullmann is a Professor of Neurology and Consultant Neurologist at the UCL Institute of Neurology and National Hospital, Queen Square. His research interests span fundamental mechanisms of synaptic transmission, computational properties of simple neural circuits, and alterations in neuronal and circuit excitability in epilepsy and other neurological disorders.

Correspondence to:
Laura Mantoan, Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK.
Email.Laura Mantoan

Pharmacoresistant epilepsy is common, and surgery to remove the epileptogenic zone is only indicated for a minority of patients referred for consideration of such treatment. Although some progress has been achieved with gene therapy in experimental models of epilepsy, this is usually considered irreversible, in that the excitability of neurons or their synaptic properties are permanently altered. Here we discuss an alternative experimental approach that potentially offers the ability to suppress seizures ‘on demand’, while leaving neuronal and synaptic functions intact the rest of the time. It relies on the ability to inhibit neurons by activating light-sensitive prokaryotic membrane proteins that act as ion channels and transporters. Substantial practical and regulatory obstacles will need to be overcome before optogenetics can be brought to the clinic. Nevertheless, it offers the prospect of temporal, regional and cellular specificity, which cannot be achieved by other treatments.

Introduction

Epilepsy affects over 50 million people worldwide, of whom only 60-70% are seizure free on medication.1 Patients who have failed to respond to adequate doses of two first-line drugs have a less than 20% expectation of achieving seizure freedom with the addition of a new antiepileptic drug.2 Pharmacoresistance is common and resective surgery is only appropriate when the epileptogenic zone does not involve eloquent cortex.3 Because seizures are intermittent, developing a method for rapid and reversible suppression of activity in a restricted area of neocortex would be an important advance, but progress in local manipulation of brain excitability has been slow, and is mainly focused on electrical brain stimulation,4 focal brain cooling5 or targeted drug delivery.6

A potentially powerful alternative way to suppress seizure activity ‘on demand’ is to photo-activate prokaryotic light-sensitive ion channels and transporters known as opsins, expressed in neurons.7,8 Opsins are a family of photosensory receptors found in all animal kingdoms, where they subserve a wide diversity of functions: from phototaxis in algae to eyesight in vertebrates. ‘Optogenetics’ is a novel technology that relies on optical control of opsins targeted to living cell membranes by gene transfer. This technique has revolutionised large areas of neuroscience in recent years, allowing specific and minimally invasive control of neuronal function that cannot be achieved with electrophysiology alone. Optogenetics has been used to manipulate the firing of specific classes of neurons in vitro9,10 and in intact brain tissue in vivo, in vertebrate11-13 and invertebrate14 models. Some recent applications have focused on opsins as potential therapeutic tools.13,15,16 Building upon these recent technological advances, we and other groups have investigated the therapeutic potential of optogenetic tools to inhibit epileptic activity in vitro and in vivo.

Opsins and optogenetic tools

Opsins are a family of proteins that combine with the vitamin-A derived chromophore retinal (or retinaldehyde). Many photosensory receptors, such as our own visual pigments, are opsins. They deliver the information carried by light to organisms by absorbing single photons, and are the molecular basis for a variety of light-sensing systems from phototaxis in flagellates to eyesight in animals. They were first successfully harnessed as a tool to control neuronal firing by G Miesenboeck’s team at Yale.17 Since then, several other groups have contributed to methodological developments and applications of this technology. Among the most prominent are those of K Deisseroth at Stanford University, G Nagel at the Max Planck Institute for Biophysics, and E Boyden at MIT.10 In a remarkable series of experiments over only a few years they developed optogenetic tools with the necessary temporal resolution to manipulate the firing of neurons with millisecond precision.18,19

The first opsins to be widely adopted as optogenetic actuators in neuroscience were Channelrhodopsin-2 (ChR2) and Halorhodopsin (NpHR). Channelrhodopsin-2 is a light-switched cation-selective ion channel found in the green flagellate alga Chlamydomonas reinhardtii.20 The absorption spectrum of ChR2 has its maximum at ~460nm. When activated by blue light, ChR2 allows positive charge into the cell, depolarising the cell membrane and functioning as an important mediator of light control of phototaxis and the photophobic response in Chlamydomonas. ChR2 was chosen to attempt genetically targeted photostimulation with fine temporal resolution because of the efficacy and speed of its natural light-transduction mechanisms. A versatile gene delivery tool is lentivirus, derived from HIV, engineered to drive ChR2 expression with an appropriate promoter, and with the yellow fluorescent protein (YFP) gene fused to the C-terminus of ChR2 to visualise the expressed protein. Lentiviruses and adeno-associated viruses (AAVs) have been successfully used to target ChR2 to mammalian neurons. Expression of ChR2 was stable over weeks and safe, as it did not alter the electrical properties or survival of neurons.10 Furthermore, ChR2 could drive neuronal depolarisation without the addition of external cofactors, as the retinal present in the mammalian brain was shown to be sufficient to constitute a functional rhodopsin.10,22 Illumination with blue light induced rapid, large amplitude depolarising currents, which rapidly deactivated after the light was switched off. We have confirmed that action potentials can be reliably elicited in hippocampal neurons recorded in acute brain slices from injected rats (Figure 1a). Pulsed optical activation of ChR2 was also able to elicit precise, repeatable spike trains in a single neuron (Figure 1b), and to drive sustained naturalistic trains of spikes in a physiologically relevant spike range (5-30Hz). Finally, ChR2 has also been shown to drive subthreshold depolarisations and to control excitatory and inhibitory synaptic transmission.10

epilepsy-fig1

Figure 1: Optogenetic experiments in vitro: Co-expression of ChR2 and NpHR allows bi-directional modulation of neuronal firing. Animals were injected with AAV-eNPAC, an adeno-associated virus carrying both ChR2 and NpHR fused with a GFP reporter gene to visualise cells expressing the opsins. (a) Sample trace of a current-clamped CA3 pyramidal neuron expressing AAV-eNPAC, showing depolarisation and action potentials elicited with 2 s pulses of 473nm laser light (irradiance 5mW/mm2). (b) 5ms laser pulses (left part of trace) reliably drove neuronal firing, and even 1ms pulses (right part of trace) elicited action potentials, albeit less reliably. (c) A hippocampal CA1 pyramidal neuron expressing AAV5-eNPAC was stimulated with a 593nm laser (13mW at 10x objective). Yellow light (400 ms pulse duration) hyperpolarised the membrane by approximately 2.5 mV (top trace), and inhibited action potentials elicited by brief current injections via the recording pipette (30pA, 20ms pulses – bottom trace). (d) Fluorescence micrograph showing the extent of expression in hippocampus injected with 1 μl AAV-eNPAC. Slices were counterstained with anti-GFP antibodies and AF 488 secondaries to amplify the GFP signal. Stratum radiatum (rad.), pyramidale (pyr.) and oriens (or).

A complementary high-speed hyperpolarising Cl–pump was discovered in the archaeon Natronomonas pharaonis (NpHR).9 NpHR has an excitation maximum in the yellow /green light spectrum near 580nm. In voltage-clamp experiments, illumination of NpHR-expressing cells with yellow light induced outward currents. We have confirmed that NpHR-mediated hyperpolarisation can abolish firing induced by depolarising current pulses (Figure 1c). Furthermore, as the absorption maxima of the two opsins are far apart, co-expression of NpHR and ChR2 in the same neurons can be combined to achieve bidirectional, independently addressable modulation of membrane potential by light of different wavelengths (Figure 1a-d).

Expression of microbial light-sensitive proteins has since been used to interrogate specific classes of neural cells, from cultured neurons to intact brain tissue in vivo.13,23 Targeting specific neuronal subpopulations has been achieved using cell-type specific promoters in viral vectors and in transgenic animals24 or cre-lox systems,25 or by combinations of these technologies.26,27 To allow optical stimulation in vivo, an integrated fibre-optic and optogenetic technology has been developed, and many laboratories now implant custom-made optical cannulae into brain areas following virus injection, or in transgenic mice, to target regions and circuits of interest.28

Applying optogenetics to epilepsy

An optimal therapeutic strategy for epilepsy would be minimally invasive, targeted to the epileptogenic zone, and only suppress neuronal activity when needed. The versatility and the electrophysiological characteristics of ChR2 and NpHR make optogenetic tools potent candidates to control neuronal firing in models of epilepsy and to provide insights into the pathophysiology of epileptic network organisation and synchronisation.

The first proof of concept that activation of NpHR could inhibit epileptiform activity came from M Kokaia’s group in Sweden, who used an in vitro model of pharmacoresistant epilepsy generated by electrical stimulation-induced burst firing in organotypic hippocampal cultures. They transduced principal neurons using a lentivirus carrying NpHR under the calcium calmodulin-binding kinase IIa (Camk2a) promoter. When NpHR was photoactivated with yellow light, neurons were hyperpolarised, preventing the generation of burst firing.15

We have recently asked whether such a strategy could be extended to suppress seizures in an established neocortical epileptic focus in vivo.29 Our long-term aim was to test the feasibility of a new approach to treatment for human focal neocortical epilepsy, and to provide the backbone for the development of other optogenetic neuromodulation therapies.

We used the tetanus toxin rat model of refractory focal neocortical epilepsy: toxin injected stereotactically to motor cortex of rodent brain is followed within a few days by spontaneous bursts of high-frequency EEG activity in the motor cortex, near the site of tetanus toxin injection, lasting over five weeks. We collaborated with K Hashemi (Brandeis University) who developed a wireless implantable EEG transmitter able to send a continuous EEG signal for several weeks.30 EEG spectral analysis revealed a large increase in high-frequency (>70Hz) power (Figure 2). Spontaneous seizures in this model are resistant to systemically delivered drugs, and are characterised by contralateral clonic movements, bilateral facial twitching, behavioural arrest, and head nodding.29,31 However, motor manifestations occurred at a frequency lower than the EEG bursts. In many respects the motor cortex tetanus toxin model resembles epilepsia partialis continua. We found that epileptogenesis was accompanied by persistent increases in the intrinsic excitability of layer five pyramidal neurons.29

epilepsy-fig3

Figure 2: The tetanus toxin model of focal epilepsy. Power values at different EEG frequency bands for control animals (n=5) and tetanus toxin (TeNT) injected animals (n=6) recorded on day 7-10 post injection (mean ± SEM): delta (0-4Hz), theta (4-8Hz), alpha (8-12Hz), beta (12-30Hz), low gamma (30-50Hz), high gamma (50-70Hz), high frequency activity (HFA) > 70Hz (displayed in two bands of HFA 70-120Hz and HFA 120-170Hz). The graph shows an increase in the HFA > 70Hz in TeNT injected animals.

In one group of experimental animals, we co-injected tetanus toxin together with 500 – 1,250nL high-titre lentivirus carrying NpHR under the Camk2a promoter to drive expression in excitatory neurons. Control animals were injected either with NpHR virus alone or with tetanus toxin together with a virus expressing only green fluorescent protein (GFP). An optical fibre was implanted above the injection site, as well as an electrode connected to the subcutaneous transmitter (Figure 3).

epilepsy-fig3

Figure 3: The optogenetic setup. Schematic of the implanted headstage for simultaneous EEG recording and optical stimulation.

We examined the effect of NpHR activation for a block of 1,000 seconds of intermittent 561nm laser light delivered via an optical fibre (20 s duration, 40 s duty cycle), and compared the EEG to a baseline 1,000 s period, and a subsequent 1,000 s after stopping the illumination. In order to quantify effects on the EEG we used several measures. Consistent results were obtained whether the data were analysed by measuring high frequency power (Figure 4), or by measuring the EEG coastline (effectively how much ink would be used to draw the EEG for a given duration), or by counting the rate of automatically detected epileptiform events (Figure 5).

Figure 4 below: Optogenetic suppression of neuronal excitability reduces high frequency activity in focal neocortical epilepsy. (a) Mean EEG power in the 120-160Hz band before, during and after laser stimulation in animals injected with tetanus toxin (TT) and NpHR lentivirus (n=6), showing a significant decrease. (b) Baseline HFA EEG power was lower in animals injected with NpHR lentivirus alone, and unaffected by laser illumination (green: mean ± SEM). (c) Laser illumination had no effect on HFA EEG power in control animals injected with TT together with either GFP-expressing control virus or fluorescent beads. (d) Representative EEG traces before, during and after 561 nm laser illumination, showing a decrease in HFA. Reproduced from ref. 29.

Figure 4 below: Optogenetic suppression of neuronal excitability reduces high frequency activity in focal neocortical epilepsy. (a) Mean EEG power in the 120-160Hz band before, during and after laser stimulation in animals injected with tetanus toxin (TT) and NpHR lentivirus (n=6), showing a significant decrease. (b) Baseline HFA EEG power was lower in animals injected with NpHR lentivirus alone, and unaffected by laser illumination (green: mean ± SEM). (c) Laser illumination had no effect on HFA EEG power in control animals injected with TT together with either GFP-expressing control virus or fluorescent beads. (d) Representative EEG traces before, during and after 561 nm laser illumination, showing a decrease in HFA. Reproduced from ref. 29.

 

Figure 5: Antiepileptic effect of NpHR assessed by coastline analysis and automated event detection. (a) Mean EEG coastline (sum of the absolute difference in voltage between consecutive sample points) length was significantly reduced by laser illumination in animals injected with TT/NpHR (symbols as in Fig. 4). (b) Baseline coastline was lower in animals injected with NpHR lentivirus alone, and unaffected by illumination. (c) EEG coastline length was unaffected by laser illumination in animals injected with TT together with GFP lentivirus or fluorescent beads. (d) Automated event classification used to detect bursts of high-frequency activity revealed a significant decrease upon laser illumination. Reproduced from ref. 29.

Figure 5: Antiepileptic effect of NpHR assessed by coastline analysis and automated event detection. (a) Mean EEG coastline (sum of the absolute difference in voltage between consecutive sample points) length was significantly reduced by laser illumination in animals injected with TT/NpHR (symbols as in Fig. 4). (b) Baseline coastline was lower in animals injected with NpHR lentivirus alone, and unaffected by illumination. (c) EEG coastline length was unaffected by laser illumination in animals injected with TT together with GFP lentivirus or fluorescent beads. (d) Automated event classification used to detect bursts of high-frequency activity revealed a significant decrease upon laser illumination. Reproduced from ref. 29.

In all cases NpHR photoactivation decreased the electrographic signature of seizures. We observed no behavioural side effects, and subsequent histological analysis confirmed that the fluorescent reporter was mainly expressed in principal cortical neurons with no evidence of abnormal cytoplasmic accumulations. No effect on the EEG was observed by laser illumination in animals injected either with the NpHR lentivirus alone or in animals injected with tetanus toxin without NpHR. These controls imply that the effect of photoactivation of NpHR was relatively selective for the abnormal high frequency activity seen in the focal epilepsy model.

Although our results provide the first evidence that focal neocortical seizures can be suppressed with optogenetics, it remains to be seen whether this effect can be harnessed to achieve a long-lasting decrease in seizure frequency or severity. Moreover, because we used a mild form of epilepsy with relatively with relatively few over motor seizures, we do not know whether generalisation of ictal activity can be prevented.

Towards a closed-loop optogenetic therapy for epilepsy

Ultimately, to take full advantage of optogenetics, the photoactivation could be triggered by the onset of a seizure, or even by an EEG signature of an impending seizure. In our study we were unable to ask if ’on demand’ seizure suppression could be achieved: the electrographic events were relatively brief, so that by the time they are detected by the automated algorithm it is too late to ask if laser activation could shorten them.

Two other groups have, however, very recently reported the development of a closed-loop system consisting of on-line detection of ictal activity coupled to lasers optically coupled to the animal. In one of these studies, from the Stanford group of J Huguenard,32 the investigators used a rat cortical stroke model that is followed by the delayed development of thalamocortical seizures resembling absence epilepsy.33 Photoactivating NpHR, expressed in thalamocortical neurons using AAV, terminated seizures and the associated behavioural arrest. The EEG signal used to trigger the laser was akin to the coastline measure that we have used, although easier to distinguish from background because of the large amplitude of the spike-wave complexes.

I Soltesz’s group at University of California Irvine, on the other hand, used a mouse model of temporal lobe epilepsy induced by unilateral intrahippocampal kainic acid injection, which is followed by the development of bilateral seizure foci. They also showed that temporal lobe seizures can be shortened either by NpHR-mediated inhibition of excitatory neurons or by activation of ChR2 in parvalbumin-positive inhibitory neurons in the hippocampus.34 In this case, the investigators exploited cre lox technology to restrict expression to one cell type or another. Interestingly, optogenetic manipulations either to the ipsilateral or to the contralateral hippocampus was successful, even when the EEG was recorded from the opposite hemisphere. This implies that the ‘mirror focus’ can be targeted with an anti-ictal effect at least in this rodent model.

Optogenetic inhibition as a future epilepsy treatment

We have demonstrated rapid and reversible suppression of epileptic EEG activity upon photoactivation of NpHR in a model of focal neocortical epilepsy.29 Other in vivo studies show that optogenetic treatment approaches for epilepsy are feasible in models of either thalamocortical32 or temporal lobe34 epilepsy. An optogenetic approach offers the prospect of aborting seizures without disrupting interictal brain function. However, when considering optogenetics as a therapeutic tool for human epilepsy, several challenges will need to be addressed. First, the safety of viral vectors needs to be established. Random insertion of lentiviral sequences into the genome in principle has the potential for mutagenesis and oncogenesis. Second, the level of transgene expression in targeted neurons can vary extensively. Third, because the opsins are non-mammalian membrane proteins they are potentially immunogenic, although there is no evidence so far that long-term expression of opsins causes an immune response,35 possibly because neurons reside in an immunologically privileged environment. Fourth, the timing and duration of illumination would need to be optimised and coupled to reliable seizure detection algorithms developed and validated in human epilepsy. Fifth, the spatial extent of viral transduction would need to be tailored to the individual, and as yet there is little agreement as to the size of the zone that generates seizures in human focal epilepsy. Finally, the hardware necessary to deliver light to the transduced area presents a substantial engineering challenge. Hitherto most of the work in vivo has used fibre-optic coupled lasers, but light-emitting diodes are showing promise, because of their size and energy efficiency. None of these challenges is insurmountable, and so we foresee the development of implantable devices analogous to automatic defibrillators that generate light pulses upon the automated electrographic detection of a seizure. This could lead to a radically new treatment alternative for a common and frequently devastating human disease.

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