Objective: Pannexin-1 (Panx1) is suspected of having a critical role in modulating neuronal excitability and acute neurological insults. Herein, we assess the changes in behavioral and electrophysiological markers of excitability associated with Panx1 via three distinct models of epilepsy. Methods Control and Panx1 knockout C57Bl/6 mice of both sexes were monitored for their behavioral and electrographic responses to seizure-generating stimuli in three epilepsy models—(1) systemic injection of pentylenetetrazol, (2) acute electrical kindling of the hippocampus and (3) neocortical slice exposure to 4-aminopyridine. Phase-amplitude cross-frequency coupling was used to assess changes in an epileptogenic state resulting from Panx1 deletion. Results: Seizure activity was suppressed in Panx1 knockouts and by application of Panx1 channel blockers, Brilliant Blue-FCF and probenecid, across all epilepsy models. In response to pentylenetetrazol, WT mice spent a greater proportion of time experiencing severe (stage 6) seizures as compared to Panx1-deficient mice. Following electrical stimulation of the hippocampal CA3 region, Panx1 knockouts had significantly shorter evoked afterdischarges and were resistant to kindling. In response to 4-aminopyridine, neocortical field recordings in slices of Panx1 knockout mice showed reduced instances of electrographic seizure-like events. Cross-frequency coupling analysis of these field potentials highlighted a reduced coupling of excitatory delta–gamma and delta-HF rhythms in the Panx1 knockout. Significance: These results suggest that Panx1 plays a pivotal role in maintaining neuronal hyperexcitability in epilepsy models and that genetic or pharmacological targeting of Panx1 has anti-convulsant effects.

Convulsive seizure behaviors are a hallmark feature of epilepsy, but automated detection of these events in freely moving animals is difficult. Here, we employed a high-resolution multi-camera array microscope with high-speed video acquisition and custom supervised machine learning (ML) for automated detection of larval zebrafish between 3- and 7-days post-fertilization (dpf). We assessed data from over 2700 zebrafish either exposed to a chemoconvulsant (pentylenetetrazole, PTZ) or genetic zebrafish lines representing Developmental Epileptic Encephalopathy (DEE) syndromes. Using eight-point skeletal body pose estimation for tracking individual larvae arrayed in a 96-well format, we report reliable, quantitative and age-dependent changes in maximum swim speed, as well as eye-, head- and tail- angle kinematics. Finally, we employed an ML-based algorithm to automatically identify normal and abnormal behaviors in an unbiased manner. Our results offer a robust framework for automated detection of zebrafish seizure-associated behaviors.

Dravet syndrome is a severe genetic epilepsy primarily caused by de novo mutations in a voltage-activated sodium channel gene (SCN1A). Patients face life-threatening seizures that are largely resistant to available anti-seizure medications. Preclinical Dravet syndrome animal models are a valuable tool to identify candidate anti-seizure medications for these patients. Among these, scn1lab mutant zebrafish, exhibiting spontaneous seizure-like activity, are particularly amenable to large-scale drug screening. Thus far, we have screened more than 3000 drug candidates in scn1lab zebrafish mutants, identifying valproate, stiripentol, and fenfluramine e.g., Food and Drug Administration approved drugs, with clinical application in the Dravet syndrome population. Successful phenotypic screening in scn1lab mutant zebrafish is rigorous and consists of two stages: (i) a locomotion-based assay measuring high-velocity convulsive swim behavior and (ii) an electrophysiology-based assay, using in vivo local field potential recordings, to quantify electrographic seizure-like events. Historically, nearly 90% of drug candidates fail during translation from preclinical models to the clinic. With such a high failure rate, it becomes necessary to address issues of replication and false positive identification. Leveraging our scn1lab zebrafish assays is one approach to address these problems. Here, we curated a list of nine anti-seizure drug candidates recently identified by other groups using preclinical Dravet syndrome models: 1-Ethyl-2-benzimidazolinone, AA43279, chlorzoxazone, donepezil, lisuride, mifepristone, pargyline, soticlestat and vorinostat. First-stage locomotion-based assays in scn1lab mutant zebrafish identified only 1-Ethyl-2-benzimidazolinone, chlorzoxazone and lisuride. However, second-stage local field potential recording assays did not show significant suppression of spontaneous electrographic seizure activity for any of the nine anti-seizure drug candidates. Surprisingly, soticlestat induced frank electrographic seizure-like discharges in wild-type control zebrafish. Taken together, our results failed to replicate clear anti-seizure efficacy for these drug candidates highlighting a necessity for strict scientific standards in preclinical identification of anti-seizure medications.

The molecular mechanisms of excitation/inhibition imbalances promoting seizure generation in epilepsy patients are not fully understood. Evidence suggests that Pannexin1 (Panx1), an ATP release channel, modulates the excitability of the brain. In this report, we performed electrophysiological, behavioral, and molecular phenotyping experiments on zebrafish larvae bearing genetic or pharmacological knockouts of Panx1a and Panx1b channels, each homologous to human PANX1. When Panx1a function is lost, or both channels are under pharmacological blockade, seizures with ictal-like events and seizure-like locomotion are reduced in the presence of pentylenetetrazol. Transcriptome profiling by RNA-seq demonstrates a spectrum of distinct metabolic and cell signaling states which correlate with the loss of Panx1a. Furthermore, the pro- and anticonvulsant activities of both Panx1 channels affect ATP release and involve the purinergic receptor P2rx7. Our findings suggest a subfunctionalization of Panx1 enabling dual roles in seizures, providing a unique and comprehensive perspective to understanding seizure mechanisms in the context of this channel.

The molecular mechanisms of excitation-inhibition imbalances promoting seizure generation in epilepsy patients are not fully understood. Experimental evidence suggests that Pannexin1 ( Panx1 ), an ATP release channel, modulates excitability of the brain. In this report, we have performed behavioral and molecular phenotyping experiments on zebrafish larvae bearing genetic or pharmacological knockouts of panx1a or panx1b channels, each highly homologous to human PANX1. When Panx1a function is lost or both channels are under pharmacological blockage, treatment with pentylenetetrazol to induce seizures causes reduced ictal-like events and seizure-like locomotion. These observations were extended by transcriptome profiling, where a spectrum of distinct metabolic and cell signaling states correlates with the loss of panx1a . The pro- and anticonvulsant activities of both Panx1 channels affects ATP release and involves the purinergic receptor p2rx7 . We propose that Panx1 zebrafish models offer opportunities to explore the molecular and physiological basis of seizures and assist anticonvulsant drug discovery.