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Rapid, nonaversive conditioning in a freshwater gastropod
BEHAVIORAL AND NEURAL BIOLOGY
36, 391-402 (1982)
Rapid, Nonaversive Conditioning in a Freshwater Gastropod II. Effects of Temporal Relationships on Learning JAMES E . ALEXANDER, JR., TERESA E . AUDESIRK, 1 AND GERALD J. AUDESIRK 2
Division of Biological Sciences, University of Missouri at Columbia, Columbia, Missouri 65211 In this paper we report that two phenomena important in vertebrate learning, the temporal distribution of training trials and the temporal relationship between the conditioned stimulus and the unconditioned stimulus, also influence learning in the pond snail Lymnaea stagnalis. The learning paradigm used in this study was nonaversive classical conditioning of feeding, in which a novel chemostimulus, amyl acete (conditioned stimulus) was paired with a phagostimulant, a mixture of sucrose and casein digest (unconditioned stimulus). The response measured was the number of stereotyped feeding movements, or rasps, made to the conditioned stimulus. We found that 15 pairings distributed over 3 days resulted in longer retention of the learned response than did 15 pairings on 1 day. We also report that, although the forward conditioning was superior, significant learning occurred utilizing simultaneous and backward conditioning procedures. Lymnaea is also susceptible to trace conditioning, both forward and backward. Demonstration of behavioral similarities between vertebrates and invertebrates strengthens the case that invertebrate preparations are suitable models for the investigation of learning and its neural basis.
In the preceding paper we demonstrated that the gastropod Lymnaea stagnalis is capable of rapid and strong acquisition of a classically conditioned feeding response. Further, we showed that learning can occur using either of two different conditioned stimuli, amyl acetate or ethyl butyrate, and that animals ranging in age from 8 to 32 weeks learn readily if properly motivated. In addition, we demonstrated that age and motivation, variables found to be important in vertebrate learning, interact to influence the acquisition and expression of the learned response. In this paper we report two phenomena in Lymnaea learning which are also characteristic 1 TO whom reprint requests and correspondence should be sent: Department of Biology, University of Colorado, Denver, 1100 14th St., Denver, Colo. 80202. z The authors wish to thank Mr. David Kovacs and Ms. Diane Voth for their technical assistance. 391 0163-1047/82 $2.00 Copyright © 1982by AcademicPress, Inc. All rightsof reproductionin any form reserved.
392
ALEXANDER, AUDESIRK, AND AUDESIRK
of vertebrate learning. First, the distribution of training trials influences the quality of learning in this gastropod, and second, the temporal relationship between the CS and UCS within individual training trials influences learning in Lymnaea. Many investigators are using invertebrates, particularly gastropods, as model systems in which to study the neural bases of learning. Demonstrations of similarities in learning between vertebrates and invertebrates greatly strengthens the case that these invertebrate preparations are indeed suitable models for neurobiological study. Previous findings include rapid and robust aversive (e.g., Sahley, Gelperin, & Rudy, 1981a) and nonaversive conditioning (Audesirk, Alexander, Audesirk, & Moyer; 1983; Sahley, Hardison, Hsuan, & Gelperin, 1982), the influence of age and motivation on conditioning (Audesirk et al., 1983), and higher-order conditioning and blocking by prior experience (Sahley, Rudy, & Gelperin, 1981b). The effects of training density and temporal order within trials reported here adds further evidence that the mechanisms underlying learning are similar in all nervous systems, regardless of design. The first experiment reported here was designed to determine whether the temporal distribution of training trials during classical conditioning has an effect on the magnitude and retention of the learned response in Lymnaea. The general finding in the vertebrate literature is that spacing the training trials of certain tasks further apart is more efficient than massed practice (Hilgard & Marquis, 1940). The second set of experiments deal with the phenomena of simultaneous and backward conditioning. There is some controversy concerning whether or not backward and simultaneous conditioning are real phenomena, due to associative rather than nonassociative processes such as sensitization. However, more recent evidence indicates that under certain conditions, true conditioning can occur with backward and simultaneous as well as forward CS-UCS pairings (e.g., Heth & Rescorla, 1973). EXPERIMENT 1
In this study, we investigated the effect of the distribution of training trials on the subsequent magnitude and retention of learning in Lymnaea.
Methods The learning protocol used in this study was nonaversive classical conditioning of feeding in the freshwater pond snail, Lymnaea stagnalis. As in the previous paper (Audesirk et al., 1983), the novel odor amyl acetate (CS) was paired with a phagostimulant, a mixture of sucrose and casein digest (UCS). The response measured was the number of stereotyped feeding movements, called rasps, made in response to the CS after training. The animals used in these studies were taken from laboratory-reared populations of Lymnaea derived from stock originating in England. They
TEMPORAL EFFECTS ON LEARNINGIN Lymnaea
393
were housed and trained in shallow plastic bins (15 x 22 x 5 cm) in 200 ml of aerated tap water. Plastic screening lined the bins to facilitate transfer of the animals with a minimum of disturbance during training. The animals were weighed and marked with an identifying number prior to the experiments. To equalize any initial differences in motivation, all groups in all experiments were allowed to feed on 1 g of TetraMin fish food for 3 min daily, starting 1 day prior to training. The basic training and testing procedures used in these studies were the same as the ones used in Experiment 1 of the preceding paper. Forty-nine animals 20 weeks of age and weighing 0.90 +_ 0.23 g (mean _+ SD) were divided into the following four groups of approximately equal average weight: Spaced Experimental (N = 11), Spaced Random Control (N = 12), Massed Experimental (N = 13), and Massed Random Control (N = 13). The two experimental groups each received 15 training trials, but the time interval between trials differed between the two groups. The Spaced Experimental group received five training trials a day for 3 consecutive days, with an intertrial interval (ITI) of 90 rain, while the Massed Experimental group received all 15 training trials on one day (the last day of training for the Spaced Experimental group), with an ITI of 45 min. The Spaced Random Control group was exposed to the CS at times ranging from 30 min before to 45 min after the presentation of the UCS, while the Massed Random Control group received the CS at times from 20 min before to 30 min after the UCS. After training, the snails were tested a total of five times over the next 11 days. Results were analyzed using the one-sided t test. Results As shown in Fig. 1, the mean response of the Spaced Experimental group was significantly greater than that of the Spaced Control on all five tests after training (p < .001), indicating that learning was retained for at least 11 days. In contrast, the score of the Massed Experimental group was significantly higher than its control on only the first three tests, up to the fifth day after training (p < .01). Although the major difference between the Massed and Spaced Experimental groups was in their retention of the learned response, both the mean values for the Experimental group and the rasp differential between the Experimental and Control groups were always greater for the snails given spaced training than for those given massed training. EXPERIMENT 2
This study was designed to study the effects of the temporal relationship between the CS and UCS on the strength of the learned response.
394
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FIG. 1. Comparison of the massed and spaced training procedures. The figure indicates the response of the massed and spaced animals of Experiment 1 after training, which consisted of 15 C S - U C S pairings. The mean of the Spaced Experimental group was significantly higher than that of the Spaced Control group throughout the experiment. The mean of the Massed Experimental group was significantly higher than the Massed Control group mean on only the first three tests. Asterisks indicate significance (p < .05, onetailed t test).
Methods The animals used in this study were 11 weeks old and weighed 0.44 + 0.14 g (mean _ SD) at the start of the experiment. A total of 60 snails were divided into six groups of 10 animals each, approximately matched in average weight. These were designated: Forward Experimental, Forward Random Control, Simultaneous Experimental, Simultaneous Random Control, Backward Experimental, and Backward Random Control. The experimental groups received 15 training trials, 5 per day for 3 consecutive days, with an ITI of 90 min. The training procedure is outlined in Table 1. The training procedure for the Forward Experimental group was identical to the procedure described in the previous paper (Audesirk et al., 1983). They received the CS 15 sec before receiving the UCS and stayed in the presence of the two stimuli for 2 min, after which they were rinsed
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and transferred. The Forward Random Control group received water followed by the UCS when the Forward Experimental group received the C S - U C S pairing. The Forward Random Control group received the CS followed by water at random times ranging from 30 min before to 45 min after the UCS. The Backward Experimental group received the UCS 15 sec before the CS, followed by rinsing and transfer back to clean home bins 2 rain later. The Backward Random Control group received the UCS followed by water when the Backward Experimental group received the U C S CS pairing. When the Forward Random Control group received the CS followed by water, the Backward Random Control group received water followed by the CS. The Simultaneous Experimental group received water followed by 50 ml of a mixture of the C S - U C S and were rinsed and transferred 2 min later. The Simultaneous Control group received the CS followed by water when the Experimental group received the C S - U C S mixture. The Simultaneous Random Control group received the CS followed by water when the other two controls received the CS. To equalize handling, the experimental groups received water followed by water when the control groups were exposed to the CS. Four tests were given over the next 9 days.
Results The results of this experiment are shown in Fig. 2. After training, all three experimental groups exhibited highly significant learning as compared to their respective controls on days 2, 4, and 6 (p < .0005, all cases). However, on the fourth test, 9 days after training, the Forward Experimental group still exhibited significant learning (p < .01), the Simultaneous Experimental group was less significant (p < .05), and the Backward Experimental group no longer showed significant learning. EXPERIMENT 3
Since considerable overlap existed between the CS And UCS in Experiment 2, it is possible that the snails were learning to equate the two stimuli, which were presented simultaneously for at least 105 sec in all cases. To eliminate the overlap and isolate the predictive aspect of the C S - U C S pairing, the following study was conducted using a trace conditioning procedure, where the CS and UCS do not overlap. The trace conditioning procedure is generally considered to be less effective than delay conditioning, where some overlap exists between the CS and UCS (Mackintosh, 1974). This experiment was designed to answer the following questions: (1) does trace conditioning result in learning in Lymnaea, and (2) do forward and backward trace conditioning both occur?
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F16. 2. Compalison of the forward, backward, and simultaneous conditioning paradigms. The figure indicates the response of the forward, backward, and simultaneous animals of Experiment 2. The means of the Forward and Simultaneous Experimental groups were significantly higher than their respective controls throughout the experiment. The Backward Experimental group mean was significantly higher than the Backward Control mean only on the first three tests after training. Asterisks indicate significance (p < .05, one-tailed t test).
Methods A total of 30 animals were split up into three groups of l0 animals each: Forward Trace Experimental, Backward Trace Experimental, and Random Control. The animals were 10 weeks old at the start of the experiment and weighed 0.32 _+ 0.06 g. The feeding protocol was the same as used in the previous experiments. The animals were housed and trained in shallow plastic bins lined with screens as described above. At each training trial (Table 1), the screens were lifted out of the home bins and the animals were transferred to new bins with 300 ml of water containing the first stimulus. The Forward Experimental group was placed in a bin containing water plus the CS, the Backward Experimental group was placed in a bin containing water plus the UCS, and the Control group was placed in 300 ml of water. The animals remained in the presence of the first stimulus for 2 min,
398
ALEXANDER, AUDESIRK, AND AUDESIRK
then were transferred to rinse bins for 15 sec. They were then transferred to bins containing the second stimulus; the Forward Experimental animals were exposed to the UCS, the Backward Experimental animals to the CS And the Random Control animals to the UCS. After 2 min, the snails were lifted out and transferred to rinse bins, and then 15 sec later back to the home bins. At times ranging from 30 min before to 45 min after the Control group was exposed to the UCS, these snails were placed in a bin containing the CS for 2 min, transferred to a rinse bin, then moved to a bin containing only water, then rinsed and placed back in the home bin. To equalize handling, both experimental groups were placed in bins containing water, rinsed, placed into other bins containing water, rinsed, and placed back into their respective home bins. Training consisted of 15 training trials, 5 trials per day for 3 consecutive days, with an ITI of 90 minutes. Results As can be seen in Fig. 3, the Forward Experimental group was significantly higher than the Control group (p < .001), as was the Backward
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FIG. 3. The response of the forward and backward trace conditioning animals. Lymnaea are capable of acquiring a learned response using both these procedures, but it is relatively short lived, with backward being less effective than forward. Asterisks indicate significance (p < .05, one-tailed t test).
TEMPORAL EFFECTS ON LEARNING IN Lymnaea
399
Experimental group (p < .0005) on the first test after training. On the second test, the mean of the Backward Experimental snails declined relative to the control (p < .01) whereas the mean of the Forward Experimental group increased relative to the control (p < .001). On the third test, however, both the Forward and Backward Experimental groups were insignificantly greater than the control group. The mean responses of the two experimental groups were compared directly and differed significantly on the second test (p < .025). DISCUSSION
Temporal distribution of training trials. It has generally been found that massed practice (training trials that follow each other closely in time) is less efficient than spaced practice, where a greater period of time separates successive trials (Hilgard and Marquis, 1940). Many examples of the superiority of spaced practice in various learning tasks can be found in the literature (for examples see Lorge, 1930; Bourne and Archer, 1956). In Lymnaea, the major effect of varying the distribution of training on learning was on retention, not on the initial magnitude of learning. In Experiment 1, greater spacing of the training trials retarded forgetting, whereas massing the trials hastened it. Immediately after training, the expression of the learned response was almost equal between the Massed and Spaced Experimental animals. The major difference occurred days later, when the mean of the Massed Experimental group fell rapidly. Temporal relationship of the CS and UCS. In pairing the two stimuli of the classical conditioning paradigm (CS and UCS) the onset of the CS can occur either before the UCS onset (forward conditioning), or after the UCS onset (backward conditioning), or the two stimuli can occur together (simultaneous conditioning). Many investigations have failed to show significant learning with the simultaneous and backward paradigms, and positive results have often been attributed to nonassociative effects such as sensitization (for reviews see Kimble, 1961; Cautela, 1965). The failure to achieve significant learning with the simultaneous and backward procedures led to the suggestion that the CS serves as a signal, enabling the organism to predict the impending occurrence of the UCS. When the onset of the CS occurs simultaneously with or after the UCS onset, the best predictor of the UCS is the UCS onset itself, the CS in these cases no longer serving as a predictor of the UCS. However, the range of training procedures used to show the failure of the simultaneous and backward conditioning has been quite narrow, as pointed out by Heth and Rescorla (1973). Short duration CSs and UCSs have invariably been used, and the UCS in almost every case has been shock. In reviewing several of the many cases of unsuccessful attempts of backward conditioning, we found that no overlap existed between the CS and UCS, a discrete amount of time separated the two.
400
ALEXANDER, AUDESIRK, AND AUDESIRK
In successful studies, such as Heth and Rescorla (1973) and Mowrer and Aiken (1954), extensive overlap existed between the UCS and CS, and the two stimuli were longer in duration. We also were successful in demonstrating simultaneous and backward conditioning with relatively long, overlapping stimuli, as seen in Experiment 2. Historically, several theories concerning reinforcement have been postulated, two of these being the theory of contiguity and the theory of expectancy (Hilgard & Marquis, 1940; Kimble, 1961). The contiguity theory of reinforcement holds that the CS is substituted for or equated with the CS after several pairings of the two. The expectancy theory is concerned with the predictive value of the CS, where the animal behaves in ways consistent with anticipated consequences. If the UCS appears after the CS, the expectation is strengthened. There are problems with both theories, however. Though the main problem with the contiguity or stimulus substitution theory has been the observation that the CR sometimes differs from the UCR (Hilgard & Marquis, 1940), a number of studies show that the animal may indeed substitute the CS for the UCS. Pavlov (1934) reported that dogs trained to salivate to a light that preceded food sometimes turned their heads to the light source and licked the light. Breland and Breland (1961) cite several similar instances in a variety of animals. The animals, after being trained to perform a particular response, drifted into behaviors similar to instinctive food obtaining patterns. Jenkins and Moore (1973) described pigeons which were autoshaped to peck at a key to obtain food or water. The pigeons' beak movements towards the key when their reward was water resemble drinking movements, while the pecks at the key for food resembled those made when eating. In this report, the rasps made by Lymnaea to the CS appear to be identical to those made to the sucrosecasein UCS. The expectancy theory, on the other hand, cannot cope with backward conditioning, because the backward procedure eliminates the predictive quality of the CS. But in the successful demonstrations of backward conditioning, some overlap existed, overlap that may enhance the equation of the two stimuli (contiguity), regardless of the temporal order of appearance of the CS and UCS. From the results of Experiment 2, it appears that Lymnaea may use both the predictive aspect of the CS and equate the CS with the UCS as well. From this, one would expect that no overlap of the two stimuli (by use of the trace conditioning procedure) would lead to weaker forward conditioning and little or no backward conditioning. The results of Experiment 3 tend to support this hypothesis, although the results are not directly comparable because slightly different training procedures were used. It is striking that significant learning did occur with both backward and forward trace conditioning procedures, although the learning was
TEMPORAL EFFECTS ON LEARNING IN Lymnaea
401
short lived, persisting for approximately 2 days. The fact that the backward trace procedure worked needs some explanation, since neither the contiguity nor the expectancy theories can account for it. Cautela (1965) presented a hypothesis that the cases of successful backward conditioning were cases of conditioning where the CS was paired with pain perception or a memory trace of the UCS. Support for this idea comes from the study of feeding in the marine mollusc Aplysia californica (Kupfermann 1974). Food arousal in Aplysia lasts for at least 5 min beyond the presentation of the initial arousing stimulation. Perhaps CNS firing to the UCS is still persisting up to the time the CS is introduced, producing a "mental overlap" of the two stimuli. As mentioned before, delay conditioning has generally been found to be superior to trace conditioning. Therefore, temporal overlap between the CS and UCS enhances the quality of learning in the classical conditioning procedures, but, as seen by the results of Experiment 3, overlap is not essential for successful learning. ff both contiguity and expectancy are assumed to contribute to learning, this could explain both the results reported here and the results of past research reported in the mammalian literature. Although the contiguity and expectancy theories of reinforcement have been cited as alternate theories (Hilgard and Marquis, 1940), we see no reason to treat them as mutually exclusive. Our findings that simultaneous and backward conditioning paradigms are effective when overlap exists between the CS and UCS are best explained by the contiguity hypothesis, whereas the superiority of the forward conditioning procedures (both delay and trace) over their backward counterparts supports the notion that the CS is also treated as a predictor of the UCS. The stimulus overlap may encourage animals to equate the two stimuli, and this equation may interact with the predictive element to produce optimal learning in both vertebrates and invertebrates alike. REFERENCES Audesirk, T. E., Alexander, J. E., Jr., Audesirk, G. J., & Moyer, C. (1983). Rapid, nonaversive conditioning in a freshwater gastropod. I. Effects of age and motivation. Behavioral and Neural Biology, 36, 379-390. Bourne, L. E., Jr., & Archer, E. J. (1956). Time continuously on target as a function of distribution of practice. Journal of Experimental Psychology, 51, 25-33. Breland, K., & Breland, M. (1961). The misbehavior of organisms. American Psychologist, 16, 681-684. Cautela, J. R. (1965). The problem of backward conditioning. Journal of Psychology, 60, 135-144. Heath, C. D., & Rescorla, R. A. (1973). Simultaneous and backward fear conditioning in the rat. Journal of Comparative and Physiological Psychology, 82, 434-443. Hilgard, E. R., & Marquis, D. G. (1940). Conditioning and Learning. New York: AppletonCentury. Jenkins, H. M., & Moore, B. R. (1973). The form of the autoshaped response with food or water reinforcers. Journal of the Experimental Analysis of Behavior, 20, 163-181.
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Kimble, G. A. (1961). Hilgard and Marguis' Conditioning and Learning. New York: Appleton-Century-Crofts. Kupfermann, I. (1974). Feeding behavior in Aplysia: A simple system for the study of motivation. Behavioral Biology, 10, 1-26. Lorg e, I. (1930). Influence of Regularly Interpolated Time Intervals upon Subsequent Learning. New York: Teachers College, Columbia University, Bureau of Publications. Mackintosh, N. J. (1974). The Psychology of Animal Learning. New York/London: Academic Press. Mowrer, O. H., & Aiken, E. G. (1954). Contiguity vs. drive-reduction in conditioned fear: temporal variations in conditioned and unconditioned stimulus. American Journal of Psychology, 67, 26-38. Pavlov, I. P. (1934). An attempt at a physiological interpretation of obsessional neurosis and paranoi. Journal of Mental Science, 80, 187-197. Sahley, C., Gelperin, A., & Rudy, J. W. (1981). One-trial associative learning modified food odor preferences of a terrestrial mollusc. Proceedings of the National Academy of Sciences USA, 78, 640-642. (a) Sahley, C., Hardison, P., Hsuan, A., & Gelperin, A. (1982). Appetitively reinforced odor conditioning modulates feeding in Limax maximus. Society for Neuroscience Abstracts, 8, 823. Sahley, C., Rudy, J. W., & Gelperin, A. (1981). An analysis of associative learning in a terrestrial mollusc. I. Higher-order conditioning, blocking, and a transient US preexposure effect. Journal of Comparative Physiology, 144, 1-8. (b)
36, 391-402 (1982)
Rapid, Nonaversive Conditioning in a Freshwater Gastropod II. Effects of Temporal Relationships on Learning JAMES E . ALEXANDER, JR., TERESA E . AUDESIRK, 1 AND GERALD J. AUDESIRK 2
Division of Biological Sciences, University of Missouri at Columbia, Columbia, Missouri 65211 In this paper we report that two phenomena important in vertebrate learning, the temporal distribution of training trials and the temporal relationship between the conditioned stimulus and the unconditioned stimulus, also influence learning in the pond snail Lymnaea stagnalis. The learning paradigm used in this study was nonaversive classical conditioning of feeding, in which a novel chemostimulus, amyl acete (conditioned stimulus) was paired with a phagostimulant, a mixture of sucrose and casein digest (unconditioned stimulus). The response measured was the number of stereotyped feeding movements, or rasps, made to the conditioned stimulus. We found that 15 pairings distributed over 3 days resulted in longer retention of the learned response than did 15 pairings on 1 day. We also report that, although the forward conditioning was superior, significant learning occurred utilizing simultaneous and backward conditioning procedures. Lymnaea is also susceptible to trace conditioning, both forward and backward. Demonstration of behavioral similarities between vertebrates and invertebrates strengthens the case that invertebrate preparations are suitable models for the investigation of learning and its neural basis.
In the preceding paper we demonstrated that the gastropod Lymnaea stagnalis is capable of rapid and strong acquisition of a classically conditioned feeding response. Further, we showed that learning can occur using either of two different conditioned stimuli, amyl acetate or ethyl butyrate, and that animals ranging in age from 8 to 32 weeks learn readily if properly motivated. In addition, we demonstrated that age and motivation, variables found to be important in vertebrate learning, interact to influence the acquisition and expression of the learned response. In this paper we report two phenomena in Lymnaea learning which are also characteristic 1 TO whom reprint requests and correspondence should be sent: Department of Biology, University of Colorado, Denver, 1100 14th St., Denver, Colo. 80202. z The authors wish to thank Mr. David Kovacs and Ms. Diane Voth for their technical assistance. 391 0163-1047/82 $2.00 Copyright © 1982by AcademicPress, Inc. All rightsof reproductionin any form reserved.
392
ALEXANDER, AUDESIRK, AND AUDESIRK
of vertebrate learning. First, the distribution of training trials influences the quality of learning in this gastropod, and second, the temporal relationship between the CS and UCS within individual training trials influences learning in Lymnaea. Many investigators are using invertebrates, particularly gastropods, as model systems in which to study the neural bases of learning. Demonstrations of similarities in learning between vertebrates and invertebrates greatly strengthens the case that these invertebrate preparations are indeed suitable models for neurobiological study. Previous findings include rapid and robust aversive (e.g., Sahley, Gelperin, & Rudy, 1981a) and nonaversive conditioning (Audesirk, Alexander, Audesirk, & Moyer; 1983; Sahley, Hardison, Hsuan, & Gelperin, 1982), the influence of age and motivation on conditioning (Audesirk et al., 1983), and higher-order conditioning and blocking by prior experience (Sahley, Rudy, & Gelperin, 1981b). The effects of training density and temporal order within trials reported here adds further evidence that the mechanisms underlying learning are similar in all nervous systems, regardless of design. The first experiment reported here was designed to determine whether the temporal distribution of training trials during classical conditioning has an effect on the magnitude and retention of the learned response in Lymnaea. The general finding in the vertebrate literature is that spacing the training trials of certain tasks further apart is more efficient than massed practice (Hilgard & Marquis, 1940). The second set of experiments deal with the phenomena of simultaneous and backward conditioning. There is some controversy concerning whether or not backward and simultaneous conditioning are real phenomena, due to associative rather than nonassociative processes such as sensitization. However, more recent evidence indicates that under certain conditions, true conditioning can occur with backward and simultaneous as well as forward CS-UCS pairings (e.g., Heth & Rescorla, 1973). EXPERIMENT 1
In this study, we investigated the effect of the distribution of training trials on the subsequent magnitude and retention of learning in Lymnaea.
Methods The learning protocol used in this study was nonaversive classical conditioning of feeding in the freshwater pond snail, Lymnaea stagnalis. As in the previous paper (Audesirk et al., 1983), the novel odor amyl acetate (CS) was paired with a phagostimulant, a mixture of sucrose and casein digest (UCS). The response measured was the number of stereotyped feeding movements, called rasps, made in response to the CS after training. The animals used in these studies were taken from laboratory-reared populations of Lymnaea derived from stock originating in England. They
TEMPORAL EFFECTS ON LEARNINGIN Lymnaea
393
were housed and trained in shallow plastic bins (15 x 22 x 5 cm) in 200 ml of aerated tap water. Plastic screening lined the bins to facilitate transfer of the animals with a minimum of disturbance during training. The animals were weighed and marked with an identifying number prior to the experiments. To equalize any initial differences in motivation, all groups in all experiments were allowed to feed on 1 g of TetraMin fish food for 3 min daily, starting 1 day prior to training. The basic training and testing procedures used in these studies were the same as the ones used in Experiment 1 of the preceding paper. Forty-nine animals 20 weeks of age and weighing 0.90 +_ 0.23 g (mean _+ SD) were divided into the following four groups of approximately equal average weight: Spaced Experimental (N = 11), Spaced Random Control (N = 12), Massed Experimental (N = 13), and Massed Random Control (N = 13). The two experimental groups each received 15 training trials, but the time interval between trials differed between the two groups. The Spaced Experimental group received five training trials a day for 3 consecutive days, with an intertrial interval (ITI) of 90 rain, while the Massed Experimental group received all 15 training trials on one day (the last day of training for the Spaced Experimental group), with an ITI of 45 min. The Spaced Random Control group was exposed to the CS at times ranging from 30 min before to 45 min after the presentation of the UCS, while the Massed Random Control group received the CS at times from 20 min before to 30 min after the UCS. After training, the snails were tested a total of five times over the next 11 days. Results were analyzed using the one-sided t test. Results As shown in Fig. 1, the mean response of the Spaced Experimental group was significantly greater than that of the Spaced Control on all five tests after training (p < .001), indicating that learning was retained for at least 11 days. In contrast, the score of the Massed Experimental group was significantly higher than its control on only the first three tests, up to the fifth day after training (p < .01). Although the major difference between the Massed and Spaced Experimental groups was in their retention of the learned response, both the mean values for the Experimental group and the rasp differential between the Experimental and Control groups were always greater for the snails given spaced training than for those given massed training. EXPERIMENT 2
This study was designed to study the effects of the temporal relationship between the CS and UCS on the strength of the learned response.
394
ALEXANDER, AUDESIRK, AND AUDESIRK
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FIG. 1. Comparison of the massed and spaced training procedures. The figure indicates the response of the massed and spaced animals of Experiment 1 after training, which consisted of 15 C S - U C S pairings. The mean of the Spaced Experimental group was significantly higher than that of the Spaced Control group throughout the experiment. The mean of the Massed Experimental group was significantly higher than the Massed Control group mean on only the first three tests. Asterisks indicate significance (p < .05, onetailed t test).
Methods The animals used in this study were 11 weeks old and weighed 0.44 + 0.14 g (mean _ SD) at the start of the experiment. A total of 60 snails were divided into six groups of 10 animals each, approximately matched in average weight. These were designated: Forward Experimental, Forward Random Control, Simultaneous Experimental, Simultaneous Random Control, Backward Experimental, and Backward Random Control. The experimental groups received 15 training trials, 5 per day for 3 consecutive days, with an ITI of 90 min. The training procedure is outlined in Table 1. The training procedure for the Forward Experimental group was identical to the procedure described in the previous paper (Audesirk et al., 1983). They received the CS 15 sec before receiving the UCS and stayed in the presence of the two stimuli for 2 min, after which they were rinsed
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and transferred. The Forward Random Control group received water followed by the UCS when the Forward Experimental group received the C S - U C S pairing. The Forward Random Control group received the CS followed by water at random times ranging from 30 min before to 45 min after the UCS. The Backward Experimental group received the UCS 15 sec before the CS, followed by rinsing and transfer back to clean home bins 2 rain later. The Backward Random Control group received the UCS followed by water when the Backward Experimental group received the U C S CS pairing. When the Forward Random Control group received the CS followed by water, the Backward Random Control group received water followed by the CS. The Simultaneous Experimental group received water followed by 50 ml of a mixture of the C S - U C S and were rinsed and transferred 2 min later. The Simultaneous Control group received the CS followed by water when the Experimental group received the C S - U C S mixture. The Simultaneous Random Control group received the CS followed by water when the other two controls received the CS. To equalize handling, the experimental groups received water followed by water when the control groups were exposed to the CS. Four tests were given over the next 9 days.
Results The results of this experiment are shown in Fig. 2. After training, all three experimental groups exhibited highly significant learning as compared to their respective controls on days 2, 4, and 6 (p < .0005, all cases). However, on the fourth test, 9 days after training, the Forward Experimental group still exhibited significant learning (p < .01), the Simultaneous Experimental group was less significant (p < .05), and the Backward Experimental group no longer showed significant learning. EXPERIMENT 3
Since considerable overlap existed between the CS And UCS in Experiment 2, it is possible that the snails were learning to equate the two stimuli, which were presented simultaneously for at least 105 sec in all cases. To eliminate the overlap and isolate the predictive aspect of the C S - U C S pairing, the following study was conducted using a trace conditioning procedure, where the CS and UCS do not overlap. The trace conditioning procedure is generally considered to be less effective than delay conditioning, where some overlap exists between the CS and UCS (Mackintosh, 1974). This experiment was designed to answer the following questions: (1) does trace conditioning result in learning in Lymnaea, and (2) do forward and backward trace conditioning both occur?
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F16. 2. Compalison of the forward, backward, and simultaneous conditioning paradigms. The figure indicates the response of the forward, backward, and simultaneous animals of Experiment 2. The means of the Forward and Simultaneous Experimental groups were significantly higher than their respective controls throughout the experiment. The Backward Experimental group mean was significantly higher than the Backward Control mean only on the first three tests after training. Asterisks indicate significance (p < .05, one-tailed t test).
Methods A total of 30 animals were split up into three groups of l0 animals each: Forward Trace Experimental, Backward Trace Experimental, and Random Control. The animals were 10 weeks old at the start of the experiment and weighed 0.32 _+ 0.06 g. The feeding protocol was the same as used in the previous experiments. The animals were housed and trained in shallow plastic bins lined with screens as described above. At each training trial (Table 1), the screens were lifted out of the home bins and the animals were transferred to new bins with 300 ml of water containing the first stimulus. The Forward Experimental group was placed in a bin containing water plus the CS, the Backward Experimental group was placed in a bin containing water plus the UCS, and the Control group was placed in 300 ml of water. The animals remained in the presence of the first stimulus for 2 min,
398
ALEXANDER, AUDESIRK, AND AUDESIRK
then were transferred to rinse bins for 15 sec. They were then transferred to bins containing the second stimulus; the Forward Experimental animals were exposed to the UCS, the Backward Experimental animals to the CS And the Random Control animals to the UCS. After 2 min, the snails were lifted out and transferred to rinse bins, and then 15 sec later back to the home bins. At times ranging from 30 min before to 45 min after the Control group was exposed to the UCS, these snails were placed in a bin containing the CS for 2 min, transferred to a rinse bin, then moved to a bin containing only water, then rinsed and placed back in the home bin. To equalize handling, both experimental groups were placed in bins containing water, rinsed, placed into other bins containing water, rinsed, and placed back into their respective home bins. Training consisted of 15 training trials, 5 trials per day for 3 consecutive days, with an ITI of 90 minutes. Results As can be seen in Fig. 3, the Forward Experimental group was significantly higher than the Control group (p < .001), as was the Backward
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TEMPORAL EFFECTS ON LEARNING IN Lymnaea
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Experimental group (p < .0005) on the first test after training. On the second test, the mean of the Backward Experimental snails declined relative to the control (p < .01) whereas the mean of the Forward Experimental group increased relative to the control (p < .001). On the third test, however, both the Forward and Backward Experimental groups were insignificantly greater than the control group. The mean responses of the two experimental groups were compared directly and differed significantly on the second test (p < .025). DISCUSSION
Temporal distribution of training trials. It has generally been found that massed practice (training trials that follow each other closely in time) is less efficient than spaced practice, where a greater period of time separates successive trials (Hilgard and Marquis, 1940). Many examples of the superiority of spaced practice in various learning tasks can be found in the literature (for examples see Lorge, 1930; Bourne and Archer, 1956). In Lymnaea, the major effect of varying the distribution of training on learning was on retention, not on the initial magnitude of learning. In Experiment 1, greater spacing of the training trials retarded forgetting, whereas massing the trials hastened it. Immediately after training, the expression of the learned response was almost equal between the Massed and Spaced Experimental animals. The major difference occurred days later, when the mean of the Massed Experimental group fell rapidly. Temporal relationship of the CS and UCS. In pairing the two stimuli of the classical conditioning paradigm (CS and UCS) the onset of the CS can occur either before the UCS onset (forward conditioning), or after the UCS onset (backward conditioning), or the two stimuli can occur together (simultaneous conditioning). Many investigations have failed to show significant learning with the simultaneous and backward paradigms, and positive results have often been attributed to nonassociative effects such as sensitization (for reviews see Kimble, 1961; Cautela, 1965). The failure to achieve significant learning with the simultaneous and backward procedures led to the suggestion that the CS serves as a signal, enabling the organism to predict the impending occurrence of the UCS. When the onset of the CS occurs simultaneously with or after the UCS onset, the best predictor of the UCS is the UCS onset itself, the CS in these cases no longer serving as a predictor of the UCS. However, the range of training procedures used to show the failure of the simultaneous and backward conditioning has been quite narrow, as pointed out by Heth and Rescorla (1973). Short duration CSs and UCSs have invariably been used, and the UCS in almost every case has been shock. In reviewing several of the many cases of unsuccessful attempts of backward conditioning, we found that no overlap existed between the CS and UCS, a discrete amount of time separated the two.
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In successful studies, such as Heth and Rescorla (1973) and Mowrer and Aiken (1954), extensive overlap existed between the UCS and CS, and the two stimuli were longer in duration. We also were successful in demonstrating simultaneous and backward conditioning with relatively long, overlapping stimuli, as seen in Experiment 2. Historically, several theories concerning reinforcement have been postulated, two of these being the theory of contiguity and the theory of expectancy (Hilgard & Marquis, 1940; Kimble, 1961). The contiguity theory of reinforcement holds that the CS is substituted for or equated with the CS after several pairings of the two. The expectancy theory is concerned with the predictive value of the CS, where the animal behaves in ways consistent with anticipated consequences. If the UCS appears after the CS, the expectation is strengthened. There are problems with both theories, however. Though the main problem with the contiguity or stimulus substitution theory has been the observation that the CR sometimes differs from the UCR (Hilgard & Marquis, 1940), a number of studies show that the animal may indeed substitute the CS for the UCS. Pavlov (1934) reported that dogs trained to salivate to a light that preceded food sometimes turned their heads to the light source and licked the light. Breland and Breland (1961) cite several similar instances in a variety of animals. The animals, after being trained to perform a particular response, drifted into behaviors similar to instinctive food obtaining patterns. Jenkins and Moore (1973) described pigeons which were autoshaped to peck at a key to obtain food or water. The pigeons' beak movements towards the key when their reward was water resemble drinking movements, while the pecks at the key for food resembled those made when eating. In this report, the rasps made by Lymnaea to the CS appear to be identical to those made to the sucrosecasein UCS. The expectancy theory, on the other hand, cannot cope with backward conditioning, because the backward procedure eliminates the predictive quality of the CS. But in the successful demonstrations of backward conditioning, some overlap existed, overlap that may enhance the equation of the two stimuli (contiguity), regardless of the temporal order of appearance of the CS and UCS. From the results of Experiment 2, it appears that Lymnaea may use both the predictive aspect of the CS and equate the CS with the UCS as well. From this, one would expect that no overlap of the two stimuli (by use of the trace conditioning procedure) would lead to weaker forward conditioning and little or no backward conditioning. The results of Experiment 3 tend to support this hypothesis, although the results are not directly comparable because slightly different training procedures were used. It is striking that significant learning did occur with both backward and forward trace conditioning procedures, although the learning was
TEMPORAL EFFECTS ON LEARNING IN Lymnaea
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short lived, persisting for approximately 2 days. The fact that the backward trace procedure worked needs some explanation, since neither the contiguity nor the expectancy theories can account for it. Cautela (1965) presented a hypothesis that the cases of successful backward conditioning were cases of conditioning where the CS was paired with pain perception or a memory trace of the UCS. Support for this idea comes from the study of feeding in the marine mollusc Aplysia californica (Kupfermann 1974). Food arousal in Aplysia lasts for at least 5 min beyond the presentation of the initial arousing stimulation. Perhaps CNS firing to the UCS is still persisting up to the time the CS is introduced, producing a "mental overlap" of the two stimuli. As mentioned before, delay conditioning has generally been found to be superior to trace conditioning. Therefore, temporal overlap between the CS and UCS enhances the quality of learning in the classical conditioning procedures, but, as seen by the results of Experiment 3, overlap is not essential for successful learning. ff both contiguity and expectancy are assumed to contribute to learning, this could explain both the results reported here and the results of past research reported in the mammalian literature. Although the contiguity and expectancy theories of reinforcement have been cited as alternate theories (Hilgard and Marquis, 1940), we see no reason to treat them as mutually exclusive. Our findings that simultaneous and backward conditioning paradigms are effective when overlap exists between the CS and UCS are best explained by the contiguity hypothesis, whereas the superiority of the forward conditioning procedures (both delay and trace) over their backward counterparts supports the notion that the CS is also treated as a predictor of the UCS. The stimulus overlap may encourage animals to equate the two stimuli, and this equation may interact with the predictive element to produce optimal learning in both vertebrates and invertebrates alike. REFERENCES Audesirk, T. E., Alexander, J. E., Jr., Audesirk, G. J., & Moyer, C. (1983). Rapid, nonaversive conditioning in a freshwater gastropod. I. Effects of age and motivation. Behavioral and Neural Biology, 36, 379-390. Bourne, L. E., Jr., & Archer, E. J. (1956). Time continuously on target as a function of distribution of practice. Journal of Experimental Psychology, 51, 25-33. Breland, K., & Breland, M. (1961). The misbehavior of organisms. American Psychologist, 16, 681-684. Cautela, J. R. (1965). The problem of backward conditioning. Journal of Psychology, 60, 135-144. Heath, C. D., & Rescorla, R. A. (1973). Simultaneous and backward fear conditioning in the rat. Journal of Comparative and Physiological Psychology, 82, 434-443. Hilgard, E. R., & Marquis, D. G. (1940). Conditioning and Learning. New York: AppletonCentury. Jenkins, H. M., & Moore, B. R. (1973). The form of the autoshaped response with food or water reinforcers. Journal of the Experimental Analysis of Behavior, 20, 163-181.
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Kimble, G. A. (1961). Hilgard and Marguis' Conditioning and Learning. New York: Appleton-Century-Crofts. Kupfermann, I. (1974). Feeding behavior in Aplysia: A simple system for the study of motivation. Behavioral Biology, 10, 1-26. Lorg e, I. (1930). Influence of Regularly Interpolated Time Intervals upon Subsequent Learning. New York: Teachers College, Columbia University, Bureau of Publications. Mackintosh, N. J. (1974). The Psychology of Animal Learning. New York/London: Academic Press. Mowrer, O. H., & Aiken, E. G. (1954). Contiguity vs. drive-reduction in conditioned fear: temporal variations in conditioned and unconditioned stimulus. American Journal of Psychology, 67, 26-38. Pavlov, I. P. (1934). An attempt at a physiological interpretation of obsessional neurosis and paranoi. Journal of Mental Science, 80, 187-197. Sahley, C., Gelperin, A., & Rudy, J. W. (1981). One-trial associative learning modified food odor preferences of a terrestrial mollusc. Proceedings of the National Academy of Sciences USA, 78, 640-642. (a) Sahley, C., Hardison, P., Hsuan, A., & Gelperin, A. (1982). Appetitively reinforced odor conditioning modulates feeding in Limax maximus. Society for Neuroscience Abstracts, 8, 823. Sahley, C., Rudy, J. W., & Gelperin, A. (1981). An analysis of associative learning in a terrestrial mollusc. I. Higher-order conditioning, blocking, and a transient US preexposure effect. Journal of Comparative Physiology, 144, 1-8. (b)