Circadian Modulation of Complex Learning in Diurnal and Nocturnal Aplysia

Circadian modulation of complex learning in diurnal and nocturnal Aplysia

Lisa C. Lyons*, Oliver Rawashdeh*, Ayelet Katzoff†, Abraham J. Susswein†, and Arnold Eskin*‡

*Department of Biology and Biochemistry, University of Houston, Houston, TX 77204-5001; and †Department of Life Sciences, Bar Ilan University, Ramat Gan 52900, Israel

Edited by Joseph S. Takahashi, Northwestern University, Evanston, IL, and approved July 26, 2005 (received for review May 9, 2005) Understanding modulation of memory, as well as the mechanisms memory formation. Researchers using fear conditioning found underlying memory formation, has become a key issue in neuro- that mice exhibit a diurnal rhythm in contextual fear condition- science research. Previously, we found that the formation of ing, with animals demonstrating greater memory during the long-term, but not short-term, memory for a nonassociative form night. However, no rhythm existed for cued fear conditioning of learning, sensitization, was modulated by the circadian clock in (6). In other research, also using fear conditioning in mice, a the diurnal Aplysia californica. To define the scope of circadian circadian rhythm of recall was observed for context and cued fear modulation of memory, we examined an associative operant conditioning, with peaks in recall seen in the early day regardless learning paradigm, learning that food is inedible (LFI). Significantly of the time of training (7). In rats, no time-of-day modulation has greater long-term memory of LFI occurred when A. californica were been observed with several different learning paradigms (8), trained and tested during the subjective day, compared with although recent research suggests that strain differences may animals trained and tested in the subjective night. In contrast, affect circadian modulation (9). Studies in humans also have animals displayed similar levels of short-term memory for LFI when yielded mixed results. Thus, using complex model systems, it has trained in either the subjective day or night. Circadian modulation been difficult to consistently demonstrate a modulatory role of of long-term memory for LFI was dependent on the time of the circadian clock on the formation of long-term memory. training, rather than the time of testing. To broaden our investi- Aplysia provides a superb opportunity to investigate circadian gation of circadian modulation of memory, we extended our modulation of long-term memory, because considerable infor- studies to a nocturnal species, Aplysia fasciata. Contrary to the mation on long-term memory formation is available at the significant memory observed during the day with the diurnal molecular, cellular, and behavioral levels (10). As a first step, we A. californica, A. fasciata showed no long-term memory for LFI demonstrated that the circadian clock modulates long-term when trained during the day. However, A. fasciata demonstrated sensitization (LTS), a nonassociative form of learning (11). In significant long-term memory when trained and tested during the circadian modulation of LTS, animals demonstrate robust long- night. Thus, the circadian clock modulates memory formation in term memory when trained during the day but little long-term phase with the animals’ activity period. The results from our memory when trained during the night. Circadian modulation of studies of circadian modulation of long-term sensitization and LFI LTS appears to occur during the induction and formation of suggest that circadian modulation of memory formation may be a long-term memory, rather than during recall. Interestingly, general phenomenon with potentially widespread implications for short-term memory for sensitization is not modulated by the many types of long-term learning. circadian clock (11). How broadly does the circadian clock affect long-term mem- biological rhythms ͉ circadian clock ͉ long-term memory ory? Are more complex forms of learning modulated? Does the neural or molecular circuitry involved in a particular type of learning limit circadian modulation of memory? To address he ability to remember an experience allows an organism to these questions, we investigated circadian modulation of learn- Tuse information gained in planning its response to future ing that food is inedible (LFI), an associative form of operant events. A variety of factors can modulate the formation and conditioning. In LFI, netted seaweed is presented to the animal. recall of memory. In fact, very few of the events occurring in the The animal responds by head-waving, biting, food entry into the environment are remembered. Health, age, motivation, previous mouth, attempts to swallow, and rejection of the food. Food experience, stress, and many other factors may modulate learn- entry into the mouth and continued failed swallowing represent ing and memory. Modulation of memory formation can affect negative reinforcement and are necessary for memory forma- how input information is processed, how memories are stored, tion. Learning for this paradigm is specific to the food presented the length of time that memories last, or even the mechanisms

during training (12). NEUROSCIENCE by which memories are recalled. Understanding the modulation We found that the circadian clock modulates long-term mem- of memory will provide insight into the cellular and molecular ory of LFI in diurnal Aplysia californica, with significantly greater processes underlying memory formation per se. The circadian long-term memory formed during the subjective day. In contrast, clock allows animals to predict when important events occur in the circadian clock did not modulate short-term memory of LFI. the environment and to anticipate changes in the environment Moreover, we found that circadian modulation of long-term LFI based on time of day. Given the enormous and widespread depended on the time of training and not the time of testing. We impact that the circadian clock has on an animal’s physiology and studied circadian modulation of long-term memory across spe- behavior, we investigated modulation of memory formation by cies by investigating a nocturnal species Aplysia fasciata (13). In the circadian clock. A. fasciata, we also found that significantly greater long-term The time of day can impact learning by becoming part of the memory occurred when animals were trained during their active context in which the learning occurs (time-stamping), or the time of day can affect the amount of memory that is formed or recalled. Time-stamping has been demonstrated in invertebrates This paper was submitted directly (Track II) to the PNAS ofﬁce. (1) and in mammals (2–5). However, in time-stamping, the Abbreviations: CT, circadian time; DD, constant darkness; LD, light–dark; LFI, learning that circadian clock is not actually modulating learning itself. food is inedible; LTS, long-term sensitization; ZT, zeitgeber time. Varying results have been obtained by using mammalian ‡To whom correspondence should be addressed. E-mail: [email protected]. models to assess the modulatory role of the circadian clock in © 2005 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0503847102 PNAS ͉ August 30, 2005 ͉ vol. 102 ͉ no. 35 ͉ 12589–12594 Downloaded by guest on October 1, 2021 phase, such that the nocturnal species exhibited robust long-term memory only when trained during the night. Thus, our research indicates that the circadian clock is a strong modulator of long-term memory for both associative and nonassociative learning paradigms and that this type of modulation extends across species such that memory formation is attenuated during an animal’s inactive phase. Materials and Methods Animal Maintenance. A. californica (100–175 g) were housed in boxes in artificial seawater (15°C) in 12:12-h light–dark (LD) cycles. Animals were fed romaine lettuce (Lactuca sativa) every second day until they were fed to satiation. For circadian experiments, animals were entrained and then transferred to constant darkness (DD). Manipulations in the dark used dim red Fig. 1. Diurnal modulation of long-term LFI in A. californica. A. californica light. As a control for non-LD-cycle cues controlling the phase were entrained to 12:12-h LD cycles, trained with netted food at ZT 3, 9, 15, or of the rhythm, some animals were entrained to a reversed-phase 21 (n ϭ 10, 9, 9, and 10, respectively), and tested 24 h after training. Memory LD cycle. Experiments were performed at 15°C. was expressed as a decrease in the total response time and a decrease in the A. fasciata (50–150 g) were collected along the Mediterranean amount of time that the netted food remained in the mouth. White bars represent data obtained in the light, whereas gray bars represent data ob- coast of Israel during the early summer and housed five to six to tained in the dark. Error bars are SEM. (A) Animals demonstrated signiﬁcantly a cage in Mediterranean seawater at 18°C (12:12-h LD). The greater decreases in total response time when trained and tested during the animals were fed one to two times weekly with Ulva lactuca (sea day, compared with animals trained and tested during the night [F(3, 34) ϭ lettuce, a green alga) and used 1 week to 1 month after 14.98, P Ͻ 0.001; Tukey’s posthoc analyses, P Ͻ 0.01 for ZT 3 vs. 15, ZT 3 vs. 21, collection. ZT 9 vs. 15, and ZT 9 vs. 21; raw data (min), ZT 3 train ϭ 17.2 Ϯ 1.4, ZT 3 test ϭ 8.0 Ϯ 0.9; ZT 9 train ϭ 17.9 Ϯ 2.0, ZT 9 test ϭ 10.7 Ϯ 0.9; ZT 15 train ϭ 22.2 Ϯ Behavioral Training and Testing. For A. californica, animals were 4.5, ZT 15 test ϭ 24.5 Ϯ 3.5; ZT 21 train ϭ 20.3 Ϯ 1.8, and ZT 21 test ϭ 20.7 Ϯ fed to satiation with laver seaweed and then starved for 5–7 days 1.7]. (B) Animals displayed signiﬁcantly greater decreases in the amount of before training. LFI training protocols were established by A.J.S. time that the netted food remained in the mouth when animals were trained and tested during the day than when trained and tested at night [F(3, 33) ϭ (12, 14–16). Animals were presented with a small piece of laver 9.277; P Ͻ 0.001; Tukey’s posthoc analyses, P Ͻ 0.05 for ZT 3 vs. 15, ZT 3 vs. 21, seaweed wrapped in netting that they could not swallow. Animals ZT 9 vs. 15, and ZT 9 vs. 21; raw data (min), ZT 3 train ϭ 7.7 Ϯ 1.1, ZT 3 test ϭ received a one-trial training paradigm that continued until the 2.8 Ϯ 0.3; ZT 9 train ϭ 7.1 Ϯ 0.7, ZT 9 test ϭ 3.9 Ϯ 0.3; ZT 15 train ϭ 10.6 Ϯ 2.2, animal stopped responding to the netted seaweed for 3 min. Two ZT 15 test ϭ 12.8 Ϯ 1.3; ZT 21 train ϭ 8.1 Ϯ 1.1, and ZT 21 test ϭ 9.7 Ϯ 1.3]. parameters were recorded: (i) the total response time and (ii) the cumulative time that the animal retained the netted seaweed in its mouth. Memory was represented as a decrease in these times. time (ZT) 3 or ZT 9 (during the day with lights on; ZT 0 is lights Testing occurred using the same procedure either 30 min later on) or at ZT 15 or ZT 21 (during the night using dim red light; for short-term memory or 24 h later for long-term memory. ZT 12 is lights off). Animals were tested 24 h after training. A. fasciata were transferred to individual cages and food- Memory was expressed as a decrease in the amount of time deprived 7 days before the start of experiments. At 24 h before required to stop responding and a decrease in the amount of time an experiment, they were transferred to 10-liter aquaria main- that the netted food remained in the mouth. Animals trained tained at 19–20°C. Because in A. fasciata the presence of a during the day and tested 24 h later showed significant reductions conspecific is needed for animals to learn that a food is inedible in both total response time and the amount of time that the food (17), a second Aplysia was added to the aquarium. The second was retained in the mouth (Fig. 1). However, when animals were animal was maintained behind a partition that allowed the flow trained and tested during the night (at ZT 15 or ZT 21), no of seawater but prevented contact between animals. Animals decreases were seen for either parameter, indicating the absence were trained and tested as previously described (12, 14–16, 18, of long-term memory for LFI. Thus, similar to our previous 19). For A. fasciata, the time the food was retained in the mouth results examining modulation of LTS (11), diurnal modulation of was measured only for the first 5 min. LFI occurred with greater long-term memory formed during the In experiments with A. californica, the person who tested the animals’ active phase. animals was not the person who trained that batch of animals. For both A. californica and A. fasciata, naı¨ve animals were Circadian Modulation of Long-Term LFI. To test whether the mod- included during long-term testing, and the individual testing the ulation of LFI was due to the circadian clock or direct effects of animals did not know which animals were previously trained. light in the LD cycle, animals were trained at various times on Statistical analysis of the data were by ANOVA with Tukey’s day 2 of DD and tested 24 h after training. Animals that were posthoc analyses (P Ͻ 0.05 was considered significant). Exper- trained and tested during the subjective (projected) day showed iments with A. fasciata were done at Bar Ilan University; significantly greater reductions in both total response time and experiments with A. californica were done at the University of the amount of time that food was retained in the mouth than did Houston. animals that were trained and tested during the subjective night (Fig. 2). These results demonstrate that the circadian clock Results modulates an associative-learning paradigm, with robust long- Diurnal Modulation of Long-Term LFI in A. californica. To investigate term memory formed in the subjective day and little or no circadian modulation of more complex forms of learning, we memory formed during the subjective night. examined an associative form of learning, LFI, in A. californica. Using this paradigm, the animals learn an association between No Diurnal or Circadian Modulation of Behavior During Training. It is netted seaweed and failed attempts at swallowing. Failure to possible that the circadian modulation of long-term LFI was consume the food acts as a negative reinforcer. To examine caused by modulation of feeding behavior per se during training diurnal modulation of long-term LFI, animals entrained to LD or testing. To test this possibility, we examined several param- cycles were trained by using the LFI paradigm at either zeitgeber eters of feeding during training. In LD conditions, there were no

12590 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0503847102 Lyons et al. Downloaded by guest on October 1, 2021 Fig. 2. The circadian clock modulated long-term LFI. Animals were entrained to 12:12-h LD cycles, switched to DD, and then subjected to LFI training at CT 3, 9, 15, or 21 (n ϭ 6 at each CT 3, 9, and 15, n ϭ 5 at CT 21) on day 2 of DD. Animals were tested 24 h later. Gray bars represent subjective day (projected time in DD), whereas black bars represent subjective night. (A) Changes in total response time varied significantly depending on when LFI training and testing occurred during the circadian cycle [F(3,19) ϭ 15.37; P Ͻ 0.001; Tukey’s posthoc analyses, P Ͻ 0.01 for CT 3 vs. 15, CT 3 vs. 21, CT 9 vs. 15, and CT 9 vs. 21; raw data (min), CT 3 train ϭ 27.2 Ϯ 3.4, CT 3 test ϭ 13.8 Ϯ 2.1; CT 9 train ϭ 31.4 Ϯ 3.6, CT 9 test ϭ 14.6 Ϯ 2.3; CT 15 train ϭ 26.7 Ϯ 2.0, CT 15 test ϭ 25.9 Ϯ 2.1; CT 21 train ϭ 31.2 Ϯ 2.6, and CT 21 test ϭ 27.6 Ϯ 1.7]. Animals demonstrated significantly greater memory when trained and tested during the subjective day, compared with animals trained and tested during the subjective night. (B) Animals displayed significantly greater decreases in the time the food was retained in the mouth when animals were trained and tested during the subjective day than during the subjective night [F ϭ 6.18; P Ͻ 0.005; (3, 19) Fig. 3. No diurnal or circadian modulation of training responses. (A1)No Ͻ Tukey’s posthoc analyses, P 0.05 for CT 9 vs. 15 and CT 9 vs. 21; raw data (min), diurnal rhythm in total response time occurred when animals were trained CT 3 train ϭ 10.9 Ϯ 1.9, CT 3 test ϭ 4.6 Ϯ 0.3; CT 9 train ϭ 12.7 Ϯ 1.7, CT 9 test ϭ at different times of day [F(3, 34) ϭ 0.769; P ϭ 0.52; n ϭ 10, 9, 9, and 10, 5.1 Ϯ 0.9; CT 15 train ϭ 13.6 Ϯ 1.9, CT 15 test ϭ 9.4 Ϯ 1.6; CT 21 train ϭ 12.3 Ϯ respectively]. (A2) No diurnal rhythm in the amount of time that the food was ϭ Ϯ 1.8, CT 21 test 9.1 1.1]. retained in the mouth occurred when animals were trained at different times of day [F(3, 34) ϭ 1.26; P ϭ 0.30]. (B1) No circadian rhythm in total response time occurred when animals were trained on the day 2 of DD [F(3, 20) ϭ 0.3487; P ϭ significant differences between animals trained in the light or 0.79; n ϭ 6 at each point]. (B2) No circadian rhythm in time the food was dark either in the total response time or in the amount of time retained in the mouth occurred when animals were trained on day 2 of DD ϭ ϭ that food was retained in the mouth (Fig. 3 A1 and A2). [F(3, 20) 0.51; P 0.68]. Furthermore, under constant conditions, analysis of baseline behavioral responses during training revealed no circadian differences (Fig. 3 B1 and B2). Although diurnal and circadian ANOVA, P ϭ 0.94). Thus, animals can form short-term memory rhythms in feeding behaviors have been found in Aplysia (20, 21), at night, whereas long-term memory for LFI cannot be formed no apparent diurnal or circadian rhythm exists in the training at night. These results are similar to our previous results for responses for LFI. sensitization (11). Although no time-of-day differences were observed in training responses on day 2 of DD, it is possible that the observed rhythms in long-term LFI were due to changes in the animals’ responses during testing on day 3 of DD due to a longer time in DD or a longer time isolated from food. To investigate these possibilities, we trained naı¨ve animals on day 3 of DD. No time-of-day differences were observed in the responses on day 3 of DD between animals trained during the subjective day and animals trained at night [circadian time (CT) 3 and CT 9; 30.6 Ϯ 3.1 min total response time, compared with CT 15 and CT 21; 29.4 Ϯ 1.5 min total response time, t ϭ 0.127, P ϭ 0.72]. Thus, circadian modulation of long-term NEUROSCIENCE LFI appears to be due to the circadian modulation of long-term memory rather than any circadian differences in baseline feeding behaviors during training or testing. Fig. 4. The circadian clock did not modulate short-term memory. Animals No Circadian Modulation of Short-Term Memory. Mechanistic dif- were entrained to 12:12-h LD cycles, switched to DD, and trained at CT 3, 9, 15, ferences exist in the formation of short-term and long-term or 21 (n ϭ 4 at CT 3 and CT 9, n ϭ 6 at CT 15 and CT 21) on day 2 of DD. Animals memory. Consequently, we tested whether the circadian clock were tested 30 min after training. Animals demonstrated robust short-term modulated short-term as well as long-term memory formation. learning during both the subjective day and subjective night. No significant A full-length single training paradigm (training continues until differences were seen with respect to time of day for either the percent the animal stops responding for 3 min) is sufficient to induce change in total response time [ANOVA, P ϭ 0.70; raw data (min): CT 3 train ϭ 22.1 Ϯ 4.6, CT 3 test ϭ 7.6 Ϯ 0.3; CT 9 train ϭ 24.0 Ϯ 3.7, CT 9 test ϭ 5.4 Ϯ 0.9; both short-term and long-term memory formation of LFI (14). ϭ Ϯ ϭ Ϯ ϭ Ϯ We trained groups of animals at different times on day 2 of DD CT 15 train 28.8 5.9, CT 15 test 6.8 0.8; CT 21 train 26.5 2.5, CT 21 test ϭ 7.1 Ϯ 1.4] (A) or the percent change in the amount of time that food was and tested the animals 30 min after the end of training. No retained in the mouth [ANOVA, P ϭ 0.94; raw data (min): CT 3 train ϭ 11.9 Ϯ circadian modulation of short-term LFI was seen for either the 3.1, CT 3 test ϭ 1.8 Ϯ 0.4; CT 9 train ϭ 12.6 Ϯ 3.0, CT 9 test ϭ 2.0 Ϯ 0.4; CT 15 total response time (Fig. 4A; ANOVA, P ϭ 0.70) or the amount train ϭ 13.3 Ϯ 3.5, CT 15 test ϭ 1.7 Ϯ 0.4; CT 21 train ϭ 10.7 Ϯ 2.4, CT 21 test ϭ of time that the food was retained in the mouth (Fig. 4B; 1.6 Ϯ 0.5] (B).

Lyons et al. PNAS ͉ August 30, 2005 ͉ vol. 102 ͉ no. 35 ͉ 12591 Downloaded by guest on October 1, 2021 Fig. 5. Time of training, rather than time of testing (recall), determined the circadian modulation of long-term LFI. (A) To determine the contribution of the time of training to the modulation in long-term LFI, animals were trained at CT 9 on day 2 of DD and then tested either 24 h later at CT 9 (day 3 of DD), 36 h later at CT 21, or 48 h later at CT 9 (day 4 of DD). Column shading indicates circadian time of testing as shown. Animals displayed robust learning when tested at either CT 9 or CT 21. No significant differences existed between animals tested at 24 h, 36 h, or 48 h after training. Results were similar for the time that food was retained in the mouth (data not shown). Thus, time of training determined whether long-term memory was formed. (B) To deter- Fig. 6. A nocturnal and a diurnal species of Aplysia exhibited opposite phases mine whether the time of testing contributed to the modulation in LFI, of diurnal modulation of long-term LFI. (A1) Nocturnal A. fasciata demon- animals were trained at CT 21 and tested 24 h later at CT 21, 36 h later at CT strated significantly greater decreases in total response time when trained 9, or 48 h later at CT 21. As shown in Fig. 3, a small amount of memory was and tested during the night, compared with animals trained and tested during observed when animals were tested 24 h after training at CT 21, but animals the day [t ϭ 31.66; P Ͻ 0.001; ZT 4, n ϭ 8; ZT 16, n ϭ 5; raw data (min), ZT 4 failed to exhibit any learning when tested 36 h after training at CT 9 (a time train ϭ 14.4 Ϯ 2.3, ZT 4 test ϭ 14.3 Ϯ 1.4, ZT 16 train ϭ 14.9 Ϯ 1.0, ZT 16 test ϭ when LFI normally occurs) or when tested 48 h later. Results were similar when 4.1 Ϯ 0.7]. Thus, the phase of the circadian modulation of LFI appears reversed the amount of time that food was retained in the mouth was analyzed (data for A. fasciata, compared with A. californica (compare Figs. 1 and 6). (A2) The not shown). time that food was retained in the mouth during the first 5 min of the training and testing sessions was modulated [t ϭ 9.25; P Ͻ 0.025; raw data (s), ZT 4 train ϭ 110.0 Ϯ 20.2, ZT 4 test ϭ 113.8 Ϯ 19.7, ZT 16 train ϭ 113.5 Ϯ 15.5, ZT Circadian Modulation of Long-Term Learning Depended on the Time 16 test ϭ 47.2 Ϯ 11.6]. (B1) As with A. californica, no diurnal rhythm in total of Training, Not the Time of Testing. The observed circadian response time was seen during training (t ϭ 0.26; P ϭ 0.876; n ϭ 8, 5). (B2) modulation of long-term LFI could be due to the modulation of Similarly, no diurnal modulation was apparent for the amount of time that the either memory consolidation or memory recall or both. To animals retained food in the mouth during training (t ϭ 0.15; P ϭ 0.905). determine whether time of training determined the circadian modulation of long-term LFI, animals were trained at CT 9 on Diurnal and Nocturnal Species of Aplysia Exhibit Opposite Phases of day 2 of DD (a time when significant learning is observed) and Modulation of Long-Term LFI. To date, our experiments examining then tested either 24 h later at CT 9, 36 h later at CT 21 (a time circadian modulation of both LFI and LTS have shown that when learning was not previously observed) or 48 h after training diurnal A. californica display significantly greater long-term at CT 9 on the day 4 of DD (Fig. 5A). Animals trained at CT 9 memory when they are trained during the day or the subjective and tested 36 h later at CT 21 showed robust learning with day. These experiments raise the question of whether circadian significant decreases observed in both total response times and modulation of long-term memory occurs with respect to partic- the amount of time that food was retained in the mouth (data not ular times within the LD cycle (e.g., light phase or projected light shown). This memory at CT 21 when animals were trained at CT phase) or whether circadian modulation occurs such that the 9 differed significantly from the responses observed when ani- phase of learning is coordinated with the animal’s inherent mals were trained and tested at CT 21 (Figs. 2 and 5A). Thus, activity pattern. Analyzing circadian modulation of long-term recall of long-term memory can occur during the subjective night memory in a nocturnal species, A. fasciata, provided the answer when animals are trained during the subjective day. These results to this question. Nocturnal A. fasciata demonstrated significantly demonstrate that the circadian clock modulates the formation of greater long-term LFI when trained and tested during the night long-term memory, rather than the recall of memory. than when trained and tested during the day (Fig. 6 A1 and A2). A complementary set of experiments was done to determine The phase of the modulation of long-term LFI for nocturnally whether circadian modulation of recall also existed. In these active A. fasciata appeared to be reversed from that seen for the experiments, animals were trained at CT 21 on the day 2 of DD diurnally active A. californica (compare Figs. 1 and 6). Thus, and then tested 24 h later at CT 21, 36 h later at CT 9, or 48 h rhythmic modulation of long-term memory formation occurs later at CT 21. When animals were trained at CT 21 and tested such that formation of long-term memory is significantly greater 36 h later at CT 9, no significant decreases were seen in either when animals are trained during their active periods irrespective total response time (Fig. 5B) or the amount of time that the food of the phase of the LD cycle or the time of occurrence of specific was retained in the mouth (data not shown). Thus, even though environmental events. animals were tested at a time (CT 9) when recall can occur (Fig. The initial training of A. fasciata also was examined to 2), no long-term memory was evident when animals were trained determine whether rhythms in baseline behavior might underlie at CT 21. These experiments demonstrate that circadian mod- the observed diurnal modulation of long-term LFI. As with ulation occurs at the time of training, presumably during the A. californica, there were no time-of-day differences in total induction and formation of long-term memory, rather than response time or the amount of time that the food was retained modulation of recall at the time of testing. in the mouth (Fig. 6 B1 and B2). Additionally, naı¨ve animals

12592 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0503847102 Lyons et al. Downloaded by guest on October 1, 2021 trained 1 day later also displayed no time-of-day differences in The question of how the circadian clock modulates the for- either training parameter (n ϭ 5 each time point; total response mation of long-term memory can be addressed at multiple levels, time, ZT 4 ϭ 16.9 Ϯ 1.1 min, ZT 16 ϭ 16.4 Ϯ 1.9 min, t ϭ 0.032, from the molecular level to the location of the circadian clock P ϭ 0.86; time in mouth, ZT 4 ϭ 96.8 Ϯ 20.7 sec, ZT 16 ϭ 107.8 Ϯ responsible for the circadian modulation of memory. In Aplysia, 10.9 sec, t ϭ 0.22, P ϭ 0.65). These results indicate that the researchers have documented only one circadian oscillator, formation of long-term memory in A. fasciata, rather than the located in the eye (22, 23). The eye contains all of the necessary feeding behavior, was modulated by time of day. components of a complete circadian system and anatomically projects through the optic nerve to the cerebral, pleural, and Discussion pedal ganglia (23, 24). Consequently, the eye may well be Our research is aimed at understanding modulation of the responsible for the circadian modulation of memory through formation of long-term memory. We have importantly expanded optic nerve projections. Circadian modulation of long-term our previous findings demonstrating circadian modulation of a memory also could occur through humoral factors as the eye has nonassociative form of long-term learning in Aplysia, LTS, by been shown to be neurosecretory (25). Alternatively, because showing that the circadian clock also modulates a more complex invertebrates, such as Drosophila (26), have independent periph- associative form of learning, LFI (Figs. 1 and 2). Moreover, as eral oscillators located throughout the organism, previously with LTS, modulation of long-term LFI was not subtle, but undetected peripheral oscillators may be responsible for circa- rather circadian modulation resulted in a major suppression of dian modulation of LFI. the formation of memory during the animals’ inactive phase. In How does the circadian clock modulate the very different contrast, the circadian clock did not modulate short-term mem- neural circuitry responsible for LTS and LFI? Sensory and motor ory for either sensitization (11) or LFI (Fig. 4). neurons involved in feeding behaviors are located primarily The circadian modulation seen in long-term LFI could be due within the buccal and cerebral ganglia (27, 28), although the to circadian rhythms in activity or feeding behaviors that might neurons specifically responsible for long-term LFI are undeter- affect the animal during training or the circadian clock could mined as of yet. The neural circuit underlying LTS resides mainly directly modulate the signaling cascades that lead to the con- in the pleural and pedal ganglia, although some facilitatory solidation of memory. In LFI, the experimenter elicits the neurons in the cerebral ganglia may be involved. One interesting feeding behaviors of the animal, so rhythms in the animal’s possibility is that the cerebral ganglion may serve as a central activity or feeding behaviors should not affect learning. In our point of modulation by the circadian clock, given the involvement experiments, no time-of-day differences occurred in the training of the cerebral ganglion in both feeding behaviors and LTS. responses for either A. californica or A. fasciata (Figs. 3 and 6). Finding additional types of memory with different neural cir- Additionally, A. californica exhibited long-term memory when cuitry that are modulated by the circadian clock might point to trained during the day and tested at night (Fig. 5), demonstrating humoral control of modulation by a central clock or perhaps the that animals are capable of displaying previous learning at night. existence of some common neuronal elements involved in the These results suggest that modulation of LFI occurs through formation of memory for different behaviors. either modulation of the formation or recall of long-term How does circadian modulation of long-term LFI occur at the memory, rather than through the modulation of the animal’s cellular level? The circadian clock could modulate the formation behavior during training or testing. Disturbing the animals at of long-term memory by (i) ‘‘gating’’ sensory input, (ii) modu- night by training during their inactive period also could be lating events involved in the induction or consolidation of responsible for the lack of long-term memory formation at night. long-term memory, or (iii) modulating the activity of motor If uninterrupted inactive time is necessary for memory consol- neurons. We find the first and third of these possibilities unlikely, idation, we would expect animals trained during the early night given that animals display short-term learning equally well at CT (ZT) 15 to show long-term memory formation, because during the day and the night, and the fact that no time-of-day these animals still have8hofuninterrupted inactive time (night differences are seen in the animals’ responses during the training time) after training. However, no long-term memory formation protocol. It appears most likely, as a conserved mechanism appears to occur for animals trained either early in the night or across behaviors, that the circadian clock modulates events late in the night. We still cannot rule out the possibility that within sensory neurons involved in the induction and consoli- circadian modulation of activity or other behaviors led to the dation of long-term, but not short-term, memory. However, it observed circadian modulation of long-term memory formation. also is possible that the clock modulates events in other neurons The molecular mechanisms underlying the formation and mod- involved in the circuit for LFI. ulation of long-term memory for LFI need to be understood What are the possible mechanisms for the circadian modula- before the contribution of activity-dependent modulation of tion of long-term learning at the molecular level? In both LTS learning can be assessed. and long-term LFI, circadian modulation of long-term learning NEUROSCIENCE Circadian modulation of long-term LFI appears to occur was dependent on the time of training, indicating that the during the induction of memory or consolidation, rather than the circadian clock modulates the formation of long-term memory. recall of memory. A. californica trained during the subjective day Consequently, the circadian clock could modulate ‘‘core steps’’ demonstrated significant long-term LFI when tested during common to the induction or formation of multiple types of either the day or the night (Fig. 5A). However, animals trained long-term memory. Thus, we hypothesize that, although the at night failed to demonstrate significant long-term memory anatomical locations of the neuronal circuits for different types when tested during either the day or the night (Fig. 5B). Thus, of long-term learning may vary, similar mechanisms for the it appears that for associative (LFI) and nonassociative forms of formation of long-term memory will be found at the molecular long-term learning (LTS), as well as appetitive (feeding) and level and that these shared processes will be putative targets for defensive (withdrawal) forms of behavior, the circadian clock circadian modulation. Indeed, recent research has shown that modulates events during the induction and consolidation of the induction and formation of long-term memory for both LTS memory, rather than events during recall. Additionally, the fact and LFI may share common features at the molecular level. that there was no difference in the amount of memory observed Changes in chromatin structure are presumed necessary for the when animals were trained during the subjective day, regardless initiation of transcription. Poly(ADP-ribosyl)ation [by poly- of whether the animals were tested 24, 36, or 48 h later (Fig. 5A), (ADP-ribose) polymerase 1 (PARP 1)] is one posttranslational demonstrates that time-stamping or contextual association was modification of nuclear proteins that can regulate the ability of not involved in these memories. transcription factors to bind DNA. In Aplysia,PARP1is

Lyons et al. PNAS ͉ August 30, 2005 ͉ vol. 102 ͉ no. 35 ͉ 12593 Downloaded by guest on October 1, 2021 activated in pleural–pedal ganglia by protocols (five pulses of modulation of memory formation is determined by the animal’s 5-hydroxytryptamine) that induce long-term facilitation in iso- internal clock, with the largest amount of memory formation lated ganglia as well as by electrical stimulation that induces LTS occurring during the animal’s active phase rather than during in the intact animal (29). Moreover, PARP 1 also is activated in some specific set of events in the environment. This research buccal and cerebral ganglia after LFI training (29). Inhibition of directly compares the effect of the circadian clock on learning in PARP 1 during training specifically blocked long-term, but not both a diurnal and a nocturnal species. Researchers studying short-term, memory. Thus, it appears that both simple and conditioned taste aversion (operant conditioning) in snails also complex forms of long-term learning and memory formation have found that higher rates of voluntary activity are linked to may have common avenues of modulation and regulation. higher levels of learning (30), and one study of fear conditioning One of the unique aspects of the current research is the ability in mice (nocturnal) demonstrated increased learning in animals to directly compare rhythmic modulation of learning between trained during the early night (6). Future experiments extending the diurnal A. californica and the nocturnal A. fasciata. Previ- our studies on circadian modulation of learning in additional ously, we suggested that circadian modulation of long-term species as well as with other learning paradigms will provide learning had adaptive significance such that long-term learning more insight into how phase is regulated by the circadian occurred to a significantly greater extent during the animals’ oscillator as well as affording us the chance to study the function active period (11). Consequently, the prediction was made that of circadian modulation of long-term memory. Comparisons of mechanisms modulating the different phases of circadian mod- nocturnal animals would display greater long-term learning ulation of learning between nocturnal and diurnal species will be during their active phase at night than during the day. As particularly interesting. predicted, the nocturnal A. fasciata demonstrated significantly greater LFI (seen in both total response time and the amount of This work was supported by National Institute of Neurological Disorders time that food was retained in the mouth) when trained and and Stroke Grant NS050589 (to A.E.), Israel Science Foundation Grant tested during the night, compared with animals trained and 357͞02-17.2 (to A.J.S.), and an Israel Institute for Psychobiology Post- tested during the day (Fig. 6A). Thus, the timing of circadian doctoral Fellowship (to A.K.).

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