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

Abstract

Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New Background 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. Methods 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. Results 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. Conclusions 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.

A vertebrate model to reveal neural substrates underlying the transitions between conscious and unconscious states

“…We used three different behavioral assays to study anesthesia in the zebrafish. First, we used loss of spontaneous movement (SPONT) as a surrogate sedation phenotype, as has been reported previously 23 , 24 . Second, we used loss of the startle reflex as a general anesthetic phenotype.…”

“…There are two ways of triggering the startle reflex, through a light stimulus or a TAP stimulus. The light stimulus has been used previously 24 and both sedative medications as well as general anesthetics caused loss of the movement response. Therefore, we decided to use the tap stimulus to be able to better differentiate between sedation and general anesthesia.…”

“…Here we show that a vertebrate model system, the larval zebrafish, can provide a simple, robust, quantitative model of drug-induced behaviors that should be easily reproduced in other laboratories. We examined motion in response to three stimuli; none (SPONT), TAP and ELEC, a paradigm of progressively more noxious stimulus 22 , 24 . Using our data, we can define a loss of SPONT as sedation, ELEC as deep sedation and TAP as general anesthesia.…”

Respiratory depression and analgesia by opioid drugs in freely-behaving larval zebrafish

“…Our novel drug discovery models allow high-throughput drug screening in a simple and amenable animal model presenting similar pharmacological and genetic profiles than humans (MacRae & Peterson, 2015). Although zebrafish has been used to identify new anesthetics and their mechanisms of actions (McGrath et al, 2020; (Yang et al, 2019), our study is the first to assess respiratory depression by opioid drugs in larval zebrafish combined with analgesia. In addition to high-throughput screening approaches, our models can also be used in combination of transgenic or knockout zebrafish to better understand the mechanisms opioid inhibition, analgesia and respiratory depression, as well as live microscopy of neural circuits of pain and respiration (Ahrens, Orger, Robson, Li, & Keller, 2013).…”

Characterization of zebrafish GABAA receptor subunits

“…Knockdown of the α2 subunit perturbed the expression of the proneural gene neurod and a GABA-synthesizing enzyme gene gad1b within 1 day of development 16 . Zebrafish larvae lacking the β3 subunit showed reduced sensitivity to anesthetic drugs such as etomidate and propofol 17 . Patch-clamp recordings of GABA A receptor-mediated miniature inhibitory postsynaptic currents from zebrafish Mauthner cells revealed three different types of gating kinetics, suggesting that zebrafish also have multiple GABA A receptor subtypes comprising different subunit combinations 18 .…”