Newly Published
Perioperative Medicine  |   September 2019
Drug-selective Anesthetic Insensitivity of Zebrafish Lacking γ-Aminobutyric Acid Type A Receptor β3 Subunits
Author Notes
  • From the Department of Anesthesiology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China (X.Y., B.Y.); the Department of Anesthesia Critical Care and Pain Medicine (X.Y., Y.J., R.J.F., S.A.F.); and the Center for Regenerative Medicine (K.M., E.C.L.), Massachusetts General Hospital, Boston, Massachusetts.
  • Some of the work presented in this article has been presented at the annual meetings of the International Society of Anesthetic Pharmacology and the American Society of Anesthesiologists, Boston, Massachusetts, October 20 to 24, 2017.
    Some of the work presented in this article has been presented at the annual meetings of the International Society of Anesthetic Pharmacology and the American Society of Anesthesiologists, Boston, Massachusetts, October 20 to 24, 2017.×
  • X.Y. and Y.J. contributed equally to this work.
    X.Y. and Y.J. contributed equally to this work.×
  • Submitted for publication January 21, 2019. Accepted for publication July 29, 2019.
    Submitted for publication January 21, 2019. Accepted for publication July 29, 2019.×
  • Correspondence: Address correspondence to Dr. Forman: Department of Anesthesia, Critical Care, and Pain Medicine, Jackson 444, Massachusetts General Hospital, Boston, Massachusetts 02114. saforman@mgh.harvard.edu. Information on purchasing reprints may be found at www.anesthesiology.org or on the masthead page at the beginning of this issue. Anesthesiology’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.
Article Information
Perioperative Medicine / Pharmacology
Perioperative Medicine   |   September 2019
Drug-selective Anesthetic Insensitivity of Zebrafish Lacking γ-Aminobutyric Acid Type A Receptor β3 Subunits
Anesthesiology Newly Published on September 24, 2019. doi:10.1097/ALN.0000000000002963
Anesthesiology Newly Published on September 24, 2019. doi:10.1097/ALN.0000000000002963
Abstract

Editor’s Perspective:

What We Already Know about This Topic:

  • β3 subunits of the γ-aminobutyric acid type A (GABAA) receptor play a central role in mediating hypnotic and sedative effects of etomidate, propofol, and pentobarbital in mice

  • Zebrafish are a vertebrate animal model amenable to high-throughput pharmacologic studies

  • The role of GABAA receptor β3 subunits in mediating the effects of anesthetic drugs in zebrafish has not been previously reported

What This Article Tells Us That Is New:

  • Zebrafish larvae lacking functional β3 subunits of the γ-aminobutyric acid type A (GABAA) receptor displayed selective insensitivity to the same anesthetic drugs (etomidate, propofol, and pentobarbital) as transgenic mice with mutated GABAA receptor β3 subunits

  • These experiments indicate phylogenetic conservation of β3 subunit-containing GABAA receptors between zebrafish and mice in mediating hypnotic and sedative components of general anesthesia

  • These observations also suggest that zebrafish can be a valuable experimental model for mechanisms of anesthesia research

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.