Psychopharmacology (2000) 149:56–62
© Springer-Verlag 2000
O R I G I N A L I N V E S T I G AT I O N
Gavan P. McNally · R. Frederick Westbrook
Acute exposure to saccharin reduces morphine analgesia in the rat: evidence for involvement of N-methyl-D-aspartate and peripheral opioid receptors Received: 12 June 1999 / Final version: 1 November 1999
Abstract Rationale: Pairings of a sweet taste and injection of morphine result in a learned avoidance of that taste and learned analgesic tolerance. This avoidance is mediated by the drug’s peripheral effect, while learned tolerance involves activation of N-methyl-D-aspartate (NMDA) receptors. Exposure to a sweet taste also reduces morphine analgesia. We studied whether this tastemediated reduction was reversed by an NMDA or peripheral opioid receptor antagonist. Objectives: To determine whether an intraoral infusion of saccharin would modulate morphine analgesia in rats, and to study the contribution of NMDA as well as peripheral opioid receptors to this modulation. Methods: Six experiments used the rat’s tail-flick response to study the effect of an intraoral infusion of a sodium saccharin solution on morphine analgesia, and the effects of the quaternary opioid receptor antagonist methylnaltrexone as well as the noncompetitive NMDA receptor antagonist MK-801 on this modulation of analgesia. Results: An intraoral infusion of saccharin reduced the analgesic effects of an intraperitoneal (i.p.) injection of morphine across a range of doses (experiment 1a), which was not attributable to an influence on tail-skin temperature (experiment 1b). This reduction was mediated by opioid receptors in the periphery and activation of NMDA receptors because morphine analgesia was reinstated by an i.p. injection of either methylnaltrexone (experiment 2a) or MK-801 (experiment 3a), which was not due to the effect of methylnaltrexone (experiment 2b) or MK-801 (experiment 3b) on morphine analgesia in the absence of saccharin. Conclusions: These results document evidence for an antagonism of morphine analgesia by actions of the drug at peripheral opioid receptors and excitatory amino-acid activity at NMDA receptors. They are discussed with referG.P. McNally · R.F. Westbrook School of Psychology, The University of New South Wales, Australia G.P. McNally (✉) Mental Health Research Institute, The University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI 48109-0720, USA e-mail:
[email protected]
ence to the aversive motivational effects of peripheral opioid receptors and pain facilitatory circuits. Key words Morphine · Opioid receptor · NMDA · Tolerance · Rat · Tail flick
Introduction Ingestion of sweet and/or calorically rich solutions modulates the analgesic efficacy of morphine in rats. For example, rats that consume a saccharin, dextrose-saccharin, glucose-saccharin, or glucose solution frequently display less analgesia when subsequently injected with morphine than rats that consume water (Lieblich et al. 1983; Bergmann et al. 1985; Klein and Green 1988; Fidler et al. 1993; D’Anci et al. 1997). Reductions in morphine analgesia have typically been detected following prolonged (3 h to >5 weeks) but not acute (e.g., 3 h) consumption of sweet and/or calorically rich solutions, and have been observed using a variety of pain tests including the hotplate and tail-flick tests. The mechanism(s) by which ingestion of these solutions modulates morphine analgesia remains unclear. It has been suggested that these reductions could be due to competitive interactions between morphine and endogenous opioids released in response to the sweet taste or the post-ingestional consequences of these solutions, or to a downregulation of opioid receptor number in response to prolonged ingestion of these solutions (Dum et al. 1983; Holder and Bolger 1988; Kanarek et al. 1991). However, there has been little direct examination of these interpretations. Consumption of saccharin followed by an injection of morphine establishes a learned avoidance of that solution (Cappell and LeBlanc 1975; Bechara and van der Kooy 1985; Hunt and Amit 1987). This learned avoidance is mediated, at least in part, by the actions of morphine on vagal afferents, because avoidance is prevented by subdiaphragmatic vagotomy, neonatal destruction of vagal afferents, or by co-administration of morphine and the quaternary opiate receptor antagonist methylnaltrexone,
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which does not readily cross the blood–brain barrier (Bechara and van der Kooy 1985; Bechara et al. 1987). The peripheral aversive effects of morphine that induce learned aversions in rats could be identified with the nausea reported by patients administered morphine for pain relief and by recreational users of heroin. These peripheral aversive effects of morphine may also contribute to reductions in the analgesic efficacy of the drug. For example, intraoral infusion of a saccharin solution, the taste of which had been rendered aversive via repeated pairings with injection of morphine, produced conditioned tolerance to morphine analgesia (as indexed by a rightward shift in the function relating morphine dose to tail-flick latency) and a conditioned hyperalgesic response when rats were tested in the absence of morphine (McNally and Westbrook 1998). Moreover, both the conditioned reduction in morphine analgesia and conditioned hyperalgesic response depended critically on excitatory amino-acid activity at spinal N- methyl-D-aspartate (NMDA) receptors because both the analgesic effectiveness of morphine and basal pain sensitivity in rats exposed to the averted saccharin solution were reinstated by systemic or intrathecal administration of an NMDA receptor antagonist (McNally and Westbrook 1998). Initially, we planned to examine the effects of prolonged exposure to a sweet taste on morphine analgesia but, in the course of these experiments, noticed that a brief exposure served to reduce morphine’s analgesic effectiveness. Thus, the aims of the experiments reported here were to determine whether acute exposure to saccharin reduces morphine analgesia in rats, and to examine the contribution of NMDA receptors and peripheral opioid receptors to this modulation. Specifically, the initial experiments studied whether an intraoral infusion of saccharin (0.3 ml) would modulate morphine analgesia in the tail-flick test (experiment 1a) and whether this modulation could be attributed to variations in tail-skin temperature (experiment 1b). The remaining experiments studied the contribution of peripheral opioid receptors (experiments 2a and 2b) and NMDA receptors (experiments 3a and 3b) to this modulation of morphine analgesia.
Experiments 1a and 1b Experiment 1a studied the analgesia produced by an injection of morphine and the modulation of this analgesia by an intraoral infusion of saccharin. Specifically, rats were tested for morphine analgesia in the tail-flick test following an intraoral infusion of either 0.3 ml saccharin, 0.3 ml salt, or no intraoral infusion. Salt was chosen as a control solution for the non-specific effects of the intraoral infusion procedure because rats do not readily express learned avoidances of a salt solution paired with an injection of morphine (Bevins et al. 1996). The design was a 3×4 factorial, in which the first factor was the type of intraoral infusion whose levels were no infusion (group morphine alone), infusion of saccharin solution
(group saccharin), and infusion of salt solution (group salt). The second factor was morphine dose, the levels of which were 0.0, 2.5, 5.0, and 10.0 mg/kg. The interpretation of data from the tail-flick test with reference to the actions of pain modulatory circuits can be confounded by an influence of tailskin temperature on tail-flick latency (Berge et al. 1988). Thus, any effect of an intraoral infusion of saccharin detected in experiment 1a could be secondary to saccharin-induced shifts in peripheral blood flow, affecting heat transfer in the tail. Accordingly, in experiment 1b, we studied the effects of morphine and an intraoral infusion of saccharin on tailskin temperatures to determine whether any modulation of performance in the tail-flick test detected in experiment 1a could be attributed to variations in tail-skin temperature. This experiment employed a 2×4 factorial design, in which the first factor was the type of intraoral infusion whose levels were no infusion (group morphine alone) or infusion of saccharin (group saccharin), and the second factor was morphine dose, the levels of which were 0.0, 2.5, 5.0, and 10.0 mg/kg morphine. Based on the results of experiment 1a, the control group receiving an intraoral infusion of salt was not included in experiment 1b. Materials and methods Subjects Subjects were experimentally naive, male Wistar rats weighing between 350 g and 480 g at the start of the experiments. They were obtained from the colony of Specific-Pathogen-Free rats maintained by the Combined Universities Laboratory Animal Services (Little Bay, Sydney, Australia). Rats were housed in plastic boxes (65 cm long × 40 cm wide × 22 cm high) with six to eight rats per box. The wire-mesh roof of each box held food and water bottles, which were continuously available. The boxes were kept in an airconditioned colony room maintained under natural lighting. The procedures used in this and subsequent experiments were approved by the Animal Care and Ethics Committee of The University of New South Wales. Apparatus The tail-flick apparatus consisted of a water bath, the temperature of which was controlled at 51°C (±0.5°C) by an open-bath thermoregulator (Ratek Instruments, Melbourne). The water bath was located in a laboratory, in which the ambient temperature was maintained between 21°C and 23°C. Tail-skin temperature was measured using a digital thermal probe (Anritsu, Tokyo). The laboratory also contained plastic buckets (26 cm diameter × 45 cm height) with air holes drilled in the lid and sides. These buckets served as chambers in which rats were kept in isolation from each other when they were brought to the laboratory. Procedure Experiment 1a. Across days 1–4 of the experiment, rats were transported to the laboratory. On arrival, rats were placed in the plastic buckets for 20 min, removed, handled, and returned to the buckets. This handling was repeated a further three times at 5-min intervals to familiarize the rats with procedures to be used on test. On day 5 of the experiment, rats were transported to the laboratory and placed in the plastic buckets for 20 min. Baseline tail-flick la-
58 tencies were determined by taking the average of the last three of four tail-flick trials spaced 5-min apart. For tail-flick testing, the distal 4-cm portion of the rat’s tail was immersed in the water bath, and latency to completely remove the tail was recorded using stopwatch. At the conclusion of each trial, the tail was wiped with a flannel cloth to prevent hot water from clinging to the tail. Five minutes following the final baseline determination, rats in group saccharin received an intraoral infusion of 0.3 ml of a 0.15% saccharin solution, while those in group salt received an intraoral infusion of a 0.9% sodium chloride solution. Specifically, solutions were infused into the rat’s mouth over a 3-s period using a 1-ml syringe. At that time, rats in group morphine alone were briefly removed, handled, and returned to the plastic buckets. Five minutes later, rats were injected i.p. with either saline, 2.5, 5.0, or 10.0 mg/kg morphine in a volume of 2 ml/kg. There were seven rats per group at doses of saline and 2.5 mg/kg morphine, and six rats per group at the remaining doses of morphine. Tail-flick testing commenced 5 min following injection of morphine and was repeated a further five times at 5-min intervals. Experiment 1b. Rats were familiarized with the plastic buckets and handling procedures as described above. On day 5, rats were transported to the laboratory and placed in the plastic buckets for 20 min. Baseline tail-skin temperatures were determined by taking the average of the last three of four tail-skin measurements spaced 5-min apart. For tail-skin temperature testing, the probe was placed on the dorsal surface of the tail 8-cm from the distal tip. Five minutes following the final baseline determination, rats in group saccharin received an intraoral infusion of 0.3 ml of 0.15% saccharin solution, whereas rats in group morphine alone were briefly removed, handled, and then returned to the plastic buckets. Five minutes later, rats were injected i.p. with either saline, 2.5, 5.0, or 10.0 mg/kg morphine in a volume of 2 ml/kg. There were six rats per group at each dose of morphine injected. Tail-skin temperature testing commenced 5-min following injection of morphine or saline.
Fig. 1 Experiment 1a mean+SEM tail-flick latencies, expressed as a percentage change from baseline, for rats injected i.p. with morphine following intraoral infusion of 0.3 ml of saccharin (group saccharin; filled square), 0.3 ml of salt (group salt; open circle), or no infusion (group morphine alone; cross)
Table 1 Experiment 1b mean±SEM tail-skin temperatures for rats injected i.p. with morphine following intraoral infusion of 0.3 ml saccharin (group saccharin) or no infusion (group morphine alone) Morphine dose (mg/kg)
Statistical analysis The average latencies from the six tail-flick tests conducted were converted to a percentage change from baseline prior to analysis in this and subsequent experiments. This conversion technique was used so that the index of nociceptive sensitivity was relative to each rat’s baseline sensitivity. In this and subsequent experiments, planned orthogonal contrasts were written to analyze differences between groups and the per-contrast error rate (alpha) was controlled at the 0.05 level using the procedure described by Hays (1972) (see Harris 1994 for review).
Results and discussion The mean and standard error of the mean (SEM) tailflick latencies expressed as percentage change from baseline for rats in experiment 1a are shown in Fig. 1. Inspection of the figure indicates that rats injected with morphine (group morphine alone) and rats that received an intraoral infusion of salt 5-min prior to injection of morphine (group salt) exhibited a dose-dependent analgesic response following i.p. injection of morphine. In contrast, rats that received an intraoral infusion of saccharin 5-min prior to injection of morphine (group saccharin) failed to show this dose-dependent analgesia. Instead, the performance of this group was characterized by little change in nociceptive sensitivity across the doses of morphine studied. These observations were confirmed by the statistical analysis. Rats in group saccharin showed significantly less morphine analgesia than
Baseline Morphine alone Baseline Saccharin
0.0
2.5
5.0
10.0
25.3±0.5 28.3±0.5 25.4±0.6 27.6±0.3
25.2±1.3 28.3±0.8 25.4±0.9 26.3±0.4
25.1±1.3 26.9±0.8 25.2±1.4 28.3±0.5
25.3±1.2 26.5±0.7 25.2±1.3 27.7±0.7
the performances shown by rats in group salt and group morphine alone (F1,60=12.3; critical F value =4.0), averaged across doses of morphine. However, there was no difference in levels of morphine analgesia between rats in group salt and rats in group morphine alone (F=2.2), averaged across dose of morphine. The analgesia produced by morphine in this experiment was dose-dependent, because there was a significant linear increase in tail-flick latencies across doses of morphine tested (F=18.2), averaged across type of intraoral infusion. There was also a significant interaction between the contrast assessing differences between group saccharin versus groups salt and morphine alone, and the contrast testing linear trend across morphine dose (F=5.6). Inspection of Fig. 1 indicates that the differences in nociceptive sensitivity between rats tested following an intraoral infusion of saccharin and rats in the remaining groups increased as the dose of morphine increased. However, there was no significant interaction between the contrast assessing differences between group salt and group morphine alone, and the contrast testing linear trend across morphine dose (F
