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ANESTHETIC PHARMACOLOGY

The Role of Nicotinic Inhibition in Ketamine-Induced Behavior

Department of Anesthesiology, Columbia University, New York, New York

Address correspondence and reprint requests to Pamela Flood, MD, Department of Anesthesiology, College of Physicians & Surgeons of Columbia University, 630 West 168th Street, New York, New York 10032. Address e-mail to pdf3{at}columbia.edu.

	Abstract

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Several anesthetic drugs are nicotinic antagonists at or below levels used for anesthesia, including ketamine and volatile anesthetics. In contrast, propofol does not inhibit nicotinic receptors. To determine the potential behavioral ramifications of nicotinic inhibition by ketamine, we determined the doses of ketamine required to induce immobility, impair the righting reflex, and cause analgesia in the absence and presence of several nicotinic ligands. Propofol was used as a control in similar experiments. When used as a sole anesthetic drug, 383 ± 22 mg/kg ketamine intraperitoneally (IP) was required for immobility and 180 ± 17 mg/kg IP impaired righting reflex. Propofol, 371 ± 34 mg/kg IP, induced immobility whereas 199 mg/kg IP inhibited the righting reflex. Nicotinic antagonists had no effect on the dose of propofol or ketamine required for either end-point. When nociceptive responses were tested at subhypnotic doses, no pronociceptive or antinociceptive phase was identified for propofol, whereas analgesia was induced at ketamine doses larger than 60 mg/kg IP. The broad-spectrum nicotinic antagonist mecamylamine enhanced the analgesic action of ketamine. These findings are different than those seen with volatile anesthetics, where nicotinic inhibition is thought to be responsible for a pronociceptive action. Such a phase is possibly obscured by analgesia induced as a result of N-methyl-d-aspartic acid antagonism by ketamine.

	Introduction

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Nicotine and other nicotinic agonists have antinociceptive effects in animal models and human studies (1,2). In our previous work, we have shown that the pronociceptive actions of volatile anesthetics are related to their actions as nicotinic antagonists (3). Volatile anesthetics have a biphasic effect on pain sensitivity; small doses up to 0.375% increase pain sensitivity, whereas larger doses decrease pain sensitivity. In contrast, we have found no evidence that nicotinic inhibition is causally related to volatile anesthetic-induced hypnosis or immobility (4,5).

Some IV anesthetics are nicotinic antagonists in vitro, whereas others are not. Ketamine is both a potent nicotinic antagonist and an antagonist at the N-methyl-d-aspartic acid (NMDA) receptor for glutamate (6–8). Propofol, in contrast, does not inhibit nicotinic receptors (9,10). Although ketamine is well known to have analgesic effects in humans (8), we sought to determine whether we could identify a discrete pronociceptive phase for ketamine or a role for nicotinic inhibition in the anesthetic end-points of hypnosis and immobility. We hypothesized that if nicotinic inhibition by ketamine played a role in any of these end-points, it would be accentuated by nicotinic antagonists. Conversely, the anesthetic end-points induced by propofol should be less affected.

	Methods

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With the approval of the Columbia University Committee on Animal Research, we studied male, C57BL/6J strain mice at 6–8 wk of age that were obtained from Jackson Laboratories (Bar Harbor, ME). We determined the dose of propofol and ketamine that were required to induce a loss of righting reflex (LORR) and for immobility using standard methods. The mice received 5 mg/kg mecamylamine (a broad-spectrum neuronal nicotinic antagonist), 2.5 mg/kg DHßE (a semi-selective nicotinic antagonist), or an equal volume of saline by intraperitoneal (IP) injection determined by random allocation. In separate experiments, mice received methyllycaconitine (MLA) 30 µg in 5 µL, -bungarotoxin (-BT) 2 µg in 5 µL, or saline in equal volume by intrathecal injection.

Immobility was determined for each mouse by a trained observer blinded as to the drug treatment as previously described (4,5). The temperature of each mouse was determined rectally and maintained between 36°C and 38°C using heating blankets. The drug dose required to induce immobility was measured as the mean doses bracketing the animal’s response and lack of response to a 1-min tail clamp. If an animal responded to tail clamp, an additional dose was given in increasing steps of approximately 10% until no response was obtained. The animals were given 6 min to reach peak dose of drug before testing (determined in previous control experiments). The sample size was chosen to be able to detect a 30% change with 80% power.

An investigator blinded as to treatment and genotype determined the dose of ketamine or propofol that resulted in LORR. The LORR was measured as the mean of the drug doses bracketing postural response and lack of response to placing the animal in a supine position. The temperature of each mouse was maintained as in immobility experiments.

Analgesic action was measured using hindpaw withdrawal latency (Harvard Apparatus, Holliston, MA) as previously described (3). The glass plate was warmed to minimize body heat loss. To diminish exploratory activity, the mice were acclimatized to this environment for at least 30 min before commencing the study. After acclimatization, a movable source of radiant heat was applied from a lamp through an aperture under the glass plate to the hindpaw of the resting mouse. The testing stimulus was 15% of maximal and caused an average increase to 42°C at movement under control conditions. An investigator who was blinded as to the treatment group measured the latency from the onset of the application of the stimulus (heat) to the time the mouse moved its hindlimb. In all studies, we obtained baseline measurements before testing with anesthetic. Each hindpaw was tested twice for a total of 4 measurements that were then averaged.

We studied the mice using two protocols. In one study, a cumulative dose response relationship was established. Propofol (120 mg/kg) was administered as 4 IP injections of 20 mg/kg every 6 min followed by 1 of 40 mg/kg. We chose this time frame based on control experiments that indicated that the peak activity of the drug occurs within 6 min of injection and lasts more than 30 min. A similar study was performed with ketamine, giving a cumulative total dose of 200 mg/kg administered as 4 IP injections of 20 mg/kg followed by 1 at 120 mg/kg with a 2-min interval between each injection. The time interval was based on pilot experiments that indicated that the peak activity of the drug occurs within 2 min and lasts more than 30 min. The above studies were performed in the presence and absence of mecamylamine, a nonspecific neuronal nicotinic antagonist. In an additional study, we observed the time course of recovery after a single injection of either 200 mg/kg IP propofol or 200 mg/kg IP ketamine. We measured withdrawal latency for 2 h to potentially identify a pronociceptive phase during withdrawal. These doses were near the EC₅₀ dose for LORR in these mice.

Because mecamylamine affected the nociceptive action of ketamine, we studied a dose response to mecamylamine in the presence and absence of a small, ineffective dose of ketamine (40 mg/kg). Mecamylamine was studied from 0.5 to 5 mg/kg. Each dose of mecamylamine was studied on a separate group of mice because the time course of effect and recovery could not be determined.

The values for MAC and LORR were calculated as the average of the largest dose tested that resulted in movement and the smallest tested that did not result in movement. The results are expressed as mean ± se and are compared with an unpaired Student’s t-test for significant differences. Latency responses were compared with analysis of variance with repeated measures. P < 0.05 was considered significant. Graphics were prepared with Microcal Origin (Microcal Inc., Northampton, MA).

	Results

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The ED₅₀ dose of propofol required to induce immobility to a 60-s tail clamp was 371 ± 34 mg/kg (Table 1a). The propofol dose required for LORR was 199 ± 25 mg/kg (Table 1b). Treatment with nicotinic antagonists, mecamylamine, DHßE, MLA, and -BT, had no significant effect on the dose of propofol required to induce these end-points.

The ED₅₀ dose of ketamine required for immobility to a 60-s tail clamp was 383 ± 22 mg/kg (Table 2a). The dose of ketamine that resulted in a LORR was 180 ± 17 mg/kg (Table 2b). Treatment with the nicotinic antagonists had no effect on the doses of ketamine required for immobility or LORR.

No pronociceptive effect was observed in response to ketamine at doses between 20 and 200 mg/kg; however, significant antinociception occurred at the maximum dose tested, 200 mg/kg (Fig. 1A). In contrast, (Fig. 1B) no pronociceptive or antinociceptive response to propofol was observed at any dose. The broad range nicotinic antagonist, mecamylamine, enhanced the antinociceptive response to ketamine but had no significant effect on the response to propofol.

View larger version (12K): [in this window] [in a new window]   Figure 1. Inhibition of nicotinic receptors by ketamine has nociceptive consequences. A, Ketamine was administered as cumulative intraperitoneal (IP) injections up to 200 mg/kg; a significant antinociceptive phase was observed after 60 mg/kg. The addition of mecamylamine 5 mg/kg enhanced the antinociceptive effect of ketamine (P < 0.001, repeated-measures analysis of variance). B, In contrast, when propofol was administered as cumulative IP injections up to 120 mg/kg (approximately half the dose that causes immobility), no pronociceptive or antinociceptive action was identified (repeated-measures analysis of variance, P > 0.05). The combination of propofol with mecamylamine had no additional effect.

Mecamylamine by itself had no antinociceptive effect (Fig. 2). Ketamine (40 mg/kg) alone did not show any significant antinociceptive action, but its antinociceptive effect was enhanced in a dose-dependent manner by mecamylamine with a maximum effect at 1 mg/kg (Fig. 2).

View larger version (13K): [in this window] [in a new window]   Figure 2. Dose-response curve for mecamylamine enhancement of ketamine antinociception. Mecamylamine enhances the antinociceptive effect of ketamine in a dose-dependent manner. There was an increase in withdrawal latency with increasing doses of mecamylamine, which peaked at a dose of 2 mg/kg (P < 0.001; repeated-measures analysis of variance). When mecamylamine was administered alone, no significant antinociceptive effect was identified.

Finally, we attempted to determine if a significant pronociceptive response could be elicited during the withdrawal phase from either ketamine or propofol (Fig. 3A, 3B). In both cases we gave a single hypnotic dose of 200 mg/kg IP. As a single dose, both drugs prolonged withdrawal latency. Withdrawal latency was measured for at least 100 min, at which time complete recovery had occurred. No significant pronociceptive effect was identified during this time period.

View larger version (15K): [in this window] [in a new window]   Figure 3. The dissipation of a single large dose of ketamine or propofol did not reveal any pronociceptive phase. A, When 200 mg/kg ketamine was administered as a single dose, no pronociceptive phase was identified during 1 h and 40 min when the animals appeared to be recovered. B, After a single injection of 200 mg/kg propofol intraperitoneally, no significant reduction from baseline withdrawal latency was observed during 2 h.

	Discussion

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Ketamine induced analgesia at doses of 60 mg/kg and larger, a dose range similar to that described by Baker et al. (11). Unlike the case with volatile anesthetics (12,13), no pronociceptive dose range for ketamine was identified. Neither was a pronociceptive phase identified during drug recovery from ketamine. These findings suggest that although ketamine is a broad-spectrum nicotinic antagonist like the volatile anesthetics, it has no identifiable pronociceptive dose range. Ketamine’s antinociceptive effects are thought to be attributable to NMDA antagonism (14). However, the combination of ketamine with the nonselective nicotinic antagonist, mecamylamine, enhanced the antinociceptive effect (Fig. 1A). The mechanism of this enhanced antinociception is unknown, but can perhaps be explained by the increased inhibition of nicotinic receptors acting presynaptically to decrease the release of glutamate (15) in addition to the postsynaptic action of ketamine to block the NMDA receptor.

In contrast, propofol had neither pronociceptive nor antinociceptive effects when administered in cumulative doses alone or with an NMDA antagonist. Although nicotinic receptors are also expressed on presynaptic terminals of inhibitory neurons, there was no nociceptive effect of nicotinic blockade. Studies by Nadeson and Goodchild (16) support our finding of a lack of pronociception induced by propofol; however, they interpreted their findings as indicating analgesia. Withdrawal from a single dose of propofol also did not provide evidence for a pronociceptive phase. These findings are not consistent with those of Wang et al. (17), who demonstrated a significant pronociceptive response to a single dose of 50 mg/kg propofol within 60 minutes of injection. However, because they tested withdrawal latency in rats rather than mice, it is possible that the difference in our findings is species related. Our findings support the hypothesis that propofol, a largely gamma-aminobutyric acid mimetic drug, does not have effects on pain sensitivity, at least when used in isolation. In contrast, ketamine has analgesic actions, but we could detect no pronociceptive action of ketamine.

Although ketamine is a potent antagonist at a variety of central nicotinic receptors in vitro (6,7,18), we have not identified any behavioral role for that inhibition. In contrast, nicotinic inhibition enhances the antinociceptive effects of ketamine, perhaps through interplay between its presynaptic and postsynaptic actions. Mecamylamine is an approved drug in the United States for the treatment of hypertension that might potentially enhance the analgesic response to ketamine.

	Footnotes

 
Accepted for publication December 14, 2004.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Udesky, J. O. Articles by Flood, P. Search for Related Content PubMed PubMed Citation Articles by Udesky, J. O. Articles by Flood, P. Related Collections Pharmacology