Abstracts

Rationale: More than one third of patients with epilepsy continue to have seizures despite treatment with anti-epileptic drugs. In this study we evaluate whether selective silencing of excitatory hippocampal neurons using the inhibitory Designer Receptor Exclusively Activated by Designer Drugs (DREADD) hM4Di leads to suppression of spontaneous hippocampal seizures in the intrahippocampal kainic acid (IHKA) mouse model for TLE. Methods: Mice (n=78) were injected with kainic acid in the right hippocampus (200 ng/50 nl, AP -2mm ML +1.5mm DV -1.8mm relative to bregma). Three weeks later, animals were injected in the KA-lesioned hippocampus with 500nl adeno-associated-viral vector carrying genes encoding hM4Di-mCherry fusion protein (4.66E+13 GC/ml, DREADD group, n=53) or mCherry only (2.08E+13 GC/ml, non-DREADD group, n=25) under transcriptional control of CamKIIalpha promotor, specific for excitatory neurons. Subsequently a bipolar recording electrode was implanted at the injection site. DREADD (n=14) and non-DREADD mice (n=11) with most frequent hippocampal seizures were selected for further testing. In the first experiment three different doses Clozapine-N-Oxide (CNO) were administered intraperitoneally (i.p.) in a first series of mice (1, 3 and 10 mg/kg, n=10, 6 and 8). In the second experiment the same mice were injected with 10 mg/kg CNO on five consecutive days (n=6). In a third experiment, other mice were injected i.p. with a subclinical, but DREADD-activating, dose of clozapine (0.1 mg/kg, n=4) and CNO (10 mg/kg). Non-DREADD mice were injected with 10mg/kg CNO (n=11) and 0.1mg/kg Clozapine (n=4). Total time in seizure during the day before treatments and during the first 8 hours post treatment were compared to evaluate seizure suppressing effects. Results: After injection of 1, 3 and 10 mg/kg CNO the total time in seizures (88 ± 34s/h, 50 ± 7s/h and 16 ± 6s/h respectively) were highly significantly reduced compared to baselines (p<0.01, 607 ± 83s/h, 695 ± 74s/h and 594 ± 104s/h respectively). These effects gradually reduced over time and were no longer present one day after injection (fig 1). Chronic treatment on five consecutive days consistently reduced total time in seizures (day 1: 123 ± 98s/h, day 2: 112 ± 40s/h, day 3: 164 ± 67s/h, day 4: 225 ± 98s/h, day 5: 277 ± 98s/h) compared to the baseline day before treatment (750 ± 104s/h). Mice treated both with 0.1 mg/kg clozapine and 10 mg/kg CNO displayed similar significant reductions in both treatments (-405 ± 71s/h and -445 ± 47s/h). In non-DREADD animals neither CNO (10 mg/kg) nor clozapine (0.1mg/kg) had effect on total time in seizures. Conclusions: These results support a crucial role for excitatory hippocampal neurons in spontaneous hippocampal seizure generation in the IHKA mouse model. This study also confirms that subclinical doses of clozapine can be used to activate DREADDs. Most importantly, this chemogenetic approach could lead to epilepsy therapy where a systemically administered drug very selectively modulates specific neurons of the seizure network without affecting other cells in the brain or body, resulting in a very potent seizure suppression without any side effects. Funding: Jana Desloovere and Robrecht Raedt received funding from the Special Research Funds of Ghent University to perform this research.