On-demand optogenetic control of spontaneous seizures in temporal lobe epilepsy

Abstract

Temporal lobe epilepsy is the most common type of epilepsy in adults, is often medically refractory, and due to broad actions and long-time scales, current systemic treatments have major negative side-effects. However, temporal lobe seizures tend to arise from discrete regions before overt clinical behaviour, making temporally and spatially specific treatment theoretically possible. Here we report the arrest of spontaneous seizures using a real-time, closed-loop, response system and in vivo optogenetics in a mouse model of temporal lobe epilepsy. Either optogenetic inhibition of excitatory principal cells, or activation of a subpopulation of GABAergic cells representing <5% of hippocampal neurons, stops seizures rapidly upon light application. These results demonstrate that spontaneous temporal lobe seizures can be detected and terminated by modulating specific cell populations in a spatially restricted manner. A clinical approach built on these principles may overcome many of the side-effects of currently available treatment options.

Figure 1. Closed-loop system design.

EEG input (blue) from the mouse hippocampus is amplified (Amp), digitized (A/D) and relayed to a PC running a custom-designed real-time seizure detection software. The signal is fed into a number of possible detection algorithms, which utilize features of signal power, spikes or frequency (sample schematics are presented here; detailed methodology is provided in Supplementary Methods). Thresholds for power and spike properties (green) are determined using tunable leaky integrators acting as low-pass filters. Top: Amplitude Correlation (purple, during an example seizure, shown in grey); Middle: spike characteristics (for example, amplitude, rate, regularity and spike width, shown in red); Bottom: power of the signal in specific frequency bands during the same seizure, with warmer colours representing higher energy. Once a seizure has been detected using the selected criteria, for 50% of the events in a random fashion (RND), the software activates the optical output (orange) delivered to the hippocampus of the mouse, via a TTL signal from the digitizer to the laser. All trigger events, however, are flagged for later off-line analysis. COMP, digital comparator. USB, universal serial bus.

Figure 2. Seizure control in mice-expressing HR in principal cells in a model of TLE.

(a) Crossing CamK-Cre and Cre-dependent HR mouse lines generated mice expressing the inhibitory opsin HR in excitatory cells (Cam-HR mice). (b) Experimental timeline. (c–e) Example electrographic seizures detected (vertical green bars), activating amber light (589 nm) randomly for 50% of events (light: amber line, example in d; no-light example in e). (f) Typical example distribution of postdetection seizure durations (5 s bin size) during light (solid amber) and no-light internal control conditions (hashed grey). Inset: first 5 s bin expanded, 1 s bin size. Note that most seizures stop within 1 s of light delivery. (g–i) Group Cam-HR data showing the per cent of seizures stopping within 5 s of detection (g), within 1 s of detection (h), and the average postdetection seizure duration (normalized to average no-light postdetection duration for each animal) (i). Note that in one animal (shown in c–e), all seizures were stopped within 1 s of light delivery. Averaged data: filled circles. Error bars represent s.e.m. Scale bars in c–e, 100 μV, 5 s.

Figure 3. Ipsilateral and contralateral control of seizures in PV-ChR2 mice.

(a) Crossing PV-Cre and Cre-dependent ChR2 mouse lines generated mice expressing the excitatory opsin ChR2 in PV-expressing GABAergic cells (PV-ChR2 mice). (b) Example electrographic seizures in a PV-ChR2 mouse (top, no-light) truncated by blue (473 nm) light delivery (bottom, blue line) to the hippocampus. (c) Distributions of postdetection seizure durations from the same animal during light and no-light conditions (solid blue: light; hashed grey: no-light), when light was delivered (blue probe) ipsilateral to the KA injection (grey hemisphere). (d) In the same animal, light delivered instead to the hippocampus contralateral to KA injection also shortens electrographic seizures. (e–f) Group data for PV-ChR2 animals with light delivered ipsilateral (e) or contralateral (f) to the KA injection site. Electrographic recordings were consistently from the hippocampus ipsilateral to KA injection. Averaged data: filled circles. Error bars represent s.e.m.

Figure 4. Extent and specificity of light-activation of opsin-expressing cells.

(a) Diagram of illumination. The fibres used in this study have a numerical aperture (NA) of 0.37 and a radius (r) of 100 μm. With an index of refraction (n) for brain tissue of 1.35, and a flat cleave tip, there is a half-angle (θ) of ~16°. Given our average power measured post hoc from the implanted fibre tip, and reported irradiance values for activating opsins, we reach a depth (d) of 0.55 mm for HR with amber light and 0.9 mm for ChR2 with blue light. Note that the diagram is not drawn to scale. (b) c-Fos expression is not induced in PV cells after 2 h of on-demand light (green diamonds), but can be induced with continuously pulsed blue light for the entire 2 h (that is, not on-demand light application, red squares) in PV-ChR2 opsin-expressing animals. Note that this c-Fos expression induced by 2 h long pulsed light application reflects both direct and indirect activation over a prolonged time period (for example, through gap-junctions and network connexions). An increase in PV-cell c-Fos expression was not observed without opsin expression (grey triangles). Light was delivered, and c-Fos expression examined, contralateral to the site of KA injections. (c) Example PV cells from PV-ChR2 opsin-expressing (top) and opsin-negative (bottom) animals in b. Green: PV. Red: c-Fos. Yellow: co-localization. Scale bar, 10 μm. (d,e) Long pulses of light (2 s on, 50 ms off; orange bars) produced robust outward (inhibitory) currents in CA1 pyramidal (d) and granule cells (e) of epileptic Cam-HR opsin-expressing animals (green traces), but not epileptic opsin-negative littermate controls (black traces). Scale bars, 1 s, 100 pA. Holding potential, −60 mV. (f) A single pulse of light (10 ms) produces robust postsynaptic currents in two CA1 pyramidal cells (dual recording) from a KA-treated PV-ChR2 opsin-expressing animal (green bolded trace, average of 15 individual sweeps, shown in light green). No-light-induced responses were seen in opsin-negative animals (bottom, average trace shown in black). Scale bars, 20 ms, 40 pA. Holding potential, −80 mV.

Figure 5. Reduction in behavioural seizure frequency.

(a) Example behavioural (stage 5) seizure (movement artifacts truncated). The vertical green bar indicates online seizure detection, prior to the start of stage 4–5 behaviour (arrow). The yellow bars under the trace highlight the theoretical window for intervention. Scale bars, 100 μV, 5 s. (b) Distribution of electrographic seizure duration after detection with (blue bars) and without (grey hashed bars) bilateral light intervention. Note that for behavioural seizure experiments in two animals light was delivered bilaterally. (c) Significant reduction in the frequency of behavioural seizures occurring during light compared with no-light (example animal; asterisk, P<0.05, two-tailed binomial). Electrographic recordings were consistently from the hippocampus ipsilateral to KA injection. Same mouse as shown in examples in Figure 3.