Psychopharmacology(1992) 109:172-176
Psychopharmacology © Springer-Verlag 1992
Effect of number of conditioning trials on the development of associative tolerance to morphine Antonio Cepeda-Benito and Stephen T. Tiffany Department of Psychological Sciences, Purdue University, West Lafayette, IN 47907, USA Received January 2, 1991 / Final version March 16, 1992
Abstract. The acquisition of associative tolerance to the analgesic effects of morphine was investigated by giving independent groups of rats 1, 3, 5, 8, 14, 20, or 30 administrations of drug either explicitly paired or unpaired with a distinctive context. Tolerance, assessed on a tail-flick device using dose-response curve (DRC) methodology, developed more rapidly and reached greater magnitude when morphine and the distinctive context were explicitly paired rather than explicitly unpaired. Tolerance magnitude in both conditions reached a maximum at eight conditioning sessions. It is argued that the tolerance found in both treatment groups was associatively controlled. The function of handling and injection cues as conditioned stimuli, and the deleterious effects of latent inhibition and partial reinforcement on conditioned excitation and conditioned inhibition are discussed. Key words: Conditioning - Tolerance - Morphine - Tailflick - Dose-response curves It is widely accepted that associative-learning processes contribute to the development of morphine tolerance (see Baker and Tiffany 1985; Goudie and Demellweek 1986; Siegel 1989). Associative tolerance models (Baker and Tiffany 1985; Siegel t975) posit that environmental cues that reliably predict the occurrence of morphine delivery function as conditioned stimuli (CSs) that elicit conditioned tolerance processes. For example, like other forms of associative learning, associative-morphine tolerance should be context specific, and subject to extinction, sensory preconditioning, overshadowing, latent inhibition, conditioned inhibition, partial reinforcement and blocking (see reviews by Baker and Tiffany 1985; Goudie and DemeUweek 1986; Siegel 1989). Although associative tolerance models would predict greater rates of tolerance acquisition when drug delivery is signalled than when it is not (Siegel 1975; Baker and Tiffany 1985), there has been little systematic research on the rate of associative-tolerance development as a funcCorrespondence to:
S.T. Tiffany
tion of the number of conditioning trials. When learning curves of tolerance development have been investigated (Siegel 1977; Tiffany et al. 1983; Dafters et al. 1988; Dafters and Odber 1989), they have typically been generated by assessing drug responsiveness during each conditioning session. Since tolerance to the analgesic effect of morphine is generally measured as the latency to make a response to a nociceptive stimulus, such studies have confounded the associative paradigm with the opportunity to practice the response under the influence of drug effects. It is generally assumed that tolerance arising from intoxicated practice represents the operation of some form of operant conditioning that produces a task-specific compensation for drug-induced disruptions in performance (Wolgin 1989). This form of tolerance should be distinguished from associative tolerance, which represents the operation of classical conditioning. Associative models of tolerance predict that animals should display a monotonically accelerated level of tolerance development over the course of conditioning trials when morphine is explicitly paired with a CS. Tiffany and his colleagues have consistently found that rats show high levels of tolerance when tested in a distinctive context that had been paired previously with eight morphine injections (Tiffany and Maude-Griffin 1988; Tiffany et al. 1991). Research from this laboratory has also found low levels of tolerance in animals that have had the distinctive test context explicitly unpaired with morphine administration (Tiffany et al. 1992). This tolerance may be controlled associatively by handling and injection cues that accompany drug administration (DaRers and Bach 1985). This is supported by the finding that this tolerance, unlike nonassociative forms (Baker and Tiffany 1985), was present after a 30-day retention interval (Tiffany et al. 1992) and could be extinguished by presenting a series of saline injections in the rats' home-cage environment over a 30-day period (Tiffany et al. 1991). When morphine administration is explicitly unpaired with a distinctive context, the context may become a conditioned inhibitor of associative tolerance effects, that is, become a CS- (LoLordo and Fairless 1985). Moreover, as predicted by the Reseorla and Wagner (1972) model of
173 classical conditioning, associative tolerance effects m a y be evident in the i n h i b i t o r y context over early c o n d i t i o n i n g trials, b u t these s h o u l d decline as c o n d i t i o n i n g p r o c e e d s (Rescorla 1985). T h e presence o f associative t o l e r a n c e effects in u n p a i r e d c o n d i t i o n s (Tiffany et al. 1991; 1992) suggests t h a t the distinctive c o n t e x t m a y n o t have acq u i r e d sufficient i n h i b i t o r y strength after eight c o n d i t i o n ing trials to c o m p l e t e l y c o u n t e r a c t the e x c i t a t o r y effects p r o d u c e d by d r u g - p a i r e d injection a n d h a n d l i n g cues. This s t u d y e x a m i n e d the effects of the n u m b e r of c o n d i t i o n i n g sessions on the d e v e l o p m e n t of tolerance to the analgesic effects of m o r p h i n e . Rats received 1, 3, 5, 8, 14, 20, o r 30 injections o f m o r p h i n e either explicitly p a i r e d o r u n p a i r e d with a distinctive c o n t e x t p r i o r to tolerance testing. A n i m a l s were n o t e x p o s e d to the nociceptive test stimulus until the t o l e r a n c e test session. As with o t h e r investigations recently c o n d u c t e d in o u r l a b o r a t o r y (Tiffany a n d M a u d e - G r i f f i n 1988; Tiffany et al. 1991; 1992), tolerance m a g n i t u d e was assessed as a shift in the d o s e - r e s p o n s e curve to the right. This research p r o v i d e d for a c o m p r e h e n s i v e investigation of the time course of associative tolerance effects u n c o n f o u n d e d by the p o t e n tial influence o f o p e r a n t contingencies.
Materials and methods Subjects. The subjects were 558 experimentally naive, male Holtzman rats 80-83 days old on the first day of conditioning. The rats were housed individually in wire mesh cages located in a colony room (on a 12/12 light/dark cycle) and maintained with continuous access to water and food in their home cages. Drugs. The concentration of morphine sulfate (expressed as the salt) used during the Tolerance Development Phase was 16 mg/ml dissolved in saline. Tolerance test doses were 1, 2, 4, 6, 8, 12, 16, 32, and 48 mg/ml dissolved in saline. The concentration of saline was adjusted so that each dose was isotonic with physiological saline. Solutions were injected intraperitoneally in a volume of 1.25 ml/kg. Analgesia assessment. Analgesia was assessed by the tail-flick method, which measures the latency for the rat to remove its tail away from a hot beam of light (e.g., Tiffany et al. 1983; Tiffany and Maude-Griffin 1988). The rat was restrained by hand and its tail was placed in a groved Plexiglass plate such that the tail was under the light source (a 125 W, prefocused General Electric bulb). When the rat moved its tail from the light-beam a photo sensitive cell tripped a timer and the tail-flick latency was automatically recorded. To avoid interactions between tail area stimulated and degree of analgesia (Yoburn et al. 1984), each assessment consisted of the average of three consecutive trails with the location of the beam varied systematically among the proximal, middle and distal third of the rat's tail. The beam intensity was adjusted such that nondrugged animals flicked at approximately 3.5 s. A 15 s limit was used to prevent damage to the tail. Habituation. To habituate the rats to handling and injection procedures, they were weighed once daily for 3 days, then weighed twice daily for 3 days and, finally, weighed and injected with saline twice daily for 8 additional days. The injections occurred at 0900 and 1600 hours. Tolerance development phase. Animals were randomly assigned to three treatment conditions: Distinctive Context (DC), Home Cage (HC), and Saline Control (SC). Animals in the DC and HC treatments were assigned to seven conditioning groups, with each group
(n ~ 30/group) receiving 1, 3, 5, 8, 14, 20, or 30 conditioning sessions. Animals in the SC treatment received 1, 3, 5, 8, or 20 conditioning sessions. All the rats were exposed to the distinctive context every 96 h, where the DC groups were injected with morphine, and the HC and SC groups received saline injections. The rats also received an injection in their home-cage environment 48 h after each distinctive-context exposure. Here, the HC groups were injected with morphine, and the DC and SC groups received saline injections. Distinctive-context exposures took place from 0800 to 1545 hours. Each rat was individually weighed in the colony room and then placed back in its home cage. Then, it was hand-carried to a distinctive-context room adjacent to the colony room. The rat was injected with either morphine or saline and placed in a wire-covered, plastic-breeding box (35 × 31 × 16 cm) with a bedding of wood shavings. The room was dimly lit, white noise was played continuously over a loudspeaker (65 dB, Scale A), and pine scented air fresheners were suspended over the boxes. After 30 rain the rat received mock tail-flick trails. On these trails the animal was restrained on the tail-flick apparatus with its tail placed to the side of the light beam. The beam was then activated for three consecutive t 5-s trials. The rat was returned to the plastic box for another 30 rain and then given a second exposure to the tail-flick procedures. After these mock tail-flicks, the rat was returned to its home cage. Injections in the home cage environment took place between 0900 and 1000 hours. For these injections, the rats were individually weighed and placed back in their home cages. Each rat was then removed from its home cage, injected with either saline or morphine, and returned to its cage. Test session. The test session occurred in the distinctive context 96 h after the last distinctive-context exposure. In order to construct dose-response curves (DRCs), each group was divided into four subgroups (n ~ 7) with each subgroup receiving a different test dose of morphine. All animals received analgesia assessments, which were conducted in lieu of mock tail-flick trials at 30 and 60 rain after the injection. Data analysis. The average of the three consecutive tail-flick trials conducted 30 min after the injections were subjected to multiple regression analysis (Cohen and Cohen 1975). Tail-flick latencies were regressed on log-dose level, group condition, and the interaction of these two variable sets. The variables were forced into the equation in the order listed. Effects for group conditions were evaluated by the use of dummy coding for pairwise comparisons. Differences between any pair of groups were indexed by a significant group effect. Parallelism of DRCs were evaluated by examination of the interaction of variables representing group-condition comparisons and dose level.
Results T a b l e 1 shows the m e a n tail-flick latencies o b t a i n e d for the different test doses used for each of the t r e a t m e n t g r o u p s at each level of conditioning. T h e D C animals, which h a d received m o r p h i n e explicitly p a i r e d with the distinctive context, s h o w e d significant D R C shifts to the right of the SC a n d H C a n i m a l s at each level of c o n d i t i o n ing. The H C animals, which h a d received m o r p h i n e explicitly u n p a i r e d with the distinctive context, showed significant shifts to the right of SC a n i m a l s after five o r m o r e c o n d i t i o n i n g sessions. All D R C shifts were parallel in the a b o v e c o m p a r i s o n s as i n d i c a t e d by nonsignificant D o s e x G r o u p interactions, F s < 2.5, Ps > 0.t0. F i g u r e 1 shows the changes in D R C s for each treatm e n t g r o u p as a function of c o n d i t i o n i n g sessions. Each p o i n t represents the log dose of m o r p h i n e n e e d e d to
174 Table 1. Mean tail-flick latencies at each test dose for treatments as a function of number of conditioning sessions Session
Treatment
1
SC a
Session
HC~ DC b 3
SC~ HC a DC b
5
SC a HC b DCc
(Dose) (TF) (Dose) (TF) (Dose) (TF)
1.25 7.15 1.25 6.3 1.25 5.83
2.5 9.33 2.5 7.39 2.5 5.96
5.0 10.0 1 1 . 7 7 13.72 5.0 10.0 11.30 12.54 5.0 10.0 8.22 11.35
(Dose) ffF) (Dose) (TF) (Dose) (TF)
1.25 5.04 1.25 4.79 2.50 4.02
2.5 6.50 2.5 5.88 5.0 5.06
5.0 6.58 5.0 7.61 10.0 10.07
10.0 13.86 10.0 13.28 20.0 12.49
(Dose) (TF) (Dose) (TF) (Dose) (TF)
1.25 4.47 2.5 4.96 5.0 4.29
2,5 5.84 5.0 6.68 10.0 7.17
5.0 8.53 10.0 10.82 20.0 10.97
10.0 11.74 20.0 14.02 40.0 13.96
8
Treatment SC a HCb DC c
14
HC a DC b
20
SC a HC b DC~
30
HC a DCb
(Dose) (TF) (Dose) (T10 (Dose) (TF)
1.25 4.45 2.5 4.73 7.5 3.92
2.5 6.56 5.0 5.22 15.0 7.64
5.0 10.0 7.00 10.86 10.0 20.0 9.15 11.02 30.0 45.0 1 0 . 3 9 13.52
(Dose) ffF) (Dose) (TF) (Dose) (TF)
2.5 5.22 10.0 5.63 1.25 5.54
5.0 6.59 20.0 9.16 2.5 6.14
10.0 7.37 40.0 12.44 5.0 8.09
20.0 12.14 60.0 13.37 10.0 12.93
(Dose) (TF) (Dose) (TF) (Dose) (TF)
2.5 5.61 10.0 6.12 5.0 5.70
5.0 6.21 20.0 7.76 10.0 5.34
10.0 8.61 40.0 13.74 20.0 11.27
20.0 13.72 60.0 14.60 40.0 12.80
(Dose) (TF)
10.0 4.14
20.0 6.68
40.0 11.74
60.0 13.21
Session = number of conditioning sessions; SC = Saline Control; HC = morphine unpaired with distinctive context; DC = morphine explicitly paired with distinctive context; Treatments with different subscripts within a given number of conditioning sessions indicate significant differences between the dose-response curves; Dose is in mg/kg; TF = tail - flick latency in seconds
Table 2. Within-treatment comparisons of changes in dose-response curves as a function of the number of conditioning sessions 1.25
Treatment
SC
.75
.5
.25 ,
,
3 5
J
8
~
r
14 CONDITIONING
20
1 vs 3*** 3 vs 5 5 vs 8 8 vs 20
Statistical analysis
sR 2 = sR 2 = sR 2 < sR 2 =
0.108, 0.008, 0.000, 0.018,
F(1, F(1, F(1, F(1,
59) = 63) = 61) < 59) =
16.2 1.47 1 2.19
53) 54) 57) 55) 56) 53)
HC
1 vs 3* 3 vs 5** 5 vs 8* 8 vs 14 14 vs 20 20 vs 30*
sR 2 = sR 2 = sR 2 = sR 2 = sR 2 = sR ~ =
0.038, 0.039, 0.039, 0.001, 0.010, 0.080,
F(1, F(1, F(1, F(1, F(1, F(t,
= = = < = =
4.46 8.52 6.47 1 1.15 9.80
DC
1 vs 3*** 3 vs 5*** 5 vs 8* 8 vs 14 14 vs 20 20 vs 30
sR 2 = sR 2 = sR 2 = sR 2 < sR 2 < sR 2 =
0.199, 0.050, 0.043, 0.000, 0.000, 0.030,
F(1, 52) = F(1, 55) F(1, 57) = F(1, 54) < F(1, 49) < F(1, 43) =
16.0 11.9 6.81 1 1 2.95
30
SESSIONS
Fig, l. Changes in dose-response curves for each treatment condition as a function of conditioning sessions. (Each point represents the tog dose of morphine needed to produce a 7.5 s tail-flick latency as calculated from the regression analysis. (O) DC = morphine explicitly paired with the distinctive context; (A) HC = morphine explicitly unpaired with the distinctive context; ([]) SC = saline control)
Session comparison
*P < 0.05; **P < 0.01; ***P < 0.001
p r o d u c e a 7.5 s tail-flick l a t e n c y as c a l c u l a t e d f r o m the r e g r e s s i o n a n a l y s i s for e a c h of t h e t r e a t m e n t g r o u p s a c r o s s all o f the c o n d i t i o n i n g sessions. R e g r e s s i o n a n a l y s e s o f these d a t a , s u m m a r i z e d in T a b l e 2, d e m o n s t r a t e d that, w i t h i n t h e S C t r e a t m e n t , the a n i m a l s t h a t h a d r e c e i v e d o n l y o n e e x p o s u r e to the d i s t i n c t i v e c o n t e x t w e r e m o r e sensitive to t h e a n a l g e s i c effects of m o r p h i n e t h a n the animals receiving three exposures. However, no further differences a p p e a r e d as a f u n c t i o n o f n u m b e r o f e x p o s u r e
sessions. T h e s e results s u g g e s t t h a t n o v e l t y a n d stress factors m a y h a v e a u g m e n t e d n o c i c e p t i v e r e s p o n d i n g in a n i m a l s r e c e i v i n g l i m i t e d e x p o s u r e to the c o n t e x t ( M a u d e Griffin a n d T i f f a n y 1989), b u t t h a t these effects d i s s i p a t e d by t h r e e e x p o s u r e s to t h e c o n t e x t . W i t h i n t h e H C t r e a t m e n t c o n d i t i o n s , the shifts in the D R C s r e v e a l e d m o d e r ate, significant d e c r e a s e s in the a n a l g e s i c effects o f m o r p h i n e f r o m o n e to e i g h t c o n d i t i o n i n g sessions, w i t h n o c h a n g e f r o m 8 t o 20 sessions, a n d a f u r t h e r significant
175
decrease between 20 and 30 sessions. Animals in the DC treatments showed rapid tolerance development, with significant increments in tolerance across one to eight conditioning sessions, but no change after that point. The data from the 60-min tail-flick tests revealed essentially the same pattern of results described for the 30-rain assessments. The overall differences between and within treatment conditions were not as pronounced at 60 rain because the analgesic effects of morphine, particularly at the lower test doses, had declined substantially by the second tail-flick assessment.
Discussion
These data show that rats that had received morphine explicitly paired with the distinctive context exhibited greater morphine tolerance than rats that had received comparable exposure to the drug explicitly unpaired with the context. Across independent groups of animals, the contextual specificity of tolerance was evident after l, 3, 5, 8, 14, 20, and 30 drug administrations and can be interpreted as clear evidence of the associative nature of the tolerance found in the DC animals. These data are consistent with past research from this laboratory (Tiffany and Maude-Griffin 1988; Tiffany et al. 1991; 1992) demonstrating that the distinctive context can acquire strong associative control over the development of tolerance to morphine's analgesic effects. The overall magnitude of the associative tolerance found in this experiment is also comparable to that of our previous studies. For example, by the eighth conditioning session, DC animals required a 5.7-fold increase in test doses to achieve the same level of analgesia produced in drug-naive animals. The parallelism of the DRCs between treatments also replicates this laboratory's past findings (Tiffany et al. 1991; 1992) that associative morphine tolerance can be characterized as a parallel shift to the right in DRCs. The development of tolerance in the DC animals was apparent after one morphine administration and increased rapidly until it reached a maximum by the eighth conditioning session. In contrast, HC rats did not begin to display tolerance, relative to the drug-naive SC rats, until the fifth morphine administration. This tolerance increased significantly with three more morphine administrations (i.e., 5 versus 8 sessions), did not change from 8 to 20 conditioning sessions, showed a further increment after 30 conditioning sessions (i.e., 20 versus 30 sessions), but never reached the level of the DC animals. The present finding that, even after 30 conditioning sessions, tolerance acquired in the HC condition remained lower than that achieved in the DC condition differs from results obtained in previous studies in which the animals practiced the test response after each conditioning session (Siegel 1977; Tiffany et al. 1983; Dafters et al. 1988; Dafters and Odber 1989). For example, Dafters and Odber (1989) reported that, although rats receiving morphine in their home-cage environment developed tolerance more slowly than animals that received morphine paired with a distinctive context, tolerance levels were comparable in both groups by the ninth administration of the drug. Their data
may describe the impact of contextual stimuli on the development of tolerance acquired through operant contingencies, and may not be relevant to an examination of associative conditioning. The tolerance found in the HC animals could represent the operation of either nonassociative or associative mechanisms. Although the results do not allow us to distinguish between these two alternatives, several considerations suggest that this tolerance is an associative effect. First, based on conventional conceptualizations of nonassociative tolerance, a 96 h inter-dose-interval (IDI) should not be conducive to the acquisition of this form of tolerance (Baker and Tiffany 1985; Tiffany et al. 1991). Furthermore, other data from our laboratory (Tiffany et al. 1991) show that the level of this tolerance is not systematically affected by IDIs ranging from 12 to 96 h. All models of nonassociative tolerance argue that the magnitude of this tolerance should show a strong, negative relationship with IDI (Baker and Tiffany 1985). Other data from our laboratory support the hypothesis that the tolerance in the HC condition may be maintained associatively by injection and handling cues (see also Dafters and Bach 1985). For example, this tolerance is less pronounced if animals are given extensive exposure to the handling and injection ritual prior to morphine administration (Drobes and Tiffany, unpublished observations). Moreover, unlike nonassociative tolerance, this HC tolerance shows nearly complete retention over a 30-day interval (Tiffany et al. 1992). Finally, repeated nonreinforced exposures to the injection and handling ritual over a 30-day retention interval appear to extinguish tolerance in HC animals (Tiffany et al. 1991). From an associative viewpoint, the lower rate and absolute magnitude of tolerance acquisition in the HC group can easily be accounted for by latent inhibition (e.g., Siegel 1977; Tiffany and Baker 1981; Dafters and Bach 1985) and partial-reinforcement effects (e.g., Krank et al. 1984; Siegel 1977). If handling and injection stimuli served as CS+s for the HC animals in the present experiment, extensive exposure to those cues prior to conditioning (latent inhibition), and administration of saline injections in the distinctive context during conditioning (partial reinforcement), should have reduced the associative strength acquired by those stimuli. There is no evidence that the distinctive context acquired the properties of a conditioned inhibitor in HC rats despite the fact that the context was unpaired with morphine administration in the presence of stimuli that, as we have argued, may have been serving as CS+s for these animals. Our conditioning procedures may have mitigated against the development of conditioned inhibition. It is widely accepted that the development of conditioned inhibition depends on the nonreinforced presentation of salient stimuli in the presence of conditioned excitation produced by other stimuli (see review by Lolordo and Fairless 1985). In the present experiment, HC rats exhibited evidence of tolerance only after five conditioning sessions. To the extent this tolerance was associatively based, these animals had several exposures to the distinctive context in the absence of conditioned excitation. Consequently, initial exposures to the distinctive context in the absence of conditioned excitation may
176 have latently inhibited the formation of conditioned inhibition (see Baker and Baker 1985, for the disruptive effects of latent inhibition in conditioned inhibition). Moreover, Fowler et al. (1985) have argued that the development of conditioned inhibition to a stimulus depends on the contiguous presentation of that stimulus with strong excitation. In this experiment, handling and injection stimuli only supported moderate levels of tolerance in the H C conditions. If the H C tolerance is associative and controlled solely by handling and injection cues, we would expect similar absolute levels of tolerance when these animals are either tested in the distinctive context or in their homecage environment. Conversely, D C animals should show less tolerance, if any, in the home-cage environment than in the distinctive context. Further research is needed to investigate this possibility. The results of this experiment support the guidelines suggested by Tiffany et al. (1991) regarding the conduct of investigations of associative tolerance. First, the results of studies that have confounded classical conditioning with operant conditioning m a y not be representative of associative tolerance effects (cf Dafters and O d b e r 1989, and the present study). As demonstrated in this study and others from this l a b o r a t o r y (Tiffany and Maude-Griffin 1988; Tiffany et al. 1991; 1992), It is not necessary to test subjects after each m o r p h i n e exposure in order to obtain robust associative tolerance effects. Second, it is apparent that injection stimuli m a y be potent cues in the support of associative tolerance effects. Consequently, tolerance that develops when drug is not explicitly paired with a distinctive context cannot, by default, be assumed to be necessarily controlled by nonassociative mechanisms. In order to attenuate this tolerance, it is advisable to extensively expose the animals to these stimuli prior to conditioning, increase the ratio of nonreinforced to reinforced presentations of such stimuli over the course of conditioning, and utilize highly salient contexts for the nominal CS ÷. Finally the data from this study continue to show that the sensitivity and reliability of D R C m e t h o d o l o g y offers clear advantages over the use of single test doses for estimates of tolerance magnitude.
Acknowledgements. Funds for this research were provided by Grant #2-R01 DA04050 from the National Institute on Drug Abuse awarded to S.T. Tiffany. We thank William Banks, Michael Denious, Anthony Gerdeman, Todd Hortman, and James Speck for their help in conducting the experiments. We also thank John Capatdi and Scott Vrana for comments on a draft of this manuscript.
References Baker AG, Baker PA (1985) Does Inhibition differ from excitation, proactive interference, contextual conditioning and extinction? In: Miller RR, Spear NE (eds) Information processing in animals: conditioned inhibition. Erlbaum, Hillsdale, New Jersey, pp 151-184 Baker TB, Tiffany ST (1985) Morphine tolerance as habituation. Psychol Rev 92 : 78.--108 Cohen J, Cohen P (1975) Applied multiple regression/correlational analysis for the behavioral sciences. Erlbaum, Hillsdale, New Jersey
Dafters RI, Bach L (1985) Absence of environment-specificity in morphine tolerance acquired in nondistinctive environments: habituation or stimulus overshadowing? Psychopharmacology 87 : 101-106 Dafters RI, Odber J (1989) Effects of dose, interdose interval and drug-signal parameters on morphine analgesic tolerance: implications for current theories of tolerance. Behav Neurosci 103: I082-1090 Dafters RI, Odber J, Miller J (1988) Associative and non-associative tolerance to morphine: support for a dual-process habituation model. Life Sci 42 : 1897-1906 Fowler H, Kleiman M, Lysle DT (1985) Factors affecting the acquisition and extinction of conditioned inhibition suggest a "slave" process. In: Miller RR, Spear NE (eds) Information processing in animals: conditioned inhibition. Erlbaum, Hillsdale, New Jersey, pp 113-150 Goudie AJ, Demellweek C (1986) Conditioning factors in drug tolerance. In: Goldberg SR, Stolerman IP (eds) Behavioral analysis of drug dependence. Academic Press, New York, pp 225-285 Krank MD, Hinson RE, Siegel S (1984) The effect of partial reinforcement on tolerance to morphine induced analgesia and weight loss in the rat. Behav Neurosci 98:79-85 LoLordo VM, Fairless JL (1985) Pavlovian conditioned inhibition: the literature since 1969. In: Miller RR, Spear NE (eds) Information processing in animals: conditioned inhibition. Erlbaum, Hillsdale, New Jersey, pp 1-50 Maude-Griffin PM, Tiffany ST (1989) Associative morphine tolerance in the rat: examination of compensatory responding and cross-tolerance with stress-induced analgesia. Behav Neur Biol 51 : 11-33 Rescorla RA (1985) Conditioned inhibition and facilitation. In: Miller RR, Spear NE (eds) Information processing in animals: conditioned inhibition. Erlbaum, Hillsdale, New Jersey, pp 299 - 326 Rescorla RA, Wagner AR (1972) A theory of Pavlovian conditioning: variations in the effectiveness of reinforcement and nonreinforcement. In: Black AH, Prokasy WF (eds) Classieal conditioning II: current research and theory. Appleton Century Crofts, New York, pp 64-99 Siegel S (1975) Evidence from rats that tolerance is a learned response. J Comp Physiol Psychol 89:323-325 Siegel S (1977) Morphine tolerance acquisition as an associative process. J Exp Psychol [Anim Behav] 3 : 1-13 Siegel S (1989) Pharmacological conditioning and drug effects. In: Goudie AJ, Emmett-Oglesby MW (eds) Psychoactive drugs: tolerance and sensitization. Humana, Clifton, New Jersey, pp t15-180 Tiffany ST, Baker TB (1981) Morphine tolerance in rats: congruence with a Pavlovian paradigm. J Comp Physiol Psychol 95 : 747-762 Tiffany ST, Drobes D, Cepeda-Benito A (1992) Contribution of associative and nonassociative processes to the development of morphine tolerance. Psychopharmacology 109:185-190 Tiffany ST, Maude-Griffin PM (I988) Tolerance to morphine in the rat: associative and nonassociative effects. Behav Neurosci 102 : 534-543 Tiffany ST, Petrie EC, Martin EM, Baker TB (1983) Drug signals enhance morphine tolerance development in hypophysectomized rats. Psychopharmacology 79:84-85 Wolgin DL (1989) The role of instrumental learning in behavioral tolerance to drugs. In: Goudie AJ, Emmett-Oglesby MW (eds) Psychoactive drugs: tolerance and sensitization. Humana, Clifton, New Jersey, pp 17-114 Yoburn BC, Morales R, Kelly DD, Inturrisi EC (1984) Constraints on the tail-flick assay: morphine analgesia and tolerance are dependent upon locus of tail stimulation. Life Sci 34:1755-1762
Psychopharmacology © Springer-Verlag 1992
Effect of number of conditioning trials on the development of associative tolerance to morphine Antonio Cepeda-Benito and Stephen T. Tiffany Department of Psychological Sciences, Purdue University, West Lafayette, IN 47907, USA Received January 2, 1991 / Final version March 16, 1992
Abstract. The acquisition of associative tolerance to the analgesic effects of morphine was investigated by giving independent groups of rats 1, 3, 5, 8, 14, 20, or 30 administrations of drug either explicitly paired or unpaired with a distinctive context. Tolerance, assessed on a tail-flick device using dose-response curve (DRC) methodology, developed more rapidly and reached greater magnitude when morphine and the distinctive context were explicitly paired rather than explicitly unpaired. Tolerance magnitude in both conditions reached a maximum at eight conditioning sessions. It is argued that the tolerance found in both treatment groups was associatively controlled. The function of handling and injection cues as conditioned stimuli, and the deleterious effects of latent inhibition and partial reinforcement on conditioned excitation and conditioned inhibition are discussed. Key words: Conditioning - Tolerance - Morphine - Tailflick - Dose-response curves It is widely accepted that associative-learning processes contribute to the development of morphine tolerance (see Baker and Tiffany 1985; Goudie and Demellweek 1986; Siegel 1989). Associative tolerance models (Baker and Tiffany 1985; Siegel t975) posit that environmental cues that reliably predict the occurrence of morphine delivery function as conditioned stimuli (CSs) that elicit conditioned tolerance processes. For example, like other forms of associative learning, associative-morphine tolerance should be context specific, and subject to extinction, sensory preconditioning, overshadowing, latent inhibition, conditioned inhibition, partial reinforcement and blocking (see reviews by Baker and Tiffany 1985; Goudie and DemeUweek 1986; Siegel 1989). Although associative tolerance models would predict greater rates of tolerance acquisition when drug delivery is signalled than when it is not (Siegel 1975; Baker and Tiffany 1985), there has been little systematic research on the rate of associative-tolerance development as a funcCorrespondence to:
S.T. Tiffany
tion of the number of conditioning trials. When learning curves of tolerance development have been investigated (Siegel 1977; Tiffany et al. 1983; Dafters et al. 1988; Dafters and Odber 1989), they have typically been generated by assessing drug responsiveness during each conditioning session. Since tolerance to the analgesic effect of morphine is generally measured as the latency to make a response to a nociceptive stimulus, such studies have confounded the associative paradigm with the opportunity to practice the response under the influence of drug effects. It is generally assumed that tolerance arising from intoxicated practice represents the operation of some form of operant conditioning that produces a task-specific compensation for drug-induced disruptions in performance (Wolgin 1989). This form of tolerance should be distinguished from associative tolerance, which represents the operation of classical conditioning. Associative models of tolerance predict that animals should display a monotonically accelerated level of tolerance development over the course of conditioning trials when morphine is explicitly paired with a CS. Tiffany and his colleagues have consistently found that rats show high levels of tolerance when tested in a distinctive context that had been paired previously with eight morphine injections (Tiffany and Maude-Griffin 1988; Tiffany et al. 1991). Research from this laboratory has also found low levels of tolerance in animals that have had the distinctive test context explicitly unpaired with morphine administration (Tiffany et al. 1992). This tolerance may be controlled associatively by handling and injection cues that accompany drug administration (DaRers and Bach 1985). This is supported by the finding that this tolerance, unlike nonassociative forms (Baker and Tiffany 1985), was present after a 30-day retention interval (Tiffany et al. 1992) and could be extinguished by presenting a series of saline injections in the rats' home-cage environment over a 30-day period (Tiffany et al. 1991). When morphine administration is explicitly unpaired with a distinctive context, the context may become a conditioned inhibitor of associative tolerance effects, that is, become a CS- (LoLordo and Fairless 1985). Moreover, as predicted by the Reseorla and Wagner (1972) model of
173 classical conditioning, associative tolerance effects m a y be evident in the i n h i b i t o r y context over early c o n d i t i o n i n g trials, b u t these s h o u l d decline as c o n d i t i o n i n g p r o c e e d s (Rescorla 1985). T h e presence o f associative t o l e r a n c e effects in u n p a i r e d c o n d i t i o n s (Tiffany et al. 1991; 1992) suggests t h a t the distinctive c o n t e x t m a y n o t have acq u i r e d sufficient i n h i b i t o r y strength after eight c o n d i t i o n ing trials to c o m p l e t e l y c o u n t e r a c t the e x c i t a t o r y effects p r o d u c e d by d r u g - p a i r e d injection a n d h a n d l i n g cues. This s t u d y e x a m i n e d the effects of the n u m b e r of c o n d i t i o n i n g sessions on the d e v e l o p m e n t of tolerance to the analgesic effects of m o r p h i n e . Rats received 1, 3, 5, 8, 14, 20, o r 30 injections o f m o r p h i n e either explicitly p a i r e d o r u n p a i r e d with a distinctive c o n t e x t p r i o r to tolerance testing. A n i m a l s were n o t e x p o s e d to the nociceptive test stimulus until the t o l e r a n c e test session. As with o t h e r investigations recently c o n d u c t e d in o u r l a b o r a t o r y (Tiffany a n d M a u d e - G r i f f i n 1988; Tiffany et al. 1991; 1992), tolerance m a g n i t u d e was assessed as a shift in the d o s e - r e s p o n s e curve to the right. This research p r o v i d e d for a c o m p r e h e n s i v e investigation of the time course of associative tolerance effects u n c o n f o u n d e d by the p o t e n tial influence o f o p e r a n t contingencies.
Materials and methods Subjects. The subjects were 558 experimentally naive, male Holtzman rats 80-83 days old on the first day of conditioning. The rats were housed individually in wire mesh cages located in a colony room (on a 12/12 light/dark cycle) and maintained with continuous access to water and food in their home cages. Drugs. The concentration of morphine sulfate (expressed as the salt) used during the Tolerance Development Phase was 16 mg/ml dissolved in saline. Tolerance test doses were 1, 2, 4, 6, 8, 12, 16, 32, and 48 mg/ml dissolved in saline. The concentration of saline was adjusted so that each dose was isotonic with physiological saline. Solutions were injected intraperitoneally in a volume of 1.25 ml/kg. Analgesia assessment. Analgesia was assessed by the tail-flick method, which measures the latency for the rat to remove its tail away from a hot beam of light (e.g., Tiffany et al. 1983; Tiffany and Maude-Griffin 1988). The rat was restrained by hand and its tail was placed in a groved Plexiglass plate such that the tail was under the light source (a 125 W, prefocused General Electric bulb). When the rat moved its tail from the light-beam a photo sensitive cell tripped a timer and the tail-flick latency was automatically recorded. To avoid interactions between tail area stimulated and degree of analgesia (Yoburn et al. 1984), each assessment consisted of the average of three consecutive trails with the location of the beam varied systematically among the proximal, middle and distal third of the rat's tail. The beam intensity was adjusted such that nondrugged animals flicked at approximately 3.5 s. A 15 s limit was used to prevent damage to the tail. Habituation. To habituate the rats to handling and injection procedures, they were weighed once daily for 3 days, then weighed twice daily for 3 days and, finally, weighed and injected with saline twice daily for 8 additional days. The injections occurred at 0900 and 1600 hours. Tolerance development phase. Animals were randomly assigned to three treatment conditions: Distinctive Context (DC), Home Cage (HC), and Saline Control (SC). Animals in the DC and HC treatments were assigned to seven conditioning groups, with each group
(n ~ 30/group) receiving 1, 3, 5, 8, 14, 20, or 30 conditioning sessions. Animals in the SC treatment received 1, 3, 5, 8, or 20 conditioning sessions. All the rats were exposed to the distinctive context every 96 h, where the DC groups were injected with morphine, and the HC and SC groups received saline injections. The rats also received an injection in their home-cage environment 48 h after each distinctive-context exposure. Here, the HC groups were injected with morphine, and the DC and SC groups received saline injections. Distinctive-context exposures took place from 0800 to 1545 hours. Each rat was individually weighed in the colony room and then placed back in its home cage. Then, it was hand-carried to a distinctive-context room adjacent to the colony room. The rat was injected with either morphine or saline and placed in a wire-covered, plastic-breeding box (35 × 31 × 16 cm) with a bedding of wood shavings. The room was dimly lit, white noise was played continuously over a loudspeaker (65 dB, Scale A), and pine scented air fresheners were suspended over the boxes. After 30 rain the rat received mock tail-flick trails. On these trails the animal was restrained on the tail-flick apparatus with its tail placed to the side of the light beam. The beam was then activated for three consecutive t 5-s trials. The rat was returned to the plastic box for another 30 rain and then given a second exposure to the tail-flick procedures. After these mock tail-flicks, the rat was returned to its home cage. Injections in the home cage environment took place between 0900 and 1000 hours. For these injections, the rats were individually weighed and placed back in their home cages. Each rat was then removed from its home cage, injected with either saline or morphine, and returned to its cage. Test session. The test session occurred in the distinctive context 96 h after the last distinctive-context exposure. In order to construct dose-response curves (DRCs), each group was divided into four subgroups (n ~ 7) with each subgroup receiving a different test dose of morphine. All animals received analgesia assessments, which were conducted in lieu of mock tail-flick trials at 30 and 60 rain after the injection. Data analysis. The average of the three consecutive tail-flick trials conducted 30 min after the injections were subjected to multiple regression analysis (Cohen and Cohen 1975). Tail-flick latencies were regressed on log-dose level, group condition, and the interaction of these two variable sets. The variables were forced into the equation in the order listed. Effects for group conditions were evaluated by the use of dummy coding for pairwise comparisons. Differences between any pair of groups were indexed by a significant group effect. Parallelism of DRCs were evaluated by examination of the interaction of variables representing group-condition comparisons and dose level.
Results T a b l e 1 shows the m e a n tail-flick latencies o b t a i n e d for the different test doses used for each of the t r e a t m e n t g r o u p s at each level of conditioning. T h e D C animals, which h a d received m o r p h i n e explicitly p a i r e d with the distinctive context, s h o w e d significant D R C shifts to the right of the SC a n d H C a n i m a l s at each level of c o n d i t i o n ing. The H C animals, which h a d received m o r p h i n e explicitly u n p a i r e d with the distinctive context, showed significant shifts to the right of SC a n i m a l s after five o r m o r e c o n d i t i o n i n g sessions. All D R C shifts were parallel in the a b o v e c o m p a r i s o n s as i n d i c a t e d by nonsignificant D o s e x G r o u p interactions, F s < 2.5, Ps > 0.t0. F i g u r e 1 shows the changes in D R C s for each treatm e n t g r o u p as a function of c o n d i t i o n i n g sessions. Each p o i n t represents the log dose of m o r p h i n e n e e d e d to
174 Table 1. Mean tail-flick latencies at each test dose for treatments as a function of number of conditioning sessions Session
Treatment
1
SC a
Session
HC~ DC b 3
SC~ HC a DC b
5
SC a HC b DCc
(Dose) (TF) (Dose) (TF) (Dose) (TF)
1.25 7.15 1.25 6.3 1.25 5.83
2.5 9.33 2.5 7.39 2.5 5.96
5.0 10.0 1 1 . 7 7 13.72 5.0 10.0 11.30 12.54 5.0 10.0 8.22 11.35
(Dose) ffF) (Dose) (TF) (Dose) (TF)
1.25 5.04 1.25 4.79 2.50 4.02
2.5 6.50 2.5 5.88 5.0 5.06
5.0 6.58 5.0 7.61 10.0 10.07
10.0 13.86 10.0 13.28 20.0 12.49
(Dose) (TF) (Dose) (TF) (Dose) (TF)
1.25 4.47 2.5 4.96 5.0 4.29
2,5 5.84 5.0 6.68 10.0 7.17
5.0 8.53 10.0 10.82 20.0 10.97
10.0 11.74 20.0 14.02 40.0 13.96
8
Treatment SC a HCb DC c
14
HC a DC b
20
SC a HC b DC~
30
HC a DCb
(Dose) (TF) (Dose) (T10 (Dose) (TF)
1.25 4.45 2.5 4.73 7.5 3.92
2.5 6.56 5.0 5.22 15.0 7.64
5.0 10.0 7.00 10.86 10.0 20.0 9.15 11.02 30.0 45.0 1 0 . 3 9 13.52
(Dose) ffF) (Dose) (TF) (Dose) (TF)
2.5 5.22 10.0 5.63 1.25 5.54
5.0 6.59 20.0 9.16 2.5 6.14
10.0 7.37 40.0 12.44 5.0 8.09
20.0 12.14 60.0 13.37 10.0 12.93
(Dose) (TF) (Dose) (TF) (Dose) (TF)
2.5 5.61 10.0 6.12 5.0 5.70
5.0 6.21 20.0 7.76 10.0 5.34
10.0 8.61 40.0 13.74 20.0 11.27
20.0 13.72 60.0 14.60 40.0 12.80
(Dose) (TF)
10.0 4.14
20.0 6.68
40.0 11.74
60.0 13.21
Session = number of conditioning sessions; SC = Saline Control; HC = morphine unpaired with distinctive context; DC = morphine explicitly paired with distinctive context; Treatments with different subscripts within a given number of conditioning sessions indicate significant differences between the dose-response curves; Dose is in mg/kg; TF = tail - flick latency in seconds
Table 2. Within-treatment comparisons of changes in dose-response curves as a function of the number of conditioning sessions 1.25
Treatment
SC
.75
.5
.25 ,
,
3 5
J
8
~
r
14 CONDITIONING
20
1 vs 3*** 3 vs 5 5 vs 8 8 vs 20
Statistical analysis
sR 2 = sR 2 = sR 2 < sR 2 =
0.108, 0.008, 0.000, 0.018,
F(1, F(1, F(1, F(1,
59) = 63) = 61) < 59) =
16.2 1.47 1 2.19
53) 54) 57) 55) 56) 53)
HC
1 vs 3* 3 vs 5** 5 vs 8* 8 vs 14 14 vs 20 20 vs 30*
sR 2 = sR 2 = sR 2 = sR 2 = sR 2 = sR ~ =
0.038, 0.039, 0.039, 0.001, 0.010, 0.080,
F(1, F(1, F(1, F(1, F(1, F(t,
= = = < = =
4.46 8.52 6.47 1 1.15 9.80
DC
1 vs 3*** 3 vs 5*** 5 vs 8* 8 vs 14 14 vs 20 20 vs 30
sR 2 = sR 2 = sR 2 = sR 2 < sR 2 < sR 2 =
0.199, 0.050, 0.043, 0.000, 0.000, 0.030,
F(1, 52) = F(1, 55) F(1, 57) = F(1, 54) < F(1, 49) < F(1, 43) =
16.0 11.9 6.81 1 1 2.95
30
SESSIONS
Fig, l. Changes in dose-response curves for each treatment condition as a function of conditioning sessions. (Each point represents the tog dose of morphine needed to produce a 7.5 s tail-flick latency as calculated from the regression analysis. (O) DC = morphine explicitly paired with the distinctive context; (A) HC = morphine explicitly unpaired with the distinctive context; ([]) SC = saline control)
Session comparison
*P < 0.05; **P < 0.01; ***P < 0.001
p r o d u c e a 7.5 s tail-flick l a t e n c y as c a l c u l a t e d f r o m the r e g r e s s i o n a n a l y s i s for e a c h of t h e t r e a t m e n t g r o u p s a c r o s s all o f the c o n d i t i o n i n g sessions. R e g r e s s i o n a n a l y s e s o f these d a t a , s u m m a r i z e d in T a b l e 2, d e m o n s t r a t e d that, w i t h i n t h e S C t r e a t m e n t , the a n i m a l s t h a t h a d r e c e i v e d o n l y o n e e x p o s u r e to the d i s t i n c t i v e c o n t e x t w e r e m o r e sensitive to t h e a n a l g e s i c effects of m o r p h i n e t h a n the animals receiving three exposures. However, no further differences a p p e a r e d as a f u n c t i o n o f n u m b e r o f e x p o s u r e
sessions. T h e s e results s u g g e s t t h a t n o v e l t y a n d stress factors m a y h a v e a u g m e n t e d n o c i c e p t i v e r e s p o n d i n g in a n i m a l s r e c e i v i n g l i m i t e d e x p o s u r e to the c o n t e x t ( M a u d e Griffin a n d T i f f a n y 1989), b u t t h a t these effects d i s s i p a t e d by t h r e e e x p o s u r e s to t h e c o n t e x t . W i t h i n t h e H C t r e a t m e n t c o n d i t i o n s , the shifts in the D R C s r e v e a l e d m o d e r ate, significant d e c r e a s e s in the a n a l g e s i c effects o f m o r p h i n e f r o m o n e to e i g h t c o n d i t i o n i n g sessions, w i t h n o c h a n g e f r o m 8 t o 20 sessions, a n d a f u r t h e r significant
175
decrease between 20 and 30 sessions. Animals in the DC treatments showed rapid tolerance development, with significant increments in tolerance across one to eight conditioning sessions, but no change after that point. The data from the 60-min tail-flick tests revealed essentially the same pattern of results described for the 30-rain assessments. The overall differences between and within treatment conditions were not as pronounced at 60 rain because the analgesic effects of morphine, particularly at the lower test doses, had declined substantially by the second tail-flick assessment.
Discussion
These data show that rats that had received morphine explicitly paired with the distinctive context exhibited greater morphine tolerance than rats that had received comparable exposure to the drug explicitly unpaired with the context. Across independent groups of animals, the contextual specificity of tolerance was evident after l, 3, 5, 8, 14, 20, and 30 drug administrations and can be interpreted as clear evidence of the associative nature of the tolerance found in the DC animals. These data are consistent with past research from this laboratory (Tiffany and Maude-Griffin 1988; Tiffany et al. 1991; 1992) demonstrating that the distinctive context can acquire strong associative control over the development of tolerance to morphine's analgesic effects. The overall magnitude of the associative tolerance found in this experiment is also comparable to that of our previous studies. For example, by the eighth conditioning session, DC animals required a 5.7-fold increase in test doses to achieve the same level of analgesia produced in drug-naive animals. The parallelism of the DRCs between treatments also replicates this laboratory's past findings (Tiffany et al. 1991; 1992) that associative morphine tolerance can be characterized as a parallel shift to the right in DRCs. The development of tolerance in the DC animals was apparent after one morphine administration and increased rapidly until it reached a maximum by the eighth conditioning session. In contrast, HC rats did not begin to display tolerance, relative to the drug-naive SC rats, until the fifth morphine administration. This tolerance increased significantly with three more morphine administrations (i.e., 5 versus 8 sessions), did not change from 8 to 20 conditioning sessions, showed a further increment after 30 conditioning sessions (i.e., 20 versus 30 sessions), but never reached the level of the DC animals. The present finding that, even after 30 conditioning sessions, tolerance acquired in the HC condition remained lower than that achieved in the DC condition differs from results obtained in previous studies in which the animals practiced the test response after each conditioning session (Siegel 1977; Tiffany et al. 1983; Dafters et al. 1988; Dafters and Odber 1989). For example, Dafters and Odber (1989) reported that, although rats receiving morphine in their home-cage environment developed tolerance more slowly than animals that received morphine paired with a distinctive context, tolerance levels were comparable in both groups by the ninth administration of the drug. Their data
may describe the impact of contextual stimuli on the development of tolerance acquired through operant contingencies, and may not be relevant to an examination of associative conditioning. The tolerance found in the HC animals could represent the operation of either nonassociative or associative mechanisms. Although the results do not allow us to distinguish between these two alternatives, several considerations suggest that this tolerance is an associative effect. First, based on conventional conceptualizations of nonassociative tolerance, a 96 h inter-dose-interval (IDI) should not be conducive to the acquisition of this form of tolerance (Baker and Tiffany 1985; Tiffany et al. 1991). Furthermore, other data from our laboratory (Tiffany et al. 1991) show that the level of this tolerance is not systematically affected by IDIs ranging from 12 to 96 h. All models of nonassociative tolerance argue that the magnitude of this tolerance should show a strong, negative relationship with IDI (Baker and Tiffany 1985). Other data from our laboratory support the hypothesis that the tolerance in the HC condition may be maintained associatively by injection and handling cues (see also Dafters and Bach 1985). For example, this tolerance is less pronounced if animals are given extensive exposure to the handling and injection ritual prior to morphine administration (Drobes and Tiffany, unpublished observations). Moreover, unlike nonassociative tolerance, this HC tolerance shows nearly complete retention over a 30-day interval (Tiffany et al. 1992). Finally, repeated nonreinforced exposures to the injection and handling ritual over a 30-day retention interval appear to extinguish tolerance in HC animals (Tiffany et al. 1991). From an associative viewpoint, the lower rate and absolute magnitude of tolerance acquisition in the HC group can easily be accounted for by latent inhibition (e.g., Siegel 1977; Tiffany and Baker 1981; Dafters and Bach 1985) and partial-reinforcement effects (e.g., Krank et al. 1984; Siegel 1977). If handling and injection stimuli served as CS+s for the HC animals in the present experiment, extensive exposure to those cues prior to conditioning (latent inhibition), and administration of saline injections in the distinctive context during conditioning (partial reinforcement), should have reduced the associative strength acquired by those stimuli. There is no evidence that the distinctive context acquired the properties of a conditioned inhibitor in HC rats despite the fact that the context was unpaired with morphine administration in the presence of stimuli that, as we have argued, may have been serving as CS+s for these animals. Our conditioning procedures may have mitigated against the development of conditioned inhibition. It is widely accepted that the development of conditioned inhibition depends on the nonreinforced presentation of salient stimuli in the presence of conditioned excitation produced by other stimuli (see review by Lolordo and Fairless 1985). In the present experiment, HC rats exhibited evidence of tolerance only after five conditioning sessions. To the extent this tolerance was associatively based, these animals had several exposures to the distinctive context in the absence of conditioned excitation. Consequently, initial exposures to the distinctive context in the absence of conditioned excitation may
176 have latently inhibited the formation of conditioned inhibition (see Baker and Baker 1985, for the disruptive effects of latent inhibition in conditioned inhibition). Moreover, Fowler et al. (1985) have argued that the development of conditioned inhibition to a stimulus depends on the contiguous presentation of that stimulus with strong excitation. In this experiment, handling and injection stimuli only supported moderate levels of tolerance in the H C conditions. If the H C tolerance is associative and controlled solely by handling and injection cues, we would expect similar absolute levels of tolerance when these animals are either tested in the distinctive context or in their homecage environment. Conversely, D C animals should show less tolerance, if any, in the home-cage environment than in the distinctive context. Further research is needed to investigate this possibility. The results of this experiment support the guidelines suggested by Tiffany et al. (1991) regarding the conduct of investigations of associative tolerance. First, the results of studies that have confounded classical conditioning with operant conditioning m a y not be representative of associative tolerance effects (cf Dafters and O d b e r 1989, and the present study). As demonstrated in this study and others from this l a b o r a t o r y (Tiffany and Maude-Griffin 1988; Tiffany et al. 1991; 1992), It is not necessary to test subjects after each m o r p h i n e exposure in order to obtain robust associative tolerance effects. Second, it is apparent that injection stimuli m a y be potent cues in the support of associative tolerance effects. Consequently, tolerance that develops when drug is not explicitly paired with a distinctive context cannot, by default, be assumed to be necessarily controlled by nonassociative mechanisms. In order to attenuate this tolerance, it is advisable to extensively expose the animals to these stimuli prior to conditioning, increase the ratio of nonreinforced to reinforced presentations of such stimuli over the course of conditioning, and utilize highly salient contexts for the nominal CS ÷. Finally the data from this study continue to show that the sensitivity and reliability of D R C m e t h o d o l o g y offers clear advantages over the use of single test doses for estimates of tolerance magnitude.
Acknowledgements. Funds for this research were provided by Grant #2-R01 DA04050 from the National Institute on Drug Abuse awarded to S.T. Tiffany. We thank William Banks, Michael Denious, Anthony Gerdeman, Todd Hortman, and James Speck for their help in conducting the experiments. We also thank John Capatdi and Scott Vrana for comments on a draft of this manuscript.
References Baker AG, Baker PA (1985) Does Inhibition differ from excitation, proactive interference, contextual conditioning and extinction? In: Miller RR, Spear NE (eds) Information processing in animals: conditioned inhibition. Erlbaum, Hillsdale, New Jersey, pp 151-184 Baker TB, Tiffany ST (1985) Morphine tolerance as habituation. Psychol Rev 92 : 78.--108 Cohen J, Cohen P (1975) Applied multiple regression/correlational analysis for the behavioral sciences. Erlbaum, Hillsdale, New Jersey
Dafters RI, Bach L (1985) Absence of environment-specificity in morphine tolerance acquired in nondistinctive environments: habituation or stimulus overshadowing? Psychopharmacology 87 : 101-106 Dafters RI, Odber J (1989) Effects of dose, interdose interval and drug-signal parameters on morphine analgesic tolerance: implications for current theories of tolerance. Behav Neurosci 103: I082-1090 Dafters RI, Odber J, Miller J (1988) Associative and non-associative tolerance to morphine: support for a dual-process habituation model. Life Sci 42 : 1897-1906 Fowler H, Kleiman M, Lysle DT (1985) Factors affecting the acquisition and extinction of conditioned inhibition suggest a "slave" process. In: Miller RR, Spear NE (eds) Information processing in animals: conditioned inhibition. Erlbaum, Hillsdale, New Jersey, pp 113-150 Goudie AJ, Demellweek C (1986) Conditioning factors in drug tolerance. In: Goldberg SR, Stolerman IP (eds) Behavioral analysis of drug dependence. Academic Press, New York, pp 225-285 Krank MD, Hinson RE, Siegel S (1984) The effect of partial reinforcement on tolerance to morphine induced analgesia and weight loss in the rat. Behav Neurosci 98:79-85 LoLordo VM, Fairless JL (1985) Pavlovian conditioned inhibition: the literature since 1969. In: Miller RR, Spear NE (eds) Information processing in animals: conditioned inhibition. Erlbaum, Hillsdale, New Jersey, pp 1-50 Maude-Griffin PM, Tiffany ST (1989) Associative morphine tolerance in the rat: examination of compensatory responding and cross-tolerance with stress-induced analgesia. Behav Neur Biol 51 : 11-33 Rescorla RA (1985) Conditioned inhibition and facilitation. In: Miller RR, Spear NE (eds) Information processing in animals: conditioned inhibition. Erlbaum, Hillsdale, New Jersey, pp 299 - 326 Rescorla RA, Wagner AR (1972) A theory of Pavlovian conditioning: variations in the effectiveness of reinforcement and nonreinforcement. In: Black AH, Prokasy WF (eds) Classieal conditioning II: current research and theory. Appleton Century Crofts, New York, pp 64-99 Siegel S (1975) Evidence from rats that tolerance is a learned response. J Comp Physiol Psychol 89:323-325 Siegel S (1977) Morphine tolerance acquisition as an associative process. J Exp Psychol [Anim Behav] 3 : 1-13 Siegel S (1989) Pharmacological conditioning and drug effects. In: Goudie AJ, Emmett-Oglesby MW (eds) Psychoactive drugs: tolerance and sensitization. Humana, Clifton, New Jersey, pp t15-180 Tiffany ST, Baker TB (1981) Morphine tolerance in rats: congruence with a Pavlovian paradigm. J Comp Physiol Psychol 95 : 747-762 Tiffany ST, Drobes D, Cepeda-Benito A (1992) Contribution of associative and nonassociative processes to the development of morphine tolerance. Psychopharmacology 109:185-190 Tiffany ST, Maude-Griffin PM (I988) Tolerance to morphine in the rat: associative and nonassociative effects. Behav Neurosci 102 : 534-543 Tiffany ST, Petrie EC, Martin EM, Baker TB (1983) Drug signals enhance morphine tolerance development in hypophysectomized rats. Psychopharmacology 79:84-85 Wolgin DL (1989) The role of instrumental learning in behavioral tolerance to drugs. In: Goudie AJ, Emmett-Oglesby MW (eds) Psychoactive drugs: tolerance and sensitization. Humana, Clifton, New Jersey, pp 17-114 Yoburn BC, Morales R, Kelly DD, Inturrisi EC (1984) Constraints on the tail-flick assay: morphine analgesia and tolerance are dependent upon locus of tail stimulation. Life Sci 34:1755-1762
