The efficacy of γ-aminobutyric acid type A receptor (GABA AR) subtype-selective positive allosteric modulators in blocking tetramethylenedisulfotetramine (TETS)-induced seizure-like behavior in larval zebrafish with minimal sedation

Abstract

The chemical threat agent tetramethylenedisulfotetramine (TETS) is a γ-aminobutyric acid type A receptor (GABA A R) antagonist that causes life threatening seizures. Currently, there is no specific antidote for TETS intoxication. TETS-induced seizures are typically treated with benzodiazepines, which function as nonselective positive allosteric modulators (PAMs) of synaptic GABA A Rs. The major target of TETS was recently identified as the GABA A R α2β3γ2 subtype in electrophysiological studies using recombinantly expressed receptor combinations. Here, we tested whether these in vitro findings translate in vivo by comparing the efficacy of GABA A R subunit-selective PAMs in reducing TETS-induced seizure behavior in larval zebrafish. We tested PAMs targeting α1, α2, α2/3/5, α6, ß2/3, ß1/2/3, and δ subunits and compared their efficacy to the benzodiazepine midazolam (MDZ). The data demonstrate that α2- and α6-selective PAMs (SL-651,498 and SB-205384, respectively) were effective at mitigating TETS-induced seizure-like behavior. Combinations of SB-205384 and MDZ or SL-651,498 and 2–261 (ß2/3-selective) mitigated TETS-induced seizure-like behavior at concentrations that did not elicit sedating effects in a photomotor behavioral assay, whereas MDZ alone caused sedation at the concentration required to stop seizure behavior. Isobologram analyses suggested that SB-205384 and MDZ interacted in an antagonistic fashion, while the effects of SL-651,498 and 2–261 were additive. These results further elucidate the molecular mechanism by which TETS induces seizures and provide mechanistic insight regarding specific countermeasures against this chemical convulsant.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) causes seizure activity in larval zebrafish via antagonism of γ-aminobutyric acid type A receptor α1β2γ2

“…Zebrafish husbandry was conducted as previously described in (Mundy et al 2021 ). All animal husbandry, spawning and experimental manipulations of zebrafish were approved by the University of California, Davis Institutional Animal Care and Use Committee and conducted in accordance with the ARRIVE guidelines (Percie du Sert et al 2020 ).…”

“…Larval zebrafish have been proposed as a valid model organism for studying chemical-induced seizures (Griffin et al 2021 ), and we and others have used this model to study seizures triggered by GABA A R antagonists (Baraban et al 2005 ; Bandara et al 2020 ). A major advantage of using zebrafish larvae is the medium-to-high-throughput capacity that enables more efficient screening of therapeutics for anti-seizure efficacy (Mundy et al 2021 ).…”

Aquatic Freshwater Vertebrate Models of Epilepsy Pathology: Past Discoveries and Future Directions for Therapeutic Discovery

“…The recurrent nature of seizures after TETS exposure in humans as well as the lack of a targeted treatment make it attractive as a seizure-induction agent for research using aquatic vertebrate models. Compared to picrotoxin, in zebrafish larvae less TETS is required to evoke seizure behavior [ 94 , 144 ]. TETS also triggered high-frequency electrical discharges which were different from electrical measurements following picrotoxin exposure.…”

Diisopropylfluorophosphate (DFP) volatizes and cross-contaminates wells in a common 96-well plate format used in zebrafish larvae toxicology studies

“…All experiments complied with the NIH Guide for the Care and Use of Laboratory Animals (National Research Council (US), 2011) and ARRIVE guidelines (du Sert et al, 2020). Fish husbandry and spawning were conducted according to previously described methods (Mundy et al, 2021). Tropical 5D wild type ZF were obtained from Sinnhuber Aquatic Research Laboratory (SARL) at Oregon State University, Corvallis, OR, and subsequent generations were raised at UC Davis.…”

“…2A). Exposure time of 1 h was chosen because 1) no significant amount of death was observed at this timepoint, and 2) it is within the reasonable range of a typical behavioral assay performed after acute exposure (i.e., 40 min photomotor assay performed after acute exposure (Mundy et al, 2021)). The experiment was done in triplicate (n = 3 larval pairs per spatial position).…”