Optogenetic stimulation of the superior colliculus suppresses genetic absence seizures

Abstract

While anti-seizure medications are effective for many patients, nearly one-third of individuals have seizures that are refractory to pharmacotherapy. Prior studies using evoked preclinical seizure models have shown that pharmacological activation or excitatory optogenetic stimulation of the deep and intermediate layers of the superior colliculus (DLSC) display multi-potent anti-seizure effects. Here we monitored and modulated DLSC activity to suppress spontaneous seizures in the WAG/Rij genetic model of absence epilepsy. Female and male WAG/Rij adult rats were employed as study subjects. For electrophysiology studies, we recorded single unit activity from microwire arrays placed within the DLSC. For optogenetic experiments, animals were injected with virus coding for channelrhodopsin-2 or a control vector, and we compared the efficacy of continuous neuromodulation to that of closed-loop neuromodulation paradigms. For each, we compared three stimulation frequencies on a within-subject basis (5, 20, 100 Hz). For closed-loop stimulation, we detected seizures in real time based on the EEG power within the characteristic frequency band of spike-and-wave discharges (SWDs). We quantified the number and duration of each SWD during each 2 h-observation period. Following completion of the experiment, virus expression and fibre-optic placement was confirmed. We found that single-unit activity within the DLSC decreased seconds prior to SWD onset and increased during and after seizures. Nearly 40% of neurons displayed suppression of firing in response to the start of SWDs. Continuous optogenetic stimulation of the DLSC (at each of the three frequencies) resulted in a significant reduction of SWDs in males and was without effect in females. In contrast, closed-loop neuromodulation was effective in both females and males at all three frequencies. These data demonstrate that activity within the DLSC is suppressed prior to SWD onset, increases at SWD onset, and that excitatory optogenetic stimulation of the DLSC exerts anti-seizure effects against absence seizures. The striking difference between open- and closed-loop neuromodulation approaches underscores the importance of the stimulation paradigm in determining therapeutic effects.

Keywords: absence epilepsy; deep brain stimulation; optogenetic; responsive neurostimulation; spike-and-wave discharge.

Figure 1

Histological verification of virus expression in the DLSC. (A) Coronal section through the deep and intermediate layers of the superior colliculus (DLSC). Red indicates mCherry fluorescence from the injected virus. Boxes indicate the regions displayed in B and C, showing higher-power magnification with arrowheads indicating ChR2-mCherry-positive somata. (D) Coronal atlas planes, modified from the Swanson Rat Brain atlas, with the DLSC filled in blue. Red ‘x’ indicates the centre of the tip of the microwire array. (E and F) Atlas planes as in D, showing the tips of the fibre-optics in male (E) and female (F) WAG/Rij rats.

Figure 2

Single-unit activity within the DLSC decreases seconds prior to SWD onset and increases during seizures. (A) Individual spike-and-wave discharges (SWDs) (16 discharges from one rat), time locked to SWD onset, as well as power spectra illustrating the characteristic 7–14 Hz peak seen in the WAG/Rij rat. Bottom: Grey trace shows the averaged signal across the individual SWDs recorded in the subject. (B) Activity was recorded from an average of 28 SWDs (range 15–61) per array; as two animals were implanted bilaterally with arrays in the deep and intermediate layers of the superior colliculus (DLSC) and recordings for these animals were performed in different sessions, we use array as the unit for this analysis. (C) Significant difference between activity pattern distribution before and after SWD start and end (χ² = 58.04, df = 6, P = 1.1 × 10⁻¹⁰; n = 89 units from 12 rats). A larger proportion of units displayed phasic decreases in firing prior to SWD start as compared to other time bins. Phasic decreases in firing were defined as firing rates that fell below the 99% confidence intervals for the baseline firing rate of the unit in the 2.5 s preceding the SWD. Baseline firing rates were defined as the average activity for the unit for the duration of the recording excluding SWDs and the 2.5 s immediately preceding the SWDs. (D) No significant sex differences in the distribution of the activity patterns (65 units from eight females, 24 units from four males). (E) No significant differences in the distribution of the activity patterns as a function of electrode placement in the deep and intermediate layers of the superior colliculus (SC) (28 units in the deep layers, 32 in the intermediate layers). (F) Baseline firing rate differed for tonic and phasic units. Both phasic increasing and phasic decreasing units had significantly higher firing rates compared to tonic units (mean + SEM; P = 0.0001 and P = 0.0018, respectively). Firing rate did not differ as a function of sex (G) or location within the SC (H). However, in both cases we detected a significant effect of time bin: unit activity, collapsed across the other variables, was higher during the baseline and SWD periods compared with the PreSWD period. *P < 0.0001, see legend to the right. (I) Raster plot (top) matched to the peri-event histogram of a unit showing a phasic decrease, (J) tonic profile and (K) phasic increase preceding seizure start (seizure start = 0). Grey shaded region indicates baseline, pink shaded region indicates the PreSWD period and purple shaded region indicates the SWD period. (L) Firing rates significantly decreased in the 2.5 s prior to SWD onset compared with baseline firing rates from the same units (P < 0.000027; estimation plot showing individual points, condition means and mean of differences, n = 35 phasic decrease units). The mean reduction in firing was 28% (11.6–8.4 Hz) (M), same units as in L, showed significant increases during the SWD compared to baseline firing rates (P < 0.0094; 18%, 11.6–13.71 Hz, n = 35 phasic decrease units). (N) Average activity in units that showed decreased firing at the time of SWD followed a biphasic response, where activity was decreased in the 2.5 s prior to seizure onset and then increased during the SWD (blue trace). Tonic units displayed no significant changes in firing rate. (Line indicates mean; shaded areas show the 95% confidence intervals). (O) Heterogeneous activity between all defined unit types at the end of the SWD. Note, in F–O, the ‘Decrease’ group indicates units that were characterized as displaying a phasic preSWD decrease in firing in C. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Open-loop optogenetic stimulation of the DLSC attenuates absence-like seizures in male WAG/Rij rats. (A) Total seizure duration and (B) number of spike-and-wave discharges (SWDs) were significantly reduced by 5, 20 and 100 Hz light delivery in ChR2-expressing WAG/Rij rats (n = 9), while (C) average SWD duration was unchanged. Note that several animals were excluded from the analysis of average SWD duration as they displayed no SWDs during the 30 min session (excluded: 5 Hz: n = 5; 20 Hz: n = 3; 100 Hz: n = 2). In control vector rats, no measure of SWDs was altered by the light delivery: total duration (D), number (E) and average duration (F) of the SWDs (n = 10). Plots show individual replicates with mean and standard error. Mean differences (treated – control, with 95% confidence intervals) are plotted below each variable. Representative EEG traces under no light delivery, 5, 20 and 100 Hz stimulation conditions in ChR2-expressing rats (G) and control-vector rats (H). *P < 0.05, ** P < 0.01. Yellow highlighting in G and H indicate individual SWDs.

Figure 4

Open-loop optogenetic stimulation of the DLSC does not decrease absence seizures in female WAG/Rij rats. Total duration, number and average duration of the spike-and-wave discharges (SWDs) did not differ as a function of light delivery in either ChR2-expressing rats (A–C, n = 10) or opsin-negative rats (D–F, n = 11). Plots show individual replicates with mean and standard error. Mean differences (treated – control, with 95% confidence intervals) are plotted below each variable. Representative EEG traces under no light delivery, 5, 20 and 100 Hz stimulation conditions in ChR2-expressing rats (G) and control-vector rats (H). Yellow highlighting in G and H indicate individual SWDs.

Figure 5

Closed-loop optogenetic stimulation of the DLSC attenuates absence seizures in male WAG/Rij rats. (A) Optogenetic stimulation of the deep and intermediate layers of the superior colliculus (DLSC) in male ChR2 WAG/Rij rats resulted in prompt termination of spike-and-wave discharges (SWDs) with 20 and 100 Hz light delivery (P = 0.029 and 0.005). There was no significant change with 5 Hz light delivery (n = 8, P = 0.082). Plots show individual replicates with mean and standard error. Mean differences (treated – control, with 95% confidence intervals) are plotted below each variable. (B–D) Cumulative distribution of seizure duration after light delivery at each frequency. Plots show the mean and 95% confidence interval for the average cumulative frequency distribution. Distributions significantly differed between stimulated and non-stimulated seizures for each frequency (P < 0.000009). (E) In control vector animals (n = 11), light delivery was without effect on within-subject seizure duration at 5 and 20 Hz but produced a small but significant increase in seizure duration with 100 Hz light delivery. (F–H) Cumulative distributions of seizure durations did not differ as a function of light delivery in control animals. (I–N) Representative SWDs for each frequency, condition and vector. Yellow shading shows the onset of seizure detection through the end of a 5 s post-detection window without light delivery. Blue shading indicates the period of light delivery. Consistent with the quantification in A–D, optogenetic stimulation of the DLSC produced fast termination of ongoing SWD activity, whereas light delivery in opsin-negative animals was without appreciable effect. ^&P = 0.08; *P < 0.05; **P < 0.01; ****P < 0.0001; n.s.= no significance.

Figure 6

Closed-loop optogenetic stimulation of the DLSC attenuates absence-like seizures in female WAG/Rij rats. (A) Optogenetic stimulation of the deep and intermediate layers of the superior colliculus (DLSC) in female ChR2 WAG/Rij rats resulted in prompt termination of spike-and-wave discharges (SWDs) at each frequency of optogenetic stimulation we tested (n = 11; 5 Hz: P = 0.022; 20 Hz: P < 0.0001; 100 Hz: P = 0.0053). Plots show individual replicates with mean and standard error. Mean differences (treated – control, with 95% confidence intervals) are plotted below each variable. (B–D) Cumulative distribution of seizure duration after light delivery at each frequency. Plots show the mean and 95% confidence interval for the average cumulative frequency distribution. Distributions significantly differed between stimulated and non-stimulated seizures for each frequency (P < 0.000000004). (E) In control vector animals (n = 14), light delivery was without effect on within-subject seizure duration at 5 and 20 Hz, but produced a small but significant increase in seizure duration with 100 Hz light delivery. (F–H) Cumulative distributions of seizure durations did not differ as a function of light delivery. (I–N) Representative SWDs for each frequency, condition, and vector. Yellow shading shows the onset of seizure detection through the end of a 5 s post-detection window without light delivery. Blue shading indicates the period of light delivery. Consistent with the quantification in (A–D) optogenetic stimulation of the DLSC produced fast termination of ongoing SWD activity, whereas light delivery in opsin-negative animals was without appreciable effect. *P < 0.05; **P < 0.01; ****P < 0.0001; n.s.= no significance.