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Effects of post-trial administration of naloxone and β-endorphin on shock-induced fighting in rats
BEHAVIORAL AND NEURAL BIOLOGY
39, 192-202 (1983)
Effects of Post-trial Administration of Naloxone and /3-Endorphin on Shock-Induced Fighting in Rats A. TAZl, R. DANTZER, P. MORMEDE, AND M. LE MOAL ) Psychobiologie des Comportements Adaptatifs INSERM-1NRA, U259, Rue Camille St-Saens, 33077 Bordeaux Cedex, France To study the involvement of endogenous opioid peptides in the development of shock-induced fighting, nalo×one (2 mg/kg) or /3-endorphin (10 ~g/kg) was administered subcutaneously immediately after the session and during nine consecutive daily sessions to rats repeatedly exposed to electric shocks./3-Endorphin blocked the development of shock-induced fighting while naloxone facilitated it but only when shock-induced fighting occurred at a low rate. The effects of/3endorphin were time dependent since when /3-endorphin was injected 90 min after the shock session instead of immediately after, its impairing effect disappeared. In addition, naloxone blocked the impairment produced by/3-endorphin. Differential postsession treatment of each member of pairs of rats with naloxone and /3endorphin resulted in a higher probability of rats treated with naloxone to be dominant over rats treated with/3-endorphin in the test situation. These results are discussed in relation with the possible involvement of endogenous opioids in the modulation of the physiological consequences of defensive behavioral responses to shock.
The administration of brief electric shocks to the feet of two rats in a testing box induces an upright posture with both animals facing each other ("boxing") striking with the forepaws, and occasionally biting (Ulrich & Azrin, 1962). This phenomenon, known as shock-induced fighting, has been used in studies of aggression in rodents since it can be produced and measured easily in the laboratory (Rodgers, 1981). A role for endogenous opioid peptides in the regulation of shockinduced fighting has been suggested on the basis that naloxone pretreatment increases the incidence of fighting (Fanselow et al., 1980; Gorelick et al., 1981). Such an increase is limited to the early portion of the session since with long duration sessions attenuated fighting may be observed during the late portion of the session (Fanselow & Sigmundi, 1982; McGivern et al., 1981). These findings are difficult to interpret since naloxone, if it is administered acutely before the session, may interfere with several important factors including, first, performance factors such /3-Endorphin was kindly donated by Dr. N. Ling and naloxone by Endo Labs.
0163-1047/83 $3.00 Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
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as sensitivity to electric shock and, second, the hierarchy of defensive responses to shock in the behavioral repertoire of the subjects. Alternatively, the amount of opioid peptides endogenously released in response to shock could influence the consequences of shock-induced fighting. It has been shown that animals shocked in pairs have less elevation of plasma ACTH than animals shocked individually (Conner et al., 1971; Weinberg et al., 1980). Shock-induced fighting would therefore be a coping response as far as it is functionally effective in reducing physiological activation and the endogenous opiate release could be a significant part of this physiological state. Endogenous opioids could also contribute to the stimulus complex of the experience accompanying exposure to electric shock. Alteration of this stimulus complex would modify the meaning of that experience and therefore its subsequent behavioral expression (Leshner et al., 1981). This latter hypothesis could be tested more directly by administering the treatment after rather than before the session, eliminating in this way any effects on performance (McGaugh and Gold, 1974). The present experiments were initiated to study whether postsession treatment with fi-endorphin and naloxone could alter the development of shock-induced fighting.
GENERAL PROCEDURE Male Sprague-Dawley albino rats obtained from IFFA-CREDO (Lyon) and weighing about 250 g were used as subjects. They were housed individually or in pairs, in plastic cages with sawdust litter. Food and tap water were available ad lib. Lighting conditions were 12-hr on/off with the light phase of the cycle beginning at 7:00 A.M. Experiments were run between 9:00 and 12:00 A.M. Shock-induced defensive fighting was assessed in a 30 × 30 × 40-cm opaque plastic chamber with a transparent cover to allow visual observation of the animals. The chamber was housed within a well-lit, sound-attenuated cabinet. Shock was delivered by a BRS-Foringer scrambled shocker programmed to administer every 45 sec, 10 trains of 10 electric shocks (2 mA, 0.5-sec on/l.0-sec off) to the stainless-steel grid floor of the chamber. The grid bars were 0.5-cm wide and spaced 1 cm apart. The animals were left undisturbed in their home cage for 12 days after arrival in the laboratory. After this, they were submitted to handling, individual exposure to the test chamber for 10 min, and subcutaneous injection with saline during three daily sessions. At the end of this habituation period, animals were assigned to weight-matched pairs for testing, with the weight difference between members of the same pair less than 10g. Test sessions lasted 10 min and were run daily for 10 consecutive days. During the test session, an observer sitting in the cabinet, classified the behavior of the animals during each train of shocks (a "trial") into one
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of three mutually exclusive categories (Stolk et al., 1974): no interaction (mainly escape attempts or freezing postures); upright position (mutual upright posture, without physical contact); fighting (mutual upright posture accompanied by striking with forepaws). The reliability of scoring was regularly checked by a second observer. Treatments were administered at the end of the first nine sessions. Naloxone hydrochloride ( a gift from Endo labs) was dissolved in isotonic saline, fl-Endorphin ( a gift from Dr. N. Ling, Salk Institute) was distributed in aliquots of 50 ~g which were stored at -70°C until use. Each aliquot was kept at -20°C on the day before use and was dissolved in saline on the day of use. Plastic tubes containing the solution were stored on ice during the course of the experiment and fresh solutions were prepared every day. All injections were given subcutaneously, in a volume of 0.2 ml/100 g. Treatments were arbitrarily coded so that the observer was naive as to the treatment received by each member of a pair. Data, expressed as number of episodes of the different behavioral categories per session, were submitted to a two-way analysis of variance with repeated measurements. Post hoc comparisons of group means were made using the Newman-Keul's test. EXPERIMENT I This experiment investigated the effects of post-trial treatment with naloxone or fi-endorphin on the development of shock-induced fighting. Methods
Animals and methods were as described in the general procedure section. Thirty rats housed individually were used. Treatments (naloxone 2 rag/ Kg, fi-endorphin 10 ~g/kg, or saline) were randomized between 15 weightmatched pairs of rats. Both members of a given pair received the same treatment which was injected immediately after the end of the test session for nine consecutive sessions. Results and Discussion
The changes over sessions of the different behavioral categories in each experimental group are shown in Fig. 1. The number of trials during which no interaction was observed decreased significantly from session to session (F(9, 108) = 3.70, p < .001) but this decrease was absent in the group treated with fi-endorphin (interaction term: F(18, 108) = 1.92, p < .05). Conversely, the incidence of fighting increased significantly with time (F(9, 108) = 11.7, p < .001) except in the fi-endorphin group (interaction term: F(18, 108) = 1.64, p = .05). The number of trials during which an upright position was scored (position score) decreased significantly with time (F(9, 108) = 5.09, p < .001) with no intergroup differences (F(18, 108) = .45).
NALOXONE AND/3-ENDORPHIN ON FIGHTING
NO
195
INTERACTION
100 c-----o Saline .L
¢ ¢ p. E N D O R P H I N .L NALOXONE
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FIGHTING 10(]
SESSION
10
FIG. 1. Effects of 2 mg/kg naloxone, 10/~g/kg/3-endorphin, or saline injected immediately after the session on behavioral categories observed in animals shocked in pairs. The curves
represent the changes over sessions of the different behavioral categories expressed as percentage of the shock trials. Each experimental group included five pairs of rats. These data indicate that the increase in the incidence of fighting episodes b e t w e e n pair-shocked rats which is observed from one session to the other o v e r the 10-day period of testing, is blocked by postsession administration of/3-endorphin and unaffected by postsession administration of naloxone. EXPERIMENT
II
The high incidence of fighting episodes observed in the first sessions o f exposure to electric shock and its rapid development over subsequent sessions could have precluded the observation of a facilitatory effect of naloxone on shock-induced fighting. Since we had fortuitously observed that animals tested early after their arrival in the laboratory displayed a lower incidence of fighting episodes when shocked in pairs, the effects of naloxone were reinvestigated under these conditions.
Methods Animals and methods were as described under General Procedure. Thirty-two rats housed individually were used. Treatments (naloxone 2
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mg/kg, or saline) were randomized between 16 weight-matched pairs. Both members of a given pair received the same treatment which was injected after the end of the test session, for nine consecutive sessions. The experiment started 5 days after arrival of animals in the laboratory. Results and Discussion The changes over sessions of the different behavioral categories scored in each experimental group are shown in Fig. 2. The number of trials during which no interaction was observed decreased significantly from session to session (F(9, 126) = 11.85, p < .001) with no difference between the groups over time (F(9, 126) = 1.68). Position scores decreased significantly with time (F(9, 126) = 10.43, p < .001) and this decrease was more marked in pairs of rats treated with naioxone (F(9, 126) = 5.30, p < .001). Fighting incidence increased significantly from session to session (F(9, 126) = 49.67, p < .001) and this increase was significantly more marked in naloxone-treated rats than in saline-treated rats (F(9, 126) = 4.36, p < .001). These data indicate that postsession administration of naloxone can
100
NO
INTERACTION
Saline
~,
A NALOXONE
0
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100
50
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o
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10 Fro. 2. Effects of 2 mg/kg naloxone or saline injected immediately after the first nine s e s s i o n s on behavioral categories observed in animals shocked in pairs. Each experimental group included eight pairs of rats. 5
SESSION
NALOXONE AND/3-ENDORPHIN ON FIGHTING
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facilitate development of shock-induced fighting when its rate of development is low. The generality of this finding needs to be tested under other conditions leading to low incidence of shock-induced fighting, e.g., low intensity of electric shock. The present results, coupled with the observation that/3-endorphin-treated rats fight less, suggest that endogenous opioids modulate the expression of shock-induced fighting in a manner similar to that reported for active or passive avoidance behavior (Izquierdo et al., 1981; Martinez & Rigter, 1982; Messing et al., 1979). E X P E R I M E N T III The post-trial effectiveness of a treatment on the development of behavioral performance suggests an effect on memory consolidation. Such a possibility may be further strengthened by the demonstration that the post-trial effects of the treatment are time dependent, in the sense that they decrease as the time between the end of session and injection is increased (McGaugh, 1966, 1973). The present experiment was initiated to study whether impairment of shock-induced fighting by post-trial administration of/3-endorphin was time dependent and also whether this effect could be antagonized by naloxone. Methods
Animals and methods were as described under General Procedure. Forty rats housed two per cage were used. Four different treatments were studied: 10 /~g/kg /3-endorphin immediately postsession; 2 mg/kg naloxone followed by 10/~g/kg/3-endorphin immediately postsession; 10 /xg/kg/3-endorphin 90 rain postsession: and saline immediately postsession. They were randomized between 20 weight-matched pairs made up of 2 animals from different cages, the animals from a same cage being allocated to the same treatment. Results and Discussion
The changes over trials of the different behavioral categories in each experimental group are shown in Fig. 3. The number of trials during which no interaction was observed decreased significantly from session to session (F(9, 144) = 8.58, p < .001) and this decrease was not affected by treatment. The incidence of fighting increased significantly with time (F(9, 144) = 30.9, p < .001) except in the pairs of rats treated immediately after the session with/3-endorphin (interaction term: F(27, 144) = 1.99, p < .001) and the overall incidence of fighting was significantly lower in this group than in other groups (F(3, 16) = 4.41, p < .05). Position scores decreased significantly with time (F(9, 144) = 5.16, p < .001) with no intergroup differences (interaction term: F(27, 144) = 0.85). These findings replicate the impairment produced by immediate posttrial administration of/3-endorphin on the development of shock-induced
198
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NO
100
INTERACTION
o-----.o S a l i n e = = ~.E (90)
-= ='
~. J$.E (0) ~ NX + ~.E
50
0 BOXING
100
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FIGHTING
10'
;
SESS,ON
,'o
Fro. 3. Time-dependent effects of/3-endorphin on shock-induced fighting and antagonism by naloxone. The curves represent the different behavioral categories observed in four experimental groups of five pairs of rats. The treatments studied were: 10/zg/kg ~-endorphin injected immediately after the first nine sessions [/3-E(0)] or 90 rain later [/3E(90)]; 2 mg/ kg naloxone followed by 10/zg/kg/3-endorphin and given immediately after the sessions (NX + B-E); and saline injected immediately after the sessions.
fighting. This effect is dependent on the interval between training and drug treatment since injection 90 min after the end of the session has no effect. In addition, the effect is blocked by an opiate antagonist, naloxone. Such results are similar to performance impairment effects of post-training injection of/3-endorphin in rats trained on active or passive avoidance learning tasks (Izquierdo et al., 1981 ; Martinez & Rigter, 1980). EXPERIMENT IV
Repeated exposure of pairs of rats to shock results in the gradual emergence of a dominance order between the two members of the pair (Reynierse, 1971). Endogenous opioids may modulate not only the development of shock-induced fighting but also the development of a dominant or submissive posture during fighting episodes. In a preliminary experiment in which pharmacological treatment was administered immediately after each session, starting from the fifth session of shock, we observed that
NALOXONE AND/3-ENDORPHIN ON FIGHTING
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simultaneous treatment of the dominant with naloxone and treatment of the subordinate with fl-endorphin did not change the rank order while the reverse combination tended to induce a dominance reversal. In the present experiment, therefore, we investigated whether a differential treatment of each member of a pair with naloxone and fl-endorphin could influence the development of the dominance order in such a way as to increase the probability that the rat treated with naloxone would be dominant over the rat treated with fl-endorphin. Methods"
Animals and methods were as described under General Procedure. Forty male rats housed individually were allocated to twenty weightmatched pairs. Naloxone (2 mg/kg) was administered immediately after the termination of the shock session to one member of each pair, in a randomized way, the other animal receiving fl-endorphin (10/xg/kg). Each treatment was administered immediately after the end of the session, starting from the first session of shock and during nine consecutive sessions. The 10th session was not followed by pharmacological treatment. Results and Discussion
Fighting did not develop in three pairs. In the remaining pairs, a dominance order could be observed usually within three to four sessions, the subordinate rat displaying a submissive-supine posture with the dominant rat arched over it. This order remained stable throughout, except in two pairs in which ambivalent postures for both animals were apparent in the early sessions. On the 10th session, naloxone-treated rats were dominant over/3-endorphin treated rats in 13 pairs out of 17 (Fisher's exact test, p = .086). The present findings suggest that the development and maintenance of the dominance order within pairs of rats exposed to shock is modulated by opiate-dependent mechanisms. The effects of post-training hormonal treatments on social behavior have been studied only on submissiveness and avoidance of attack by mice exposed to trained fighters and in the context of the pituitary-adrenal axis hormones (Leshner et al., 1981) so the generality of the present results is difficult to evaluate. In addition, no attempt has been made here to separate the effects of fl-endorphin on one rat from those of naloxone on the other, and the investigation of a possible effect of either treatment per se on the development of dominance still awaits further study. GENERAL DISCUSSION The present experiments have been initiated to determine whether postsession treatment with/3-endorphin or naloxone could alter the development of shock-induced fighting in rats repeatedly exposed to electric
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shock. Experiment I showed that/3-endorphin blocked the development of shock-induced fighting, but that naloxone had no effect. In Experiment 2, naloxone was found to increase shock-induced fighting when its rate of occurrence was low. Experiment 3 showed that the effects of /3endorphin were time dependent and blocked by naloxone. Finally, in Experiment 4, postsession administration of/3-endorphin to one member of each pair of rats and postsession administration of naloxone to the other animal resulted in a higher incidence of submissiveness in/3-endorphin-treated rats than in naloxone-treated rats. These findings point to a modulatory role of opioids in the development of shock-induced fighting and the dominance order resulting from fighting episodes and suggest that normal physiological consequences of the behavioral response to shock are important in modulating the storage and later retention of the specific information provided by the experience. The observation that shock-induced fighting is sensitive to interference by postsession treatments in a time-dependent manner is in contrast with the commonly held view that this behavior is reflexive and involves little learning. The reference to "pain-induced reflexive fighting" (Stolk et al., 1974; Ulrich and Azrin, 1962; Payne et al., 1970) illustrates this point well. Under optimal conditions (i.e., a limited amount of space and scrambled shock intensity over 2 mA), fighting often occurs at more than 90% of the shock presentations (Ulrich and Azrin, 1962). Under less drastic conditions, however, response frequency typically increases with successive exposures to shock (e.g., Payne et al., 1970) and there is a gradual qualitative change in the nature of the aggressive posture exhibited by the animals, with the emergence of a dominance order in paired rats (Reynierse, 1971). These findings suggest that the behavior of animals shocked in pairs is not stereotyped as initially believed but somewhat flexible and adaptive. The results of the present experiments provide more direct evidence in favor of a learning component in both the development of shock-induced fighting and the apparition of a dominant or submissive posture in fighting episodes. The mechanisms behind such effects are still largely unknown. The simplest explanation would be that/3-endorphin affects the performance in a nonspecific way, e.g., it decreases sensitivity to electric shock, alters emotional reactivity, or has general debilitating effects. However, such an explanation is not likely, in view of the time-dependent effects of the peptide. Based on an extensive series of studies on the effects of endogenous opioids and naloxone on shuttle avoidance and habituation of a rearing response to a tone, Izquierdo and colleagues (1981) suggested that endogenous opioids are part of an amnesic mechansim which enables the animal to forget adventitious responding that occurs during any form of training and interferes with it. According to this interpretation, low doses of/3-endorphin should facilitate learning but high doses should impair
NALOXONE AND/3-ENDORPHIN ON FIGHTING
201
performance, since, with high doses, the endogenous amnesic mechanism should spread to the main behavioral response required by the task. Although our results would be consistent with this interpretation, in the absence of a study of dose-response relationships and the absence of the measurement of endogenously released opioids, such an hypothesis is difficult to support in the present case. Rather than acting on memory storage,/3-endorphin or naloxone could serve as a stimulus producing what has been called "state-dependent retention" (cf. Riccio and Concannon, 1981). The treatment would alter directly or indirectly the physiological state of the animal between memory processing and retention testing, and cause the state prevalent after a training session to be different from the state prevailing during the next session. However, this interpretation cannot readily account for the enhancement of performance produced by naloxone. A related possibility which does not assume any direct interference with learning or memory processes, would be that the physiological response to the experience of shock contributes to some part of the stimulus complex of the experience. In cases where this stimulus complex is made more salient or, inversely, if certain cues are minimized or altered, then the meaning of that experience is correlatively altered, more meaningful experiences being remembered better than less meaningful experiences (Lesbner et al., 1981). The stimulatory effect of naloxone when administered alone to rats shocked in pairs, suggests that endogenous opioids could normally contribute to the stimulus complex of the experience. Such an effect could be centrally or peripherally mediated. In the later case, the chronic administration of/3-endorphin and naloxone might interfere with the normal neuroendocrine consequences of shock-induced fighting (Conner et al., 1971; Weiss et al., 1976). This hypothesis is currently under investigation. REFERENCES Conner, R. L., Vernikos-Danellis, J., & Levine, S. (1971). Stress, fighting and neuroendocrine function. Nature (London), 234, 564-566. Fanselow, M. S., & Sigmundi, R. A. (1982). The enhancement and reduction of defensive fighting by naloxone pretreatment. Physiological Psychology, 10, 313-316. Fanselow, M. S., Sigmundi, R. A., & Bolles, R. C. (1980). Naloxone pretreatment enhances shock elicited aggression. Physiological Psychology, 8, 369-371. Gold, P. E., & Delanoy, R. L. (1981). ACTH modulation of memory storage processing. In J. L. Martinez, Jr., R. A. Jensen, R. B. Messing, H. Rigter, & J. L. McGaugh (Eds.), Endogenous Peptides and Learning and Memory Processes, pp. 79-97. New York: Academic Press. Gorelick, D. A., Elliot, M. L., & Sbordone, R. J. (1981). Naloxone increases shock-elicited aggression in rats. Research Communications in Substance Abuse, 2, 419-422. Izquierdo, I., Perry, M. L., Dias, R. D., Souza, D. O., Elisabetsky, E., Carrasco, M. A., Orsingher, O. O., & Netto, C. A. (1981). Endogenous opioids, memory modulation and state dependency. In J. L. Martinez, Jr., R. A. Jensen, R. B. Messing, H. Rigter,
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& J. L. McGaugh (Eds.), Endogenous Peptides and Learning and Memory Processes, pp. 269-290. New York: Academic Press. Leshner, A. I., Merkle, D. A., & Mixon, J. F. (1981). Pituitary-adrenocortical effects on learning and memory in social situations. In J. L. Martinez, Jr., R. A. Jensen, R. B. Messing, H. Rigter, & J. L. McGaugh (Eds.), Endogenous Peptides and Learning and Memory Processes, pp. 159-180. New York: Academic Press. McGaugh, J. L. (1966). Time-dependent processes in memory storage. Science, 153, 13511358. McGaugh, J. L. (1973). Drug facilitation of learning and memory. Annual Review of Pharmacology, 13, 229-241. McGaugh, J. L., & Gold, P. E. (1974). Conceptual and neurobiological issues in studies of treatments affecting memory storage. In G. H. Bower (Ed.), The Psychology of Learning and Motivation, pp. 233-262. New York: Academic Press. McGivern, R. F., Lobaugh, N. J., & Collier, A. (1981). Effect of naloxone and housing conditions on shock-elicited reflexive fighting: Influence of immediate prior stress. Physiological Psychology, 9, 251-256. Martinez J. L., Jr., & Rigter, H. (1980) Endorphins alter acquisition and consolidation of an inhibitory avoidance response in rats. Neuroscience Letters, 18, 197-201. Martinez, J. L., & Rigter, H. (1982). Enkephalin actions on avoidance conditioning may be related to adrenal medullary function. Behav. Brain Res., 6, 289-299. Messing, R. B., Jensen, R. A., Martinez, J. L., Spiehler, V. R., Vasquez, B. J., SoumireuMourat, B., Liang, K. C., & McGaugh, J. L. (1979). Naloxone enhancement of memory. Behavioral and Neural Biology, 27, 266-275. Payne, R., Anderson, D. C., & Murcurio, J. (1970). Preshock-produced alterations in painelicited fighting. Journal of Comparative and Physiological Psychology, 71, 258-266. Reynierse, J. H. (1971). Submissive postures during shock-elicited aggression. Animal Behaviour, 19, 102-107. Riccio, D. C., & Concannon, J. T. (1981). ACTH and the reminder phenomena. In J. L. Martinez, Jr., R. A. Jensen, R. B. Messing, H. Rigter, & J. L. McGaugh (Eds.), Endogenous Peptides and Learning and Memory Processes, pp. 117-142. New York: Academic Press. Rodgers, R. J., (1981). Pain and aggression. In P. F. Brain and D. Benton (Eds.), The Biology of Aggression, pp. 519-527. Alphen: Sijthoff and Noordhoff. Stolk, J. M., Conner, R. L., Levine, S., & Barchas, J. D. (1974). Brain norepinephrine metabolism and shock-induced fighting behavior in rats: Differential effects of shock and fighting on the neurochemical response to a common footshock stimulus. Journal of Pharmacology and Experimental Therapy, 190, 193-209. Ulrich, R. E., & Azrin, N. H. (1962). Reflexive fighting in response to aversive stimulation. Journal ~f Experimental and Analytical Behavior, 5, 511-520. Weinberg, J., Erskine, M., & Levine, S. (1980). Shock-induced fighting attenuates the effects of prior shock experience in rats. Physiology and Behavior, 25, 9-16. Weiss, J. M., Pohoreeky, L. A., Salman, S., & Gruenthal, M. (1976). Attenuation of gastric lesions by psychological aspects of aggression in rats. Journal of Comparative and Physiological Psychology, 90, 252-259.
39, 192-202 (1983)
Effects of Post-trial Administration of Naloxone and /3-Endorphin on Shock-Induced Fighting in Rats A. TAZl, R. DANTZER, P. MORMEDE, AND M. LE MOAL ) Psychobiologie des Comportements Adaptatifs INSERM-1NRA, U259, Rue Camille St-Saens, 33077 Bordeaux Cedex, France To study the involvement of endogenous opioid peptides in the development of shock-induced fighting, nalo×one (2 mg/kg) or /3-endorphin (10 ~g/kg) was administered subcutaneously immediately after the session and during nine consecutive daily sessions to rats repeatedly exposed to electric shocks./3-Endorphin blocked the development of shock-induced fighting while naloxone facilitated it but only when shock-induced fighting occurred at a low rate. The effects of/3endorphin were time dependent since when /3-endorphin was injected 90 min after the shock session instead of immediately after, its impairing effect disappeared. In addition, naloxone blocked the impairment produced by/3-endorphin. Differential postsession treatment of each member of pairs of rats with naloxone and /3endorphin resulted in a higher probability of rats treated with naloxone to be dominant over rats treated with/3-endorphin in the test situation. These results are discussed in relation with the possible involvement of endogenous opioids in the modulation of the physiological consequences of defensive behavioral responses to shock.
The administration of brief electric shocks to the feet of two rats in a testing box induces an upright posture with both animals facing each other ("boxing") striking with the forepaws, and occasionally biting (Ulrich & Azrin, 1962). This phenomenon, known as shock-induced fighting, has been used in studies of aggression in rodents since it can be produced and measured easily in the laboratory (Rodgers, 1981). A role for endogenous opioid peptides in the regulation of shockinduced fighting has been suggested on the basis that naloxone pretreatment increases the incidence of fighting (Fanselow et al., 1980; Gorelick et al., 1981). Such an increase is limited to the early portion of the session since with long duration sessions attenuated fighting may be observed during the late portion of the session (Fanselow & Sigmundi, 1982; McGivern et al., 1981). These findings are difficult to interpret since naloxone, if it is administered acutely before the session, may interfere with several important factors including, first, performance factors such /3-Endorphin was kindly donated by Dr. N. Ling and naloxone by Endo Labs.
0163-1047/83 $3.00 Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
192
NALOXONE AND fl-ENDORPHIN ON FIGHTING
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as sensitivity to electric shock and, second, the hierarchy of defensive responses to shock in the behavioral repertoire of the subjects. Alternatively, the amount of opioid peptides endogenously released in response to shock could influence the consequences of shock-induced fighting. It has been shown that animals shocked in pairs have less elevation of plasma ACTH than animals shocked individually (Conner et al., 1971; Weinberg et al., 1980). Shock-induced fighting would therefore be a coping response as far as it is functionally effective in reducing physiological activation and the endogenous opiate release could be a significant part of this physiological state. Endogenous opioids could also contribute to the stimulus complex of the experience accompanying exposure to electric shock. Alteration of this stimulus complex would modify the meaning of that experience and therefore its subsequent behavioral expression (Leshner et al., 1981). This latter hypothesis could be tested more directly by administering the treatment after rather than before the session, eliminating in this way any effects on performance (McGaugh and Gold, 1974). The present experiments were initiated to study whether postsession treatment with fi-endorphin and naloxone could alter the development of shock-induced fighting.
GENERAL PROCEDURE Male Sprague-Dawley albino rats obtained from IFFA-CREDO (Lyon) and weighing about 250 g were used as subjects. They were housed individually or in pairs, in plastic cages with sawdust litter. Food and tap water were available ad lib. Lighting conditions were 12-hr on/off with the light phase of the cycle beginning at 7:00 A.M. Experiments were run between 9:00 and 12:00 A.M. Shock-induced defensive fighting was assessed in a 30 × 30 × 40-cm opaque plastic chamber with a transparent cover to allow visual observation of the animals. The chamber was housed within a well-lit, sound-attenuated cabinet. Shock was delivered by a BRS-Foringer scrambled shocker programmed to administer every 45 sec, 10 trains of 10 electric shocks (2 mA, 0.5-sec on/l.0-sec off) to the stainless-steel grid floor of the chamber. The grid bars were 0.5-cm wide and spaced 1 cm apart. The animals were left undisturbed in their home cage for 12 days after arrival in the laboratory. After this, they were submitted to handling, individual exposure to the test chamber for 10 min, and subcutaneous injection with saline during three daily sessions. At the end of this habituation period, animals were assigned to weight-matched pairs for testing, with the weight difference between members of the same pair less than 10g. Test sessions lasted 10 min and were run daily for 10 consecutive days. During the test session, an observer sitting in the cabinet, classified the behavior of the animals during each train of shocks (a "trial") into one
194
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of three mutually exclusive categories (Stolk et al., 1974): no interaction (mainly escape attempts or freezing postures); upright position (mutual upright posture, without physical contact); fighting (mutual upright posture accompanied by striking with forepaws). The reliability of scoring was regularly checked by a second observer. Treatments were administered at the end of the first nine sessions. Naloxone hydrochloride ( a gift from Endo labs) was dissolved in isotonic saline, fl-Endorphin ( a gift from Dr. N. Ling, Salk Institute) was distributed in aliquots of 50 ~g which were stored at -70°C until use. Each aliquot was kept at -20°C on the day before use and was dissolved in saline on the day of use. Plastic tubes containing the solution were stored on ice during the course of the experiment and fresh solutions were prepared every day. All injections were given subcutaneously, in a volume of 0.2 ml/100 g. Treatments were arbitrarily coded so that the observer was naive as to the treatment received by each member of a pair. Data, expressed as number of episodes of the different behavioral categories per session, were submitted to a two-way analysis of variance with repeated measurements. Post hoc comparisons of group means were made using the Newman-Keul's test. EXPERIMENT I This experiment investigated the effects of post-trial treatment with naloxone or fi-endorphin on the development of shock-induced fighting. Methods
Animals and methods were as described in the general procedure section. Thirty rats housed individually were used. Treatments (naloxone 2 rag/ Kg, fi-endorphin 10 ~g/kg, or saline) were randomized between 15 weightmatched pairs of rats. Both members of a given pair received the same treatment which was injected immediately after the end of the test session for nine consecutive sessions. Results and Discussion
The changes over sessions of the different behavioral categories in each experimental group are shown in Fig. 1. The number of trials during which no interaction was observed decreased significantly from session to session (F(9, 108) = 3.70, p < .001) but this decrease was absent in the group treated with fi-endorphin (interaction term: F(18, 108) = 1.92, p < .05). Conversely, the incidence of fighting increased significantly with time (F(9, 108) = 11.7, p < .001) except in the fi-endorphin group (interaction term: F(18, 108) = 1.64, p = .05). The number of trials during which an upright position was scored (position score) decreased significantly with time (F(9, 108) = 5.09, p < .001) with no intergroup differences (F(18, 108) = .45).
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INTERACTION
100 c-----o Saline .L
¢ ¢ p. E N D O R P H I N .L NALOXONE
50
,.._, 0 BOXING '--'100
0 Lu 5t
FIGHTING 10(]
SESSION
10
FIG. 1. Effects of 2 mg/kg naloxone, 10/~g/kg/3-endorphin, or saline injected immediately after the session on behavioral categories observed in animals shocked in pairs. The curves
represent the changes over sessions of the different behavioral categories expressed as percentage of the shock trials. Each experimental group included five pairs of rats. These data indicate that the increase in the incidence of fighting episodes b e t w e e n pair-shocked rats which is observed from one session to the other o v e r the 10-day period of testing, is blocked by postsession administration of/3-endorphin and unaffected by postsession administration of naloxone. EXPERIMENT
II
The high incidence of fighting episodes observed in the first sessions o f exposure to electric shock and its rapid development over subsequent sessions could have precluded the observation of a facilitatory effect of naloxone on shock-induced fighting. Since we had fortuitously observed that animals tested early after their arrival in the laboratory displayed a lower incidence of fighting episodes when shocked in pairs, the effects of naloxone were reinvestigated under these conditions.
Methods Animals and methods were as described under General Procedure. Thirty-two rats housed individually were used. Treatments (naloxone 2
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mg/kg, or saline) were randomized between 16 weight-matched pairs. Both members of a given pair received the same treatment which was injected after the end of the test session, for nine consecutive sessions. The experiment started 5 days after arrival of animals in the laboratory. Results and Discussion The changes over sessions of the different behavioral categories scored in each experimental group are shown in Fig. 2. The number of trials during which no interaction was observed decreased significantly from session to session (F(9, 126) = 11.85, p < .001) with no difference between the groups over time (F(9, 126) = 1.68). Position scores decreased significantly with time (F(9, 126) = 10.43, p < .001) and this decrease was more marked in pairs of rats treated with naioxone (F(9, 126) = 5.30, p < .001). Fighting incidence increased significantly from session to session (F(9, 126) = 49.67, p < .001) and this increase was significantly more marked in naloxone-treated rats than in saline-treated rats (F(9, 126) = 4.36, p < .001). These data indicate that postsession administration of naloxone can
100
NO
INTERACTION
Saline
~,
A NALOXONE
0
BOXING uJ (3 0 I a. w
100
50
0
o
FIGHTING
100
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|
10 Fro. 2. Effects of 2 mg/kg naloxone or saline injected immediately after the first nine s e s s i o n s on behavioral categories observed in animals shocked in pairs. Each experimental group included eight pairs of rats. 5
SESSION
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facilitate development of shock-induced fighting when its rate of development is low. The generality of this finding needs to be tested under other conditions leading to low incidence of shock-induced fighting, e.g., low intensity of electric shock. The present results, coupled with the observation that/3-endorphin-treated rats fight less, suggest that endogenous opioids modulate the expression of shock-induced fighting in a manner similar to that reported for active or passive avoidance behavior (Izquierdo et al., 1981; Martinez & Rigter, 1982; Messing et al., 1979). E X P E R I M E N T III The post-trial effectiveness of a treatment on the development of behavioral performance suggests an effect on memory consolidation. Such a possibility may be further strengthened by the demonstration that the post-trial effects of the treatment are time dependent, in the sense that they decrease as the time between the end of session and injection is increased (McGaugh, 1966, 1973). The present experiment was initiated to study whether impairment of shock-induced fighting by post-trial administration of/3-endorphin was time dependent and also whether this effect could be antagonized by naloxone. Methods
Animals and methods were as described under General Procedure. Forty rats housed two per cage were used. Four different treatments were studied: 10 /~g/kg /3-endorphin immediately postsession; 2 mg/kg naloxone followed by 10/~g/kg/3-endorphin immediately postsession; 10 /xg/kg/3-endorphin 90 rain postsession: and saline immediately postsession. They were randomized between 20 weight-matched pairs made up of 2 animals from different cages, the animals from a same cage being allocated to the same treatment. Results and Discussion
The changes over trials of the different behavioral categories in each experimental group are shown in Fig. 3. The number of trials during which no interaction was observed decreased significantly from session to session (F(9, 144) = 8.58, p < .001) and this decrease was not affected by treatment. The incidence of fighting increased significantly with time (F(9, 144) = 30.9, p < .001) except in the pairs of rats treated immediately after the session with/3-endorphin (interaction term: F(27, 144) = 1.99, p < .001) and the overall incidence of fighting was significantly lower in this group than in other groups (F(3, 16) = 4.41, p < .05). Position scores decreased significantly with time (F(9, 144) = 5.16, p < .001) with no intergroup differences (interaction term: F(27, 144) = 0.85). These findings replicate the impairment produced by immediate posttrial administration of/3-endorphin on the development of shock-induced
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NO
100
INTERACTION
o-----.o S a l i n e = = ~.E (90)
-= ='
~. J$.E (0) ~ NX + ~.E
50
0 BOXING
100
! O 50 m a.
FIGHTING
10'
;
SESS,ON
,'o
Fro. 3. Time-dependent effects of/3-endorphin on shock-induced fighting and antagonism by naloxone. The curves represent the different behavioral categories observed in four experimental groups of five pairs of rats. The treatments studied were: 10/zg/kg ~-endorphin injected immediately after the first nine sessions [/3-E(0)] or 90 rain later [/3E(90)]; 2 mg/ kg naloxone followed by 10/zg/kg/3-endorphin and given immediately after the sessions (NX + B-E); and saline injected immediately after the sessions.
fighting. This effect is dependent on the interval between training and drug treatment since injection 90 min after the end of the session has no effect. In addition, the effect is blocked by an opiate antagonist, naloxone. Such results are similar to performance impairment effects of post-training injection of/3-endorphin in rats trained on active or passive avoidance learning tasks (Izquierdo et al., 1981 ; Martinez & Rigter, 1980). EXPERIMENT IV
Repeated exposure of pairs of rats to shock results in the gradual emergence of a dominance order between the two members of the pair (Reynierse, 1971). Endogenous opioids may modulate not only the development of shock-induced fighting but also the development of a dominant or submissive posture during fighting episodes. In a preliminary experiment in which pharmacological treatment was administered immediately after each session, starting from the fifth session of shock, we observed that
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simultaneous treatment of the dominant with naloxone and treatment of the subordinate with fl-endorphin did not change the rank order while the reverse combination tended to induce a dominance reversal. In the present experiment, therefore, we investigated whether a differential treatment of each member of a pair with naloxone and fl-endorphin could influence the development of the dominance order in such a way as to increase the probability that the rat treated with naloxone would be dominant over the rat treated with fl-endorphin. Methods"
Animals and methods were as described under General Procedure. Forty male rats housed individually were allocated to twenty weightmatched pairs. Naloxone (2 mg/kg) was administered immediately after the termination of the shock session to one member of each pair, in a randomized way, the other animal receiving fl-endorphin (10/xg/kg). Each treatment was administered immediately after the end of the session, starting from the first session of shock and during nine consecutive sessions. The 10th session was not followed by pharmacological treatment. Results and Discussion
Fighting did not develop in three pairs. In the remaining pairs, a dominance order could be observed usually within three to four sessions, the subordinate rat displaying a submissive-supine posture with the dominant rat arched over it. This order remained stable throughout, except in two pairs in which ambivalent postures for both animals were apparent in the early sessions. On the 10th session, naloxone-treated rats were dominant over/3-endorphin treated rats in 13 pairs out of 17 (Fisher's exact test, p = .086). The present findings suggest that the development and maintenance of the dominance order within pairs of rats exposed to shock is modulated by opiate-dependent mechanisms. The effects of post-training hormonal treatments on social behavior have been studied only on submissiveness and avoidance of attack by mice exposed to trained fighters and in the context of the pituitary-adrenal axis hormones (Leshner et al., 1981) so the generality of the present results is difficult to evaluate. In addition, no attempt has been made here to separate the effects of fl-endorphin on one rat from those of naloxone on the other, and the investigation of a possible effect of either treatment per se on the development of dominance still awaits further study. GENERAL DISCUSSION The present experiments have been initiated to determine whether postsession treatment with/3-endorphin or naloxone could alter the development of shock-induced fighting in rats repeatedly exposed to electric
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shock. Experiment I showed that/3-endorphin blocked the development of shock-induced fighting, but that naloxone had no effect. In Experiment 2, naloxone was found to increase shock-induced fighting when its rate of occurrence was low. Experiment 3 showed that the effects of /3endorphin were time dependent and blocked by naloxone. Finally, in Experiment 4, postsession administration of/3-endorphin to one member of each pair of rats and postsession administration of naloxone to the other animal resulted in a higher incidence of submissiveness in/3-endorphin-treated rats than in naloxone-treated rats. These findings point to a modulatory role of opioids in the development of shock-induced fighting and the dominance order resulting from fighting episodes and suggest that normal physiological consequences of the behavioral response to shock are important in modulating the storage and later retention of the specific information provided by the experience. The observation that shock-induced fighting is sensitive to interference by postsession treatments in a time-dependent manner is in contrast with the commonly held view that this behavior is reflexive and involves little learning. The reference to "pain-induced reflexive fighting" (Stolk et al., 1974; Ulrich and Azrin, 1962; Payne et al., 1970) illustrates this point well. Under optimal conditions (i.e., a limited amount of space and scrambled shock intensity over 2 mA), fighting often occurs at more than 90% of the shock presentations (Ulrich and Azrin, 1962). Under less drastic conditions, however, response frequency typically increases with successive exposures to shock (e.g., Payne et al., 1970) and there is a gradual qualitative change in the nature of the aggressive posture exhibited by the animals, with the emergence of a dominance order in paired rats (Reynierse, 1971). These findings suggest that the behavior of animals shocked in pairs is not stereotyped as initially believed but somewhat flexible and adaptive. The results of the present experiments provide more direct evidence in favor of a learning component in both the development of shock-induced fighting and the apparition of a dominant or submissive posture in fighting episodes. The mechanisms behind such effects are still largely unknown. The simplest explanation would be that/3-endorphin affects the performance in a nonspecific way, e.g., it decreases sensitivity to electric shock, alters emotional reactivity, or has general debilitating effects. However, such an explanation is not likely, in view of the time-dependent effects of the peptide. Based on an extensive series of studies on the effects of endogenous opioids and naloxone on shuttle avoidance and habituation of a rearing response to a tone, Izquierdo and colleagues (1981) suggested that endogenous opioids are part of an amnesic mechansim which enables the animal to forget adventitious responding that occurs during any form of training and interferes with it. According to this interpretation, low doses of/3-endorphin should facilitate learning but high doses should impair
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performance, since, with high doses, the endogenous amnesic mechanism should spread to the main behavioral response required by the task. Although our results would be consistent with this interpretation, in the absence of a study of dose-response relationships and the absence of the measurement of endogenously released opioids, such an hypothesis is difficult to support in the present case. Rather than acting on memory storage,/3-endorphin or naloxone could serve as a stimulus producing what has been called "state-dependent retention" (cf. Riccio and Concannon, 1981). The treatment would alter directly or indirectly the physiological state of the animal between memory processing and retention testing, and cause the state prevalent after a training session to be different from the state prevailing during the next session. However, this interpretation cannot readily account for the enhancement of performance produced by naloxone. A related possibility which does not assume any direct interference with learning or memory processes, would be that the physiological response to the experience of shock contributes to some part of the stimulus complex of the experience. In cases where this stimulus complex is made more salient or, inversely, if certain cues are minimized or altered, then the meaning of that experience is correlatively altered, more meaningful experiences being remembered better than less meaningful experiences (Lesbner et al., 1981). The stimulatory effect of naloxone when administered alone to rats shocked in pairs, suggests that endogenous opioids could normally contribute to the stimulus complex of the experience. Such an effect could be centrally or peripherally mediated. In the later case, the chronic administration of/3-endorphin and naloxone might interfere with the normal neuroendocrine consequences of shock-induced fighting (Conner et al., 1971; Weiss et al., 1976). This hypothesis is currently under investigation. REFERENCES Conner, R. L., Vernikos-Danellis, J., & Levine, S. (1971). Stress, fighting and neuroendocrine function. Nature (London), 234, 564-566. Fanselow, M. S., & Sigmundi, R. A. (1982). The enhancement and reduction of defensive fighting by naloxone pretreatment. Physiological Psychology, 10, 313-316. Fanselow, M. S., Sigmundi, R. A., & Bolles, R. C. (1980). Naloxone pretreatment enhances shock elicited aggression. Physiological Psychology, 8, 369-371. Gold, P. E., & Delanoy, R. L. (1981). ACTH modulation of memory storage processing. In J. L. Martinez, Jr., R. A. Jensen, R. B. Messing, H. Rigter, & J. L. McGaugh (Eds.), Endogenous Peptides and Learning and Memory Processes, pp. 79-97. New York: Academic Press. Gorelick, D. A., Elliot, M. L., & Sbordone, R. J. (1981). Naloxone increases shock-elicited aggression in rats. Research Communications in Substance Abuse, 2, 419-422. Izquierdo, I., Perry, M. L., Dias, R. D., Souza, D. O., Elisabetsky, E., Carrasco, M. A., Orsingher, O. O., & Netto, C. A. (1981). Endogenous opioids, memory modulation and state dependency. In J. L. Martinez, Jr., R. A. Jensen, R. B. Messing, H. Rigter,
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& J. L. McGaugh (Eds.), Endogenous Peptides and Learning and Memory Processes, pp. 269-290. New York: Academic Press. Leshner, A. I., Merkle, D. A., & Mixon, J. F. (1981). Pituitary-adrenocortical effects on learning and memory in social situations. In J. L. Martinez, Jr., R. A. Jensen, R. B. Messing, H. Rigter, & J. L. McGaugh (Eds.), Endogenous Peptides and Learning and Memory Processes, pp. 159-180. New York: Academic Press. McGaugh, J. L. (1966). Time-dependent processes in memory storage. Science, 153, 13511358. McGaugh, J. L. (1973). Drug facilitation of learning and memory. Annual Review of Pharmacology, 13, 229-241. McGaugh, J. L., & Gold, P. E. (1974). Conceptual and neurobiological issues in studies of treatments affecting memory storage. In G. H. Bower (Ed.), The Psychology of Learning and Motivation, pp. 233-262. New York: Academic Press. McGivern, R. F., Lobaugh, N. J., & Collier, A. (1981). Effect of naloxone and housing conditions on shock-elicited reflexive fighting: Influence of immediate prior stress. Physiological Psychology, 9, 251-256. Martinez J. L., Jr., & Rigter, H. (1980) Endorphins alter acquisition and consolidation of an inhibitory avoidance response in rats. Neuroscience Letters, 18, 197-201. Martinez, J. L., & Rigter, H. (1982). Enkephalin actions on avoidance conditioning may be related to adrenal medullary function. Behav. Brain Res., 6, 289-299. Messing, R. B., Jensen, R. A., Martinez, J. L., Spiehler, V. R., Vasquez, B. J., SoumireuMourat, B., Liang, K. C., & McGaugh, J. L. (1979). Naloxone enhancement of memory. Behavioral and Neural Biology, 27, 266-275. Payne, R., Anderson, D. C., & Murcurio, J. (1970). Preshock-produced alterations in painelicited fighting. Journal of Comparative and Physiological Psychology, 71, 258-266. Reynierse, J. H. (1971). Submissive postures during shock-elicited aggression. Animal Behaviour, 19, 102-107. Riccio, D. C., & Concannon, J. T. (1981). ACTH and the reminder phenomena. In J. L. Martinez, Jr., R. A. Jensen, R. B. Messing, H. Rigter, & J. L. McGaugh (Eds.), Endogenous Peptides and Learning and Memory Processes, pp. 117-142. New York: Academic Press. Rodgers, R. J., (1981). Pain and aggression. In P. F. Brain and D. Benton (Eds.), The Biology of Aggression, pp. 519-527. Alphen: Sijthoff and Noordhoff. Stolk, J. M., Conner, R. L., Levine, S., & Barchas, J. D. (1974). Brain norepinephrine metabolism and shock-induced fighting behavior in rats: Differential effects of shock and fighting on the neurochemical response to a common footshock stimulus. Journal of Pharmacology and Experimental Therapy, 190, 193-209. Ulrich, R. E., & Azrin, N. H. (1962). Reflexive fighting in response to aversive stimulation. Journal ~f Experimental and Analytical Behavior, 5, 511-520. Weinberg, J., Erskine, M., & Levine, S. (1980). Shock-induced fighting attenuates the effects of prior shock experience in rats. Physiology and Behavior, 25, 9-16. Weiss, J. M., Pohoreeky, L. A., Salman, S., & Gruenthal, M. (1976). Attenuation of gastric lesions by psychological aspects of aggression in rats. Journal of Comparative and Physiological Psychology, 90, 252-259.