Excitation, but not inhibition, of the fastigial nucleus provides powerful control over temporal lobe seizures

Abstract

Key points: On-demand optogenetic inhibition of glutamatergic neurons in the fastigial nucleus of the cerebellum does not alter hippocampal seizures in a mouse model of temporal lobe epilepsy. In contrast, on-demand optogenetic excitation of glutamatergic neurons in the fastigial nucleus successfully inhibits hippocampal seizures. With this approach, even a single 50 ms pulse of light is able to significantly inhibit seizures. On-demand optogenetic excitation of glutamatergic fastigial neurons either ipsilateral or contralateral to the seizure focus is able to inhibit seizures. Selective excitation of glutamatergic nuclear neurons provides greater seizure inhibition than broadly exciting nuclear neurons without cell-type specificity.

Abstract: Temporal lobe epilepsy is the most common form of epilepsy in adults, but current treatment options provide limited efficacy, leaving as many as one-third of patients with uncontrolled seizures. Recently, attention has shifted towards more closed-loop therapies for seizure control, and on-demand optogenetic modulation of the cerebellar cortex was shown to be highly effective at attenuating hippocampal seizures. Intriguingly, both optogenetic excitation and inhibition of cerebellar cortical output neurons, Purkinje cells, attenuated seizures. The mechanisms by which the cerebellum impacts seizures, however, are unknown. In the present study, we targeted the immediate downstream projection of vermal Purkinje cells - the fastigial nucleus - in order to determine whether increases and/or decreases in fastigial output can underlie seizure cessation. Though Purkinje cell input to fastigial neurons is inhibitory, direct optogenetic inhibition of the fastigial nucleus had no effect on seizure duration. Conversely, however, fastigial excitation robustly attenuated hippocampal seizures. Seizure cessation was achieved at multiple stimulation frequencies, regardless of laterality relative to seizure focus, and even with single light pulses. Seizure inhibition was greater when selectively targeting glutamatergic fastigial neurons than when an approach that lacked cell-type specificity was used. Together, these results suggest that stimulating excitatory neurons in the fastigial nucleus may be a promising approach for therapeutic intervention in temporal lobe epilepsy.

Keywords: cerebellum; fastigial nucleus; optogenetics; temporal lobe epilepsy.

Figure 1.

Fastigial intervention in VGluT2-HR mice. A-B) Schematics of experimental design. Note that seizures are recorded from the hippocampus and light is delivered in an on-demand fashion to the fastigial nucleus during the chronic phase of the disorder. C) Example seizure events detected on-line (denoted by purple bar) that were either randomly selected not to receive light (top trace) or receive light (bottom trace, 3 seconds of light delivery denoted by amber box). Scale bar: 5s, 0.05mV. D-F) 3 seconds of long light pulses (1000ms on, 50ms off) to inhibit the fastigial nucleus produces no significant change in seizure duration (D, example animal; amber bars: events receiving light intervention; hashed bars: no-light internal controls; top trace illustrates pulsed light delivery paradigm) when light (589nm) is delivered to the contralateral (E) or ipsilateral (F) fastigial nucleus (each gray point represents data from one animal, black points represent mean). Similarly, 3 seconds of shorter light pulses (G-I) at 7Hz (50ms on, 100ms off), or (J-L) 10 Hz (50ms on, 50ms off), produce no significant change in seizure duration.

Figure 2.

On-demand optogenetic excitation of the fastigial nucleus contralateral to KA injection. A) Spontaneous seizures are recorded from the KA-injected hippocampus and blue (473nm) light is delivered in an on-demand fashion to the contralateral fastigial nucleus in VGluT2-ChR animals. B) Example seizure events detected on-line (denoted by purple bar) that were either randomly selected not to receive light (top trace) or receive light (bottom trace, 3 seconds of light delivery denoted by blue box). Scale bar: 5s, 0.05mV. Three seconds of pulsed light delivery (1000ms on, 50ms off) significantly reduces seizure duration. C) Post-detection seizure duration distributions for an example animal (93% reduction, p < 0.001, two sample Kolmogorov-Smirnov test). Blue bars: events receiving light intervention; hashed bars: no-light internal controls. Top trace illustrates pulsed light delivery paradigm. Inset: first 5s bin expanded, 1s bin size. D) No effect of light delivery on seizure duration in an opsin negative animal (p = 0.896, two sample Kolmogorov-Smirnov test). E) Light delivery produces a significant reduction of seizure duration in opsin positive VGluT2-ChR mice (each gray data point represents one animal, black data points represent mean). Similarly, 3 seconds of shorter light pulses (F-H) at 7Hz (50ms on, 100ms off), or (I-K) 10 Hz (50ms on, 50ms off), produce a significant reduction in seizure duration.

Figure 3.

On-demand optogenetic excitation of the fastigial nucleus ipsilateral to KA injection. A) Example seizure events detected on-line (denoted by purple bar) that were either randomly selected not to receive light (top trace) or receive light (bottom trace, 3 seconds of light delivery denoted by blue box). Scale bar: 5s, 0.05mV. Three seconds of pulsed blue light delivery (1000ms on, 50ms off) significantly reduces seizure duration. B) Post-detection seizure duration distribution for an example VGluT2-ChR animal; blue bars: events receiving light intervention; hashed bars: no-light internal controls; top trace illustrates pulsed light delivery paradigm (57% reduction, p < 0.001, two sample Kolmogorov-Smirnov test). Inset: first 5s bin expanded, 1s bin size. C) No effect of light delivery on seizure duration in an opsin negative animal (p = 0.423, two sample Kolmogorov-Smirnov test). D) Light delivery produces a significant reduction of seizure duration in opsin positive VGluT2-ChR mice (each gray data point represents one animal, black data points represent mean). Similar results are seen for 7Hz (E) and 10Hz (F) stimulation.

Figure 4.

A single 50ms pulse of light significantly reduces seizure duration in VGluT-ChR animals. A) Data from an example opsin-positive animal; blue bars: events receiving blue (473nm) light intervention; hashed bars: no-light internal controls; top trace illustrates pulsed light delivery paradigm (50% reduction, p < 0.001, two sample Kolmogorov-Smirnov test). Inset: first 5s bin expanded, 1s bin size. B) No effect of blue (473nm) light delivery in an opsin negative animal (p = 0.237, two sample Kolmogorov-Smirnov test). Single pulses of light in opsin positive VGluT2-ChR animals significantly reduce seizure duration when targeting either contralateral (C) or ipsilateral (D) fastigial nucleus (each gray data point represents one animal, black data points represent mean).

Figure 5.. Selective excitation of fastigial glutamatergic neurons provides robust seizure control.

A) Schematic of experimental design and timeline. Viral approaches allowed for the targeting of nuclear neurons broadly, including nuclear glutamatergic, GABAergic, and glycinergic neurons (B), or selective targeting of glutamatergic nuclear neurons (shown in dark blue in the figure). (C) Selective expression in glutamategic neurons is achieved following injection of cre-dependent viruses in VGluT2-cre transgenic mice (Top- Green: GFP, Middle- Red: VGluT2 immunohistochemistry, Bottom- overlay, scale bar 70μm). D) GFP expression in nuclear neurons following injection in a Black-6 mouse. E) GFP expression in nuclear neurons following injection of cre-dependent virus in a VGluT2-cre mouse. Scale bars for D and E: 500μm. Green: GFP. Blue: DAPI. F) Example seizure events detected on-line (denoted by purple bar) that were either randomly selected not to receive light (top trace) or receive light (bottom trace, 3 seconds of blue (473nm) light delivery denoted by blue box). Scale bar: 5s, 0.05mV. G-H) Light delivery significantly reduces seizure duration in virally injected Black-6 (G, 44% reduction, p = 0.001, two sample Kolmogorov-Smirnov test) and VGluT2-cre (H, 81% reduction, p < 0.001, two sample Kolmogorov-Smirnov test) mice expressing channelrhodopsin. Blue bars: events receiving light intervention; hashed bars: no-light internal controls; top trace illustrates pulsed light delivery paradigm. I) No effect of light delivery in a mouse injected with AAV9-CAG-GFP control vector (p = 0.705, two sample Kolmogorov-Smirnov test). J-K) Stimulation significantly reduces the duration of hippocampal seizures across the population of channelrhodospin-expressing virally injected Black-6 (J) and VGluT2 (K) mice. Each open circle represents one animal. Black data points represent mean. L) Selective targeting of glutamatergic neurons in the fastigial nucleus produces significantly greater seizure attenuation than targeting fastigial neurons more broadly (p = 0.026, Mann-Whitney test).