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

Etomidate, barbiturates, alphaxalone, and propofol are anesthetics that allosterically modulate GABA_A receptors via distinct sets of molecular binding sites. Two-state concerted co-agonist 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.

We 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 co-agonist interactions in α1β3γ2L receptors activated by GABA vs. anesthetics [log(d, AN):log(d, GABA) ratio]. Anesthetic interactions at concentrations relevant to zebrafish were assessed in receptors activated with low GABA.

In zebrafish larvae, etomidate interacted additively with both propofol and the barbiturate R-mTFD-MPAB (mean ± SD α = 1.0 ± 0.07 and 0.96 ± 0.11 respectively, where 1.0 indicates additivity), while the four other drug pairs displayed synergy (mean α 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 alphaxalone comparably modulated receptors activated by GABA or etomidate [log(d) ratio = 0.87 ± 0.056]. With low GABA activation, etomidate combined with alphaxalone was supra-additive (n = 6; P = 0.023 by paired t-test), but etomidate plus R-mTFD-MPAB or propofol was not.

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 co-agonist models and support alternatives where different anesthetics may stabilize distinct receptor conformations, altering the effects of other drugs.