Abstracts

Rationale: Padsevonil (UCB0942, PSL) is a new drug candidate for treatment of focal (partial-onset) seizures in patients with drug-resistant epilepsy. PSL was designed to bind with high affinity to SV2 isoforms (SV2A, B and C) and with moderate affinity to the GABA-A receptor where it acts as a partial agonist. This pre- and post-synaptic mechanism of PSL translated into an improved efficacy profile across a large panel of epilepsy models including drug-resistant epilepsy (Leclercq et al. 2017, 2018). We further characterized the mode of action of PSL by exploring effects of PSL on neuronal network activity using all-optical electrophysiology (Optopatch, optical transmembrane potential) recordings in rat primary hippocampal neurons. Methods: Rat primary hippocampal neurons were prepared from E18 embryos and used at DIV7-15. Optopatch recordings were performed using a genetically encoded channelrhodopsin CheRiff (actuator) and fluorescent voltage sensor QuasAr (reporter). Genetic vectors were expressed in hippocampal neurons at DIV 7 using lentiviral transduction. Blue light (470 nm) was used to produce neuronal depolarization and red light (635 nm) to excite the fluorescent voltage sensor to record sub- and supra-threshold electrical activity. For synaptic transmission experiments, a CreOFF-CheRiff and CreON-QuasAr system was used to ensure specific stimulation of pre-synaptic cells and recording in post-synaptic cells. A dlx1/2-nucGFP marker detected pre- and post-synaptic inhibitory GABAergic neurons. Results: Spontaneous and intrinsic excitability was recorded by Optopatch across hundreds of neurons. Light-evoked stimulation (using step and ramp protocols) led to robust detection of individual spiking neurons. PSL (30 nM-10 µM) did not modulate spontaneous and intrinsic spiking activity of neurons. Carbamazepine (1-300 µM, +ve control) produced a dose-dependent inhibition of spontaneous and light-evoked neuronal spiking activity. Synaptic transmission experiments, using the CRE recombinase-dependent system, led to reliable measurement of inhibitory (IPSP) and excitatory post-synaptic (EPSP) potentials. We further validated pharmacological response to benzodiazepine ligands on IPSPs and to cyclothiazide (AMPA receptor modulator) on EPSPs. PSL, designed as a low affinity GABA-A receptor allosteric modulator, dose-dependently increased IPSP amplitude, rise-time and area with an EC50 value of ~3 µM. Expression of the dlx1/2-GFP marker indicated that only a low percentage of hippocampal neurons express dlx1/2 (2-5%), which could be reliably recorded by Optopatch. PSL increased IPSPs in post-synaptic dlx(+) and dlx(-) neurons. Conclusions: In this study, we explored the combination of Optopatch with CRE recombinase-dependent systems for the measurement of neuronal network activity. Optopatch recordings confirm that PSL does not modulate intrinsic neuronal excitability further strengthening its differentiation from classical antiepileptic drugs. This study also validates the post-synaptic mechanism of PSL mediated by the low affinity interaction and allosteric modulation of post-synaptic GABA-A receptors in dlx(+) and dlx(-) neurons. Funding: UCB Pharma-sponsored