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ACTH 4–10 on the classically-conditioned rabbit nictitating membrane
Peptides. Vol. 3, pp. 715-719, 1982. Printed in the U.S.A.
Effects of M S H / A C T H A l0 on the Classically-Conditioned Rabbit Nictitating Membrane CATHERINE
A. S C H A E F F E R , 2 D O U G L A S C R A I G F. C E G A V S K E
C. N A T H A N S O N ,
Department of Psychology and Center for Neurobehavioral Sciences State University of New York, Binghamton, N Y 13901 AND RICHARD
A. R O E M E R
Department of Psychiatr3', Temple University Henry Avenue and Abbott~sford Road, Philadelphia, PA 19129 R e c e i v e d 22 M a r c h 1982 SCHAEFFER, C. A., D. C. NATHANSON, C. F. CEGAVSKE AND R. A. ROEMER. Effects of MSH/ACTH 4-10 on the classically-conditioned rabbit nictitating membrane. PEPTIDES 3(5) 715-719, 1982.--Subjects were conditioned/extinguished under four experimental conditions using either MSH/ACTH 4-10 (A) or diluent (D): D/D, D/A, A/D. and A/A. The major question investigated was whether or not the peptide has an effect on this classically-conditioned behavior similar to that reported for instrumental conditioning paradigms. The results indicated that it does not. An effect was seen on performance, not on learning or attentional processes. Animals treated with the peptide performed more poorly (i.e., displayed fewer conditioned responses) during both acquisition and extinction. In addition, there was an apparent residual effect of the peptide that lasted 24 but not 48 hours. MSH ACTH 4-10 Nictitating membrane
Peptides
Attention
Learning
T H E 4-10 fragments of melanocyte stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) produce at least one consistent behavioral effect in mammals. The substances prolong the extinction of learned responses in instrumental conditioning paradigms (see e.g., [1, 3, 4]. The effect of these substances has not been demonstrated in classically-conditioned behaviors. We report here the effects of M S H / A C T H 4--10 on such a behavior in rabbits. The behavioral parameters of the rabbit nictitatingmembrane (NM) preparation have been extensively studied by Gormezano and his associates (see e.g., [5, 6, 7]). These parameters are well understood and reflect many of the advantages that this preparation has for studying the neurophysiological and neuroanatomical substrates of the conditioned behavior (see [2] and [10] for discussions). Consequently, if a peptide effect could be demonstrated in this preparation, it might be possible eventually to discover the sites of peptide action on the conditioned behavior.
Classical conditioning
Rabbit
In addition, Sandman et al. [9] have proposed that the prolonged extinction effects seen in instrumental conditioning paradigms may be a result of the peptide enhancing attention. If this is correct, then one would expect a behaviorally opposite effect in the extinction phase of classical conditioning. In the instrumental situation, the subject, being more attentive to the CS and its consequences, would respond more consistently to the CS and therefore discover less rapidly that the reinforcer (UCS) was no longer being presented. On the other hand, in a classical-conditioning paradigm, the CS and UCS are always presented, so the subject would quickly discover that the UCS was no longer being given and the extinction phase would be shortened. Thus, there were two reasons why it was of interest to determine if M S H / A C T H 4--10 has an effect on the behavior of the classically-conditioned rabbit NM preparation: If an effect could be demonstrated, (1) The NM preparation might allow sites of peptide action to be discovered, and (2)
~Requests for reprints should be addressed to Craig Cegavske, whose current address is Department of Biology, State University of New York, Binghamton. NY 13901. 2These data were submitted in partial fulfillment of the requirements for C. A. Schaeffer's M. A. thesis from SUNY-Binghamton. Her current address is 309 Fleming Road. Cincinnati, OH 45215.
C o p y r i g h t ~ 1982 A N K H O
I n t e r n a t i o n a l Inc.--0196-9781/82/050715-05503.00/0
S C H A E F F E R ET AL.
716 I00,
M S H / A C T H 4-10 might allow additional information to be gained about inferred processes (e.g., attention) at work in the rabbit NM preparation.
90
80
•
ACTH 4-10
o
DILUENT
METHOD 70
The design of the present study was a simple 2×2 factorial to assess the effect of M S H / A C T H 4-10 during acquisition and extinction of the classically conditioned response.
A nim a ls Thirty-two male, New Zealand white rabbits weighing 1.8-3.2 kg were used as subjects. They were housed individually under a 12-hour light-dark cycle (lights on at 0800) with ad lib access to food and water. The subjects were divided into four groups and trained/ extinguished on M S H / A C T H 4-10 (A) or diluent (D): D/D (n=8), D/A (n=9), A/D (n=8), and A/A (n=7).
Apparatus The apparatus and procedure used for conditioning were similar to those described in detail by Gormezano [5]. Animals were conditioned or extinguished three at a time in three separate lighted and ventilated chambers. Inside the chambers each subject was restrained in a Plexiglas box and fitted with a leather head harness which held the airpuff delivery system and the nictitating membrane transducer. Eyeclips were used to hold the eyelids open during training. The conditioned stimulus (CS) was a 350 msec, I kHz, 85 dB tone; the unconditioned stimulus (UCS) was an airpuff gated from a source pressurized to 5 psi. The nozzle was positioned about 10 mm from the cornea. The UCS overlapped the final 100 msec of the CS providing a 250 msec interstimulus interval (ISI). The intertrial interval (ITI) was a pseudorandom sequence of 50, 60, or 70 seconds, with an average of 60 seconds.
Conditioning Procedure Prior to training, the left eye was anesthetized with a local anesthetic (Opthaine) and a snap (for transducer attachment) was sutured to the NM. After suturing, animals were allowed at least 24 hr recovery time. They were then placed in the experimental chamber, with all apparatus connected, for two hours in order to habituate to the setting and apparatus. The following day acquisition training was started. Two days of acquisition training (paired CS and UCS) were followed by two days of extinction training CS and (CS alone). There were 104 trials per day. Approximately 30 minutes prior to training, each animal was injected subcutaneously in the back with 80/zg/kg of M S H / A C T H 4-10 dissolved in 0.4 cc of diluent (0.01 M acetic acid in 0.9% saline), or with 0.4 cc of diluent alone. After the injection the animals were left alone in the experimental chambers for about 20 minutes. The transducer and eyeclips were then attached, the airpuff nozzle positioned properly, and the training session started. Running time was approximately 2V2-3 hours from injection to the end of the session for that day.
Scoring Criterion The data were manually scored from the polygraph records. One-half millimeter of NM closure was defined as a
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FIG. 1. A c o m p a r i s o n of the acquisition of conditioned behavior for
animals treated with MSH/ACTH 4-10 (groups A/D and A/A combined) and diluent (groups D/D and D/A combined). In this and subsequent figures, each trial block contains 8 trials.
response, and latencies were measured from a CS-onset marker pulse to this point on the NM response curve. During acquisition a response was classified as "'conditioned" only if it occmTed within 250 msec of CS onset (CS to UCS interval was 250 h,sec). During extinction, any response occurring within 500 msec of the CS marker was considered " c o n d i t i o n e d " since no UCS was given.
Dependent Variable For analysis purposes, trials were grouped into blocks of 8 to produce 13 trial-blocks per day. Trials with pre-CS activity of the NM were considered uncodeable, and were deleted from the analysis. A small number of trials were uncodeable because of equipment failure, and were also deleted. In all, 3% of the trials were discarded. The number of conditioned responses (CRs) in each block was divided by the number of codeable responses in that block to produce a percent-CR measure. RESULTS
Acquisition and Extinction A major result of the present study was that animals treated with M S H / A C T H 4-10 performed more poorly than diluent-treated controls during both acquisition and extinction. Figure 1 shows the CR acquisition curves for the diluent and the M S H / A C T H 4-10 treated groups. Throughout the two days of training, the peptide group produced a lower percentage of conditioned responses than the diluent treated group. We performed a three-way split-plot A N O V A on the acquisition data with the treatment (diluent or peptide) as the between subjects factor; and two levels of within subjects factors, one factor being days (one and two), and the second factor the thirteen trial-blocks per day. This analysis yielded a significant treatment effect, F(1,30)=5.36, p<0.03. As expected, there was also a significant days effect, a trial block effect, and a days by trial-block interaction, but there were
MSH/ACTH 4-10 AND RABBIT NM C O N D I T I O N I N G
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no two or three level interactions which were related to the drug treatment. To evaluate the effects of the peptide on extinction of the conditioned response, we pooled all animals receiving MSH/ACTH 4--10 during extinction (D/A and A/A groups) and compared them to the animals receiving diluent during acquisition and extinction (D/D). (The A/D group was not pooled with the D/D group because of the apparent carryover effect of the peptide discussed below.) Prior to performing such an ANOVA, we tested to confirm that this pooling was statistically justified. A two-way split plot ANOVA comparing extinction performance of animals trained and extinguished on the peptide (A/A) with those trained on diluent and extinguished on the peptide (D/A) yielded a nonsignificant between subjects F(l, 14)= 1.052. Figure 2 depicts the extinction curves for days one and two of the D/D and the pooled A/A and D/A groups. There was an immediate drop in the level of conditioned responding with administration of the peptide at the beginning of extinction (the drop occurred on the first trial of the block), and this difference was maintained throughout extinction. A three-factor split-plot ANOVA yielded a significant treatment (D vs A) effect, F(I,22)=10.938, p=0.003, both days and trial-block main effects, and a significant treatment by trial-block interaction, F(12,264)= 1.85, p =0.04. Unlike the acquisition data, a marginally significant difference in rate was present in extinction. The peptide group extinguished slightly slower than the diluent group. Examination of the day-two data revealed that this was probably the result of a floor effect operating in the peptide group on day two (see Figs. 2 and 3). To aid in interpreting this interaction, two-factor split-plot ANOVAs (treatment by trial-block) were performed separately for days one and two. Each ANOVA revealed a main effect of treatment but only on day two was there a treatment by trial-block interaction, F(12,264)=1.823, p=0.045. Therefore, the treatment by trial-block interaction in the three-way analysis and the daytwo analysis may not reflect a peptide effect.
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DAY I
DAY 2
E X T I N C T I O N T R I A L BLOCKS FIG. 3. The residual effect of MSH/ACTH 4-10 is illustrated. Both groups trained under the peptide (A/D and A/A) start extinction at a level lower than the group trained with diluent (D/D) regardless of the treatment given in extinction. The group switched from the peptide in acquisition to diluent during extinction (A/D) does not extinguish as fast as the group remaining on the peptide (A/A); and, by the end of day 2, is indistinguishable from the group trained and extinguished on the peptide (D/D).
Caro,-Over Lffect of MSH/A CTH 4-10 The data presented in Fig. 3 show the A/D group beginning extinction with a response rate similar to that of the A/A group. Then, during the course of extinction, the response rate appears to have shifted away from that of the peptide treated group and toward the rate of the diluent treated group (D/D). When a mean (percent CRs) of all trials during the first day of extinction was taken for each group shown in Fig. 3, the A/D group was found to lie about halfway between the other two (the means were 72.6 for D/D, 53.9 for A/D, and 37.8 for A/A). No significant overall extinction differences were present between the D/D and the A/D groups. A twoway split-plot ANOVA yielded a nonsignificant between subjects F(1,14)=0.58. However, an additional split-plot ANOVA for the first four extinction-trial blocks was performed with the between groups factor being subjects treated with either diluent (D/D) or MSH/ACTH 4-10 (A/D) during acquisition. The analysis yielded a marginally significant result, F(1,14)=4.602, p<0.05. These data suggest that the peptide had a residual effect on response performance lasting at least 24, but less than 48, hours. Although there was an immediate drop in responding from acquisition to extinction in the D/A group, no comparable increase in responding, indicating a release from peptide effects, was seen in the A/D group (see Table l).
Latency of Conditioned Responses During acquisition, there were no significant CR latency differences between the diluent and peptide treated groups. The slight trend toward shorter latencies by the diluent treated group seen in Fig. 4 was expected as these animals showed higher levels of conditioned responding (see Fig. 1). It is characteristic, in this preparation, for CR latencies to shorten as conditioned behavior improves.
718
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PERCENT AND
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FOR FOUR
LAST
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BLOCKS
SCHAEFFER ET AL.
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ACQUISITION
o DILUENT
EXTINCTION
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Extinction
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During extinction there were insufficient responses to allow valid statistical evaluation. However, inspection of the data indicated no unexpected CR latency differences as a function of peptide treatment.
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DAY 2 T R I A L BLOCKS
DISCUSSION
The results of the present experiment indicate that M S H / A C T H 4-10 had an effect on performance factors alone rather than on learning or attention. In contrast to previous reports employing instrumental avoidance paradigms, CR performance in extinction was depressed, not raised, in the present classical-conditioning situation. In addition, the peptide depressed performance during acquisition. A number of observations suggest that these changes were due to the peptide's action, but that the effect was n,,t on learning or attentional processes. If learning had been effected, a difference in the rate t~f acquisition should have been seen. No such difference was observed. The rate of acquisition of CRs was the same for both groups after a delayed onset in the peptide group Therefore, it can be inferred that the actual mecharisr, ',:i underlying the learning of the conditioned behavior ,~as (were) not being affected by the drug during the learning process. In addition, both groups given extinction training on diluent (D/D and A/D in Fig. 3) had the same final level of CR performance at the end of extinction. This also suggests that the actual level of learning was similar for the two groups at the end of acquisition despite the lower level of performance by the A/D group at that time. A clear and immediate decrease in performance was seen in the subjects extinguished on the peptide following training on diluent. Scrutiny of the individual trials within the first block of extinction confirmed this. The immediate loss of conditioned responding after peptide administration strongly suggests that the peptide had its effect on performance not on the learning process per se. This is compelling for two reasons: (1) The level of CR performance at the end of acquisition was almost identical in the two groups trained on diluent (D/D and D/A in Table 1), and (2) the subsequent rate of decrement of CR performance was similar for subjects treated with peptide and diluent (see Fig. 2) indicating that actual extinction was progressing at about the same rate. There was no reciprocal change in the group released from the peptide (A/D). That group did not immediately begin performing at the level of the D/D group. In fact, there was little difference between the peptide and diluent treated groups (excluding D/D) in the first four blocks of extinction (see Table 1). A performance effect is, again, suggested because the final level of extinction was the same for the D/D and A/D groups. The rate of CR performance was depressed
FIG. 4. The latencies of group responses are compared for groups trained on MSH/ACTH 4-10 (groups A/D and A/A combined) and diluent (groups D/D and D/A combined).
during acquisition under the peptide and this depressi~m was carried over into extinction. The group that was rcleas~'d from the peptide during extinction (A/D) showed a much slower ~ate of extinction during day one than did the A/A group or the D/D group (Fig. 3). But on day 2, t h A / D group end, d at the ,,ame performance level as the D/I) ,.~up. "I his r c ,lit can most easily be explained by assuming a "'wearing ~)ff" of some effect of the peptide i~ ~:veal the true level of :xtinction. The conditioned behavior must have been learned despite the M S H / A C T H 4-10 treatment during acquisition. If increased attention to the stimuli had operated, one might have expected an accelerated rate of acquisition and/or an earlier onset of CR behavior. Either of these effects could produce a relatively higher level of final CR performance if the level of attention were maintained throughout the training session. The data suggest that this did not occur: the rate of acquisition was no different from controls. and the onset of CR performance was delayed, not made earlier, leading to a lower level of final performance at the end of acquisition. If attention levels were increased by the peptide in extinction, an increase in the rate-of-loss of CR performance should have been observed. This did not occur within the first 8 trials or even during the first day. The extinction rate was essentially the same for both the experimental and control groups on day 1. There appeared to be an accelerated rate during the first few blocks of day 2 of extinction. However, this was partly a result of one animal in the A/A group which responded at a high rate in the first block and thereby raised that data point. Even ignoring this, the result could not be easily explained as an attentional effect without explaining why it did not happen on day 1 of extinction as well. All of the data reported here argue against the idea that MSH/ACTH 4-10 improves the learning process or increases levels of attention in this classically-conditioned preparation. How the peptide might affect performance variables is not clear. It could have caused some sort of malaise in the animals. However, casual observation of the animals during
MSH/ACTH 4-10 AND RABBIT NM CONDITIONING
the e x p e r i m e n t s u g g e s t e d no d i f f e r e n c e s in e i t h e r t h e i r app e a r a n c e or general b e h a v i o r ( a p p a r e n t a l e r t n e s s , a c t i v i t y level, etc.). A s e c o n d possibility w o u l d be a d o s e effect. It is not k n o w n w h e r e the peptide effects r e p o r t e d here w o u l d fall on a d o s e - r e s p o n s e c u r v e . T h e d o s a g e used in o t h e r s p e c i e s m a y not be a p p r o p r i a t e for the rabbit. A d d i t i o n a l r e s e a r c h will be r e q u i r e d to clarify this issue. In s u m , M S H / A C T H 4 - 1 0 d o e s h a v e an effect o n t h e b e h a v i o r o f t h e classically c o n d i t i o n e d r a b b i t N M p r e p a r a tion, a n d t h a t effect is a r e d u c t i o n in the p e r f o r m a n c e of CRs. It r e m a i n s to be d e t e r m i n e d if the peptide will p r o l o n g ext i n c t i o n in this p r e p a r a t i o n if the t r a i n i n g p r o c e d u r e is c h a n g e d f r o m classical to i n s t r u m e n t a l c o n d i t i o n i n g . If so,
719 t h e peptide m a y s e r v e as a useful tool in this p r e p a r a t i o n for u n c o v e r i n g d i f f e r e n c e s b e t w e e n the s u b s t r a t e s of classical and instrumental conditioning. ACKNOWLEDGMENTS This research was supported in part by grants from the SUNY/Research Foundation, University Awards Program; by BRSG grant S07RR07149-08 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health; and by NIMH grants MH-12507 and MH-32154. The authors express their appreciation to Dr. Lyle H. Miller for early discussions about the experiment and for providing the peptide.
REFERENCES 1. Beckwith, B. E. and C. A. Sandman. Behavioral influences of the neuropeptides ACTH and MSH: A methodological review. Neurosci. Biobehav. Rev. 2:311-338, 1978. 2. Cegavske, C. F., T. A. Harrison and Y. Torigoe. The neuroanatomical substrates of the unconditioned response in the classically conditioned rabbit nictitating membrane preparation. In: Classical Conditioning 111: Behavioral, Neurophysiological, and Nearochemical Studies in the Rabbit, edited by !. Gormezano, W. F. Prokasy and R. F. Thompson. 1982, in press. 3. DeWied, D. Inhibitory effect of ACTH and related peptides on extinction of conditioned avoidance behavior in rats. Proc. Soc. exp. Biol. Med. 122: 28-32, 1966. 4. Garrud, P., J. A. Gray and D. DeWied. Pituitary adrenal hormones and extinction of rewarded behavior in the rat. Physiol. Behav. 12:109-119, 1974.
5. Gormezano, I. Classical conditioning. In: Experimental Methods and Instrumentation in Psychology, edited by J. B. Sidowski. New York: McGraw Hill, 1966. 6. Gormezano, I. Investigations of defense and reward conditioning in the rabbit. In: Classical Conditioning, vol. 2, Current Research and Theory, edited by A. H. Black and W. F. Prokasy. New York: Appleton-Century-Crofts, 1972. 7. Gormezano, 1. and J. W. Moore. Classical conditioning. In: Learning Processes, edited by M. H. Marx. London: Macmillan, 1969. 8. Kirk. R. E. Experimental Design: Procedures ft," the Behavioral Sciences. Belmont, CA: Brooks-Cole, 1968. 9. Sandman, C. A., L. H. Miller, A. J. Kastin and A. V. Schally. Neuroendocrine influence on attention and memory. J. comp. physiol. Psvchol. 80: 54-58, 1972. 10. Thompson, R. F., T. W. Berger, C. F. Cegavske, M. M. Patterson, R. A. Roemer, T. J. Teyler and R. A. Young. The search for the engram. Am. Psych,I. 31: 209-227, 1976.
Effects of M S H / A C T H A l0 on the Classically-Conditioned Rabbit Nictitating Membrane CATHERINE
A. S C H A E F F E R , 2 D O U G L A S C R A I G F. C E G A V S K E
C. N A T H A N S O N ,
Department of Psychology and Center for Neurobehavioral Sciences State University of New York, Binghamton, N Y 13901 AND RICHARD
A. R O E M E R
Department of Psychiatr3', Temple University Henry Avenue and Abbott~sford Road, Philadelphia, PA 19129 R e c e i v e d 22 M a r c h 1982 SCHAEFFER, C. A., D. C. NATHANSON, C. F. CEGAVSKE AND R. A. ROEMER. Effects of MSH/ACTH 4-10 on the classically-conditioned rabbit nictitating membrane. PEPTIDES 3(5) 715-719, 1982.--Subjects were conditioned/extinguished under four experimental conditions using either MSH/ACTH 4-10 (A) or diluent (D): D/D, D/A, A/D. and A/A. The major question investigated was whether or not the peptide has an effect on this classically-conditioned behavior similar to that reported for instrumental conditioning paradigms. The results indicated that it does not. An effect was seen on performance, not on learning or attentional processes. Animals treated with the peptide performed more poorly (i.e., displayed fewer conditioned responses) during both acquisition and extinction. In addition, there was an apparent residual effect of the peptide that lasted 24 but not 48 hours. MSH ACTH 4-10 Nictitating membrane
Peptides
Attention
Learning
T H E 4-10 fragments of melanocyte stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) produce at least one consistent behavioral effect in mammals. The substances prolong the extinction of learned responses in instrumental conditioning paradigms (see e.g., [1, 3, 4]. The effect of these substances has not been demonstrated in classically-conditioned behaviors. We report here the effects of M S H / A C T H 4--10 on such a behavior in rabbits. The behavioral parameters of the rabbit nictitatingmembrane (NM) preparation have been extensively studied by Gormezano and his associates (see e.g., [5, 6, 7]). These parameters are well understood and reflect many of the advantages that this preparation has for studying the neurophysiological and neuroanatomical substrates of the conditioned behavior (see [2] and [10] for discussions). Consequently, if a peptide effect could be demonstrated in this preparation, it might be possible eventually to discover the sites of peptide action on the conditioned behavior.
Classical conditioning
Rabbit
In addition, Sandman et al. [9] have proposed that the prolonged extinction effects seen in instrumental conditioning paradigms may be a result of the peptide enhancing attention. If this is correct, then one would expect a behaviorally opposite effect in the extinction phase of classical conditioning. In the instrumental situation, the subject, being more attentive to the CS and its consequences, would respond more consistently to the CS and therefore discover less rapidly that the reinforcer (UCS) was no longer being presented. On the other hand, in a classical-conditioning paradigm, the CS and UCS are always presented, so the subject would quickly discover that the UCS was no longer being given and the extinction phase would be shortened. Thus, there were two reasons why it was of interest to determine if M S H / A C T H 4--10 has an effect on the behavior of the classically-conditioned rabbit NM preparation: If an effect could be demonstrated, (1) The NM preparation might allow sites of peptide action to be discovered, and (2)
~Requests for reprints should be addressed to Craig Cegavske, whose current address is Department of Biology, State University of New York, Binghamton. NY 13901. 2These data were submitted in partial fulfillment of the requirements for C. A. Schaeffer's M. A. thesis from SUNY-Binghamton. Her current address is 309 Fleming Road. Cincinnati, OH 45215.
C o p y r i g h t ~ 1982 A N K H O
I n t e r n a t i o n a l Inc.--0196-9781/82/050715-05503.00/0
S C H A E F F E R ET AL.
716 I00,
M S H / A C T H 4-10 might allow additional information to be gained about inferred processes (e.g., attention) at work in the rabbit NM preparation.
90
80
•
ACTH 4-10
o
DILUENT
METHOD 70
The design of the present study was a simple 2×2 factorial to assess the effect of M S H / A C T H 4-10 during acquisition and extinction of the classically conditioned response.
A nim a ls Thirty-two male, New Zealand white rabbits weighing 1.8-3.2 kg were used as subjects. They were housed individually under a 12-hour light-dark cycle (lights on at 0800) with ad lib access to food and water. The subjects were divided into four groups and trained/ extinguished on M S H / A C T H 4-10 (A) or diluent (D): D/D (n=8), D/A (n=9), A/D (n=8), and A/A (n=7).
Apparatus The apparatus and procedure used for conditioning were similar to those described in detail by Gormezano [5]. Animals were conditioned or extinguished three at a time in three separate lighted and ventilated chambers. Inside the chambers each subject was restrained in a Plexiglas box and fitted with a leather head harness which held the airpuff delivery system and the nictitating membrane transducer. Eyeclips were used to hold the eyelids open during training. The conditioned stimulus (CS) was a 350 msec, I kHz, 85 dB tone; the unconditioned stimulus (UCS) was an airpuff gated from a source pressurized to 5 psi. The nozzle was positioned about 10 mm from the cornea. The UCS overlapped the final 100 msec of the CS providing a 250 msec interstimulus interval (ISI). The intertrial interval (ITI) was a pseudorandom sequence of 50, 60, or 70 seconds, with an average of 60 seconds.
Conditioning Procedure Prior to training, the left eye was anesthetized with a local anesthetic (Opthaine) and a snap (for transducer attachment) was sutured to the NM. After suturing, animals were allowed at least 24 hr recovery time. They were then placed in the experimental chamber, with all apparatus connected, for two hours in order to habituate to the setting and apparatus. The following day acquisition training was started. Two days of acquisition training (paired CS and UCS) were followed by two days of extinction training CS and (CS alone). There were 104 trials per day. Approximately 30 minutes prior to training, each animal was injected subcutaneously in the back with 80/zg/kg of M S H / A C T H 4-10 dissolved in 0.4 cc of diluent (0.01 M acetic acid in 0.9% saline), or with 0.4 cc of diluent alone. After the injection the animals were left alone in the experimental chambers for about 20 minutes. The transducer and eyeclips were then attached, the airpuff nozzle positioned properly, and the training session started. Running time was approximately 2V2-3 hours from injection to the end of the session for that day.
Scoring Criterion The data were manually scored from the polygraph records. One-half millimeter of NM closure was defined as a
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FIG. 1. A c o m p a r i s o n of the acquisition of conditioned behavior for
animals treated with MSH/ACTH 4-10 (groups A/D and A/A combined) and diluent (groups D/D and D/A combined). In this and subsequent figures, each trial block contains 8 trials.
response, and latencies were measured from a CS-onset marker pulse to this point on the NM response curve. During acquisition a response was classified as "'conditioned" only if it occmTed within 250 msec of CS onset (CS to UCS interval was 250 h,sec). During extinction, any response occurring within 500 msec of the CS marker was considered " c o n d i t i o n e d " since no UCS was given.
Dependent Variable For analysis purposes, trials were grouped into blocks of 8 to produce 13 trial-blocks per day. Trials with pre-CS activity of the NM were considered uncodeable, and were deleted from the analysis. A small number of trials were uncodeable because of equipment failure, and were also deleted. In all, 3% of the trials were discarded. The number of conditioned responses (CRs) in each block was divided by the number of codeable responses in that block to produce a percent-CR measure. RESULTS
Acquisition and Extinction A major result of the present study was that animals treated with M S H / A C T H 4-10 performed more poorly than diluent-treated controls during both acquisition and extinction. Figure 1 shows the CR acquisition curves for the diluent and the M S H / A C T H 4-10 treated groups. Throughout the two days of training, the peptide group produced a lower percentage of conditioned responses than the diluent treated group. We performed a three-way split-plot A N O V A on the acquisition data with the treatment (diluent or peptide) as the between subjects factor; and two levels of within subjects factors, one factor being days (one and two), and the second factor the thirteen trial-blocks per day. This analysis yielded a significant treatment effect, F(1,30)=5.36, p<0.03. As expected, there was also a significant days effect, a trial block effect, and a days by trial-block interaction, but there were
MSH/ACTH 4-10 AND RABBIT NM C O N D I T I O N I N G
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no two or three level interactions which were related to the drug treatment. To evaluate the effects of the peptide on extinction of the conditioned response, we pooled all animals receiving MSH/ACTH 4--10 during extinction (D/A and A/A groups) and compared them to the animals receiving diluent during acquisition and extinction (D/D). (The A/D group was not pooled with the D/D group because of the apparent carryover effect of the peptide discussed below.) Prior to performing such an ANOVA, we tested to confirm that this pooling was statistically justified. A two-way split plot ANOVA comparing extinction performance of animals trained and extinguished on the peptide (A/A) with those trained on diluent and extinguished on the peptide (D/A) yielded a nonsignificant between subjects F(l, 14)= 1.052. Figure 2 depicts the extinction curves for days one and two of the D/D and the pooled A/A and D/A groups. There was an immediate drop in the level of conditioned responding with administration of the peptide at the beginning of extinction (the drop occurred on the first trial of the block), and this difference was maintained throughout extinction. A three-factor split-plot ANOVA yielded a significant treatment (D vs A) effect, F(I,22)=10.938, p=0.003, both days and trial-block main effects, and a significant treatment by trial-block interaction, F(12,264)= 1.85, p =0.04. Unlike the acquisition data, a marginally significant difference in rate was present in extinction. The peptide group extinguished slightly slower than the diluent group. Examination of the day-two data revealed that this was probably the result of a floor effect operating in the peptide group on day two (see Figs. 2 and 3). To aid in interpreting this interaction, two-factor split-plot ANOVAs (treatment by trial-block) were performed separately for days one and two. Each ANOVA revealed a main effect of treatment but only on day two was there a treatment by trial-block interaction, F(12,264)=1.823, p=0.045. Therefore, the treatment by trial-block interaction in the three-way analysis and the daytwo analysis may not reflect a peptide effect.
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DAY I
DAY 2
E X T I N C T I O N T R I A L BLOCKS FIG. 3. The residual effect of MSH/ACTH 4-10 is illustrated. Both groups trained under the peptide (A/D and A/A) start extinction at a level lower than the group trained with diluent (D/D) regardless of the treatment given in extinction. The group switched from the peptide in acquisition to diluent during extinction (A/D) does not extinguish as fast as the group remaining on the peptide (A/A); and, by the end of day 2, is indistinguishable from the group trained and extinguished on the peptide (D/D).
Caro,-Over Lffect of MSH/A CTH 4-10 The data presented in Fig. 3 show the A/D group beginning extinction with a response rate similar to that of the A/A group. Then, during the course of extinction, the response rate appears to have shifted away from that of the peptide treated group and toward the rate of the diluent treated group (D/D). When a mean (percent CRs) of all trials during the first day of extinction was taken for each group shown in Fig. 3, the A/D group was found to lie about halfway between the other two (the means were 72.6 for D/D, 53.9 for A/D, and 37.8 for A/A). No significant overall extinction differences were present between the D/D and the A/D groups. A twoway split-plot ANOVA yielded a nonsignificant between subjects F(1,14)=0.58. However, an additional split-plot ANOVA for the first four extinction-trial blocks was performed with the between groups factor being subjects treated with either diluent (D/D) or MSH/ACTH 4-10 (A/D) during acquisition. The analysis yielded a marginally significant result, F(1,14)=4.602, p<0.05. These data suggest that the peptide had a residual effect on response performance lasting at least 24, but less than 48, hours. Although there was an immediate drop in responding from acquisition to extinction in the D/A group, no comparable increase in responding, indicating a release from peptide effects, was seen in the A/D group (see Table l).
Latency of Conditioned Responses During acquisition, there were no significant CR latency differences between the diluent and peptide treated groups. The slight trend toward shorter latencies by the diluent treated group seen in Fig. 4 was expected as these animals showed higher levels of conditioned responding (see Fig. 1). It is characteristic, in this preparation, for CR latencies to shorten as conditioned behavior improves.
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DISCUSSION
The results of the present experiment indicate that M S H / A C T H 4-10 had an effect on performance factors alone rather than on learning or attention. In contrast to previous reports employing instrumental avoidance paradigms, CR performance in extinction was depressed, not raised, in the present classical-conditioning situation. In addition, the peptide depressed performance during acquisition. A number of observations suggest that these changes were due to the peptide's action, but that the effect was n,,t on learning or attentional processes. If learning had been effected, a difference in the rate t~f acquisition should have been seen. No such difference was observed. The rate of acquisition of CRs was the same for both groups after a delayed onset in the peptide group Therefore, it can be inferred that the actual mecharisr, ',:i underlying the learning of the conditioned behavior ,~as (were) not being affected by the drug during the learning process. In addition, both groups given extinction training on diluent (D/D and A/D in Fig. 3) had the same final level of CR performance at the end of extinction. This also suggests that the actual level of learning was similar for the two groups at the end of acquisition despite the lower level of performance by the A/D group at that time. A clear and immediate decrease in performance was seen in the subjects extinguished on the peptide following training on diluent. Scrutiny of the individual trials within the first block of extinction confirmed this. The immediate loss of conditioned responding after peptide administration strongly suggests that the peptide had its effect on performance not on the learning process per se. This is compelling for two reasons: (1) The level of CR performance at the end of acquisition was almost identical in the two groups trained on diluent (D/D and D/A in Table 1), and (2) the subsequent rate of decrement of CR performance was similar for subjects treated with peptide and diluent (see Fig. 2) indicating that actual extinction was progressing at about the same rate. There was no reciprocal change in the group released from the peptide (A/D). That group did not immediately begin performing at the level of the D/D group. In fact, there was little difference between the peptide and diluent treated groups (excluding D/D) in the first four blocks of extinction (see Table 1). A performance effect is, again, suggested because the final level of extinction was the same for the D/D and A/D groups. The rate of CR performance was depressed
FIG. 4. The latencies of group responses are compared for groups trained on MSH/ACTH 4-10 (groups A/D and A/A combined) and diluent (groups D/D and D/A combined).
during acquisition under the peptide and this depressi~m was carried over into extinction. The group that was rcleas~'d from the peptide during extinction (A/D) showed a much slower ~ate of extinction during day one than did the A/A group or the D/D group (Fig. 3). But on day 2, t h A / D group end, d at the ,,ame performance level as the D/I) ,.~up. "I his r c ,lit can most easily be explained by assuming a "'wearing ~)ff" of some effect of the peptide i~ ~:veal the true level of :xtinction. The conditioned behavior must have been learned despite the M S H / A C T H 4-10 treatment during acquisition. If increased attention to the stimuli had operated, one might have expected an accelerated rate of acquisition and/or an earlier onset of CR behavior. Either of these effects could produce a relatively higher level of final CR performance if the level of attention were maintained throughout the training session. The data suggest that this did not occur: the rate of acquisition was no different from controls. and the onset of CR performance was delayed, not made earlier, leading to a lower level of final performance at the end of acquisition. If attention levels were increased by the peptide in extinction, an increase in the rate-of-loss of CR performance should have been observed. This did not occur within the first 8 trials or even during the first day. The extinction rate was essentially the same for both the experimental and control groups on day 1. There appeared to be an accelerated rate during the first few blocks of day 2 of extinction. However, this was partly a result of one animal in the A/A group which responded at a high rate in the first block and thereby raised that data point. Even ignoring this, the result could not be easily explained as an attentional effect without explaining why it did not happen on day 1 of extinction as well. All of the data reported here argue against the idea that MSH/ACTH 4-10 improves the learning process or increases levels of attention in this classically-conditioned preparation. How the peptide might affect performance variables is not clear. It could have caused some sort of malaise in the animals. However, casual observation of the animals during
MSH/ACTH 4-10 AND RABBIT NM CONDITIONING
the e x p e r i m e n t s u g g e s t e d no d i f f e r e n c e s in e i t h e r t h e i r app e a r a n c e or general b e h a v i o r ( a p p a r e n t a l e r t n e s s , a c t i v i t y level, etc.). A s e c o n d possibility w o u l d be a d o s e effect. It is not k n o w n w h e r e the peptide effects r e p o r t e d here w o u l d fall on a d o s e - r e s p o n s e c u r v e . T h e d o s a g e used in o t h e r s p e c i e s m a y not be a p p r o p r i a t e for the rabbit. A d d i t i o n a l r e s e a r c h will be r e q u i r e d to clarify this issue. In s u m , M S H / A C T H 4 - 1 0 d o e s h a v e an effect o n t h e b e h a v i o r o f t h e classically c o n d i t i o n e d r a b b i t N M p r e p a r a tion, a n d t h a t effect is a r e d u c t i o n in the p e r f o r m a n c e of CRs. It r e m a i n s to be d e t e r m i n e d if the peptide will p r o l o n g ext i n c t i o n in this p r e p a r a t i o n if the t r a i n i n g p r o c e d u r e is c h a n g e d f r o m classical to i n s t r u m e n t a l c o n d i t i o n i n g . If so,
719 t h e peptide m a y s e r v e as a useful tool in this p r e p a r a t i o n for u n c o v e r i n g d i f f e r e n c e s b e t w e e n the s u b s t r a t e s of classical and instrumental conditioning. ACKNOWLEDGMENTS This research was supported in part by grants from the SUNY/Research Foundation, University Awards Program; by BRSG grant S07RR07149-08 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health; and by NIMH grants MH-12507 and MH-32154. The authors express their appreciation to Dr. Lyle H. Miller for early discussions about the experiment and for providing the peptide.
REFERENCES 1. Beckwith, B. E. and C. A. Sandman. Behavioral influences of the neuropeptides ACTH and MSH: A methodological review. Neurosci. Biobehav. Rev. 2:311-338, 1978. 2. Cegavske, C. F., T. A. Harrison and Y. Torigoe. The neuroanatomical substrates of the unconditioned response in the classically conditioned rabbit nictitating membrane preparation. In: Classical Conditioning 111: Behavioral, Neurophysiological, and Nearochemical Studies in the Rabbit, edited by !. Gormezano, W. F. Prokasy and R. F. Thompson. 1982, in press. 3. DeWied, D. Inhibitory effect of ACTH and related peptides on extinction of conditioned avoidance behavior in rats. Proc. Soc. exp. Biol. Med. 122: 28-32, 1966. 4. Garrud, P., J. A. Gray and D. DeWied. Pituitary adrenal hormones and extinction of rewarded behavior in the rat. Physiol. Behav. 12:109-119, 1974.
5. Gormezano, I. Classical conditioning. In: Experimental Methods and Instrumentation in Psychology, edited by J. B. Sidowski. New York: McGraw Hill, 1966. 6. Gormezano, I. Investigations of defense and reward conditioning in the rabbit. In: Classical Conditioning, vol. 2, Current Research and Theory, edited by A. H. Black and W. F. Prokasy. New York: Appleton-Century-Crofts, 1972. 7. Gormezano, 1. and J. W. Moore. Classical conditioning. In: Learning Processes, edited by M. H. Marx. London: Macmillan, 1969. 8. Kirk. R. E. Experimental Design: Procedures ft," the Behavioral Sciences. Belmont, CA: Brooks-Cole, 1968. 9. Sandman, C. A., L. H. Miller, A. J. Kastin and A. V. Schally. Neuroendocrine influence on attention and memory. J. comp. physiol. Psvchol. 80: 54-58, 1972. 10. Thompson, R. F., T. W. Berger, C. F. Cegavske, M. M. Patterson, R. A. Roemer, T. J. Teyler and R. A. Young. The search for the engram. Am. Psych,I. 31: 209-227, 1976.