A Behavioral and Electroencephalographic Study of Anesthetic State Induced by MK-801 Combined with Haloperidol, Ketamine and Riluzole in Mice

Abstract

Background: Ketamine is an intravenous anesthetic that acts as a channel blocker on the N-methyl- d -aspartate (NMDA) receptor, a glutamate receptor subtype. MK-801 is the most potent compound among noncompetitive NMDA receptor antagonists. Ketamine induces loss of the righting reflex (LORR) in rodents, which is one of the indicators of unconsciousness, whereas high doses of MK-801 produce ataxia, but not LORR. In contrast, we previously reported that MK-801 combined with a low dose of the dopamine receptor antagonist haloperidol-induced LORR in mice. To assess a neurophysiologically distinct brain state and demonstrate unconsciousness, electroencephalograms (EEG) need to be examined together with LORR. Therefore, we herein investigated EEG changes after the systemic administration of MK-801 alone or in combination with haloperidol, and compared them with those induced by ketamine, the glutamate release inhibitor riluzole, and the γ-aminobutyric acid type A receptor agonist propofol.

Methods: All drugs were intraperitoneally administered to adult male ddY mice (n = 168). General anesthesia was evaluated based on the righting reflex test. Animals who exhibited no righting for more than 30 seconds were considered to have LORR. In a separate group of mice, EEG of the primary visual cortex was recorded before and after the administration of MK-801 (3.0 mg/kg) alone or in combination with haloperidol (0.2 mg/kg), ketamine (150 mg/kg), riluzole (30 mg/kg), or propofol (240 mg/kg). The waveforms recorded were analyzed using EEG power spectra and spectrograms.

Results: The high dose of MK-801 alone did not induce LORR, whereas MK-801 combined with haloperidol produced LORR in a dose-dependent manner. Ketamine, riluzole, and propofol also dose-dependently induced LORR. In the EEG study, MK-801 alone induced a significant increase in δ power, while MK-801 plus haloperidol exerted similar effects on not only δ, but also θ and α power during LORR, suggesting that increases in δ, θ, and α power were necessary for LORR. The results obtained on MK-801 plus haloperidol were similar to those on ketamine in the behavioral and EEG studies, except for an increase in γ power by ketamine during LORR. Propofol significantly increased δ, θ, α, and β power during LORR. However, the EEG results obtained using riluzole, which produced a unique pattern of lower amplitude activity spanning most frequencies, markedly differed from those with the other drugs.

Conclusions: This study revealed differences in EEG changes induced by various sedatives. The results obtained on MK-801 alone and MK-801 plus haloperidol suggest the importance of dopamine transmission in maintaining the righting reflex.

Figure 1.

Time-course effects of MK-801 alone (A) or in combination with haloperidol (A and B), ketamine (C), riluzole (D), and propofol (E) on righting reflex scores in mice. All drugs were i.p. administered. MK-801 alone at a dose of 3.0 mg/kg was administered 30 min after the i.p. injection of vehicle saline (n = 7; A). Haloperidol at 0.2 mg/kg was injected 30 min before the administration of 3.0 mg/kg MK-801 (n = 6; A). Various doses of MK-801 (0.5, 1.0, 1.5, and 5.0 mg/kg, n = 6) were administered 30 min after the injection of haloperidol at 0.2 mg/kg (B). Various doses of ketamine (50, 60, 80, and 150 mg/kg, n = 5; C), riluzole (15, 20, 25, and 30 mg/kg, n = 7; D), and propofol (70, 100, 150, and 240 mg/kg, n = 5; E) were administered immediately after recording. The righting reflex was assessed every 2 min for 3 h after the injection of MK-801, ketamine, riluzole, or propofol using righting reflex scores. A score of 0 indicated a normal righting reflex; +1 indicated that the mouse righted itself within 2 s in all 3 trials (slightly impaired righting reflex); +2 indicated that the time to righting in the best response among 3 trials was >2 s, but <10 s (moderately or severely impaired righting reflex); +3 corresponded to the absence of the righting reflex (no righting within 10 s in all 3 trials). Each point represents the mean. i.p. indicates intraperitoneal.

Figure 2.

Effects of MK-801 in combination with haloperidol (A), ketamine (B), riluzole (C), and propofol (D) on the righting reflex in mice. All drugs were i.p. administered. Various doses of MK-801 (0.5, 1.0, 1.5, 2.0, 3.0, and 5.0 mg/kg, n = 6) were administered 30 min after the injection of haloperidol at 0.2 mg/kg (A). Various doses of ketamine (50, 60, 70, 80, 100, and 150 mg/kg, n = 5; B), riluzole (15.0, 20.0, 22.5, 25.0, 27.5, and 30.0 mg/kg, n = 7; C), and propofol (50, 70, 100, 150, 200, and 240 mg/kg, n = 5; D) were administered immediately after recording. The righting reflex was assessed every 2 min for 3 h after the injection of MK-801, ketamine, riluzole, or propofol using the righting reflex. The righting reflex was considered to be lost in an animal that exhibited no righting for more than 30 s. Each point represents the percent effect of 5 to 7 animals per dose. The dose response was fit to a sigmoidal curve to obtain the 50% effective dose (ED₅₀). Hill slopes and their corresponding 95% confidence limits were calculated using Prism version 7.03. i.p. indicates intraperitoneal.

Figure 3.

Representative recording showing an electroencephalographic spectrogram after i.p. administration of MK-801 alone (A) or in combination with haloperidol (B), ketamine (C), riluzole (D), or propofol (E). All drugs were i.p. administered. MK-801 alone at a dose of 3.0 mg/kg was administered 30 min after the i.p. injection of vehicle saline. Haloperidol at 0.2 mg/kg was injected 30 min before the administration of 3.0 mg/kg MK-801. Ketamine, riluzole, and propofol were administered at doses of 150, 30, and 240 mg/kg, respectively. i.p. indicates intraperitoneal.

Figure 4.

Effects of MK-801 alone (A) or in combination with haloperidol (B) on EEG power in δ (a), θ (b), α (c), β (d), and γ (e) frequency bands, and EMG power in integrated myoelectric potential (f) in mice. All drugs were i.p. administered. MK-801 alone at a dose of 3.0 mg/kg was administered 30 min after the i.p. injection of vehicle saline (n = 4; A). Haloperidol at 0.2 mg/kg was injected 30 min before the administration of 3.0 mg/kg MK-801 (n = 4; B). Total powers in each band for 30 min before the administration of MK-801 alone (A) or haloperidol (B) were divided by 3, and averaged 10 min powers were considered to be the baseline. Data on each 10–min cumulative power after MK-801 or haloperidol were normalized as percent (%) changes from baseline values. EMG power was analyzed in the same manner as EEG power. Each bar represents the mean ± SEM (n = 4). *P < .05, **P < .01 significantly different from the baseline (time −10 to 0), a 2-way analysis of variance followed by Dunnett’s test. EEG indicates electroencephalograms; EMG, electromyographic; i.p., intraperitoneal; SEM, standard error of the mean.

Figure 5.

Effects of ketamine (A), riluzole (B), and propofol (C) on EEG power in δ (a), θ (b), α (c), β (d), and γ (e) frequency bands, and EMG power in integrated myoelectric potential (f) in mice. Ketamine, riluzole, and propofol were intraperitoneally administered at doses of 150 mg/kg (n = 4; A), 30 mg/kg (n = 7; B), and 240 mg/kg (n = 4; C), respectively, at least 30 min after recordings. Total power in each band for 30 min before the administration of ketamine or riluzole was divided by 3, and averaged 10 min power was considered to be the baseline. Data on each 10–min cumulative power after ketamine or riluzole were normalized as percent (%) changes from baseline values. EMG power was analyzed in the same manner as EEG power. Each bar represents the mean ± SEM (n = 4–7). *P < .05, **P < .01 significantly different from the baseline (time −10 to 0), a 2-way analysis of variance followed by Dunnett’s test. EEG indicates electroencephalograms; EMG, electromyographic; SEM, standard error of the mean.