Anim Cogn (2013) 16:177–186 DOI 10.1007/s10071-012-0561-3
ORIGINAL PAPER
Cue competition effects in the planarian Jose Prados • Beatriz Alvarez • Joanna Howarth • Katharine Stewart • Claire L. Gibson • Claire V. Hutchinson Andrew M. J. Young • Colin Davidson
•
Received: 2 May 2012 / Revised: 28 August 2012 / Accepted: 29 August 2012 / Published online: 14 September 2012 Ó Springer-Verlag 2012
Abstract The learning abilities of planarian worms (Dugesia tigrina) were assessed by using a number of Pavlovian conditioning paradigms. Experiment 1 showed that planaria were susceptible to basic conditioning in that they readily developed a conditioned response to a change in ambient luminance when it was consistently paired with an electric shock over a number of trials. In Experiment 2, the change in luminance was presented in a compound with a vibration stimulus during conditioning. Subsequent tests revealed poor conditioning of the elements compared with control groups in which the animals were conditioned in the presence of the elements alone, an instance of overshadowing. In Experiment 3, pre-training of one of the elements before compound conditioning resulted in blocking of learning about the other element. These results add to other studies that have reported cue competition effects in animal species belonging to different phyla (chordate, mollusk, arthropod), suggesting that learning in these phyla could be ruled by similar principles. The results are discussed adopting an evolutionarycomparative approach.
J. Prados (&) B. Alvarez J. Howarth K. Stewart C. L. Gibson C. V. Hutchinson A. M. J. Young School of Psychology, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK e-mail:
[email protected] B. Alvarez Facultad de Psicologı´a, Universidad de Oviedo, Oviedo, Spain C. Davidson Basic Medical Sciences, St George’s University of London, London, UK
Keywords Invertebrate learning Pavlovian conditioning Overshadowing Blocking
Introduction Learning from experience is an important ability that allows individuals to adapt to the changing conditions of their environments. Although it has been documented throughout the animal kingdom, it is uncertain whether learning in different phyla is ruled by the same general principles. The research reported below was designed to examine this important question by assessing the learning abilities of the platyhelminth planaria (Dugesia tigrina). Platyhelminth is the most distant phylum from chordata in the phylogenetic tree with a bilateral centralized nervous system (Agata et al. 1998). Planaria’s nervous system presents anatomical (e.g., Sarnat and Netsky 1985) and neurochemical (e.g., Eriksson and Panula 1994; Rawls et al. 2006; Umeda et al. 2005) similarities with the nervous system of vertebrates. Because of its simplified nervous system and vertebrate-like neurotransmitters, it has been used, for example, as a model for the effects of abused drugs (e.g., Raffa and Valdez 2001). Planaria exposed to an electric shock display a characteristic shrinking unconditioned response (UR). Thompson and McConnell (1955) paired a relatively neutral stimulus, a light, with a shock and observed the development of a conditioned response (CR): Animals showed an increased tendency to shrink and/or suddenly turn when presented with the light conditioned stimulus (CS). In three control groups (light alone, shock alone, and a naı¨ve group with neither a CS nor a US), the rate of CR did not change throughout the experimental sessions. This early demonstration of Pavlovian conditioning in an invertebrate model
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initially attracted a lot of criticism due to the inadequacy of the control groups used. Rather than Pavlovian conditioning, it was frequently argued, increased responsiveness to the light is an instance of sensitization: The presentation of the shock-US could sensitize the animals at the onset of the training phase increasing the responsiveness to any stimulation (e.g., Jensen 1965). The basic procedure developed by Thompson and McConnell was widely used during the 1960s with improved control groups, and a significant body of evidence was reported that univocally characterized planaria’s behavioral plasticity as Pavlovian conditioning. Baxter and Kimmel (1963), for example, compared the progress of the CR in animals that were given either paired or unpaired presentations of light and shock—equating the experience with the CS and the US in both groups. All the animals were initially responsive to the light, a result that could be attributed to sensitization. However, the number of trials in which a CR was observed significantly increased in the paired group, but steadily decreased in the control unpaired group. This seems to suggest that animals in the paired group were learning the causal relationship between the CS and the US rather than showing a nonspecific increased tendency to respond. However, even accepting that CS–US pairings result in excitatory conditioning, the usage of the unpaired control group is not free of problems: In the unpaired group, the CS could potentially become an inhibitor of the US (a signal for the absence of the US). During the non-signaled presentations of the shock, the context might become an effective CS; in that case, presentations of the light-CS would take place in an excitatory context leading, according to standard associative learning theory, to the establishment of the light as an inhibitor of the US. If that was the case, differences in responsiveness to the CS could be attributed to a decrease in the responsiveness of the control group rather than an increase of the responsiveness in the experimental group. To get around this problem, the experimental paired group has been compared with a truly random control group in which, although the CS and the US can be presented together occasionally, the CS has no informative value about the occurrence of the US (Rescorla 1967). The results have shown that the CR develops in the experimental, but not in the random control group (Levison and Gavurin 1979). Further evidence that animals specifically respond to the designated CS was reported by Jacobson et al. (1967) in a study in which planarian worms were trained in a simple discrimination in which a light was paired with a shock and a vibration was presented alone. (Light and vibration were counterbalanced.) During the training phase, the animals responded more to the light than to the vibration. In a subsequent test, the relationship between the CSs and the shock was reversed: That is, the vibration was now
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followed by the shock, whereas the light was presented alone. It was found that the animals’ behavior adapted to the new reinforcement contingency, showing high levels of CR to the vibration and relatively low levels of responding to the light. This shows that conditioning is specific to the stimulus used as the CS, a characteristic of true conditioning that distinguishes it from pseudo-conditioning or sensitization. Other studies reported evidence to support the notion that, in some respects, conditioning in planaria resembles conditioning in vertebrate species. Baxter and Kimmel (1963) reported that the CR developed during conditioning rapidly extinguishes when the CS is presented alone for a number of trials, and reappears after some rest after the extinction has been completed (an instance of spontaneous recovery, Pavlov 1927). In a similar study, Kimmel and Yaremko (1966) compared two groups of planarian worms given equal numbers of CS and US presentations. For one group, the US always followed the presentation of the CS whereas in the other, the US only followed the presentation of the CS in fifty percent of the trials. When the CS was presented alone for a number of trials during the test, it was found that the CR was more resistant to extinction in the group that was given partial than continuous reinforcement, an instance of the partial reinforcement extinction effect routinely observed in vertebrate conditioning preparations. Pavlovian conditioning in planaria has been reported in preparations that made use of an altogether different set of stimuli. In a study by Wisenden and Millard (2001), a neutral stimulus, fish odor, was found to have no effect upon the activity of planaria; exposure to planaria injuryreleased chemicals, however, elicited a strong avoidance behavior. After paired presentations of the two stimuli, Wisenden and Millard found that the fish odor reliably produced an avoidance response, suggesting the establishment of an association between the fish odor and the injuryreleased chemical (between the CS and the US). The odor was used by the planarian worms as an indicator of danger, and the authors of the study claimed that platyhelminthes possess the simplest nervous system known to be capable of learned risk association. The available data on planarian learning show that a CR develops to the CS specifically when paired with a US, and extinguishes when the US is omitted after the presentation of the CS. These data, however, are not informative about the nature of the principles that rule conditioning in planaria, which might depend upon a set of very basic learning mechanisms which would link, in a Hebbian way (Hebb 1949), the conditioned stimulus (CS) and the unconditioned stimulus (US) whenever they are presented together (or very closely) in the organism’s environment. This would fail to capture some essential features of the learning process which is known to rule Pavlovian conditioning in
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vertebrate species. For example, when two stimuli are presented in a compound conditioned stimulus (CS1 and CS2-US), one of the elements, CS1, can control the CR at the expense of the other, CS2; however, if CS2 is presented alone as a predictor of the US (CS2–US), it fully controls the conditioned response. The prevalence of CS1 over CS2 in conditioning is known as overshadowing and was first reported by Pavlov (1927) in experiments using dogs. Furthermore, training of one of the elements of the compound (CS1–US) before training of the compound (CS1 and CS2–US) fails to establish the CS2 as an effective conditioned stimulus. This blocking effect (first reported by Kamin 1969 in experiments using rats) clearly shows that co-occurrence of two events (CS2 and US in this case) is not enough to promote Pavlovian conditioning. Overshadowing and blocking have been influential in the formulation of modern learning theories. Early theories of overshadowing and blocking assumed learning to depend on the establishment of associations between events during conditioning, a process that is governed by an error-correcting rule that leads to stimuli being in competition for the control they acquire over behavior. Whenever two stimuli are presented simultaneously as signals for the same outcome, the more salient or predictive of the stimuli gains associative strength—or predictive value—at the expense of the other. According to these theories, the CR to a target cue reflects its associative strength; from that view, therefore, the CR deficits observed in overshadowing and blocking are regarded as acquisition deficit effects (e.g., Mackintosh 1975; Pearce and Hall 1980; Rescorla and Wagner 1972; Wagner 1981). Other theories assume that during compound conditioning (CS1 and CS2–US), associations of each element with the US will form independently (there is no acquisition deficit). Rather than a reflection of the associative strength of the target cue (CS2), the CR is mediated at the time of test by the retrieved memory of competing cues: The target cue that is present at the time of test, CS2, and an absent cue that has been associated with both the target cue and the US, the CS1 or comparator stimulus. These theories propose that, at the time of test, the product of the association between the comparator and the target (CS1– CS2) and the association between comparator and the US (CS1–US) reduces excitation elicited by the target cue’s direct association with the US (CS2–US). Therefore, according to this view, commonly known as the comparator theory, overshadowing and blocking are regarded as performance effects (e.g., Miller and Matzel 1987; Stout and Miller 2007). A different approach to the cue competition effects assumes learning to depend upon the spatiotemporal distribution of events rather than upon the establishment of associations: The animal learns, for example, a temporal
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map—the times of onset and offset of the CS and the US— and uses this information in the decisions that determine their conditioned behavior. The learning mechanism allows the individual to detect redundant information and calculate a differential rate of reinforcement for the relevant and irrelevant (redundant) signals. If the expected time for the next presentation of the US in the presence of CS1 (in the absence of CS2) is equal to the expected time for reward in the presence of the CS1–CS2 compound, then the individual will assign high predictive value—reinforcement rate—to CS1 and low or null predictive value to the redundant CS2. This would account for the blocking effect. Similarly, if two stimuli are simultaneously presented before the onset of the US, an overshadowing procedure, the learning mechanism will act to reduce the predictive value of one of the elements in favor of the other (e.g., Balsam and Gallistel 2009; Gallistel and Gibbon 2000). Other processes have been proposed that can account for cue competition effects by reference to a controlled rational-like process that helps the individual realize the redundancy of the target cue. These high-level processes of rational inference have been said to account for some instances of blocking in human predictive learning (e.g., De Houwer and Beckers 2003; Lovibond 2003; Mitchell and Lovibond 2002) and even in some instances of forward blocking in the rat (e.g., Beckers et al. 2006; Blaisdell et al. 2006). In spite of their differences, all these theories are able to predict the response deficit observed when testing the target CS in the overshadowing and blocking procedures. One aspect they have in common is that the learning mechanisms they advocate would help the individual discount redundant information at the time of acquisition or test. These mechanisms would therefore play an important role in helping individuals optimally adapting their behavior to the prevalent conditioning contingencies. Overshadowing and blocking have been observed in invertebrate species like the arthropod honeybee (e.g., Couvillon and Bitterman 1989; Couvillon et al. 1997; Smith and Cobey 1994), and the mollusks garden snail (e.g., Acebes et al. 2009; Loy et al. 2006) and slug (Sahley et al. 1981), suggesting that learning in invertebrate species is similar to learning in vertebrate species in that they also can turn down redundant information at the time of acquisition or test. The experiments reported below aimed to assess overshadowing and blocking in the platyhelminth planaria. A successful demonstration of overshadowing and blocking in brown planaria could be taken to be evidence that animals in a number of phyla including chordates, mollusks, arthropods, and platyhelminthes learn in similar ways. The implications of these findings for an evolutionary-comparative approach to the learning phenomenon will be discussed in the general discussion.
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General methods Subjects Sixty brown planaria, D. tigrina, supplied by Blades Biological Ltd. (Kent, UK), were used in the experiments reported below. Flatworms were held in a plastic aquarium (25 9 30 9 20 cm) filled with 5 liters of dechlorinated tap water. Water temperature was 20 °C (±2°), and the aquarium was held in a room with natural light. The flatworms were fed raw chicken meat every 2 days, and the water was changed after each feeding. Apparatus In Experiment 1, animals were kept in a double-cubical plastic container (2 9 2 9 2 cm each compartment) during the experimental sessions. In Experiments 2 and 3, animals were kept in a truncated squared pyramidal plastic container (6.5 cm high, 4.5 cm sided at the base, and 7 cm sided at the top). The plastic containers were filled to a depth of 0.5 cm with dechlorinated water. These plastic containers were fixed to a semitransparent polystyrene base, which was 24 cm over a table. The room background light during the experimental sessions was provided by a small desk lamp with a red bulb in a distant corner of the room; the background luminance was 0.14 cd/m2 as measured over the plastic container where the animal was kept during the experiments. Underneath the semitransparent base, a standard 60-W blue bulb was located (the top of the bulb was 9 cm from the polystyrene base). The light conditioned stimulus (CS) used in the experiments was presented by switching on the blue bulb for 10 s; presentation of the light stimulus resulted in an increase in the luminance of 186.66 cd/m2 as measured immediately over the plastic container where the animal was kept. A second CS, a mechanical vibration (mean f = 5 Hz) applied by the experimenter by gently hitting the plastic container for 10 s with a piece of plastic, could be presented. Pilot tests carried out in our laboratory had shown that both the light and the vibration were effective CSs—both resulted in the development of a CR when paired with a US. The unconditioned stimulus (US) used in the experiments was a 4.5-v electric shock delivered for 0.5 s through an Adjustable DC Power Supply (SkyTronic Ltd., Manchester, UK). The experimenter delivered the US by introducing the positive and negative poles (which were separated by 1.5 cm) in the water in the front and rear of the worm. This invariably resulted in the immediate display of the shrinking UR. The experimental trials were recorded by using a digital video camera (Sony DCR SR57) located 30 cm over the top of the plastic containers (see Fig. 1).
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Fig. 1 Schematic representation of the apparatus used in the present research
Procedure The animals were tested individually—except in Experiment 1, in which animals were tested in pairs in separate compartments (see details below, in the description of Experiment 1). The subjects were given a 10-min adaptation period before the onset of the experimental session. Planarian worms typically alternate periods of activity, in which they are elongated and swim around the container, with periods of inactivity in which they stick to the wall of the receptacle in a ball. When the animal is inactive, it is impossible to observe any UR to the shock-US or CR to the CS. Therefore, a prerequisite for starting a conditioning or test trial was that the animal should be active and at full length. If the animal was inactive before the onset of the next trial, the experimenter would administer a squirt of water using a small 1-ml syringe to encourage activity. This intervention was required on a similar number of occasions for animals in the different groups. There were two different types of trials in the experiments reported below: conditioning and test. During a conditioning trial, the conditioned stimulus was presented alone for 10 s; during the last half a second of the CS, the animals were presented with the shock-US. During a test trial, the CS was presented alone for 10 s. The inter-trial interval (ITI)
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was 5 min. The delivery of the shock results in a longitudinal contraction; the conditioned response was defined as a longitudinal contraction, a sudden turning, or a longitudinal contraction accompanied by a sudden turning in response to the CS. All the trials were videotaped, and the conditioning and test trials were all rated by two independent observers and compared using Cohen’s kappa analyses to assess for inter-observer consistency. These analyses showed moderate to strong inter-observer agreement (minimum kappa = 0.60). The data reported below correspond to the mean of the 2 independent registers.
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such a way that it was equally likely at any point during the ITI. Although there was a CS–US pairing (when the shock was presented at the onset of the first 15-s interval of the ITI), the US presentations were not correlated with the CS presentations. In the control Random group, animals could be sensitized by the presentations of the shock-US to the same extent as animals given paired presentation of light and shock. Conditioning would be observed if animals in the Paired group developed a conditioned response to the light to a higher extent than animals in the Random group. Results and discussion
Experiment 1: Pavlovian conditioning A type 1 error rate of 0.05 was adopted for all reported statistical comparisons throughout the article. Figure 2 depicts the percentage of trials in which the CR was observed in the presence of the light-CS in the two groups (Paired and Random) of Experiment 1 across four blocks of five conditioning trials. One ANOVA with group and blocks of trials as factors showed a significant group 9 blocks of trials interaction, F3,30 = 8.35. Neither the group nor the block factors was significant, maximum F1,10 = 3.01. An analysis of the group 9 blocks of trials interaction (simple main effects) showed a significant effect of group in the last two blocks of conditioning trials, Fs1,10 C 7.15. Animals in the Paired group showed a significant increase in the level of conditioned responses over the four blocks of conditioning trials, F3,15 = 4.18; contrastingly, animals in the Random group showed a significant decrease in the level of responding over the blocks of trials, F3,15 = 6.78.
100 Paired Random
90 80 70
% of CR
Experiment 1 aimed to assess Pavlovian conditioning in brown planaria. Demonstrations of conditioning in planaria abound in the literature (e.g., Jacobson et al. 1967; Thompson and McConnell 1955; Wisenden and Millard 2001); however, the procedures traditionally used to assess the learning abilities of flatworms in the past substantially differ from the procedure used in our laboratory. For example, in the Jacobson et al.’s (1967) study, animals were exposed to a 3-s light during the last second of which shock was also presented; the ITI was 20 s, and animals were given four training sessions which consisted of up to seventy five trials each. In the present experiment, animals were given 20 conditioning trials spaced 5 min apart in a single session. In each conditioning trial, a 10-s light-CS was presented; during the last 0.5 s of the light-CS, the shock-US was presented. Twelve planaria were used (Paired group, n = 6; and Random group, n = 6); animals were tested in pairs (one animal belonging to the Paired group and one belonging to the Random group) by using the small double-cubical container described above—they occupied adjacent independent compartments. We tested the animals in pairs to explicitly equate the experience with all the aspects of the procedure except the CS–US relationship (as described below). (Having both animals simultaneously ready for the next conditioning trial—active, elongated, and swimming—proved occasionally to be challenging, so we abandoned this procedure in subsequent experiments in which each animal was conditioned individually.) In each conditioning trial, the two animals were given the light stimulus simultaneously. Following the presentation of the light, animals in the Paired group were always shocked during training. We established twenty 15-s intervals between each two presentations of the light-CS, and all the animals in the Random group were shocked once at the onset of each such interval, chosen at random. Each interval was used once during the session. Therefore, the shock was presented at random throughout the session in
60 50 40 30 20 10 0 1
2
3
4
Blocks of five trials Fig. 2 Mean group percentage of trials in which a conditioned response was observed (±SEM) across blocks of five trials during the conditioning phase of Experiment 1
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Animals in the Paired group showed a significant increase in the level of responding to the light-CS, whereas animals in the Random group showed a significant decrease in the level of responding during the conditioning training. It is worth mentioning that the animals did not show the characteristic CR during the ITI. Pilot studies in our laboratory showed that several presentations of the light stimulus alone did not result in systematic longitudinal contraction or sudden turning (the CR). The initial relatively high level of responding observed in both the Paired and the Random groups could therefore be characterized as an instance of sensitization due to the presentation of the arousing shock-US. This tendency to respond to the light, however, subsequently habituated in the Random group, suggesting that the sustained increase in the level of response to the light observed in the Paired group is a reliable index of Pavlovian conditioning. However, as we have already discussed above, linking a CS with a US does not exhaust the possibilities of Pavlovian conditioning. This learning strategy is known to allow animals to anticipate the occurrence of relevant events, but it can do so in such a way that lets the animal manage the information very efficiently, selectively attending to the more informative stimulus when more than one stimulus is present at the time of conditioning. Experiments 2 and 3 assessed whether brown planaria are capable of showing evidence for the usage of these rich learning strategies, which are supposed to underlie Pavlovian conditioning in vertebrate species.
Anim Cogn (2013) 16:177–186 Table 1 Design of Experiments 2 and 3
Experiment 2
Experiment 3
Group
Single conditioning
Single-L
L?
Single-V
V?
L V L and V ?
L
Compound-V
L and V ?
V
Control-L
L and V ?
L
Control-V
L and V ?
V
L and V ? L and V ?
L V
Blocking-L Blocking-V
V? L?
L and V represent the light and the vibration conditioned stimuli. ? represents the shock unconditioned stimulus
the vibration (see Table 1). The experiment was carried out in a single session. Overshadowing would be observed if animals in the Compound groups show less responding during the test than animals in the Single groups. Results and discussion Figure 3 depicts the percentage of conditioned responses to the CS in the four groups of Experiment 2 across blocks of five conditioning trials. During the conditioning phase of the experiment, animals in the Compound groups, trained with a compound consisting of the light and the vibration
100
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Test
Compound-L
Experiment 2: Overshadowing
Single-V
Compound-V
Single-L
Compound-L
90 80 70
% of CR
Overshadowing is observed when two stimuli are presented together in a compound as predictors of a US; under these conditions, less conditioning is observed to each of the elements of the compound as compared to the case in which they are the sole predictors of the outcome (e.g., Pavlov 1927). This phenomenon has been observed in vertebrate species (pigeon, rat, rabbit, human, etc.), as well as in arthropods (honeybee, Couvillon and Bitterman 1989) and mollusks (garden snail, Loy et al. 2006). In Experiment 2, animals in the overshadowing groups were given twenty conditioning trials with a compound CS consisting of the simultaneous presentation of light and vibration (groups Compound-L and Compound-V, n = 6). Animals in the control groups were given twenty conditioning trials in the presence of the light-CS (group SingleL, n = 6) or the vibration-CS (group Single-V, n = 6). Following conditioning, animals in the Compound-L and the Single-L groups were given ten test trials in the presence of the light, and animals in the Compound-V and Single-V groups were given ten test trials in the presence of
Compound conditioning
60 50 40 30 20 10 0
C1
C2
C3
C4
T1
T2
Blocks of five trials Fig. 3 Mean group percentage of trials in which a conditioned response was observed (±SEM) across blocks of five trials during the conditioning and the test phases of Experiment 2 (C conditioning, T test)
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stimuli, showed a higher percentage of conditioned response than animals in the Single groups, trained with a simple stimulus, either the light or the vibration. One ANOVA with training (Compound vs. Single), test stimulus (light vs. vibration) and blocks of trials as factors showed a significant effect of training, F1,20 = 11.29, and blocks of trials, F3,60 = 4.28. None of the remaining main factors and interactions were significant, maximum F3,60 = 1.49. During the test phase of the experiment, animals in the Compound groups showed a lower level of response than animals in the Single groups. An ANOVA with training (Compound vs. Single), test stimulus (light vs. vibration) and blocks of trials as factors showed a significant effect of training, F1,20 = 6.69. The remaining factors and interactions were all non-significant, maximum F1,20 = 1.74. Animals in the Compound groups showed a higher level of response during conditioning than animals in the Single groups. This is hardly surprising, given that animals in the Compound groups were trained in the presence of a compound of two stimuli (light and vibration), whereas animals in the Single groups were trained in the presence of a single stimulus (either light or vibration). The salience of the compound was undoubtedly higher than the salience of the single stimuli, and it is well established in the vertebrate learning literature that salience modulates the acquisition of conditioned responses during conditioning (e.g., Kamin 1965). The same seems to apply to planaria; we have observed in our laboratory that planaria respond differently to stimuli of different intensity or salience: For example, a photonegative response was of higher magnitude in response to an intense than to a relatively weak luminous source (Davidson et al. 2011). In spite of the fact that animals in the Compound groups responded to a higher level than those in the Control groups during conditioning, they showed less responding to the target stimulus during the test, a clear demonstration of overshadowing. Interestingly, the present results did not show any significant differences in the effectiveness of the light and the vibration as CSs. During the conditioning phase, animals in the Single-L and Single-V groups showed a similar level of CR. During the test phase, animals in the Compound-V group showed a slightly lower level of response to the vibration than animals in group Compound-L, tested with the light. This might suggest that the light was more effective in overshadowing the vibration than the other way around. However, statistical analysis revealed the absence of significant differences. These results suggest that vibration and light were equally effective in overshadowing each other, an instance of reciprocal overshadowing. The outcome of the present experiment parallels the effect typically observed in vertebrates using standard conditioning tasks (e.g., Mackintosh 1976).
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Experiment 3: Blocking Blocking is observed when a novel stimulus is presented accompanied by an effective conditioned stimulus as predictors of a US. Under these conditions, less conditioning is observed to the novel stimulus than in the case in which it is accompanied by a neutral stimulus (e.g., Kamin 1969). This phenomenon has been observed in vertebrate species (pigeon, rat, rabbit, human, etc.), arthropods (honeybee, Couvillon et al. 1997; Smith and Cobey 1994) and mollusks (garden snail, Acebes et al. 2009; slug, Sahley et al. 1981) and offers, together with the overshadowing effect, compelling evidence that mere contiguity between the CS and the US does not suffice to promote effective Pavlovian conditioning. There were three phases in Experiment 3 that took place over 2 days: single conditioning (day 1), compound conditioning and test (day 2). During the single conditioning phase of the experiment, animals in the Blocking groups (n = 6) were given twenty conditioning trials in the presence of vibration (group Blocking-L) or light (group Blocking-V). Animals in the Control groups were not treated during day 1. The following day, animals in the Blocking and the Control groups (Control-L, n = 6; and Control-V, n = 6) were given twenty conditioning trials with a compound CS consisting of the simultaneous presentation of light and vibration. Following compound conditioning, animals in the Blocking-L and the Control-L groups were given ten test trials in the presence of the light, whereas animals in the groups Blocking-V and Control-V were given ten test trials in the presence of the vibration stimulus. Blocking would be observed if animals in the Blocking groups show less responding during the test than animals in the Control groups. Results and discussion Figure 4 depicts the percentage of conditioned responses to the CS in the four groups of Experiment 2 across blocks of five conditioning trials. During the single conditioning phase of the experiment, animals in the Blocking groups, trained in the presence of a single CS (light or vibration), showed a significant increase in the percentage of conditioned responses across the blocks of trials. An ANOVA with stimulus (light vs. vibration) and blocks of trials showed a significant effect of blocks of trials, F3,30 = 10.04. Neither the stimulus factor nor the stimulus 9 blocks of trials interaction was significant. During the compound conditioning phase, in which all the animals were trained in the presence of a compound consisting of the light and the vibration stimuli, animals in the Blocking groups showed a higher percentage of conditioned response than animals in the Control groups. One ANOVA with
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100 90 80
% of CR
70 60 50 40 30 20 10 0
S1
S2
S3
Blocking-V
Control-V
Blocking-L
Control-L
S4
C1
C2
C3
C4
T1
T2
Blocks of five trials Fig. 4 Mean group percentage of trials in which a conditioned response was observed (±SEM) across blocks of five trials during the simple conditioning, compound conditioning, and test phases of Experiment 3 (S simple conditioning; C compound conditioning, T test)
training (Blocking vs. Control), test stimulus (light vs. vibration) and blocks of trials as factors showed a significant effect of training, F1,20 = 15.72. None of the remaining main factors and interactions were significant, maximum F1,20 = 1.91. During the test trials, animals in the Blocking groups showed a lower level of responding than animals in the Control groups. An ANOVA with training (Blocking vs. Control), test stimulus (light vs. vibration) and blocks of trials showed a significant effect of training, F1,20 = 10.39, and a significant training 9 blocks of trials interaction, F1,20 = 8.91. The remaining factors and interactions were all non-significant, Fs \ 1. An analysis of the training 9 blocks of trials interaction (simple main effects) showed a significant effect of training in the second block of test trials, F1,23 = 35.69. Although animals in the Control groups maintained a relatively high level of response across the two blocks of trials, animals in the Blocking groups showed a significant reduction in the level of conditioned response, F1,11 = 4.90. The present experiment replicates some of the main results observed in Experiment 2. During the single conditioning phase, animals trained in the presence of the light and the vibration showed a similar level of conditioned responding (replicating, with slightly higher levels of response, the outcome observed in the Single groups of Experiment 2). During the compound conditioning phase, animals in the Control groups (equivalent to the Compound groups of Experiment 2) showed levels of conditioned responding similar to the ones observed for the Compound
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groups in Experiment 2 (between 70 and 80 % of CR in the last block of conditioning trials). Animals in the Blocking groups showed higher levels of conditioned response than animals in the Control groups in compound conditioning; this is hardly surprising, given that in the Blocking groups, the light–vibration compound contained one element that had already been trained as an effective CS. During the test phase, the two Control groups showed slightly different levels of response, with the group tested in the presence of the vibration showing a slightly inferior level of response than the animals tested with the light. These differences were not significant, however, replicating the outcome observed in the Compound groups of Experiment 2. The more interesting result is, however, the comparison between the Blocking and Control groups during test. Animals in the Blocking groups, which were trained in the presence of a novel and an effective CS presented in a compound, showed less responding to the test stimulus than animals in the Control groups, trained in the presence of two novel stimuli presented in a compound. Interestingly, the results showed that whereas animals in the Control groups maintained a relatively high level of CR, animals in the Blocking groups showed a clear and significant tendency to decrease—in other words, the weaker (compared with the Control groups) CR shown by animals in the Blocking groups was also more susceptible to extinction.
Discussion The results of the present Experiment 1 offer evidence that brown planaria are capable of showing simple Pavlovian conditioning—the animals showed increased responsiveness to the light-CS when it was reliably paired with the US. Thus brown planaria would join an abundant group of animals which has been shown to be able to associate environmental events. This Pavlovian conditioning might depend upon a set of very basic learning mechanisms which would link, in a Hebbian way, the CS and the US whenever they are presented together (or very closely) in the organism’s environment. This set of basic learning mechanisms would be utterly insufficient to account for the cue competition effects observed in Experiments 2 and 3. In Experiment 2, training with a light–vibration compound resulted in less conditioning to each element than that observed in the control Single groups, trained in the presence of the individual elements (either light or vibration). In Experiment 3, pre-training with one of the elements (either light or vibration) blocked conditioning of the other during compound conditioning. In both cases, merely pairing the target stimulus with the unconditioned stimulus did not suffice to establish this element as an effective CS.
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These results characterize a learning mechanism which goes beyond the Hebbian blind association between events that co-occur. Overshadowing and blocking in the platyhelminth planaria add to previous studies that reported these cue competition effects in invertebrate species like the arthropod honeybee (e.g., Couvillon et al. 1997; Couvillon and Bitterman 1989; Smith and Cobey 1994), and the mollusks garden snail (e.g., Acebes et al. 2009; Loy et al. 2006) and slug (Sahley et al. 1981), suggesting that learning in these phyla is ruled by learning mechanisms which are able to efficiently process the available information by responding less to the redundant cues. The fact that cue competition can be observed in species only distantly related like rats, honeybees, snails, and planaria could be of interest to understand the evolutionary history of learning. If, for example, the same learning mechanism can be concluded to underlie cue competition in a number of species closely related, we could infer that this learning mechanism had been inherited from a common ancestor. We might also obtain evidence that different groups of species have different underlying mechanisms producing cue competition, suggesting that they evolved different solutions for the same problem, the need for efficient information processing. The problem is that we cannot be certain yet of the nature of the learning mechanisms responsible for cue competition in the species so far evaluated. As discussed above, overshadowing and blocking can be explained in different ways. Interestingly, there is evidence in the literature for a number of mechanisms that can modulate compound conditioning in the same vertebrate species. When a rat, for example, is presented with a compound of two events preceding the appearance of a US, it can disregard irrelevant information in different ways (e.g., Cole and McNally 2007; Dickinson et al. 1976; Pearce et al. 2012); similarly, research has shown that cue competition in causal learning in humans depends under certain conditions—low memory load tasks—on rational inference processes and under other conditions—high memory load tasks—on uncontrolled automatic processes predicted by associative learning theory (Le Pelley et al. 2005). This means that overshadowing and blocking are complex multi-determined phenomena. The analysis of these complex phenomena in species located far away from vertebrates in the phylogenetic scale has just begun, and although we have compelling demonstrations of the cue competition effects in several invertebrate species, there is no unequivocal evidence for any particular learning mechanism in these species. Future research should unveil the actual mechanisms responsible for cue competition effects in planaria and other invertebrate species, assessing, for example, the effect of additional reinforced or non-reinforced presentations of the
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blocking stimulus on the CR elicited by the target CS, a manipulation that produces changes that can be easily predicted by the comparator theory (Miller and Matzel 1987; Stout and Miller 2007), but not by the standard associative learning theories (e.g., Rescorla and Wagner 1972), or assessing whether the blocking effects reported in different species are flexible and sensitive to constraints of causal inference—such as the violation of additivity—as suggested by Beckers et al. (2006). Information about the actual mechanisms that modulate cue competition in animal species located far away from each other in the phylogenetic scale would be instrumental for reconstructing the evolutionary history of learning. Acknowledgments The present research was supported by an NC3Rs-LASA (UK) Small Award to the authors.
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