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Tiger salamanders’ (Ambystoma tigrinum) response retention and usage of visual cues following brumation
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Behavioural Processes journal homepage: www.elsevier.com/locate/behavproc
Tiger salamanders’ (Ambystoma tigrinum) response retention and usage of visual cues following brumation ⁎
Shannon M.A. Kundey , Anne Lessard, Aleyna Fitz, Manika Panwar Hood College, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Salamander Brumation Memory Amphibian
Brumation enables tiger salamanders (Ambystoma tigrinum) to survive changes in temperature. However, it is unclear how this affects memory retention. We explored how brumation impacted salamanders’ retention of a learned response to a visual cue through two experiments. We hypothesized salamanders would retain information across this state. However, we also hypothesized that retention could be manipulated through cold temperature exposure timing. We hypothesized that cold temperature exposure immediately after reactivation of a memory would decrease retention of that memory following brumation. Our results indicate that salamanders can respond utilizing visual cues and that performance can be retained across this state. However, our results also indicate that if exposure to cold temperatures occurs directly following a recall experience, memory for the information that was recalled just prior to cold temperature exposure can be disrupted. This suggests that the timing of the recalling of information and the exposure to the cold temperatures inherent to brumation is important to memory retention through this state. Future studies should investigate the impact of the timing of extreme temperature exposure on retention over other torpor states, including hibernation and aestivation. Additionally, the mechanism underlying such impaired retention should be explored.
1. Introduction Long-term torpor states such as brumation, hibernation, and aestivation enable animals to survive harsh environmental conditions. These processes differ substantially between warm-blooded and cold-blooded animals. For example, while warm-blooded animals that hibernate move from hibernation to sleep throughout the hibernation period, cold-blooded animals depend on their surroundings and remain torpid until the environmental temperature increases (e.g., Healy and Jones, 2002). Studies of torpor states to date have centered on examining physiological relationships such as body temperature and oxygen usage (e.g., Healy and Jones, 2002). It remains unclear how such states affect memory retention and, more generally, cognitive function—especially in cold-blooded animals. Retention of learned information across such conditions would obviously be adaptive for any species. For example, the ability to remember information, such as areas of high food availability or increased danger, would likely increase fitness. However, torpor states pose a formidable memory retention challenge. While little research exists regarding the effect of torpor states on cognitive function generally, several studies have been performed investigating how hibernation impacts warm-blooded animals.
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Research suggests that hibernation for mammals leads to a drastic decrease in the number of synapses (Van der Ohe et al., 2007). Additionally, since neuronal activity is critical for maintaining neuronal function, one might hypothesize that decreased brain activity due to hibernation might negatively impact memory retention. Research to date, however, conflicts regarding the actual impact of hibernation. Some studies suggest significant memory loss (e.g., Millesi et al., 2001; Weltzin et al., 2006) while others suggest significant retention (e.g., Nowakowski et al., 2009; Ruczynski and Siemers, 2011). Other studies suggest that impairment may be transient (e.g., Thompson et al., 2013). Work regarding torpor states’ impact on cold-blooded animals’ memory retention are few. However, Wilkinson et al. (2017) recently investigated the impact of brumation on fire salamanders’ (Salamandra salamandra) ability to retain a spatial discrimination in a T-maze. All subjects learned to make the initial discrimination response. After training, half brumated while the other half did not. Later testing showed that subjects from both the brumation and no brumation conditions retained the learned response. However, it remains unclear whether memory for other types of tasks would survive brumation. Additionally, it is not clear when the memory must be consolidated or reconsolidated relative to the beginning of the brumation period in order to be retained.
Corresponding author at: Hood College, Department of Psychology, 401 Rosemont Avenue, Tatem 324, Frederick, MD 21701, USA. E-mail address: [email protected] (S.M.A. Kundey).
https://doi.org/10.1016/j.beproc.2018.06.008 Received 16 February 2018; Received in revised form 11 June 2018; Accepted 15 June 2018 0376-6357/ © 2018 Published by Elsevier B.V.
Please cite this article as: Kundey, S.M.A., Behavioural Processes (2018), https://doi.org/10.1016/j.beproc.2018.06.008
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performance following the delay (e.g., McGaugh and Herz, 1972). Thus, we hypothesized that retention following brumation would vary as a function of when exposure to cold temperatures began, with delayed exposure to cold temperatures associated with greater retention due to less interference with reconsolidation processes.
To continue to clarify some of these issues, we explored the impact of brumation on tiger salamanders’ (Ambystoma tigrinum) retention of a response they learned to make previously to a visual cue as they moved about in a maze. We used a paradigm that had been previously successful in our laboratory (Kundey et al., 2016). In that earlier series of experiments, we explored tiger salamanders’ ability to learn to make responses within a maze as visual cues that were located within the maze changed. We found that salamanders could learn to make turning responses based on visual cues placed in the maze. In the last experiment of this series, we investigated if a compound visual cue (a foreground diamond on a contrasting background) could control salamanders’ behaviour when it was the only predictor of how to obtain reinforcement. In this experiment, we varied the start position of the salamander within the maze by placing it in a pseudorandomly determined maze arm at the beginning of each trial. We also varied whether the cue was placed on the right or left side of the alley that the salamander walked down prior to making a turning response. Salamanders did learn to use the placement of this visual cue within the maze to make turning responses that would lead to reinforcement. Here, we built upon this paradigm to confirm the results of that third experiment and to explore the impact of brumation on salamanders’ retention of a learned response to a visual cue. Brumation is of interest because it occurs in cold temperatures. Cold temperatures are interesting because much research suggests that consolidation of new memories and reconsolidation of older memories can be disrupted by various agents, including hypothermia (for review, see Riccio et al., 2006), if those agents are presented near enough in time to the information that is to be remembered (in the case of consolidation) or that has been recently recalled (in the case of reconsolidation). In short, a window exists after the creation or reactivation of a memory during which it is vulnerable to modification by new information, neural interventions, environmental conditions, or pharmacological interventions. Thus, exposure to the cold temperatures inherent in brumation with respect to memory reactivation (i.e., through recall) and reconsolidation processes is especially interesting because prior work in the memory literature suggests that cold temperatures may disrupt reconsolidation processes if exposure to cold temperatures occurs near enough in time to the reactivating event (e.g., McGaugh and Herz, 1972; Mactutus et al., 1979; Riccio et al., 2006). In other words, if a memory is reactivated and followed by exposure to cold temperatures quickly (e.g., exposure to cold temperatures occurs immediately after reactivation), the exposure to the cold temperatures may interfere with memory reconsolidation, leading to difficulty recalling that reactivated memory in the future because reconsolidation has not concluded prior to the cold temperature exposure. If, on the other hand, a memory is reactivated and reconsolidated prior to cold temperature exposure (e.g., exposure to cold temperatures occurs 24 h after the reactivating event), the reactivated memory is more likely to be recalled successfully at a future date because reconsolidation processes had concluded prior to cold temperature exposure. Prior work with rats indicates that nervous system perturbations such as body cooling can produce difficulties in recalling information either when new information has been learned but not yet consolidated or when previously learned information has been recalled or reactivated but not yet reconsolidated (as reviewed by Riccio et al., 2006). Additionally, research suggest that reconsolidation processes may be more prone to interference from cold temperatures than initial consolidation processes (Mactutus et al., 1979). Overall, we hypothesized that salamanders would maintain memory for the response to the visual cue across brumation as evidenced by their performance after brumation. However, we hypothesized that exposure to cold temperatures immediately following reactivation of a memory through requiring the salamanders to work with that memory could interfere with retention of that memory, potentially through interference with reconsolidation processes, and thus decrease
2. Experiment 1 The primary goal of Experiment 1 was to investigate if salamanders could retain information across a 90-day brumation period. We trained all salamanders to turn toward a compound proximal visual cue (square tile with a colored background and a large diamond in the foreground) as start position and cue location varied on each trial in a cross-maze as we did in Kundey et al. (2016) and as described in section 2.1. After learning to turn correctly in the direction indicated by the cue, we separated the foreground and background components of the cue. In a forced-choice procedure the next day, we then evaluated each salamander for turning preference towards the foreground or background of the visual cue. Next, we divided salamanders into three groups for a delay of 90 days: maintenance in their home cage under normal laboratory conditions (maintenance), immediate brumation following training (immediate brumation), or delayed brumation after a 24-hour delay (delay brumation). Following the delay, we evaluated if turning toward the compound proximal visual cue was maintained and repeated foreground versus background testing to evaluate whether the preference after the delay matched the preference displayed during the test trial prior to the delay. We hypothesized that salamanders would maintain memory for the response to the proximal compound visual cue across brumation by continuing to turn toward the proximal compound visual cue after the delay at comparable rates across groups. We did not expect retention of turning towards the proximal compound visual cue to be affected for any group since it always occurred on the day prior to preference testing and thus did not occur immediately before exposure to cold temperatures in any group. However, we hypothesized that exposure to cold temperatures to induce brumation immediately following activation of the preference memory through the foreground/background preference test might interfere with the reconsolidation of the preference memory in the immediate brumation group relative to the other groups. Thus, we hypothesized that the preference displayed just prior to exposure to cold temperatures would not match the preference displayed after brumation in the immediate brumation group but would match in the delayed brumation and the no brumation groups. 2.1. Method 2.1.1. Subjects The subjects were 28 adult male salamanders obtained from Haha Reptiles, Honey Grove, TX. (Females were unavailable at the time of purchase.). Salamanders were maintained throughout training and testing in their home cages at 23 °C ( ± 2 °C) and 70% humidity in group aquariums with coconut fibre bedding in a dimly lit laboratory with access to freeze-dried insects twice weekly. All procedures accorded with Hood College’s Institutional Animal Care and Use regulations. For the brumation groups, a fridge (General Electric model WMR03GAZABB) without illumination was maintained at 3.89 °C ( ± 2 °C) and 60% humidity was employed. Throughout brumation, salamanders were individually housed in plastic 64 oz rectangular containers with moist coconut fibre bedding. The fridge was opened every 3 days for 5 min to allow for air exchange. 2.1.2. Apparatus A cross-maze (114.3 cm X 114.3 cm X 7.62 cm) as shown in Fig. 1 was constructed from square plastic tiles painted a uniform silver colour 2
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Fig. 1. Depicts the apparatus used in the experiment as well as the training and test stimuli.
top of the background stimulus in training was removed for testing.
(see Kundey et al., 2016). We utilized one tile to block off the entrance to the arm directly opposite the start arm on each trial, which varied across trials. Thus, three arms were open to the salamanders during training and testing and which three arms were opened varied across trials. Two timeout containers (15.25 cm X 25.25 cm X 9 cm) were similarly constructed. The timeout containers were separate from the maze and placed relative to the open arms of the maze for each trial, one on the left and the other on the right. The timeout containers were bright and dry. We did not surround the maze with a larger exterior wall or exterior curtain outside the maze to block extramaze cues. This was unnecessary because only the compound visual cue was predictive of reinforcement since starting position varied across trials (as described below) and the turn required to receive reinforcement was not predictive of reinforcement. We covered two training tiles (7.62 cm X 7.62 cm; depicted in Fig. 1) with compound stimuli (red diamond foreground on yellow square background, yellow diamond foreground on red square background; background: 7.62 cm X 7.62 cm, foreground: 5.08 cm X 5.08 cm, square background area: 11.48cm2, diamond foreground area: 11.48cm2). These were placed at the maze junction at the cross of the maze during training as described in 2.1.3. Four test tiles were also constructed (red diamond foreground, yellow square background, yellow diamond foreground, red square background; identical dimensions to the training tiles were used). There were placed (as described in 2.1.3.) at the maze junction during testing. The diamond foreground area was 11.48cm2; the square background area (with the diamond now removed) was 22.86 cm2. This area was larger simply because the foreground stimulus that was previously on
2.1.3. Procedure During trials, the room was brightly lit by overhead fluorescent lighting. The maze was cleaned between trials to counter scent cues. The experimenters stood distant from the maze out of the subjects’ view. Experimenters noted responses in real time using a monitor showing an overhead view of the maze. Interrater reliability was obtained for 10% of responses; raters agreed on all responses. During training, we placed salamanders individually within the start area of the maze and allowed for 5 min to complete the maze by performing a left or right turn at the maze junction (apex of the T; see Fig. 1). We defined a turn as crossing a line 15 cm from the maze’s centre in either direction within the apex of the ‘T’ composed of the open maze arms with any part of the body; most often, this was the head. When correct, the salamander was returned to its home cage until the next experimental day. Previously, we found this to be reinforcing to the salamanders, likely because the home cage environment was moister and allowed for burrowing. If the turn was incorrect, we removed the salamander to the timeout box on the side of the incorrect choice for 1 min. The timeout box did not allow for burrowing and was dry and bright. In our prior work, this did not appear to be reinforcing for the salamanders. After 1 min, the salamander moved on to the next scheduled trial. We did not employ correction trials. If salamanders made an error, they went on to the next scheduled trial for the day without receiving information regarding the correct response on the trial that they had just completed. We introduced new trials until the salamander initially made a correct choice on a trial or completed a maximum of 10 trial attempts/day. If the 3
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test trial per day. On the 91 st day, we evaluated if turning toward the compound proximal visual cue was maintained (hereafter, referred to as compound cue testing). We used the same procedure as in training with the exception that each animal only received one trial instead of up to 10 trials each day. This test let us evaluate if the salamanders remembered to turn towards the compound visual cue from training. On the 92nd day, we repeated foreground versus background preference testing using the same procedure used just before the delay. This test let us evaluate if their preference for the foreground or background was maintained across the various delay conditions.
salamander made an error on the 10th trial of the day, it was placed in the timeout box prior to being returned to its home aquarium. If a salamander did not execute a turn within 5 min, the salamander was removed to the timeout box for 1 min. Subsequently, trials were continued until it made a correct choice on the first attempt at a trial (maximum of 10 trial attempts/day). Thus, although 10 trials were scheduled each day, a salamander could complete fewer trials per day if it executed the correct response on the first or second attempted trial of the day. In our prior study, we found that salamanders learned more quickly about visual cues when the start arm of the maze varied over trials. Thus, the arm in which we placed the salamander to start a trial changed over the course of training. We varied the start arm of the maze pseudorandomly from trial to trail for each salamander to make sure that a salamander did not start in the same arm more than twice in a row. We placed one of the two compound visual cues at the maze junction (where the start arm intersected with the tangential choice arms) on either side of the alley to show which turn direction would be reinforced on that trial. The placement of the cue on either side of the alley (right or left) varied pseudorandomly across trials to make sure that the cue was not placed on the same side more than twice in a row. We did this to help make sure that the salamanders attended to the features of the cue itself rather than whether it was on the right or left side of the ally. For each subject, the compound visual cue used was consistent for the entire experiment; we ensured that half of the animals were trained with one compound visual cue, and the remaining half were trained with the other compound visual cue. Thus, both the compound visual cue location and the start arm location varied on each trial. This made sure that the only consistent predictor of reinforcement was the compound visual cue itself. We counterbalanced compound visual cue assignment (red diamond foreground on yellow square background, yellow diamond foreground on red square background) across subjects. However, assignment was consistent for each animal. In this experiment, the criterion for moving from training trials to test trials was the completion of 3 consecutive days of turning towards the reinforced compound proximal visual cue on the first training trial attempt of the day. Since each subject had a 50% chance of turning correctly on each trial by chance, subjects had a 12.5% chance of executing the correct choice by chance over the 3 days in combination. The salamander moved on to a test trial to evaluate preference for foreground or background of the compound proximal visual cue the following day after criterion was met. This arbitrary, but relatively low threshold, is consistent with that of our prior work (Kundey et al., 2016). The test trial for preference following initial training resembled training except that the test tiles that corresponded to the compound stimulus’s dissociated foreground and background were used. The test tiles were placed at the maze junction, one on the right and the other on the left of the alley, immediately before the intersection. The start arm for testing varied in the same manner as in training. Following this test trial, salamanders were removed from the maze. Then, they were randomly placed into one of three delay conditions that lasted for 90 days: maintenance in their home cage under normal laboratory conditions, immediate brumation following training, or delayed brumation after a 24-hour delay spent in the home cage under normal laboratory conditions. Thus, salamanders in the immediate brumation group were exposed to cold temperatures right after they completed the preference testing for the foreground or the background. Salamanders in the delayed brumation group also were exposed to cold temperatures, but this began 24 h after they completed the preference testing. Salamanders in the maintenance group were never exposed to cold temperatures. After the 90-day delay, all subjects resided in their home cages for the remainder of the experiment. On days 91 and 92, we conducted one
2.2. Results In total, 23 of the 28 salamanders beginning the experiment completed it. Only the data from salamanders that completed the entire experiment are included in the analyses. Of the 5 that did not complete the experiment, 3 died during the experiment (1 prior to brumation and 2 during brumation) and 2 were eliminated due to errors by the experimenters. Of the salamanders that completed all portions of the experiment, 8 were in the normal maintenance group, 7 were in the immediate brumation group, and 8 were in the delayed brumation group. All of the 23 salamanders that completed the experiment reached the criterion of accumulating 3 successive days of turning in the correct direction on their first attempted trial of the day. The number of attempted trials that were required to reach this criterion ranged from 3–215, with a mean of 53.78 attempted trials (SD = 52.07), completed over a range of 3–94 days, with a mean of 26.52 days (SD = 25.58). These data are comparable to those obtained by Kundey et al. (2016) during training. The number of attempted trials and number of days to criterion did not differ significantly over groups. During the preference test trial prior to the delay, 16 of the 23 salamanders turned towards the foreground test tile while 7 turned towards the background test tile. We used a one-tailed binomial test to evaluate the significance of turning toward the foreground tile, as our prior work (Kundey et al., 2016) had indicated a preference by this species of salamander for the foreground tile. The test indicated that salamanders turned towards the foreground tile significantly more often than predicted by chance (p = 0.047) in this experiment. However, this preference was not associated with speed of learning. Following the 90-day delay, we first tested whether salamanders would remember to turn towards the compound cue. These data are depicted in Fig. 2. All salamanders in the no brumation maintenance group and the delayed brumation group turned toward the compound cue that was reinforced during training. Six of 7 salamanders in the immediate brumation group also turned toward the compound cue that was reinforced during training. We used a two-tailed Freeman-Halton extension of the Fisher exact probability test to examine whether turning toward the proximal compound visual cue differed across the groups (p = 1). The results indicated that no significant difference existed between the groups with respect to turning towards the proximal compound visual cue following the 90-day delay. We used a one-tailed binomial test to evaluate if salamanders turned toward the compound proximal visual cue more often than expected by chance, as we predicted that salamanders would remember the turning response to the compound proximal visual cue as it occurred at least 24 h prior to exposure to cold temperatures in the brumation groups. The one-tailed binomial test (p < 0.001) indicated that salamanders turned toward the proximal compound visual cue significantly more often than expected by chance. Next, we evaluated whether exposure to cold temperatures immediately following preference testing interfered with later retention of that preference. In effect, we evaluated whether the preference shown prior to the 90-day delay matched the preference shown after the 90day delay. These data are also depicted in Fig. 2. Seven of 8 4
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finding that salamanders could retain information over brumation. As a result, we only included the delayed brumation and immediate brumation groups in Experiment 2. Additionally, we counterbalanced the day of the compound proximal visual cue test and the preference test after the 90-day delay. 3.1. Method 3.1.1. Subjects The subjects were 44 adult male salamanders obtained and maintained as in Experiment 1 (Females were unavailable at the time of purchase.). Naïve animals were used in Experiment 2. During brumation, salamanders were housed in groups of 4–5 animals/container. 3.1.2. Apparatus The same materials used in Experiment 1 were employed in Experiment 2.
Fig. 2. Depicts performance of salamanders in the no brumation, delayed brumation and immediate brumation groups in Experiment 1 following brumation. The black bar indicates the percentage of salamanders in each group that retained the turning response to the proximal compound visual cue. The white bar indicates the percentage of salamanders in each group who evidenced the same preference in the foreground versus background preference test prior to and following brumation.
3.1.3. Procedure The procedure was exactly the same as Experiment 1 except that salamanders were divided into only two groups for the delay: immediate brumation and delayed brumation. Following the 90-day delay, all subjects were moved to their home cages. On the 91 st and 92nd days, we evaluated whether turning toward the compound proximal visual cue was maintained and repeated foreground versus background testing. Which test was performed on which day was counterbalanced across animals to help alleviate the concern that potential effects might be driven by order of test presentation. Care was taken to ensure that 50% of the animals in each group completed the compound proximal visual cue test on day 91 and the preference test on day 92 and the remaining 50% completed the preference test on day 91 and the compound proximal visual cue test on day 92.
salamanders in the normal maintenance group maintained their preference for the foreground or background cue following the delay. Additionally, 7 of 8 salamanders in the delayed brumation group also maintained their preference for the foreground or background cue following the delay. However, only 3 salamanders in the immediate brumation group maintained their preference for the foreground or background cue following the delay. We used a two-tailed FreemanHalton extension of the Fisher exact probability test to examine whether the maintaining of preference across the 90-day delay varied across groups (p = 0.169). The results indicated that no significant difference existed between the groups with respect to maintaining their preference of turning towards the foreground or background of the compound proximal visual cue following the 90-day delay.
3.2. Results In total, 38 of the 44 salamanders beginning the experiment completed it. Only the data from salamanders that completed the entire experiment are included in the analyses. Four animals that did not complete the experiment died during brumation. An additional two animals are not included in the analyses due to experimenter error. Of the salamanders that completed all portions of the experiment, 18 were in the immediate brumation group, and 20 were in the delayed brumation group. All of the 38 salamanders that completed the experiment reached the criterion of accumulating 3 successive days of turning in the correct direction on their first attempted trial of the day. The number of attempted trials that were required to reach this criterion ranged from 7–211, with a mean of 64.76 attempted trials (SD = 56.11), completed over a range of 5–94 days, with a mean of 32.87 days (SD = 30.18). These data are comparable to those obtained by Kundey et al. (2016) and during training for Experiment 1. The number of attempted trials and number of days to criterion did not differ significantly over groups. During the preference test trial prior to the delay, 25 of the 38 salamanders turned towards the foreground test tile while 13 turned towards the background test tile. As in Experiment 1, we used a onetailed binomial test to evaluate the significance of turning toward the foreground tile. The test indicated that salamanders turned towards the foreground tile significantly more often than predicted by chance (p = 0.036) in this experiment. However, this preference was not associated with speed of learning. Following the 90-day delay, we tested whether salamanders would remember to turn towards the compound proximal visual cue and their preference for the foreground versus the background tile on separate days, counterbalanced across days. These data are depicted in Fig. 3. Of the salamanders in the delayed brumation group, 19 of 20 turned toward the compound cue that was reinforced during training. Of the
2.3. Discussion As in our prior work, these results indicate that the visual cue that was reinforced during training gained control over salamanders’ turning behaviour in the maze when the start positions and cue placement locations were randomized. We think that the compound proximal visual cue gained control in this experiment because it was the only consistent predictor of reinforcement. As in our prior work, although geometric cues were available, they were not good predictors of reinforcement due to the randomized start position and visual cue location (Please see Kundey et al., 2016 for theories regarding why salamanders might have preferred the foreground component of the stimulus.). These results suggest that following brumation, salamanders maintained the general information about how to respond to the proximal compound cue that had been trained. This finding fits well with the recent work by Wilkinson et al. (2017) showing that at least some forms of learning are largely retained by salamanders over brumation. In Experiment 1, we failed to find an effect of timing of exposure to cold temperatures and memory retention. However, only a small number of animals were included in Experiment 1. Thus, this initial experiment was likely underpowered with respect to exploring the impact of the timing of exposure to cold temperatures on a recently reactivated memory across brumation. 3. Experiment 2 The primary goal of Experiment 2 was to examine whether timing of exposure to cold temperatures interfered with retention processes in salamanders with more statistical power as well as to replicate the 5
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exhibited the same preference for the foreground or background during testing before and after brumation. Why did the salamanders in the immediate brumation group not evidence the same preference for the foreground or background after brumation that they had prior to brumation? The ability of the delayed brumation group to retain their preference over the delay suggests that the difference in performance is not merely due to a lengthy delay, as if this was the case then individuals in both groups should have shown a mismatch in their choices just after training and following brumation. Additionally, the ability of the delayed brumation group to retain their foreground or background preference over the delay suggests that the difference in performance is not merely due to exposure to cold temperatures, as if this was the case then individuals in both the delayed and immediate brumation groups should have shown a mismatch between their choices before and after brumation. Instead, we suspect that the timing of exposure to cold temperatures relative to when the preference for foreground or background testing was conducted prior to the brumation period affected the retention of the displayed preference for the foreground or the background cue after brumation. While the preference was likely formed throughout the course of training as salamanders learned to make the correct turning response to the compound proximal visual cue, we forced salamanders to work with this memory directly during the preference test trial by exposing them to the foreground and the background cues as they moved down the ally and made a turning response. For the immediate brumation group, this was just prior to exposure to the cold temperature needed to induce brumation. Although we cannot point to the exact mechanism for this difference in retention from this study alone, prior studies with rats suggest that cooling the body and thus the nervous system can produce amnesia, perhaps by disrupting reconsolidation (e.g., McGaugh and Herz, 1972; Riccio et al., 2006). Since the delayed brumation group did not experience the cold temperature needed to induce brumation until 24 h after they had completed the initial preference testing prior to the delay, they would have had more time to reconsolidate the memory of their preference, leading to a maintained preference after the brumation period. However, alternative explanations exist, including the retrieval failure hypothesis, which suggests that the amnestic agent, in this case cold temperatures, affects the coding of memories to make them harder to recover later (Riccio and Richardson, 1984). This hypothesis would suggest that the original preference that the salamanders in the immediate brumation group displayed prior to brumation should be recoverable after brumation given the right test conditions or cuing. We do have some evidence regarding this account. In Experiment 1, all animals completed the compound proximal visual cue test on day 91 and the foreground vs. background test on day 92. In Experiment 2, we counterbalanced the order of the testing after brumation such that half of the animals completed the compound proximal visual cue test on day 91 and the foreground vs. background test on day 92; the remaining half completed the compound proximal visual cue test on day 92 and the foreground vs. background test on day 91. The compound proximal visual cue test could be viewed as a reminder treatment, as the compound proximal visual cue contains both the foreground superimposed on the background. The retrieval failure hypothesis suggests that reminder treatments should facilitate recall of information that was learned prior to a nervous system perturbation such as cooling. However, salamanders that were immediately exposed to cold temperatures following the preference test continued to show reduced preference matching between the before brumation and after brumation time periods compared to salamanders that were not immediately exposed to cold temperatures. It remains possible, however, that a more powerful reminder treatment could be conducted that would lead to results consistent with a retrieval failure account. Additionally, it is possible that placement of the salamanders in the fridge immediately after the foreground/background preference test in
Fig. 3. Depicts performance of salamanders in the delayed brumation and immediate brumation groups in Experiment 2 following brumation. The black bar indicates the percentage of salamanders in each group that retained the turning response to the proximal compound visual cue. The white bar indicates the percentage of salamanders in each group who evidenced the same preference in the foreground versus background preference test prior to and following brumation.
salamanders in the immediate brumation group, 16 of 18 salamanders turned toward the compound cue that was reinforced during training. We used a two-tailed Fisher’s exact test to examine whether turning toward the proximal compound visual cue differed across the groups (p = 0.595). The results indicated that no significant difference existed between the groups with respect to turning towards the proximal compound visual cue following the 90-day delay. A one-tailed binomial (p < 0.001) indicated that salamanders turned toward the proximal compound visual cue significantly more often than expected by chance. Next, we evaluated whether exposure to cold temperatures immediately following preference testing interfered with later retention of that preference. In effect, we evaluated whether the preference shown prior to brumation matched the preference shown after brumation. These data are also depicted in Fig. 3. Of the salamanders in the delayed brumation group, 18 of 20 maintained their preference for the foreground or background cue following the delay. Of those in the immediate brumation group, 10 of 18 maintained their preference for the foreground or background cue following the delay. We used a twotailed Fisher’s exact test to examine whether turning toward the proximal compound visual cue differed across the groups (p = 0.027). The results indicated that a significant difference existed between the groups with respect to maintaining their preference of turning towards the foreground or background of the compound proximal visual cue following brumation, with the delayed brumation group being significantly more likely to maintain the preference demonstrated prior to the delay than the immediate brumation group. 3.3. Discussion As in Experiment 1, these results suggest that following brumation, salamanders maintained their ability to respond correctly to the proximal compound visual cue that had been trained prior to brumation. Importantly exposure to cold temperatures to start the brumation period occurred at least 24 h after the last turning response to the compound proximal visual cue for both groups. These results accord with Experiment 1 and suggest that following brumation, the salamanders remembered how to respond to the proximal compound cue that had been trained 90 days earlier prior to brumation. However, the salamanders in the immediate brumation group, which were exposed to cold temperatures directly following the test for their preference for the foreground or background prior to the 90-day brumation period, did not maintain their preference after the brumation period. Salamanders in the delayed brumation group, who were exposed to cold temperatures 24 h after the preference testing, 6
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Author note
the immediate brumation group constituted a punishment trial for this group. However, we hypothesize that if this were the case that these salamanders in the immediate brumation group would have exhibited more no-choice trials in the foreground/background preference test or would have systematically chosen oppositely in the foreground/background test after brumation compared to before brumation (i.e., pick the foreground prior to brumation and the background after brumation). However, the salamanders in this group did make choices in the foreground/background preference test and no systematic change in preference for the foreground or the background was observed when comparing before and after brumation test results of the preference test. Regardless of the explanation, our results suggest exposure to cold temperatures near in time to when a memory is reactivated in salamanders disrupts later retention following brumation. Although we only tested adult male salamanders in this experiment, we hypothesize that similar results would be obtained for female salamanders. It would be of interest for future experiments to confirm this finding in female salamanders as well as to explore the impact of brumation on memory at various points in the lifecycle of salamanders.
The authors would like to thank Beth Reiten for her help in preparing this manuscript. This work was supported by a Summer Research Institute grant from Hood College and the Faculty Development Fund at Hood College. These studies comply with the ethical standards of the United States for the use of animals as research subjects. We have no conflicts of interest to report. References Healy, S.D., Jones, C.M., 2002. Animal learning and memory: an integration of cognition and ecology. Zool 105, 321–327. http://dx.doi.org/10.1078/0944-2006-00071. Kundey, S.A., Millar, R., McPherson, J., Gonzalez, M., Fitz, A., Allen, C., 2016. Tiger salamanders’ (Ambystoma tigrinum) response learning and usage of visual cues. Anim. Cogn. 19, 533–541. http://dx.doi.org/10.1007/s10071-016-0954-9. Mactutus, C.F., Riccio, D.C., Ferek, J.M., 1979. Retrograde amnesia for old (reactivated) memory: some anomalous characteristics. Science 204, 1319–1320. http://dx.doi. org/10.1126/science.572083. McGaugh, J.L., Herz, M.J., 1972. Memory Consolidation. Albion, San Francisco. Millesi, E., Prossinger, H., Dittami, J.P., Fieder, M., 2001. Hibernation effects on memory in European ground squirrels (Spermophilus citellus). J. Biol. Rhythms 16, 264–271. http://dx.doi.org/10.1177/074873040101600309. Nowakowski, S.G., Swoap, S.J., Sandstrom, N.J., 2009. A single bout of torpor in mice protects memory processes. Physiol. Behav. 97, 115–120. http://dx.doi.org/10.1016/ j.physbeh.2009.02.013. Riccio, D.C., Richardson, R., 1984. The status of memory following experimentally induced amnesias: gone, but not forgotten. Physiol. Psychol. 12, 59–72. http://dx.doi. org/10.3758/bf03332169. Riccio, D.C., Millin, P.M., Bogart, A.R., 2006. Reconsolidation: a brief history, a retrieval view, and some recent issues. Learn. Mem. 13, 536–544. http://dx.doi.org/10.1101/ lm.290706. Ruczynski, I., Siemers, B., 2011. Hibernation does not affect memory retention in bats. Biol. Lett. 7, 153–155. http://dx.doi.org/10.1098/rsbl.2010.0585. Spear, N.E., Riccio, D.C., 1994. Memory: Phenomena and Principles. Allyn and Bacon, Boston. Thompson, A.B., Montiglio, P., Humphries, M.M., 2013. Behavioural impacts of torpor expression: a transient effect in captive eastern chipmunks (Tamias striatus). Physiol. Behav. 110–111, 115–121. http://dx.doi.org/10.1016/j.physbeh.2013.01.005. Van der Ohe, C.G., Garner, C.C., Darian-Smith, C., Heller, H.C., 2007. Synaptic protein dynamics in hibernation. J. Neurosci. 27, 84–92. http://dx.doi.org/10.1523/ JNEUROSCI.4385-06.2007. Weltzin, M.M., Zhao, H.W., Drew, K.L., Bucci, D.J., 2006. Arousal from hibernation alters contextual learning and memory. Behav. Brain Res. 167, 128–133. http://dx.doi.org/ 10.1016/j.bbr.2005.08.021. Wilkinson, A., Hloch, A., Mueller-Paul, J., Huber, L., 2017. The effect of brumation on memory retention. Sci. Rep. 7, 40079. http://dx.doi.org/10.1038/srep40079.
4. Conclusion In conclusion, our results indicate that salamanders can learn to make responses utilizing compound proximal visual cues in a maze and that this performance can be retained across a 90-day brumation. However, retention of information can be impaired if exposure to cold temperatures occurs immediately after reactivation of a memory. Future experiments could explore the mechanism(s) underlying this memory impairment brought on by exposure to cold temperatures near in time to memory reactivation, perhaps by attempting to find conditions under which the memory of the preference demonstrated just prior to brumation could be recovered. If the information was not reconsolidated successfully before brumation, it should not be possible to recover it. If, however, the information was reconsolidated successfully prior to brumation but is now difficult to retrieve, an appropriate reminder cue might be able to be used to reactive the memory after brumation (e.g., Riccio and Richardson, 1984; Spear and Riccio, 1994). Additionally, future studies could investigate how much time must elapse between activation/reactivation of a memory and exposure to cold temperatures to preserve later performance when cold temperatures remit.
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Contents lists available at ScienceDirect
Behavioural Processes journal homepage: www.elsevier.com/locate/behavproc
Tiger salamanders’ (Ambystoma tigrinum) response retention and usage of visual cues following brumation ⁎
Shannon M.A. Kundey , Anne Lessard, Aleyna Fitz, Manika Panwar Hood College, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Salamander Brumation Memory Amphibian
Brumation enables tiger salamanders (Ambystoma tigrinum) to survive changes in temperature. However, it is unclear how this affects memory retention. We explored how brumation impacted salamanders’ retention of a learned response to a visual cue through two experiments. We hypothesized salamanders would retain information across this state. However, we also hypothesized that retention could be manipulated through cold temperature exposure timing. We hypothesized that cold temperature exposure immediately after reactivation of a memory would decrease retention of that memory following brumation. Our results indicate that salamanders can respond utilizing visual cues and that performance can be retained across this state. However, our results also indicate that if exposure to cold temperatures occurs directly following a recall experience, memory for the information that was recalled just prior to cold temperature exposure can be disrupted. This suggests that the timing of the recalling of information and the exposure to the cold temperatures inherent to brumation is important to memory retention through this state. Future studies should investigate the impact of the timing of extreme temperature exposure on retention over other torpor states, including hibernation and aestivation. Additionally, the mechanism underlying such impaired retention should be explored.
1. Introduction Long-term torpor states such as brumation, hibernation, and aestivation enable animals to survive harsh environmental conditions. These processes differ substantially between warm-blooded and cold-blooded animals. For example, while warm-blooded animals that hibernate move from hibernation to sleep throughout the hibernation period, cold-blooded animals depend on their surroundings and remain torpid until the environmental temperature increases (e.g., Healy and Jones, 2002). Studies of torpor states to date have centered on examining physiological relationships such as body temperature and oxygen usage (e.g., Healy and Jones, 2002). It remains unclear how such states affect memory retention and, more generally, cognitive function—especially in cold-blooded animals. Retention of learned information across such conditions would obviously be adaptive for any species. For example, the ability to remember information, such as areas of high food availability or increased danger, would likely increase fitness. However, torpor states pose a formidable memory retention challenge. While little research exists regarding the effect of torpor states on cognitive function generally, several studies have been performed investigating how hibernation impacts warm-blooded animals.
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Research suggests that hibernation for mammals leads to a drastic decrease in the number of synapses (Van der Ohe et al., 2007). Additionally, since neuronal activity is critical for maintaining neuronal function, one might hypothesize that decreased brain activity due to hibernation might negatively impact memory retention. Research to date, however, conflicts regarding the actual impact of hibernation. Some studies suggest significant memory loss (e.g., Millesi et al., 2001; Weltzin et al., 2006) while others suggest significant retention (e.g., Nowakowski et al., 2009; Ruczynski and Siemers, 2011). Other studies suggest that impairment may be transient (e.g., Thompson et al., 2013). Work regarding torpor states’ impact on cold-blooded animals’ memory retention are few. However, Wilkinson et al. (2017) recently investigated the impact of brumation on fire salamanders’ (Salamandra salamandra) ability to retain a spatial discrimination in a T-maze. All subjects learned to make the initial discrimination response. After training, half brumated while the other half did not. Later testing showed that subjects from both the brumation and no brumation conditions retained the learned response. However, it remains unclear whether memory for other types of tasks would survive brumation. Additionally, it is not clear when the memory must be consolidated or reconsolidated relative to the beginning of the brumation period in order to be retained.
Corresponding author at: Hood College, Department of Psychology, 401 Rosemont Avenue, Tatem 324, Frederick, MD 21701, USA. E-mail address: [email protected] (S.M.A. Kundey).
https://doi.org/10.1016/j.beproc.2018.06.008 Received 16 February 2018; Received in revised form 11 June 2018; Accepted 15 June 2018 0376-6357/ © 2018 Published by Elsevier B.V.
Please cite this article as: Kundey, S.M.A., Behavioural Processes (2018), https://doi.org/10.1016/j.beproc.2018.06.008
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performance following the delay (e.g., McGaugh and Herz, 1972). Thus, we hypothesized that retention following brumation would vary as a function of when exposure to cold temperatures began, with delayed exposure to cold temperatures associated with greater retention due to less interference with reconsolidation processes.
To continue to clarify some of these issues, we explored the impact of brumation on tiger salamanders’ (Ambystoma tigrinum) retention of a response they learned to make previously to a visual cue as they moved about in a maze. We used a paradigm that had been previously successful in our laboratory (Kundey et al., 2016). In that earlier series of experiments, we explored tiger salamanders’ ability to learn to make responses within a maze as visual cues that were located within the maze changed. We found that salamanders could learn to make turning responses based on visual cues placed in the maze. In the last experiment of this series, we investigated if a compound visual cue (a foreground diamond on a contrasting background) could control salamanders’ behaviour when it was the only predictor of how to obtain reinforcement. In this experiment, we varied the start position of the salamander within the maze by placing it in a pseudorandomly determined maze arm at the beginning of each trial. We also varied whether the cue was placed on the right or left side of the alley that the salamander walked down prior to making a turning response. Salamanders did learn to use the placement of this visual cue within the maze to make turning responses that would lead to reinforcement. Here, we built upon this paradigm to confirm the results of that third experiment and to explore the impact of brumation on salamanders’ retention of a learned response to a visual cue. Brumation is of interest because it occurs in cold temperatures. Cold temperatures are interesting because much research suggests that consolidation of new memories and reconsolidation of older memories can be disrupted by various agents, including hypothermia (for review, see Riccio et al., 2006), if those agents are presented near enough in time to the information that is to be remembered (in the case of consolidation) or that has been recently recalled (in the case of reconsolidation). In short, a window exists after the creation or reactivation of a memory during which it is vulnerable to modification by new information, neural interventions, environmental conditions, or pharmacological interventions. Thus, exposure to the cold temperatures inherent in brumation with respect to memory reactivation (i.e., through recall) and reconsolidation processes is especially interesting because prior work in the memory literature suggests that cold temperatures may disrupt reconsolidation processes if exposure to cold temperatures occurs near enough in time to the reactivating event (e.g., McGaugh and Herz, 1972; Mactutus et al., 1979; Riccio et al., 2006). In other words, if a memory is reactivated and followed by exposure to cold temperatures quickly (e.g., exposure to cold temperatures occurs immediately after reactivation), the exposure to the cold temperatures may interfere with memory reconsolidation, leading to difficulty recalling that reactivated memory in the future because reconsolidation has not concluded prior to the cold temperature exposure. If, on the other hand, a memory is reactivated and reconsolidated prior to cold temperature exposure (e.g., exposure to cold temperatures occurs 24 h after the reactivating event), the reactivated memory is more likely to be recalled successfully at a future date because reconsolidation processes had concluded prior to cold temperature exposure. Prior work with rats indicates that nervous system perturbations such as body cooling can produce difficulties in recalling information either when new information has been learned but not yet consolidated or when previously learned information has been recalled or reactivated but not yet reconsolidated (as reviewed by Riccio et al., 2006). Additionally, research suggest that reconsolidation processes may be more prone to interference from cold temperatures than initial consolidation processes (Mactutus et al., 1979). Overall, we hypothesized that salamanders would maintain memory for the response to the visual cue across brumation as evidenced by their performance after brumation. However, we hypothesized that exposure to cold temperatures immediately following reactivation of a memory through requiring the salamanders to work with that memory could interfere with retention of that memory, potentially through interference with reconsolidation processes, and thus decrease
2. Experiment 1 The primary goal of Experiment 1 was to investigate if salamanders could retain information across a 90-day brumation period. We trained all salamanders to turn toward a compound proximal visual cue (square tile with a colored background and a large diamond in the foreground) as start position and cue location varied on each trial in a cross-maze as we did in Kundey et al. (2016) and as described in section 2.1. After learning to turn correctly in the direction indicated by the cue, we separated the foreground and background components of the cue. In a forced-choice procedure the next day, we then evaluated each salamander for turning preference towards the foreground or background of the visual cue. Next, we divided salamanders into three groups for a delay of 90 days: maintenance in their home cage under normal laboratory conditions (maintenance), immediate brumation following training (immediate brumation), or delayed brumation after a 24-hour delay (delay brumation). Following the delay, we evaluated if turning toward the compound proximal visual cue was maintained and repeated foreground versus background testing to evaluate whether the preference after the delay matched the preference displayed during the test trial prior to the delay. We hypothesized that salamanders would maintain memory for the response to the proximal compound visual cue across brumation by continuing to turn toward the proximal compound visual cue after the delay at comparable rates across groups. We did not expect retention of turning towards the proximal compound visual cue to be affected for any group since it always occurred on the day prior to preference testing and thus did not occur immediately before exposure to cold temperatures in any group. However, we hypothesized that exposure to cold temperatures to induce brumation immediately following activation of the preference memory through the foreground/background preference test might interfere with the reconsolidation of the preference memory in the immediate brumation group relative to the other groups. Thus, we hypothesized that the preference displayed just prior to exposure to cold temperatures would not match the preference displayed after brumation in the immediate brumation group but would match in the delayed brumation and the no brumation groups. 2.1. Method 2.1.1. Subjects The subjects were 28 adult male salamanders obtained from Haha Reptiles, Honey Grove, TX. (Females were unavailable at the time of purchase.). Salamanders were maintained throughout training and testing in their home cages at 23 °C ( ± 2 °C) and 70% humidity in group aquariums with coconut fibre bedding in a dimly lit laboratory with access to freeze-dried insects twice weekly. All procedures accorded with Hood College’s Institutional Animal Care and Use regulations. For the brumation groups, a fridge (General Electric model WMR03GAZABB) without illumination was maintained at 3.89 °C ( ± 2 °C) and 60% humidity was employed. Throughout brumation, salamanders were individually housed in plastic 64 oz rectangular containers with moist coconut fibre bedding. The fridge was opened every 3 days for 5 min to allow for air exchange. 2.1.2. Apparatus A cross-maze (114.3 cm X 114.3 cm X 7.62 cm) as shown in Fig. 1 was constructed from square plastic tiles painted a uniform silver colour 2
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Fig. 1. Depicts the apparatus used in the experiment as well as the training and test stimuli.
top of the background stimulus in training was removed for testing.
(see Kundey et al., 2016). We utilized one tile to block off the entrance to the arm directly opposite the start arm on each trial, which varied across trials. Thus, three arms were open to the salamanders during training and testing and which three arms were opened varied across trials. Two timeout containers (15.25 cm X 25.25 cm X 9 cm) were similarly constructed. The timeout containers were separate from the maze and placed relative to the open arms of the maze for each trial, one on the left and the other on the right. The timeout containers were bright and dry. We did not surround the maze with a larger exterior wall or exterior curtain outside the maze to block extramaze cues. This was unnecessary because only the compound visual cue was predictive of reinforcement since starting position varied across trials (as described below) and the turn required to receive reinforcement was not predictive of reinforcement. We covered two training tiles (7.62 cm X 7.62 cm; depicted in Fig. 1) with compound stimuli (red diamond foreground on yellow square background, yellow diamond foreground on red square background; background: 7.62 cm X 7.62 cm, foreground: 5.08 cm X 5.08 cm, square background area: 11.48cm2, diamond foreground area: 11.48cm2). These were placed at the maze junction at the cross of the maze during training as described in 2.1.3. Four test tiles were also constructed (red diamond foreground, yellow square background, yellow diamond foreground, red square background; identical dimensions to the training tiles were used). There were placed (as described in 2.1.3.) at the maze junction during testing. The diamond foreground area was 11.48cm2; the square background area (with the diamond now removed) was 22.86 cm2. This area was larger simply because the foreground stimulus that was previously on
2.1.3. Procedure During trials, the room was brightly lit by overhead fluorescent lighting. The maze was cleaned between trials to counter scent cues. The experimenters stood distant from the maze out of the subjects’ view. Experimenters noted responses in real time using a monitor showing an overhead view of the maze. Interrater reliability was obtained for 10% of responses; raters agreed on all responses. During training, we placed salamanders individually within the start area of the maze and allowed for 5 min to complete the maze by performing a left or right turn at the maze junction (apex of the T; see Fig. 1). We defined a turn as crossing a line 15 cm from the maze’s centre in either direction within the apex of the ‘T’ composed of the open maze arms with any part of the body; most often, this was the head. When correct, the salamander was returned to its home cage until the next experimental day. Previously, we found this to be reinforcing to the salamanders, likely because the home cage environment was moister and allowed for burrowing. If the turn was incorrect, we removed the salamander to the timeout box on the side of the incorrect choice for 1 min. The timeout box did not allow for burrowing and was dry and bright. In our prior work, this did not appear to be reinforcing for the salamanders. After 1 min, the salamander moved on to the next scheduled trial. We did not employ correction trials. If salamanders made an error, they went on to the next scheduled trial for the day without receiving information regarding the correct response on the trial that they had just completed. We introduced new trials until the salamander initially made a correct choice on a trial or completed a maximum of 10 trial attempts/day. If the 3
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test trial per day. On the 91 st day, we evaluated if turning toward the compound proximal visual cue was maintained (hereafter, referred to as compound cue testing). We used the same procedure as in training with the exception that each animal only received one trial instead of up to 10 trials each day. This test let us evaluate if the salamanders remembered to turn towards the compound visual cue from training. On the 92nd day, we repeated foreground versus background preference testing using the same procedure used just before the delay. This test let us evaluate if their preference for the foreground or background was maintained across the various delay conditions.
salamander made an error on the 10th trial of the day, it was placed in the timeout box prior to being returned to its home aquarium. If a salamander did not execute a turn within 5 min, the salamander was removed to the timeout box for 1 min. Subsequently, trials were continued until it made a correct choice on the first attempt at a trial (maximum of 10 trial attempts/day). Thus, although 10 trials were scheduled each day, a salamander could complete fewer trials per day if it executed the correct response on the first or second attempted trial of the day. In our prior study, we found that salamanders learned more quickly about visual cues when the start arm of the maze varied over trials. Thus, the arm in which we placed the salamander to start a trial changed over the course of training. We varied the start arm of the maze pseudorandomly from trial to trail for each salamander to make sure that a salamander did not start in the same arm more than twice in a row. We placed one of the two compound visual cues at the maze junction (where the start arm intersected with the tangential choice arms) on either side of the alley to show which turn direction would be reinforced on that trial. The placement of the cue on either side of the alley (right or left) varied pseudorandomly across trials to make sure that the cue was not placed on the same side more than twice in a row. We did this to help make sure that the salamanders attended to the features of the cue itself rather than whether it was on the right or left side of the ally. For each subject, the compound visual cue used was consistent for the entire experiment; we ensured that half of the animals were trained with one compound visual cue, and the remaining half were trained with the other compound visual cue. Thus, both the compound visual cue location and the start arm location varied on each trial. This made sure that the only consistent predictor of reinforcement was the compound visual cue itself. We counterbalanced compound visual cue assignment (red diamond foreground on yellow square background, yellow diamond foreground on red square background) across subjects. However, assignment was consistent for each animal. In this experiment, the criterion for moving from training trials to test trials was the completion of 3 consecutive days of turning towards the reinforced compound proximal visual cue on the first training trial attempt of the day. Since each subject had a 50% chance of turning correctly on each trial by chance, subjects had a 12.5% chance of executing the correct choice by chance over the 3 days in combination. The salamander moved on to a test trial to evaluate preference for foreground or background of the compound proximal visual cue the following day after criterion was met. This arbitrary, but relatively low threshold, is consistent with that of our prior work (Kundey et al., 2016). The test trial for preference following initial training resembled training except that the test tiles that corresponded to the compound stimulus’s dissociated foreground and background were used. The test tiles were placed at the maze junction, one on the right and the other on the left of the alley, immediately before the intersection. The start arm for testing varied in the same manner as in training. Following this test trial, salamanders were removed from the maze. Then, they were randomly placed into one of three delay conditions that lasted for 90 days: maintenance in their home cage under normal laboratory conditions, immediate brumation following training, or delayed brumation after a 24-hour delay spent in the home cage under normal laboratory conditions. Thus, salamanders in the immediate brumation group were exposed to cold temperatures right after they completed the preference testing for the foreground or the background. Salamanders in the delayed brumation group also were exposed to cold temperatures, but this began 24 h after they completed the preference testing. Salamanders in the maintenance group were never exposed to cold temperatures. After the 90-day delay, all subjects resided in their home cages for the remainder of the experiment. On days 91 and 92, we conducted one
2.2. Results In total, 23 of the 28 salamanders beginning the experiment completed it. Only the data from salamanders that completed the entire experiment are included in the analyses. Of the 5 that did not complete the experiment, 3 died during the experiment (1 prior to brumation and 2 during brumation) and 2 were eliminated due to errors by the experimenters. Of the salamanders that completed all portions of the experiment, 8 were in the normal maintenance group, 7 were in the immediate brumation group, and 8 were in the delayed brumation group. All of the 23 salamanders that completed the experiment reached the criterion of accumulating 3 successive days of turning in the correct direction on their first attempted trial of the day. The number of attempted trials that were required to reach this criterion ranged from 3–215, with a mean of 53.78 attempted trials (SD = 52.07), completed over a range of 3–94 days, with a mean of 26.52 days (SD = 25.58). These data are comparable to those obtained by Kundey et al. (2016) during training. The number of attempted trials and number of days to criterion did not differ significantly over groups. During the preference test trial prior to the delay, 16 of the 23 salamanders turned towards the foreground test tile while 7 turned towards the background test tile. We used a one-tailed binomial test to evaluate the significance of turning toward the foreground tile, as our prior work (Kundey et al., 2016) had indicated a preference by this species of salamander for the foreground tile. The test indicated that salamanders turned towards the foreground tile significantly more often than predicted by chance (p = 0.047) in this experiment. However, this preference was not associated with speed of learning. Following the 90-day delay, we first tested whether salamanders would remember to turn towards the compound cue. These data are depicted in Fig. 2. All salamanders in the no brumation maintenance group and the delayed brumation group turned toward the compound cue that was reinforced during training. Six of 7 salamanders in the immediate brumation group also turned toward the compound cue that was reinforced during training. We used a two-tailed Freeman-Halton extension of the Fisher exact probability test to examine whether turning toward the proximal compound visual cue differed across the groups (p = 1). The results indicated that no significant difference existed between the groups with respect to turning towards the proximal compound visual cue following the 90-day delay. We used a one-tailed binomial test to evaluate if salamanders turned toward the compound proximal visual cue more often than expected by chance, as we predicted that salamanders would remember the turning response to the compound proximal visual cue as it occurred at least 24 h prior to exposure to cold temperatures in the brumation groups. The one-tailed binomial test (p < 0.001) indicated that salamanders turned toward the proximal compound visual cue significantly more often than expected by chance. Next, we evaluated whether exposure to cold temperatures immediately following preference testing interfered with later retention of that preference. In effect, we evaluated whether the preference shown prior to the 90-day delay matched the preference shown after the 90day delay. These data are also depicted in Fig. 2. Seven of 8 4
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finding that salamanders could retain information over brumation. As a result, we only included the delayed brumation and immediate brumation groups in Experiment 2. Additionally, we counterbalanced the day of the compound proximal visual cue test and the preference test after the 90-day delay. 3.1. Method 3.1.1. Subjects The subjects were 44 adult male salamanders obtained and maintained as in Experiment 1 (Females were unavailable at the time of purchase.). Naïve animals were used in Experiment 2. During brumation, salamanders were housed in groups of 4–5 animals/container. 3.1.2. Apparatus The same materials used in Experiment 1 were employed in Experiment 2.
Fig. 2. Depicts performance of salamanders in the no brumation, delayed brumation and immediate brumation groups in Experiment 1 following brumation. The black bar indicates the percentage of salamanders in each group that retained the turning response to the proximal compound visual cue. The white bar indicates the percentage of salamanders in each group who evidenced the same preference in the foreground versus background preference test prior to and following brumation.
3.1.3. Procedure The procedure was exactly the same as Experiment 1 except that salamanders were divided into only two groups for the delay: immediate brumation and delayed brumation. Following the 90-day delay, all subjects were moved to their home cages. On the 91 st and 92nd days, we evaluated whether turning toward the compound proximal visual cue was maintained and repeated foreground versus background testing. Which test was performed on which day was counterbalanced across animals to help alleviate the concern that potential effects might be driven by order of test presentation. Care was taken to ensure that 50% of the animals in each group completed the compound proximal visual cue test on day 91 and the preference test on day 92 and the remaining 50% completed the preference test on day 91 and the compound proximal visual cue test on day 92.
salamanders in the normal maintenance group maintained their preference for the foreground or background cue following the delay. Additionally, 7 of 8 salamanders in the delayed brumation group also maintained their preference for the foreground or background cue following the delay. However, only 3 salamanders in the immediate brumation group maintained their preference for the foreground or background cue following the delay. We used a two-tailed FreemanHalton extension of the Fisher exact probability test to examine whether the maintaining of preference across the 90-day delay varied across groups (p = 0.169). The results indicated that no significant difference existed between the groups with respect to maintaining their preference of turning towards the foreground or background of the compound proximal visual cue following the 90-day delay.
3.2. Results In total, 38 of the 44 salamanders beginning the experiment completed it. Only the data from salamanders that completed the entire experiment are included in the analyses. Four animals that did not complete the experiment died during brumation. An additional two animals are not included in the analyses due to experimenter error. Of the salamanders that completed all portions of the experiment, 18 were in the immediate brumation group, and 20 were in the delayed brumation group. All of the 38 salamanders that completed the experiment reached the criterion of accumulating 3 successive days of turning in the correct direction on their first attempted trial of the day. The number of attempted trials that were required to reach this criterion ranged from 7–211, with a mean of 64.76 attempted trials (SD = 56.11), completed over a range of 5–94 days, with a mean of 32.87 days (SD = 30.18). These data are comparable to those obtained by Kundey et al. (2016) and during training for Experiment 1. The number of attempted trials and number of days to criterion did not differ significantly over groups. During the preference test trial prior to the delay, 25 of the 38 salamanders turned towards the foreground test tile while 13 turned towards the background test tile. As in Experiment 1, we used a onetailed binomial test to evaluate the significance of turning toward the foreground tile. The test indicated that salamanders turned towards the foreground tile significantly more often than predicted by chance (p = 0.036) in this experiment. However, this preference was not associated with speed of learning. Following the 90-day delay, we tested whether salamanders would remember to turn towards the compound proximal visual cue and their preference for the foreground versus the background tile on separate days, counterbalanced across days. These data are depicted in Fig. 3. Of the salamanders in the delayed brumation group, 19 of 20 turned toward the compound cue that was reinforced during training. Of the
2.3. Discussion As in our prior work, these results indicate that the visual cue that was reinforced during training gained control over salamanders’ turning behaviour in the maze when the start positions and cue placement locations were randomized. We think that the compound proximal visual cue gained control in this experiment because it was the only consistent predictor of reinforcement. As in our prior work, although geometric cues were available, they were not good predictors of reinforcement due to the randomized start position and visual cue location (Please see Kundey et al., 2016 for theories regarding why salamanders might have preferred the foreground component of the stimulus.). These results suggest that following brumation, salamanders maintained the general information about how to respond to the proximal compound cue that had been trained. This finding fits well with the recent work by Wilkinson et al. (2017) showing that at least some forms of learning are largely retained by salamanders over brumation. In Experiment 1, we failed to find an effect of timing of exposure to cold temperatures and memory retention. However, only a small number of animals were included in Experiment 1. Thus, this initial experiment was likely underpowered with respect to exploring the impact of the timing of exposure to cold temperatures on a recently reactivated memory across brumation. 3. Experiment 2 The primary goal of Experiment 2 was to examine whether timing of exposure to cold temperatures interfered with retention processes in salamanders with more statistical power as well as to replicate the 5
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exhibited the same preference for the foreground or background during testing before and after brumation. Why did the salamanders in the immediate brumation group not evidence the same preference for the foreground or background after brumation that they had prior to brumation? The ability of the delayed brumation group to retain their preference over the delay suggests that the difference in performance is not merely due to a lengthy delay, as if this was the case then individuals in both groups should have shown a mismatch in their choices just after training and following brumation. Additionally, the ability of the delayed brumation group to retain their foreground or background preference over the delay suggests that the difference in performance is not merely due to exposure to cold temperatures, as if this was the case then individuals in both the delayed and immediate brumation groups should have shown a mismatch between their choices before and after brumation. Instead, we suspect that the timing of exposure to cold temperatures relative to when the preference for foreground or background testing was conducted prior to the brumation period affected the retention of the displayed preference for the foreground or the background cue after brumation. While the preference was likely formed throughout the course of training as salamanders learned to make the correct turning response to the compound proximal visual cue, we forced salamanders to work with this memory directly during the preference test trial by exposing them to the foreground and the background cues as they moved down the ally and made a turning response. For the immediate brumation group, this was just prior to exposure to the cold temperature needed to induce brumation. Although we cannot point to the exact mechanism for this difference in retention from this study alone, prior studies with rats suggest that cooling the body and thus the nervous system can produce amnesia, perhaps by disrupting reconsolidation (e.g., McGaugh and Herz, 1972; Riccio et al., 2006). Since the delayed brumation group did not experience the cold temperature needed to induce brumation until 24 h after they had completed the initial preference testing prior to the delay, they would have had more time to reconsolidate the memory of their preference, leading to a maintained preference after the brumation period. However, alternative explanations exist, including the retrieval failure hypothesis, which suggests that the amnestic agent, in this case cold temperatures, affects the coding of memories to make them harder to recover later (Riccio and Richardson, 1984). This hypothesis would suggest that the original preference that the salamanders in the immediate brumation group displayed prior to brumation should be recoverable after brumation given the right test conditions or cuing. We do have some evidence regarding this account. In Experiment 1, all animals completed the compound proximal visual cue test on day 91 and the foreground vs. background test on day 92. In Experiment 2, we counterbalanced the order of the testing after brumation such that half of the animals completed the compound proximal visual cue test on day 91 and the foreground vs. background test on day 92; the remaining half completed the compound proximal visual cue test on day 92 and the foreground vs. background test on day 91. The compound proximal visual cue test could be viewed as a reminder treatment, as the compound proximal visual cue contains both the foreground superimposed on the background. The retrieval failure hypothesis suggests that reminder treatments should facilitate recall of information that was learned prior to a nervous system perturbation such as cooling. However, salamanders that were immediately exposed to cold temperatures following the preference test continued to show reduced preference matching between the before brumation and after brumation time periods compared to salamanders that were not immediately exposed to cold temperatures. It remains possible, however, that a more powerful reminder treatment could be conducted that would lead to results consistent with a retrieval failure account. Additionally, it is possible that placement of the salamanders in the fridge immediately after the foreground/background preference test in
Fig. 3. Depicts performance of salamanders in the delayed brumation and immediate brumation groups in Experiment 2 following brumation. The black bar indicates the percentage of salamanders in each group that retained the turning response to the proximal compound visual cue. The white bar indicates the percentage of salamanders in each group who evidenced the same preference in the foreground versus background preference test prior to and following brumation.
salamanders in the immediate brumation group, 16 of 18 salamanders turned toward the compound cue that was reinforced during training. We used a two-tailed Fisher’s exact test to examine whether turning toward the proximal compound visual cue differed across the groups (p = 0.595). The results indicated that no significant difference existed between the groups with respect to turning towards the proximal compound visual cue following the 90-day delay. A one-tailed binomial (p < 0.001) indicated that salamanders turned toward the proximal compound visual cue significantly more often than expected by chance. Next, we evaluated whether exposure to cold temperatures immediately following preference testing interfered with later retention of that preference. In effect, we evaluated whether the preference shown prior to brumation matched the preference shown after brumation. These data are also depicted in Fig. 3. Of the salamanders in the delayed brumation group, 18 of 20 maintained their preference for the foreground or background cue following the delay. Of those in the immediate brumation group, 10 of 18 maintained their preference for the foreground or background cue following the delay. We used a twotailed Fisher’s exact test to examine whether turning toward the proximal compound visual cue differed across the groups (p = 0.027). The results indicated that a significant difference existed between the groups with respect to maintaining their preference of turning towards the foreground or background of the compound proximal visual cue following brumation, with the delayed brumation group being significantly more likely to maintain the preference demonstrated prior to the delay than the immediate brumation group. 3.3. Discussion As in Experiment 1, these results suggest that following brumation, salamanders maintained their ability to respond correctly to the proximal compound visual cue that had been trained prior to brumation. Importantly exposure to cold temperatures to start the brumation period occurred at least 24 h after the last turning response to the compound proximal visual cue for both groups. These results accord with Experiment 1 and suggest that following brumation, the salamanders remembered how to respond to the proximal compound cue that had been trained 90 days earlier prior to brumation. However, the salamanders in the immediate brumation group, which were exposed to cold temperatures directly following the test for their preference for the foreground or background prior to the 90-day brumation period, did not maintain their preference after the brumation period. Salamanders in the delayed brumation group, who were exposed to cold temperatures 24 h after the preference testing, 6
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Author note
the immediate brumation group constituted a punishment trial for this group. However, we hypothesize that if this were the case that these salamanders in the immediate brumation group would have exhibited more no-choice trials in the foreground/background preference test or would have systematically chosen oppositely in the foreground/background test after brumation compared to before brumation (i.e., pick the foreground prior to brumation and the background after brumation). However, the salamanders in this group did make choices in the foreground/background preference test and no systematic change in preference for the foreground or the background was observed when comparing before and after brumation test results of the preference test. Regardless of the explanation, our results suggest exposure to cold temperatures near in time to when a memory is reactivated in salamanders disrupts later retention following brumation. Although we only tested adult male salamanders in this experiment, we hypothesize that similar results would be obtained for female salamanders. It would be of interest for future experiments to confirm this finding in female salamanders as well as to explore the impact of brumation on memory at various points in the lifecycle of salamanders.
The authors would like to thank Beth Reiten for her help in preparing this manuscript. This work was supported by a Summer Research Institute grant from Hood College and the Faculty Development Fund at Hood College. These studies comply with the ethical standards of the United States for the use of animals as research subjects. We have no conflicts of interest to report. References Healy, S.D., Jones, C.M., 2002. Animal learning and memory: an integration of cognition and ecology. Zool 105, 321–327. http://dx.doi.org/10.1078/0944-2006-00071. Kundey, S.A., Millar, R., McPherson, J., Gonzalez, M., Fitz, A., Allen, C., 2016. Tiger salamanders’ (Ambystoma tigrinum) response learning and usage of visual cues. Anim. Cogn. 19, 533–541. http://dx.doi.org/10.1007/s10071-016-0954-9. Mactutus, C.F., Riccio, D.C., Ferek, J.M., 1979. Retrograde amnesia for old (reactivated) memory: some anomalous characteristics. Science 204, 1319–1320. http://dx.doi. org/10.1126/science.572083. McGaugh, J.L., Herz, M.J., 1972. Memory Consolidation. Albion, San Francisco. Millesi, E., Prossinger, H., Dittami, J.P., Fieder, M., 2001. Hibernation effects on memory in European ground squirrels (Spermophilus citellus). J. Biol. Rhythms 16, 264–271. http://dx.doi.org/10.1177/074873040101600309. Nowakowski, S.G., Swoap, S.J., Sandstrom, N.J., 2009. A single bout of torpor in mice protects memory processes. Physiol. Behav. 97, 115–120. http://dx.doi.org/10.1016/ j.physbeh.2009.02.013. Riccio, D.C., Richardson, R., 1984. The status of memory following experimentally induced amnesias: gone, but not forgotten. Physiol. Psychol. 12, 59–72. http://dx.doi. org/10.3758/bf03332169. Riccio, D.C., Millin, P.M., Bogart, A.R., 2006. Reconsolidation: a brief history, a retrieval view, and some recent issues. Learn. Mem. 13, 536–544. http://dx.doi.org/10.1101/ lm.290706. Ruczynski, I., Siemers, B., 2011. Hibernation does not affect memory retention in bats. Biol. Lett. 7, 153–155. http://dx.doi.org/10.1098/rsbl.2010.0585. Spear, N.E., Riccio, D.C., 1994. Memory: Phenomena and Principles. Allyn and Bacon, Boston. Thompson, A.B., Montiglio, P., Humphries, M.M., 2013. Behavioural impacts of torpor expression: a transient effect in captive eastern chipmunks (Tamias striatus). Physiol. Behav. 110–111, 115–121. http://dx.doi.org/10.1016/j.physbeh.2013.01.005. Van der Ohe, C.G., Garner, C.C., Darian-Smith, C., Heller, H.C., 2007. Synaptic protein dynamics in hibernation. J. Neurosci. 27, 84–92. http://dx.doi.org/10.1523/ JNEUROSCI.4385-06.2007. Weltzin, M.M., Zhao, H.W., Drew, K.L., Bucci, D.J., 2006. Arousal from hibernation alters contextual learning and memory. Behav. Brain Res. 167, 128–133. http://dx.doi.org/ 10.1016/j.bbr.2005.08.021. Wilkinson, A., Hloch, A., Mueller-Paul, J., Huber, L., 2017. The effect of brumation on memory retention. Sci. Rep. 7, 40079. http://dx.doi.org/10.1038/srep40079.
4. Conclusion In conclusion, our results indicate that salamanders can learn to make responses utilizing compound proximal visual cues in a maze and that this performance can be retained across a 90-day brumation. However, retention of information can be impaired if exposure to cold temperatures occurs immediately after reactivation of a memory. Future experiments could explore the mechanism(s) underlying this memory impairment brought on by exposure to cold temperatures near in time to memory reactivation, perhaps by attempting to find conditions under which the memory of the preference demonstrated just prior to brumation could be recovered. If the information was not reconsolidated successfully before brumation, it should not be possible to recover it. If, however, the information was reconsolidated successfully prior to brumation but is now difficult to retrieve, an appropriate reminder cue might be able to be used to reactive the memory after brumation (e.g., Riccio and Richardson, 1984; Spear and Riccio, 1994). Additionally, future studies could investigate how much time must elapse between activation/reactivation of a memory and exposure to cold temperatures to preserve later performance when cold temperatures remit.
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