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Rapid Associative Learning and Stable Long-Term Memory in the Squid Euprymna scolopes.

Abstract. Learning and memory in cephalopod molluscs have received intensive study because of cephalopods' complex behavioral repertoire and relatively accessible nervous systems. While most of this research has been conducted using octopus and cuttlefish species, there has been relatively little work on squid. Euprymna scolopes Berry, 1913, a sepiolid squid, is a promising model for further exploration of cephalopod cognition. These small squid have been studied in detail for their symbiotic relationship with bioluminescent bacteria, and their short generation time and successful captive breeding through multiple generations make them appealing models for neurobiological research. However, little is known about their behavior or cognitive ability. Using the well-established "prawn-in-the-tube" assay of learning and memory, we show that within a single 10-min trial E. scolopes learns to inhibit its predatory behavior, and after three trials it can retain this memory for at least 12 d. Rapid learning and very long-term retention were apparent under two different training schedules. To our knowledge, this study is the first demonstration of learning and memory in this species as well as the first demonstration of associative learning in any squid.

Introduction

Molluscan models have played an instrumental role in advancing our understanding of the cognitive, neuronal, and molecular mechanisms that underlie learning and memory. The role played by potassium and sodium ions in action potentials was discovered in the squid Loligo pealeii (Hodgkin and Huxley, 1952). In the sea slug Aplysia californica, researchers connected changes in both pre- and postsynaptic cells to habituation, a simple form of learning (reviewed by Glanzman, 2010). More complex cognitive processes, including problem solving and observational learning, have been studied in Octopus vulgaris (Fiorito and Scotto, 1992; Richter et al., 2016). Here, we explore the capacity for associative learning in the squid Euprymna scolopes Berry, 1913, a species of sepiolid squid that has proven to be a promising laboratory model.

Euprymna scolopes is well studied for its symbiotic relationship with the bioluminescent bacteria, Vibrio fischeri (Nyholm and McFall-Ngai, 2004). As a result of this mutualism, E. scolopes is relatively common in research laboratories, yet literature concerning its behavior, including its capacity for learning and memory, is scarce. To characterize these understudied aspects of their behavior, we conducted a behavioral assay of learning and memory that is well validated in another cephalopod, the cuttlefish: the "prawn-in-the-tube" experiment (Messenger, 1971; Agin et al., 2003, 2006a, b; Cartron et al., 2013). This method takes advantage of the cephalopod prey drive by introducing shrimp to the subject in a clear glass tube. Initially, the squid attacks these prey items but learns that the shrimp are inaccessible over the course of training trials. Long-term memory retention is tested during retention trials occurring at minutes to hours after training. Although this experiment has been performed using cuttlefish, neither it nor any other assay has been used to measure learning and memory in E. scolopes.

In this study, we compared two training schedules (spaced vs. massed; see Materials and Methods) to determine whether long-term memory retention in E. scolopes was similar to durations seen in other cephalopod species. It is well established (including in other cephalopods) that spaced training protocols, where reinforcement occurs over a longer period, is more effective at promoting long-term memory than is massed training, where reinforcement events occur in quick succession (Long et al., 1989; Mauelshagen et al., 1998; Menzel et al., 2001; Agin et al., 2006b; Cartron et al., 2013). Our results suggest that E. scolopes learns rapidly under either reinforcement schedule and that long-term memory of suppression of tentacle strikes is retained for multiple days, periods comparable to those observed in other cephalopods.

Materials and Methods

Animals

Squid were bred from wild-caught Euprymna scolopes collected in the waters surrounding O'ahu, Hawai'i. Subjects were reared from birth in the laboratory, in continuously circulating artificial seawater maintained at a temperature of 24.5 [degrees]C. Squid were fed ad libitum live grass shrimp (Palaemonetes spp.) once per day and were fed only after the conclusion of trials on experiment days. Euprymna scolopes is short lived, growing to sexual maturity around 50-60 d post-hatching and entering senescence between 90 and 120 d (Lee et al., 2009b). All squid used in this study were adults, ranging from 42 to 88 d old.

Squid were housed in groups of 4-6 in round enclosures 26 cm in diameter. About 2.5 cm of sand covered the mesh bottom of the enclosure, and water level was maintained at about 14 cm, flowing in from an overhead pipe and out through the mesh bottom. For both the training and the tests, a single squid was transferred by beaker to an enclosure identical to the main housing tubs, except that the mesh bottom was bare to prevent burying.

General procedure

Five large grass shrimp (Palaemonetes spp.; carapace length, 1-1.5 cm) were enclosed in a 400-ml beaker. The beaker was sealed on the top with mesh to prevent shrimp from escaping and was placed in the test chamber under a water inflow of 520 ml [min.sup.-1]. Prior to introducing the subjects, the beaker was covered with an opaque sleeve.

One squid was transferred directly from its home enclosure and given 1 min to acclimate to the test chamber. Animals typically settled promptly on the mesh base and remained still during acclimation. At the end of the 60-s acclimation period, the opaque sleeve covering the beaker was lifted, allowing the squid to see the enclosed shrimp. All trials were recorded by a remote-control camcorder (HDR-SR 12; Sony, Tokyo, Japan) mounted 75 cm above the arena.

Training protocols

In this study, we compared learning and memory in two different training protocols, hereafter termed "massed" and "spaced."

For massed conditioning, subjects (n = 9) received three 10-min training trials in a single day, with each trial separated by a 10-min intertrial interval during which the beaker was covered with the opaque sleeve. Animals remained in the training arena throughout the 50-min training sequence. Memory tests, consisting of a single 10-min trial identical to the first training trial, were conducted 4 d after the training day and again 12 d after the training day. A relatively small sample of animals was available for this study; thus, all animals received both the day 4 and the day 12 test, making the day 4 test effectively a "reminder training" for the final day 12 test (for a timeline, see Fig. 1A).

For spaced conditioning, subjects (n = 16) received a single 10-min training trial once per day for 3 consecutive days. Memory tests were conducted 4 d after the final training trial for a subset of animals (n = 9) and 10 d after the final training trial (i.e., 12 d after the first training trial) for the remaining animals (n = 7) (for a timeline, see Fig. 1B, C). The long-term memory test was conducted 10 d post-training for the spaced conditioning group (vs. 12 d for the massed conditioning group) because of a procedural error; however, we note that both the 10- and the 12-d retention test far exceeded the typical "long-term" retention interval (24 h) used in similar protocols.

Behavioral analysis and statistical procedures

All trials were videotaped, and data were recorded from playback by multiple trained observers, with each trial scored by at least two independent observers. Each 10-min trial was divided into 60-s intervals, and strikes made at prey in each minute-long interval were counted.

For the first training trial only, we denoted the first 60-s interval during which the squid made its first strike as "minute 1," designating the interval during which the learning experience of hitting the beaker with the tentacles first began. This allowed us to control for squid that did not begin attending to the beaker immediately after it was exposed. Only one squid made no strikes on prey during the first 10-min exposure; its data were excluded.

All trials subsequent to the first training trial were recorded from the first minute of beaker exposure, irrespective of whether the squid struck during the first minute of that trial.

Behavioral variables

An attempted attack on the confined shrimp was counted as a strike if the following conditions were met: (1) prior to impact with the glass, the squid carried its arms and tentacles in a hunting posture, in which its arms were pointed in a conical shape oriented at the glass (see Fig. 2A-C), and (2) the squid subsequently made contact with the beaker (see Fig. 2D). A strike was usually, but not always, accompanied by a change in chromatophore coloration from a deep red to a lighter beige. Some interactions contained multiple strikes but were considered as such only if the squid detached its arms and tentacles from the beaker completely between strikes. If the squid did not detach completely, it was considered a single strike. An interaction was not considered a strike if the squid contacted the beaker with its mantle or the side of its body, contacted the beaker with its arms lowered, or did not touch the beaker.

Statistical analyses

Each video was scored by two independent observers. If there was disagreement of more than one strike per minute, a third observer rescored that trial, and the final count was obtained by consensus among all three observers.

For statistical analysis, strike counts were summed across the first and last 5 min of each trial. Counts were normally distributed. We compared strikes made between the first and second half of each trial using paired Bonferroni-corrected t tests, and comparisons of counts across trials were made using independent-sample Bonferroni-corrected t tests. Because animals were group housed and not individually marked and because in the spaced training protocol different animals were used for tests at the two retention intervals, it was not appropriate to use paired t tests when comparing among trials conducted on different days. Analyses were conducted in Prism software (ver. 7; GraphPad, San Diego, CA). We considered P < 0.05 after Bonferroni correction to indicate statistical significance. For a subset of trials, we tracked animal movement and activity using EthoVision software (Noldus, Wageningen, Netherlands) and generated heat maps of squid movement during the first and last minute of the first training trial.

Results

Massed conditioning produces rapid inhibition of predatory striking and stable long-term memory

Euprymna scolopes learned rapidly to avoid striking at the confined prey. The number of strikes made at the beaker during the second half of the first training trial was significantly lower than the number of strikes made during the first half (P = 0.001), indicating rapid, learned inhibition of predatory striking (Fig. 3A). Reduction in predatory strikes was also accompanied by a reduction in attention to the beaker (for an example, see Fig. 4A, B). After a single 10-min learning trial, short-term memory was retained for at least 10 min, with the number of strikes made during the first half of the second trial significantly reduced compared with that made during the first 5 min of the first trial (P = 0.001). Strike counts continued to decline in the two subsequent training trials but were not significantly lower than those observed during the late interval of the first training trial.

We tested long-term memory retention under this brief learning experience at two intervals: 4 and 12 d post-training. Strike counts observed during the early interval of the 4-d post-test were significantly lower than those observed during the early interval of the first training trial (P = 0.001) and were not significantly higher than those observed during the late interval of the third training trial (P = 0.22). Similarly, at 12 d post-training strike counts observed during the early interval of the memory test were significantly lower than those observed during the early interval of the first training trial (P = 0.001) but were not significantly higher than those observed during the last minute of the third training trial (P = 0.16).

Spaced conditioning produces slower learning and stable long-term memory

During the first training trial, squid rapidly learned to inhibit their strikes on confined prey. Strikes made during the late interval of the first training trial were significantly reduced compared with those made during the early half of the trial (P = 0.001; Fig. 3B).

On the second training day, the number of strikes made during the first 5 min was significantly lower than the number made during the early stage of training on day 1 (P = 0.001) but was not different from the number made during the late interval on the previous day (P = 0.001). Strikes declined further from the early to the late interval on day 2 (P = 0.006).

On training day 3, strike counts were not significantly different from those for the late interval of the previous day (P = 0.87), and those for the late interval of the day 3 training were not significantly different from those for the early interval (P = 0.66).

We tested long-term memory retention at the same intervals as for spaced conditioning: at 4 and 12 d post-training. Strike counts observed during the early interval of the 4-d post-test were significantly lower than those observed during the early interval of the first training trial (P = 0.001) and were not significantly higher than those observed during the late interval of the final training trial (P = 0.99). Similarly, at 12 d post-training strike counts observed during the early interval of the memory test were significantly lower than those observed during the early interval of the first training trial (P = 0.001) but were not significantly higher than those observed during the last minute of the third training trial (P = 0.99).

Discussion

Squid have received little study at the behavioral level, in part because they are challenging to keep in typical laboratory environments. Here, we show robust performance of an experimentally tractable squid, Euprymna scolopes, in a validated operant conditioning task used extensively in cuttlefish (Messenger, 1971, 1973; Chichery and Chichery, 1992; Agin et al., 2006a, b). To our knowledge, this is the first demonstration of learning and memory in this species as well as the first demonstration of associative learning in any squid, although both habituation and sensitization have been shown in loligonid squid previously (Long et al., 1989; Crook et al., 2011; Oshima et al., 2016). We demonstrate both that E. scolopes shows learning capabilities equivalent to those of cuttlefish when presented with an identical task and that squid show impressive long-term memory of the association even after a single day of massed training.

In our study, animals in our massed training group made fewer (although not significantly fewer) strikes overall compared with those in the spaced training group. Because of changes in husbandry over the course of the experiment, animals in the massed training group were less accustomed to daytime feedings, and this likely affected their hunting motivation in the afternoons, when trials were conducted. Animals in the two training groups were also of slightly different ages; although all subjects were sexually mature adults, those in the group that received spaced training were slightly larger and may have had higher hunting motivation at the outset of each day's trials. In operant conditioning paradigms, learning requires that the animal perform the behavior to be modified (Reynolds, 1975); thus, animals in the group receiving massed conditioning received fewer overall "learning experiences" (i.e., strikes on prey that went unrewarded) because they made fewer strikes at the beaker, despite all other aspects of the experimental setup being identical. However, the rate of learning and duration of memory for the massed and spaced training conditions were similar, with roughly linear declines in strikes made across successive training trials.

Many similar studies using cuttlefish have shown a deficit in recall of the learned association around 8-20 min after training, which is the period between the decay of short-term memory and the appearance of intermediate-term memory (Messenger, 1971; Agin et al., 2003, 2006a). In our study, our massed training protocol captured this period at the outset of the second training trial, which occurred 10 min after the end of the first training trial. Interestingly, we saw no evidence of recovery of the normal striking response at this time, as strike counts were identical to those observed during the last 5 min of the previous trial. Thus, although we did not set out to explicitly test whether a biphasic retention curve occurs in E. scolopes, as it does in cuttlefish, it appears that this biphasic retention either does not occur or differs substantially in its temporal dynamics.

Another striking finding in our study is the very long duration of long-term memory displayed by E. scolopes for both training conditions. To our knowledge, the longest reported retention of the "prawn-in-the-tube" procedure in cuttlefish is 24 h (see Agin et al., 2003). We tested long-term memory at 12 d after the beginning of training in the spaced conditioning group and at 12 d after training (although this occurred 8 d after a single reminder training) in the massed conditioning group and found very strong retention of memory in both groups. Memory lasting more than 24 h is certainly "long-term"; in E. scolopes, long-term memory seems to be exceptionally stable. This is an interesting finding given the nature of the task itself--our observations of both cuttlefish (E. A. Zepeda, R. J. Veline, and R. J. Crook, unpubl. data) and E. scolopes strikes suggest that cuttlefish strike from a greater distance and with a higher tentacle club speed than E. scolopes, suggesting that the impact on the glass is more violent for cuttlefish. Although there continues to be considerable debate about what precise cues are learned in this task (Agin et al., 2006b; Cartron et al., 2013), we hypothesize that the noxious impact of the tentacle clubs with the glass may contribute to learning, although this remains unresolved. Additional reinforcement comes from the unrewarded efforts to capture prey that is never obtained. If aversive sensory input does play a role in learning and memory in this task, it is somewhat counterintuitive that the presumably less noxious impact made by E. scolopes contributes to longer-term memory than that of cuttlefish, although undoubtedly many other biological and ecological factors influence memory duration. We did not attempt to ascertain what aspects of the experimental apparatus contribute to learning. It is plausible that the squid learn contextual cues from the arena or the presence of the beaker (which may be visible to squid), or they may learn from visual or chemical cues emitted by the shrimp.

While squid remain fairly uncommon models for learning and memory, we suggest that E. scolopes--with its small size, short generation time, ability to breed through multiple generations in the laboratory (Hanlon et al., 1997; Lee et al., 2009b), relative ease of husbandry, and strongly modifiable behaviors--represents a promising emerging model for behavioral and neurobiological research. Here, for the first time, we show the highly tractable nature of this species for behavioral research. Coupled with the large existing literature on its bacterial symbiosis (McFall-Ngai, 2008; Lee et al., 2009a; McFall-Ngai etal., 2012; Collins etal, 2015; Kerwin and Nyholm, 2017), the rapidly expanding volume of information on cephalopod genomes (Zhang et al., 2012; Albertin et al., 2015), and the strong (although controversial) pressure to replace vertebrate models with invertebrate models as part of the Three Rs (Crook and Walters, 2011), we propose that E. scolopes has considerable promise as a novel behavioral and neurobiological model organism.

Acknowledgments

We thank the Nyholm Laboratory at the University of Connecticut for supplying eggs of Euprymna scolopes and Eric Koch of the University of Hawai'i for supplying adults of E. scolopes. We thank Bret Grasse from the Monterey Bay Aquarium and Roger Hanlon from the Marine Biological Laboratory for advice on E. scolopes husbandry. Members of the Crook Laboratory assisted with animal husbandry and data analysis. Funding for this study was provided to RJC from San Francisco State University and NIH MARC award to EAZ (T34-GM008574).

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EMILY A. ZEPEDA (*), ROBERT J. VELINE (*), AND ROBYN J. CROOK ([dagger])

Department of Biology, San Francisco State University, 1600 Holloway Avenue, San Francisco, California 94132

Received 21 April 2017; Accepted 12 June 2017; Published online 5 September 2017.

(*) Each of these authors contributed equally to this work.

([dagger]) To whom correspondence should be addressed. E-mail: rcrook@sfsu.edu.
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Author:Zepeda, Emily A.; Veline, Robert J.; Crook, Robyn J.
Publication:The Biological Bulletin
Article Type:Report
Date:Jun 1, 2017
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