Neurobiology of Learning and Memory 123 (2015) 217–224

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Place memory retention in Drosophila Daniela Ostrowski, Lily Kahsai, Elizabeth F. Kramer, Patrick Knutson, Troy Zars ⇑ 114 Lefevre Hall, Division of Biological Sciences, University of Missouri, Columbia, MO 65211, United States

a r t i c l e

i n f o

Article history: Received 18 May 2015 Revised 25 June 2015 Accepted 26 June 2015 Available online 2 July 2015 Keywords: Place memory Memory phases High temperature Intermittent training Genetic mutation Drosophila

a b s t r a c t Some memories last longer than others, with some lasting a lifetime. Using several approaches memory phases have been identified. How are these different phases encoded, and do these different phases have similar temporal properties across learning situations? Place memory in Drosophila using the heat-box provides an excellent opportunity to examine the commonalities of genetically-defined memory phases across learning contexts. Here we determine optimal conditions to test place memories that last up to three hours. An aversive temperature of 41 °C was identified as critical for establishing a long-lasting place memory. Interestingly, adding an intermittent-training protocol only slightly increased place memory when intermediate aversive temperatures were used, and slightly extended the stability of a memory. Genetic analysis of this memory identified four genes as critical for place memory within minutes of training. The role of the rutabaga type I adenylyl cyclase was confirmed, and the latheo Orc3 origin of recognition complex component, the novel gene encoded by pastrel, and the small GTPase rac were all identified as essential for normal place memory. Examination of the dopamine and ecdysone receptor (DopEcR) did not reveal a function for this gene in place memory. When compared to the role of these genes in other memory types, these results suggest that there are genes that have both common and specific roles in memory formation across learning contexts. Importantly, contrasting the timing for the function of these four genes, plus a previously described role of the radish gene, in place memory with the temporal requirement of these genes in classical olfactory conditioning reveals variability in the timing of genetically-defined memory phases depending on the type of learning. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Some memories last a lifetime, while others are already forgotten within a few seconds. By altering learning conditions and molecular genetic manipulations, memory phase complexity can be revealed. In addition to humans, animals ranging in nervous system complexity from primates and rodents to simpler organisms like the fly Drosophila and the nematode Caenorhabditis elegans are capable of forming memories with various levels of temporal complexity (Burne et al., 2011; Glanzman, 2010). Our understanding of how different memories are more or less stable is far from complete. Drosophila can be operantly trained to learn and remember a safe place (Foucaud, Burns, & Mery, 2010; Ofstad, Zuker, & Reiser, 2011; Zars, 2010). The heat box provides a relatively simple learning task to study place memory dynamics (Kahsai & Zars, 2011; Ostrowski & Zars, 2014; Wustmann, Rein, Wolf, & Heisenberg, 1996; Zars, Wolf, Davis, & Heisenberg, 2000). In this apparatus, a fly is placed in an elongated rectangular chamber in which the ⇑ Corresponding author. E-mail address: [email protected] (T. Zars). http://dx.doi.org/10.1016/j.nlm.2015.06.015 1074-7427/Ó 2015 Elsevier Inc. All rights reserved.

temperature is regulated by the behavior of the fly. Flies can be conditioned within minutes in this paradigm by pairing a rising temperature with a part of the chamber. If a fly chooses to spend time in a safe part of the chamber, the temperature falls to a preferred temperature of 24 °C (Hamada et al., 2008; Kahsai & Zars, 2011; Sayeed & Benzer, 1996; Zars, 2001). So far the memory dynamics have not been extensively studied in this learning paradigm (LaFerriere, Speichinger, Stromhaug, & Zars, 2011; Putz & Heisenberg, 2002). With the advent of identifying potential common and unique mechanisms that are important for maintaining memory performance in the fly, we characterized multiple training and genetic parameters that influence place memory stability. We trained flies with reinforcing temperatures of 33°, 37° and 41 °C with training durations of 4, 10, 15 and 20 min. Memory performance was tested up to several hours after training. Our results show that conditioning with 37 °C does not result in any measurable lasting place memory. Conditioning using 41 °C for 4 min results in place memory that lasts for at least one hour. However, simply extending the training duration did not delay memory decay. Additionally, intermittent training, which usually results in more stable memories, only slightly improved memory stability. Finally, the roles of the rutabaga type I adenylyl cyclase,

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the latheo Orc3 origin of recognition complex component, the novel gene encoded by pastrel, the small GTPase rac, and the dopamine and ecdysone receptor (DopEcR) in promoting the stability of a place memory were examined. 2. Material and methods 2.1. Animals Drosophila melanogaster were raised on cornmeal-based fly food media and maintained on a 12 h/12 h day/night cycle at 24 °C and 60% relative humidity. For behavioral experiments wild-type Canton S (CS) flies at the age of 2–5 days were used. Prior to the behavioral experiments flies were provided 16–24 h on new fly food. The Canton S and cantonized w1118 mutant flies stem from the Martin Heisenberg laboratory stocks. The elav-C155, tublin-Gal80ts and UAS-Drac1(N17) stocks were obtained from the Bloomington Stock Center. These experimental lines were outcrossed to the cantonized w1118 line for six generations and had the first chromosomes replaced with a wild-type version. The rutabaga adenylyl cyclase (rut2080), latheo (lat6), pastrel (pst1), and the dopamine/ecdysone receptor (DopEcRPB1), provided by Drs. Paul Shaw (Washington University in St. Louis) and Toshihiro Kitamoto (University of Iowa), are in a Canton S background (Boynton & Tully, 1992; Dubnau et al., 2003; Ishimoto, Wang, Rao, Wu, & Kitamoto, 2013; Levin et al., 1992). Standard genetic crosses were used to generate experimental groups. Female flies were used for all rac experiments. Crosses for the rac experiments were raised at 18 °C and progeny were divided into two groups. The induced group was transferred to a 30 °C incubator for 3 days, whereas the control group was kept at 18 °C. Both groups were allowed to recover at 24 °C for at least 3 h before behavioral experiments. Heterozygous controls were raised under identical conditions and in parallel to exclude influence of the temperature shift on fly performance. 2.2. Behavioral experiments Place memory was tested using the heat-box apparatus. The heat-box consists of multiple rectangular chambers in which single flies are allowed to walk freely back and forth (Ostrowski & Zars, 2014; Zars, 2009, 2010). The position of a single fly within each chamber is recorded throughout an experiment. Fast temperature changes within the chambers are provided by Peltier-elements on top and bottom. A computer coordinates rising temperatures with position of the fly. Before each training session flies are provided a pre-test phase (30 s) at constant 24 °C to determine any potential spontaneous side preference. During conditioning (the training phases) one chamber half is defined as the side associated with high temperature and the other as not. Every time the fly enters the high temperature associated side the whole chamber heats up to an aversive temperature (33–41 °C). The return of the fly to the other side quickly cools down the chamber to a non-aversive temperature (24 °C) (Sayeed & Benzer, 1996; Zars, 2001). The following 3 min post-test measures place preference while the chamber is kept at the same non-aversive temperature. A performance index (PI) is calculated by the difference in time a fly spent in either chamber half (unpunished side vs. punished side) divided by the total time within a session. The PI can vary from 1.0 to 1.0. Zero indicates that on average the flies spent equal time on both sides of the chamber, whereas 1.0 shows a perfect side preference of the fly for the unpunished chamber half. Flies were conditioned with different reinforcing temperatures (33, 37 and 41 °C) and training durations of 4, 10, 15 or 20 min. Training duration was either massed or intermittent with intervals of rest. For intermittent training each 2 min training interval was

followed by a 1 min rest period; e.g., a 20 min training was interrupted by 9  1 min rest periods. During the rest periods flies remained within the chambers that were kept at 24 °C. Memory performance was either tested immediately after training or after time intervals as listed in Section 3. During the time intervals flies were taken out of the heat-box chambers and put together in a tube with fresh food. The time intervals include handling of flies, so that the rest period starts with the end of the training period and ends with the beginning of the post-test. Post-tests included short reminder training for 30 s using 41 °C as the reinforcing temperature. Olfactory Memory: Undiluted 4-methylcyclohexanol (MCH) and 3-octanol (OCT) were used as odorants with protocols previously described (Krashes & Waddell, 2008; Zars, Fischer, Schulz, & Heisenberg, 2000). To test for aversive olfactory memories, memory was tested after training as described in Section 3. Flies were held in fly food vials in the longer retention intervals. Flies were trained by pairing either MCH or OCT with 12, 100 V electric shocks. Flies were given 1 min to choose between converging odorant streams in a T-maze for the memory tests. A PI was calculated for the memory experiments. This score was calculated by subtracting the number of flies choosing the control odorant from the number of flies choosing the shock-associated odorant, divided by the total number of flies in a ‘‘half test.’’ An average PI was calculated from a pair of half-test PIs, where each half came from conditioning of one of the two odors. 2.3. Data analysis Position of flies within the chambers is recorded by a custom made program and spatial preference (PI) of individual flies during post-test is automatically calculated. Flies that were inactive during pre-test or did not experience heat during training were automatically discarded. Data are shown as mean ± SEM. Unless otherwise noted each group consists of at least 120 flies. The olfactory memory experiments used at least 5 experiments per genotype. For statistical analysis Sigmaplot 12 or Statistica was used. Data were analyzed for their normal distribution using Shapiro–Wilk test (data not shown). Results from tests of normal distribution varied. Therefore, non-parametric tests have been used to test for significant differences in place memory. Parametric ANOVA were used for the olfactory memory tests. P < 0.05 was considered to be statistically significant. 3. Results 3.1. Place memory performance Flies were trained with reinforcing temperatures of 33, 37 or 41 °C for training durations of 4, 10, 15 or 20 min (Fig. 1A). A low reinforcing temperature of 33 °C with extended training of at least 10 min revealed a change in place preference of flies when tested during the post-test phase (Fig. 1A). Prolonged training duration with higher reinforcing temperatures strengthened place memory performance. For each training session of 4, 10 or 20 min, higher temperatures induced higher memory scores (Fig. 1A), largely consistent with previous results (Diegelmann, Zars, & Zars, 2006; Zars & Zars, 2006). However, while it was previously shown that 4 min of training at 33 °C can induce a place memory (Diegelmann et al., 2006), the experiments described here did not induce a significant place memory. This difference might reflect variability in the ability to detect a low-level place memory under these modest conditioning parameters. Nevertheless, the general relationship of higher temperature and increased training duration giving rise to higher levels of place memory is maintained. There is a maximum in memory performance level flies reach with extended training duration and temperatures. For

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Fig. 1. Memory performance of flies after place learning using different training conditions. (A) Flies were trained with reinforcing temperatures of 33°, 37° or 41 °C for training durations of 4, 10, 15 or 20 min. Memory performance (PI) was calculated by a 3 min post-test immediately after training. A low training temperature and duration (33 °C for 4 min) does not induce place memory (Z = 0.493, P = 0.623). Longer training duration and higher reinforcing temperatures result in memory levels up to 0.9 (H = 387.1, P 6 0.05 = ⁄). (B) Selected training conditions with intermittent training were performed. Compared to training with no rest intervals (massed training) only one training condition (37 °C at 20 min) had a significant increase in memory levels with intermittent training (U-test = 7676.5, P < 0.001 = ⁄⁄⁄).

conditioning with 33 and 37 °C the maximum place memory is reached after 10 min of training; an increase in training duration to 20 min had no additional effect on place memory performance. For conditioning with 41 °C maximum performance is reached after 15 min of training (Fig. 1A) (Diegelmann et al., 2006; Zars & Zars, 2006). Intermittent training sessions strengthens memory performance. Selected training conditions were used in further experiments to analyze the impact of intermittent training on place memory (Fig. 1B). For intermittent training each 2 min training interval was followed by a 1 min rest period. During this rest period flies remained within the chambers that were kept at 24 °C. Only with 20 min intermittent training using 37 °C was place memory higher than with massed training. This enhancement is similar to previous results, which showed an increase in immediate place memory with intermittent training (Putz & Heisenberg, 2002). Intermittent training with the high temperature of 41 °C, by contrast, does not increase immediate memory compared to massed training conditions, which seems to already be maximal under both conditions. 3.2. The stability of a place memory depends on training temperature Persistent place memory decreases within minutes to negligible levels in the absence of the high temperature (Diegelmann et al.,

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2006; Putz & Heisenberg, 2002; Zars & Zars, 2009; Zars, Wolf, et al., 2000). However, if a fly is allowed a rest period and then given short reminder training, it can display a savings from previous conditioning (Putz & Heisenberg, 2002). So far, this type of memory savings has only been analyzed with one type of training. We therefore analyzed in more detail how different training protocols can influence extended place memory. Massed training conditions of 4 min at 41 °C as well as 10 min at 37 °C or 20 min at 37 °C all result in similar place memory levels (Fig. 1B). We analyzed extended place memory for these conditions and asked whether training conditions that differ in duration and temperature but result in equal direct memories have the same memory retention properties. Flies’ memory was initially tested after a rest period of 5 min. During that time flies were kept in a fresh food vial. A short reminder training of 30 s at 41 °C was given before the 3 min memory test. Four min at 41 °C training induced an immediate place memory of about 0.5 (Fig. 2). When place memory was tested 5 min after training and with a reminder, flies still showed a place memory significantly different from zero (Fig. 2). By contrast, 5 min memory after conditioning with 10 min of training at 37 °C was at negligible levels (Fig. 2). Even with prolonged and intermittent training of 20 min at 37 °C place memory decreased to zero within the 5 min interval (Fig. 2). Thus, the 37 °C reinforced memory is remarkably short-lived. Flies trained intermittently for 20 min and tested for memory after removing and re-inserting the flies within 30 s still did not have a significant place memory after a reminder (PI = 0.04 ± 0.03, N = 121, U-test against zero; Z = 0.962, P = 0.34). Thus, although various training protocols using 37 °C results in significant immediate place memory, this temperature is not enough to induce an extended place memory. 3.3. Place memory retention after different training conditions A higher temperature can induce a more stable place memory. Control animals with either 4 or 20 min to run in the chambers without changing temperatures had very low PIs after ‘reminder’ training (Fig. 3). Impressively, extended place memory was induced by just four min of training with 41 °C for at least one hour compared to control flies (Fig. 3). Under these conditions, memory at 3 h after training was not different from control groups (Fig. 3). Extended training of 20 min with 41 °C increased place preference of flies when tested directly after training, but did not slow down

Fig. 2. Training conditions using 37 °C does not give rise to extended place memory. Although training conditions of 4 min/41 °C, 10 min/37 °C and 20 min/ 37 °C result in similar place memory performance when tested immediately after training (white bars), 5 min place memory was only induced in flies trained with 41 °C (Z = 5.4, P < 0.001 = ⁄⁄⁄). Using 37 °C as the aversive temperature did not induce a lasting memory with 10 min massed training (Z = 0.327, P = 0.74). An increase in training duration (10 vs. 20 min) did not induce an extended memory (Z = 1.018, P = 0.31). Intermittent training also failed to extend a memory using 37 °C (Z = 0.136, P = 0.89).

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Fig. 3. Memory retention for training conditions with 41 °C as reinforcing temperature. Flies were trained with 41 °C reinforcing temperature for 4 min (black diamond), 20 min (dark gray square), or 20 min with intermittent rest periods during training (light gray triangle). Memory performance is compared to control flies that spend the same time within the heat-box as trained flies but with no heat experience (dark circle = 4 min; white circle = 20 min experience). Place memory levels are tested 5, 10, 60, and 180 min after training. Between training and test phases flies were allowed to rest in a food tube. A short reminder (30 s) is given before the 3 min post-test. With 4 min training memory can be measured up to 1 h after training (Z5min = 8963, Z10min = 7497, Z1h = 7895, Z3h = 7765, P < 0.05 = ⁄). An extended training of 20 min increases memory performance when tested directly after training (Z = 4024.5, P < 0.001) but is not increased compared to 4 min training duration when tested 5, 10, 60 or 180 min after training (Z5min = 10412.5, Z10min = 7483, Z1h = 8146, Z 3h = 7852, P’s > 0.05). Twenty minutes of intermittent training induces memory levels that are slightly higher than flies mass trained for 20 min (Z5min = 13463.5, Z10min = 8301, Z3h = 6325, P’s > 0.05), but memory tested 3 h after training are still significantly different compared to control performance (Z = 6323, P = 0.01).

fly lines for defects in place memory. In two sets of experiments, the susceptibility of the memory directly after training and an extended memory after training was assessed by mutation of five different genes. In the first set, flies mutant for rutabaga (rut), latheo (lat), pastrel (pst), and the dopamine/ecdysone receptor (DopEcR) were tested (Boynton & Tully, 1992; Dubnau et al., 2003; Ishimoto et al., 2013; Levin et al., 1992). All of these genes when mutated have been shown previously to reduce memory levels at different time points after training, mostly after olfactory learning. The DopEcR gene has been examined in courtship conditioning memory. In the second more extended analysis, the small GTPase rac was examined (Shuai et al., 2010). The rac-manipulations have been shown previously to increase memory performance in olfactory learning experiments. Four genes show differential effects on place memory dynamics. In tests for memory directly after 4 min of 41 °C training, rut, lat, and pst all showed a significantly lower place memory compared to wild-type flies. The rut phenotype is similar to previous results, although they were tested here with a higher temperature and different conditioning times, where these flies have been shown to have a strongly reduced place memory (Fig. 4A) (Diegelmann et al., 2006; Zars, Wolf, et al., 2000). Both the lat and pst mutant flies also had a significantly reduced memory score compared to wild-type flies (Fig. 4). The DopEcR mutant flies had a similar place

memory decay compared to the shorter training conditions. After even a 5 min rest, place memory decreased to about 0.25, similar to flies trained for just 4 min with this temperature (Fig. 3). A comparison of place memory between flies trained 4 min and flies trained with 20 min revealed no significant difference for all rest time intervals tested from 5 to 180 min. Since other types of learning with intermittent training can induce a days-long memory (Isabel, Pascual, & Preat, 2004; Perisse, Burke, Huetteroth, & Waddell, 2013; Tully, Preat, Boynton, & Del Vecchio, 1994) we examined place memory retention after intermittent training conditions. Memory performance tested right after training did not show a further increase compared to massed training conditions (Figs. 1B and 3). Flies tested 5–180 min later after training showed a slightly higher place memory, which was not significantly different to flies with 20 min massed training. However, flies trained with intermittent training have a significant place memory 3 h after training. Place memory after 20 min of intermittent training was not detectable 5 h after training (PI = 0.04 ± 0.02; U test against zero; Z = 1.560; P = 0.1, N = 134). Memory after very long training was also tested, with three phases each of 20 min intermittent training and 30 min breaks between training sessions. In this case, flies showed essentially no gain in memory performance at 10 min after this over-training (PI = 0.30 ± 0.05; U-test = 6126.5, P = 0.9 compared to normal intermittent training for 20 min with 41 °C; N = 80).

3.4. Genetics of place memory Differential susceptibility of memory after training to mutations in several genes has been used to classify memory phases after classical olfactory learning (e.g., Isabel et al., 2004; Krashes & Waddell, 2008; Tully et al., 1994). To examine whether some of these genes might also alter place memory, which has a much shorter time-line than olfactory learning, we tested several mutant

Fig. 4. Some genetically altered flies have reduced place memory. Flies mutant for the rutabaga adenylyl cyclase (rut), latheo (lat), pastrel (pst), and the dopamine/ ecdysone receptor (DopEcR) were tested. (A) For place memory tested directly after 4 min of training at 41 °C, the rut, lat, and pst flies had a significantly lower place memory compared to wild-type flies (H = 89.8, P < 0.05 = ⁄, P < 0.001 = ⁄⁄⁄). (B) Both the pst and DopEcR flies were tested for a memory defect 60 min after training. In this case, only the pst flies had a significant place memory deficit (H = 9.36, P < 0.05 = ⁄).

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memory as the wild-type level (Fig. 4). The somewhat reduced immediate memory of pst mutant flies and normal immediate memory for DopEcR flies prompted testing of a memory after a 60 min rest followed by a short reminder training. In this case, the pst mutant flies again had a lower memory compared to wild-type flies; the DopEcR flies were not altered (Fig. 4B). Thus, three genes that have been implicated in olfactory memory at different time-points after training all altered place memory within minutes of training. The DopEcR mutant flies, which have reduced courtship conditioning, did not alter place memory under the conditions examined here. The rac small G protein is important in forgetting mechanisms after aversive olfactory learning (Shuai et al., 2010). That is, reduction in Rac activity leads to an increase in olfactory memory levels. Here we tested whether inhibiting Rac activity would also prolong place memory retention. A dominant-negative form of the rac protein (UAS-Drac1(N17)) was expressed by a pan-neuronal elav-Gal4 driver in combination with Gal80ts (elav-Gal4/+;Gal80ts/ +;UAS-Drac1(N17)/+). With this approach Rac function was normal when flies were kept at 18 °C (normal Rac function) but inhibited after incubating flies at 30 °C for 3 days (inhibited Rac function). No difference was found between flies with normal or reduced Rac function directly after 4 min of 41 °C training (Fig. 5A). Although transgenic rac flies were repeatedly outcrossed to the wild-type Canton S (CS) strain, the memory performance of both induced and uninduced flies was low compared to CS flies. It may be that low levels of dominant negative rac that could occur even in the absence of transgenic induction could be lowering memory shortly after training. Memory retention was also analyzed 10, 60, 180 and 300 min after training and a short reminder training (Fig. 5A). Place memory of flies with normal Rac function decays similar to CS flies (Fig. 5A). In these genotypes, flies’ memory 3 h after training reduced to levels near zero. However, flies with decreased Rac function had a much slower memory decay compared to both control and CS flies (Fig. 5A). For the first 60 min after training the performance of Rac inhibited flies remains as high as memory tested directly after training. Furthermore, place memory 3 and 5 h after training was still significantly different from control animals’ memory (Fig. 5A). Genetic control flies were analyzed with respect to memory both directly after training and after a 60 min rest (Fig. 5B). Also, to exclude an impact of the temperature shift on memory performance all flies were incubated at either 18 °C or 30 °C. Place memory tested directly after training varies somewhat between different genetic groups, but 60 min after training the genetic control groups show similar low memory scores compared to the Rac inhibited flies. A thermosensitivity test was done to determine if there were changes in response to temperatures in genetically altered flies (Zars, 2001, 2010; Zars, Wolf, et al., 2000). Half of the chamber is warmed to 41 °C while the other half of the chamber is kept at the preferred 24 °C in this test. No significant differences were evident between wild-type CS and rut, lat, pst, DopEcR, or rac flies (Table 1). Thus, the data strongly suggest that the mutant flies with memory changes do so independently of changes in responsiveness to 41 °C. Rac inhibited flies were trained and tested in aversive olfactory learning in a second set of control experiments (Fig. 5C). Flies were trained then tested either directly after training or 2 h later. Memory tested directly after training is similar between flies with normal rac expression and flies with impaired Rac function. Aversive olfactory memory 2 h after training is lower in flies from both control groups. By contrast, Rac inhibited flies show no memory decay 2 h after the training (Fig. 5C).

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Fig. 5. Flies with inhibited Rac activity have a slower memory decay. (A) For inhibition of Rac in the nervous system a dominant-negative form of Rac (UASDrac1(N17)) was expressed by a pan-neuronal elav-Gal4 driver in combination with Gal80ts (elav-Gal4;Gal80ts;UAS-Drac1(N17)). To inhibit Rac function flies were shifted from the rearing temperature of 18 °C to 30 °C for 3 days. Control flies were kept at 18 °C. Acquisition of place memory after 4 min of 41 °C training is not different between flies with inhibited Rac function (black square) and control flies (gray diamond; Z = 7783, P = 0.1). Both transgenic groups had lower place memory compared to CS (H = 32.9, P < 0.001). Flies with inhibited Rac function show a slower memory decay compared to flies with normal Rac function (Z10min = 5848.5, Z60min = 3471, Z3h = 6311.5, Z5h = 7734.5, P’s < 0.05 = ⁄). (B) Genetic control flies were analyzed with respect to memory at 0 and 60 min retention intervals. To also examine a potential impact of temperature shift on memory performance genetic control flies were incubated at either 18 °C or 30 °C. Although place memory tested directly after training (0 min) varies between different genetic groups, within each genotype the temperature exposure did not alter place memory (ZDrac1/+ = 8177, Zelav/+ = 7867, ZDrac1/elav = 7783, P’s > 0.1). Flies with impaired Rac function (black bar, 30 °C) had a higher memory level at 60 min compared to all other fly groups, H = 25.416, P < 0.01 = ⁄⁄). (C) rac function is important in regulating memory decline after aversive olfactory conditioning. Olfactory memory tested directly after training is not different between CS flies and flies with normal or inhibited Rac function (ANOVA, P > 0.05, N = 5). Memory performance tested 2 h after training was higher in Rac inhibited flies compared to CS and normal Rac function (ANOVA, P’s < 0.05 = ⁄, N’s = 5).

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Table 1 Thermosensitivity tests for 41 °C. Genotype

N

41 °C

CS rut lat pst DopEcR normal Rac inhibited Rac

105 136 129 142 142 138 140

0.79 ± 0.03 0.75 ± 0.02 0.79 ± 0.03 0.78 ± 0.02 0.78 ± 0.02 0.81 ± 0.02 0.82 ± 0.02

Flies were allowed to choose between a warm and cool temperature chamber half for one minute. Tests for significant differences were found (H (6,N = 932) = 85.2, P < 0.001. All comparisons to CS were non-significant).

4. Discussion Temperature as an aversive reinforcer interacts with training conditions to induce place memories of different stabilities. Previous work showed that intermittent training for Drosophila in space and place memory increases memory performance up to two hours after training (Galili et al., 2014; LaFerriere, Speichinger, et al., 2011; Ofstad et al., 2011; Putz & Heisenberg, 2002). Shown here is that temperatures at or above 41 °C are needed for induction of this longer lasting memory. That is, 37 °C and below can act as an aversive reinforcer and condition flies to avoid a part of the training chamber, but continued avoidance decays within minutes of training (Fig. 2). It is only with a temperature of 41 °C that an hours-long memory is induced with massed and intermittent training (Fig. 3) (Putz & Heisenberg, 2002). This abrupt difference in the length of the memory after training with the higher temperature may reflect a threshold of some sort, the steepness of which is currently unknown. This could arise from a differential input to the reinforcing circuit from separate sensory systems, like the Trp family of receptors, or from altered output from one of these sensory systems (Galili et al., 2014; Gallio, Ofstad, Macpherson, Wang, & Zuker, 2011; Hamada et al., 2008; Ni et al., 2013; Tracey, Wilson, Laurent, & Benzer, 2003). Future studies on different temperature responsive proteins may differentiate between these possibilities. Genetic analysis challenges the use of time as a critical factor in determining a memory phase. Memory phases in the fly were initially examined after classical olfactory conditioning where an odorant is typically paired with an aversive electric shock or a rewarding sugar (Tully and Quinn, 1985; McGuire, Deshazer, & Davis, 2005; Perisse et al., 2013; Zars, 2010). Four different memory phases have been classified based roughly on time after training and genetic/pharmacological manipulations (McGuire et al., 2005; Perisse et al., 2013; Tully et al., 1994; Zars, 2010). Short-term memory after olfactory learning is measured within minutes of training; long-term memory and anesthesia resistant memory start to be active within hours and are increasingly important for memories at the 24 h range and longer. An intermediate memory is thought to be important in the interval between short-term and long-term memories. That time alone is a critical factor in determining these phases loses support when comparing flies with different mutations in aversive and rewarded olfactory memory. For example, the long-known mutant radish was originally shown to be important in the hours-long range after aversive olfactory training and genetically classified the anesthesia-resistant memory (Folkers, Drain, & Quinn, 1993; Folkers, Waddell, & Quinn, 2006; Krashes & Waddell, 2008; LaFerriere, Speichinger, et al., 2011; Tully et al., 1994). Interestingly, this gene is important within minutes of training in rewarded olfactory memory (Krashes & Waddell, 2008; LaFerriere, Speichinger, et al., 2011). Several genes that are important for early to late phases of classical olfactory conditioning are critical on a finer time scale in place

memory. Mutation of both the rut and lat genes leads to reduced aversive olfactory memory tested immediately after training, as well as longer time points (Boynton & Tully, 1992; Levin et al., 1992; Schwaerzel, Heisenberg, & Zars, 2002). Although it is currently unclear when during the life-cycle these genes are important for place memory, mutation of rut and lat reduces memory directly after training (Fig. 4). Furthermore, both the rut and lat products have been implicated in synaptic plasticity at the neuromuscular junction (NMJ), which suggests a role for these genes in early stages of learning and memory (Rohrbough, Pinto, Mihalek, Tully, & Broadie, 1999; Zhong & Wu, 1991). It is pretty straight-forward that the rut-encoded type I adenylyl cyclase is also acting early on in associative processes in place learning. The lat gene encoding a subunit of the origin of replication (orc3) is also localized to the pre-synaptic specializations at the NMJs (Pinto et al., 1999; Rohrbough et al., 1999; Zhong & Wu, 1991). The lat-orc3 also acts early-on in associative processes for place learning (Fig. 4). How the lat-orc3 product is related to regulation of cAMP levels is, however, not as clear. The rut and lat results add to our understanding of an apparently common set of short-term changes in memory between olfactory and place memory, which include a common function of the S6 kinase II, an atypical tribbles kinase, and the arouser EPS8L3 (Laferriere, Ostrowski, Guarnieri, & Zars, 2011; LaFerriere, Speichinger, et al., 2011; Putz, Bertolucci, Raabe, Zars, & Heisenberg, 2004). And, the recently identified role of the foxp transcription factor specifically in operant learning, as tested in a flight simulator, suggests another set of genes that could be important for operant place memory in the minutes range (Mendoza et al., 2014). Late memory phases in classical olfactory conditioning depend on a set of genes that are important for place memory within minutes. The first challenge to a common timing of a memory phase came from the radish gene. In contrast to a role in the hours range after olfactory learning, radish mutant flies have a deficit in operant place memory within minutes of training (LaFerriere, Speichinger, et al., 2011). Furthermore, the pst gene (cg8588), encoding a novel product, has been previously shown to have a specific defect in aversive olfactory memory 24 h after spaced training (Akalal, Yu, & Davis, 2011; Dubnau et al., 2003). That is, the pst mutant flies have a normal short-term olfactory memory but a defective memory 1 day later. Interestingly, in the heat-box pst mutant flies already show a significant decrement in place memory immediately after training (Fig. 4). This place memory defect seems to get worse within the first hour after training, reduced to 50% of normal after 60 min. Thus, this ‘long-term memory gene’ is also involved in a memory within minutes of training in a second learning situation. Using the classical aversive olfactory learning paradigm the rac small GTPase has been identified as a key regulator in memory retention (Shuai et al., 2010). Inhibition of Rac activity slows early olfactory memory decay, leading to elevated memory levels one hour after training, but becoming increasingly important 2 h after training. There does not appear to be an effect of Rac inhibition in olfactory memory in the minutes range after training. Transgenic flies with inhibited Rac function also have an increase in memory retention after place memory training. However, the first evidence of an increase in memory performance is within 10 min. Impressively, significant place memory was still evident up to 5 h after training (Fig. 5), far beyond the range that can be typically measured in wild-type flies (LaFerriere, Speichinger, et al., 2011; Ofstad et al., 2011; Putz & Heisenberg, 2002). Thus, while rac has a more general role in stabilizing memories, the timing of this function depends again on the type of memory trace that is formed. Not all memory genes first identified in other contexts, however, play a significant role in place memory. The DopEcR gene

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has been implicated in several behaviors, including a 30 min memory after courtship conditioning (Inagaki et al., 2012; Ishimoto et al., 2013). This G-protein linked receptor is responsive to both dopamine and the steroid hormone ecdysone. Remarkably, DopEcR has been shown to interact with the cAMP cascade through double mutant and pharmacological tests. Using conditions that induce a robust and lasting place memory, the DopEcR mutant flies do not show a defect in memory directly after training or at 1 h post-training. This is despite the fact that the rut and cAMP-phosphodiesterase genes (dunce) are critical for place memory (Kahsai & Zars, 2011; Ostrowski & Zars, 2014; Wustmann et al., 1996; Zars, Wolf, et al., 2000). It may be that DopEcR is not required for this type of learning and would be consistent with the independence of place memory from dopamine signaling (Sitaraman et al., 2008). Alternatively, other redundant pathways may compensate for the reduction in DopEcR function caused by the DopEcRPB1 allele. One might further speculate that other types of behavioral plasticity, such as reversal learning or memory enhancement after unpredicted high temperature exposures in the heat-box might be more sensitive to DopEcR changes (Sitaraman & Zars, 2010; Sitaraman, Zars, & Zars, 2007; Zars & Zars, 2009). Future experiments will determine if this is the case. Memory stability across learning contexts in Drosophila has some common genetic mechanisms, but the timing for gene action depends on the type of learning (Kahsai & Zars, 2011; LaFerriere, Speichinger, et al., 2011; Ostrowski & Zars, 2014; Zars, 2010). That we add several genes here, including lat, pst, and rac as regulators of memory stability in operant place memory suggests that there are at least some common molecular processes in memory stability across different learning types. However, the timing of these genetically-defined phases depends on what is learnt. We speculate that an ideal system to regulate memory stability would be one that activates its own decline. That is, a given memory type should activate the process of decreasing memory expression. This might work with the recruitment of a reinforcing pathway, like the dopaminergic signal that is important for both the acquisition of an associative olfactory memory and the active process of forgetting that association (Aso et al., 2012; Berry, Cervantes-Sandoval, Nicholas, & Davis, 2012; Berry & Davis, 2014; Placais et al., 2012). In this case an odor associated with shock gives rise to a memory trace in mushroom body neurons that depends on a set of dopamine neurons that is important for both memory acquisition and decline. Whether this type of aminergic-based system applies to other forms of memory is not yet known. However, if an aminergic-based signal is common in memory decline, as appears to be the case with the rac-based mechanism, differences in the types of aminergic neurons or innervation targets could give rise to the altered stabilities of behaviorally expressed memories. Author contributions D.O., L.K., E.K., and P.K. conducted the experiments. D.O., L.K., and T.Z. analyzed the data. D.O., L.K., and T.Z. wrote the paper. Acknowledgments We thank Paul Shaw and Toshihiro Kitamoto for generously providing fly lines. This study was supported by the Alexander von Humboldt Society (D.O.), MU Future Faculty Post-doctoral Program (L.K.), and the MU Research Board. References Akalal, D. B., Yu, D., & Davis, R. L. (2011). The long-term memory trace formed in the Drosophila alpha/beta mushroom body neurons is abolished in long-term memory mutants. Journal of Neuroscience, 31, 5643–5647.

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