pii: sp-00579-13

http://dx.doi.org/10.5665/sleep.3992

CHARACTERIZATION OF SLEEP IN APLYSIA CALIFORNICA

Characterization of Sleep in Aplysia californica Albrecht P.A. Vorster, Dipl Biol1; Harini C. Krishnan, BSc2; Chiara Cirelli, MD, PhD3; Lisa C. Lyons, PhD4 Department of Biological Science, Program in Neuroscience, Florida State University, Tallahassee, FL; Present Address: Department of Medical Psychology and Behavioral Neurobiology and Center for Integrative Neuroscience CIN, University of Tübingen, Tübingen, Germany; 2Department of Biological Science, Program in Neuroscience, Florida State University, Tallahassee, FL; 3Department of Psychiatry, University of Wisconsin-Madison, Madison, WI; 4Department of Biological Science, Program in Neuroscience, Florida State University, Tallahassee, FL 1

Study Objective: To characterize sleep in the marine mollusk, Aplysia californica. Design: Animal behavior and activity were assessed using video recordings to measure activity, resting posture, resting place preference, and behavior after rest deprivation. Latencies for behavioral responses were measured for appetitive and aversive stimuli for animals in the wake and rest states. Setting: Circadian research laboratory for Aplysia. Patients or Participants: A. californica from the Pacific Ocean. Interventions: N/A. Measurements and Results: Aplysia rest almost exclusively during the night in a semi-contracted body position with preferential resting locations in the upper corners of their tank. Resting animals demonstrate longer latencies in head orientation and biting in response to a seaweed stimulus and less frequent escape response steps following an aversive salt stimulus applied to the tail compared to awake animals at the same time point. Aplysia exhibit rebound rest the day following rest deprivation during the night, but not after similar handling stimulation during the day. Conclusions: Resting behavior in Aplysia fulfills all invertebrate characteristics of sleep including: (1) a specific sleep body posture, (2) preferred resting location, (3) reversible behavioral quiescence, (4) elevated arousal thresholds for sensory stimuli during sleep, and (5) compensatory sleep rebound after sleep deprivation. Keywords: Aplysia, circadian rhythm, invertebrate sleep, mollusk, sleep, sleep deprivation Citation: Vorster AP, Krishnan HC, Cirelli C, Lyons LC. Characterization of sleep in Aplysia californica. SLEEP 2014;37(9):1453-1463.

INTRODUCTION Sleep appears conserved in animal species across phylogenies, suggesting evolutionary ancient and preserved functions.1-4 However, much remains unknown about the underlying molecular and cellular processes. Although initially sleep was studied primarily in mammalian systems, sleep research using lower vertebrate and invertebrate species including zebrafish,5 cockroach,6 bees,7–10 Drosophila,11,12 crayfish,13,14 mollusks,15–17 and the nematode Caenorhabditis elegans18 has significantly furthered the field and demonstrated the phylogenetic conservation of the physiological need for sleep. Despite the prevalence of sleep across phyla, the functions and mechanisms of sleep remain elusive. To identify conserved processes and functions of sleep, the need persists for a simple invertebrate model in which sleep can be investigated at single-cell molecular and electrophysiological resolution within identified neuronal circuits. Although long studied as models for learning and memory and circadian research, the use of mollusks as models for sleep research has been relatively recent. The phylum Mollusca, consisting of the cephalopods, gastropods, and bivalves, is one of the largest phyla. Cephalopods with well-developed and relatively large brains such as octopus, cuttlefish, and squid demonstrate complex behavior and higher order learning and memory similar to mammalian species. In the gastropods, the freshwater pond snail Lymnaea, the sea hare Aplysia, and the Submitted for publication September, 2013 Submitted in final revised form March, 2014 Accepted for publication March, 2014 Address correspondence to: Lisa C. Lyons, PhD, Department of Biological Science, Florida State University, Tallahassee, FL 32306-4295; Tel:(850) 645-8255; Fax: (850) 645-8447; E-mail: [email protected]

predatory sea slug Pleurobranchaea are frequently used as models for memory and behavior studies to correlate behavior with neuronal plasticity because of the anatomically distributed central nervous system and relatively small number of neurons. The conserved need for sleep is evident as sleeplike states are seen in both groups. Octopus vulgaris demonstrate robust nocturnal activity patterns with significant rebound rest observed when animals are deprived of rest.16 The cuttlefish Sepia demonstrate both changes in behavioral activity and brain activity patterns associated with sleep.15,19 However, the complexity and development of the cephalopod nervous system limits the potential for sleep to be investigated at the level of single neurons within identifiable neuronal circuits using these models. With a relatively simple nervous system and anatomically distinct ganglia, the pond snail Lymnaea also demonstrates a sleeplike state, although quiescence occurs infrequently in brief spontaneous intervals totaling less than 10% of the overall time observed.17 Furthermore, quiescence in Lymnaea does not appear to be regulated by either time of day or through homeostatic regulation, highlighting differences with most observations in invertebrate and mammalian sleep.17 A sleeplike condition of inactivity has been proposed for Pleurobranchaea, along with differences in behavioral responses to either a tactile stimulus or a food stimulus between still and active states.20 However, the sleeplike state or circadian activity rhythms have not been further studied in Pleurobranchaea. Until now sleep has not been systematically characterized in Aplysia. To date only anecdotal observations made during early studies of locomotion and activity patterns in different species of Aplysia suggest a sleeplike state.21–25 Aplysia californica exhibit strong diurnal and circadian rhythms in locomotor activity with high levels of activity restricted to the day.22,23,26,27 Further indications of state-dependent behavioral differences in

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Aplysia arose from studies on feeding behavior that identified state dependent behavioral differences in response to a food stimulus between active animals and quiet animals in either a behaviorally quiescent “balled” state, or a quiescent “still” state.28 As these studies were performed after tank cleaning in the morning at times when animals are normally active, potentially the “balled” state may have been indicative of a stress response in some animals. Thus, although previous studies have suggested that Aplysia do rest, no systematic study of Aplysia sleep has been performed. As Aplysia has proven a powerful system for the dissection of the underlying cellular mechanisms of neural plasticity in learning and memory, nociception and injury, and circadian rhythms, 29–32 we characterized sleep in A. californica. The relatively simple distributed nervous system of Aplysia, composed of approximately 20,000 large, pigmented, and identifiable neurons, facilitates the correlation of cellular activity with behavior. Moreover, the high degree of conservation of signaling pathways across species has ensured that the research with this model has clear relevance and applicability for mechanisms underlying behavior in higher organisms. Invertebrate sleep is usually defined through behavioral characterizations including: (1) elevated arousal threshold, (2) reversible behavioral quiescence,33 (3) compensatory rebound sleep after sleep deprivation,6 (4) specific sleep posture,34 and (5) a preferred resting location.4,35–37 To determine if the Aplysia rest state meets these recognized criteria for sleep, we systematically evaluated rest in Aplysia for each of the aforementioned characteristics. Through analysis of rest-waking activity in multiday video recordings, we found that Aplysia exhibited robust diurnal rhythms in locomotor and rest activity with locomotion mainly during the day and sustained periods of rest during the night. Animals also demonstrated a strong resting place preference and exhibited differences in body posture between wake and rest states. To determine whether Aplysia rest is accompanied by changes in sensory arousal threshold, behavioral responses to aversive and appetitive stimuli were assessed for awake and resting animals. Waking animals showed significantly greater responses to sensory stimuli compared to resting animals at the same time point. Finally, we found that rest deprivation during the night produced compensatory rebound rest during the following day, in agreement with the presence of homeostatic regulation. Overall, therefore, we find that the Aplysia rest state fulfills all of the standard behavioral criteria for sleep in invertebrates,1,2,4 establishing Aplysia as a valid new model for sleep research. METHODS Animal Maintenance A. californica (100-175 g; South Coast Bio-Marine, San Pedro, CA) were maintained in individual boxes in 110-gallon circulating tanks in artificial seawater (Instant Ocean, Aquarium Systems, Mentor, OH) at 15°C with a 12 h light/12 h dark (LD) cycle. Animals were fed romaine lettuce (~5 g) every 1-2 days. Feeding times were varied. Activity Analysis For video surveillance (30 frames per sec recording), single animals were housed in 40 × 40 × 15 cm compartments within a 135-gallon circulating seawater tank. Infrared light was used for

illumination for video recording during the dark. Activity was scored via video analysis for each 1-min period with an activity score indicating resting, stationary movements, or locomotion. Animals were scored as resting when they demonstrated no movements for at least 1 min (1,798 frames) with the exception of slight rhinophore movements or respiratory pumping and siphon movement. Animals were scored as stationary moving when body position remained stationary for more than 5 min with the animal exhibiting head and neck movements. Rest Deprivation Animals were transferred at Zeitgeber time (ZT) 13 in the dark to individual, chilled, and aerated (Aquatic Gardens air pump 1000, Aquatic Gardens) 40 × 40 cm plastic containers filled with 3 L of artificial seawater. Each container was equipped with different contexts (big stones, small stones, plastic grid, terrarium liner). During the first hour, animals actively explored the new contexts. Starting at ZT 14, animals were placed in a new context approximately every 30 min and were handled irregularly every 3-6 min based on whether animals were mobile or immobile until the end of the dark period (ZT 24). Rest deprivation procedures were all performed in the dark using dim red light. To facilitate comparisons between sleep deprived and non-sleep deprived control animals, experiments were performed with a counterbalanced measures design in which animals subjected to sleep deprivation in one experiment served as non-sleep deprived controls in a later experiment and vice versa. As a control for the stress of physically handling the animals on rest/activity behavior, in subsequent experiments the same procedure was undertaken with the animals during the light period beginning at ZT 0 and continued for 10 h. As with the sleep deprivation experiments, each animal was recorded with handling in one experiment and as a nonhandled control in another experiment. Sensory Arousal Testing Animals were transferred at ZT 17 or ZT 21 to 2-L plastic bowls and allowed to acclimate for 1 h prior to behavioral testing at ZT 18 or ZT 22. Testing occurred for resting animals following at least 5 min of continuous rest. Animals exhibiting either stationary movements or locomotion were classified as awake. Testing responses to appetitive stimulus: Animals were presented with a 4 mm² piece of dried seaweed (Dulse, Maine Coast Sea Vegetables, Franklin, Maine ) brushed against the rhinophores and then held motionless in the water. Latency to the first lip contact and the first biting pattern elicited was measured. Measurements were stopped if no reaction occurred for more than 2 min. Individual animals were tested at least four times at each time point. Testing responses to aversive stimulus: After acclimation, 5 µL of saturated sodium chloride solution (6.14 M) was pipetted against the tip of the tail. The latency to tail withdrawal and latency for the first step escape response > 1cm if elicited during a 2-min period was measured. Only one behavioral test was performed on each animal during a single night with experiments repeated on multiple nights to obtain complete data for each animal.

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Statistical Analysis Statistical analysis of the data used Student t-test when two groups were compared for parametric data or Kruskal-Wallis Sleep in Aplysia—Vorster et al.

analysis with Dunn multiple comparison post hoc tests or Mann-Whitney U test for nonparametic data. The specific statistical analysis used is indicated with results. Significance was defined as * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical analysis was performed using GraphPad Prism (Graphpad Software, Inc, La Jolla, California).

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RESULTS Activity Analysis and Characterization of Rest As a first step we analyzed individual animal activity patterns throughout the LD cycle for a period of 3 days at 1-min intervals, classifying activity as either locomotion, stationary movements, or rest (see Video 1 for an example of 24-h time lapse video). Immobility was scored as rest when animals were not showing movements for more than 1 min analogous to the “0” behavioral state defined by Kupfermann.38 As previously shown,22,23,26,27 Aplysia demonstrate robust diurnal rhythms in locomotor activity (Figure 1). Animals anticipated the light period with locomotor activity initiated 71.8 ± 40.6 min before dawn, with the predawn activity uninterrupted by resting bouts. Animals actively moved for the greater portion of the light period (548 ± 47 min, n = 8) with the remaining portion (~ 24% of the time) spent engaged in stationary movements including head waving and eating (Figure 1A3). Stationary movements were evident throughout the day and the night with increased stationary activity observed during light transitions. Aplysia exhibit sustained periods of rest only during the night (Figure 1; min of rest per 12-h dark period: 438 ± 44, mean ± standard error of the mean, n = 8). In comparison, rest during the light period was rare and occurred only during immediate anticipation of the dark period in a subset of animals (17 ± 9 min, n = 3). The remaining animals (n = 5) initiated rest shortly after light offset (57 ± 30 min). Aplysia show a strong resting place preference with animals spending 75% of their resting time at the upper ceiling of the tank close to the water surface, especially in the upper corners (62%; Figure 2). Aplysia rested primarily at the sides of their cage. Animals spent only 10% of the time resting in the middle of the tank attached to only a single wall (Figure 2B). Animals seldom changed their resting place during the night (mean 1.9 ± 1.2 location changes per night, n = 8 animals over a 3-day period) and often returned to the original resting location reinforcing the resting location preference. To further characterize Aplysia rest, we analyzed the structure of resting bouts exhibited during the night, as previously done for other organisms (Lymnaea,17 Drosophila,11,39 bees,40 and zebrafish41). We classified the resting bouts in five categories: < 10 min, 10–30 min, 31–60 min, 61–100 min, > 100 min. We found that Aplysia rest during the night was separated in 10 (± 3) bouts with an average duration of 46 (± 49) min. Interestingly, the length of the rest bouts varied during the night. Toward the beginning of the night animals rest primarily in medium-long bouts with the longest nightly rest bouts > 100 min observed in the first half of the night. Afterward, bout length declines toward the end of the night and awakenings are more frequent, consistent with homeostatic regulation (Figure 3C). Long bouts over 60 min account for most of the total resting time, whereas short bouts of less than 10 min account for only 2% of the resting time (Figure 3B). Although SLEEP, Vol. 37, No. 9, 2014 1455 Downloaded from https://academic.oup.com/sleep/article-abstract/37/9/1453/2416887 by guest on 14 January 2018

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Figure 1—Aplysia exhibit robust diurnal rhythms in activity and rest. Activity plot of Aplysia californica (n = 8) for 3 consecutive days on a 12:12 h light-dark cycle. Activity was assessed for each animal individually via video recordings with 1 min resolution and is plotted for 30-min periods. (A1) Locomotor activity defined as active movements with changes in body position. (A2) Resting activity as defined by no movement except for minor spontaneous twitches. (A3) Stationary movements are defined as head waving or movements without changes in location. (B) Bars represent mean (± standard error of the mean) for the sum total of the three types of activity during the light and dark periods.

most rest occurs during long bouts, short rest bouts are more frequent (Figure 3A). Characteristic Resting Posture A specific resting body posture is another characteristic of invertebrate sleep4 that can be widely observed with examples including octopus,16 cuttlefish,15 flies,11 honeybees,42,43 moths,44 and scorpions.45 Quiescent body postures (balled and still states) have also previously been observed in Aplysia28 and the ragged sea hare Bursatella.46 We found that Aplysia exhibited Sleep in Aplysia—Vorster et al.

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resting bouts per type

12%

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resting time per bout type 2% 100 min.

21%

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61-100 min.

resting bouts >100 min.

Figure 2—Aplysia demonstrate resting place preferences during the dark period. (A) Diagram of an individual Aplysia cage housed within a larger aquarium. Numbers in (A) correspond to areas shown in (B). (B) Percentage of time spent in different areas of the tank during resting. n = 8, pooled for 3 consecutive days.

a characteristic resting posture with the entire body more contracted compared to wake animals during the day (Figure 4). During rest, the animal’s neck and tail were retracted, yet the rhinophores and the tentacles appeared relaxed. Occasionally, rhinophores retracted while resting, but the rhinophores did not appear to be actively probing the environment. Furthermore, during rest, the animal’s rhinophores were frequently observed in a crossed yet relaxed position, a body posture that was never seen during the animal’s wake or active states. During rest, parapodia were folded tightly together, yet the siphon remained open with occasional respiratory pumping patterns occurring spontaneously. Overall, the few movements observed during rest did not seem to be part of active motor behavior. Reduced Sensory Response Behavioral quiescence qualifies as sleep only in connection with a concomitant increase in arousal threshold, i.e., the observed decrease in sensory responses to external stimuli.2,4,36 To test for differences in arousal threshold and changes in behavioral responses to sensory stimuli between wake and rest states, we performed two series of experiments testing the responses to an appetitive and an aversive stimulus between awake and resting animals. The circadian clock regulates sensory responses as observed in many species.47–50 To control for possible circadian or diurnal regulation in sensory responses, we compared wake and resting animals at the same time point. We presented animals in the wake and resting states at ZT 18 with appetitive stimuli by brushing the rhinophore of the animal with a 4 mm² piece of Dulse seaweed. This appetitive

Figure 3—Aplysia resting bout length varies between the early and late night. (A) Percentage of the number of rest bouts according to bout type. (B) Percentage of the resting time according to the duration of a sleep bout (bout type). (C) Mean (± standard error of the mean) number of long rest bouts > 100 min during the first half of the dark period (ZT 12-18) and the second half of the dark period (ZT 18-24). n = 8, data pooled from 3 consecutive days and analyzed by Student t-test ** P < 0.01.

stimulus represents an ethologically relevant compound stimulus consisting of tactile and chemosensory input and elicited behavioral responses in more than 96% of the time in resting animals. Tactile stimulation alone using filter paper was insufficient to induce biting responses in awake animals or to arouse resting animals (data not shown). Following presentation of the seaweed, we measured the latency for the animal to turn the head with the lips oriented toward the seaweed (an appetitive behavior) and the latency for the animals to bite at the seaweed (a consummatory behavior) \ (Video 2). Experiments were repeated a minimum of four times on different nights to measure response times between the wake and rest states within individual animals (Figure 5). We found that awake animals had significantly shorter latencies for both orientation to the seaweed (Figure 5A1) and for the biting response compared to resting animals (Figure 5A2). Similar results were found when animals were tested during a late night time point at ZT 22 (Figure 5B). These results demonstrate that resting animals have significantly decreased behavioral responses to appetitive

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parapodia siphon tail

rhinophores neck

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Figure 4—Aplysia exhibit a characteristic posture during rest. (A) During wake, the tail and neck are elongated and the tentacles slightly lifted from the floor, probing the environment. Parapodia are partially open, sometimes flaring to allow water fluctuation. (B) During rest, the body as well as the tail and neck is contracted, the parapodia are closed around the body, and the foot is well attached to the floor. Tentacles rest flat on the floor while the rhinophores are found relaxed and sometimes crossed. Tentacles and rhinophores do not appear to be actively probing the environment.

stimuli (Figure 5C; latency to first lip contact: Kruskal-Wallis test, H = 56.20, P < 0.0001, latency to biting response: KruskalWallis test, H = 58.06, P < 0.0001, Dunn post hoc comparisons showed significant differences between wake and rest states). Although responses to appetitive stimuli were decreased during rest, the behavioral responses to aversive or noxious stimuli could be differentially affected by rest as aversive stimuli induce defensive behaviors. To determine if responses to aversive stimuli are affected by the resting state, we used a mild aversive stimulus consisting of 5 µL of saturated sodium chloride solution (6.1 M) applied to the tail that elicits a local withdrawal of the tail followed by an escape step51 (Video 3). As in the aforementioned experiments, the response to the salt stimulus was tested in awake and resting animals at ZT 18 and ZT 22. We measured the latency for tail withdrawal as well as the latency to initiate the first step of more than 1 cm (an escape step). Interestingly, the latency for tail withdrawal was the same between wake and resting animals with no significant differences observed (Figure 6A; Kruskal-Wallis test, H = 2.024, P = 0.57), whereas significant differences were observed in the escape response (Figure 6B; Kruskal-Wallis test H = 61.76, P < 0.0001, Dunn multiple comparison test showed significant differences between wake and rest states). Awake animals responded at ZT 18 or ZT 22 with an escape step occurring 88.4% and 93.7% of the time, respectively, whereas less than 27.0% / 21.7% of the resting animals took an escape step (Figure 6B). Increasing the amount of salt solution (15 µL) applied to the tail increased the percentage of resting animals responding with an escape step to ~ 45% (data not shown), suggesting that rest at night affected sensory arousal thresholds. The increased responses of resting animals to the 15-µL volume appeared due to the increased salt stimulus and not to potential changes in water turbulence as a

Figure 5—Resting Aplysia exhibit longer latencies in response to an appetitive stimulus. Response to a seaweed stimulus was measured at two time points during the middle of the dark period at ZT 18 and during the late night at ZT 22. Animals were assigned to a behavioral state based upon whether they were awake (wake) or resting (rest) for more than 5 min. Animals responded to the presentation of seaweed at the rhinophores in two discrete stages: (1) head orientation to the stimulus to make lip contact with the seaweed; and (2) first bite. (A) Mean response latency at ZT 18 for individual animals for lip contact (A1) and biting (A2), n = 17. (B) Mean response latency at ZT 22 for individual animals for lip contact (B1) and biting (B2), n = 26. (C) Mean (± standard error of the mean) latency to lip contact (C1) and for biting (C2) at ZT 18 (n = 17) and ZT 22 (n = 26). Each animal was tested only once per night and was tested at least four times on different nights for each time point. Data for each behavioral state per time point was averaged for individual animals. ** P < 0.01, *** P < 0.001, Kruskal-Wallis with Dunn post hoc comparisons.

result of the higher stimulus volume, because no reactions were observed in resting animals treated with 500–1000 µL volumes of low concentration salt solutions (data not shown). Because only a small percentage of resting animals took an escape step, we pooled the data between ZT 18 and ZT 22 to determine whether there was a difference in the latency to take the escape step between awake and resting animals. We found that the

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latency to escape was significantly greater in resting animals compared to awake animals (Figure 6C; Mann-Whitney U test, U = 346.0, P < 0.01). These experiments demonstrate that rest behavior increases the sensory arousal thresholds for appetitive and aversive stimuli in Aplysia and when combined with the preceding results strongly suggest that Aplysia rest behavior meets the criteria for sleep.

Figure 6—Resting Aplysia display decreased escape behavior in response to an aversive stimulus. Response toward an aversive stimulus of 5 µL sodium chloride (NaCl) solution (6.1 M) pipetted to the tail was measured at two time points during the dark period (ZT 18 and ZT 22). Animals were assigned to a behavioral state for testing on the basis of waking (wake) or resting (rest) and tested after more than 5 continuous min in that state. Each animal was tested at least four times on different nights for each time point. Data for each behavioral state was averaged for individual animals. (A) Latency for tail withdrawal. (B) Fraction of animals for which an escape response was elicited. (C) Latency to escape response of animals shown in (B) with data pooled between time points. N is indicated at the base of each bar. ** P < 0.01, *** P < 0.001, Kruskal-Wallis analysis with Dunn post hoc comparisons for multiple groups (B), Mann-Whitney U test for two groups (C).

Rest Deprivation Triggers Rest Rebound Increased rest after enforced waking is an established criterion to demonstrate that sleep is under homeostatic control.4–6,8,12,16,19,45 To assess whether sleeplike rest is homeostatically regulated in Aplysia, we deprived animals of rest for 11 h beginning at ZT 13 by placing them in individual containers equipped with different contexts in addition to manual stimulation dependent on the animals’ activity. Subsequent activity was monitored. Rest deprived animals displayed decreased locomotor activity the day following the rest deprivation and significant morning periods of rest (Figure 7A). The rest deprivation procedure induced rebound rest in all animals ranging from 62 min to 285 min with a mean of 156 ± 73 min during the first 9 h of the subsequent light period. All nonhandled control animals displayed no rest during the same period. At the end of the following night rest deprived animals exhibited a shorter period of anticipatory activity before dawn: Rest deprived animals initiated activity 44 ± 33 min, whereas non-handled control animals 90 ± 47 min before dawn (P = 0.0078 nonparametric Wilcoxon matched-pair test) consistent with a higher sleep need in the rest-deprived animals. In the second night following rest deprivation, resting behavior returned to normal levels with no significant differences in activity observed between experimental and control animals. The increased rest observed during the day subsequent to sleep deprivation could be caused by nonfatigue related stress induced by the manual handling of the animal. To determine whether the observed sleep rebound was caused by handling stress instead of a homeostatic drive for sleep, we used the same handling and context stimulation used for sleep deprivation to animals during the light period. Animals were handled from ZT 0 - ZT 10 during the day leaving 2 h before light offset to assess whether rest periods during the light period were elicited when animals are usually not engaged in resting activity. Although handling was performed during the day with time of day potentially causing variation in stress responses, in previous studies52 no diurnal or circadian differences were found in the necessary threshold for an electrical stimulus to evoke baseline tail and siphon withdrawal responses, suggesting that defensive behavioral responses to mild stress do not vary with time of day. Although daytime-handled animals showed no significant advance in rest compared to nonhandled control animals, we found that six of the daytime-handled animals anticipated rest before light offset. However, this short period of rebound rest during the late day was followed by increased activity during the second half of the night, with a significant decrease in rest spanning ZT 18 through ZT 22, a negative rebound. Overall, rest in daytime-handled animals was reduced in comparison with animals that were rest deprived during the night. On the day following handling, no significant differences were observed for either the light or dark period between handled and nonhandled

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Figure 7—Aplysia exhibit a homeostatic drive for rest with rebound rest apparent after rest deprivation. Amount of rest is plotted for consecutive 30-min periods. (A) Animals were deprived of rest for 11 h by manual handling during the first dark period (Rest deprived) and then allowed to rest. No stimulation was given to a control group (Control). (B) As a control experiment for handling stress, animals were comparably stimulated during the light period for 10 h and then allowed to rest (Stimulated) and compared with an unstimulated control group (Control). Data points represent mean (± standard error of the mean), n = 8. Each time point was analyzed by Student t-test * P < 0.05, ** P < 0.01.

animals. Thus, handling itself did not appear to increase fatigue or the pressure for rest (Figure 7B). Together these experiments suggest that Aplysia rest follows a homeostatic drive for rebound sleep similar to that observed in other animals. DISCUSSION Insufficient sleep, sleep deprivation, and sleep disorders have become increasingly prevalent in society, affecting between 50 to 70 million U.S. adults. Insufficient sleep adversely affects public health through the increased incidence of automobile accidents, industrial accidents, and occupational errors. Additionally, sleep disorders lead to additional individual health problems including metabolic disorders, hypertension, cardiovascular disorders, immune system complaints, decrements in cognitive functioning, and mood disorders.53–62 Thus, there is a critical need to fully understand the functions of sleep and the SLEEP, Vol. 37, No. 9, 2014 1459 Downloaded from https://academic.oup.com/sleep/article-abstract/37/9/1453/2416887 by guest on 14 January 2018

underlying neural and molecular mechanisms to develop future treatments and therapies for sleep disorders. The advent of ground-breaking research establishing sleep in Drosophila11,12 has fundamentally changed perceptions such that sleep is regarded as an evolutionarily conserved physiological state essential to an animal’s well-being and survival.2,4,36,63 Despite the advances in sleep research and molecular insights gained from the genetic tractability of Drosophila, the need still exists for fundamental research on sleep using models with relatively simple nervous systems to track down the function of sleep to individual neurons. The past decade has ushered in an era of sleep research in simple invertebrate models, including the nematode Caenorhabditis elegans18 and the freshwater pond snail Lymnaea stagnalis.17 To identify a model with a relatively simple nervous system in which to study sleep at a single neuronal level, we Sleep in Aplysia—Vorster et al.

investigated sleep in the marine mollusk Aplysia californica, a well-established neuroscience model that has been historically successful in the identification of conserved molecular mechanisms in learning and memory. Although early research by Strumwasser22,23 mentioned sleep in Aplysia while describing the robust diurnal activity patterns of Aplysia with restlike immobility at night, no studies to date have been performed specifically to determine whether Aplysia rest fulfills the requirements of sleep. In our experiments as in previous research,22,23,26,27 we found that Aplysia exhibit strong diurnal rhythms in locomotor activity. Aplysia were active almost exclusively during the day, with rest evident throughout the night. During the night, Aplysia displayed periods of immobility lasting up to 100 min, with longer bouts of rest more evident in the first half of the night. This last result points to a homeostatic regulation of sleep, similar to what has been observed in flies39,64 and mammals, including humans, in which the duration and/or intensity of sleep increase with time spent awake and decrease with time spent asleep.65,66 Aplysia is a monophasic sleeping animal similar to humans and rarely exhibits rest bouts during the day, raising the possibility that studies of sleep in Aplysia could provide mechanistic and functional insights regarding sleep applicable to human behavior. Stationary activity was equally distributed throughout day and night with the animals actively head waving but not shifting overall body position, perhaps for periodic assessment of the environment or in response to muscle fatigue. Aplysia also exhibited dawn anticipatory locomotor activity, a component of locomotor activity consistent with circadian regulation of behavior. Arguably the most important of the criteria to qualify invertebrate resting behavior as sleep is the observed increase in the necessary threshold and response times for sensory arousal during sleep as observed in fruit flies,11 honeybees,42,43 scorpions,45 cockroaches,6 and mosquitoes.67 We found that resting Aplysia display significantly greater latencies in head orientation and biting responses to appetitive stimuli compared to animals in the wake state. When comparing responses within individual animals as opposed to overall population behavior, we also found increased response times during rest compared to the wake state. Our results are consistent with earlier findings by Preston and Lee28 that identified state-dependent behavioral differences in Aplysia in response to food stimuli, although it should be noted that in these earlier studies the quiet state was not defined as either rest or sleep. Previous research in Aplysia identified a diurnal rhythm in the latency to adopt a feeding posture after stimulation with seaweed with longer latencies observed during the night.68 Because this previous research used animals showing either no movement or only stationary movements, the reported diurnal rhythm may be explained by sleeping behavior at night as the latency to adopt a feeding posture at night was similar to the response times we found for sleeping animals, but not awake animals at night. However, regulation of behavioral responses to a sensory stimulus can vary between appetitive and aversive stimuli. For Lymnaea, animals in a quiescent state display increased latencies in response to an appetitive stimulus, but in response to aversive stimuli the animals exhibit a longer, more sustained

response.17 To test whether Aplysia respond differently to an aversive stimulus than an appetitive stimulus in a resting sleeplike state, we used a saturated salt solution applied to the tail that invokes a two-step behavioral response.51 Application of a saturated salt solution to the tail of an awake animal resulted in an immediate contraction of the tail followed by an escape step in which the animal physically changed its body position, moving away from the aversive stimulus. The tail elicited–tail withdrawal reflex is a defensive reflex with a monosynaptic reflex circuitry encompassing the tail sensory-motor neuron synapse in the pedal ganglion with contributing interneurons in the pleural ganglion.69,70 As demonstrated using in vitro reduced preparations, the tail elicited–tail withdrawal reflex consists of a centrally mediated and a peripherally mediated component.70,71 We hypothesize that the animal’s immediate contraction of the tail in response to the aversive stimulus in both wake and sleep states represents local contractions mediated through the site of stimulation and primarily involves peripheral neuronal activity. The second response, the escape locomotion, is based on a polysynaptic circuit, requiring additional neuronal signaling and cerebral-pleural connectivity.72–74 Lesions of the cerebropleural (C-PL) connectives abolish escape locomotion, whereas tail withdrawal can still be elicited.72,74 We found that waking and resting animals responded with similar latency for the tail elicited–tail withdrawal reflex, but only 30% of the resting animals initiated an escape response. Interestingly, in the small percentage of resting animals that displayed an escape response the latency for the escape response was significantly longer than that observed in awake animals. As previous research concluded that the presence of an escape response was dependent upon stimulus intensity,72 we hypothesize that the absence of an escape response in most resting animals was caused by an increased arousal threshold. Alternatively, during rest, connectivity between the ganglia may be reduced or altered as has been recently suggested in humans for connectivity between brain areas.75,76 We conclude that the peripherally mediated defensive response remains consistent between wake and rest states, but that centrally mediated responses to an aversive stimulus are significantly different between the wake and rest states. In mammals, the neuronal mechanisms by which sensory stimuli fail to trigger behavioral responses and are not consciously perceived in nonrapid eye movement and rapid eye movement states are poorly understood. Thus, Aplysia may become a useful model to identify the basis for the disconnection during sleep. Given that Aplysia demonstrate significantly different latencies in response to an appetitive stimulus and differential responses to an aversive stimulus between the wake and rest states, we conclude that Aplysia rest is accompanied by an increased arousal threshold, a strong indication of a sleep state. The regulation of sleep occurs dually through circadian and homeostatic regulation.77,78 The homeostatic drive for sleep can be observed in the propensity or pressure for sleep that accumulates following a period of prolonged wakefulness.79 Experimentally, we tested the homeostatic drive for sleep and the propensity for rebound rest by depriving animals of rest using context changes and manual stimulation during the night. We found that Aplysia deprived of rest at night demonstrated increased rest the following morning, reflecting a homeostatic

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drive for sleep, whereas animals similarly handled during the day showed no sign of increased sleep pressure. In line with results from sleep deprivation studies in other animals and humans, the magnitude (duration) of the observed rebound rest did not total the amount of deprived rest. Converging evidence in many species indicates that sleep has two dimensions, duration and intensity, that can be regulated independently.80 Intensity varies across sleep stages and differences in sleep intensity also occur as a result of prior activity.81,82 Differences in sleep intensity are not confined to higher organisms. Intensity as a regulated dimension of sleep has been recently characterized in Drosophila,64 with earlier research suggesting differences in sleep intensity in cockroaches83 and honeybees.8 Potentially, differences in sleep intensity account for the duration of rebound rest observed in Aplysia. Interestingly, animals handled during the day showed a short rebound before light offset, but significantly decreased amounts of rest during the night, possibly because of handling-induced increases in arousal. The diurnal regulation and observed rebound sleep in rest-deprived Aplysia contrasts with the regulation of resting behavior in the freshwater pond snail. In Lymnaea, rest does not appear to be regulated by the circadian clock and researchers to date have not identified a rebound sleep mechanism in Lymnaea,17 indicating that different mechanisms regulate rest behavior in Lymnaea, as has recently been suggested using computer simulations.84 Thus, compared to other simple invertebrate models, Aplysia sleep more closely resembles sleeping behavior observed in higher organisms. The increased vulnerability of sleeping animals because of decreased responsiveness to external stimuli necessitates careful selection of resting place location. Across species, animals display preferences in location choice for resting or sleeping.1 The preference for a particular resting location may reflect a defensive mechanism or satisfy a physiological need such as temperature conservation. For example, the soft-bodied cuttlefish display a preference for resting location in the laboratory by resting on or burying themselves within gravel substrates perhaps reflecting a defensive camouflage behavior.15 Coral reef teleosts retreat within the corals during the night because of predators’ attacks, although the mortality rate is still higher at night.85,86 In our experiments, we found that Aplysia prefer to rest in the upper corners of their cages near the water surface. Resting in corners or cracks was also noted previously by Susswein et al.24 and might signal a defensive behavior, leaving less space for attacks from predators or rough sea movements. The semicontracted body posture of the animal during rest with less body surface exposed to predators may act as an additional defensive mechanism as has been suggested by studies of lobster and Aplysia interactions.87 Alternatively, the preferred resting location in the upper part of the tank near the water surface may be indicative of a physiological need for higher water oxygen levels, possibly because of less water flow across the gills caused by inactivity and the parapodia closed around the body. The lack of muscle movement at night might also lower the circulation of hemolymph throughout the animal, leading to lower oxygen availability. Our results are consistent with previous laboratory observations of another Aplysiidae family member, the ragged sea hare Bursatella leachii, in which animals classified behaviorally as “still” were most

commonly observed in the upper corner of the aquaria near the return bubble stream.46 Although considerable neuroanatomical differences occur between invertebrates and mammals, invertebrate models have been exceedingly useful for defining the functions and mechanisms of complex behaviors and correlating behaviors with changes in individual neurons and neuronal circuits. Comparing sleep across organisms with different activity patterns and different nervous system and brain organization has been proposed as an optimal method for furthering understanding of the neuronal changes and functions underlying sleep.63,88 The results reported in this study establish Aplysia, with its relatively simple nervous system and easily identifiable neurons, as a valid model for sleep research, making it an excellent model for phylogenetic comparisons. ACKNOWLEDGMENTS The authors thank Dr. Edgar T. Walters for helpful suggestions and Emily Dehning for the drawings in Figure 4. DISCLOSURE STATEMENT This was not an industry supported study. Financial support was provided by a National Institute of Mental Health grant R01MH81012 and a grant from the Center of Research and Creativity at the Florida State University to Dr. Lyons. The work was performed at Florida State University. The authors have indicated no financial conflicts of interest.

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REFERENCES

1. Campbell SS, Tobler I. Animal sleep: a review of sleep duration across phylogeny. Neurosci Biobehav Rev 1984;8:269–300. 2. Tobler I. Phylogeny of sleep regulation. In: Kryger MH, Roth T, Dement WC, eds. Principles and Practice of Sleep Medicine, 5th ed. St Louis, MO: Elsevier Saunders, 2011:112–25. 3. Lesku JA, Rattenborg NC, Amlaner CJ. The evolution of sleep: a phylogenetic approach. In: Lee-Chiong TL, ed. Sleep: A Comprehensive Handbook. Hoboken, NJ: John Wiley & Sons, Inc, 2005:49–61. 4. Hartse KM. The phylogeny of sleep. Handb Clin Neurol 2011;98:97–109. 5. Zhdanova IV, Wang SY, Leclair OU, Danilova NP. Melatonin promotes sleep-like state in zebrafish. Brain Res 2001;903:263–8. 6. Tobler I. Effect of forced locomotion on the rest-activity cycle of the cockroach. Behav Brain Res 1983;8:351–60. 7. Kaiser W, Steiner-Kaiser J. Neuronal correlates of sleep, wakefulness and arousal in a diurnal insect. Nature 1983;301:707–9. 8. Sauer S, Herrmann E, Kaiser W. Sleep deprivation in honey bees. J Sleep Res 2004;13:145–52. 9. Klein BA, Olzsowy KM, Klein A, Saunders KM, Seeley TD. Castedependent sleep of worker honey bees. J Exp Biol 2008;211:3028–40. 10. Eban-Rothschild AD, Bloch G. Differences in the sleep architecture of forager and young honeybees (Apis mellifera). J Exp Biol 2008;211:2408–16. 11. Hendricks JC, Finn SM, Panckeri KA, et al. Rest in Drosophila is a sleeplike state. Neuron 2000;25:129–38. 12. Shaw PJ, Cirelli C, Greenspan RJ, Tononi G. Correlates of sleep and waking in Drosophila melanogaster. Science 2000;287:1834–7. 13. Ramon F, Hernandez-Falcon J, Nguyen B, Bullock TH. Slow wave sleep in crayfish. Proc Natl Acad Sci U S A 2004;101:11857–61. 14. Mendoza-Angeles K, Hernandez-Falcon J, Ramon F. Slow waves during sleep in crayfish. Origin and spread. J Exp Biol 2010;213:2154–64. 15. Frank MG, Waldrop RH, Dumoulin M, Aton S, Boal JG. A preliminary analysis of sleep-like states in the cuttlefish Sepia officinalis. PLoS One 2012;7:e38125. 16. Brown ER, Piscopo S, De Stefano R, Giuditta A. Brain and behavioural evidence for rest-activity cycles in Octopus vulgaris. Behav Brain Res 2006;172:355–9.

Sleep in Aplysia—Vorster et al.

17. Stephenson R, Lewis V. Behavioural evidence for a sleep-like quiescent state in a pulmonate mollusc, Lymnaea stagnalis (Linnaeus). J Exp Biol 2011;214:747–56. 18. Raizen DM, Zimmerman JE, Maycock MH, et al. Lethargus is a Caenorhabditis elegans sleep-like state. Nature 2008;451:569–72. 19. Duntley SP, Uhles M, Feren S. Sleep in the cuttlefish Sepia pharaonis. Sleep 2002;25(Abstract Suppl):A159–60. 20. Lee RM, Palovcik RA. Behavioral states and feeding in the gastropod Pleurobranchaea. Behav Biol 1976;16:251–66. 21. Leonard JL, Lukowiak K. The behavior of Aplysia californica Cooper (Gastropoda; Opisthobranchia): I. Ethogram. Behaviour 1986;98:320–60. 22. Strumwasser F. The cellular basis of behavior in Aplysia. J Psychiatr Res 1971;8:237–57. 23. Strumwasser F. Seventeenth Bowditch lecture. Neural and humoral factors in the temporal organization of behavior. Physiologist 1973;16:9–42. 24. Susswein AJ, Gev S, Feldman E, Markovich S. Activity patterns and time budgeting of Aplysia fasciata under field and laboratory conditions. Behav Neural Biol 1983;39:203–20. 25. Ziv I, Lustig C, Ben-Zion M, Susswein AJ. Daily variation of multiple behaviors in Aplysia fasciata: integration of feeding, reproduction, and locomotion. Behav Neural Biol 1991;55:86–107. 26. Kupfermann I. A circadian locomotor rhythm in Aplysia Californica. Physiol Behav 1968;3:179-81. 27. Jacklet JW. Circadian locomotor activity in Aplysia. J Comp Physiol A 1972;79:325–41. 28. Preston RJ, Lee RM. Feeding behavior in Aplysia californica: role of chemical and tactile stimuli. J Comp Physiol Psychol 1973;82:368–81. 29. Kandel ER. The molecular biology of memory storage: A dialogue between genes and synapses. Science 2001;294:1030–8. 30. Lee YS, Bailey CH, Kandel ER, Kaang BK. Transcriptional regulation of long-term memory in the marine snail Aplysia. Mol Brain 2008;1:3. 31. Michel M, Gardner JS, Green CL, Organ CL, Lyons LC. Protein phosphatase-dependent circadian regulation of intermediate-term associative memory. J Neurosci 2013;33:4605–13. 32. Walters ET, Moroz LL. Molluscan memory of injury: evolutionary insights into chronic pain and neurological disorders. Brain Behav Evol 2009;74:206–18. 33. Piéron H. Le problème physiologique du sommeil. Paris: Masson et Cie, 1913. 34. Flanigan WF, Jr. Sleep and wakefulness in iguanid lizards, Ctenosaura pectinata and Iguana iguana. Brain Behav Evol 1973;8:401–36. 35. Bruce Durie D. Sleep in animals. In: Wheatley D, British Association for Psychopharmacology, eds. Psychopharmacology of Sleep. New York: Raven Press, 1981. 36. Cirelli C, Tononi G. Is sleep essential? PLoS Biol 2008;6:e216. 37. Zimmerman JE, Naidoo N, Raizen DM, Pack AI. Conservation of sleep: insights from non-mammalian model systems. Trends Neurosci 2008;31:371–6. 38. Kupfermann I. Feeding behavior in Aplysia: A simple system for the study of motivation. Behav Biol 1974;10:1–26. 39. Huber R, Hill SL, Holladay C, Biesiadecki M, Tononi G, Cirelli C. Sleep homeostasis in Drosophila melanogaster. Sleep 2004;27:628–39. 40. Hussaini SA, Bogusch L, Landgraf T, Menzel R. Sleep deprivation affects extinction but not acquisition memory in honeybees. Learn Mem 2009;16:698–705. 41. Yokogawa T, Marin W, Faraco J, et al. Characterization of sleep in zebrafish and insomnia in hypocretin receptor mutants. PLoS Biol 2007;5:e277. 42. Kaiser W. Busy Bees need rest, too–behavioral and electromyographical sleep signs in honeybees. J Comp Physiol A Sens Neural Behav Physiol 1988;163:565–84. 43. Kaiser W. Rest at night in some solitary bees–a comparison with the sleep-like state of honey-bees. Apidologie 1995;26:213–30. 44. Andersen FS. Sleep in moths and its dependence on the frequency of stimulation in Anagasta kuehniella. Opuscula Entomologica 1968;33:15–24. 45. Tobler I, Stalder J. Rest in the scorpion - a sleep-like state. J Comp Physiol A Sens Neural Behav Physiol 1988;163:227–35. 46. Ramos LJ, Lopez Rocafort JL, Miller MW. Behavior patterns of the aplysiid gastropod Bursatella leachii in its natural habitat and in the laboratory. Neurobiol Learn Mem 1995;63:246–59.

SLEEP, Vol. 37, No. 9, 2014 1462 Downloaded from https://academic.oup.com/sleep/article-abstract/37/9/1453/2416887 by guest on 14 January 2018

47. Granados-Fuentes D, Tseng A, Herzog ED. A circadian clock in the olfactory bulb controls olfactory responsivity. J Neurosci 2006;26:12219–25. 48. Krishnan B, Dryer SE, Hardin PE. Circadian rhythms in olfactory responses of Drosophila melanogaster. Nature 1999;400:375–8. 49. Mazzoni EO, Desplan C, Blau J. Circadian pacemaker neurons transmit and modulate visual information to control a rapid behavioral response. Neuron 2005;45:293–300. 50. Page TL, Koelling E. Circadian rhythm in olfactory response in the antennae controlled by the optic lobe in the cockroach. J Insect Physiol 2003;49:697–707. 51. Walters ET, Erickson MT. Directional control and the functional organization of defensive responses in Aplysia. J Comp Physiol A 1986;159:339–51. 52. Fernandez RI, Lyons LC, Levenson J, Khabour O, Eskin A. Circadian modulation of long-term sensitization in Aplysia. Proc Natl Acad Sci U S A 2003;100:14415–20. 53. Breslau N, Roth T, Rosenthal L, Andreski P. Sleep disturbance and psychiatric disorders: A longitudinal epidemiological study of young adults. Biol. Psychiatry 1996;39:411–8. 54. Burgos I, Richter L, Klein T, et al. Increased nocturnal interleukin-6 excretion in patients with primary insomnia: a pilot study. Brain Behav Immun 2006;20:246–53. 55. Grandner MA, Jackson NJ, Pak VM, Gehrman PR. Sleep disturbance is associated with cardiovascular and metabolic disorders. J Sleep Res 2012;21:427–33. 56. Goel N, Rao H, Durmer JS, Dinges DF. Neurocognitive consequences of sleep deprivation. Semin Neurol 2009;29:320–39. 57. Leproult R, Van Cauter E. Role of sleep and sleep loss in hormonal release and metabolism. Endocr Dev 2010;17:11–21. 58. Benca RM, Peterson MJ. Insomnia and depression. Sleep Med 2008;1:S3–9. 59. Hsieh SD, Muto T, Murase T, Tsuji H, Arase Y. Association of short sleep duration with obesity, diabetes, fatty liver and behavioral factors in Japanese men. Intern Med 2011;50:2499–502. 60. Neckelmann D, Mykletun A, Dahl AA. Chronic insomnia as a risk factor for developing anxiety and depression. Sleep 2007;30:873–80. 61. Suka M, Yoshida K, Sugimori H. Persistent insomnia is a predictor of hypertension in Japanese male workers. J Occup Health 2003;45:344–50. 62. Vgontzas AN, Liao D, Bixler EO, Chrousos GP, Vela-Bueno A. Insomnia with objective short sleep duration is associated with a high risk for hypertension. Sleep 2009;32:491–7. 63. Piscopo S. Sleep and its possible role in learning: a phylogenetic view. Front Biosci (Schol Ed) 2009;1:437–47. 64. van Alphen B, Yap MH, Kirszenblat L, Kottler B, van Swinderen B. A dynamic deep sleep stage in Drosophila. J Neurosci 2013;33:6917–27. 65. Borbely AA, Tobler I. Manifestations and functional implications of sleep homeostasis. Handb Clin Neurol 2011;98:205–13. 66. Tobler I. Is sleep fundamentally different between mammalian species? Behav Brain Res 1995;69:35–41. 67. Haufe WO. Ethological and statistical aspects of a quantal response in mosquitoes to environmental stimuli. Behaviour 1963;20:221–41. 68. Levenson J, Byrne JH, Eskin A. Levels of serotonin in the hemolymph of Aplysia are modulated by light/dark cycles and sensitization training. J Neurosci 1999;19:8094–103. 69. Cleary LJ, Byrne JH. Identification and characterization of a multifunction neuron contributing to defensive arousal in Aplysia. J Neurophysiol 1993;70:1767–76. 70. Walters ET, Byrne JH, Carew TJ, Kandel ER. Mechanoafferent neurons innervating tail of Aplysia. I. Response properties and synaptic connections. J Neurophysiol 1983;50:1522–42. 71. Philips GT, Sherff CM, Menges SA, Carew TJ. The tail-elicited tail withdrawal reflex of Aplysia is mediated centrally at tail sensory-motor synapses and exhibits sensitization across multiple temporal domains. Learn Mem 2011;18:272–82. 72. Fredman SM. Recovery of escape locomotion following a CNS lesion in Aplysia. Behav Neural Biol 1988;49:261–79. 73. Hening WA, Walters ET, Carew TJ, Kandel ER. Motorneuronal control of locomotion in Aplysia. Brain Res 1979;179:231–53. 74. Jahan-Parwar B, Fredman SM. Neural control of locomotion in Aplysia: role of the central ganglia. Behav Neural Biol 1979;27:39–58.

Sleep in Aplysia—Vorster et al.

75. Esser SK, Hill S, Tononi G. Breakdown of effective connectivity during slow wave sleep: investigating the mechanism underlying a cortical gate using large-scale modeling. J Neurophysiol 2009;102:2096–111. 76. Massimini M, Ferrarelli F, Huber R, Esser SK, Singh H, Tononi G. Breakdown of cortical effective connectivity during sleep. Science 2005;309:2228–32. 77. Borbely AA. A two process model of sleep regulation. Hum Neurobiol 1982;1:195–204. 78. Borbely AA, Achermann P. Sleep homeostasis and models of sleep regulation. J Biol Rhythms 1999;14:557–68. 79. Porkka-Heiskanen T. Sleep homeostasis. Curr Opin Neurobiol 2013;23:799–805. 80. Achermann P, Borbely AA. Mathematical models of sleep regulation. Front Biosci 2003;8:s683–93. 81. Huber R, Tononi G, Cirelli C. Exploratory behavior, cortical BDNF expression, and sleep homeostasis. Sleep 2007;30:129–39.

SLEEP, Vol. 37, No. 9, 2014 1463 Downloaded from https://academic.oup.com/sleep/article-abstract/37/9/1453/2416887 by guest on 14 January 2018

82. Hanlon EC, Faraguna U, Vyazovskiy VV, Tononi G, Cirelli C. Effects of skilled training on sleep slow wave activity and cortical gene expression in the rat. Sleep 2009;32:719–29. 83. Tobler II, Neuner-Jehle M. 24-h variation of vigilance in the cockroach Blaberus giganteus. J Sleep Res 1992;1:231–9. 84. Stephenson R. Sleep homeostasis: progress at a snail’s pace. Commun Integr Biol 2011;4:446–9. 85. Goldshmid R, Holzman R, Weihs D, Genin A. Aeration of corals by sleepswimming fish. Limnol Oceanogr 2004;49:1832–9. 86. Holbrook SJ, Schmitt RJ. Competition for shelter space causes densitydependent predation mortality in damselfishes. Ecology 2002;83:2855–68. 87. Watkins AJ, Goldstein DA, Lee LC, et al. Lobster attack induces sensitization in the sea hare, Aplysia californica. J Neurosci 2010;30:11028–31. 88. Hendricks JC, Sehgal A, Pack AI. The need for a simple animal model to understand sleep. Prog Neurobiol 2000;61:339–51.

Sleep in Aplysia—Vorster et al.