Potentiation of GABAA receptor activity by volatile anaesthetics is reduced by α5GABAA receptor-preferring inverse agonists

Abstract

Background: Animal studies have shown that memory deficits in the early post-anaesthetic period can be prevented by pre-treatment with an inverse agonist that preferentially inhibits α5 subunit-containing γ-aminobutyric acid type A (α5GABA(A)) receptors. The goal of this in vitro study was to determine whether inverse agonists that inhibit α5GABA(A) receptors reduce anaesthetic potentiation of GABAA receptor activity.

Methods: Cultures of hippocampal neurones were prepared from Swiss white mice, wild-type mice (genetic background C57BL/6J and Sv129Ev) and α5GABA(A)receptor null mutant (Gabra5-/-) mice. Whole-cell voltage clamp techniques were used to study the effects of the α5GABA(A) receptor-preferring inverse agonists L-655,708 and MRK-016 on anaesthetic potentiation of GABA-evoked currents.

Results: L-655,708 (50 nM) reduced sevoflurane potentiation of GABA-evoked current in wild-type neurones but not Gabra5-/- neurones, and produced a rightward shift in the sevoflurane concentration-response plot [sevoflurane EC50: 1.9 (0.1) mM; sevoflurane+L-655,708 EC(50): 2.4 (0.2) mM, P<0.05]. Similarly, L-655,708 (50 nM) reduced isoflurane potentiation of GABA-evoked current [isoflurane: 4.0 (0.6) pA pF(-1); isoflurane+L-655,708: 3.1 (0.5) pA pF(-1), P<0.01]. MRK-016 also reduced sevoflurane and isoflurane enhancement of GABA-evoked current [sevoflurane: 1.5 (0.1) pA pF(-1); sevoflurane+MRK-016 (10 nM): 1.2 (0.1) pA pF(-1), P<0.05; isoflurane: 3.5 (0.3) pA pF(-1); isoflurane+MRK-016 (1 nM): 2.9 (0.2) pA pF(-1), P<0.05].

Conclusions: L-655,708 and MRK-016 reduced the potentiation by inhaled anaesthetics of GABAA receptor activated by a low concentration of GABA. Future studies are required to determine whether this effect contributes to the memory preserving properties of inverse agonists after anaesthesia.

Keywords: anaesthetics volatile, sevoflurane, isoflurane; brain, GABA; electrophysiology.

Figures

Fig 1

L-655,708 inhibits currents evoked by low, but not high, concentrations of GABA in neurones from Swiss white mice. Summarized data show that L-655,708 (L6) has a concentration-dependent inhibitory effect on currents evoked by GABA (0.1 and 1 µM) but no effect on currents evoked by GABA (10 and 100 µM) (n=5–6). **P<0.01 vs GABA. Data are mean (sem).

Fig 2

L-655,708 inhibits GABA-evoked currents in neurones from wild-type but not Gabra5−/− mice. (a) and (b) The effects of L-655,708 (L6) on currents evoked by GABA (0.5 µM). (c) and (d) Summarized data showing the concentration-dependent effects of L-655,708 on GABA-evoked current (n=6). **P<0.01 vs GABA. Data are mean (sem).

Fig 3

L-655,708 attenuates the potentiating effects of sevoflurane in neurones from wild-type but not Gabra5−/− mice. (a) and (b) The effects of L-655,708 (L6) on sevoflurane (SEVO, 266 μM)-mediated enhancement of currents evoked by GABA (0.5 μM). (c) and (d) The concentration-dependent effects of L-655,708 on sevoflurane-mediated enhancement of GABA-evoked current are shown (n=6). **P<0.01 vs GABA+SEVO. Data are mean (sem).

Fig 4

L-655,708 decreases the potency of sevoflurane for increasing GABAA receptor activity in neurones from Swiss white mice. (a) The effects of L-655,708 (L6, 50 nM) on sevoflurane (SEVO)-mediated enhancement of currents evoked by GABA (0.5 µM). As shown previously, a tail current was observed during drug washout at higher concentrations of sevoflurane (6, 8, and 10 mM). (b) The corresponding concentration–response plots for sevoflurane potentiation of GABA-evoked current in the absence or presence of L-655,708 (L6). L-655,708 increased the EC50 from 1.9 (0.1) to 2.4 (0.2) (P<0.05, n=6) without affecting the maximal response. Application of sevoflurane (10 mM) caused a decrease in current, which is consistent with previous studies. Data points for sevoflurane (10 mM) were excluded from the curve-fitting analyses. Data are mean (sem).

Fig 5

L-655,708 inhibits the potentiating effects of isoflurane on GABA currents in neurones from Swiss white mice. (a) The effects of L-655,708 (L6) on isoflurane (ISO, 250 µM)-mediated enhancement of currents evoked by GABA (0.5 µM). (b) Summarized data showing the concentration-dependent inhibitory effects of L-655,708 on isoflurane-mediated enhancement of GABA-evoked currents (n=6). **P<0.01, ***P<0.001 vs GABA+ISO. Data are mean (sem).

Fig 6

MRK-016 attenuates the potentiating effects of sevoflurane and isoflurane in neurones from Swiss white mice. (a) and (b) The effects of MRK-016 (MRK) on sevoflurane (SEVO, 266 μM) and isoflurane (ISO, 250 μM) enhancement of currents evoked by GABA (0.5 μM). (c) and (d) Summarized data showing the concentration-dependent inhibitory effects of MRK-016 on sevoflurane- and isoflurane-mediated enhancement of GABA current (n=6). *P<0.05, **P<0.01, ***P<0.001 vs GABA+SEVO or GABA+ISO. Data are mean (sem).

Fig 7

L-655,708 and MRK-016 inhibit GABA-evoked currents in neurones from Swiss white mice. (a) and (b) The effects of L-655,708 (L6) and MRK-016 (MRK) on currents evoked by GABA (0.5 µM). (c) and (d) Summarized data showing the concentration-dependent inhibitory effects of L-655,708 and MRK-016 on GABA-evoked current (n=6). *P<0.05, **P<0.01, and ***P<0.001 vs GABA. Data are mean (sem).