Nitrous oxide (N(2)O) requires the N-methyl-D-aspartate receptor for its action in Caenorhabditis elegans

Abstract

Nitrous oxide (N(2)O, also known as laughing gas) and volatile anesthetics (VAs), the original and still most widely used general anesthetics, produce anesthesia by ill-defined mechanisms. Electrophysiological experiments in vertebrate neurons have suggested that N(2)O and VAs may act by distinct mechanisms; N(2)O antagonizes the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors, whereas VAs alter the function of a variety of other synaptic proteins. However, no genetic or pharmacological experiments have demonstrated that any of these in vitro actions are responsible for the behavioral effects of either class of anesthetics. By using genetic tools in Caenorhabditis elegans, we tested whether the action of N(2)O requires the NMDA receptor in vivo and whether its mechanism is shared by VAs. Distinct from the action of VAs, N(2)O produced behavioral defects highly specific and characteristic of that produced by loss-of-function mutations in both NMDA and non-NMDA glutamate receptors. A null mutant of nmr-1, which encodes a C. elegans NMDA receptor, was completely resistant to the behavioral effects of N(2)O, whereas a non-NMDA receptor-null mutant was normally sensitive. The N(2)O-resistant nmr-1(null) mutant was not resistant to VAs. Likewise, VA-resistant mutants had wild-type sensitivity to N(2)O. Thus, the behavioral effects of N(2)O require the NMDA receptor NMR-1, consistent with the hypothesis formed from vertebrate electrophysiological data that a major target of N(2)O is the NMDA receptor.

Figures

Fig. 2.

Effect of loss-of-function mutations in glr-1 and nmr-1 on N2O sensitivity. Rate of reversing direction in air and in 70% N2O of the wild-type strain N2, glr-1(ky176lf), nmr-1(ak4lf), and nmr-1(ak4) transformed with wild-type nmr-1 (rescued ak4). Unlike the data in Table 1, these data were collected after a 10-min incubation on the plate, which increased the baseline frequency of reversing direction to improve the sensitivity of detecting a reduction in reversal frequency by N2Ointhe glr-1 and nmr-1 mutant animals. All data are expressed as means ± SD. n > 10 animals for all conditions. *, different from air at P < 0.01, two-tailed unpaired t test.

Fig. 4.

Effect of glutamate receptor mutations on VA and aldicarb sensitivity. (A and B) EC50 values were calculated from a minimum of eight values. The ky176 rescued strain is VM1494 (see Methods). *, different from N2 by nonlinear regression analysis at P < 0.01. †, different from glr-1(ky176). (C) Fraction moving versus incubation time on agar plates containing 0.35 mM aldicarb. At least 30 animals were scored for each strain. The fraction moving of nmr-1(ak4), glr-1(ky176), and nmr-1(ak4);glr-1(ky176) were significantly greater at all time points between 2 and 3.5 h (P < 0.01, unpaired two-tailed t test). (D) Identical to C, except that the agar plates contained 0.1 mM levamisole. No values were significantly different than those of wild type.

Fig. 1.

Concentration-dependent effect of N2O against forward foraging behavior in wild type. Cylinders containing 20%, 40%, 50%, 60%, and 70% ± 0.5% N2OinO2 (Puritan Medical Products) were used to flush assay chambers with >25 vol of gas; assays were then performed as described in Methods. Data points are expressed as means ± SEM of values normalized to 0% control values obtained on the same day. n for both measurements = 111 (0%), 22 (20%), 24 (40%), 21 (50%), 19 (60%), and 35 (70%). Curve fits to y = minimum + (maximum – min)/[1 + ([VA]/EC50)–k]. Minimum was assumed to be the measured response of nmr-1(ak4 null) mutant – 6.6% for the percentage of time backwards, 13.0% for reversals per min. EC50 (percentage of time backwards) = 38.9 ± 3.6 vol%, 95% confidence interval = 10.1, k = 2.45 ± 0.6. EC50 (reversals per min) = 38.1 ± 2.7 vol%, 95% confidence interval = 7.4, k = 2.47 ± 0.5. P values for the percentage of time backwards and the reversals per min were by one-tailed unpaired t test versus air controls performed in parallel on the same day = 20% (0.40, 0.32); 40% (0.01, 0.007); 50% (0.003, 0.0001); 60% (4 × 10–5, 0.0001); and 70% (0.0002, 3 × 10–6).

Fig. 3.

Sensitivity of VA-resistant mutants to N2O and of N2O-resistant nmr-1(ak4) to VAs. (A) Rate of reversing direction of wild-type and two VA-resistant mutants.*, different from air at P < 0.01, unpaired two-tailed t test. (B–E) Sensitivity of N2 (▪) and nmr-1(ak4) (•) to isoflurane and halothane in two locomotion assays. Compared with N2, nmr-1(ak4) is not significantly resistant to either VA as measured by the ability to disperse across an agar plate (B and D) or by the rate of body bends (C and E). Significance threshold was P < 0.01 by nonlinear regression analysis (63, 64).