Cerebellar Directed Optogenetic Intervention Inhibits Spontaneous Hippocampal Seizures in a Mouse Model of Temporal Lobe Epilepsy

Abstract

Temporal lobe epilepsy is often medically refractory and new targets for intervention are needed. We used a mouse model of temporal lobe epilepsy, on-line seizure detection, and responsive optogenetic intervention to investigate the potential for cerebellar control of spontaneous temporal lobe seizures. Cerebellar targeted intervention inhibited spontaneous temporal lobe seizures during the chronic phase of the disorder. We further report that the direction of modulation as well as the location of intervention within the cerebellum can affect the outcome of intervention. Specifically, on-demand optogenetic excitation or inhibition of parvalbumin-expressing neurons, including Purkinje cells, in the lateral or midline cerebellum results in a decrease in seizure duration. In contrast, a consistent reduction in spontaneous seizure frequency occurs uniquely with on-demand optogenetic excitation of the midline cerebellum, and was not seen with intervention directly targeting the hippocampal formation. These findings demonstrate that the cerebellum is a powerful modulator of temporal lobe epilepsy, and that intervention targeting the cerebellum as a potential therapy for epilepsy should be revisited.

Keywords: Purkinje; cerebellum; epilepsy; hippocampus; optogenetics; seizure.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Cover Figure

Krook-Magnuson et al. report a bidirectional functional connectivity between the hippocampus and the cerebellum in a mouse model of temporal lobe epilepsy, and demonstrate that cerebellar directed on-demand optogenetic intervention can stop seizures recorded from the hippocampus.

Figure 1

Lateral cerebellar intervention in PV-ChR2 mice. a, In PV-Cre animals, Cre is expressed in cerebellar neurons, including Purkinje neurons. Layers indicated: M, molecular; P, Purkinje; G, granular. b, Blue light produces action potentials in Purkinje neurons in PV-ChR2 animals (top) but not in opsin negative animals (bottom trace). c, In vivo juxtacellular recording of a Purkinje cell (black trace), illustrating an increase in firing rate (50 ms sliding window; red trace) with light delivery (blue bar). Portion of trace expanded in d is indicated by a thick black bar. d, Detected spikes illustrated by green trace. Asterisk indicates a complex spike. e, On-line detection of spontaneous temporal lobe seizures allows on-demand light delivery during the chronic phase of the disorder. f, The EEG is recorded from the hippocampus ipsilateral to previous kainate injection (KA), and light is delivered to the cerebellum. g, Example detected seizures not receiving light (top trace) and receiving cerebellar directed light-intervention (bottom). Gray bars denote seizure detection. Blue box denotes 3 s of pulsed light delivery. In opsin-expressing PV-ChR2 animals, light delivered to the ipsilateral (h) or contralateral (i) cerebellum produces a significant reduction in seizure duration, but there is no effect of light in an opsin-negative animal (j). k, Three seconds of pulsed light delivery produces a significant reduction in seizure duration in opsin-expressing animals with both short pulses (50 ms on, 100 ms off) and long pulses (1000 ms on, 50 ms off). *p < 0.05. Scale bars: a, 200 µm; b, 50 ms, 20m V; c, 1 s; 1 mV or 100 Hz change in firing rate; d, 50 ms; 1 mV or 100 Hz change in firing rate; g, 5 s, 0.05 mV.

Figure 2

On-demand optogenetic intervention in PV-HR animals. a, Crossing PV-Cre mice with mice expressing in a Cre-dependent manner the inhibitory opsin halorhodopsin (HR) produces mice expressing HR in PV-expressing neurons, including cerebellar Purkinje cells. Light delivery to cerebellar slices from PV-HR mice produces strong inhibition, capable of inhibiting Purkinje cell firing induced by direct current injected. b, Light delivery inhibits spontaneous firing of a Purkinje cell recorded juxtacellularly in vivo. Orange bars denote light delivery. Green trace indicates detected spikes. Blue trace illustrates the change in firing frequency (50 ms sliding window). The asterisk marks a complex spike. In vivo on-demand intervention in PV-HR animals directed to the cerebellar cortex ipsilateral (c) or contralateral (d) to previous hippocampal kainate injection reduced postdetection seizure duration in opsin-expressing, but not opsin-negative (e) animals. f, Both short and long light pulses were effective at stopping seizures. g, In contrast, light delivery to the hippocampus in PV-HR animals did not affect seizure duration. dur red, Duration reduction. Scale bars: a, 500 ms, 20 mV; b, 50 ms; top, 4 mV; bottom, 100 Hz change in firing rate.

Figure 3

Optogenetic intervention targeting the midline cerebellum. On-demand optogenetic intervention with 3 s of pulsed light delivered to the midline cerebellum (vermis) in a PV-ChR2 mouse reduces seizure duration (a) and increases time to next seizure (b). Blue bars and traces indicate events receiving light. Gray indicates events not receiving light. Inset, Box and whisker plot indicating median, 25th and 75th percentiles, and 25th and 75th percentile ± 1.5 times the interquartile range. c, With 3 s of short light pulses (50 ms on, 100 ms off), there is an increase in time to next seizure with vermal directed intervention in PV-ChR2 animals. CB, Cerebellum; hipp, hippocampus; ipsilat or ipsi, ipsilateral to KA; contrlat, contralateral.

Figure 4

On-demand optogenetic intervention with Purkinje cell specific expression. a, In Pcp-Cre mice, cerebellar Cre is selectively expressed in Purkinje neurons. Cre expression (white) was visualized by crossing with a reporter strain. Bottom, blue, DAPI fluorescent stain. b, Crossing Pcp-Cre mice with mice expressing ChR2 in a Cre-dependent manner produces mice with ChR2 expressed selectively in Purkinje neurons. Light delivery to cerebellar slices from Pcp-ChR2 animals induces firing in Purkinje neurons. c, In vivo on-demand optogenetic intervention targeting the lateral cerebellum produces an inhibition of seizure duration. d, Light delivery to the hippocampus (which does not express opsin in Pcp-ChR2 animals) does not inhibit seizures. Light delivery to the midline cerebellum is capable of reducing seizure duration (e) and increasing time to next seizure (f). Scale bars: a, top, 500 µm; bottom, 50 µm; b, 100 ms, 20 mV.

Figure 5.

Temporal lobe seizures modulate cerebellar activity. An example temporal lobe seizure recorded in the hippocampus (a), which produced changes in the cerebellar EEG (b) and in the firing rate of a juxtacellularly recorded Purkinje cell over the course of the seizure (c) as well as on a shorter time scale (d). Green trace denotes detected Purkinje cell spikes. Scale bars: a, 1 mV; b, 0.5 mV; a−c, 10 s; d, 0.5 mV or 0.1 kHz change in firing rate, 0.1 s.