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Does conditioned taste aversion learning in the pond snail Lymnaea stagnalis produce conditioned fear?

Introduction

Conditioned taste aversion (CTA) is thought to be an adaptive trait that enables the organism to avoid poisonous substances. To cause CTA in the pond snail Lymnaea stagnalis, a sucrose solution, which elicits feeding behavior (i.e., biting), is used as the conditional stimulus (CS), and a KC1 solution, which evokes the whole-body withdrawal response and thus inhibits feeding, is used as the unconditional stimulus (US) (Ito et al., 1999; Kojima et al.; 2001; Sadamoto et al., 2010). After 10 paired presentations of the CS and US, the CS (i.e., sucrose) no longer acts as an appetitive stimulus (i.e., it no longer elicits the feeding response). Following this training, the CTA persists as a long-term memory (LTM) for more than a month (Kojima et al., 1996, 1997). Additionally, with more intense training (i.e., > 50 pairings of the CS-US), the CS not only no longer elicits biting but now elicits the withdrawal of the snail into its shell (i.e., the whole-body withdrawal response; Kojima et al., 1996). Thus, we hypothesize that with the more intensive training procedure the CS gains the ability not only to inhibit feeding but to elicit "fear" in snails.

In English, the word "fear" has, according to the Second Edition of the Oxford English Dictionary (1989; updated draft edition 2007), been accepted since the 14th century to mean "the internal state or emotion denoting alarm, terror, fright or dread." We define fear in Lymnaea as a state of heightened alarm, arousal, or fright in response to a perceived threatening stimulus. Fear is a basic survival mechanism that is observed in almost all organisms and is elicited by stimuli that signal the threat of danger (e.g., Adolphs, 2008; Bishop, 2008; Rodrigues et al., 2009). Conditioned fear in vertebrates such as rodents is often brought about by pairing an aversive stimulus (the US)--for example, an electric shock-- with a neutral stimulus (the CS)--for example, a tone (see Maren, 2001). Eventually with pairing of the CS and US, the neutral stimulus (i.e., the CS) alone elicits the fear response. Conditioned fear in rodents is often measured by how long the rat or mouse remains immobile (freezes) on presentation of the CS after conditioning. The freeze response (being "scared stiff") is a reaction mediated by the autonomic nervous system that occurs when mammals face an overwhelming threat. Changes in the heart rate or the respiratory rate, both also mediated by the autonomic nervous system, can also be used to assess fear in mammals and non-mammals (Sacchetti et al., 2005, 2009; Ehrlich et al., 2009; Sehlmeyer et al., 2009; Yoshida et al., 2009). Fear conditioning is thus a form of associative learning and the subsequent formation of memory.

Research groups utilizing various invertebrate model systems, including gastropod molluscs, have also posited conditioned fear. For example, if one considers the internal state or emotion of fear as eliciting a "flight or fright" response, then the early work of Willows on the swim escape response triggered in Tritonia by predator detection certainly qualifies (Willows and Hoyle, 1969). Sometime later, one of the earliest papers examining the question of fear in molluscs was that examining whether Aplysia could be classically conditioned (Carew et al., 1981; Walters et al., 1981). In fact, the behavioral paper in this series was entitled "Associative learning in Aplysia: evidence for conditioned fear in an invertebrate." Fear conditioning has also been posited in crabs, as Hermitte and Maldonado (2006) showed fear conditioning using both escape behavior and alterations in cardiac activity as indices for fear in the crab Chasmagnathus.

We here hypothesize that in Lymnaea after the establishment of CTA with 50 or more pairings of the CS-US, the CS (sucrose) would elicit a fear response rather than an appetitive behavior (biting). Thus, the presentation of sucrose would elicit the whole-body withdrawal response because the CS now signals a threat rather than a taste. Here we were interested in whether, with only a single pairing of the CS (sucrose) and US (KC1), the CS would begin to elicit fear in snails. An easily observable behavior that can give us an indication of fear in snails is heart rate. Heart rate has a further advantage in that it can be measured in a noninvasive manner. The heart of Lymnaea, as it is in mammals, is myogenic. But both its rate and force of contraction can be modulated depending on the requirements of the organism. In Lymnaea, the rate and force contraction are modulated by a number of identified neurons in the central nervous system (Buckett et al., 1990a, b; Willoughby et al., 1999a, b).

If, as we hypothesize, the CS begins to elicit a fear state in the snail after a single pairing of the CS-US in the CTA training procedure, there might be a significant alteration in heart activity. Thus our first hypothesis was that the heart rate is changed in the conditioned snails by application of the CS. Moreover, in human, stress, ischemia, hypoxia, etc., are known to cause a premature ventricular contraction that is perceived as a skipped heartbeat (e.g., Guyton and Hall, 2005). Classically conditioned fear and excitement evoke ventricular arrhythmias in monkeys (Randall and Hasson, 1981). Monkeys also acquire a conditioned bradycardia with a US consisting of a loud noise coupled with a stream of compressed air aimed at the face (Kalin et al., 1996). In humans, the heart rate during unpleasant pictures shows the expected greater deceleration (Avezedo et al., 2005). These studies suggest that fear causes a transient change in heartbeat in animals (also see the review by Alboni et al., 2008). Therefore, our second hypothesis was that if the CS comes to signal fear in Lymnaea there will be a "skipped" heartbeat.

Two issues remain to be resolved before embarking on experiments to test the aforementioned hypotheses. The first of these concerns the establishment of an adequate control group. Fortunately, we have found that when we train snails using our CTA training procedure, there are always a number of snails that are categorized as poor learners (Sugai et al., 2007). In these snails, the CS continues to elicit feeding behavior, even though the CS has been paired with the aversive US. We are uncertain why these snails do not have the capacity to learn this procedure (that is a whole other line of research), but we can make use of their poor learning ability in the present study. We predict that even though these so-called poor learners experience the exact same conditioning sequence as the good learners, the presentation of the CS will not alter their heart activity. The second issue concerns the whole-body withdrawal response (Ferguson and Benjamin, 1991a, b; Syed and Winlow, 1991; Winlow et al., 1991). We performed pilot experiments in which we applied KC1 to the snail to elicit the whole-body withdrawal response while we monitored heart rate. When the whole-body withdrawal response was elicited by the KC1, the heart stopped beating. These pilot data confirmed the observations, which had been known to many researchers using Lymnaea but not published, that activation of the whole-body withdrawal response results in the suppression of heartbeat.

To satisfy the two just-mentioned issues, we subjected snails to a one-trial CTA (i.e., only one pairing of CS-US) training procedure (Sugai et al, 2007). One-trial training procedures leading to the formation of LTM have been well documented in Lymnaea for both classical and operant conditioning (Alexander et al., 1984; Kemenes et al., 2002; Fulton et al., 2005; Martens et al., 2007). We previously found that a single pairing of the CS and US resulted in about 40% of snails exhibiting LTM (Sugai et al., 2007). Most interestingly, use of the one-trial CTA training procedure on these snails did not result in the CS eliciting the whole-body withdrawal response. Therefore, we can use the one-trial CTA training procedure to determine if the CS elicited an alteration in heart rate after conditioning. If our hypotheses are correct, the CS should alter heart rate or produce a skipped heartbeat only in the good learners and not in the poor learners.

Materials and Methods

Snails

Specimens of Lymnaea stagnalis (L.) with shell lengths of 20-25 mm were obtained from our snail-rearing facility (original stocks from Vrije Universiteit Amsterdam). All snails were maintained in dechlorinated tapwater (i.e., pond water) at 18-24 [degrees]C and fed lettuce.

Snails were deprived of food for 1 day before training. Data from our previous experiments demonstrated that 1 day of food deprivation before the one-trial CTA training procedure was necessary to obtain consistent results (Sugai et al., 2007). This deprivation did not appear to alter such easily observable behaviors as egg laying, aerial respiration, or heart rate (pers. obs.), and did not result in any change in the general health of snails, such as evidenced by a change in mortality rate.

One-trial CTA training procedure

Snails were trained for CTA in a 35-mm (diameter) petri dish. The CS used was 2 ml of 50 mmol [1.sup.-1] sucrose solution, and the US was 2 ml of 10 mmol [1.sup.-1] KC1. The CS elicits reliable biting (number of bites [min.sup.-1]) from snails deprived of food for 1 day, whereas the US reliably causes the snails to stop biting (Wagatsuma et al., 2004; Sugai et al., 2006).

The heart was observed visually in all snails for 20 s in the petri dish filled with distilled water (DW) before any training commenced (Fig. 1). If the number of heartbeats was <5 per 20 s, the snail was not used because it was judged not to be healthy. Next, all the remaining snails were given a pretest session to determine their biting rate in response to the CS. Snails that did not show an adequate biting response ([greater than or equal to] 10 bites [min.sup.-1]) following the presentation of the CS were also excluded from the study. In the pretest session, snails were given the CS for 20 s. During this time period, the number of heartbeats in each snail was counted, and we determined if there were any "skipped" heartbeats. A "skip" is defined as an interruption in the ongoing cardiac rhythm. The CS was then rinsed from the petri dish with DW. The number of bites made by the snails in DW was counted over the course of the next 1 min. The pretest session was given to the snails at least 10 min before the one-trial CTA training.

[FIGURE 1 OMITTED]

In the one-trial CTA training procedure, snails were first exposed to the CS for 15 s, then exposed to the US for 15 s, and then the US was rinsed out with DW. The change of solutions was done using a micropipette. A post-test session, identical to the pretest session, was performed 9 min 30 s after the one-trial CTA training session. In the post-test session, the number of heartbeats was again counted during the 20-s period of CS application. We started the CS application irrespective of the state of heart contraction. We also monitored whether there were any skipped heartbeats. Finally, the number of bites was counted during the period of DW application.

Two different control experiments were also performed to demonstrate that the significant change in biting in the post-test session of CTA-trained snails was the result of associative learning. In the first control experiment, referred to as the backward conditioning control, the temporal presentation of the CS and US was reversed. That is, snails were first exposed to the US for 15 s and then exposed to the CS for a further 15 s. These snails were also given both the pre- and post-test sessions. In the second control experiment, referred to as the naive control, snails were exposed only to DW. The DW was poured over the snails at exactly the same time and for exactly the same interval as the CS and US in the other two groups. The naive control snails were also given both the pre- and the post-test sessions with the CS.

On the basis of our previously published data (Sugai et al., 2007), we set a performance boundary to distinguish between "good" and "poor" learners. A snail possessing LTM (a good learner) is expected not to open its mouth following presentation of the CS. However, some snails open their mouths by chance (i.e., spontaneously) in the absence of any delivered stimulus (Kojima et al., 1996). Such spontaneous openings occur at a rate of about one per min. Thus, we defined a good learner as a snail that made 0-1 bite [min.sup.-1] during the post-test session in response to the CS. Poor learners were therefore defined as snails that made [greater than or equal to]2 bites [min.sup.-1] in response to the CS during the post-test session. All conditioning and testing procedures described above were performed using a blind protocol.

Photographs and raster diagrams for heartbeats

The beating heart in Lymnaea is visible to the naked eye in all the experiments. The photographs of the beating heart were taken using a stereo microscope (SZX16; Olympus, Tokyo, Japan) equipped with a CCD camera (CS230B; Olympus, Tokyo, Japan) and a camcorder (Handycam DCR-HC90; Sony, Tokyo, Japan) (Fig. 2). Although we could easily locate the heart without a microscope, it was difficult to identify the beating heart using photographs alone. Therefore, the video images were processed using ImageJ software (ver. 1.40g; National Institutes of Health, Bethesda, MD, USA) to reduce the background noise and enhance the intensity of the heart region.

[FIGURE 2 OMITTED]

To better provide clear examples of skipped heartbeats, we recorded the beating heart using a video camera (GZ-MS230; JVC, Tokyo, Japan) and converted the video signal to a raster diagram. The end-diastolic point of the beating heart was used as the timing for a raster. The sampling time was 0.5 s. All image processing described above was also performed using a blind protocol.

Statistical analysis

Data are expressed as the mean [+ or -] s.e.m. Statistical significance (P < 0.05) was determined by a one-way analysis of variance (ANOVA) followed by the post hoc Scheffe test. The Student t-test was used for comparison between two groups. The [[chi].sup.2] test was also used to determine significant differences in the skip of a heartbeat between the good and poor learners.

Results

Biting and heartbeat in one-trial CTA training

One hundred sixty snails met our minimum criteria and were therefore used in the one-trial CTA training procedure. That is, the snails subjected to the one-trial CTA training procedure exhibited the necessary bite rate in response to the CS presentation in the pretest session (12.2 [+ or -] 0.1 bites [min.sup.-1]. Fig. 3A) and exhibited the necessary number of heartbeats in DW and in the CS over the 20-s measurement period (7.6 [+ or -] 0.1 and 7.6 [+ or -] 0.1, respectively; Fig. 4A, B). We found that 24 of the 160 snails (15%) spontaneously exhibited a skipped heartbeat in DW and the CS pretest session. We considered this to be our background noise.

[FIGURE 3 OMITTED]

Of the 160 snails that received the one-trial CTA training procedure, 29% (46 snails) were classified as good learners. That is, in the post-test session (9 min and 30 s after the one-trial CTA training procedure), the CS elicited a bite rate of either 0 or 1 bite [min.sup.-1] in these snails (0.2 [+ or -] 0.1 bites [min.sup.-1]; Fig. 3B). In the pretest session, the CS elicited a response of 12.3 [+ or -] 0.1 bites [min.sup.-1] in these 46 snails. This level of responsiveness to the CS in the pretest session was not significantly different from the pretest session response values (12.2 [+ or -] 0.2 bites [min.sup.-1]) elicited by the CS in the 114 snails that were not classified as good learners (i.e., they were classified as poor learners). Thus, at the start of the experiment (in the pretest session), all snails performed similarly in response to the CS. In the 114 snails that we classified as poor learners, the CS in the post-test session elicited 8.2 [+ or -] 0.3 bites [min.sup.-1] (Fig. 3B). This level of responsiveness ([greater than or equal to] 2 bites [min.sup.-1]) did not meet the criterion of a good learner. Thus, about 1 out of 3 snails met the criterion to be classified as a good learner following the one-trial CTA training procedure.

We next had to demonstrate that the significant difference in the bite rate in response to the CS in the post-test session was a bona fide example of associative learning. It is possible that this significant decrease in biting by the good learners was due to repeated stimulation of the snails by the changes in solution or was the result of a non-associative effect of the exposure to the aversive KC1 stimulus. Thus, we used two control groups of snails: a backward conditioning control group (n = 160) and a naive control group (n = 160). Snails in the backward conditioning control group received the CS and US in the reverse order (i.e., US-CS), whereas snails in the naive control group were exposed only to DW changes.

All snails in the two control groups met the response criteria outlined above. We found that in neither group was there a significant difference in the biting elicited by the CS between the pre- and post-test sessions (i.e., P > 0.05 for all tests; Fig. 3A, B), nor did any of these snails meet the good learner criterion. Thus, we conclude that the significant decrease in the biting elicited by the CS in the post-test session of the one-trial CTA trained snails, at least the good learners, was the result of associative learning.

We next examined the total number of heartbeats in the 20-s recording epochs in the experimental (i.e., the one-trial CTA good and poor learners) and control (i.e., backward conditioning and naive) snails. As shown in Figure 4, the number of heartbeats in the preliminary screen test (20 s in DW; Fig. 4A), the pretest session (Fig. 4B; 20 s in sucrose), and the post-test session (Fig. 4C; 20 s in sucrose) were not significantly different between any of the groups. Thus, our first hypothesis that the presentation of the CS following the one-trial CTA learning in good learners would signal fear, as evidenced by a change, was rejected. These data suggested to us that we should examine the heart rate in each individual snail and not just compare the total number of heartbeats for the 20-s interval.

[FIGURE 4 OMITTED]

Skipped heartbeat

We therefore examined whether there would be any difference between the groups (good learners, poor learners, backward conditioning control, and naive control) in the number of skipped heartbeats in the post-test session (Fig. 5). That is, would the CS elicit a fear response, as evidenced by an increased probability of a skipped heartbeat, in the good learners in the post-test session? In the backward conditioning control and naive control groups, there was no significant difference in the number of skipped heartbeats between the pretest and the post-test CS applications. In the pretest session, 20 of 160 backward conditioning control snails exhibited a skipped heartbeat (i.e., 13%); whereas in the post-test session, 22 of the 160 snails (14%) exhibited a skipped heartbeat. Similar data were obtained in the naive control group. In this group, 23 of the 160 naive control snails (14%) exhibited a skipped heartbeat in the pretest session, whereas 21 of the 160 snails (14%) skipped a heartbeat in the post-test session. All these values were near the background level (15%). That is, there were no significant differences in the ratio of skipped heartbeats among the two control groups and the background level. Here we note that when a skipped heartbeat occurred, it was observed immediately after the CS application. In other words, there were no data showing that a skipped heartbeat occurred > 5 s later after the start of CS application. Further, as mentioned in the Materials and Methods, we started the CS application irrespective of the state of heart contraction.

[FIGURE 5 OMITTED]

We next turned our attention to all the one-trial CTA trained snails (Tables 1 and 2). In the pretest session, 24 of 160 snails (15%) exhibited a skipped heartbeat that is the same as the background rate observed in all groups (Table 1). However, after the one-trial CTA training, 51 of the 160 snails (32%) exhibited a skipped heartbeat in the post-test session (Table 2). This is clearly above the background rate.
Table 1
Skipped heart beat by CS application before one-trial CTA training
procedure

                            Number of    Number of poor learners
                          good learners

Skipped                          6                  18
Not skipped                     40                  96
Total number of learners        46                 114

P > 0.05 (not significant) by [[chi].sup.2] test.

Table 2
Skipped heart beat by CS application after one-trial CTA training
procedure

                            Number of    Number of poor learners
                          good learners

Skipped                         29                   22
Not skipped                     17                   92
Total number of learners        46                  114

P < 0.01 by [[chi].sup.2] test.


We then split the one-trial CTA trained snails into good (i.e., exhibited associative learning) and poor (i.e., did not exhibit associative learning) learners based on their feeding response elicited by the CS in the post-test session. In the pretest session, 6 of the 46 (13%) snails that would come to be classified as good learners exhibited a skipped heartbeat in the presence of the CS (Table 1). Similarly, in the pretest session, 18 of the 144 snails (16%) that would come to be classified as poor learners exhibited a skipped heartbeat in the presence of the CS (Table 1). These rates are in line with the spontaneous rate and the rates in the pretest sessions of the backward conditioning control and naive control groups.

In the post-test session of the poor learners, 22 of the 114 snails (19%) skipped a heartbeat in the presence of the CS (Table 2). Again, this value is close to the background value seen in the two control groups. However, in marked contrast to all other groups, in the post-test session of the good learners, 29 of the 46 snails (63%) exhibited a skipped heartbeat in the presence of the CS (Table 2, Fig. 5). That is, the probability of a skipped heartbeat elicited by the CS was clearly increased in only the good learners. These results suggest that we have to conclude that the CS in the good learners comes to elicit "fear." That is, our second hypothesis that the conditioned fear is demonstrated as a skipped heartbeat in Lymnaea was confirmed.

We subjected all the data regarding skipped heartbeats to statistical analyses. In the poor learners, the backward conditioning control group, and the naive control group, there was statistically no significant change in the probability of a skipped heartbeat in the post-test session compared to the pretest session. Moreover, the probability of a skipped heartbeat was not statistically different from the background spontaneous rate in any of the pre- or post-test sessions. On the other hand, in the good learners, the probability of a skipped beat in the presence of the CS in the post-test session was statistically significant (P < 0.01). That is, in the post-test session in the presence of the CS, snails classified as good learners skipped a heartbeat more often. Thus, we conclude that, in the good learners, one-trial CTA training results in the CS becoming competent to elicit fear.

Discussion

Skipped heartbeat in good learners by fear stimulus

The data presented above show that with a one-trial CTA training procedure, it was possible to produce CTA in a subset (ca. 30%) of snails. We classified these snails as "good learners." The remaining 70% of the snails did not acquire CTA. That is, in the poor learners, the CS in the post-test session continued to elicit a feeding response. However, in the good learners, the CS in the post-test session did not elicit a feeding response. These data confirm that it is possible to use a one-trial CTA training procedure to result in associative learning involving feeding in Lymnaea. Thus, in Lymnaea, like most other organisms studied (e.g.. Dere et al., 2007), a certain percentage of subjects have the capacity to form an association between two stimuli with only a single trial.

However, the main objective of the present study was not to confirm whether snails can undergo associative learning for CTA using a single CS-US pairing. Rather, we were interested in determining whether the CS would become capable of eliciting a fear response in snails through the one-trial CTA training procedure. We found that the CS could elicit fear--but only in the good learners--as evidenced by a statistically significant increase in the number of individuals exhibiting a skipped heartbeat in the presence of the CS after the one-trial CTA training. The CS did not gain the ability to alter the number of skipped heartbeats in the post-test session of the poor learners or snails in either of the two control groups. We thus conclude that our second hypothesis--that the CS can elicit conditioned fear--was demonstrated by a skipped heartbeat in the snails retaining CTA memory.

If we only calculated the total number of heartbeats in the post-test session in the presence of the CS, we found that there was no statistical difference between any of the groups (Fig. 4). Thus, our first hypothesis that the overall heart rate would be altered as a result of CTA conditioning was unsubstantiated. This result may have been expected because Orr et al. (2007) found no overall change in heart rate when Lymnaea was exposed to a predator scent. The same predator scent did, however, elicit a suite of behavioral changes that the authors labeled as vigilance behaviors. Among them was a greater tendency for a shadow stimulus to elicit the whole snail withdrawal response into its shell. Because this is the snails' ultimate escape, or defense, response, the data indicated that snails show a heightened arousal or fear to predator detection. In the Orr et al. (2007) study, they did not look to see if predator detection caused a skipped heartbeat.

It was only when we looked at the individual response to the CS in the post-test session of each snail that we saw any difference between the groups in whether the CS increased the probability of a skipped heartbeat. This is not the first time that the data from individuals has been used to demonstrate that experimental treatments alter the ability of Lymnaea to learn and form memory. For example, in the Rosenegger et al. (2004) study, all snails were graded individually on how well they formed memory. In examining over 2300 snails, it was determined that about 33% of these snails showed a learning grade of "A" with the specific training procedure used, whereas 24% did not form memory. Thus, looking at individual performances can be very revealing. Orr and Lukowiak (2008) demonstrated that predator detection also enhanced the formation of LTM. Unpublished data from that study showed that if individual marks for snails, as in the Rosenegger et al. (2004) study, were analyzed, the number of A grades increased to over 66%, while the number of failures decreased to about 10% (Lukowiak, unpubl. obs.).

The hypothesis tested here was that the CS would elicit a fear state in the good learners after one-trial CTA training. Aversive, stressful stimuli in Lymnaea elicit the whole-body withdrawal response. That is, those stimuli are perceived as a threat, and the snail responds in an appropriate manner: it withdraws into its shell and all non-compatible behaviors, including copulation, feeding, and aerial respiration (Inoue et al., 1996), are inhibited. The whole-body withdrawal response typically results in the snail retracting into its shell completely. This behavior, however, is not an all-or-none response, and is actually graded depending on how aversive the stimulus is. For example, placing a snail into a small watch glass containing differing concentrations of KC1 (e.g., 5 to 100 mmol [l.sup.-1]) produces results from incomplete withdrawal into the shell at 5 mmol [l.sup.-1] to complete withdrawal for long periods of time at concentrations greater than 25 mmol [l.sup.-1] (Martens et al., 2007). Thus, aversive stimuli that elicit the whole-body withdrawal response can be considered to be fear-evoking stimuli. In the present study, we chose a concentration of KCl as the US that did not elicit the complete whole-body withdrawal response. However, the US was sufficiently aversive that when it was paired with sucrose as the CS it could elicit CTA in many snails with only a single pairing. Importantly, as shown here in these good learners, the CS elicited a fear response, as evidenced by an increased probability of a skipped heartbeat.

The distinction between responses elicited by fear and those elicited by an aversive stimulus

Even though we posit that Lymnaea perceives fear with the presentation of the CS after one-trial CTA training, we cannot dismiss the possibility that the snails simply detect an aversive stimulus and respond appropriately, with no fear involved. This problem is partly dependent on the brief duration of what we term the fear state. That is, the skipped heartbeat, our index of fear, elicited by the CS after one-trial CTA training, is brief. It only persists for a period of about 2 s (Fig. 5). In mammals, including humans and monkeys, fear causes a similar, brief premature ventricular contraction that is perceived as a skipped heartbeat or it evokes ventricular arrhythmias (Randall and Hasson, 1981; Guyton and Hall, 2005). That is, fear elicits a transient change in heartbeat in these mammals. Thus, the brief duration of the change in cardiac behavior elicited by the CS in Lymnaea should not be considered to rule out the existence of a fear response. However, these autonomic responses have not been established in snails, so cannot be used to complement our findings regarding alterations in cardiac activity. Thus we need more careful observations to finally determine whether Lymnaea experiences emotional fear or simply detects an aversive stimulus and responds appropriately, with no fear involved.

Fear conditioning in vertebrates and invertebrates

Fear conditioning in mammalian model systems (e.g., rodents) has contributed greatly to our understanding of not only how fear is mediated at the neuronal level but how learning, memory, extinction, and reconsolidation are formed. It is beyond the scope of this discussion to go over these findings in detail. Interested readers are directed to a number of recent studies and reviews (e.g., LeDoux, 2000; Gogolla et al., 2009; Monfils et al., 2009; Schiller et al., 2010). However, in fear conditioning, a neutral CS following a pairing with an aversive US can elicit fear. Most often this is associated with cessation of motion, or freezing (Ehrlich et al., 2009), but it can also be seen in changes to heart rate (Bryant, 2006) or to other indices of change in sympathetic nerve activity (e.g., an increase in the galvanic skin response; Dunsmoor and Schmajuk, 2009).

As mentioned in the Introduction, previous studies have attempted to establish conditioned fear in invertebrates (Carew et al., 1981; Walters et al., 1981; Hermitte and Maldonado, 2006). In Lymnaea, alterations in cardiac activity (e.g., a 20% decrease in heart rate) have been observed as a result of placing snails in a hypoxic environment for hours (Taylor et al., 2003). Changes in heart rate have also been reported in Aplysia in response to noxious stimuli such as increases in ambient temperature and hypoxia (Dieringer et al., 1978; Getting, 1985). All these studies indicate that changes in cardiac activity may signal a response to aversive stimuli that could be interpreted as threatening, stressful, or fear-inducing. As mentioned above, Orr et al. (2007) reported that the overall heart rate was not changed in Lymnaea by exposure to a predictor scent that does elicit specific anti-predator responses that are indicative of a stressful or fearful state. Our data presented here are all consistent with the Orr et al. (2007) data showing that the CS in snails exhibiting CTA memory does not induce a change in overall heart rate (Fig. 4). To our knowledge, researchers studying fear or the behavioral responses elicited by stressful stimuli in molluscs have not previously examined whether such stimuli elicit a skipped heartbeat. The benefit of our present findings in Lymnaea is that they may lead to determination of the mechanism at the level of identified neurons (i.e., those controlling the heart or other higher-order neurons) by which this fear state is expressed and, thereby, to elucidation of a neural correlate of fear.

Fear as a conscious experience

Many researchers believe that fear is a conscious experience (Panksepp, 2005); if we posit that Lymnaea exhibits fear, then are we also positing that Lymnaea possesses consciousness? Consciousness has been discussed previously in molluscs. For example, Edelman and colleagues attempted to address this question in octopus (Edelman et al., 2005). Their data suggest that the possibility of consciousness in these molluscs cannot be ruled out. In another invertebrate, the honey bee, individuals use a waggle dance to inform their nestmates of the location and quality of flowers they have visited (von Frisch, 1993; Seeley, 2003; Okada et al., 2008). Griffin and Speck (2004) commented that
  many find it difficult even to conceive of the possibility that honey
  bees could be conscious to any degree at all. But the dance
  communication system provides us with the same general kind of
  evidence that we routinely use to infer human conscious experiences.
  Of course the content of any conscious experiences of bees must be
  very different from any human thoughts. But if this category of
  evidence is valid for us, it must, in principle, be valid for other
  animals as well.


These examples go to show that the present state of neuroscience research has not determined in invertebrates, or for that matter in mammals, what the neural basis of consciousness is. Thus, as there is a problem with the mention of fear in an invertebrate at present, we leave it to the readers to determine whether or not the data suggest to them that fear exists. What we show are that snails alter their heart rate in the anticipation of a stimulus that to many signals fear.

Acknowledgments

This study was partly supported by KAKENHI from the JSPS (Nos. 19370039 and 21657022 to EI) and a grant from CIHR (No. MOP 64339 to K.L.).

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SERINA KITA (1), RYUJI HASHIBA (1), SAYA UEKI (1), YUKARI KIMOTO (1), YOSHITO ABE (1), YUTA GOTODA (1), RYOKO SUZUKI (1), ERIKO URAKI (1), NAOHISA NARA (1), AKIRA KANAZAWA (2), DAI HATAKEYAMA (3), RYO KAWAI (3), YUTAKA FUJITO (4), KEN LUKOWIAK (5), AND ETSURO ITO (3), *

(1) Science Club, Hokkaido Shiraoihigashi High School, 5-17-3 Hinodemachi, Shiraoi-Cho 059-0903, Japan; (2) Shiribeshi District Office, Hokkaido Board of Education, Kita 1-jo, Higashi 2-chrome, Kutchan-Cho, Abuta-Gun 044-8544, Japan; (3) Laboratory of Functional Biology, Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, 1314-1 Shido, Sanuki 769-2193, Japan; (4) Department of System Neuroscience, School of Medicine, Sapporo Medical University, Minami 1-jo, Nishi 17-chome, Chuo-ku, Sapporo 060-8556, Japan; and (5) Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Dr. NW, Calgary, AB T2N 4N1, Canada

Received 6 October 2010; accepted 14 December 2010.

* To whom correspondence should be addressed. E-mail: eito@kph.bunri-u.ac.jp

Abbreviations: CS, conditional stimulus; CTA, conditioned taste aversion; LTM, long-term memory; US, unconditional stimulus.
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Author:Kita, Serina; Hashiba, Ryuji; Ueki, Saya; Kimoto, Yukari; Abe, Yoshito; Gotoda, Yuta; Suzuki, Ryoko;
Publication:The Biological Bulletin
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Date:Feb 1, 2011
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