Behavioral Neuroscience Copyright 1987 by the American Psychological Association, I nc 1987. Vol. 101, No. 6. 854-857 0735-7044/~7/$(X) 75

Brief Communications

Conditioned Hyperalgesia Depends on the Pain Sensitivity Measure

Marvin D. Krank Mount Allison University

Sackville, New Brunswick, Canada

Conflicting reports about the acquisition of conditioned hyperalgesia during the development of conditioned morphine tolerance have led researchers to suggest that tolerance reflects a reduction of stimulus processing rather than a compensatory response interaction, l tested conditioned hyperalgesia on both the hot-plate and tail-flick tests in the same animals. In accordance with previous reports, the tail-flick responses in drug-free animals failed to reveal a conditioned compensatory hyperalgesia. Conditioning effects in the tail-flick test were found only when the animals were challenged with a low dose of morphine. However, the hot-plate responses in drug- free animals replicated earlier demonstrations of conditioned hyperalgesia. The results suggest that the measurement of conditioned responses in drug-free animals depends on characteristics of the assessment procedure. These findings are consistent with accounts of morphine tolerance that depend on compensatory response interactions.

In recent studies of drug tolerance and sensitization, re- searchers have acknowledged the contribution of drug-asso- ciated cues through processes of Pavlovian conditioning. In a typical drug administration procedure, environmental cues predictive of drug administration serve as conditioned stimuli (CSs), and the physiological impact of drug administration serves as the unconditioned stimulus (UCS). The consequence of this association between the environmental CS and drug UCS is often the acquisition of a physiological conditioned response (CR). Such drug CRs may be measured in drug-free animals that are exposed to drug-associated cues and given a placebo injection. Often in these studies the CR is opposite in direction to the measured response to drug administration (for reviews, see Obfil, 1966; Siegel, 1983: Siegel, Krank, & Hinson, 1987; for a discussion of factors that influence the direction of the CR, see Eikelboom & Stewart, 1982). Because such drug-opposite CRs occurring in anticipation of drug administration would be expected to cancel out or compen- sate for the measured effects of the drug, Siegel (1975, 1977, 1979) suggested that their presence would contribute to drug tolerance that is defined by a reduced effect of the drug.

Critical predictions derived from the associative account of opiate tolerance have largely been confirmed in the past few years (for recent reviews, see Siegel, 1983; Siegel et al., 1987: Siegel & Macrae, 1984). It is well documented, for example, that the presence or absence of previously drug-associated cues often determines the magnitude of tolerance to an opi- ate's effects (e.g. Siegel, Hinson, & Krank, 1978, 1981 : Siegel,

This research was supported by a National Sciences and Engineer- ing Council (NSERC) grant to Marvin D. Krank.

The author acknowledges the assistance of Susan O'Neill, Anne- Marie Wall and Tracy Estabrooks in collection and analysis of the data.

Correspondence concerning this article should be addressed to Marvin D. Krank, Department of Psychology, Mount Allison Uni- versity, Sackville, New Brunswick. Canada E0A 3C0.

Hinson, Krank, & McCully, 1982). In addition, according to the associative model, various conditioning procedures with the environmental CS present at testing should affect the magnitude of drug tolerance. Evidence to date is that tolerance development responds as predicted to many conditioning procedures including latent inhibition (Siegel, 1977). extinc- tion (Siegel, 1977; Siegel, Sherman, & Mitchell, 1980). dis- crimination training (Hinson & Siegel, 1983: Siegel et al.. 1982), partial reinforcement (Krank, Hinson, & Siegel. 1984; Siegel, 1977), inhibitory training (Fanselow & German, 1982: Siegel et al., 1981), and blocking and sensory preconditioning (Dafters, Hetherington, & McCartney, 1983). Taken together. these data strongly support a role for Pavlovian conditioning in opiate tolerance.

One prediction of Siegel's compensatory response model, however, has been challenged by recent observations: At- tempts to measure compensatory CRs have not been uni- formly successful. Failure to measure compensatory. CRs in the absence of drug stimulation has been most notable in studies in which changes in pain sensitivity in response to a signal for morphine are measured. Although Siegel (1975: Krank, Hinson, & Siegel, 1981) found evidence for a com- pensatory hyperalgesic CR elicited by a signal for morphine administration, Tiffany, Petrie, Baker, and Dahl (1983) con- sistently did not find a hyperalgesia CR elicited by signals for morphine administration despite the fact that these signals control differential levels of tolerance to morphine's analgesic effects.

Failure to find compensatory CRs under conditions in which context-specific tolerance has been demonstrated has led to the development of associative models of tolerance that do not rely on the response interaction proposed by Siegel. For example, Baker and Tiffany (1985) proposed an adapta- tion of Wagner's (1976) priming model of habituation to morphine tolerance. Baker and Tiffany's model relies on a reduction of stimulus processing rather than an antagonistic response interaction. The priming approach to context-spe-

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cific tolerance postulates that associative activation of the representation of the drug UCS by the CS reduces subsequent stimulus processing when the UCS actually occurs and thus results in a decrement in the UCR. The reduced stimulus processing explanation of morphine tolerance does not de- pend on the presence of a compensatory CR in a nondrugged animal and is consistent with failures to find such responses.

More recently, Palletta and Wagner (1986), adapting Wag- ner's( 1981 ) SOP (Sometimes Opponent Process) model, pro- posed a dual-process account of morphine tolerance that includes a role for both response interactions and reduced stimulus processing. According to the SOP model, like its predecessor, context-specific habituation occurs because of reduced stimulus processing of the UCS resulting from pre- representation of the UCS by the CS. In this model, however, it is also acknowledged that any CR elicited by the CS will inevitably interact with the UCR. Depending on the direction of the CR, this interaction may either diminish or augment the UCR. Drug CRs that mimic the drug UCR would aug- ment the response magnitude. If the magnitude of the drug- mimicking CR is larger than the decrement in response magnitude resulting from reduced stimulus processing, then the net result would be sensitization. Otherwise, the net result would be a reduction in response magnitude. Compensatory CRs would always add to the decrement in response magni- tude resulting from reduced stimulus processing. In such cases, two separate processes would contribute to tolerance. Palletta and Wagner's model is consistent with both the failure to find compensatory CRs in some preparations in which conditioned tolerance is reported and the demonstration of compensatory CRs in others.

Given the possible contribution of two separable processes to tolerance development, it is important to identify the situations in which compensatory conditioning contributes to tolerance and those in which no evidence for such condition- ing can be found. Although many procedural differences may be present, the primary difference between successful dem- onstrations of conditioned hyperalgesia (Krank et al., 1981 ; Siegel, 1975) and demonstrations of no conditioned hyperal- gesia (Tiffany et al., 1983) is the response measure used by the experimenter, the hot-plate test versus the tail-flick test, respectively. In this experiment, I used a within-subject design to test whether differences in response measures could account for the discrepancies between the results of previous assess- ments of conditioned hyperalgesia.

Method

Subjects and Apparatus

The subjects were 36 male Sprague-Dawley (CD) rats obtained from Canadian Breeding Farms, St. Constant, Quebec. The animals weighed 300-350 g at the start of the experiment and were allowed free access to food and water except during the experimental sessions (described in the Procedure section).

Pain sensitivity was measured in one of two methods: tail-flick tests and hot-plate tests. For tail-flick tests, restrained animals had their tails dropped in a constant-temperature hot-water bath (48 ~ Haake Model #D1-13) to a depth of 5 cm. The time until the animal lifted its tail from the water (tail-flick latency, or TFL) was recorded

by two independent observers who were blind to the experimental conditions of the animals. Trials were terminated at 30 s if no response had occurred. For hot-plate tests, the animals were placed in a round stainless steel tray (diameter = 21 cm) that had been submerged in the hot-water bath at a temperature of 52 ~ The tray was encased by a round Plexiglas cylinder to a height of 31 cm and capped with a Plexiglas lid. Two independent observers, who were blind to the experimental conditions of the animals, recorded the time until the animal either licked its paw or jumped off the hot plate (paw-lick latency, or PLL). A third observer recorded the number of times that the animal reared during the hot-plate trial. Hot-plate trials were terminated at 30 s if no response had occurred. Discrepancies between the observers (which were generally small) were resolved by means of averaging the two scores.

Procedure

Tolerance development phase. During tolerance development, all animals received ten exposures to a "distinctive room" for 60 rain. The distinctive room was characterized by a higher level of overhead illumination than the animal's colony room. a different cage (Plexiglas vs. wire mesh), and a background level of white noise (74 db). Fifteen minutes into each distinctive-room exposure, all animals were re- moved from the Plexiglas cage, injected, and returned to the Plexiglas cage for the remainder of the period. At the end of the distinctive- room exposure, the animals were returned to their colony room. Successive distinctive-room exposures were separated by intervals of 2-4 days. On the day after distinctive-room exposure, all animals received an injection in the colony environment.

The animals were randomly assigned to one of three groups that differed only during the tolerance development phase of the experi- ment. The three experimental groups of the animals differed only with respect to the solutions injected in the distinctive room and in the colony room. Group paired animals were injected with 5 mg/kg of a 5-mg/cc solution of morphine sulphate in each exposure to the distinctive room. These animals received equivalent volume saline injections in the colony 24 hr later. Group unpaired animals were injected with saline in the distinctive room, but received 5 mg/kg injections of morphine sulphate in the colony 24 hr later. Group control animals were injected with saline both in the distinctive room and in the colony room.

Tail-flick CR test. In the 1 lth exposure to the distinctive room, animals in each condition were given a saline injection. Thirty minutes later, pain sensitivity was assessed via the tail-flick measure. After the test, the animals were returned to the Plexiglas cages for the remainder of the exposure.

Morphine challenge test. In the 12th exposure to the distinctive room, all animals received a 1 mg/kg dose of morphine. Thirty minutes later, tolerance to the analgesic effect of this low dose of morphine was assessed via the tail-flick method. After the analgesia test, the animals were returned to the Plexiglas cages, where they remained for the rest of the distinctive-room exposure.

Hot-plate CR test. In the 13th exposure to the distinctive room, all animals were again given an injection of saline. Thirty minutes later, their pain sensitivity was assessed via the hot-plate method.

Data analysis. Group scores on the tolerance test. the tail-flick CR test, and the hot-plate CR test were analyzed via separate one- way analyses of variance (ANOVAS). Planned orthogonal comparisons between group paired versus group unpaired and group paired versus the group control were analyzed for evidence of conditioning effects.

Resul ts

Table 1 shows the mean and standard errors for the three groups on each of the three pain sensitivity tests. The tail-

856 BRIEF COMMUNICATIONS

Table 1 Mean Response Latencies on Pain Sensitivity Tests

Tail flick Paw lick:

Training Saline Morphine Saline

Paired M 5.65 6.81 a 10.56 a SEM 0.68 1.06 2.08

Unpaired M 5.81 9.66 15.71 SEM 0.61 1.31 2.06

Control M 5.41 12.49 18.62 SEu 0.72 2.78 2.70

Note. Values represent the mean latency to emit the pain-sensitive response. Lower values indicate greater pain sensitivity. Differs from the unpaired (p < .05) and control (p < .01) groups.

flick test with saline revealed no reliable differences between the groups, F(2, 33) < 1, ns. However, the tail-flick test with a low dose of morphine showed that the groups differed reliably on this measure, F(2, 33) = 3.40, p < 05. Comparisons among the means revealed that group paired tolerance was greater (shown by faster TFLs) than either group unpaired tolerance (p < .05) or group control tolerance (p < .01). Analysis of the difference between the saline tail-flick test scores and morphine tail-flick test scores supported this con- clusion. Higher TFLs on the morphine test than on the saline test were found for both the unpaired group, t(11) = 2.08, p < .05, and the control group, t ( l l ) = 3.00, p < .01 (one- tailed). Group paired, on the other hand, did not have signif- icantly higher TFLs on the morphine test, t(11) = 0.66, ns. Last, the three groups differed on the saline hot-plate measure, F(2, 33) = 3.98, p < .05. The paired group exhibited faster PLLs than either the unpaired group (p < .05) or the control group (p < .01 ). The group means for rearing behavior during the hot-plate test were 2.16 for group paired, 2.58 for group unpaired, and 3.08 for group control. The ANOVA indicated that the groups did not differ reliably on this measure, F(2, 33) < 1, ns. Rearing and paw-lick latencies were not signifi- cantly correlated (Pearson product-moment correlation p = .06, ns).

Discussion

The pattern of these results is consistent with previous tests Of conditioned hyperalgesia. As in previous studies with the tail-flick test (Palletta & Wagner, 1986; Tiffany et al., 1983), conditioning with morphine did not result in a compensatory response on a CR test in drug-free animals. Only when the animals were challenged with a low dose of morphine did the tail-flick measure reveal the effects of conditioning. However, a subsequent test of pain sensitivity with saline via the hot- plate method in these same animals revealed a hyperalgesia CR. Conditioned hyperalgesia found in the hot-plate method of assessment is consistent with previous successful demon- strations of compensatory CRs (Krank et al., 1981; Siegel, 1975). These observations demonstrate within individual an- imals that the type of response measure used by the experi-

menter is a critical determinant of whether a compensatory CR will be observed.

Recently, Palletta and Wagner (1986) proposed a dual- process theory of tolerance in which both compensatory CRs and reduced stimulus processing contribute to tolerance. They suggested that compensatory CRs can be measured and con- tribute to tolerance only in biphasic response systems. Bi- phasic response measures are defined as those in which the initial drug effect gives way to a secondary effect in the opposite direction (cf. opponent process; Solomon & Corbit, 1974). According to the model, CRs in biphasic response systems mimic this secondary reaction to the UCS. In support of their model, Palletta and Wagner (1986) demonstrated that the time course of morphine's effects on the tail-flick measure does not reveal such a biphasic effect (only analgesia was observed), whereas activity measures do reveal a biphasic effect (hypoactivity followed by hyperactivity was observed). In accordance with their expectations, tail-flick tests did not provide evidence for compensatory CRs in drug-free animals, but activity tests did demonstrate compensatory CRs in drug- free animals.

Several investigators (Mucha, Volkovskis, & Kalant, 1981; Palletta & Wagner, 1986; Tiffany et al., 1983) have proposed that compensatory responses are found on the hot-plate mea- sure of analgesia because it is confounded with activity effects. According to this interpretation, the compensatory hyperac- tivity CR, which occurs on activity measures, produces ap- parent hyperalgesia CR on the hot-plate test. Although plau- sible, this explanation is not consistent with the rearing be- havior found during the hot-plate test in this experiment. There is no suggestion in the group means for rearing that a general increase in activity accounts for the differences in specific defensive reactions to nociception. Nor is there any correlation between rearing behavior and paw-lick latencies. Although rearing behavior may not be a good indicator of conditioned activity, this observation at least illustrates the possibility that paw-lick behavior may not be directly depend- ent on a general increase in behavior. Until data demonstrate that the hot-plate measure of pain sensitivity is positively correlated with other activity measures, the contribution of conditioned activity to the hyperalgesic CR remains an open question. Research on the precise relationship between paw- lick responses and activity is necessary in order to decide whether conditioned hyperalgesia is merely a reflection of conditioned activity or a compensatory CR.

An alternative explanation of my findings is that compen- satory CRs may be difficult to measure in certain response systems (Hinson, Poulos, & Cappell, 1982; Siegel & Macrae, 1984). This interpretation is similar to that of Palletta and Wagner (1986) except the emphasis here is on the ability to measure the CR rather than on its contribution to drug tolerance. Although hyperalgesic CRs cannot be measured across a wide range of temperatures in the tail-flick test (cf. Palletta & Wagner, 1986; Tiffany et al., 1983), these obser- vations may demonstrate only that the tail-flick response system does not react to conditioned hyperalgesia; that is, the tail-flick may be a unidirectional response measure, which responds primarily to manipulations that produce decreases in pain sensitivity. The presence of a compensatory CR may

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be revealed only if such a system is challenged (cf. Hinson et al., 1982) or if another response is measured. My results are consistent with the view that conditioned tolerance to mor- phine's effects on the tail-flick test are based on a hyperalgesic CR that can be measured on the hot-plate test but not on the tail-flick test.

This study is not unique in demonstrating differential sen- sitivity of these two response systems to analgesic and hyper- algesic events. A variety of pharmacological manipulations also produce divergent effects on different measures of pain sensitivity (e.g., Fasmer, Berge, Tveiten, & Hole, 1986; Ogren & Berge, 1984: Post, Minor, Davies, & Archer, 1986). These findings indicate that different tests measure different types of pain sensitivity. Specifically, the tail-flick response is me- diated by a spinal withdrawal reflex, whereas the paw-lick response involves the supra-spinal integration of several motor response systems. The pharmacological and physiological dif- ferentiation of these two response systems is consistent with the view that failure to observe conditioned hyperalgesia in tail-flick tests reflects an inherent measurement property of this response rather than the absence of a CR.

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Received January 7, 1987 Revision June 9, 1987

Accepted June 18, 1987 9