The authors have declared that no competing interests exist.
Developed the model, conducted the experiments and wrote the first draft of the paper: IS. Performed immunohistochemical staining and part of the experiments: VRR. Designed and built the acquisition system: AMC SSC. Developed and contributed the vector: KD CR VG. Discussed the results and implications and reviewed and commented on the manuscript: IS AMC OJA VRR VG CR KD AKM SSC. Conceived and designed the experiments: SSC IS AKM. Performed the experiments: IS VRR. Analyzed the data: IS AMC OJA SSC. Contributed reagents/materials/analysis tools: KD CR VG. Wrote the paper: IS SSC AMC OJA.
Epilepsy is a devastating disease, currently treated with medications, surgery or electrical stimulation. None of these approaches is totally effective and our ability to control seizures remains limited and complicated by frequent side effects. The emerging revolutionary technique of optogenetics enables manipulation of the activity of specific neuronal populations
Epilepsy is one of the most common neurological disorders and has severe and devastating manifestations. It is characterized by recurrent seizures and is presently treated by medications, surgery or electrical stimulation. Unfortunately, our ability to control seizures with current therapies remains limited. Medications are the mainstay of treatment, but approximately 30% of patients continue to have seizures despite maximal medical management
New therapies that precisely act on specific circuit elements during seizures may be more effective than current options, and would minimize adverse side effects. Dramatic developments in using genetics for optical control of neuronal activity have raised the possibility of an entirely new method for studying and controlling seizures
In this exploratory study we tested the feasibility of the optogenetic approach in an
All procedures were performed in accordance with institutional and national guidelines for animal care and use for research purposes and the study protocol was approved by the Massachusetts General Hospital institutional review board (IACUC, protocol # 2009N000051).
Recombinant adeno-associated virus (AAV) vector carrying Halorhodopsin (eNpHR) gene tagged with a fluorescent marker protein (yellow fluorescent protein, YFP) under the control of the CaMKIIα promotor was prepared at the University of North Carolina Vector Core Facility (rAAV5/CaMKIIα-eNpHR3.0-eYFP). The titer of the concentrated virus for
In order to deliver the virus and to manipulate transduced neurons by light while recording neuronal activity we developed a custom designed injection-recording system. It consisted of a pedestal, electrodes and optical fiber guide, through which the viral vector and optical fiber were introduced during the experiment. A custom made plastic pedestal with 6 openings (Plastics One) was secured to the skull using dental acrylic cement (GlassLute, Pulpdent). Five openings were used to connect the reference, two cortical and two hippocampal electrodes. A 20 ga guide cannula was molded into the sixth opening to act as an optical fiber guide. The tip of the twisted electrode used to record from the hippocampus was aimed at the location of the injection. To inject the virus, a 26 ga injection cannula was lowered through the guide cannula into the hippocampus and the virus was deposited. During the experiment, an optical fiber (crossection 200 µm, BFL37–200, ThorLabs) was lowered through this same guide cannula to the stereotaxic position of the virus injection to allow illumination of transduced neurons.
Electrodes were connected to a multi-channel USB Biosignal amplifier (Guger Technologies) via the pedestal using a five channel custom cable (Plastics One) secured to the optical fiber. The optical fiber was coupled to a 561 nm laser (CrystaLaser) through an FC/PC connector. The laser was gated by a digital computer pulse (TTL) delivered via the parallel port of a laptop computer. EEG was recorded at a sampling rate of 256 or 512 Hz with bandpass filtering from 0.1 to 100 Hz. Video recordings were also taken of each experiment for behavioral analysis.
Adult male Sprague-Dawley rats were used in this study. Rats were anaesthetized with pentobarbital (50 mg/kg). The top of the head was shaved, and the animal was immobilized in a stereotaxic frame. The skin was opened to expose the skull. Three small burr holes were drilled in the skull: two 1 mm anterior to bregma, 1.5 mm lateral to the midline over each hemisphere, and one 4 mm posterior to bregma, 2.5 mm lateral to the midline on the right side. Surface recording electrodes (two stainless steel jeweler’s screws, shaft diameter 1.2 mm, length 1.6 mm, Plastics One) were inserted in the right and left frontal locations. A twisted–wire electrode coupled to the guide cannula was lowered into the hippocampus to a depth of 3 mm according to the atlas of Paxinos and Watson via the third hole
Following the recovery period, seizures were induced using the lithium-pilocarpine model. Animals received 127 mg/kg lithium hydrochloride i.p. 18–24 hours prior to injection of pilocarpine for seizure induction. On the experimental day scopolamine methyl bromide 1 mg/kg was injected i.p. 30 min before injection of pilocarpine to prevent peripheral cholinergic-agonist induced side effects. Electrodes were connected through the head pedestal to an EEG amplifier system, and the recording was started. To initiate seizures pilocarpine, a muscarinic cholinergic agonist, (30 mg/kg) was injected i.p. and the recording continued. After seizures developed the recording continued for another 20–30 min and then pentobarbital (100 mg/kg i.p.) was used to stop the seizure. Animals were then perfused transcardially with phosphate buffered saline (PBS) followed by 10% formalin, and the brains were dissected and processed for histology.
EEG recordings were downsampled, when necessary, to a common sampling rate of 512 Hz to allow for standardized analyses to be performed across recordings. To obtain time-frequency representations of the seizures, a short-time fast Fourier transform was performed using a sliding Hamming window 1000 ms long, overlapped by 500 ms. Line-length
To characterize the dynamics and evolution of the seizures, cumulative line-length was computed for 25 minutes from the start of each seizure. The cumulative line length,
Behavioral seizure manifestations were rated on the Racine scale in 30 second epochs based on review of videorecordings of the experiments
Spectral analysis of the hippocampal LFP was performed using the Chronux toolbox for Matlab
Animals in this group were implanted with electrodes and guide cannula (for optical fiber introduction), but no virus was injected. Animals were allowed to recover for 9–14, 16–21, or 31–35 days after electrode implantation (n = 4, n = 5, n = 4 respectively). Following the recovery period, seizures were induced using the lithium-pilocarpine model.
This group is designed to control for the possible effect of the viral construct
This group is designed to control for the possible effect of light. Eight animals were implanted with electrodes and guide cannula (for optical fiber introduction), but no virus was injected. Following the recovery period (10–29 days), seizures were induced using the lithium-pilocarpine model, and light delivered via the optical fiber similar to the experimental group.
Experimental attempts to control seizures by light were conducted 13–23 days following electrode implantation and virus injection. Electrodes and the optical fiber were connected to the recording system/laser as discussed above. The laser output power was set to 35 mW (fiber output power 18 mW as measured using a power meter (PM100D, ThorLabs)), corresponding approximately to light intensity of 570 mW/mm2. We based light manipulation parameters on those found in literature in other
Five to ten minutes after injecting pilocarpine (8±0.9 min), irrespective of visualized electrographic activity but designed to be 1–2 min before earliest anticipated seizure start according to our control group data, the laser was continuously activated until the seizure was terminated with pentobarbital overdose.
In a different set of experiments illumination was initiated when a change in the EEG suggestive of early evolving seizure activity or a change in the behavior of the animal was observed. In this circumstance the laser was turned on for periods of 1 to 2 min, with at least 1 minute between light pulses, until the seizure was stopped with pentobarbital overdose. This non-continuous triggered illumination began approximately 7–8 min after injecting pilocarpine (7.4±0.2 min).
Animals from experimental and control groups were alternated. Final histological confirmation of injection and eNpHR expression success was done post-experimentally, therefore investigator was blinded to the experimental outcome.
According to Gradinaru et al. (2009), light power sufficient to activate eNpHR (1 mW/mm2) is present at least within 1.5 mm distance of the fiber tip using 30 mW light source. We used a laser output power of 35 mW, with measured fiber power output of 18 mW with a 200 µm diameter fiber, corresponding approximately to a light intensity of 570 mW/mm2. Based on the light penetration curve for 561 nm light
We attempted to control acute seizures in awake-behaving rats in the lithium-pilocarpine model of elicited seizures using an optogenetic approach. Hippocampal pyramidal neurons were transduced with a virus carrying eNpHR
The tolerability of the implant used for recordings, virus injection and light delivery to the hippocampus was assessed in a control group of animals. Following the implantation, tissue damage was limited to the immediate proximity of the electrodes (200–400 µm) with no regions devoid of neurons present elsewhere in the hippocampus and cortex at any of the survival time points (12–35 days; n = 12), showing that the implant was well tolerated. In addition, in virally-transduced animals, pyramidal neurons located in the area surrounding the implant expressed eNpHR, suggesting that these neurons were healthy as evidenced by intact cellular machinery.
In animals transduced with AAV, the transduction extent was verified in histological sections of the brain following the experiment. Hippocampal neurons were transduced successfully and virus was expressed predominantly in pyramidal cells and granule cells in dentate gyrus, as demonstrated by fluorescent signal from YFP. The full medio-lateral extent of hippocampal pyramidal layer neurons was labeled (CA1, CA2), with labeling extending to CA3 in the dorso-ventral dimension. Transduced neurons extended over 3.5 mm antero-posteriorly, with minimal spread of at least 2 mm (
We first characterized the course of seizure development in a sub-group of sham control animals which were not transduced with eNpHR. After gathering this initial information we conducted experiments with experimental and the different control groups interleaved. Pilocarpine injection reliably induced seizures in all of the control rats. Seizure onsets occurred 15.2±1.1 min following pilocarpine injection (n = 13,
Experimental group is significantly different from the control group, (p = 0.007, one way ANOVA followed by Holm-Sidak method for multiple comparisons versus the control group). Error bars represent SEMs.
The seizure was terminated by pentobarbital administration in each case (arrowhead). Color spectrogram represents time-frequency distribution (scale is 0 to 50 Hz) and blue line represents the EEG recording (µV, scale 0 to 600 µV). Black vertical lines mark pilocarpine administration at time 0, vertical blue lines mark the beginning of the seizure. Yellow shading under the time axis indicates illumination with yellow light. Blue traces overlaid on the spectrograms show windowed line-length over the course of the seizure.
Behavioral manifestations of the seizure were delayed in experimental animals as compared to the controls. Area shaded in gray marks the timepoints at which group scores differed significantly (non-parametric, cluster-based statistical test, p<0.05).
Two additional control groups were designed to assess for possible effects of the NpHR transgene and the light. In the first, hippocampal neurons were transduced with the virus, the optical fiber was introduced, but no light manipulation was applied during the course of the experiment (NpHR control). The delay before seizure start in this group was similar to that of the original control group with no virus injected (17.3±1.3 min, n = 7, p = 0.4, one way ANOVA followed by Holm-Sidak method for multiple comparisons versus the control group). In the additional control group (light control) no virus was injected and light was delivered via the optical fiber similar to the experimental group. Seizure onset delay in this group was 16.3±1.7 (n = 8) similar to the sham control group (p = 0.66, one way ANOVA followed by Holm-Sidak method for multiple comparisons versus the control group).
To confirm inhibition of transduced hippocampal neurons by light and to control for the effect of prolonged inhibition of pyramidal hippocampal neurons and possible rebound activity after light discontinuation, illumination was tested in animals expressing eNpHR in the absence of seizures. Our experimental design did not allow recording from single units to confirm inhibition directly, therefore we used high frequency oscillations as a surrogate for spiking activity. In particular, high-gamma oscillations in the 70–110 Hz range are strongly correlated to spiking activity
We used two protocols for light-mediated inhibition of seizures: a continuous illumination and an intermittent illumination in which light was turned on for 1–2 minutes at 1 to 3 min intervals. In both protocols the illumination was started 5–10 minutes following pilocarpine injection, a few minutes before the earliest time of the anticipated seizure start according to our control group data. Both modes of illumination delayed the electrographic and behavioral onset of the seizure and changed the dynamic of seizure evolution in the same way. In these light illumination experiments, electrographic seizures started 21±1.8 min following administration of pilocarpine (n = 16,
In addition to affecting the time to onset of electrographic and behavioral (
To quantify the dynamics of seizure development we calculated the cumulative line-length for cortical and hippocampal channels as a measure of the amplitude and frequency dynamics of each seizure
Cumulative line-length, or path-length, of the cortical EEG recording in control (red, N = 14, from sham and additional control groups) and experimental (blue, N = 16) rats from onset to 25 minutes into the seizure, reflect changes in power and frequency on the standardized scale (Y axis). Areas with shallow slopes indicate lower seizure activity, while steep slopes indicate increased amounts of seizure activity as measured by line-length.
Interestingly, analysis of the spectral content of the LFP recorded from the hippocampus in experimental animals (expressing eNpHR, when light was turned on following seizure induction by pilocarpine) revealed that even in the presence of pilocarpine exerting its proconvulsive effects, 10 out of 12 animals showed significantly reduced high-gamma power during the first 5 seconds of illumination (p<0.004, Mann-Whitney U test;
We successfully introduced optogenetic probes into pyramidal neurons of the hippocampus and subsequently recorded from and directed light into those structures in a freely behaving animal. Inhibiting activity in primary excitatory hippocampal neurons with light delayed status epilepticus onset in the lithium-pilocarpine epilepsy model. This result suggests that seizure activity originating in the hippocampus may be crucial for seizure development and subsequent propagation to the cerebral cortex in this model
High-gamma power reflects neuronal spiking activity and its suppression following illumination suggests inhibition of transduced hippocampal neurons by light, as has been demonstrated directly by electrophysiological
The NpHR light-driven inhibition acts by shifting the membrane potential and hyperpolarizing neurons; Zhang et al
The fact that unilateral inactivation was sufficient to produce this result raises the possibility that each hippocampus plays a role in the network of activated structures necessary for seizure evolution. Removing one portion of this network alters the ictogenic process. These results are in line with findings reported for kainate model of epilepsy in mice
Two illumination protocols, tested for efficiency and safety reasons, produced similar results, suggesting that the inhibition produced in both modes is sufficient to delay the seizure onset, likely by delaying development of activity to the level necessary to evolve into seizure, while not causing obvious adverse behavioral or electroencephalographic effects. Slower dynamics of early seizure evolvement in experimental animals as demonstrated by cumulative line-length also points toward suppression of neuronal activity and delay in its development leading to interruption in “orchestration” of activity between neuronal populations into high amplitude oscillations. Controls further confirm that seizure delay is due to light-mediated inhibition of the hippocampal neurons expressing eNpHR. First, eNpHR expression by itself does not affect seizure development time, as animals which expressed eNpHR, but were not exposed to light, showed no delay of seizure onset. Second, illumination of transduced hippocampal neurons in the absence of seizure resulted in no rebound activity after light discontinuation. These finding are in line with Tonnesen et al
The modest scale of seizure attenuation in this study is in accordance with the extent of the hippocampal area affected by light and experimental design used. This may have contributed to the fact that illumination did not produce a visible change in cross-correlation or phase synchrony between the electrodes. The study was designed as an exploratory study of applying optogenetic approach for control of seizures, therefore we applied a minimal intervention approach and affected circuitry involved in seizures unilaterally. Even unilateral intervention was powerful enough to produce seizure delay. Since the epilepsy model used in our study is not focal, many of the neurons that contribute to the seizure are beyond the range of the unilateral optical fiber light inhibition. In addition, in this model pilocarpine used to induce the seizure remains present in the system and continues to exert proconvulsant activity long after the start of the light triggered inhibition
A number of other limitations could be addressed by future experiments. Epilepsy is thought to result from an imbalance in excitatory and inhibitory activity
Further steps will include optimizing the timing of light manipulation with regard to prospective seizure onset
In summary, our results demonstrate that inhibition of excitatory drive in hippocampus can delay seizure onset. This provides a proof-of-principle that this approach could be used in the future for treatment of seizures and the dissection of epilepsy mechanisms.
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We thank Drs. Andrew Cole, Matt Bianchi, and members of Dr. Cash’s laboratory for their insightful comments on the manuscript, especially Justine Cormier.