Photomotor Responses in Zebrafish and Electrophysiology Reveal Varying Interactions of Anesthetics Targeting Distinct Sites on gamma-Aminobutyric Acid Type A Receptors

Background: Etomidate, barbiturates, alfaxalone, and propofol are anesthetics that allosterically modulate gamma-aminobutyric acid type A (GABA(A)) receptors via distinct sets of molecular binding sites. Two-state concerted coagonist models account for anesthetic effects and predict supra-additive interactions between drug pairs acting at distinct sites. Some behavioral and molecular studies support these predictions, while other findings suggest potentially complex anesthetic interactions. We therefore evaluated interactions among four anesthetics in both animals and GABA(A) receptors. Methods: The authors used video assessment of photomotor responses in zebrafish larvae and isobolography to evaluate hypnotic drug pair interactions. Voltage clamp electrophysiology and allosteric shift analysis evaluated coagonist interactions in alpha 1 beta 3 gamma 2L receptors activated by gamma-aminobutyric acid (GABA) versus anesthetics [log(d, AN):log(d, GABA) ratio]. Anesthetic interactions at concentrations relevant to zebrafish were assessed in receptors activated with low GABA. Results: In zebrafish larvae, etomidate interacted additively with both propofol and the barbiturate R-5-allyl-1-methyl m-trifluoromethyl mephobarbital (R-mTFD-MPAB; mean +/- SD alpha = 1.0 +/- 0.07 and 0.96 +/- 0.11 respectively, where 1.0 indicates additivity), while the four other drug pairs displayed synergy (mean alpha range 0.76 to 0.89). Electrophysiologic allosteric shifts revealed that both propofol and R-mTFD-MPAB modulated etomidate-activated receptors much less than GABA-activated receptors [log(d, AN):log(d, GABA) ratios = 0.09 +/- 0.021 and 0.38 +/- 0.024, respectively], while alfaxalone comparably modulated receptors activated by GABA or etomidate [log(d) ratio = 0.87 +/- 0.056]. With low GABA activation, etomidate combined with alfaxalone was supra-additive (n = 6; P = 0.023 by paired t test), but etomidate plus R-mTFD-MPAB or propofol was not. Conclusions: In both zebrafish and GABA(A) receptors, anesthetic drug pairs interacted variably, ranging from additivity to synergy. Pairs including etomidate displayed corresponding interactions in animals and receptors. Some of these results challenge simple two-state coagonist models and support alternatives where different anesthetics may stabilize distinct receptor conformations, altering the effects of other drugs.