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Context Extinction and Associative Learning in Lymnaea
Neurobiology of Learning and Memory 78, 23–34 (2002) doi:10.1006/nlme.2001.4041
Context Extinction and Associative Learning in Lymnaea Chloe McComb,1 Susan Sangha,1 Syed Qadry, James Yue, Andi Scheibenstock, and Ken Lukowiak Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1 Published online March 14, 2002
Aerial respiratory behavior in the pond snail Lymnaea was operantly conditioned so that snails learned not to perform aerial respiration in a hypoxic environment. Snails were trained in either the standard context (no food odorant) or a carrot (foododorant) context. An operant training procedure of two 45-min training sessions with a 1-h interval between the sessions followed by a third 45-min training session 18 h later was sufficient to produce associative learning and long-term memory (LTM) that persisted for at least 5 days. If, however, following the third operant training session snails received three 45-min extinction training sessions, with each extinction session separated by at least a 1-h interval, LTM was not observed when tested the following day. That is, the memory was extinguished. Extinction, however, did not occur if the context of the extinction training was different from the context of the associative training. That is, in the snails trained in the standard context, extinction did not occur if the extinction training sessions were performed in the food-odorant context and vice versa. 䉷 2002 Elsevier Science (USA) Key Words: associative learning; long-term memory; concurrent context learning; extinction; Lymnaea.
INTRODUCTION Learning and memory are two distinct but related processes, each with its own forms and rules (Milner et al., 1998; Martin et al., 2000). The acquisition or alteration of behavior as a result of training is what we mean by learning, and the retention of the changed behavior is what we define as memory. Depending on a number of factors, for example, This work was supported by a grant from the CIHR to K.L., J.Y., and S.Q. were recipients of AHFMR summer studentships. We thank Drs. Q. Pittman, N. Syed, G. Spencer, and R. Hawkes for discussions and comments on earlier drafts of the manuscript. Address reprint requests to Dr. Ken Lukowiak, Department of Physiology and Biophysics, Neuroscience Research Group, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada, T2N 4N1. Fax: 403 283 2700. E-mail: [email protected]. 1 These authors contributed equally to this paper. 23
1074-7427/02 $35.00 䉷 2002 Elsevier Science (USA) All rights reserved.
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the duration and the interval between training sessions, the persistence of the memory varies significantly (McGaugh, 2000). It is also possible to extinguish the learned behavior by extinction training (i.e., withholding the presentation of the reinforcing stimulus) (Pavlov, 1927). The behavioral phenotype following extinction training resembles the naı¨ve state. Lymnaea have the ability to associatively learn and to consolidate that learning into memories lasting hours to weeks (reviewed by Benjamin et al., 2000). We have focused our attention on aerial respiratory behavior in Lymnaea and have developed training procedures that result in operant conditioning (a form of associative learning) and longlasting memory of at least 1 month duration (Lukowiak et al., 1996, 1998, 2000). Briefly, Lymnaea respire both cutaneously and via a respiratory orifice, the pneumostome, to a rudimentary lung (i.e., aerial respiration). Under eumoxic conditions cutaneous respiration predominates but in hypoxic conditions the respiratory requirements of the snail are met primarily by aerial respiration (Lukowiak et al., 1996). We have chosen to study aerial respiratory behavior in Lymnaea because a three-neuron network, whose necessity and sufficiency have been demonstrated, mediates this behavior (Syed et al., 1990, 1992). Further, we have found neural correlates of this associative learning and its memory encoded in these three neurons in both isolated ganglionic and semi-intact preparations (Spencer et al., 1999, 2001). Lymnaea also demonstrate context-specific learning and long-term memory (LTM) (Haney & Lukowiak, 2001). For example, if the snail is trained in a hypoxic environment that includes a food odorant (i.e., the food-odorant context), LTM is only demonstrated if the food odorant is present at the time of the memory test (Haney & Lukowiak, 2001). The modern concept of extinction can be traced back to the experiments initiated by Pavlov at the beginning of the 20th century (Pavlov, 1927). There are a number of conflicting hypotheses that have been formulated to explain the process of extinction (Mackintosh, 1974). Further, extinction has been hypothesized to be an active process (Kimble, 1961), but it has proven difficult to directly test this. We show here that extinction of the learned behavior occurs under the appropriate experimental conditions. However, extinction, just as learning, is context dependent. That is, if the extinction training is given in a context different from the context in which the learning and the memory occurred, extinction does not occur. Recent experiments in both humans and rodents have also shown that extinction is context dependent (Holt & Maren, 1999; Lovibond et al., 2000; Harris et al., 2000). In demonstrating that extinction is context dependent we also showed that snails have the ability to learn and establish LTM concurrently in two different contexts. MATERIALS AND METHODS Lymnaea Lymnaea originally obtained from Vrije Universiteit in Amsterdam were laboratory bred in our snail facility at the University of Calgary. Snails were 25–30 mm in length, the same length (and thus age) that has been used in our previously cited learning and memory experiments. All animals used in these studies had continuous access to food (lettuce) in their home eumoxic aquaria.
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Procedures for Training and Memory Testing Associative training. A 1-liter beaker filled with 500 ml of eumoxic pond water was first made hypoxic by bubbling N2 through it for 20 min. Individually marked snails were placed into the hypoxic pond water for a 10-min period of acclimatization. During this period they were free to open and close their pneumostome at the water surface in order to perform aerial respiration. At the end of this period all snails were gently pushed under the water and the training begins. Two different contexts were used to train snails, the “standard context” and the “food-odorant context.” The only difference between the two contexts was that in the food-odorant context the N2 gas was first bubbled through a 750-ml Erlenmeyer flask containing cut up carrot. Full details are given in Haney and Lukowiak (2001). In the standard training procedure and the food-odorant procedure snails were subjected to three 45-min training sessions given over a 1.5-day period. The first two sessions, separated by a 1-h rest interval, were given on the first day. The third training session was given 18 h later on the second day. The memory test session was given 5 days later. Since we determine whether memory is present by comparing the number of tactile stimuli delivered to the pneumostome as it begins to open (i.e., an attempted pneumostome opening; see below), the tactile stimulus is applied in the operant training and memory test sessions. In all sessions the tactile stimulus was delivered to the snail as it began to open its pneumostome at the air–water interface. This caused the immediate closure of the pneumostome and did not result in the snail withdrawing its body into the shell. Following closure of the pneumostome snails continued to stay at the air–water interface or crawl down along the side of the beaker. A full description of the snails’ response to pneumostome stimulation is given in Lukowiak et al. (1996). The time of each stimulus was recorded and then tabulated. Extinction training. The three extinction training sessions (45 min duration) were given in the same temporal sequence as the operant conditioning training sessions. They were the same as the associative training sessions; however, the snail was allowed to perform aerial respiration and no tactile stimuli were delivered to the pneumostome area. The first extinction training session was given to the snails 1 h after the last associative training session, with the second session given 1 h later. The third extinction training session was given on the following day. The memory test session was performed 1 h after the last extinction training session (i.e., approximately 26 h after the final associative training session). Extinction training sessions were performed with either the standard or food-odorant hypoxic procedure. “Blind” extinction training and memory test procedures. The experimenter performing the extinction training did so “blindly,” not knowing in what context the snails had been trained in. Moreover, this experimenter did not reveal to the other experimenters which context was used for extinction training. In fact, in some experiments no extinction training was given. Thus when the subsequent memory test was performed on the snails the different experimenter did not know (1) whether the snail had received extinction training or (2) if extinction training had been given, in what context it was given. Only after all the results were tabulated did we know the outcome of the various experiments.
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Learning and memory in two different contexts. In these experiments snails were initially operantly trained in one context for three 45-min sessions on the first day, with at least a 1-h interval between the sessions. On the following day, they received another three operant training sessions but in the other context (i.e., if the standard context was used first then the odorant context was used second and vice versa). Again, the three sessions were each 45 min in duration with at least a 1-h interval between them. Five days later the snails received a memory test session for each context (45 min duration). There was at least a 1-h interval between the two memory test sessions (the standard and the food-odorant sessions). Yoked control experiments. Yoked control experiments were performed as previously described (Lukowiak et al., 1996, 2000; Haney & Lukowiak, 2001). Briefly, on the first day (the Pretest Session) snails were placed in the hypoxic test beaker, just as the experimental snails and the number of attempted pneumostome openings was determined. Each time the snail attempted to open its pneumostome it received a tactile stimulus to its pneumostome (one 45-min session is not sufficient to produce LTM, Lukowiak et al., 2000). One day later, these snails were again placed in the hypoxic test beaker. However, now the tactile stimulus was applied to the pneumostome area not when the snail attempted to open its pneumostome but when the snail to which it was “yoked” attempted to open its pneumostome. The “yoked” snails thus received three “training sessions” over the 1.5day period. On the following day after the third yoked control session, the yoked snails were again placed into the beaker. Now each time they opened their pneumostome they received a tactile stimulus to the pneumostome. This was the Posttest Session. We compared the number of tactile stimuli in the “Post-session” to the “Pre-session” and found that they were statistically insignificant from each other (paired t test; p ⬎ .05). Thus, just receiving the tactile stimuli does not result in a diminution of the response (i.e., no learning occurs). Because of this we have not presented any graphs of these data under Results. Operational definitions of learning and memory. We have operationally defined learning and memory as we have previously (Lukowiak et al., 1996, 2000; Spencer et al., 1999). Briefly, associative learning is defined as a significant effect of training on the number of attempted pneumostome openings (one-way ANOVA, p ⬍ .05; followed by a post-hoc Fisher’s LSD protected t test, p ⬍ .05 within each separate group). The number of attempted pneumostome openings in the final training session had to be significantly less than the number of attempted pneumostome openings in the first training session. LTM was defined as being present if : (1) the number of attempted pneumostome openings in the memory test session was not significantly different from the number of attempted openings in the last training session and (2) the number of attempted openings in the memory test session was significantly less than the number of attempted openings in Session 1.
RESULTS Two 45-min training sessions with a 1-h interval between sessions were delivered to the snails (N ⫽ 20) on day 1. Eighteen hours later a third 45-min training session was given. This procedure (Fig. 1A) produces associative learning and LTM that persists for
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FIG. 1. Learning, long-term memory (LTM), and extinction in “standard context” experiments. (A) Twenty naı¨ve snails received operant conditioning training and these snails exhibited learning. That is, there was no significant difference (NSD) in the number of attempted pneumostome openings between the memory test session and Session 3. (ANOVA F(19,2) ⫽ 16.255, p ⬍ .001; Session 3 was significantly less than Session 1, p ⬍ .01). When a memory test was performed 5 days after the last training session, LTM was present. The memory test session was not significantly different from Session 3, p ⬎ .05, and the memory test session was significantly less than Session 1, p ⬍ .01). (B) Another cohort of nai¨ve snails (N ⫽ 21) were operantly conditioned in the “standard context” (ANOVA F(20,2) ⫽ 12.9195, p ⬍ .001; Session 3 was significantly less than Session 1, p ⬍ .01) and then received extinction training in the standard context. Extinction training resulted in LTM not being observed when tested (memory test) 26 h after the last operant conditioning training session. That is, the number of attempted pneumostome openings in the memory test session was significantly greater than the number of attempted openings in Session 3.
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5 days. Yoked control animals did not exhibit learning and thus did not exhibit LTM (data not shown; see Materials and Methods). We next showed that we could extinguish the associative learned behavior by employing the extinction training procedure (see Materials and Methods) after learning and memory consolidation had occurred. Briefly, 1 h after the last training session (Session 3) the snails were placed in the standard hypoxic training apparatus for a 45-min session. However, in this and two ensuing 45-min sessions the reinforcing stimulus (a tactile stimulus delivered to the pneumostome area as the pneumostome opens) was not presented to the snail. Thus, the snail was able to perform aerial respiratory behavior. We tested for LTM 1 h after the final extinction session in the memory test session (i.e., 26 h after the last operant training session). The results of these experiments (N ⫽ 21) show that we are able to extinguish the learned behavior (Fig. 1B). The three associative learning training sessions resulted in learning. However, following the three extinction training sessions LTM could not be demonstrated. That is, the number of attempted pneumostome openings in the memory test session was significantly greater than the number of attempted pneumostome openings in Session 3 ( p ⬍ .01). Moreover, the number of attempted openings in the memory test session was not significantly different from the number of attempted openings in Session 1 ( p ⬎ .05). As we saw from the data in Fig. 1A, the memory should have persisted for at least 5 days after the last training session. However, with extinction training the memory does not persist longer than 26 h following the last training session. We conclude therefore that it is possible to extinguish the learned associative behavior. Operant conditioning of aerial respiration in Lymnaea is context dependent (Haney & Lukowiak, 2001). We therefore wished to determine if extinction was also context dependent. To test this hypothesis we first had to show that using the training procedure in Fig. 1A snails could also learn and form LTM in a food-odorant (carrot) context. Using the carrot-odor procedure (see Materials and Methods) we established that snails (N ⫽ 19) could learn and exhibit LTM. These snails (Fig. 2A) could be associatively conditioned in the food-odorant context. Moreover, when tested 5 days later (memory test session), these snails exhibited LTM. Thus snails were capable of associative learning and LTM that persisted for at least 5 days when trained in either context with the three 45-min training sessions. We next determined if the food-odor context LTM could also be extinguished. In the following experiments the “blind” procedure described under Materials and Methods was used for training and testing the snails. In another cohort of snails (N ⫽ 20) we used the extinguishing procedure for the food-odorant context described under Materials and Methods. Thus, following the third training session, three 45-min extinction training sessions (in the food-odorant context) were given as for the standard context extinction experiment (Fig. 2B). As can be seen, learning occurred over the three associative learning training sessions, but following the extinction training sessions LTM was not observed (the memory test session was significantly greater than Session 3, p ⬍ .01, but was not significantly different than Session 1, p ⬎ .05). To determine if extinction was context dependent, two further sets of experiments were performed. In the first of these (Fig. 3A) we associatively conditioned snails (N ⫽ 20) with the standard hypoxic procedure (i.e., no carrot odor). As can be seen, learning occurred. Following the last training session, the snails were now given extinction training
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FIG. 2. Learning, LTM, and extinction in “food-odorant context” experiments. (A) Data as in Fig. 1A except that the snails were trained in the food-odorant context. (ANOVA F(18,2) ⫽ 41.4915, p ⬍ .001; Session 3 was significantly less than Session 1, p ⬍ .01). These snails exhibited LTM. That is, the number of attempted pneumostome openings in the memory test session was not significantly different from the number of attempted openings in Session 3 ( p ⬎ .05) but was significantly less than the number of attempted openings in Session 1 ( p ⬍ .01). (B) As in Fig. 1B except that both the operant conditioning training (ANOVA F(19,2) ⫽ 25.5488, p ⬍ .001; Session 3 was significantly less than Session 1, p ⬍ .01) and the extinction training were in the food-odorant context.
but with the food-odorant (carrot) procedure. Extinction training in a different context did not result in extinction. That is, in the memory test session LTM was observed (the memory test session was not significantly different than Session 3, p ⬎ .05, but was significantly different from Session 1). Similar results were obtained (Fig. 3B) when we first trained snails (N ⫽ 20) using the food-odorant procedure and then switched to the standard procedure for extinction training. That is, LTM was still observed (the memory test session was not significantly different than Session 3, p ⬎ .05, but was significantly
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FIG. 3. Extinction is context dependent. (A) A naı¨ve group of snails (N ⫽ 20) were operantly trained in the standard context (ANOVA F(19,2) ⫽ 29.5651, p ⬍ .001; Session 3 was significantly less than Session 1, p ⬍ .01) and then received extinction training in the food-odorant context. The extinction training did not significantly alter LTM, as it was still present in the memory test session. (B) As in A, except the snails were operantly conditioned in the food-odorant context (ANOVA F(19,2) ⫽ 24.9663, p ⬍ .001; Session 3 was significantly less than Session 1, p ⬍ .01) and received extinction training in the standard context. Again, LTM was still observed.
different from Session 1), even though the reinforcing stimulus was not delivered to the open pneumostome during the extinction training sessions. We hypothesized that extinction is a form of learning (i.e., “unlearning” of the learned behavior). Thus it could be argued that the reason that extinction does not occur in a different context is that snails do not have the capacity to learn and remember concurrently in two different contexts. That is, snails are not “smart” enough to learn and remember at the same time in the two different contexts. To test whether snails have the capacity to learn and remember concurrently in two different contexts we first trained snails in one context, allowed memory consolidation and then trained them in the second context. We then tested for LTM for both contexts 5 days later. These data are shown in Fig. 4. Snails (N ⫽ 10) were first associatively trained with the standard hypoxic procedure (Sessions 1–3) and learning was demonstrated. On the following day these snails were
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FIG. 4. Lymnaea have the capacity to learn and form LTM in two different contexts concurrently. A cohort (N ⫽ 10) of naı¨ve snails first received operant conditioning training in (a) the standard context (blank bars). On the following day these same snails received similar training but in (b) the food-odorant context (filled bars). Learning was observed in both the standard (ANOVA F(9,2) ⫽ 9.2425, p ⬍ .001; Session 3 was significantly less than Session 1, p ⬍ .01) and food-odorant (ANOVA F(9,2) ⫽ 10.2895, p ⬍ .001; Session 3 was significantly less than Session 1, p ⬍ .01) contexts. When tested 5 days later LTM was shown to be present for both learning contexts. That is, the number of attempted pneumostome openings in the standard procedure memory test (c) was not significantly different from the number of attempted pneumostome openings in Standard Session 3 ( p ⬎ .05), but was significantly less than the number of attempted openings in Standard Session 1 ( p ⬍ .01). Moreover, the number of attempted pneumostome openings in the food-odorant memory test was not significantly different from the number of attempted openings in the food-odorant third training session ( p ⬎ .05), but was significantly less than that in the food-odorant first training session ( p ⬍ .01).
next associatively trained with the food-odorant procedure. Again these snails exhibited learning. Notice, however, that the response of the snails in first training session in the food-odorant context was not significantly different from the initial session with the standard context (paired t test; p ⬎ .05). That is, memory was context specific. Five days later, when tested first in the standard hypoxic procedure memory test and then in the food-odorant procedure memory test, LTM was shown to be present for both learning contexts. Another cohort of 10 snails was also trained in the two different contexts; however, in these experiments the food-odorant context was presented first, followed by the standard procedure. As the results from this experiment were the same as those presented in Fig. 4 they have not been shown here. Thus we conclude that snails have the capacity to associatively learn and exhibit LTM concurrently in two different contexts. DISCUSSION The results of the experiments reported here illustrate that it is possible to extinguish associatively learned behavior in Lymnaea; that extinction is context dependent and, that the central nervous system (CNS) of Lymnaea has the capacity to associatively learn and remember concurrently in at least two different contexts. Previously we used a number of different training procedures to associatively train snails not to perform aerial respiration in a hypoxic environment where this form of respiration predominates. In the hypoxic environment there is normally a significant
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increase in the number of bouts of aerial respiration compared to the eumoxic environment (Lukowiak et al., 1996). Both yoked control procedures and hypoxic control procedures have shown that the alteration in respiratory behavior (i.e., learning) produced by the various operant training procedures is a true form of associative learning (Lukowiak et al., 1996, 1998, 2000; Spencer et al., 1999, 2001; Haney & Lukowiak, 2001). We demonstrate here that another training procedure, utilizing three 45-min training sessions over a 1.5-day period, is also sufficient to produce associative learning and LTM that persists for at least 5 days. We further found that we were able to reverse or subordinate the processes that lead to the establishment of LTM by inserting three extinction training sessions following the final operant conditioning training procedure. Instead of the LTM phenotype, administration of the extinction training resulted in a behavioral phenotype similar to that of the naı¨ve animal. However, just allowing the snails to perform aerial respiration in a hypoxic environment without reinforcement was not in itself sufficient to prevent LTM. That is, if the context of the extinction training was different from the context of operant training, LTM was still observed (Fig. 3). Thus, extinction as well as operant conditioning is context dependent. These data are consistent with the hypothesis that extinction is a form of learning. That is, if the process of extinction were solely the result of opening the pneumostome and not receiving a tactile stimulus to the pneumostome area, then the snails shown in Fig. 3 should have undergone the extinction process. However, this was not the case because the context in which they received extinction training was not the same as the context in which they formed the learned association. They could only “unlearn” a context-specific learned behavior. Thus, the snails associatively trained with the standard hypoxic procedure did not exhibit extinction of LTM when given extinction training with the food-odorant procedure and vice versa. Similar data regarding the specificity of extinction (i.e., context-specific extinction) have been obtained in both human and rodent studies where context was an important factor in determining whether extinction of a learned response occurred (Holt & Maren, 1999; Lovibond et al., 2000; Harris et al., 2000). All of these data are consistent with the hypothesis that extinction is another form of learning in which the subject unlearns the specific behavior (Mackintosh, 1974; Squire & Kandel, 1999). Since learning is context dependent it should not be too surprising to find that extinction of that learned behavior is also context dependent. If extinction was not context dependent then important learned behavior that has been consolidated into LTM could more easily be lost. If extinction is itself a form of learning a different behavior (Kimble, 1961; Squire & Kandel, 1999) then it is arguable that the component of the Lymnaea nervous system responsible for controlling aerial respiratory behavior, the respiratory CPG, may possess only a limited capacity for learning. Thus, a different behavior (i.e., extinction in a different context) cannot be concomitantly learned. However, this is not the case, as the neural network was demonstrated here (Fig. 4) to have the capacity to be trained and establish LTM concurrently in at least two different contexts (standard and carrot food odorant). This finding is not too surprising since previous results from a number of different molluscan preparations, although not specific to learning in two different contexts, demonstrated that the molluscan nervous systems is competent at mediating more complex forms of associative learning (reviewed recently in Sahley & Crow, 1998). For example, molluscs
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are capable of second order classical conditioning (Sahley et al., 1981) and even observational learning (Fiorito & Scotto, 1992). Thus, higher order forms of associative learning are not the sole domains of vertebrates. The data shown here also illustrate an interesting feature of the Lymnaea respiratory CPG; it is a “hard-wired” neuronal circuit that possesses a remarkable amount of plasticity. Evidence for this has been obtained in cell culture experiments. The CPG neurons only establish appropriate synapses (i.e., those found in the living organism) and do not form synapses with inappropriate (i.e., synapses not seen in the living snail) partners, even though the inappropriate neurons possess the necessary transmitter receptors (Syed et al., 1990; Hamakawa et al., 1999; Woodin et al., 1999; Lukowiak & Syed, 1999; Taylor & Lukowiak, 2000). In contrast, we have seen here that employing the appropriate behavioral training procedures can associatively modify the behavior mediated by the CPG. In addition, “plastic” changes in neuronal activity and synaptic connectivity that are correlated with associative learning and its memory have been seen in the CPG neurons in isolated ganglionic and semi-intact preparations (Spencer et al., 1999, 2001). We can now begin to examine the neuronal mechanisms that underlie the processes of extinction and multicontext learning by using semi-intact preparations from trained snails. By using semi-intact preparations it is possible to simultaneously record pneumostome activity (i.e., behavior) and the intracellular activity of CPG neurons (Spencer et al., 2001). The results from these experiments should enhance our understanding of how neurons can learn and remember two different context situations at the same time. As well, these data also should give us insight into how the activity of identified neurons is altered by extinction training. These data may allow us to formulate hypotheses to explain how forgetting occurs at the level of individual neurons. REFERENCES Benjamin, P., Staras, K., & Kemenes, G. (2000). A systems approach to the cellular analysis of associative learning in the pond snail Lymnaea. Learning and Memory, 7, 124–131. Fiorito, G., & Scotto, P. (1992). Observational learning in Octopus vulgaris. Science, 256, 545–547. Hamakawa, T., Woodin, M., Bjorgum, M., Painter, S., Takaski, M., Lukowiak, K., Nagle, G., & Syed, N. (1999). Excitatory synaptogenesis between identified Lymnaea neurons in vitro requires extrinsic trophic factors and is mediated by receptor kinases. Journal of Neuroscience, 19, 9306–9312. Haney, J., & Lukowiak, K. (2001). Context learning and effect of context on memory retrieval in Lymnaea. Learning and Memory, 8, 35–43. Harris, J., Jones, M., Bailey, G., & Westbrook, R. (2000). Contextual control over conditioned responding in an extinction paradigm. J. Exp. Psychol. Anim. Behav. Process, 26, 174–185. Holt, W., & Maren, S. (1999). Muscimol inactivation of the dorsal hippocampus impairs contextual retrieval of fear memory. Journal of Neuroscience, 19, 9054–9062. Kimble, G. (1961). Hilgard and Marquis’ conditioning and learning. New York: Appleton–Century–Crofts. Lovibond, P., Davis, N., & O’Flaherty, A. (2000). Protection from extinction in human fear conditioning. Behav. Res. Ther., 38, 967–983. Lukowiak, K., Ringseis, E., Spencer, G., Wildering, W., & Syed, N. (1996). Operant conditioning of aerial respiratory behaviour in Lymnaea stagnalis. Journal of Experimental Biology, 199, 683–91. Lukowiak, K., Cotter, R., Westley, J., Ringseis, E., Spencer, G., & Syed, N. (1998). Long term memory of an operantly conditioned respiratory behaviour in Lymnaea stagnalis. Journal of Experimental Biology, 199, 683–691.
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Lukowiak, K., Adatia, A., Krygier, D., & Syed, N. (2000). Operant conditioning in Lymnaea: Evidence for intermediate and long-term memory. Learning and Memory, 7, 140–150. Mackintosh, N. (1974). The psychology of animal learning. New York: Academic Press. Martin, S. J., Grimwood, P. D., & Morris, R. G. M. (2000). Synaptic plasticity and memory: An evaluation of the hypothesis. Annual Review of Neuroscience, 23, 649–711. McGaugh, J. (2000). Memory, a century of consolidation. Science, 287, 248–251. Milner, B., Squire, L., & Kandel, E. (1998). Cognitive neuroscience and the study of memory. Neuron, 20, 445–468. Pavlov, I. (1927). Conditioned reflexes (translated by G. V. Anrep). London: Oxford Univ. Press. Rosenzweig, M., Bennett, E., Colombo, P., Lee, D., & Serrano, P. (1993). Short-term, intermediate term and long term memories. Behaviour Brain Research, 57, 193–198. Spencer, G., Syed, N., & Lukowiak, K. (1999). Neural changes after operant conditioning of the aerial respiratory behavior in Lymnaea stagnalis. Journal of Neuroscience, 19, 1836–1843. Spencer, G., Kazmi, M., Syed, N., & Lukowiak, K. (2001). Changes in the activity of a central pattern generator neuron following the reinforcement of an operantly conditioned behavior in Lymnaea (submitted for publication). Sahley, C., & Crow, T. (1998). Invertebrate learning: Current perspectives. In J. Martinez & R. Kesner (Eds.), Neurobiology of learning and memory (pp. 177–209). San Diego: Academic Press. Sahley, C., Rudy, J., & Gelperin, A. (1981). An analysis of associative learning in a terrestrial mollusk. I. Higher-order conditioning, blocking, and a US—pre-exposure effect. Journal of Comparative Physiology, 144, 1–8. Squire, L., & Kandel, E. (1999). Memory: From molecules to mind. New York: Sci. Am. Library. Syed, N. I., Bulloch, A. G. M., & Lukowiak, K. (1990). In vitro reconstruction of the respiratory central pattern generator of the mollusk Lymnaea. Science, 250, 282–285. Syed, N. I., Ridgway, R., Lukowiak, K., & Bulloch, A. G. M. (1992). Transplantation and functional integration of an identified respiratory interneuron in Lymnaea stagnalis. Neuron, 8, 767–774. Taylor, B., & Lukowiak, K. (2000). The respiratory central pattern generator of Lymnaea: a model, measured and malleable. Respiratory Physiology, 122, 197–207. Woodin, M., Hamakawa, T., Takaski, M., Lukowiak, K., & Syed, N. (1999). Trophic factor-induced plasticity of synaptic connections between identified Lymnaea neurons. Learning and Memory, 6, 307–316.
Context Extinction and Associative Learning in Lymnaea Chloe McComb,1 Susan Sangha,1 Syed Qadry, James Yue, Andi Scheibenstock, and Ken Lukowiak Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1 Published online March 14, 2002
Aerial respiratory behavior in the pond snail Lymnaea was operantly conditioned so that snails learned not to perform aerial respiration in a hypoxic environment. Snails were trained in either the standard context (no food odorant) or a carrot (foododorant) context. An operant training procedure of two 45-min training sessions with a 1-h interval between the sessions followed by a third 45-min training session 18 h later was sufficient to produce associative learning and long-term memory (LTM) that persisted for at least 5 days. If, however, following the third operant training session snails received three 45-min extinction training sessions, with each extinction session separated by at least a 1-h interval, LTM was not observed when tested the following day. That is, the memory was extinguished. Extinction, however, did not occur if the context of the extinction training was different from the context of the associative training. That is, in the snails trained in the standard context, extinction did not occur if the extinction training sessions were performed in the food-odorant context and vice versa. 䉷 2002 Elsevier Science (USA) Key Words: associative learning; long-term memory; concurrent context learning; extinction; Lymnaea.
INTRODUCTION Learning and memory are two distinct but related processes, each with its own forms and rules (Milner et al., 1998; Martin et al., 2000). The acquisition or alteration of behavior as a result of training is what we mean by learning, and the retention of the changed behavior is what we define as memory. Depending on a number of factors, for example, This work was supported by a grant from the CIHR to K.L., J.Y., and S.Q. were recipients of AHFMR summer studentships. We thank Drs. Q. Pittman, N. Syed, G. Spencer, and R. Hawkes for discussions and comments on earlier drafts of the manuscript. Address reprint requests to Dr. Ken Lukowiak, Department of Physiology and Biophysics, Neuroscience Research Group, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada, T2N 4N1. Fax: 403 283 2700. E-mail: [email protected]. 1 These authors contributed equally to this paper. 23
1074-7427/02 $35.00 䉷 2002 Elsevier Science (USA) All rights reserved.
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the duration and the interval between training sessions, the persistence of the memory varies significantly (McGaugh, 2000). It is also possible to extinguish the learned behavior by extinction training (i.e., withholding the presentation of the reinforcing stimulus) (Pavlov, 1927). The behavioral phenotype following extinction training resembles the naı¨ve state. Lymnaea have the ability to associatively learn and to consolidate that learning into memories lasting hours to weeks (reviewed by Benjamin et al., 2000). We have focused our attention on aerial respiratory behavior in Lymnaea and have developed training procedures that result in operant conditioning (a form of associative learning) and longlasting memory of at least 1 month duration (Lukowiak et al., 1996, 1998, 2000). Briefly, Lymnaea respire both cutaneously and via a respiratory orifice, the pneumostome, to a rudimentary lung (i.e., aerial respiration). Under eumoxic conditions cutaneous respiration predominates but in hypoxic conditions the respiratory requirements of the snail are met primarily by aerial respiration (Lukowiak et al., 1996). We have chosen to study aerial respiratory behavior in Lymnaea because a three-neuron network, whose necessity and sufficiency have been demonstrated, mediates this behavior (Syed et al., 1990, 1992). Further, we have found neural correlates of this associative learning and its memory encoded in these three neurons in both isolated ganglionic and semi-intact preparations (Spencer et al., 1999, 2001). Lymnaea also demonstrate context-specific learning and long-term memory (LTM) (Haney & Lukowiak, 2001). For example, if the snail is trained in a hypoxic environment that includes a food odorant (i.e., the food-odorant context), LTM is only demonstrated if the food odorant is present at the time of the memory test (Haney & Lukowiak, 2001). The modern concept of extinction can be traced back to the experiments initiated by Pavlov at the beginning of the 20th century (Pavlov, 1927). There are a number of conflicting hypotheses that have been formulated to explain the process of extinction (Mackintosh, 1974). Further, extinction has been hypothesized to be an active process (Kimble, 1961), but it has proven difficult to directly test this. We show here that extinction of the learned behavior occurs under the appropriate experimental conditions. However, extinction, just as learning, is context dependent. That is, if the extinction training is given in a context different from the context in which the learning and the memory occurred, extinction does not occur. Recent experiments in both humans and rodents have also shown that extinction is context dependent (Holt & Maren, 1999; Lovibond et al., 2000; Harris et al., 2000). In demonstrating that extinction is context dependent we also showed that snails have the ability to learn and establish LTM concurrently in two different contexts. MATERIALS AND METHODS Lymnaea Lymnaea originally obtained from Vrije Universiteit in Amsterdam were laboratory bred in our snail facility at the University of Calgary. Snails were 25–30 mm in length, the same length (and thus age) that has been used in our previously cited learning and memory experiments. All animals used in these studies had continuous access to food (lettuce) in their home eumoxic aquaria.
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Procedures for Training and Memory Testing Associative training. A 1-liter beaker filled with 500 ml of eumoxic pond water was first made hypoxic by bubbling N2 through it for 20 min. Individually marked snails were placed into the hypoxic pond water for a 10-min period of acclimatization. During this period they were free to open and close their pneumostome at the water surface in order to perform aerial respiration. At the end of this period all snails were gently pushed under the water and the training begins. Two different contexts were used to train snails, the “standard context” and the “food-odorant context.” The only difference between the two contexts was that in the food-odorant context the N2 gas was first bubbled through a 750-ml Erlenmeyer flask containing cut up carrot. Full details are given in Haney and Lukowiak (2001). In the standard training procedure and the food-odorant procedure snails were subjected to three 45-min training sessions given over a 1.5-day period. The first two sessions, separated by a 1-h rest interval, were given on the first day. The third training session was given 18 h later on the second day. The memory test session was given 5 days later. Since we determine whether memory is present by comparing the number of tactile stimuli delivered to the pneumostome as it begins to open (i.e., an attempted pneumostome opening; see below), the tactile stimulus is applied in the operant training and memory test sessions. In all sessions the tactile stimulus was delivered to the snail as it began to open its pneumostome at the air–water interface. This caused the immediate closure of the pneumostome and did not result in the snail withdrawing its body into the shell. Following closure of the pneumostome snails continued to stay at the air–water interface or crawl down along the side of the beaker. A full description of the snails’ response to pneumostome stimulation is given in Lukowiak et al. (1996). The time of each stimulus was recorded and then tabulated. Extinction training. The three extinction training sessions (45 min duration) were given in the same temporal sequence as the operant conditioning training sessions. They were the same as the associative training sessions; however, the snail was allowed to perform aerial respiration and no tactile stimuli were delivered to the pneumostome area. The first extinction training session was given to the snails 1 h after the last associative training session, with the second session given 1 h later. The third extinction training session was given on the following day. The memory test session was performed 1 h after the last extinction training session (i.e., approximately 26 h after the final associative training session). Extinction training sessions were performed with either the standard or food-odorant hypoxic procedure. “Blind” extinction training and memory test procedures. The experimenter performing the extinction training did so “blindly,” not knowing in what context the snails had been trained in. Moreover, this experimenter did not reveal to the other experimenters which context was used for extinction training. In fact, in some experiments no extinction training was given. Thus when the subsequent memory test was performed on the snails the different experimenter did not know (1) whether the snail had received extinction training or (2) if extinction training had been given, in what context it was given. Only after all the results were tabulated did we know the outcome of the various experiments.
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Learning and memory in two different contexts. In these experiments snails were initially operantly trained in one context for three 45-min sessions on the first day, with at least a 1-h interval between the sessions. On the following day, they received another three operant training sessions but in the other context (i.e., if the standard context was used first then the odorant context was used second and vice versa). Again, the three sessions were each 45 min in duration with at least a 1-h interval between them. Five days later the snails received a memory test session for each context (45 min duration). There was at least a 1-h interval between the two memory test sessions (the standard and the food-odorant sessions). Yoked control experiments. Yoked control experiments were performed as previously described (Lukowiak et al., 1996, 2000; Haney & Lukowiak, 2001). Briefly, on the first day (the Pretest Session) snails were placed in the hypoxic test beaker, just as the experimental snails and the number of attempted pneumostome openings was determined. Each time the snail attempted to open its pneumostome it received a tactile stimulus to its pneumostome (one 45-min session is not sufficient to produce LTM, Lukowiak et al., 2000). One day later, these snails were again placed in the hypoxic test beaker. However, now the tactile stimulus was applied to the pneumostome area not when the snail attempted to open its pneumostome but when the snail to which it was “yoked” attempted to open its pneumostome. The “yoked” snails thus received three “training sessions” over the 1.5day period. On the following day after the third yoked control session, the yoked snails were again placed into the beaker. Now each time they opened their pneumostome they received a tactile stimulus to the pneumostome. This was the Posttest Session. We compared the number of tactile stimuli in the “Post-session” to the “Pre-session” and found that they were statistically insignificant from each other (paired t test; p ⬎ .05). Thus, just receiving the tactile stimuli does not result in a diminution of the response (i.e., no learning occurs). Because of this we have not presented any graphs of these data under Results. Operational definitions of learning and memory. We have operationally defined learning and memory as we have previously (Lukowiak et al., 1996, 2000; Spencer et al., 1999). Briefly, associative learning is defined as a significant effect of training on the number of attempted pneumostome openings (one-way ANOVA, p ⬍ .05; followed by a post-hoc Fisher’s LSD protected t test, p ⬍ .05 within each separate group). The number of attempted pneumostome openings in the final training session had to be significantly less than the number of attempted pneumostome openings in the first training session. LTM was defined as being present if : (1) the number of attempted pneumostome openings in the memory test session was not significantly different from the number of attempted openings in the last training session and (2) the number of attempted openings in the memory test session was significantly less than the number of attempted openings in Session 1.
RESULTS Two 45-min training sessions with a 1-h interval between sessions were delivered to the snails (N ⫽ 20) on day 1. Eighteen hours later a third 45-min training session was given. This procedure (Fig. 1A) produces associative learning and LTM that persists for
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FIG. 1. Learning, long-term memory (LTM), and extinction in “standard context” experiments. (A) Twenty naı¨ve snails received operant conditioning training and these snails exhibited learning. That is, there was no significant difference (NSD) in the number of attempted pneumostome openings between the memory test session and Session 3. (ANOVA F(19,2) ⫽ 16.255, p ⬍ .001; Session 3 was significantly less than Session 1, p ⬍ .01). When a memory test was performed 5 days after the last training session, LTM was present. The memory test session was not significantly different from Session 3, p ⬎ .05, and the memory test session was significantly less than Session 1, p ⬍ .01). (B) Another cohort of nai¨ve snails (N ⫽ 21) were operantly conditioned in the “standard context” (ANOVA F(20,2) ⫽ 12.9195, p ⬍ .001; Session 3 was significantly less than Session 1, p ⬍ .01) and then received extinction training in the standard context. Extinction training resulted in LTM not being observed when tested (memory test) 26 h after the last operant conditioning training session. That is, the number of attempted pneumostome openings in the memory test session was significantly greater than the number of attempted openings in Session 3.
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5 days. Yoked control animals did not exhibit learning and thus did not exhibit LTM (data not shown; see Materials and Methods). We next showed that we could extinguish the associative learned behavior by employing the extinction training procedure (see Materials and Methods) after learning and memory consolidation had occurred. Briefly, 1 h after the last training session (Session 3) the snails were placed in the standard hypoxic training apparatus for a 45-min session. However, in this and two ensuing 45-min sessions the reinforcing stimulus (a tactile stimulus delivered to the pneumostome area as the pneumostome opens) was not presented to the snail. Thus, the snail was able to perform aerial respiratory behavior. We tested for LTM 1 h after the final extinction session in the memory test session (i.e., 26 h after the last operant training session). The results of these experiments (N ⫽ 21) show that we are able to extinguish the learned behavior (Fig. 1B). The three associative learning training sessions resulted in learning. However, following the three extinction training sessions LTM could not be demonstrated. That is, the number of attempted pneumostome openings in the memory test session was significantly greater than the number of attempted pneumostome openings in Session 3 ( p ⬍ .01). Moreover, the number of attempted openings in the memory test session was not significantly different from the number of attempted openings in Session 1 ( p ⬎ .05). As we saw from the data in Fig. 1A, the memory should have persisted for at least 5 days after the last training session. However, with extinction training the memory does not persist longer than 26 h following the last training session. We conclude therefore that it is possible to extinguish the learned associative behavior. Operant conditioning of aerial respiration in Lymnaea is context dependent (Haney & Lukowiak, 2001). We therefore wished to determine if extinction was also context dependent. To test this hypothesis we first had to show that using the training procedure in Fig. 1A snails could also learn and form LTM in a food-odorant (carrot) context. Using the carrot-odor procedure (see Materials and Methods) we established that snails (N ⫽ 19) could learn and exhibit LTM. These snails (Fig. 2A) could be associatively conditioned in the food-odorant context. Moreover, when tested 5 days later (memory test session), these snails exhibited LTM. Thus snails were capable of associative learning and LTM that persisted for at least 5 days when trained in either context with the three 45-min training sessions. We next determined if the food-odor context LTM could also be extinguished. In the following experiments the “blind” procedure described under Materials and Methods was used for training and testing the snails. In another cohort of snails (N ⫽ 20) we used the extinguishing procedure for the food-odorant context described under Materials and Methods. Thus, following the third training session, three 45-min extinction training sessions (in the food-odorant context) were given as for the standard context extinction experiment (Fig. 2B). As can be seen, learning occurred over the three associative learning training sessions, but following the extinction training sessions LTM was not observed (the memory test session was significantly greater than Session 3, p ⬍ .01, but was not significantly different than Session 1, p ⬎ .05). To determine if extinction was context dependent, two further sets of experiments were performed. In the first of these (Fig. 3A) we associatively conditioned snails (N ⫽ 20) with the standard hypoxic procedure (i.e., no carrot odor). As can be seen, learning occurred. Following the last training session, the snails were now given extinction training
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FIG. 2. Learning, LTM, and extinction in “food-odorant context” experiments. (A) Data as in Fig. 1A except that the snails were trained in the food-odorant context. (ANOVA F(18,2) ⫽ 41.4915, p ⬍ .001; Session 3 was significantly less than Session 1, p ⬍ .01). These snails exhibited LTM. That is, the number of attempted pneumostome openings in the memory test session was not significantly different from the number of attempted openings in Session 3 ( p ⬎ .05) but was significantly less than the number of attempted openings in Session 1 ( p ⬍ .01). (B) As in Fig. 1B except that both the operant conditioning training (ANOVA F(19,2) ⫽ 25.5488, p ⬍ .001; Session 3 was significantly less than Session 1, p ⬍ .01) and the extinction training were in the food-odorant context.
but with the food-odorant (carrot) procedure. Extinction training in a different context did not result in extinction. That is, in the memory test session LTM was observed (the memory test session was not significantly different than Session 3, p ⬎ .05, but was significantly different from Session 1). Similar results were obtained (Fig. 3B) when we first trained snails (N ⫽ 20) using the food-odorant procedure and then switched to the standard procedure for extinction training. That is, LTM was still observed (the memory test session was not significantly different than Session 3, p ⬎ .05, but was significantly
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FIG. 3. Extinction is context dependent. (A) A naı¨ve group of snails (N ⫽ 20) were operantly trained in the standard context (ANOVA F(19,2) ⫽ 29.5651, p ⬍ .001; Session 3 was significantly less than Session 1, p ⬍ .01) and then received extinction training in the food-odorant context. The extinction training did not significantly alter LTM, as it was still present in the memory test session. (B) As in A, except the snails were operantly conditioned in the food-odorant context (ANOVA F(19,2) ⫽ 24.9663, p ⬍ .001; Session 3 was significantly less than Session 1, p ⬍ .01) and received extinction training in the standard context. Again, LTM was still observed.
different from Session 1), even though the reinforcing stimulus was not delivered to the open pneumostome during the extinction training sessions. We hypothesized that extinction is a form of learning (i.e., “unlearning” of the learned behavior). Thus it could be argued that the reason that extinction does not occur in a different context is that snails do not have the capacity to learn and remember concurrently in two different contexts. That is, snails are not “smart” enough to learn and remember at the same time in the two different contexts. To test whether snails have the capacity to learn and remember concurrently in two different contexts we first trained snails in one context, allowed memory consolidation and then trained them in the second context. We then tested for LTM for both contexts 5 days later. These data are shown in Fig. 4. Snails (N ⫽ 10) were first associatively trained with the standard hypoxic procedure (Sessions 1–3) and learning was demonstrated. On the following day these snails were
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FIG. 4. Lymnaea have the capacity to learn and form LTM in two different contexts concurrently. A cohort (N ⫽ 10) of naı¨ve snails first received operant conditioning training in (a) the standard context (blank bars). On the following day these same snails received similar training but in (b) the food-odorant context (filled bars). Learning was observed in both the standard (ANOVA F(9,2) ⫽ 9.2425, p ⬍ .001; Session 3 was significantly less than Session 1, p ⬍ .01) and food-odorant (ANOVA F(9,2) ⫽ 10.2895, p ⬍ .001; Session 3 was significantly less than Session 1, p ⬍ .01) contexts. When tested 5 days later LTM was shown to be present for both learning contexts. That is, the number of attempted pneumostome openings in the standard procedure memory test (c) was not significantly different from the number of attempted pneumostome openings in Standard Session 3 ( p ⬎ .05), but was significantly less than the number of attempted openings in Standard Session 1 ( p ⬍ .01). Moreover, the number of attempted pneumostome openings in the food-odorant memory test was not significantly different from the number of attempted openings in the food-odorant third training session ( p ⬎ .05), but was significantly less than that in the food-odorant first training session ( p ⬍ .01).
next associatively trained with the food-odorant procedure. Again these snails exhibited learning. Notice, however, that the response of the snails in first training session in the food-odorant context was not significantly different from the initial session with the standard context (paired t test; p ⬎ .05). That is, memory was context specific. Five days later, when tested first in the standard hypoxic procedure memory test and then in the food-odorant procedure memory test, LTM was shown to be present for both learning contexts. Another cohort of 10 snails was also trained in the two different contexts; however, in these experiments the food-odorant context was presented first, followed by the standard procedure. As the results from this experiment were the same as those presented in Fig. 4 they have not been shown here. Thus we conclude that snails have the capacity to associatively learn and exhibit LTM concurrently in two different contexts. DISCUSSION The results of the experiments reported here illustrate that it is possible to extinguish associatively learned behavior in Lymnaea; that extinction is context dependent and, that the central nervous system (CNS) of Lymnaea has the capacity to associatively learn and remember concurrently in at least two different contexts. Previously we used a number of different training procedures to associatively train snails not to perform aerial respiration in a hypoxic environment where this form of respiration predominates. In the hypoxic environment there is normally a significant
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increase in the number of bouts of aerial respiration compared to the eumoxic environment (Lukowiak et al., 1996). Both yoked control procedures and hypoxic control procedures have shown that the alteration in respiratory behavior (i.e., learning) produced by the various operant training procedures is a true form of associative learning (Lukowiak et al., 1996, 1998, 2000; Spencer et al., 1999, 2001; Haney & Lukowiak, 2001). We demonstrate here that another training procedure, utilizing three 45-min training sessions over a 1.5-day period, is also sufficient to produce associative learning and LTM that persists for at least 5 days. We further found that we were able to reverse or subordinate the processes that lead to the establishment of LTM by inserting three extinction training sessions following the final operant conditioning training procedure. Instead of the LTM phenotype, administration of the extinction training resulted in a behavioral phenotype similar to that of the naı¨ve animal. However, just allowing the snails to perform aerial respiration in a hypoxic environment without reinforcement was not in itself sufficient to prevent LTM. That is, if the context of the extinction training was different from the context of operant training, LTM was still observed (Fig. 3). Thus, extinction as well as operant conditioning is context dependent. These data are consistent with the hypothesis that extinction is a form of learning. That is, if the process of extinction were solely the result of opening the pneumostome and not receiving a tactile stimulus to the pneumostome area, then the snails shown in Fig. 3 should have undergone the extinction process. However, this was not the case because the context in which they received extinction training was not the same as the context in which they formed the learned association. They could only “unlearn” a context-specific learned behavior. Thus, the snails associatively trained with the standard hypoxic procedure did not exhibit extinction of LTM when given extinction training with the food-odorant procedure and vice versa. Similar data regarding the specificity of extinction (i.e., context-specific extinction) have been obtained in both human and rodent studies where context was an important factor in determining whether extinction of a learned response occurred (Holt & Maren, 1999; Lovibond et al., 2000; Harris et al., 2000). All of these data are consistent with the hypothesis that extinction is another form of learning in which the subject unlearns the specific behavior (Mackintosh, 1974; Squire & Kandel, 1999). Since learning is context dependent it should not be too surprising to find that extinction of that learned behavior is also context dependent. If extinction was not context dependent then important learned behavior that has been consolidated into LTM could more easily be lost. If extinction is itself a form of learning a different behavior (Kimble, 1961; Squire & Kandel, 1999) then it is arguable that the component of the Lymnaea nervous system responsible for controlling aerial respiratory behavior, the respiratory CPG, may possess only a limited capacity for learning. Thus, a different behavior (i.e., extinction in a different context) cannot be concomitantly learned. However, this is not the case, as the neural network was demonstrated here (Fig. 4) to have the capacity to be trained and establish LTM concurrently in at least two different contexts (standard and carrot food odorant). This finding is not too surprising since previous results from a number of different molluscan preparations, although not specific to learning in two different contexts, demonstrated that the molluscan nervous systems is competent at mediating more complex forms of associative learning (reviewed recently in Sahley & Crow, 1998). For example, molluscs
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are capable of second order classical conditioning (Sahley et al., 1981) and even observational learning (Fiorito & Scotto, 1992). Thus, higher order forms of associative learning are not the sole domains of vertebrates. The data shown here also illustrate an interesting feature of the Lymnaea respiratory CPG; it is a “hard-wired” neuronal circuit that possesses a remarkable amount of plasticity. Evidence for this has been obtained in cell culture experiments. The CPG neurons only establish appropriate synapses (i.e., those found in the living organism) and do not form synapses with inappropriate (i.e., synapses not seen in the living snail) partners, even though the inappropriate neurons possess the necessary transmitter receptors (Syed et al., 1990; Hamakawa et al., 1999; Woodin et al., 1999; Lukowiak & Syed, 1999; Taylor & Lukowiak, 2000). In contrast, we have seen here that employing the appropriate behavioral training procedures can associatively modify the behavior mediated by the CPG. In addition, “plastic” changes in neuronal activity and synaptic connectivity that are correlated with associative learning and its memory have been seen in the CPG neurons in isolated ganglionic and semi-intact preparations (Spencer et al., 1999, 2001). We can now begin to examine the neuronal mechanisms that underlie the processes of extinction and multicontext learning by using semi-intact preparations from trained snails. By using semi-intact preparations it is possible to simultaneously record pneumostome activity (i.e., behavior) and the intracellular activity of CPG neurons (Spencer et al., 2001). The results from these experiments should enhance our understanding of how neurons can learn and remember two different context situations at the same time. As well, these data also should give us insight into how the activity of identified neurons is altered by extinction training. These data may allow us to formulate hypotheses to explain how forgetting occurs at the level of individual neurons. REFERENCES Benjamin, P., Staras, K., & Kemenes, G. (2000). A systems approach to the cellular analysis of associative learning in the pond snail Lymnaea. Learning and Memory, 7, 124–131. Fiorito, G., & Scotto, P. (1992). Observational learning in Octopus vulgaris. Science, 256, 545–547. Hamakawa, T., Woodin, M., Bjorgum, M., Painter, S., Takaski, M., Lukowiak, K., Nagle, G., & Syed, N. (1999). Excitatory synaptogenesis between identified Lymnaea neurons in vitro requires extrinsic trophic factors and is mediated by receptor kinases. Journal of Neuroscience, 19, 9306–9312. Haney, J., & Lukowiak, K. (2001). Context learning and effect of context on memory retrieval in Lymnaea. Learning and Memory, 8, 35–43. Harris, J., Jones, M., Bailey, G., & Westbrook, R. (2000). Contextual control over conditioned responding in an extinction paradigm. J. Exp. Psychol. Anim. Behav. Process, 26, 174–185. Holt, W., & Maren, S. (1999). Muscimol inactivation of the dorsal hippocampus impairs contextual retrieval of fear memory. Journal of Neuroscience, 19, 9054–9062. Kimble, G. (1961). Hilgard and Marquis’ conditioning and learning. New York: Appleton–Century–Crofts. Lovibond, P., Davis, N., & O’Flaherty, A. (2000). Protection from extinction in human fear conditioning. Behav. Res. Ther., 38, 967–983. Lukowiak, K., Ringseis, E., Spencer, G., Wildering, W., & Syed, N. (1996). Operant conditioning of aerial respiratory behaviour in Lymnaea stagnalis. Journal of Experimental Biology, 199, 683–91. Lukowiak, K., Cotter, R., Westley, J., Ringseis, E., Spencer, G., & Syed, N. (1998). Long term memory of an operantly conditioned respiratory behaviour in Lymnaea stagnalis. Journal of Experimental Biology, 199, 683–691.
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