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Effects of naloxone on pavlovian conditioning of eyeblink and heart rate responses in rabbits
L i f e S c i e n c e s , V o l . 27, p p . 863-869 P r i n t e d i n t h e U.S.A.
Pergamon P r e s s
EFFECTS OF NALOXDNEON PAVLOVIAN CONDITIONINGOF EYEBLINK AND HEARTRATE RESPONSES IN RABBITS Linda L. llern6ndez and D. A. Powell Neuroscience Laboratory, Wm. Jennings Bryan Dorn Veterans' Hospital Columbia, SC 29201 and Dept. Psychology, University of South Carolina, Columbia, SC 29208 (P.eceived i n f i n a l
form June 30, 1980)
Summar~ Albino rabbits were subjected to aversive Pavlovian conditioning and extinction of eyeblink and heart rate responses. Naloxone administration had no effect on acquisition of the eyeblink response ~ut increased responding during extinction. Naloxone also attenuated the bradycardiac heart rate CR, suggesting that endogenous opioids may be involved in mediating this response. Several recent experiments have suggested that endogenousopioid systems may be involved in learning and/or memory ( I - 8 ) . Such an involvement is consistent with the fact that exogenous opiates affect learning (9,10), and that endogenous opioids and opiate receptors in the brain are localized in areas which are known to be involved in learning (11-14). Many of these same brain areas have been implicated in the control of c l a s s i c a l l y conditioned somatic and autonomic responses in rabbits (15-18). I t therefore seemed reasonable to inquire whether endogenous opioid systems are involved in this control. I f opioid systems participate in Pavlovian conditioning processes in the rabbit, then administration of specific opiate antagonists, such as naloxone, should produce behavioral effects in this paradigm. The present experiment was undertaken to investigate this possibility, by examining the effects of systemically administered naloxone on aversive Pavlovian conditioning and extinction of eyeblink (EB) and heart rate (HR) responses in r a b b i t s . In addition, the present experiment was designed to assess possible statedependent learning (19) following naloxone and to examine the effects of naloxone treatment during extinction on subsequent performance of conditioned EB and HR responses. Method
Subjects. Forty-one experimentally naive male albino rabbits (Oryctolagus cunlcu]us) obtained from Hayes Rabbitry, West Columbia, SC were used as subjects. All animals werekept drug-free for at least f i v e days p r i o r to the beginning of behavioral testing, which was conducted between 8 a.m. and l p.m. Apparatus. Behavioral testing occured in four v e n t i l a t e d , sound and l i g h t attenuating animal enclosures equipped with overhead speakers for d e l i v e r y of auditory s t i m u l i . During testing the animals were restrained in standard 0024-3205/80/360863-07502.00/0
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plexiglass rabbit restrainers. All experimental events were controlled from an adjoining room by a DEC-PDP 11/10 minicomputer, which also collected and summarized the behavioral data directly from a Grass Model 7-D polygraph. Procedure: Each conditioning t r i a l consisted of the presentation of the conditioning stimulus (CS), immediately followed by the unconditioned stimulus (US). The CS was a 1216 Hz, 75 dB square-wave tone, 500 msec in duration. The US was a 3 mA, 500 msec, AC paraorbital electric shock. In each session the US was omitted on seven evenly-spaced "test" t r i a l s during which HR was measured. The i n t e r t r i a l interval was 45 seconds. The EB response (consisting of eyelid closure, eyeball retraction, and/or nict i t a t i n g membrane extension) was measured on each t r i a l via stainless-steel electrodes inserted underneath the eyelids. An EB response was defined as a change of at least 200 uV from pre-CS baseline across the recording electrodes (2 mm pen deflection), occurring during the 500 msec CS i n t e r v a l . HR was recorded for 4 secs prior to and eight secs after CS onset via stainless-steel safety pins inserted subcutaneously over the right shoulder and left flank. At the end of each daily session mean EB amplitude and latency, percent t r i a l s on which EB responses occurred, t r i a l s - t o - c r i t e r i o n , and mean HR for pre-CS and post-CS seconds were calculated for each S and stored for later analysis. During acquisition each session was continued until Ss reached a c r i t e r i o n of lO consecutive t r i a l s on which an EB response occur'~ed, or u n t i l lO0 t r i a l s were delivered. Daily acquisition sessions were conducted until Ss attained a multiple two-day c r i t e r i o n which consisted of: (a) reaching--the IO-EB CR criterion within lO0 t r i a l s on the f i r s t day, and (b) reaching the IO-EB CR criterion within the f i r s t 30 t r i a l s on the following day. Animals f a i l i n g to reach this two-day criterion within lO consecutive days were dropped from the experiment. One S was dropped for this reason. On the day following a t t a i n ment of the a c q u ~ i t i o n c r i t e r i o n Ss were tested for 30 e x t i n c t i o n t r i a l s during which the tone, unpaired with--the US, was presented. On the following day Ss again received up to lO0 paired tone-shock t r i a l s to assess whether nal~one treatment during extinction would affect subsequent performance of EB and HR responses. During acquisition two groups of 20 Ss each received either naloxone or saline prior to each session. During extinTtion these two groups were divided into 4 sub-groups of 10 Ss each; half the Ss in each drug group received naloxone and half received salTne. On the day ~llowing extinction each S received the same drug administered during acquisition. The drug injections ~nsisted of either (a) l.O mg/kg of naloxone HCl (Endo) in a volume of 0.33 ml/kg (approximately 1.0 ml per rabbit) or (b) an equivalent volume of 0.9% (w/v) NaCI ( s a l i n e ) . Intravenous injections were given into the lateral ear vein, each ear being used on alternate days. The time between injection and the f i r s t t r i a l of the experimental session was held constant at 15 min. The EB data for all acquisition sessions (2-10 sessions per S) were averaged to yield an overall acquisition score on each dependent variab~. Thus, one observation per S on each dependent measure for acquisition and extinction were subjected to sTparate, mixed-design ANOVAs (acquisition drug x extinction drug x subject). HR data for each S for each experimental session were f i r s t transformed into change-from-baselin--e scores, i . e . mean pre-CS HR was subtracted from each of the eight, one-sec post-CS blocks. Acquisition data were then averaged as above and acquisition and extinction data were subjected to separate, mixed-design ANOVAs (acquisition drug x extinction drug x block x subj e c t ) . EB and HR data from the post-extinction session were compared to those
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from the session immediately preceeding extinction, using mixed-design AHOVAs which included session as a variable. Results Acquisition: During acquisition naloxone administration had no significant effect on percent EB, EB amplitude, EB latency, or t r i a l s to reach the 2-day EB CR criterion. However, heart rate change-from-baseline, v i z . the HR CR, was affected by the drug treatment in that naloxone attenuated the bradycardiac HR CR [F(1,36)=4.00, p=O.05 for the main effect of acquisition drug]. As can be seen in Fig. l , naloxone-treated animals showed smaller decelerations in later blocks (blocks 3-8) due to an earlier return toward baseline beginning 3-4 sec following CS onset; the HR CR in saline-treated animals reached i t s maximum bradycardia at about this time. This drug-induced difference in HR topography was s t a t i s t i c a l l y significant [F(7,252)= 2.26, p
olld~ I
III,"I01oli
0
I+ .
-110
FIG. I. Moan change-from-baseline HR following CS onset during acquisition, as a function of drug treatment.
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FIG. 2 Mean percent of t r i a l s (± 1 SEM) on which EB CR's occurred during acquisition and extinction as a function of acquisition and extinction drug. Open and shaded bars indicate data collected following saline and naloxone administration, respectively.
effect occurred both in animals treated with saline and in those treated with naloxone during acquisition. These differences are shown in Fig. 2. Statistical analysis of percent EB data showed that these differences were reliable [F(1,36)=5.26, p
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Discussion Administration of naloxone during acquisition failed to affect development of the EB CR ( t r i a l s - t o - c r i t e r i o n ) , percent blinks, or the topography of the EB response (amplitude or latency). However, naloxone treatment resulted in a higher EB CR percentage during e x t i n c t i o n . This effect occurred both in animals that had received naloxone and in animals that had received saline injections during acquisition, indicating that i t was not due to state-dependent learning (19). Naloxone treatment during e x t i n c t i o n did not, however, d i f f e r e n t i a l l y affect EB performance on the following day, suggesting that extinction of the EB CR occurred in naloxone-treated animals despite enhanced responding during the extinction session. The results concerning the concomitantly conditioned HR CR show that naloxone administration attenuated the bradycardiac HR CR accompanying aversive Pavlovian EB conditioning. Thefailure of naloxone to affect baseline HR supports the contention that naloxone affects the HR response (or HR change) and not cardiovascular a c t i v i t y in general. This finding has previously been reported for rabbits (20) as well as other species (21-23). The persistence of the naloxone-induced attenuation of bradycardia throughout late as well as early acquisition, and during the post-extinction session, further suggests that the effect of naloxone was not restricted to the large HR decelerations which occur in response to i n i t i a l presentations of the CS (viz., the cardiac component of the orienting response; 24). Moreover, attenuation of the bradycardiac HR CR by naloxone was not accompanied by accelerated development of tachycardiac "defense" responses (24), which may occur in the present behavioral s i t u a t i o n after EB responding has reached a s j ~ t o t i c levels (25,26). This r e s u l t would seem to argue against interpretations of the naloxone-induced effect on HR in terms o~ sensitization. However, this p o s s i b i l i t y cannot be ruled out on the basis of the present data since controls for pseudoconditioning and s e n s i t i z a tion (e.g., groups of animals exposed to unpaired tone-shock presentations) were not employed. The finding that the HR CR in naloxone-treated animals did not decrease in magnitude between the last acquisition session and the postextinction session is also noteworthy in t h i s context. The present results thus suggest that naloxone specifically affected conditioned HR deceleration, and may indicate an involvement of endogenousopioid systems in mediating t h i s conditioned response. Further investigations w i l l obviously be necessary to verify these findings. The present results regarding somatomotor conditioning are consistent with previous reports of the effects of opiate agonists and antagonists on aversively motivated learned responses. Gallagher & Kapp (4) reported that p o s t - t r i a l administration of naloxone into the amygdala facilitated retention of a passive avoidance task in rats, whereas, administration of levorphanol decreased retention. Messing et al. (7) also reported that systemically administered naloxone f a c i l i t a t e d retention of both passive and active avoidance learning in r a t s , and that the effect of naloxone on passive avoidance could be antagonized by morphine. These investigators also found no effects of naloxone during acquisition of active avoidance, in accordance with the present results. Izquierdo (8) has also reported f a c i l i t a t i o n of retention of several tasks by posttraining administration of naloxone, including aversively-motivated shuttle-box responses acquired using a classical conditioning procedure. The f a c i l i t a t o r y effects of post-training naloxone administration have been interpreted as a f a c i l i t a t i o n of memory consolidation (4,7,8). The present results demonstrate that naloxone can also enhance performance of a classically conditioned somatic response when administered prior to extinction testing, suggesting that endogenous opioids may be involved in more than one aspect of learned behavioral
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change. Facilitation of extinction of active avoidance by Y-endorphin has also been reported (6,27), although other opioid peptides delayed e x t i n c t i o n . I t is of note here that a related peptide, ACTH 4-10, i n h i b i t s e x t i n c t i o n of active avoidance and f a c i l i t a t e s retention of passive avoidance (3,28). ACTH 4-I0 has also been found to have specific opiate antagonist properties (29). Fanselow (30) has shown that naloxone attenuates rats' preference for signalled vs unsignalled shock when administered p r i o r to t r a i n i n g , and has suggested that a conditioned release of endogenous opioids might occur in anticipation of an aversive stimulus and act to attenuate its aversiveness. While this hypothesis might explain increased EB responding in animals treated with naloxone only during extinction, i t is not clear why similar increased responding also occurred in animals treated with naloxone during both acquisition and e x t i n c tion. Similarly, the hypothesis that naloxone increased responding due to increased reactivity of the animals to the US during acquisition does not appear consistent with the lack of effect of naloxone on acquisition of the EB response; i . e . , a f a c i l i t a t i o n of EB conditioning due to a f u n c t i o n a l l y more intense US might be expected. Investigation of the effects of chronic naloxone treatment, and of naloxone's effects on s e n s i t i v i t y to shock and overall responsivity in the rabbit, will therefore be required before firm conclusions can be reached regarding the specific nature of naloxone-induced enhancement of somatic responding during extinction. Acknowledgements This work was supported by VA I n s t i t u t i o n a l Research funds. We thank S h i r l e y L. Buchanan f o r t e c h n i c a l assistance and c o n s u l t a t i o n , and James B. Appel for generously providing the naloxone. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
A.J. KASTIN, E.L. SCOLLAN, M.G. KING, A.V. SCHALLY & D.H. COY, Pharmacol. Biochem. & Behav. 5691-695 (1976). J.D. BELLUZZI & L.--STEIN, Soc. Neurosci. Abstr. 3230 (1977). D. DE WIED, B. BOHUS, J.M. VAN REE & I. URBAN, 7. Pharmacol. Exp. Ther. 204570-580 (1978). l~T-.GALLAGHER & B.S. KAPP, Life Sci. 231973-1978 (1978). H. RIGTER, Science 20083-85 (1978) M. LE MOAL, G.F. IE=OI)B & F.E. BLOOM, Life Sci. 241631-1636 (1979). R.B. MESSING, R.A. JENSEN, J.L. MARTINEZ, V.R. SPT[HLER, B.J. VASQUEZ, B. SOUMIREU-MOURAT, K.C. LIANG & J.L. MC GAUGH, Behav. Neural Biol. 2__77 266-275 (197g). I. IZQUIERDO, Psychopharmacol. 66199-203 (1979). N. WHITE, R. MAJOR& J. SIEGEL,~ife Sci. 231967-1972 (1978). M.D. MORRIS & G.F. GEBHART, Psychopharmaco1.---57267-27] (1978). J. ROSSIER, T. VARGO, S. MINICK, N. LING, F. ~OOM & R. GUILLEMIN, Proc. Natl. Acad. Sci. USA 745162-5165 (1977). C. GROS, P. PRADELLES~--,C. ROUGET, O. BEPOLDIN, F. DRAY, M.C. FOURNIEZALUSKA, B.P. ROQUES, H. POLLARD, C. LLORENS-CORTES, & J.C. SCHWARTZ, J. Neurochem. 3129-39 (1978). O. JOHANSS'071, T. HOKFELT, R.P. ELDE, M. SCHULTZBERG, & L. TERENIUS, Advances in Biochemical Ps~chopharmacology, 1851-70 (1978). H.Y.T. YANG, J.S. HONG, W. FRA1-FA& E. COSTA-~-Advances in Biochemical Ps~chopharmacolo~ 18149-15g (1978). B.S. KAPP, R.C. TITYSINGER, M. GALLAGHER& A.J. BRETSCHNEIDER, Soc. Neurosci. Abstr. 3236 (1977). J. FRANCIS, S-~ BUCHANAN & D.A. POWELL, Soc. Neurosci. Abstr. 3232 (1977).
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17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
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D.A. POWELL, D. MANKOWSKI & S.L. BUCHANAN, Physiol. & Behav. 20 143-150 (1978). n D.A. POWELL, W.L. MILLIGAN & S.L. BUCHANAN, Physiol. & Behav. 17 955-962 (1976). D . A . OVERTON, Psychopharmacology, A Review of Progress, 1957-1967~ D.H. Efron, J.O. Cole, J. Levine & R. Wittenborn (eds) U.S. Gov't Printing Office, Washington, D.C. 918-930 (1968). J.C. SCHADT & D.H. YORK, Fed. Proc. 39 843 (1980). G.L. BELENKY & J.W. HOLADAY, Brain R~. 177 414-417 (1979). W.L. DEWEY, Advances in Biochemical Ps~cIT~narmacolo~ 8 263-272 (1973). J.C. WILLER, F. BOUREAU, C. DAUTHIER & M. BONORA, ~europharmaco] I_88 469-472 (1979). F.K. GRAHAM& R.K. CLIFTON, Psych. Bull. 65, 305-320 (1966). D.A. POWELL, M. LIPKIN & W.L. MILLIGAN, L~rn. & Motiv. 5 532-547 (1974). D.A. POWELL & E. KAZIS, Psychophysiology 13 441-447 (19%). D. DE WIED, G.L. KOVACS, B. BOHUS, J.M. ~ N REE & H.M. GREVEN, Eur. J. Pharmacol. 49 427-436 (1978)~ F. RAMAEKER~,H. RIGTER & B.E. LEONARD, Pharmacol. Biochem. & Behav. 8 547-551 (1978). F.C. COLPAERT, C.J. NIEMEGEERS, P.A. JANSSEN, J.M. VAN REE & D. DE WIED, Psychoneuroendocrinology 3 203-210 (1978). M.S. FANSELOW, Physiol. PTychol. ~.70-74 (Ig79).
Pergamon P r e s s
EFFECTS OF NALOXDNEON PAVLOVIAN CONDITIONINGOF EYEBLINK AND HEARTRATE RESPONSES IN RABBITS Linda L. llern6ndez and D. A. Powell Neuroscience Laboratory, Wm. Jennings Bryan Dorn Veterans' Hospital Columbia, SC 29201 and Dept. Psychology, University of South Carolina, Columbia, SC 29208 (P.eceived i n f i n a l
form June 30, 1980)
Summar~ Albino rabbits were subjected to aversive Pavlovian conditioning and extinction of eyeblink and heart rate responses. Naloxone administration had no effect on acquisition of the eyeblink response ~ut increased responding during extinction. Naloxone also attenuated the bradycardiac heart rate CR, suggesting that endogenous opioids may be involved in mediating this response. Several recent experiments have suggested that endogenousopioid systems may be involved in learning and/or memory ( I - 8 ) . Such an involvement is consistent with the fact that exogenous opiates affect learning (9,10), and that endogenous opioids and opiate receptors in the brain are localized in areas which are known to be involved in learning (11-14). Many of these same brain areas have been implicated in the control of c l a s s i c a l l y conditioned somatic and autonomic responses in rabbits (15-18). I t therefore seemed reasonable to inquire whether endogenous opioid systems are involved in this control. I f opioid systems participate in Pavlovian conditioning processes in the rabbit, then administration of specific opiate antagonists, such as naloxone, should produce behavioral effects in this paradigm. The present experiment was undertaken to investigate this possibility, by examining the effects of systemically administered naloxone on aversive Pavlovian conditioning and extinction of eyeblink (EB) and heart rate (HR) responses in r a b b i t s . In addition, the present experiment was designed to assess possible statedependent learning (19) following naloxone and to examine the effects of naloxone treatment during extinction on subsequent performance of conditioned EB and HR responses. Method
Subjects. Forty-one experimentally naive male albino rabbits (Oryctolagus cunlcu]us) obtained from Hayes Rabbitry, West Columbia, SC were used as subjects. All animals werekept drug-free for at least f i v e days p r i o r to the beginning of behavioral testing, which was conducted between 8 a.m. and l p.m. Apparatus. Behavioral testing occured in four v e n t i l a t e d , sound and l i g h t attenuating animal enclosures equipped with overhead speakers for d e l i v e r y of auditory s t i m u l i . During testing the animals were restrained in standard 0024-3205/80/360863-07502.00/0
864
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plexiglass rabbit restrainers. All experimental events were controlled from an adjoining room by a DEC-PDP 11/10 minicomputer, which also collected and summarized the behavioral data directly from a Grass Model 7-D polygraph. Procedure: Each conditioning t r i a l consisted of the presentation of the conditioning stimulus (CS), immediately followed by the unconditioned stimulus (US). The CS was a 1216 Hz, 75 dB square-wave tone, 500 msec in duration. The US was a 3 mA, 500 msec, AC paraorbital electric shock. In each session the US was omitted on seven evenly-spaced "test" t r i a l s during which HR was measured. The i n t e r t r i a l interval was 45 seconds. The EB response (consisting of eyelid closure, eyeball retraction, and/or nict i t a t i n g membrane extension) was measured on each t r i a l via stainless-steel electrodes inserted underneath the eyelids. An EB response was defined as a change of at least 200 uV from pre-CS baseline across the recording electrodes (2 mm pen deflection), occurring during the 500 msec CS i n t e r v a l . HR was recorded for 4 secs prior to and eight secs after CS onset via stainless-steel safety pins inserted subcutaneously over the right shoulder and left flank. At the end of each daily session mean EB amplitude and latency, percent t r i a l s on which EB responses occurred, t r i a l s - t o - c r i t e r i o n , and mean HR for pre-CS and post-CS seconds were calculated for each S and stored for later analysis. During acquisition each session was continued until Ss reached a c r i t e r i o n of lO consecutive t r i a l s on which an EB response occur'~ed, or u n t i l lO0 t r i a l s were delivered. Daily acquisition sessions were conducted until Ss attained a multiple two-day c r i t e r i o n which consisted of: (a) reaching--the IO-EB CR criterion within lO0 t r i a l s on the f i r s t day, and (b) reaching the IO-EB CR criterion within the f i r s t 30 t r i a l s on the following day. Animals f a i l i n g to reach this two-day criterion within lO consecutive days were dropped from the experiment. One S was dropped for this reason. On the day following a t t a i n ment of the a c q u ~ i t i o n c r i t e r i o n Ss were tested for 30 e x t i n c t i o n t r i a l s during which the tone, unpaired with--the US, was presented. On the following day Ss again received up to lO0 paired tone-shock t r i a l s to assess whether nal~one treatment during extinction would affect subsequent performance of EB and HR responses. During acquisition two groups of 20 Ss each received either naloxone or saline prior to each session. During extinTtion these two groups were divided into 4 sub-groups of 10 Ss each; half the Ss in each drug group received naloxone and half received salTne. On the day ~llowing extinction each S received the same drug administered during acquisition. The drug injections ~nsisted of either (a) l.O mg/kg of naloxone HCl (Endo) in a volume of 0.33 ml/kg (approximately 1.0 ml per rabbit) or (b) an equivalent volume of 0.9% (w/v) NaCI ( s a l i n e ) . Intravenous injections were given into the lateral ear vein, each ear being used on alternate days. The time between injection and the f i r s t t r i a l of the experimental session was held constant at 15 min. The EB data for all acquisition sessions (2-10 sessions per S) were averaged to yield an overall acquisition score on each dependent variab~. Thus, one observation per S on each dependent measure for acquisition and extinction were subjected to sTparate, mixed-design ANOVAs (acquisition drug x extinction drug x subject). HR data for each S for each experimental session were f i r s t transformed into change-from-baselin--e scores, i . e . mean pre-CS HR was subtracted from each of the eight, one-sec post-CS blocks. Acquisition data were then averaged as above and acquisition and extinction data were subjected to separate, mixed-design ANOVAs (acquisition drug x extinction drug x block x subj e c t ) . EB and HR data from the post-extinction session were compared to those
Vol. 27, No. i0, 1980
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from the session immediately preceeding extinction, using mixed-design AHOVAs which included session as a variable. Results Acquisition: During acquisition naloxone administration had no significant effect on percent EB, EB amplitude, EB latency, or t r i a l s to reach the 2-day EB CR criterion. However, heart rate change-from-baseline, v i z . the HR CR, was affected by the drug treatment in that naloxone attenuated the bradycardiac HR CR [F(1,36)=4.00, p=O.05 for the main effect of acquisition drug]. As can be seen in Fig. l , naloxone-treated animals showed smaller decelerations in later blocks (blocks 3-8) due to an earlier return toward baseline beginning 3-4 sec following CS onset; the HR CR in saline-treated animals reached i t s maximum bradycardia at about this time. This drug-induced difference in HR topography was s t a t i s t i c a l l y significant [F(7,252)= 2.26, p
olld~ I
III,"I01oli
0
I+ .
-110
FIG. I. Moan change-from-baseline HR following CS onset during acquisition, as a function of drug treatment.
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FIG. 2 Mean percent of t r i a l s (± 1 SEM) on which EB CR's occurred during acquisition and extinction as a function of acquisition and extinction drug. Open and shaded bars indicate data collected following saline and naloxone administration, respectively.
effect occurred both in animals treated with saline and in those treated with naloxone during acquisition. These differences are shown in Fig. 2. Statistical analysis of percent EB data showed that these differences were reliable [F(1,36)=5.26, p
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Discussion Administration of naloxone during acquisition failed to affect development of the EB CR ( t r i a l s - t o - c r i t e r i o n ) , percent blinks, or the topography of the EB response (amplitude or latency). However, naloxone treatment resulted in a higher EB CR percentage during e x t i n c t i o n . This effect occurred both in animals that had received naloxone and in animals that had received saline injections during acquisition, indicating that i t was not due to state-dependent learning (19). Naloxone treatment during e x t i n c t i o n did not, however, d i f f e r e n t i a l l y affect EB performance on the following day, suggesting that extinction of the EB CR occurred in naloxone-treated animals despite enhanced responding during the extinction session. The results concerning the concomitantly conditioned HR CR show that naloxone administration attenuated the bradycardiac HR CR accompanying aversive Pavlovian EB conditioning. Thefailure of naloxone to affect baseline HR supports the contention that naloxone affects the HR response (or HR change) and not cardiovascular a c t i v i t y in general. This finding has previously been reported for rabbits (20) as well as other species (21-23). The persistence of the naloxone-induced attenuation of bradycardia throughout late as well as early acquisition, and during the post-extinction session, further suggests that the effect of naloxone was not restricted to the large HR decelerations which occur in response to i n i t i a l presentations of the CS (viz., the cardiac component of the orienting response; 24). Moreover, attenuation of the bradycardiac HR CR by naloxone was not accompanied by accelerated development of tachycardiac "defense" responses (24), which may occur in the present behavioral s i t u a t i o n after EB responding has reached a s j ~ t o t i c levels (25,26). This r e s u l t would seem to argue against interpretations of the naloxone-induced effect on HR in terms o~ sensitization. However, this p o s s i b i l i t y cannot be ruled out on the basis of the present data since controls for pseudoconditioning and s e n s i t i z a tion (e.g., groups of animals exposed to unpaired tone-shock presentations) were not employed. The finding that the HR CR in naloxone-treated animals did not decrease in magnitude between the last acquisition session and the postextinction session is also noteworthy in t h i s context. The present results thus suggest that naloxone specifically affected conditioned HR deceleration, and may indicate an involvement of endogenousopioid systems in mediating t h i s conditioned response. Further investigations w i l l obviously be necessary to verify these findings. The present results regarding somatomotor conditioning are consistent with previous reports of the effects of opiate agonists and antagonists on aversively motivated learned responses. Gallagher & Kapp (4) reported that p o s t - t r i a l administration of naloxone into the amygdala facilitated retention of a passive avoidance task in rats, whereas, administration of levorphanol decreased retention. Messing et al. (7) also reported that systemically administered naloxone f a c i l i t a t e d retention of both passive and active avoidance learning in r a t s , and that the effect of naloxone on passive avoidance could be antagonized by morphine. These investigators also found no effects of naloxone during acquisition of active avoidance, in accordance with the present results. Izquierdo (8) has also reported f a c i l i t a t i o n of retention of several tasks by posttraining administration of naloxone, including aversively-motivated shuttle-box responses acquired using a classical conditioning procedure. The f a c i l i t a t o r y effects of post-training naloxone administration have been interpreted as a f a c i l i t a t i o n of memory consolidation (4,7,8). The present results demonstrate that naloxone can also enhance performance of a classically conditioned somatic response when administered prior to extinction testing, suggesting that endogenous opioids may be involved in more than one aspect of learned behavioral
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change. Facilitation of extinction of active avoidance by Y-endorphin has also been reported (6,27), although other opioid peptides delayed e x t i n c t i o n . I t is of note here that a related peptide, ACTH 4-10, i n h i b i t s e x t i n c t i o n of active avoidance and f a c i l i t a t e s retention of passive avoidance (3,28). ACTH 4-I0 has also been found to have specific opiate antagonist properties (29). Fanselow (30) has shown that naloxone attenuates rats' preference for signalled vs unsignalled shock when administered p r i o r to t r a i n i n g , and has suggested that a conditioned release of endogenous opioids might occur in anticipation of an aversive stimulus and act to attenuate its aversiveness. While this hypothesis might explain increased EB responding in animals treated with naloxone only during extinction, i t is not clear why similar increased responding also occurred in animals treated with naloxone during both acquisition and e x t i n c tion. Similarly, the hypothesis that naloxone increased responding due to increased reactivity of the animals to the US during acquisition does not appear consistent with the lack of effect of naloxone on acquisition of the EB response; i . e . , a f a c i l i t a t i o n of EB conditioning due to a f u n c t i o n a l l y more intense US might be expected. Investigation of the effects of chronic naloxone treatment, and of naloxone's effects on s e n s i t i v i t y to shock and overall responsivity in the rabbit, will therefore be required before firm conclusions can be reached regarding the specific nature of naloxone-induced enhancement of somatic responding during extinction. Acknowledgements This work was supported by VA I n s t i t u t i o n a l Research funds. We thank S h i r l e y L. Buchanan f o r t e c h n i c a l assistance and c o n s u l t a t i o n , and James B. Appel for generously providing the naloxone. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
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