Page 1 of 9 Impulse: The Premier Undergraduate Neuroscience Journal 2013 Restless Legs Syndrome Model melanogaster Show Successful Olfactory Learning and 1-day Retention of the Acquired Memory

Mika F. Asaba1, Adrian A. Bates1, Hoa M. Dao1, Mika J. Maeda1 1Wellesley College, Wellesley, Massachusetts 02481

Restless Legs Syndrome (RLS) is a prevalent but poorly understood disorder that is characterized by uncontrollable movements during sleep, resulting in sleep disturbance. Olfactory memory in has proven to be a useful tool for the study of cognitive deficits caused by sleep disturbances, such as those seen in RLS. A recently generated Drosophila model of RLS exhibited disturbed sleep patterns similar to those seen in humans with RLS. This research seeks to improve understanding of the relationship between cognitive functioning and sleep disturbances in a new model for RLS. Here, we tested learning and memory in wild type and dBTBD9 mutant flies by Pavlovian olfactory conditioning, during which a shock was paired with one of two odors. Flies were then placed in a T-maze with one odor on either side, and successful associative learning was recorded when the flies chose the side with the unpaired odor. We hypothesized that due to disrupted sleep patterns, dBTBD9 mutant flies would be unable to learn the shock-odor association. However, the current study reports that the recently generated Drosophila model of RLS shows successful olfactory learning, despite disturbed sleep patterns, with learning performance levels matching or better than wild type flies.

Abbreviations: LTM - long-term memory, MCH - 4-methylcyclohexanol, OCT - 3-octanol, REM – rapid eye movement, RLS - Restless Legs Syndrome, WT - wild type

Keywords: sleep disorder; olfactory conditioning

Introduction Sleep is known to play an important role controls showed both improved task in learning and memory (Stickgold, 2005). performance and increased functional Sleep deprivation after learning a task connectivity between brain regions that support negatively impacts the consolidation of newly consolidation, such as the superior temporal formed memories in humans (Stickgold and sulcus and the (Maquet et al., 2003). Walker, 2007), specifically in working memory Thus it is apparent that sleep enhances learning (Chee et al., 2006), spatial memory (Graves et and memory consolidation, while sleep al., 2003), declarative memory (Backhaus et al., deprivation negatively affects consolidation in 2006), and visuomotor memory (Maquet et al., humans. 2003) tasks. REM sleep has been strongly A disorder for which the effects of implicated in the consolidation of procedural chronic sleep loss on cognitive functioning can motor learning (Nitsche et al., 2010). This be studied is Restless Legs Syndrome. Restless pattern is proposed to reflect the offline Legs Syndrome (RLS) is a sensorimotor processing of newly formed memories and leads neurological disorder characterized by an urge to improved performance on the task the to move the legs, motor restlessness, worsened following day (Maquet et al., 2003). Compared symptoms or exclusively present at rest, and to sleep-deprived patients, typically-sleeping worsened symptoms at night (Allen et al., 2003; Page 2 of 9 Restless Legs Syndrome Model Drosophila melanogaster Show Successful Olfactory Learning and 1-day Retention 2013 Byrne et al., 2006). Symptoms can be partially Genome-wide association studies have or temporarily relieved with movement (Byrne reported that a polymorphism in an intron of the et al., 2006). Sleep fragmentation, disrupted BTBD9 of is associated non-REM/REM sleep cycles, is often with an increased risk for RLS (Freeman et al., characteristic of RLS (Trenkwalder and Paulus, 2012). The Drosophila gene CG1826 is the 2010). However, the pathophysiology of RLS is homolog to the human gene dBTBD9. Freeman still poorly understood. Trenkwalder and Paulus et al. (2012) found that the deletion of CG1826 (2010) report that the expression of RLS could in Drosophila disturbs sleep similarly in flies as involve dopamine neuromodulation, but did not RLS does in human patients (Freeman et al., specify where dopaminergic dysregulation is 2012). Prior to the dBTBD9 mutants, there was observed in the brain. They found that no Drosophila model for Restless Legs dopaminergic agents are effective at reducing Syndrome (Freeman et al., 2012). The flies symptoms in low to moderate doses display disrupted sleep and increased motor (Trenkwalder and Paulus, 2010). Unfortunately, activity (Freeman et al., 2012). This motor it is difficult to dissociate the effects of sleep restlessness is associated with sleep disturbances deprivation from the effects of neurotransmitter in a similar fashion to what is observed in dysregulation on cognition. Future research humans (Freeman et al., 2012). should localize changes in dopamine levels to More generally, Drosophila is a specific brain regions to understand the promising model species in which to study relationships between the dopaminergic system, sleep, as they exhibit rest/wake patterns similar sleep disturbances, and cognition. to those of humans (Cirelli and Bushey, 2008). Given the observed chronic sleep Sleep in Drosophila has been defined as disruption in RLS patients, past research has behavioral immobility for at least five minutes identified differences in cognitive performance (Li et al., 2009). This sleep state is regulated by between patients with RLS and typically- both circadian and homeostatic processes sleeping controls (Pearson et al., 2006) and (Cirelli and Bushey, 2008). Flies are active sleep-restricted controls (Gamaldo et al., 2008). during the day and at night, displaying periods According to Pearson et al. (2006), participants of immobility and increased arousal threshold with RLS did significantly worse on two Cirelli and Bushey, 2008). An increased arousal cognitive tasks, a letter fluency test, and trail- threshold means that higher stimulus intensities making test, than typically sleeping controls. are necessary to produce motor responses; this The trail-making test required the subject to characteristic distinguishes sleep from quiet connect a set of 25 numbered dots, providing wakefulness (Cirelli and Bushey, 2008). Thus, information about visual search speed, mental Drosophila dBTBD9 is a potentially useful flexibility, and executive functioning. These model for RLS because they display motor tasks were chosen in order to activate the restlessness and sleep fragmentation similar to prefrontal cortex, which is particularly sensitive humans to sleep loss (Pearson et al., 2006). However, In this study, we compared the Gamaldo et al. (2008) found that RLS subjects performance of Drosophila dBTBD9 mutant performed significantly better on an array of flies to wild type (WT) flies, Canton-S strain, in cognitive tasks, such as a letter fluency and an olfactory conditioning task. Sleep category fluency task, when compared to age- fragmentation and sleep deficits in humans have matched sleep-restricted controls. The resulted in impaired performance on cognitive discrepancy between these two studies is likely tasks compared to subjects with more typical due to the population that RLS patients are sleeping patterns (Pearson et al., 2006). We compared to—typically sleeping controls vs. hypothesized that fragmented sleep loss impairs sleep-restricted subjects. Thus, it has been associative learning and memory retention in the proposed that RLS may be associated with an dBTBD9 mutant when compared to typically- enhanced physiological level of alertness that sleeping controls. While there has been evidence compensates for long-term sleep loss (Gamaldo of higher cognitive performance in RLS patients et al., 2008). when compared to sleep-restricted controls Page 3 of 9 Impulse: The Premier Undergraduate Neuroscience Journal 2013 (Gamaldo et al., 2008), this study used typically (Ejima and Griffith, 2007). Training was sleeping controls. Here, we extend previous performed using the protocol described by Tully research on the effects of sleep disturbances on and Quinn (1985), which has successfully been cognitive functioning in a new model of RLS. used in many Drosophila olfactory conditioning paradigms (Li et al., 2009). Flies were exposed to two consecutive aversive odors (3-octanol, Material and Methods OCT and 4-methylcyclohexanol, MCH) for 60 seconds with a 45 second rest between the Drosophila were cultured on a standard presentations of each odor. During exposure to medium (Bloomington Fly Stock, 66-112, the first odor (either OCT or MCH: trials were Bloomington, IN)) at 25°C and 60% relative counterbalanced), the odor was paired with a humidity in a 12h light/dark cycle. mechanical shock produced by vortex at Approximately 30 flies were reared in one food 2000rpm in 1.5s pulses for 60 seconds (Lagasse vial and transferred to fresh vials once every et al., 2012). After 60s of rest, the flies were three days. The dBTBD9 mutant flies were presented with the other odor. The flies rested provided by Dr. A. Freeman of Emory for 2 minutes after which the conditioning was University. The Canton-S strain (WT) was repeated two more times. After the conditioning obtained from Bloomington Stock Center procedure, flies were transferred into the initial (DBst0000001). chamber of the T-maze where they were allowed

to choose between the side with the shock- Drosophila Activity Measurements paired odor, the side with the unpaired odor, or Activity of 24 WT and 20 dBTBD9 not choose a side by staying in the initial mutant flies was measured with the TriKinetics chamber. Flies that learned the task and avoided Drosophila Activity Monitor (Waltham, MA), the paired odor went through a retention test the using DAMSystem3038. Activity was recorded following day, similar to what has been used between 7 and 9 pm. The light was set to turn previously (Tully and Quinn, 1985). The off at 8 pm so that flies were subjected to 1 hour retention test consisted of the same T-maze as of light and 1 hour of darkness. on the training day with the paired odor on one side and the unpaired odor on the other side. Odor Discrimination Test Flies that avoided the paired odor and instead Flies were tested for initial odor chose to go to the unpaired odor were counted as discrimination to ensure that both odors were having successful long-term memory (LTM) aversive. Both groups of flies, WT and dBTBD9 retention for the paradigm. mutants, were kept in the same conditions and transferred at three days. Flies were put in an Data Analysis initial chamber of a T-maze and then allowed to Activity measurements are presented as stay in the chamber or move to the choice point 24 data points per fly, with each data point and choose between a neutral odor and one indicating the activity for a 5-minute bin. Any aversive odor (either 3-octanol, OCT or 4- activity in a 5-minute period would indicate methylcyclohexanol, MCH) (Sigma-Aldrich; 0.5 wakefulness for that time and, thus, no activity µL) (Tully and Quinn, 1985). Flies were kept in would be considered as a period of sleep. the T-maze for three to four minutes. The choice (Schaper et al., 2012). The sleep ratio analyzed point was defined to be the place where the T- was the number of sleep bouts within the total maze split into the two sides. In addition, a test number of bins for each phase. Actograms were was performed in which flies had to choose made with ActogramJ (Schmid et al., 2011). between the two aversive odors, OCT and MCH. Activity data were analyzed with a repeated- measures ANOVA (SPSS). Pavlovian Olfactory Conditioning Data are presented as mean ± standard Behavioral tests were conducted in a deviation (SD) for odor testing. For odor room at 25°C temperature and 60% humidity discrimination, preference ratios were calculated under red light in order to remove visual input per test. Each test was performed with 4-14 Page 4 of 9 Restless Legs Syndrome Model Drosophila melanogaster Show Successful Olfactory Learning and 1-day Retention 2013 flies. In the neutral-aversive test, each fly was F(1,42) = 3.077, p = 0.087), indicating that given a score of 1 for neutral, 0 for aversive, and while both groups behaved similarly during the 0.5 for no choice. For choosing between the two light phase, dBTBD9 mutant flies sleep less than aversive odors, each fly was given a score of 1 WT flies in the dark phase. for OCT, 0 for MCH, and 0.5 for no choice. These ratings were converted into a percentage of the total number of subjects per test, and tests Wild Type dBTBD9 were counterbalanced for the left and right side of the T-maze. To determine if flies showed a significant preference for the neutral odor, preference scores were tested using a one sample t-test against 0.5 (not having a preference for one of the odors). Preferences for choosing between the two aversive stimuli was tested using Student’s t-test. For the olfactory conditioning task, chi-squared tests were performed for WT and dBTBD9 mutant flies. Flies that did not choose the paired or unpaired side (i.e., stayed in the initial chamber) were excluded from analysis for the olfactory conditioning task tests. Figure 1: Actograms show that RLS D. melanogaster sleep less. Representative examples of actograms from Statistics one WT (a, b) and one dBTBD9 mutant (c,d) fly are Repeated-measures analyses on sleep shown. The gray bar on top indicates the first hour when activity was measured during the light phase; the ratios were performed in SPSS (Version 19). To black bar on top indicates the second hour, when compare preference ratios, analyses were activity was measured during the dark phase. performed in Excel. Conditioning tasks were evaluated by calculating the chi-squared values. All statistical analyses utilized an alpha level of 0.05.

Results Actograms of Drosophila To compare sleep patterns between WT and dBTBD9 mutants, flies were subjected to both light and dark treatments, and activity was measured at a resolution of 5 minutes. Sleep is defined as 5-minute periods of inactivity (Schaper et al., 2012) (Figure 1). On average, the sleep ratios during the light phase were 0.27 ± 0.06 for WT and 0.30 ± 0.08 for dBTBD9 mutant flies. For the dark phase, the ratios were Figure 2: dBTBD9 mutant flies (light grey), but not 0.52 ± 0.07 for WT and 0.39 ± 0.09 for dBTBD9 WT (dark grey), continue to be active in the dark. Mean sleep ratios (± SEM) for WT flies (N = 24) and mutant flies (Figure 2). There was a significant dBTBD9 mutant flies (N = 20) are shown during the effect of ‘Treatment’ (Light versus Dark; light phase (left two bars) and the dark phase (right two repeated-measures ANOVA, F(1,42) = 12.780, bars). p = 0.001). There was a trend that approached significance for the interaction effect between Group*Treatment (repeated- measures ANOVA, Page 5 of 9 Impulse: The Premier Undergraduate Neuroscience Journal 2013 WT and dBTBD9 mutant flies are able to discriminate odors We tested flies on their ability to discriminate odors. When choosing between a neutral odor and an aversive odor, dBTBD9 mutant flies as well as WT flies had a significant preference for the neutral odor compared to the aversive odor (WT: t(2) = 10.661, p = 0.009; N = 30; dBTBD9 mutant: t(2) = 6.638, p = 0.022; N= 24). There were no significant differences between WT and dBTBD9 mutant flies (Figure Figure 3: Olfactory discrimination. WT and dBTBD9 3). mutant flies were put in a T-maze containing a neutral When choosing between the two odor and one aversive odor (either OCT or MCH). Flies either moved to the choice point and chose a side, aversive odors, both strains exhibited no or stayed in the initial chamber. Preference ratios were preference for either odor (WT: t(1) = 1, p > calculated per test, with N = 4-14 flies per trial, and 0.05; N = 20; dBTBD9 mutant: t(1) = 1, p > two trials per condition. Both WT and dBTBD9 mutant 0.05; N = 21). There were no significant flies showed a significant preference for the neutral differences in preference ratio between WT and odor over the aversive odor. There were no significant differences in preference ratio between WT and dBTBD9 mutant flies (Figure 4). dBTBD9 mutant flies. The dashed line indicates the 0.5 chance level. All scores are presented as the mean dBTBD9 mutant flies are able to learn shock preference ratio ± SD. The asterisk represents p < 0.05. association

We examined learning immediately after flies were trained using Pavlovian olfactory conditioning. Significant differences in learning were found between the WT group (Canton-S) and the dBTBD9 mutant fly group. A chi- squared test was performed to examine the observed effects after training. Flies that did not choose a side were excluded from the analysis.

WT flies did not choose the unpaired odor significantly more often than the paired odor, although a trend towards significance was Figure 4: Aversive olfactory avoidance. WT and found, X2 (1, N = 62) = 3.630, p = 0.0568 dBTBD9 mutant flies were put in a T-maze containing (Figure 5). However, dBTBD9 mutant flies did two aversive odors (OCT and MCH). Flies either moved show a learned association and avoided the to the choice point and chose a side, or stayed in the initial chamber. Preference ratios were calculated per test, paired odor significantly more often, X2 (1, N = with N = 6-12 flies per trial, and two trials per condition. 36) = 13.444, p = 0.0002 (Figure 5). There was no significant preference for either of the aversive odors in both strains. There were no significant

differences in preference ratio between WT and dBTBD9 mutant flies. The dashed line indicates the 0.5 chance

level. All scores are expressed as the mean preference

ratio ± SD.

Page 6 of 9 Restless Legs Syndrome Model Drosophila melanogaster Show Successful Olfactory Learning and 1-day Retention 2013 Long-Term Memory Tests We also tested the LTM of the flies after 1 day. Many flies, both WT and dBTBD9 mutants, died overnight and were thus excluded from our analysis. Flies that learned the task went through the same T-maze the following day without any additional training. A chi- squared test was performed to examine retention of the learned association. The result showed that the shock association was retained for the dBTBD9 mutant flies, X2 (1, N = 12) = 7.143, p = 0.0075 (Figure 6). Although all WT flies Figure 6: dBTBD9 mutants flies retain association the chose the unpaired odor side (see Figure 6), the following day. WT and dBTBD9 mutant flies that number of flies used (N = 3) for this task was exhibited learning of the conditioning task were placed in the same t-maze the following day without any not large enough to detect a significant additional training. A chi-square test was performed for preference. the WT (N = 3) and RLS (N = 14) flies. dBTBD9 mutant flies showed a significant avoidance of the paired odor (p = 0.0075). Since N = 3 for the WT flies, statistical tests could not be performed. The asterisk represents p < 0.05.

Discussion The newly developed dBTBD9 mutant of Drosophila has recently become a model for the study of the cognitive functioning in human Restless Legs Syndrome (RLS) patients. The mutant was created by disruption of the gene BTBD9, which is hypothesized to play a role in the regulation of sleep. Mutation of this gene in flies caused phenotypic sleep disruption similar Figure 5: dBTBD9 mutant flies are able learn shock to human expression of RLS (Freeman et al. association. WT and dBTBD9 mutant flies were trained 2012). The current study shows that dBTBD9 on the olfactory conditioning task in which one odor mutant flies are capable of learning a Pavlovian (OCT or MCH) was paired with a shock and one odor olfactory association with an aversive odor was unpaired. The flies were then put in a T-maze with (Figure 5), and that this memory persists after the paired odor on one side and the unpaired odor on the one day (Figure 6). This study provides clues to other side. The flies were considered to have learned the cognition in RLS model flies, extending upon shock association if they avoided the paired odor. A chi- what is already known about RLS in humans. square test was performed for the WT (N = 62) and RLS As expected, both WT and dBTBD9 (N = 36) flies. dBTBD9 mutant flies significantly avoided mutant flies were more active during the light- the paired odor (p = 0.0002) by going to the unpaired phase. Furthermore, dBTBD9 mutant flies may odor side. Significant differences in choosing the paired not sleep as much as WT flies during the dark- vs. unpaired odor were not found for the WT flies (p = phase. Although this trend was not significant, a 0.0568). The asterisk represents p < 0.05. bigger sample size or better techniques to

prolong the period of observation may change this result. Many of the flies did not survive for more than several hours during the night. It is Page 7 of 9 Impulse: The Premier Undergraduate Neuroscience Journal 2013 possible that the hyperactivity of dBTBD9 short-term memory deficits (Seugent et al., mutant flies caused fatalities due to exhaustion 2009). Similar to the aS fly model, decreased (four out of the original 24 dBTBD9 mutant died cognitive performance has been seen in human during experimentation, versus none in the WT patients with Parkinson’s (Hietanen and group). In our experiment, none of our flies Teräväinen, 1986). Current theories propose that survived through the entire night, whereas the sleep disturbances seen in Hyperkinetic and previous experiments have measured sleep Shaker flies are a main component of the activity over 2-3 days (Gilestro, 2012). cognitive deficits seen in each mutant. While Although sleep is defined as 5-minute periods of dBTBD9 mutant flies may exhibit shorter inactivity, the current analysis only includes one durations of sleep (Freeman et al., 2012), the possible timeframe of this bin length (Schaper et present study does not report cognitive deficits al., 2012). Bins of shorter durations might in associative learning in dBTBD9 mutant flies. represent more accurate sleep bouts. Future research should compare associative Interestingly, our findings suggest that learning in dBTBD9 mutant flies with dBTBD9 mutant flies are able to learn an odor- Hyperkinetic and Shaker flies to determine shock association and retain this learned specifically which aspects of each disease affect material at a higher rate than WT flies despite cognitive abilities. Our study was limited by the their fragmented sleep (Freeman et al., 2012). small number of flies that survived in our task This result is particularly interesting because it (Fig. 6). Further research should focus on is in contrast to predictions that sleep characterizing performance of WT flies in fragmentation limits an animal’s ability to learn similar cognitive batteries in order to draw more and retain associations. It is possible that the solid conclusions. ability to learn an odor-shock association in We should also consider the cellular dBTBD9 mutant flies may be a result of the mechanisms related to RLS in order to physical characteristics of the strain: understand how sleep fragmentation may affect specifically, physical weakness in comparison to cognitive functioning. Dysfunction of the WT flies (Freeman et al., 2012). One dopaminergic system has been implicated in the explanation of these findings is that the dBTBD9 symptoms of RLS (Montplaisir et al., 1991; mutant flies may have been more affected by the Rye, 2004), and has been studied in Drosophila shock, resulting in a stronger association models of Parkinson’s Disease (Seugent et al., between the shock and odor. In human subjects, 2009). Fly strains in which dopamine was Gamaldo et al. (2008) found that patients with genetically reduced showed age-dependent loss RLS performed significantly better on several of DA neurons characteristic of the progression cognitive tasks than sleep-restricted controls. of Parkinson’s disease in humans (Seugent et al., This suggests that the physiological alertness 2009). dBTBD9 mutant flies treated with the level may be higher in RLS patients in order to drug Pramipexole, which targets D2-like compensate for sleep fragmentation. The results receptors, show improvements in sleep from this study suggest that dBTBD9 mutant fragmentation (Freeman et al., 2012). Taken flies might show stronger associative learning together, these results implicate dopamine as a through increased physical and physiological main component of RLS symptoms. responses from a shock. Restless Legs Syndrome affects nearly The dBTBD9 mutant flies’ success on 10% of adults in the United States. the associative task in this study is in strong Unfortunately, the molecular pathways contrast to past research done with other sleep- underlying development of the disorder remain disturbed flies. Similar to dBTBD9 mutant unclear (Trotti and Rye, 2011). We found that flies, hyperkinetic Drosophila display decreased dBTBD9 mutant flies are able to learn using the duration of sleep episodes as compared to WT olfactory conditioning paradigm and retain the flies (Bushey, 2007). Additionally, related association the next day. 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