Chemical-genetic attenuation of focal neocortical seizures

Abstract

Focal epilepsy is commonly pharmacoresistant, and resective surgery is often contraindicated by proximity to eloquent cortex. Many patients have no effective treatment options. Gene therapy allows cell-type specific inhibition of neuronal excitability, but on-demand seizure suppression has only been achieved with optogenetics, which requires invasive light delivery. Here we test a combined chemical-genetic approach to achieve localized suppression of neuronal excitability in a seizure focus, using viral expression of the modified muscarinic receptor hM4Di. hM4Di has no effect in the absence of its selective, normally inactive and orally bioavailable agonist clozapine-N-oxide (CNO). Systemic administration of CNO suppresses focal seizures evoked by two different chemoconvulsants, pilocarpine and picrotoxin. CNO also has a robust anti-seizure effect in a chronic model of focal neocortical epilepsy. Chemical-genetic seizure attenuation holds promise as a novel approach to treat intractable focal epilepsy while minimizing disruption of normal circuit function in untransduced brain regions or in the absence of the specific ligand.

Figure 1. Expression and tolerability of HA-hM4D_i-mCitrine.

(a) Expression of HA-hM4D_i-mCitrine in deeper layers of right primary motor cortex (M1), visualized with anti-HA antibody. Scale, 100 μm. (b) Colocalization of CamKIIα-HA-hM4D_i-mCitrine, visualized with anti-HA (green) and anti-CamKIIα (red) antibodies. Scale, 20 μm. (c) Motor coordination with (right) and without (left) intraperitoneal CNO in rats receiving sham injection (red) or HA-hM4D_i-mCitrine in right M1 (blue). Foot faults for the left forelimb (contralateral to HA-hM4D_i-mCitrine) were compared with the right (ipsilateral) forelimb (paired t-test) and with foot faults of the left forelimb of sham-injected animals (unpaired t-test; mean±s.e.m., n=5 sham-operated and 7 hM4-injected rats). NS: P>0.05.

Figure 2. Chemical–genetic silencing of pilocarpine-induced seizures.

(a) Morlet-wavelet EEG spectra from a rat administered intracortical pilocarpine (time 0), with either intraperitoneal vehicle (top) or CNO (bottom). (b) EEG segment from (a, top), showing simple spikes (SS), complex spikes (CS) and runs of intermediate frequency (IF) activity (expanded below). (c) Behavioural seizures correlated with EEG. SS activity was associated with no motor seizures (severity score 0) or twitching of limb, head or body (score 1), while IF was associated with repetitive head or body shaking (score 2) or rearing, retrograde locomotion and generalized convulsions (3). Six hundred consecutive 4-s-intervals per rat were assessed in three rats (numbered 1–3). (d) Detection of Intermediate Frequency (IF)-activity by Fourier transformation: Fourier transform (right) of EEG segments in one rat showing either SS (black) or IF (red) activity (two 5-s periods shown to the left) induced by pilocarpine. The IF Fourier transform shows a peak around 7 Hz (and a harmonic at 14 Hz). (e) Temporal evolution of spike frequency (left), 4–14 Hz power (middle), and number of IF runs (right), in vehicle (red) and CNO (blue) trials (consecutive 10-min-intervals before (-10–0 min) and after pilocarpine and vehicle/CNO injection). N=6 rats (10 pairs of trials, averaged within rat where repeated, data are shown as mean±s.e.m.). (f) Same data as in e, but plotted as cumulative electrographic seizure metrics (frequency, power, number of IF events), comparing vehicle versus CNO for each animal (indicated by colour). Symbols indicate consecutive cumulative metrics at 10-min-intervals. The 45-degree line (grey) indicates equivalence of CNO and vehicle.

Figure 3. Chemical–genetic silencing of picrotoxin-induced seizures.

(a) Morlet-wavelet power spectra of EEG in an animal injected with picrotoxin at time 0 (1 mm below pia, 10 mM, 300 nl), together with intraperitoneal vehicle (top) or 1 mg ml⁻¹ CNO in vehicle (bottom) on consecutive days. (b) EEG activity. Bottom, expanded sections from times indicated (2-s-duration) showing SS, CS and IF activity. (c) Motor seizures were more severe in association with IF activity than with SS activity, and intermediate with CS activity (severity scale as in Fig. 2c). Six hundred consecutive 4-s-intervals per rat were assessed in three rats (numbered 1–3). (d) Fourier transform (right) of 5-s-traces displayed (left, middle) containing SS (black) and IF (4–14 Hz, peak around 11.5 Hz; red) activity induced by picrotoxin. (e) Temporal evolution of spike frequency (left), 4–14 Hz power (middle), and number of IF runs (right), in vehicle (red) and CNO (blue) trials (consecutive 10-min-intervals before (−10–0 min) and after picrotoxin and vehicle/CNO injection). N=5 rats (12 pairs of trials, averaged within rat where repeated, mean±s.e.m.). (f) Same data as in e, but plotted as cumulative electrographic seizure metrics for each animal as in Fig. 2e.

Figure 4. Effect of CNO on chemically induced seizures in control rats.

Two distinct control groups per condition were tested: rats injected with an AAV5-CamKIIα-ArchT-GFP virus, and rats which were not injected with a virus (but otherwise underwent the same surgery and implantations as the other groups). For panels (a) and (c) data from both groups were pooled, while in panels (b) and (d) data from all rats and groups are shown individually. (a) Temporal evolution of spike frequency (left), 4–14 Hz power (middle), and number of IF runs (right), in vehicle (grey) and CNO (black) trials (consecutive 10-minute intervals before (—10–0 min) and after pilocarpine and vehicle/CNO injection. N=6 ArchT-virus transfected and five untransfected rats (two pairs of trials per animal, which were averaged within each rat; mean±s.e.m.). (b) Same data as in a, but plotted as cumulative seizure metrics for each individual animal (indicated by colour; blue hues with triangle symbols indicate untransfected animals, red hues with circles indicate ArchT-transfected animals; display as in Fig. 2f). (c) Temporal evolution of spike frequency (left), 4–14 Hz power (middle), and number of IF runs (right), in vehicle (grey) and CNO (black) trials (consecutive 10-min-intervals before and after picrotoxin and vehicle/CNO injection. N=5 ArchT-transfected and six untransfected rats (two pairs of trials averaged within each rat; one animal in the ArchT-group contributed only one pair of trials; mean±s.e.m.). (d) Same data as in c, but plotted as cumulative electrographic seizure metrics colour-coded as in b.

Figure 5. Chemical–genetic silencing of picrotoxin-induced motor seizures.

(a,b) Pair-wise comparison of the number of episodes with severe motor seizures (class 3, as rated for Fig. 3c, see Methods) between vehicle and CNO trials in hM4D_i-transfected (a, n=7) and untransfected (b, n=6) rats. Two pairs of trials were conducted per animal and the counts averaged within each animal. Orange bar indicates the decrease (%, right axis), where significant (paired t-test; P<0.05). (c) The absolute difference in the number of severe motor seizures for hM4D_i-transfected (black) and untransfected rats (grey; error bars indicate s.e.m.; asterisk indicates statistical significance, P=0.031, one-tailed unpaired t-test).

Figure 6. Chemical–genetic silencing of focal neocortical epilepsy.

(a) Sample EEG traces from a tetanus toxin injected animal, showing background activity (1) and four types of epileptiform activity: ‘long events of low amplitude’ (2), ‘short events of high amplitude’ (3), ‘long event of high amplitude’ (4), and ‘high amplitude plus intermittent spikes’ (5) (see ref. 7). (b–d) Pair-wise comparison of the frequency of epileptiform events (b), coastline (c) and high-frequency power (d) between vehicle and CNO trials. N=6 rats; 15 pairs of trials, averaged within animal where repeated. Orange bars indicate the decrease (%, right axis), where significant (Wilcoxon test; P<0.05).

Figure 7. Chemical–genetic inhibition of synaptic transmission.

(a) Experimental configuration; a stimulation electrode was placed in stratum radiatum to activate Schaffer collateral fibres expressing HA-hM4D_i-mCitrine. The resulting fEPSP, evoked every 30 s, was recorded further medial in CA1 with an extracellular electrode. (b) Sample traces showing a stimulation artifact, fibre volley and subsequent fEPSP under baseline condition (black), during CNO wash-in (10 μM; red) and after washout (blue). (c) Average normalized slope of evoked fEPSPs (black) and average normalized amplitude of evoked fibre volleys over time before (6 min), during (10 min) and after (6 min) CNO (10 μM) across slices (n=9). Slices were only included if mCitrine fluorescence was visible in CA3 and CA1. Error bars show s.e.m.