Drug-selective Anesthetic Insensitivity of Zebrafish Lacking γ-Aminobutyric Acid Type A Receptor β3 Subunits.

Abstract

Transgenic mouse studies suggest that γ-aminobutyric acid type A (GABAA) receptors containing β3 subunits mediate important effects of etomidate, propofol, and pentobarbital. Zebrafish, recently introduced for rapid discovery and characterization of sedative-hypnotics, could also accelerate pharmacogenetic studies if their transgenic phenotypes reflect those of mammals. The authors hypothesized that, relative to wild-type, GABAA-β3 functional knock-out (β3) zebrafish would show anesthetic sensitivity changes similar to those of β3 mice. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 mutagenesis was used to create a β3 zebrafish line. Wild-type and β3 zebrafish were compared for fertility, growth, and craniofacial development. Sedative and hypnotic effects of etomidate, propofol, pentobarbital, alphaxalone, ketamine, tricaine, dexmedetomidine, butanol, and ethanol, along with overall activity and thigmotaxis were quantified in 7-day postfertilization larvae using video motion analysis of up to 96 animals simultaneously. Xenopus oocyte electrophysiology showed that the wild-type zebrafish β3 gene encodes ion channels activated by propofol and etomidate, while the β3 zebrafish transgene does not. Compared to wild-type, β3 zebrafish showed similar morphology and growth, but more rapid swimming. Hypnotic EC50s (mean [95% CI]) were significantly higher for β3 versus wild-type larvae with etomidate (1.3 [1.0 to 1.6] vs. 0.6 [0.5 to 0.7] µM; P < 0.0001), propofol (1.1 [1.0 to 1.4] vs. 0.7 [0.6 to 0.8] µM; P = 0.0005), and pentobarbital (220 [190 to 240] vs. 130 [94 to 179] μM; P = 0.0009), but lower with ethanol (150 [106 to 213] vs. 380 [340 to 420] mM; P < 0.0001) and equivalent with other tested drugs. Comparing β3 versus wild-type sedative EC50s revealed a pattern similar to hypnosis. Global β3 zebrafish are selectively insensitive to the same few sedative-hypnotics previously reported in β3 transgenic mice, indicating phylogenetic conservation of β3-containing GABAA receptors as anesthetic targets. Transgenic zebrafish are potentially valuable models for sedative-hypnotic mechanisms research.