Peptides, Vol. 13, pp. 885-889, 1992
0196-9781/92 $5.00 + .00 Copyright© 1992PergamonPressLtd.
Printed in the USA.
D-Pen2-[D-PenS]Enkephalin Impairs Acquisition and Enhances Retention of a One-Way Active Avoidance Response in Rats JOE L. M A R T I N E Z , JR., 1 R U B E N V. H E R N A N D E Z A N D S U S A N B. R O D R I G U E Z
University o f California, Department o f Psychology, Berkeley, CA 94720 Received 3 F e b r u a r y 1992 MARTINEZ, J. L., JR., R. V. HERNANDEZ AND S. B. RODRIGUEZ. D-Pen2-[D-PenS]Enkephalin impairs acquisition and enhances retention of a one-way active avoidance response in rats. PEPTIDES 13(5) 885-889, 1992.--We reported previously that D-Pen2-[D-PenS]enkephalin(DPDPE), a 6-opioid receptor selective analog of Leu-enkephalin, impairs acquisition of an automated jump-up avoidance response in rats and acquisition of a one-way active avoidance response in mice. In the present study we investigated the effects of DPDPE on one-way avoidance conditioning in rats. The rats received two escape-onlytrials on day 1 and eight additional training trials on day 2. DPDPE (1.16 ~g/kg IP) administered prior to training on day 2 impaired acquisition of the avoidance response. On the other hand, DPDPE (0.332 #g/kg IP) administered following presentation of the two escape-onlytrials on day 1 significantlyenhanced retention, as measured by improved one-wayactive avoidance performance on day 2. These results indicate that activation of 6-opioid receptors by DPDPE has a modulatory effect on acquisition and retention of aversively motivated performance. D-Pen2-[D-PenS]Enkephalin
Activeavoidance
Acquisition
BOTH Met- and Leu-enkephalin are reported to enhance and impair acquisition of new learning (pretraining injections), and to enhance and impair retention of recently acquired responses (posttraining injections). For example, Met-enkephalin enhances acquisition ofa 12-choice maze in rats (7) and it impairs retention of a discriminated Y-maze shock escape task in mice (34). Leuenkephalin enhances acquisition of a passive avoidance response in rats (26) and it impairs retention of an appetitively motivated Y-maze in mice (12). These effects do not appear to be species or task dependent [for review see (27)]. The ability of opioid peptides and other agents both to enhance and impair learning and memory can be explained within the context of modulation (17). According to this view, memory storage mechanisms have two fundamental physiological components, the memory trace itself and a modulatory system that conveys the importance of the event for the organism and hence influences associative strength. Sometimes it is important for the organism to remember an event well (enhanced acquisition or retention), and sometimes it is more advantageous to store the memory of the event, particularly of a relatively unimportant event (6), with less fidelity (impaired acquisition or retention). Memory systems appear to be constructed such that memory traces can be made either stronger or weaker as a result of the actions of the modulatory inputs to them. Opioid peptides ap-
Retention
6-opioidreceptor
parently act both to increase and decrease the strength of memories. The pharmacological tools available for investigating how opioid peptides influence acquisition and retention of learned responses have improved with the recent development of drugs selective for one of the three kinds of opioid receptors (2-4,911,19). A highly selective agonist for the 6-opioid receptor, DPen2-[D-PenS]enkephalin (DPDPE), impairs acquisition of a jump-up avoidance response in mice (30), while a highly selective 6-opioid receptor antagonist, ICI 174,864, enhances acquisition of a one-way active avoidance task in mice (28). Because ICI 174,864 blocks the effects of Leu-enkephalin on one-way active avoidance conditioning in mice, we suggested that Leu-enkephalin mediates its effects on conditioning through the 6-opioid receptor (28). In the present study we extended our original observation that DPDPE impairs acquisition of one-way avoidance conditioning in mice and jump-up avoidance conditioning in rats (30,32) to demonstrate that DPDPE both impairs acquisition of a one-way active avoidance response in rats when it is administered prior to training and enhances retention of the same response as measured by avoidance responding on day 2 in animals that received the drug immediately after presentation of two escape-only trials on day 1. The results are discussed within
J Requests for reprints should be addressed to Joe L. Martinez, Jr., Ph.D., Psychology Department, 3210 Tolman Hall, University of California, Berkeley, CA 94720.
885
886
MARTINEZ, HERNANDEZ AND RODRIGUEZ
the context of our theory of the modulation of learning and memory ( 17,18). METHOD
Subjects The subjects were male Sprague-Dawley rats (Simonsen Labs, Gilroy, CA) weighing 260-280 g on arrival at our vivarium. The animals were housed individually, with ad lib access to food and water, in accordance with N | H guidelines. All animal use and testing procedures were approved in advance by the Institutional Animal Care and Use Committee at the University of California at Berkeley.
One-Way Active Avoidance Task The apparatus is a two-chamber box with a floor comprised of two metal plates (15). The chamber design requires the animal to make contact with both plates at all times. Testing on day 1 consisted o f two escape-only trials, which began by placing the animal in the darkened start compartment. After 10 s the interconnecting door was opened, and an unscrambled constant current footshock (900 uA) was delivered across the metal floor plates. The shock was terminated when the animal escaped into the safe white compartment. Moving to the white compartment required the animal to act against its natural tendency to remain in the dark. One animal failed to escape within 30 s and was eliminated from further study. The intertrial interval was 30 s. On all trials on day 2 the animal could make either an avoidance or an escape response. For these trials the interconnecting door was opened at the start of the trial and the animal was given l0 s to move into the safe compartment (avoidance response) before the shock was activated and subsequently terminated by an escape response or the passage of 30 s. Animals that failed to escape within 30 s were moved by the experimenter to the safe compartment. Animals remained in the safe compartment throughout each 30-s intertrial interval and then were moved by the experimenter to the start compartment to begin the next trial. Each animal completed eight trials on day 2. The two animals that failed to escape on three consecutive trials on day 2 were eliminated from further study. The number of avoidances made by the remaining 141 animals on day 2 was used to measure performance. Each animal was given an IP injection of saline or D P D P E (0.332, 1.16, 3.32, or 11.6 #g/kg) either 2 min prior to testing on day 2 (acquisition condition) or 30 s after completion of the two escape-only trials on day 1 (retention condition).
1. acquisition: saline, 1.16 ~g/kg DPDPE, and 11.6 /~g/kg DPDPE: 2. retention: saline, 0.332 #g/kg DPDPE, and 3.32 ~g/kg DPDPE; 3. retention: saline, 1.16 #g/kg DPDPE, and 11.6 tzg/kg DPDPE. The significance of differences between combined saline and drug treatment means obtained within each experiment was tested with a single degree of freedom A N O V A (8). RESULTS
Effects o[DPDPE on Acquisition ~f a Ono Way Avoidance Response Figure I depicts the effects of D P D P E (1.16 and 11.6 #g/kg IP) in the first experiment, administered on day 2 prior to presentation of eight training trials, on subsequent one-way avoidance responding. When compared with the saline control treatment, the 1.16 ug/kg dose of D P D P E produced a significant impairment of avoidance performance, F(1,29) - 4.34, p < 0.05, while the 11.6 ug/kg D P D P E dose was without significant effect, F(I, 29) = 1.78, p > 0.05.
Effects o['DPDPE on Retention o[One-Way Avoidance Conditioning Figure 2 depicts the effects of D P D P E (0.332 and 3.32 ug/ kg IP), administered on day 1 immediately after presentation of two escape-only trials, on subsequent day 2 one-way avoidance responding in the second experiment. When compared with the saline control treatment, the 0.332 ~g/kg dose of D P D P E produced a significant enhancement of avoidance performance, F( 1, 30) - 4.42, p < 0.05, while the 3.32 ~g/kg DPDPE dose was without significant effect, F( 1, 31 ) - 0.78, p > 0.05. In the third experiment neither 1.16 nor 11.6 ug/kg of D P D P E had a significant effect on retention performance [mean number of avoidances + SEM: saline, 3.9 _+ 0.58; DPDPE 1.16 #g/kg, 4.9
~9
7
Z < D
6
6
5
>
5
n." '" rn
4
D
3
Z Z
0.05; saline vs. DPDPE 11.6 #g/kg, F(1, 29) = 0.97, p > 0.05]. Visual examination of the individual saline control groups (compare Figs. I and 2) indicated that the level of avoidance performance in these animals appears to be different between the acquisition and retention conditions. An ANOVA comparing the saline group from the acquisition experiment with the combined saline groups from the two retention experiments revealed that the saline groups differed significantly, F(2, 44) = 3.235, p < 0.05. DISCUSSION
We found in this study that DPDPE impaired one-way avoidance responding when administered prior to training on day 2. Because these animals were presented with two escapeonly training trials on day 1 but received drug treatment only on day 2, we interpret the DPDPE effect in this experiment to be an impairment of response acquisition. This result agrees with our previous finding that DPDPE impairs acquisition of a jump-up avoidance response in rats (32) and of a one-way avoidance response in mice (30). The effective 1.16 ug/kg DPDPE dose is equimolar to a 1/~g/kg dose of Leu-enkephalin, which impairs significantly both one-way active avoidance response (16) and jump-up avoidance response (32) acquisition in the rat. Furthermore, because Leu-enkephalin is highly selective for the 6-opioid receptor (24,25), and because DPDPE has a sixteenfold greater sensitivity than does Leu-enkephalin for this same receptor (9), we suggest that the impairment of avoidance acquisition produced by both Leu-enkephalin and DPDPE is mediated through 6-opioid receptors. It is unlikely that the impairing action of Leu-enkephalin is attributable to alterations in locomotor activity or to production of analgesia, because we and others determined previously that this peptide does not affect either locomotor activity or analgesia at doses that affect conditioning (14,22,26,28,30). D-Pen2-[D-
887
PenS]Enkephalin does not alter locomotor activity or produce analgesia in mice at doses that impair acquisition of one-way active avoidance responding (28). Taken together, these data suggest that neither Leu-enkephalin nor DPDPE influences acquisition of avoidance conditioning through actions on performance variables unrelated to learning. The DPDPE administered after escape training on day 1 enhanced avoidance performance on day 2. Previously we showed that two escape-only training trials are sufficient for mice to show enhanced retention on day 2, as evidenced by an increased number of avoidances, as compared to mice that received zero or one training trial on day 1 (29). We suggest that the DPDPE acted on neural processes initiated by training on day 1 to affect subsequent performance on day 2; this is commonly considered to be an effect on consolidation (18,20). Also, given the high selectivity of DPDPE for 6-opioid receptors (1,2,5,23), we suggest that DPDPE's enhancing effect is mediated through 6-opioid receptors. The dose-response curves for both the impairment and the enhancement of avoidance performance produced by DPDPE in the present study were U-shaped. In the case of impairment, a 1.16 #g/kg dose of DPDPE was effective, while a higher 11.6 ug/kg dose was without effect. Because there necessarily must be a lower dose of DPDPE that also is ineffective, the doseresponse curve for DPDPE's effect on avoidance response acquisition must be U-shaped. Similarly, in the case of enhancement, a 0.332 ug/kg dose of DPDPE was effective, whereas higher doses up to 11.6 ug/kg were without effect. U-shaped dose-response curves are a common finding in studies of drug effects on learning and memory, in contrast to the hyperbolic doseresponse functions more commonly seen in other types of pharmacological research. A U-shaped dose-response function indicates involvement of opposing processes, each of which mediates a different response and is maximally stimulated by a different concentration of drug (31 ). Martinez et al. (17) suggest that U-shaped curves indicate that the drug in question affects learning and memory through a modulatory mechanism that is capable of regulating (increasing or decreasing) associative strength, rather than having a direct (presumably unidirectional) effect on the memory trace itself. Martinez et al. (17) also suggest that modulatory substances are characterized by an ability both to enhance and impair learning and memory. They propose that modulatory substances such as Leu-enkephalin and DPDPE influence learning and memory processes in the brain through an effect mediated by the neural pathways that also convey information concerning unconditioned stimuli (UCS). Because the relationship between UCS intensity and performance also is a U-shaped function (33), Martinez et al. (17) suggest that drugs that have U-shaped doseresponse functions act to modulate learning through a merging in a common locus of afferent stimulus information that results from the experimental treatment (Leu-enkephalin or DPDPE) and the information provided by the UCS (footshock). D-Pen2[D-PenS]Enkephalin appears to be such a modulatory substance, because it can produce opposing effects (impairment or enhancement) on learning and memory and because the doseresponse curves for both effects are U-shaped, even though the effective dose differs for these two effects. That a single substance may have opposing effects on avoidance performance as a result of differences in the time of its administration is consistent with the theory of modulation outlined above. There is another possible interpretation for the opposing effects of DPDPE on avoidance performance when it is given before vs. after training. Most psychologists believe that memory involves at least two functionally distinct stages, which are termed
888
MARTINEZ, HERNANDEZ AND RODRIGUEZ
short-term and long-term memory. It is reasonable to suggest that these processes might have different neurological bases that might be differentially sensitive to modulation by drug treatment. If a drug were to enhance one stage of m e m o r y and impair the other, then treatments administered immediately after training on day 1 vs. immediately before training on day 2, such as was done in the present study, would produce opposite effects on day 2 performance. Similarly, if acquisition and consolidation processes are subserved by different neural mechanisms, then it would not be surprising that a given drug has one effect on acquisition and a different or opposite effect on consolidation. That different doses and treatment regimens of D P D P E have opposing effects would agree with this interpretation. Additionally, when animals are trained while drug is on board, the drug may alter such variables as performance, motivation, and attention that can themselves influence production of the measured response. By contrast, when animals are trained and tested in a drug-free state, these intervening variables do not influence response production. Again, it is reasonable to postulate that different mechanisms may be involved in these two situations and that these mechanisms may be differentially sensitive to modulation by drug treatment. Interestingly, as discussed in Martinez (13), most drugs that affect learning and m e m o r y produce the same effect when given before or after training; D P D P E is unusual in this regard. The procedures we used to assess drug effects on acquisition vs. retention of learning produced different baseline levels of performance. The animals that received a saline injection immediately after training on day 1 showed enhanced performance relative to that of animals that received an injection 2 min prior to training on day 2. This effect is consistent with our view that
many aspects of the experimental treatment, including the experience of handling and injection as well as the drug treatment itself, can act as unconditioned stimuli that can each contribute to changes in conditioned performance, which we interpret as changes in associative strength, through actions mediated by a single c o m m o n pathway (17). We believe that the close temporal contiguity between training and treatment, in the case of posttraining injections, facilitated the action of the injection procedure itself as a UCS. Thus, the group that received the posttraining injection had enhanced performance relative to that of animals that received an injection 2 min pretraining on day 2. Similar reinforcing effects of posttraining treatments were reported by Rigter et al. (26). In the Rigter et al. study, animals that received footshock and handling performed better than animals that received an injection and handling [see Fig. 2 in (26)], although both groups of animals learned the passive avoidance response. The results reported here confirm and extend our own findings and those of others reported in the literature. As reviewed above, both Met- and Leu-enkephalin enhance and impair acquisition of new learning, and enhance and impair retention of recently acquired responses. The finding that D P D P E has opposite effects on acquisition and consolidation processes agrees quite well with other results reported for the enkephalins and suggests that all of these peptides are modulatory substances for learning and m e m o r y (21). ACKNOWLEDGEMENTS Supported by NIDA #DA04195 to LL.M., Jr. and NIMH #T32MH18882 to R.V.H.
REFERENCES 1. Corbett, A. D.,; Gillan, M. G. C.; Kosterlitz, H. W.; McKnight, A. T.; Paterson, S. J.; Robson, L. E. Selectivities of opioid peptide analogues as agonists and antagonists at the delta-receptor. Br. J. Pharmacol. 83:271-279; 1984. 2. Cotton, R.; Kosterlitz, H. W.; Paterson, S. J.; Rance, M. J.; Traynor, J. R. The use of [3H]-[D-Pen2,D-PenS]enkephalin as a highly selective ligand for the S-binding site. Br. J. Pharmacol. 84:927-932; 1985. 3. Goldstein, A.; James, I. F. Site-directed alkylation of multiple opioid receptors: II. Pharmacological selectivity. Mol. Pharmacol. 25:343348; 1984. 4. Hayes, A. G.; Birch, P. J.; Hayward, N. J.; Sheehan, M. J.; Rogers, H.; Tyers, M. B.; Judd, D. B.; Scopes, D. I. C.; Naylor, A. A series of novel, highly potent and selective agonists for the k-opioid receptor. Br. J. Pharmacol. 101:944-948; 1990. 5. Hruby, V. J. Design ofconformationally constrained cyclic peptides with high delta and mu opioid receptor specificities. NIDA Res. Monogr. 69:128-147; 1986. 6. lzquierdo, I. B-endorphin and forgetting. Trends Pharmacol. Sci. 15:119-121; 1982. 7. Kastin, A. J.; Scollan, E. L.; King, M. G.; Schally, A. V.; Coy, D. H. Enkephalin and a potent analog facilitate maze performance after intraperitoneal administration in rats. Pharmacol. Biochem. Behav. 5:691-695; 1976. 8. Keppel, G. Design and analysis: A researcher's handbook. Englewood Cliffs, N J: Prentice-Hall; 1991. 9. Kosterlitz, H. W.; Corbett, A. D.; Gillan, M. G. C.; McKnight, A. T.; Paterson, S. J.; Robson, L. E. Recent developments in bioassay using selective ligands and selective in vitro preparations. In: Rapaka, R. S.; Hawks, R. L., eds. NIDA Research Monograph 70: Opioid peptides: Molecular pharmacology, biosynthesis, and analysis. RockviUe, MD: National Institute on Drug Abuse; 1986:223-236. 10. Kosterlitz, H. W.; Robson, L. E.; Paterson, S. J. Opioid peptides and their receptors. In: Weiner, H.; Florin, I.; Murison, R.; Hell-
11. 12. 13. 14. 15.
16. 17.
18.
19.
hammer, D., eds. Frontiers of stress research. Toronto: Hans Huber Publishers; 1989:302-308. Leslie, F. M. Methods used for the study ofopioid receptors. Pharmacol. Rev. 39:197-249; 1987. Linden, D.; Martinez, J. L., Jr. Leu-enkephalin impairs memory of an appetitive maze response in mice. Behav. Neurosci. 100:33-38; 1986. Martinez, J. L., Jr. Memory: Drugs and hormones. In: Martinez, J. L., Jr.; Kesner, R., eds. Learning and memory: A biological view. Orlando: Academic Press; 1986:127-163. Martinez, J. L., Jr.'; Conner, P.; Dana, R. C. Central versus peripheral actions of leu-enkephalin on acquisition of a one-way avoidance response in mice. Brain Res. 327:37-43; 1985. Martinez, J. L., Jr.; de Graaf, J. S. Quarternary naloxone enhances acquisition of a discriminated Y-maze escape and a one-way active avoidance task in mice. Psychopharmacology (Berlin) 87:410-413; 1985. Martinez, J. L., Jr.; Rigter, H. Enkephalin actions on avoidance conditioning may be related to adrenal medullary function. Behav. Brain Res. 6:282-299; 1982. Martinez, J. L., Jr.; Schulteis, G.; Weinherger, S. B. How to increase and decrease the strength of memory traces: The effects of drugs and hormones. In: Martinez, J. L., Jr.; Kesner, R., eds. Learning and memory: A biological view. Orlando, FL: Academic Press; 1991: 149-198. Martinez, J. L., Jr.; Weinberger, S. B.; Janak, P. H.; Schulteis, G.; Shibanoki, S.; Ishikawa, K. Peripheral signaling of the brain: How hormones influence learning and memory. In: Frederickson, R. C. A.; McGaugh, J. L.; Felten, D. L., eds. Peripheral signaling of the brain: Role in neural-immune interactions, learning and memory. Toronto: Hogrefe and Huber; 1991:365-378. Martinka, G. P.; Jhamandas, K.; Sabourin, L.; Lapierre, C.; Lemaire, S. Dynorphin A-(l- 13)-TyrJ4-Leul%Phelr-AsnlT-GlylS-Pro19: A po-
DPDPE AND ONE-WAY AVOIDANCE RESPONSE
20. 21.
22. 23.
24. 25. 26.
27.
tent and selective k opioid peptide. Eur. J. Pharmacol. 196:161167; 1991. McGaugh, J. L. Time-dependent processes in memory storage. Science 153:1351-1358; 1966. McGaugh, J. L.; Martinez, J. L., Jr. Learning modulatory hormones: An introduction to endogenous peptides and learning and memory processes. In: Martinez, J. L., Jr.; Jensen, R. A.; Messing, R. B.; Rigter, H.; McGaugh, J. L., eds. Endogenous peptides and learning and memory processes. New York: Academic Press; 1981 : 1-3. Mens, W. B. J.; Van Ree, J. M. Influence of/3-1ipotropin fragments on responsiveness of rats to electric footshock. Pharmacol. Biochem. Behav. 15:27-32; 1981. Mosberg, H. I.; Hurst, R.; Hruby, V. J.; Gee, K.; Yamamura, H. I.; Galligan, J. J.; Burks, T. F. Bis-penicillamine enkephalins possess highly improved specificity toward ~ opioid receptors. Proc. Natl. Acad. Sci. USA 80:5871-5874; 1983. Pasternak, G. W. The opiate receptors. Clifton, N J: Humana Press; 1988. Paterson, S. J.; Robson, L. E.; Kosterlitz, H. W. Classification of opioid receptors. Br. Med. Bull. 39:25-30; 1983. Rigter, H.; Jensen, R. A.; Martinez, J. L., Jr.; Messing, R. B.; Vasquez, B. J.; Liang, K. C.; McGaugh, J. L. Enkephalin and fear-motivated behavior. Proc. Natl. Acad. Sci. USA 77:37293732; 1980. Schulteis, G.; Janak, P. H.; Derrick, B. E.; Martinez, J. L., Jr. Endogenous opioid peptides and learning and memory. In: Szekely,
889
28. 29.
30.
31. 32.
33. 34.
J. I.; Ramabadran, K., eds. Opioid peptides volume IV: Biochemistry and applied physiology. Boca Raton, FL: CRC Press; 1990:189219. Schulteis, G.; Martinez, J. L., Jr. ICI 174,864, a selective delta opioid antagonist, reverses the learning impairment produced by [leu]enkephalin. Psychopharmacology (Berlin) 100:102-109; 1990. Schulteis, G.; Martinez, J. L., Jr. Post-training administration of [leu]enkephalin and its metabolite, Tyr-Gly-Gly, impairs subsequent performance in an active avoidance paradigm. Pharmacol. Biochem. Behav. (in press). Schulteis, G.; Martinez, J. L., Jr.; Hruby, V. J. Stimulation and antagonism of opioid fi-receptors produce opposite effects on active avoidance conditioning in mice. Behav. Neurosci. 102:678-686; 1988. Solomon, R. L. Acquired motivation and affective opponent-processes. In: Madden J., IV, ed. Neurobiology of learning, emotion and affect. New York: Raven Press; 1991:307-347. Weinberger, S. B.; Gehrig, C. A.; Martinez, J. L., Jr. Dpen 2[DPenS]enkephalin, a delta opioid receptor-selective analog of [Leu]enkephalin, impairs avoidance learning in an automated shelfjump task in rats. Regul. Pept. 26:323-329; 1989. Yerkes, R. M.; Dodson, J. D. The relation of strength of stimulus to rapidity of habit-formation. J. Comp. Neurol. Psychol. 18:459482; 1908. Zhang, S.-Y.; McGaugh, J. L.: Juler, R. G.; Introini-Collison, I. B. Naloxone and [met]enkephalin effects on retention: Attenuation by adrenal denervation. Eur. J. Pharmacol 138:37; 1987.
0196-9781/92 $5.00 + .00 Copyright© 1992PergamonPressLtd.
Printed in the USA.
D-Pen2-[D-PenS]Enkephalin Impairs Acquisition and Enhances Retention of a One-Way Active Avoidance Response in Rats JOE L. M A R T I N E Z , JR., 1 R U B E N V. H E R N A N D E Z A N D S U S A N B. R O D R I G U E Z
University o f California, Department o f Psychology, Berkeley, CA 94720 Received 3 F e b r u a r y 1992 MARTINEZ, J. L., JR., R. V. HERNANDEZ AND S. B. RODRIGUEZ. D-Pen2-[D-PenS]Enkephalin impairs acquisition and enhances retention of a one-way active avoidance response in rats. PEPTIDES 13(5) 885-889, 1992.--We reported previously that D-Pen2-[D-PenS]enkephalin(DPDPE), a 6-opioid receptor selective analog of Leu-enkephalin, impairs acquisition of an automated jump-up avoidance response in rats and acquisition of a one-way active avoidance response in mice. In the present study we investigated the effects of DPDPE on one-way avoidance conditioning in rats. The rats received two escape-onlytrials on day 1 and eight additional training trials on day 2. DPDPE (1.16 ~g/kg IP) administered prior to training on day 2 impaired acquisition of the avoidance response. On the other hand, DPDPE (0.332 #g/kg IP) administered following presentation of the two escape-onlytrials on day 1 significantlyenhanced retention, as measured by improved one-wayactive avoidance performance on day 2. These results indicate that activation of 6-opioid receptors by DPDPE has a modulatory effect on acquisition and retention of aversively motivated performance. D-Pen2-[D-PenS]Enkephalin
Activeavoidance
Acquisition
BOTH Met- and Leu-enkephalin are reported to enhance and impair acquisition of new learning (pretraining injections), and to enhance and impair retention of recently acquired responses (posttraining injections). For example, Met-enkephalin enhances acquisition ofa 12-choice maze in rats (7) and it impairs retention of a discriminated Y-maze shock escape task in mice (34). Leuenkephalin enhances acquisition of a passive avoidance response in rats (26) and it impairs retention of an appetitively motivated Y-maze in mice (12). These effects do not appear to be species or task dependent [for review see (27)]. The ability of opioid peptides and other agents both to enhance and impair learning and memory can be explained within the context of modulation (17). According to this view, memory storage mechanisms have two fundamental physiological components, the memory trace itself and a modulatory system that conveys the importance of the event for the organism and hence influences associative strength. Sometimes it is important for the organism to remember an event well (enhanced acquisition or retention), and sometimes it is more advantageous to store the memory of the event, particularly of a relatively unimportant event (6), with less fidelity (impaired acquisition or retention). Memory systems appear to be constructed such that memory traces can be made either stronger or weaker as a result of the actions of the modulatory inputs to them. Opioid peptides ap-
Retention
6-opioidreceptor
parently act both to increase and decrease the strength of memories. The pharmacological tools available for investigating how opioid peptides influence acquisition and retention of learned responses have improved with the recent development of drugs selective for one of the three kinds of opioid receptors (2-4,911,19). A highly selective agonist for the 6-opioid receptor, DPen2-[D-PenS]enkephalin (DPDPE), impairs acquisition of a jump-up avoidance response in mice (30), while a highly selective 6-opioid receptor antagonist, ICI 174,864, enhances acquisition of a one-way active avoidance task in mice (28). Because ICI 174,864 blocks the effects of Leu-enkephalin on one-way active avoidance conditioning in mice, we suggested that Leu-enkephalin mediates its effects on conditioning through the 6-opioid receptor (28). In the present study we extended our original observation that DPDPE impairs acquisition of one-way avoidance conditioning in mice and jump-up avoidance conditioning in rats (30,32) to demonstrate that DPDPE both impairs acquisition of a one-way active avoidance response in rats when it is administered prior to training and enhances retention of the same response as measured by avoidance responding on day 2 in animals that received the drug immediately after presentation of two escape-only trials on day 1. The results are discussed within
J Requests for reprints should be addressed to Joe L. Martinez, Jr., Ph.D., Psychology Department, 3210 Tolman Hall, University of California, Berkeley, CA 94720.
885
886
MARTINEZ, HERNANDEZ AND RODRIGUEZ
the context of our theory of the modulation of learning and memory ( 17,18). METHOD
Subjects The subjects were male Sprague-Dawley rats (Simonsen Labs, Gilroy, CA) weighing 260-280 g on arrival at our vivarium. The animals were housed individually, with ad lib access to food and water, in accordance with N | H guidelines. All animal use and testing procedures were approved in advance by the Institutional Animal Care and Use Committee at the University of California at Berkeley.
One-Way Active Avoidance Task The apparatus is a two-chamber box with a floor comprised of two metal plates (15). The chamber design requires the animal to make contact with both plates at all times. Testing on day 1 consisted o f two escape-only trials, which began by placing the animal in the darkened start compartment. After 10 s the interconnecting door was opened, and an unscrambled constant current footshock (900 uA) was delivered across the metal floor plates. The shock was terminated when the animal escaped into the safe white compartment. Moving to the white compartment required the animal to act against its natural tendency to remain in the dark. One animal failed to escape within 30 s and was eliminated from further study. The intertrial interval was 30 s. On all trials on day 2 the animal could make either an avoidance or an escape response. For these trials the interconnecting door was opened at the start of the trial and the animal was given l0 s to move into the safe compartment (avoidance response) before the shock was activated and subsequently terminated by an escape response or the passage of 30 s. Animals that failed to escape within 30 s were moved by the experimenter to the safe compartment. Animals remained in the safe compartment throughout each 30-s intertrial interval and then were moved by the experimenter to the start compartment to begin the next trial. Each animal completed eight trials on day 2. The two animals that failed to escape on three consecutive trials on day 2 were eliminated from further study. The number of avoidances made by the remaining 141 animals on day 2 was used to measure performance. Each animal was given an IP injection of saline or D P D P E (0.332, 1.16, 3.32, or 11.6 #g/kg) either 2 min prior to testing on day 2 (acquisition condition) or 30 s after completion of the two escape-only trials on day 1 (retention condition).
1. acquisition: saline, 1.16 ~g/kg DPDPE, and 11.6 /~g/kg DPDPE: 2. retention: saline, 0.332 #g/kg DPDPE, and 3.32 ~g/kg DPDPE; 3. retention: saline, 1.16 #g/kg DPDPE, and 11.6 tzg/kg DPDPE. The significance of differences between combined saline and drug treatment means obtained within each experiment was tested with a single degree of freedom A N O V A (8). RESULTS
Effects o[DPDPE on Acquisition ~f a Ono Way Avoidance Response Figure I depicts the effects of D P D P E (1.16 and 11.6 #g/kg IP) in the first experiment, administered on day 2 prior to presentation of eight training trials, on subsequent one-way avoidance responding. When compared with the saline control treatment, the 1.16 ug/kg dose of D P D P E produced a significant impairment of avoidance performance, F(1,29) - 4.34, p < 0.05, while the 11.6 ug/kg D P D P E dose was without significant effect, F(I, 29) = 1.78, p > 0.05.
Effects o['DPDPE on Retention o[One-Way Avoidance Conditioning Figure 2 depicts the effects of D P D P E (0.332 and 3.32 ug/ kg IP), administered on day 1 immediately after presentation of two escape-only trials, on subsequent day 2 one-way avoidance responding in the second experiment. When compared with the saline control treatment, the 0.332 ~g/kg dose of D P D P E produced a significant enhancement of avoidance performance, F( 1, 30) - 4.42, p < 0.05, while the 3.32 ~g/kg DPDPE dose was without significant effect, F( 1, 31 ) - 0.78, p > 0.05. In the third experiment neither 1.16 nor 11.6 ug/kg of D P D P E had a significant effect on retention performance [mean number of avoidances + SEM: saline, 3.9 _+ 0.58; DPDPE 1.16 #g/kg, 4.9
~9
7
Z < D
6
6
5
>
5
n." '" rn
4
D
3
Z Z
0.05; saline vs. DPDPE 11.6 #g/kg, F(1, 29) = 0.97, p > 0.05]. Visual examination of the individual saline control groups (compare Figs. I and 2) indicated that the level of avoidance performance in these animals appears to be different between the acquisition and retention conditions. An ANOVA comparing the saline group from the acquisition experiment with the combined saline groups from the two retention experiments revealed that the saline groups differed significantly, F(2, 44) = 3.235, p < 0.05. DISCUSSION
We found in this study that DPDPE impaired one-way avoidance responding when administered prior to training on day 2. Because these animals were presented with two escapeonly training trials on day 1 but received drug treatment only on day 2, we interpret the DPDPE effect in this experiment to be an impairment of response acquisition. This result agrees with our previous finding that DPDPE impairs acquisition of a jump-up avoidance response in rats (32) and of a one-way avoidance response in mice (30). The effective 1.16 ug/kg DPDPE dose is equimolar to a 1/~g/kg dose of Leu-enkephalin, which impairs significantly both one-way active avoidance response (16) and jump-up avoidance response (32) acquisition in the rat. Furthermore, because Leu-enkephalin is highly selective for the 6-opioid receptor (24,25), and because DPDPE has a sixteenfold greater sensitivity than does Leu-enkephalin for this same receptor (9), we suggest that the impairment of avoidance acquisition produced by both Leu-enkephalin and DPDPE is mediated through 6-opioid receptors. It is unlikely that the impairing action of Leu-enkephalin is attributable to alterations in locomotor activity or to production of analgesia, because we and others determined previously that this peptide does not affect either locomotor activity or analgesia at doses that affect conditioning (14,22,26,28,30). D-Pen2-[D-
887
PenS]Enkephalin does not alter locomotor activity or produce analgesia in mice at doses that impair acquisition of one-way active avoidance responding (28). Taken together, these data suggest that neither Leu-enkephalin nor DPDPE influences acquisition of avoidance conditioning through actions on performance variables unrelated to learning. The DPDPE administered after escape training on day 1 enhanced avoidance performance on day 2. Previously we showed that two escape-only training trials are sufficient for mice to show enhanced retention on day 2, as evidenced by an increased number of avoidances, as compared to mice that received zero or one training trial on day 1 (29). We suggest that the DPDPE acted on neural processes initiated by training on day 1 to affect subsequent performance on day 2; this is commonly considered to be an effect on consolidation (18,20). Also, given the high selectivity of DPDPE for 6-opioid receptors (1,2,5,23), we suggest that DPDPE's enhancing effect is mediated through 6-opioid receptors. The dose-response curves for both the impairment and the enhancement of avoidance performance produced by DPDPE in the present study were U-shaped. In the case of impairment, a 1.16 #g/kg dose of DPDPE was effective, while a higher 11.6 ug/kg dose was without effect. Because there necessarily must be a lower dose of DPDPE that also is ineffective, the doseresponse curve for DPDPE's effect on avoidance response acquisition must be U-shaped. Similarly, in the case of enhancement, a 0.332 ug/kg dose of DPDPE was effective, whereas higher doses up to 11.6 ug/kg were without effect. U-shaped dose-response curves are a common finding in studies of drug effects on learning and memory, in contrast to the hyperbolic doseresponse functions more commonly seen in other types of pharmacological research. A U-shaped dose-response function indicates involvement of opposing processes, each of which mediates a different response and is maximally stimulated by a different concentration of drug (31 ). Martinez et al. (17) suggest that U-shaped curves indicate that the drug in question affects learning and memory through a modulatory mechanism that is capable of regulating (increasing or decreasing) associative strength, rather than having a direct (presumably unidirectional) effect on the memory trace itself. Martinez et al. (17) also suggest that modulatory substances are characterized by an ability both to enhance and impair learning and memory. They propose that modulatory substances such as Leu-enkephalin and DPDPE influence learning and memory processes in the brain through an effect mediated by the neural pathways that also convey information concerning unconditioned stimuli (UCS). Because the relationship between UCS intensity and performance also is a U-shaped function (33), Martinez et al. (17) suggest that drugs that have U-shaped doseresponse functions act to modulate learning through a merging in a common locus of afferent stimulus information that results from the experimental treatment (Leu-enkephalin or DPDPE) and the information provided by the UCS (footshock). D-Pen2[D-PenS]Enkephalin appears to be such a modulatory substance, because it can produce opposing effects (impairment or enhancement) on learning and memory and because the doseresponse curves for both effects are U-shaped, even though the effective dose differs for these two effects. That a single substance may have opposing effects on avoidance performance as a result of differences in the time of its administration is consistent with the theory of modulation outlined above. There is another possible interpretation for the opposing effects of DPDPE on avoidance performance when it is given before vs. after training. Most psychologists believe that memory involves at least two functionally distinct stages, which are termed
888
MARTINEZ, HERNANDEZ AND RODRIGUEZ
short-term and long-term memory. It is reasonable to suggest that these processes might have different neurological bases that might be differentially sensitive to modulation by drug treatment. If a drug were to enhance one stage of m e m o r y and impair the other, then treatments administered immediately after training on day 1 vs. immediately before training on day 2, such as was done in the present study, would produce opposite effects on day 2 performance. Similarly, if acquisition and consolidation processes are subserved by different neural mechanisms, then it would not be surprising that a given drug has one effect on acquisition and a different or opposite effect on consolidation. That different doses and treatment regimens of D P D P E have opposing effects would agree with this interpretation. Additionally, when animals are trained while drug is on board, the drug may alter such variables as performance, motivation, and attention that can themselves influence production of the measured response. By contrast, when animals are trained and tested in a drug-free state, these intervening variables do not influence response production. Again, it is reasonable to postulate that different mechanisms may be involved in these two situations and that these mechanisms may be differentially sensitive to modulation by drug treatment. Interestingly, as discussed in Martinez (13), most drugs that affect learning and m e m o r y produce the same effect when given before or after training; D P D P E is unusual in this regard. The procedures we used to assess drug effects on acquisition vs. retention of learning produced different baseline levels of performance. The animals that received a saline injection immediately after training on day 1 showed enhanced performance relative to that of animals that received an injection 2 min prior to training on day 2. This effect is consistent with our view that
many aspects of the experimental treatment, including the experience of handling and injection as well as the drug treatment itself, can act as unconditioned stimuli that can each contribute to changes in conditioned performance, which we interpret as changes in associative strength, through actions mediated by a single c o m m o n pathway (17). We believe that the close temporal contiguity between training and treatment, in the case of posttraining injections, facilitated the action of the injection procedure itself as a UCS. Thus, the group that received the posttraining injection had enhanced performance relative to that of animals that received an injection 2 min pretraining on day 2. Similar reinforcing effects of posttraining treatments were reported by Rigter et al. (26). In the Rigter et al. study, animals that received footshock and handling performed better than animals that received an injection and handling [see Fig. 2 in (26)], although both groups of animals learned the passive avoidance response. The results reported here confirm and extend our own findings and those of others reported in the literature. As reviewed above, both Met- and Leu-enkephalin enhance and impair acquisition of new learning, and enhance and impair retention of recently acquired responses. The finding that D P D P E has opposite effects on acquisition and consolidation processes agrees quite well with other results reported for the enkephalins and suggests that all of these peptides are modulatory substances for learning and m e m o r y (21). ACKNOWLEDGEMENTS Supported by NIDA #DA04195 to LL.M., Jr. and NIMH #T32MH18882 to R.V.H.
REFERENCES 1. Corbett, A. D.,; Gillan, M. G. C.; Kosterlitz, H. W.; McKnight, A. T.; Paterson, S. J.; Robson, L. E. Selectivities of opioid peptide analogues as agonists and antagonists at the delta-receptor. Br. J. Pharmacol. 83:271-279; 1984. 2. Cotton, R.; Kosterlitz, H. W.; Paterson, S. J.; Rance, M. J.; Traynor, J. R. The use of [3H]-[D-Pen2,D-PenS]enkephalin as a highly selective ligand for the S-binding site. Br. J. Pharmacol. 84:927-932; 1985. 3. Goldstein, A.; James, I. F. Site-directed alkylation of multiple opioid receptors: II. Pharmacological selectivity. Mol. Pharmacol. 25:343348; 1984. 4. Hayes, A. G.; Birch, P. J.; Hayward, N. J.; Sheehan, M. J.; Rogers, H.; Tyers, M. B.; Judd, D. B.; Scopes, D. I. C.; Naylor, A. A series of novel, highly potent and selective agonists for the k-opioid receptor. Br. J. Pharmacol. 101:944-948; 1990. 5. Hruby, V. J. Design ofconformationally constrained cyclic peptides with high delta and mu opioid receptor specificities. NIDA Res. Monogr. 69:128-147; 1986. 6. lzquierdo, I. B-endorphin and forgetting. Trends Pharmacol. Sci. 15:119-121; 1982. 7. Kastin, A. J.; Scollan, E. L.; King, M. G.; Schally, A. V.; Coy, D. H. Enkephalin and a potent analog facilitate maze performance after intraperitoneal administration in rats. Pharmacol. Biochem. Behav. 5:691-695; 1976. 8. Keppel, G. Design and analysis: A researcher's handbook. Englewood Cliffs, N J: Prentice-Hall; 1991. 9. Kosterlitz, H. W.; Corbett, A. D.; Gillan, M. G. C.; McKnight, A. T.; Paterson, S. J.; Robson, L. E. Recent developments in bioassay using selective ligands and selective in vitro preparations. In: Rapaka, R. S.; Hawks, R. L., eds. NIDA Research Monograph 70: Opioid peptides: Molecular pharmacology, biosynthesis, and analysis. RockviUe, MD: National Institute on Drug Abuse; 1986:223-236. 10. Kosterlitz, H. W.; Robson, L. E.; Paterson, S. J. Opioid peptides and their receptors. In: Weiner, H.; Florin, I.; Murison, R.; Hell-
11. 12. 13. 14. 15.
16. 17.
18.
19.
hammer, D., eds. Frontiers of stress research. Toronto: Hans Huber Publishers; 1989:302-308. Leslie, F. M. Methods used for the study ofopioid receptors. Pharmacol. Rev. 39:197-249; 1987. Linden, D.; Martinez, J. L., Jr. Leu-enkephalin impairs memory of an appetitive maze response in mice. Behav. Neurosci. 100:33-38; 1986. Martinez, J. L., Jr. Memory: Drugs and hormones. In: Martinez, J. L., Jr.; Kesner, R., eds. Learning and memory: A biological view. Orlando: Academic Press; 1986:127-163. Martinez, J. L., Jr.'; Conner, P.; Dana, R. C. Central versus peripheral actions of leu-enkephalin on acquisition of a one-way avoidance response in mice. Brain Res. 327:37-43; 1985. Martinez, J. L., Jr.; de Graaf, J. S. Quarternary naloxone enhances acquisition of a discriminated Y-maze escape and a one-way active avoidance task in mice. Psychopharmacology (Berlin) 87:410-413; 1985. Martinez, J. L., Jr.; Rigter, H. Enkephalin actions on avoidance conditioning may be related to adrenal medullary function. Behav. Brain Res. 6:282-299; 1982. Martinez, J. L., Jr.; Schulteis, G.; Weinherger, S. B. How to increase and decrease the strength of memory traces: The effects of drugs and hormones. In: Martinez, J. L., Jr.; Kesner, R., eds. Learning and memory: A biological view. Orlando, FL: Academic Press; 1991: 149-198. Martinez, J. L., Jr.; Weinberger, S. B.; Janak, P. H.; Schulteis, G.; Shibanoki, S.; Ishikawa, K. Peripheral signaling of the brain: How hormones influence learning and memory. In: Frederickson, R. C. A.; McGaugh, J. L.; Felten, D. L., eds. Peripheral signaling of the brain: Role in neural-immune interactions, learning and memory. Toronto: Hogrefe and Huber; 1991:365-378. Martinka, G. P.; Jhamandas, K.; Sabourin, L.; Lapierre, C.; Lemaire, S. Dynorphin A-(l- 13)-TyrJ4-Leul%Phelr-AsnlT-GlylS-Pro19: A po-
DPDPE AND ONE-WAY AVOIDANCE RESPONSE
20. 21.
22. 23.
24. 25. 26.
27.
tent and selective k opioid peptide. Eur. J. Pharmacol. 196:161167; 1991. McGaugh, J. L. Time-dependent processes in memory storage. Science 153:1351-1358; 1966. McGaugh, J. L.; Martinez, J. L., Jr. Learning modulatory hormones: An introduction to endogenous peptides and learning and memory processes. In: Martinez, J. L., Jr.; Jensen, R. A.; Messing, R. B.; Rigter, H.; McGaugh, J. L., eds. Endogenous peptides and learning and memory processes. New York: Academic Press; 1981 : 1-3. Mens, W. B. J.; Van Ree, J. M. Influence of/3-1ipotropin fragments on responsiveness of rats to electric footshock. Pharmacol. Biochem. Behav. 15:27-32; 1981. Mosberg, H. I.; Hurst, R.; Hruby, V. J.; Gee, K.; Yamamura, H. I.; Galligan, J. J.; Burks, T. F. Bis-penicillamine enkephalins possess highly improved specificity toward ~ opioid receptors. Proc. Natl. Acad. Sci. USA 80:5871-5874; 1983. Pasternak, G. W. The opiate receptors. Clifton, N J: Humana Press; 1988. Paterson, S. J.; Robson, L. E.; Kosterlitz, H. W. Classification of opioid receptors. Br. Med. Bull. 39:25-30; 1983. Rigter, H.; Jensen, R. A.; Martinez, J. L., Jr.; Messing, R. B.; Vasquez, B. J.; Liang, K. C.; McGaugh, J. L. Enkephalin and fear-motivated behavior. Proc. Natl. Acad. Sci. USA 77:37293732; 1980. Schulteis, G.; Janak, P. H.; Derrick, B. E.; Martinez, J. L., Jr. Endogenous opioid peptides and learning and memory. In: Szekely,
889
28. 29.
30.
31. 32.
33. 34.
J. I.; Ramabadran, K., eds. Opioid peptides volume IV: Biochemistry and applied physiology. Boca Raton, FL: CRC Press; 1990:189219. Schulteis, G.; Martinez, J. L., Jr. ICI 174,864, a selective delta opioid antagonist, reverses the learning impairment produced by [leu]enkephalin. Psychopharmacology (Berlin) 100:102-109; 1990. Schulteis, G.; Martinez, J. L., Jr. Post-training administration of [leu]enkephalin and its metabolite, Tyr-Gly-Gly, impairs subsequent performance in an active avoidance paradigm. Pharmacol. Biochem. Behav. (in press). Schulteis, G.; Martinez, J. L., Jr.; Hruby, V. J. Stimulation and antagonism of opioid fi-receptors produce opposite effects on active avoidance conditioning in mice. Behav. Neurosci. 102:678-686; 1988. Solomon, R. L. Acquired motivation and affective opponent-processes. In: Madden J., IV, ed. Neurobiology of learning, emotion and affect. New York: Raven Press; 1991:307-347. Weinberger, S. B.; Gehrig, C. A.; Martinez, J. L., Jr. Dpen 2[DPenS]enkephalin, a delta opioid receptor-selective analog of [Leu]enkephalin, impairs avoidance learning in an automated shelfjump task in rats. Regul. Pept. 26:323-329; 1989. Yerkes, R. M.; Dodson, J. D. The relation of strength of stimulus to rapidity of habit-formation. J. Comp. Neurol. Psychol. 18:459482; 1908. Zhang, S.-Y.; McGaugh, J. L.: Juler, R. G.; Introini-Collison, I. B. Naloxone and [met]enkephalin effects on retention: Attenuation by adrenal denervation. Eur. J. Pharmacol 138:37; 1987.
