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. Author manuscript; available in PMC: 2014 May 8.
Published in final edited form as: Genes Brain Behav. 2009 Feb 11;8(4):377–389. doi: 10.1111/j.1601-183X.2009.00483.x

Aversive phototaxic suppression: evaluation of a short-term memory assay in Drosophila melanogaster

L Seugnet †,1, Y Suzuki †,1, R Stidd ‡, P J Shaw †,*
PMCID: PMC4014202  NIHMSID: NIHMS564348  PMID: 19220479

Abstract

Drosophila melanogaster is increasingly being used to model human conditions that are associated with cognitive deficits including fragile-X syndrome, Alzheimer’s disease, Parkinson’s disease, sleep loss, etc. With few exceptions, cognitive abilities that are known to be modified in these conditions in humans have not been evaluated in fly models. One reason is the absence of a simple, inexpensive and reliable behavioral assay that can be used by laboratories that are not expert in learning and memory. Aversive phototaxic suppression (APS) is a simple assay in which flies learn to avoid light that is pairedwith an aversive stimulus (quinine/humidity). However, questions remain about whether the change in the fly’s behavior reflects learning an association between light and quinine/humidity or whether the change in behavior is because of nonassociative effects of habituation and/or sensitization. We evaluated potential effects of sensitization and habituation on behavior in the T-maze and conducted a series of yoked control experiments to further exclude nonassociative effects and determine whether this task evaluates operant learning. Together these experiments indicate that a fly must associate the light with quinine/humidity to successfully complete the task. Next, we show that five classic memory mutants are deficient in this assay. Finally, we evaluate performance in a fly model of neurodegenerative disorders associated with the accumulation of Tau. These data indicate that APS is a simple and effective assay that can be used to evaluate fly models of human conditions associated with cognitive deficits.

Keywords: Learning, Drosophila, short-term memory, phototaxis, mushroom body

Response inhibition and short-term memory are important for maintaining adaptive behavior and are likely to be altered in flies that are used to model human conditions associated with cognitive deficits including fragile-X syndrome, Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, sleep loss, etc. (Iijima et al. 2004; Inlow & Restifo 2004; Mershin et al. 2004; Restifo 2005; Seugnet et al. 2008). Functional insights into these conditions are likely to be enhanced by combining molecular genetic investigations with behavioral assays that can assess relevant aspects of cognitive behavior (Restifo 2005). Frequently, labs that specialize in elucidating the underlying mechanism and pathophysiology of the aforementioned conditions may not have the additional resources to evaluate cognitive behavior. In this context, a learning assay would only be intermittently utilized once sufficient progress had been made in identifying a candidate pathway. As a consequence, the assay should be simple, inexpensive and reliable. Ideally, the apparatus should not require dedicated space and thus should be easily stored. Moreover, as the severity of many of the conditions described above progress with age, it may be important to be able to evaluate the behavior of an individual fly, and on more than one occasion.

In 2002, Le Bourg developed a learning assay, aversive phototaxis suppression (APS), that appears to meet these criteria (Le Bourg & Buecher 2002). Flies are instinctively phototaxic (Hirsch & Boudreau 1958), and when given a choice between a light or a dark alley, flies choose the lighted alley more frequently (Le Bourg & Badia 1995). Quinine, on the other hand, induces avoidance behavior and is frequently used as a negative reinforcer (Fresquet & Medioni 1993; Hendel et al. 2005; Le Bourg 2004; Meunier et al. 2003; Quinn et al. 1974). Flies are individually placed into a T-maze and allowed to choose between a lighted and a darkened chamber. Filter paper is wetted with quinine solution and placed into the lighted chamber such that the quinine/humidity provide an aversive association (Fig. 1a,b). The number of visits to the dark chamber is tabulated during four blocks of four trials where the quinine and light appear equally on the left and right side of the apparatus; there are no delays between trials and the entire task takes ~15 min to complete. Flies learn to select the dark alley more frequently over the course of the 16 trials (Le Bourg & Buecher 2002). In theory, the performance of a fly in this assay depends upon both short-term memory and the fly’s ability to inhibit their attraction toward light. However, questions remain about whether the change in the fly’s behavior reflects learning, that is an association between light and quinine/humidity or whether the change in behavior is because of nonassociative effects of habituation and/or sensitization etc. As APS depends, in part, upon response inhibition, it is not possible to exclude nonassociative effects using the differential conditioning protocols (e.g. CS1+US, CS2 and CS2+US and CS1) developed for olfactory conditioning (Tully & Quinn 1985). Thus, alternative strategies must be used to determine the nature of learning in this assay. Given the potential benefit of this learning paradigm to many disciplines, we have conducted a series of experiment to determine whether the change in behavior is because of associative or nonassociative effects.

Figure 1. Experimental apparatus for testing phototaxic suppression.

Figure 1

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(a) Flies are placed in a T-maze and allowed to choose between a lighted and darkened alley. Quinine is then placed into the lighted alley to provide an aversive association. The number of visits to the dark alley is tabulated during four blocks of four trials. (b)Female Cs flies (n = 100) learn to select the dark alley more frequently over the course of the 16 trials. A high score indicates learning (performance); one-way anova for blocks (F3,396 = 132.26, P = 2.3E−59). (c) Frequency histograms for the number of photonegative choices during block 1 and block 4 trials show that scores in block 4 are normally distributed. (d) Flies rarely visit the dark chamber in the absence of quinine (black, n = 12); when quinine is present in both the dark and light alleys (light gray, n = 10) or when quinine is present only in the dark alley (dark gray, n = 10). However, flies visit the dark chamber when quinine is present only in the lighted alley (white, n = 10). One-way anova for condition (F3,38 = 32.01, P = 1.7E−10). ‘Q’ indicates vials containing quinine * P<0.05.

Methods

Fly stocks

Canton-S (Cs),w1118,w1118; Df(2R)lio2, pigeon2 drl2 (lio2), Df(2R)vg-B and Df(3L)pbl-X1 flies were obtained from the Bloomington Drosophila stock center (Bloomington, IN, USA). We obtained rut2080 and UAS-shits1 from M. Heisenberg (University of Wurzburg, Wurzburg, Germany), pastrel1 from Josh Dubnau (Cold Spring Harbor Laboratories,-Cold Spring Harbor, NY, USA), latheop1 from Tim Tully (Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, USA), MBSwitch from Ron Davis (Baylor School of Medicine, Houston, TX, USA), dunce1(dnc1), dnc1;UAS-dunce1 and dnc1;c309-GAL4 from Bruno Van Swinderen (University of Queensland, Brisbane, Australia), and UAS-TauWT from Mel Feaney (Harvard University, Cambridge, MA, USA). Flies were cultured at 25°C, 50–60% humidity, in 12 h:12 h light:dark cycle, on a standard food containing yeast, dark corn syrup, molasses, dextrose and agar. Newly enclosed adult flies were collected from culture vials daily under CO2 anesthesia. Flies were evaluated between 3 and 8 days of age. Female flies were used for testing, unless otherwise specified.

Learning test

The learning paradigm was adapted from an assay developed by Benzer and colleagues (Quinn et al. 1974) and has been previously described (Le Bourg and Buecher 2002). The alleys of the maze (2 mm wide and 1.4 mm high) are carved in an opaque plastic block. Each alley exits to a vertical vial, either lighted or dark. The ceiling of the maze is covered by a clear Plexiglas surface and a red filter, allowing monitoring of the fly behavior. Flies are blind to red light, and the only source of visible light in the maze comes from the lighted alley, at the choice point of the T-maze. Both dark and lighted vials are covered with filter paper. The filter paper in the lighted vial is wetted with 320 µl of a 10−1 m quinine hydrochloride solution (Sigma, St. Louis, MO, USA). Using a syringe, a fly is placed at the entrance of the maze. After entering the dark or lighted vial, the choice is recorded and the fly is quickly removed from the vial, put back in the syringe and placed back at the entrance of the maze. One passage through the maze takes on average 45 seconds and the entire testing session is completed within 10–20 min. During the test, the light and quinine appear equally on both the right and left side of the apparatus. The percentage of times the fly enters the dark vial constitutes the performance score for each block of four trials (i.e., 0 visits = 0%, 1 visit = 25%, 2 visits = 50%, 3 visits = 75% and 4 visits = 100%). The final score for each block is obtained by averaging the score of all the participating flies. A high score indicates learning. Filter papers and vials are changed between each fly. Unless otherwise stated, all flies were tested in the morning between Zeitgeber Time (ZT) 0 and ZT4. Statistical analyses were performed using Systat (Systat 7, Chicago, IL, USA). Differences in performance during block 4 were assessed using either a Student’s t-test or analyses of variance (anova), which were followed by planned pairwise comparisons with a Tukey correction. Unless stated otherwise, n ≥ 8 in all experiments.

Photosensitivity

Photosensitivity was evaluated in the T-maze over 10 trials in the absence of filter paper. The lightened and darkened chambers appeared equally on both the left and right. The average proportion of photopositive choices during 10 trials was calculated for each individual fly. The final phototaxis index is the average of the scores obtained for five flies ± SEM. Table S1 provides the data obtained for each genotype.

Quinine sensitivity

Sensitivity to quinine was evaluated as in Le Bourg and Buecher (2002) with the following modifications: five flies were individually placed at the bottom of a 14-cm transparent cylindrical tube that was uniformly lighted and maintained horizontal after the introduction of the animal. Each half of the apparatus contained separate pieces of filter paper that could be wetted with quinine or kept dry. The quinine sensitivity index (QSI) was determined by calculating the time in seconds that the fly spent on the dry side of the tube when the other side had been wetted with quinine, during a 5-min period. In the absence of quinine when both sides of the tube are dry, flies quickly move to the side opposite their point of entry and stay there. Indeed, under dry conditions, flies never reside formore than 1 min on the side of the chamber where theywere placed initially. However, in the presence of quinine, flies briefly cross from the dry end of the tube into the quinine portion of the apparatus and then quickly come back to the dry side of the chamber. Table S1 shows the data obtained for each genotype.

Electric shock

An electrifiable copper grid was placed into the lighted vial. Upon entering the lighted vial, the flies were exposed to 1.5 seconds pulses of 70 V DC electric shock at 5-second intervals for 30 seconds. There was a small area at the bottom of the vial that could not be electrified. If a fly remained at the bottom of the vial, the experimenter would gently tap the apparatus on the bench to initiate climbing. Flies were then removed, put in the syringe and placed back at the entrance of themaze. As above, the flies were trained for 16 trials and the number of times the fly enters the dark tube is tabulated during four blocks of four trials. Shock avoidance was evaluated by placing six groups of five flies into a transparent cylindrical tube for 1 min. In the tube, one half of the surface was covered with an electrifiable copper grid that presented shock pulses of 70 V DC. The number of flies that avoided the copper grid after 1 min was tabulated.

Yoked tests

Yoked control experiments were modeled after protocols described by Putz and Heisenberg (2002) and Brembs andWiener (2006). In the first experiment, yoked control flies were exposed to quinine/humidity or dark/dry vials at the same time and for the same amount of time as a fly performing the task in a fashion similar to that reported by Putz and Heisenberg (2002). Yoked control flies were manually placed into vials containing wetted quinine solution or into a dry dark vial for 5 seconds according to the behavior of an experimental fly. After each 5-second exposure, yoked control flies were then removed from the vial and placed into a syringe that is used to introduce the flies into the maze. The yoked control remained in the syringe while the experimental fly completed the next trial. Thus, the yoked controls experienced dark/dry quinine/humidity/light at the same frequency, duration and interval as experimental flies. The yoked control flies were then immediately tested for one block of four trials using the standard protocol. In the second experiment, flies were run through 16 trials in the T-maze. On 10 of these trials, the ‘yoked control’ fly encountered the same pairing (light/quinine or dark/dry) as an experimental fly. However, on six trials (one trial in block 1, two trials in block 2, one trial in block 3 and two trials in block 4), the choice of the yoked control fly was reversed so that the outcome would match that of the experimental fly. For example, when the yoked control fly entered the dark vial on trial 5, they would encounter quinine and the dark cover would be immediately removed so that the fly would be exposed to light/quinine although they had chosen the dark vial. In the final yoked experiment, the ‘yoked control’ flies were also run through 16 trials with 10 trials being concordant (light/quinine or dark/dry) and six trials being discrepant (e.g. dry/quinine or light/dry) based upon data from experimental flies. In contrast to the experiment described above, the lighting conditions remained the same for a given trial, the only difference being that the fly would unexpectedly encounter quinine in the dark vial or dry in the lighted vial when they had made a dark or light choice, respectively. That is, they were exposed to dark and quinine the same number of times and at the same interval as an experimental fly.

Sleep

Three-day-old flies were individually placed into 65-mm glass tubes so that sleep parameters can be continuously evaluated using the Trikinetics activity monitoring system as previously described (Shaw et al. 2000) (www.Trikinetics.com). Flies were sleep deprived using an automated sleep deprivation apparatus that has been found to produce waking without non-specifically activating stress responses. The sleep nullifying apparatus tilts asymmetrically from −60° to +60° such that sleeping flies are displaced during the downward movement 10 times per min. This stimulus is effective presumably because it initiates a geotactic response.

Drugs

RU486 (mifepristone; Sigma) was diluted in ethanol at a concentration of 50 mg/ml and then diluted to 100 µg/ml final concentration in regular food. Flies were fed RU486 containing food for 48 h before being tested.

Immunohistochemistry

Fly brains were dissected in cold phosphate-buffered saline (PBS), fixed for 20 min in a 4% paraformaldehyde PBS solution, washed in 3% Triton-X-100 PBS (PBS-T) and blocked for at least 45 min in 3% goat serum PBS-T. Anti-fas2 (Hybridoma Bank, University of Iowa, Iowa City, Iowa, USA) was used at 1:100. After primary antibody incubation, brains were washed in PBS-T and incubated with an Alexa 488 conjugated anti-mouse immunoglobulin G (Molecular Probes, Carlsbad, CA, USA). Brains were mounted in hard-set vectashield and imaged using a Fluoview confocal microscope (Olympus, Center Valley, PA, USA). Confocal stacks were processed using Metamorph 6.2 software.

Results

As mentioned above, flies are placed into a T-maze and allowed to choose between a lighted and a darkened chamber. When filter paper is wetted with 10−1 m quinine hydrochloride solution and placed into the lighted chamber, flies learn to select the dark alley more frequently over the course of the 16 trials (Fig. 1b). The minimal number of trials that yields a reliable score is 4 and thus the number of visits to the dark chamber is tabulated during four blocks of four trials (Le Bourg & Buecher 2002; Seugnet et al. 2008). To provide a more detailed view of behavior in this assay, we calculated frequency histograms for the number of photonegative choices made by 100 flies during block 1 and block 4 (Fig. 1c). Note that no fly visits the dark vial twice during block 1. In contrast, 87% of the flies enter the dark vial 2 or more times during block 4. Interestingly, a decrease in photonegative choices is reliably observed for block 3. The number of visits to the dark vial reaches a maximum during the last four trials of the test and does not improve with additional training (Le Bourg & Buecher 2002; Seugnet et al. 2008). For each analysis, a one-way anova revealed a significant main effect for block. In addition, scores in block 4 are the most significantly affected by experimental or genetic manipulation. Thus, to simplify the presentation of the data and to facilitate comparisons among multiple groups, we calculate the performance index as the percentage of visits to the dark vial during the last block of 4 trials of the 16 trial test.

In the standard test, quinine/humidity only appears in the lighted vial and the dark vial is always dry. To determine whether the association between light and quinine is required to observe an increase in visits to the dark vial, we altered the location of quinine with respect to the light and dark vial. To begin, we evaluated behavior over 16 trials in the absence of quinine. As seen in Fig. 1d, flies are strongly phototaxic and, during block 4, only choose the dark vial 10% of the time. Because flies do not increase their visit to the dark vial, these data indicate that flies do not sensitize or habituate to light over 16 trials. We then placed quinine simultaneously in both the light and dark alley to prevent an association between quinine and either chamber. Again, flies rarely chose the dark vial although they were exposed to both light and quinine/humidity. Not surprisingly, the number of photonegative choices remains low when quinine only appears in the dark chamber (Fig. 1d) and was not significantly different from the one obtained in the absence of quinine (P = 0.38). However, when quinine is presented only in the lighted chamber, flies display a fivefold increase in the number of visits to the dark alley (Fig. 1b,d).

To determine whether flies can desensitize or habituate to the aversive stimulus, we preexposed flies to filter paper wetted with quinine for 10 min and then evaluated learning over 16 trials as normal (preincubation). During the standard test, flies that select the lighted vial are only exposed to quinine/humidity for ≈5 seconds before being removed for the next trial. Thus, flies are exposed to quinine/humidity for a total of 45–60 seconds during the course of 16 trials. Despite a 10-fold increase in quinine/humidity preexposure, performance was identical to controls (Fig. 2a). Importantly, the behavior in block 1, immediately following forced quinine exposure was identical to naive flies (data not shown). Together these data indicate that exposure to quinine produced no immediate or long-term changes in preference for the dark vial.

Figure 2. Control experiments to evaluate the role of desensitization/habituation.

Figure 2

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(a) Learning score for female Cs flies using the standard protocol (base; n = 9) vs. flies preexposed to a filter paper wetted with quinine for 10 min before the beginning of the 16-trial test (preinc.; n = 9). Normal learning is observed in flies preexposed to quinine; t(16) = 1.74, P = 0.28. (b) Flies experienced 16 unpaired trials after which performance was evaluated for an additional four trials using the standard protocol. For a given trial in the unpaired test, vials were either both dark or both light, and quinine was randomly distributed to either the right or left vial. Thus, the flies would be exposed to dark quinine and light quinine. Performance remained low in flies exposed to the unpaired protocol (n = 8) vs. trained flies (n = 8); t(15) = −6.53, P = 9.8E−06. (c) Flies were trained for three blocks and then transferred to a clean maze for the final four trials to assess potential confounds of chemical traces. Learning during block 4 was similar in transferred (n = 8) and control flies (n = 10); t(16) = 0.4, P = 0.69. (d) Each yoked control fly was exposed to light/quinine or dark/dry for 16 trials at the same interval (50 seconds) and for the same duration (5 seconds) as an experimental fly performing the task (normal training) and then tested for an additional four trials using the standard protocol. Performance remained low in yoked control flies (n = 8) compared with trained flies (n = 10); t(16) = −5.89, P = 2.28E−05. (e) Flies were trained for 16 trials. During 10 of these trials, the yoked control’ fly encountered the same pairing (light/quinine or dark/dry) as during a normal test. However, on six trials (one trial in block 1, two trials in block 2, one trial in block 3 and two trials in block 4) light conditions were immediately reversed after the fly made its choice (n = 11) so that a dark choice would result in light/quinine and vice versa. Flies were then tested for an additional four blocks using the standard protocol. Few visits to the dark vial were observed in yoked control flies compared with controls (n = 8); t(19) = −3.43, P = 0.001. (f) As above, light conditions were modified on 6 of 16 trials. However, in this protocol, the fly would unexpectedly encounter quinine in the dark or dry in the lighted vial when they had made a dark or light choice, respectively. Performance remained low in yoked controls (n = 8) compared with trained siblings (n = 8); t(16) = −3.61, P = 0.001. *P < 0.05, n.s.: non significant, all tests were conducted between ZT0-ZT3:59.

Next, we removed the contingency between the light vial and quinine/humidity while exposing the flies to the same amount of sensory stimulation as during training (unpaired test). Instead of having a single trial with both a light and dark vial as is the case during learning, in this new protocol both vials in the T-maze would be the same for a given trial (both vials light or both vials dark). During each trial, quinine would appear in one of the vials randomly. Thus, the flies would be exposed to dark quinine and light quinine. Flies tested for 16 trials with this unpaired protocol enter the quinine vial 42 ± 2% of the trials. Following the unpaired protocol, flies were immediately tested for an additional block of four trials using the standard test. The score obtained was identical to naive flies; thus, no learning was observed (Fig. 2b; P = 0.22 compared with naive flies). Finally, we tested whether any chemical deposited in the maze could contribute to the observed performance. This is an unlikely possibility because the light and dark vial appear equally on the left and right side of the maze. Nonetheless, flies were tested for 12 trials in one maze and then transferred to a new (clean) maze for the final four trials (trials 13–16). As anticipated, transferring flies during block 4 did not affect performance in the final block (Fig. 2c).

To determine whether learning in this task is because of operant conditioning, we conducted three yoked control experiments (Brembs & Heisenberg 2000; Putz & Heisenberg 2002). In the first protocol, yoked control flies were exposed to light/quinine/humidity or dark/dry vials at the same time and for the same amount of time as a fly performing the task (Fig. 2d, inset, and Methods). Thus, the yoked controls experienced dark/dry quinine/humidity/light at the same frequency, duration and interval as experimental flies. The yoked control flies were then immediately tested for one block of four trials using the standard protocol. The score obtained for yoked controls was not significantly different from the one obtained by naive flies; thus, no learning was observed (Fig. 2d, P = 0.44 compared with naive flies).

The second yoked control experiments were modeled after the protocol outlined by Brembs and Wiener (2006) for flies trained in the flight arena. It is important to note that our assay does not permit the kind of temporal control of the conditioned stimulus or unconditioned stimulus that can be achieved by the flight arena. Flies were run through 16 trials. On 10 of these trials, behavior of the yoked control fly was concordant with the experimental fly and thus they encountered the same pairing (light/quinine or dark/dry) as an experimental fly. However, on six trials (one trial in block 1, two trials in block 2, one trial in block 3 and two trials in block 4), the choice of the yoked control fly was forced to match that of the experimental fly regardless of the yoked controls’ initial choice (Fig. 2e, inset, and Methods). Using this protocol, the yoked control experienced the same number of light/ quinine and dark/dry exposures, and learning was significantly impaired (Fig. 2e). In the third experiment, yoked control flies were also run through 16 trials with 10 trials being concordant (light/quinine or dark/dry), and six trials being discrepant (e.g. dry/quinine or light/dry), based upon data from experimental flies. In contrast to the experiment described above, the lighting conditions remained the same for a given trial, the only difference being that the fly would unexpectedly encounter quinine in the dark vial or dry in the lighted vial when they had made a dark or light choice, respectively. As above, these flies had significantly reduced learning scores (Fig. 2f).

Classic memory mutants

Olfactory conditioning protocols have been successfully used to identified mutants that are deficient in both acquisition and short-term memory, including linotte (lio2), latheo (latp1), pastrel (pst1), dunce (dnc1), rutabaga (rut2080) and the Drosophila D1 receptor (dumb2) (Boynton & Tully 1992; Dubnau et al. 2003; Dura et al. 1995; Han et al. 1992; Kim et al. 2007). Although there is no a priori reason to believe that any particular mutant that performs poorly in olfactory conditioning would also be deficient in an independent learning assay, consistency between assays would increase the confidence that short-term memory is indeed required for APS. Because flies mutant for lio2 have structural brain deficits and also show impairments in the acquisition of olfactory memory, we began by assessing their performance in the APS. Not surprisingly, lio2 mutants are learning impaired (Fig. 3a). lio2 mutants exhibited normal photosensitivity [photosensitivity index (PI) percentage of photopositive choices in 10 trials in the absence of quinine] and quinine sensitivity (QSI time in seconds flies reside on the nonquinine side of a chamber), indicating that the impairment was not because of alterations in sensory thresholds (Table S1).We next evaluated latp1 and pst1 mutants and found them to be impaired, as is also the case for olfactory conditioning (Fig. 3b,c;Boynton&Tully 1992;Dubnau et al. 2003). As with lio2 mutants, performance deficits could not be explained by changes in photosensitivity or quinine sensitivity (control metrics). To confirm that this phenotype maps to either the lat and pst locus, respectively, we crossed each line with the appropriate deficiency Df(2R)vg-B and Df(3L)pbl-X1 and found them to be impaired, indicating that the deficits were because of disruption of lat and pst. Interestingly, while both male and female dnc1 mutants performed poorly in the APS, only male rut2080 mutants are learning impaired (Fig. 3d,e). Female rut2080 mutants achieve the same learning score and display the same rate of acquisition as wild-type females over 16 trials (Fig. 3e,f). These data provide independent confirmation for sexually dimorphic learning behavior in the APS for rut2080 mutants (Perisse et al. 2007). Learning impairment in male dnc1 mutants can be rescued by expressing wild-type dunce in the MBs using c309-GAL4 (Fig. 3g). Similarly, the learning impairments seen in male rut2080 mutants can be rescued by expressing wild-type rut in the mushroom bodies (MBs) using either the 247-GAL4 or c309-GAL4 drivers (Fig. 3h,i). Recent studies have shown that flies mutant for the Drosophila dopamine 1 receptor (dumb2) are impaired in both olfactory conditioning and in the APS (Kim et al. 2007; Seugnet et al. 2008). Importantly, performance in both olfactory conditioning and the APS in dumb2 mutants could be rescued when the dDA1 receptor was expressed in the MBs using 247-GAL4. Together these data indicate that several mutants deficient in olfactory conditioning also show deficits in APS and that, like olfactory conditioning, the MBs play an important role in APS.

Figure 3. Phototaxic suppression in memory mutants.

Figure 3

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(a) Female lio2 mutants (n = 9) are learning impaired compared with controls (n = 10); t(16) = −3.91, P = 0.0005. As previously described, lio2 mutants have abnormal mushroom bodies with reduced or absent α lobes and fused β/γ lobes. (b) Female latp1 flies (n = 8) show performance decrements compared with controls (n = 9). Impairments are still observed in latp1/Df(2R)vg-B (n = 7); one-way anova for genotype (F2,21 = 4.74, P = 0.019). (c) Females flies mutant for pastrel (pst1, n = 6) and pst1/Df(3L)pbl-X1 (n = 8) perform significantly worse than controls (n = 10); one-way anovas for genotype (F2,21 = 8.98, P = 0.001). (d) Both male (n = 8) and female (n = 9) dnc1 mutants are learning impaired compared with their respective controls (n = 8). A 2(sex) × 2(genotype) anova yielded significant main effect for genotype (F1,29 = 8.35, P = 0.006). (e) Male rut2080 mutants (n = 10) show impairments in learning, whereas rut2080 females (n = 10) show normal performance. A 2(sex) × 2(genotype) anova yielded significant sex × genotype interaction (F1,36 = 10.22, P = 0.003). (f) Cumulative visits to the dark vial over four blocks is similar in female Cs and rut2080 flies indicating that they do not differ in the rate of acquisition. (g) Learning impairments can be restored in male dnc1 mutant flies by expressing a UAS-dnc construct in the MB with the c309 GAL4 driver; one-way ANOVA for genotype (F2,34 = 3.29, P = 0.049). (h, i) rut2080 males flies bearing the 247-GAL4 driver (rut;247, n = 6) or the UAS-rut construct alone (rut;UAS-rut, n = 8) show learning impairments. rut;UAS-rut/247 flies (n = 11) and rut;UAS-rut/c309 (n = 8) express wild-type rut in the MB and show normal learning; one-way anova for genotype (F3,49 = 2.37, P = 0.04). *Planned comparisons with Tukey correction (P < 0.05). n.s.: non significant. All tests were conducted between ZT0-ZT3:59.

The role of the MBs on retention, acquisition and retrieval in APS

A role for the MBs in memory acquisition, retention and retrieval has been firmly established using olfactory conditioning (Heisenberg 2003). Hydroxylurea ablation of MB suggests that they are also involved in APS (Seugnet et al. 2008). However, the role of the MBs in memory retention and retrieval for APS remains poorly understood. The APS depends, in part, upon response inhibition, and thus contextual cues are strong determinants of behavior in the T-maze. For example, eliminating quinine also removes the contextual cues provided by humidity such that trained flies immediately behave like naive flies when tested in the T-maze in the absence of quinine (Le Bourg 2005; Le Bourg & Buecher 2002). This observation precludes one from testing memory in the APS various times after training in the absence of the unconditioned stimulus as is typically the case for olfactory conditioning (Tully & Quinn 1985). Thus, memory is tested in the APS by training flies for 16 trials, inserting a delay of various durations, and then testing performance during four trials using the standard training conditions. It is important to emphasize that a block of four trials is the minimal unit that can be used to assess performance; a single trial is insufficient to evaluate behavior (Le Bourg & Buecher 2002; Seugnet et al. 2008). Using this protocol, each ‘test’ trial is also an additional training trial. Similar protocols have been employed for courtship conditioning, where it has been suggested that reproducing the conditions of reinforcement during subsequent testing may result in better retention than in tests without reinforcement (Kamyshev et al. 1999; Keleman et al. 2007). Flies complete each trial in ≈45 seconds and rarely visit the dark alley on more than two consecutive occasions. These data suggest that a fly must be able to remember the association between light and quinine for a minimum of 45–90 seconds. To test this hypothesis, we evaluated retention in those Cs and rut2080 females that learned after 16 trials (performance score of 0.5). To identify the retention interval, we retested learning in flies after a delay of 2, 5 or 15 min. Wild-type Cs flies maintained performance following 2- and 5-min delays and were significantly degraded after 15 min (Fig. 4a). Performance in Cs flies was similar to naive flies (no memory) after 1 h, indicating that this procedure does not induce long-term memories (data not shown). In contrast, rut2080 flies were significantly impaired after a 2-min delay and performed similar to naive flies by 5 min (Fig. 4a). Note that both Cs and rut2080 females exhibit the same rate of learning acquisition (Fig. 3f) and thus the learning score achieved after the delay cannot be attributed simply to different rates of relearning. Thus, the APS requires the animal to remember the association between light and an aversive stimulus for a short interval of <2 min.

Figure 4. Retention, acquisition and retrieval.

Figure 4

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(a) Performance requires <2 min of memory retention: Cs and rut2080 flies that achieved a performance score of 0.5 during block 4were retested after a 2-, 5- or 15-min delay. A 2(genotype: Cs, rut2080) × 3(delay: 2, 5, 15 min) × 2 (test: test, retest) repeated measures anova showed a significant genotype × test interaction (F1,32 = 22.87, P = 3.73E−5). Cs flies retained memory of the association for up to 15 min. rut2080 flies retain the association for no more than 2 min. (b) Blocking neurotransmitter release in a large subset of Kenyon cells by expressing the UAS-shits1 transgene under the control of MBswitch does not impair learning: 2(RU486 vs. ETOH) × 3(genotype) (F2,43 = 0.21, P = 0.81). Both RU486 (RU+) and ETOH (vehicle, RU−) fed flies were transferred to the nonpermissive temperature (34°C) for 15 min and then tested at 34°C. (c) MBSwitch>UAS-shits1 flies fed RU+ (n = 5) and RU− (n = 7) were trained at 34°C and evaluated for retrieval after a 5-min delay at 34°C. Blocking neurotransmitter during retrieval impairs performance; t(10) = −1.86, P = 0.046 (left). Both parental lines, MBSwitch/+ (n = 5) and UAS-shits1/+ (n = 5) displayed normal retrieval at 34°C; t(8) = −0.53, P = 0.6 (right). (d) MBSwitch>UAS-shits1 flies fed RU+ (n = 5) and RU− (n = 5) flies were trained at 34°C and evaluated for retrieval after a 5-min delay at 24°C. Retrieval was identical for RU+ and RU− flies at 24°C; t(8) = 0, P = 1. *Planned comparisons with Tukey correction (P < 0.05). n.s.: non significant. All tests were conducted between ZT0-ZT3:59.

Previous studies have shown that blocking neurotransmitter release from MBs αβ neurons during training does not interfere with the acquisition of olfactory learning but prevents retrieval (Dubnau et al. 2001; Krashes et al. 2007; McGuire et al. 2001; Schwaerzel et al. 2003). To determine whether this might also be true for our learning paradigm, we expressed a temperature-sensitive allele of the Drosophila dynamin gene shibire (UAS-shits1) in the MBs using the GeneSwitch system (MBSwitch). MBSwitch is highly expressed in αβγ lobes (Mao et al. 2004), and vesicle recycling can be reversibly blocked in tissues expressing UAS-shits1 when the temperature is raised above 31°C. We have previously shown that RU486 (RU+) does not affect the ability of Cs flies to learn in this assay (Seugnet et al. 2008), and previous reports show that RU does not alter olfactory conditioning, phototaxis, geotaxis, locomotion, the escape response, sleep or sleep homeostasis (Joiner et al. 2006; Mao et al. 2004). Thus, we evaluated acquisition when flies were trained at 34°C. As seen in Fig. 4b, MBSwitch>UAS-shits1 flies acquired the association between light and quinine at 34°C although neurotransmitter release was blocked in a third of the Kenyon cells. We then tested retrieval in MBSwitch>UAS-shits1 flies that were trained at 34°C and then tested 5 min later at either the nonpermissive or permissive temperature. As seen in Fig. 4c, RU+ flies exhibited significantly lower scores than their RU siblings at 34°C (Fig. 4c). However, MBSwitch>UAS-shits1 flies trained at 34°C and tested after a 5-min delay at 24°C retrieved the association as previously reported. Parental lines (MBSwitch/+ and UAS-shits1/+) fed RU486 retrieved the association when trained and tested at 34°C (Fig. 4c). Together, these data suggest that the MBs play a role in both memory acquisition and retrieval in this paradigm as reported for other established learning assays.

Utility of APS for identifying learning deficits

The APS is a particularly sensitive assay for identifying learning deficits following sleep disruption (Seugnet et al. 2008). A mutation may alter both sleep regulation and sensory thresholds. To determine whether it is possible to evaluate learning in flies with reduced photosensitivity, we evaluated performance in Cs flies at two different light levels. At high light levels (9500 lx), flies obtain a PI of 82%, while at low light levels (73 lx) the PI was 64%. Although baseline learning is shifted slightly higher at low light levels, both groups show significant reductions in learning following sleep loss during block 4 (Fig. 5a,b). Importantly, the magnitude of the percent change in learning between baseline and sleep deprivation during block 4 is similar in flies with high and low PIs (−22 ± 9% and −18 ± 5%, respectively). To determine whether a low preference for light is equivalent to an inability to detect light, we evaluated blind flies (Stark et al. 1993). Totally blind flies would be expected to choose light in a random manner, and thus obtain a score of 0.5 regardless of the block. This is effectively the case, as seen in Fig. 5c: males mutant for no receptor potential A (NorpA36) make 50% of photonegative choices in all blocks, thus showing no evidence of learning. NorpA36 males’ performance is not altered by sleep loss. Thus, reduced photosensitivity produces a much different outcome than an inability to detect light. Moreover, by measuring the difference between baseline and sleep deprivation, the APS can be used to evaluate how sleep loss impacts learning across flies with a range of photosensitivities.

Figure 5. Detection of cognitive impairments using phototaxic suppression.

Figure 5

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(a) Reduced photosensitivity does not impair learning and does not prevent sleep-deprivation-induced cognitive impairments. Female Cs flies with a PI of 0.82 (9500 lx, n = 10) learn as well as flies with a PI of 0.64 (73 lx, n = 9); male blind flies NorpA36 (n = 10) do not change their behavior over 16 trials. Sleep deprivation only results in a significant learning impairment in flies with vision. A 3(vision) × 2(base, SD) × 4(blocks) repeated measures anova shows a significant vision×blocks interaction (F6,159 = 6.13, P = 8.42E−6). (b) Female Cs flies learn to suppress phototaxis when electric shock is used as the negative reinforcer (n = 17); electric shock does not produce such a strong association as to prevent learning impairments following sleep loss (n = 15) t(30) = 2.24, P = 0.016. (c) Female flies expressing human tau (c155;UAS-tauwt, n = 13) show significant reductions in learning compared with parental lines (c155/+ n = 10 and UAS-tauwt, n = 7 F2,29 = 4.00, P = 0.029). (d) Learning in 23- to 25-day-old MBSwitch>UAS-Tauwt (n = 14) flies fed RU486 (RU+) was impaired compared with age-matched siblings fed vehicle (RU−) and 25- to 30-day-old Cs flies (n = 10) (F2,33 = 3.48, P = 0.042). *Planned comparisons with Tukey correction (P < 0.05). All tests were conduced between ZT0-ZT3:59.

Although quinineworks effectively as a negative reinforcer in the APS, we wanted to know whether other, more traditional, negative reinforcers could substitute for quinine/humidity in the APS. Thus, we acquired the copper grids used for olfactory conditioning and placed one into the lighted vial to provide an electric shock (see Methods). As seen in Fig. 5d, flies learn to inhibit their photopositive tendencies and choose the dark vial more frequently when electric shock is used as a negative reinforcer. Importantly, electric shock is not so strong as to prevent learning impairments following sleep loss. Shock avoidance was evaluated by placing six groups of five flies into a transparent cylindrical tube for 1 min and tabulating the number of flies that avoided the half of the tube covered by the copper grid. Under baseline conditions 90 ± 4% of flies avoided the copper grid after 1 min and avoidance was not changed by sleep deprivation 90 ± 6%. Thus electric shock can be used in the APS as a negative reinforcer.

As mentioned previously, labs that do not have the resources to routinely use olfactory conditioning may wish to evaluate an aspect of cognitive behavior. Is the APS sensitive enough to measure changes in performance in fly models of human conditions associated with cognitive deficits? Recently, several labs have shown that mutations for genes that model aspects of Alzheimer’s disease are deficient in olfactory conditioning. Deficits have been found in flies carrying an artificial mutation in human amyloid-b-42, flies mutant for presenilin and flies expressing a wild-type version of the microtubule associated protein Tau (UAS-Tauwt), to name a few (Iijima et al. 2008; Knight et al. 2007; Mershin et al. 2004). Interestingly, the learning deficits frequently precede other signs of pathology (Iijima et al. 2008; Mershin et al. 2004). Consistent with previous reports, we show that young flies expressing UAS-Tauwt throughout the brain using elav-GAL4 (C155) show learning impairments (Fig. 5e). Both parental lines were unaffected and all lines showed normal PI and QSI. As directed expression of Tau to the MBs has been shown to disrupt olfactory conditioning (Mershin et al. 2004), we evaluated behavior in the APS when wild-type Tau was expressed in the MBs using the GeneSwitch system. Female MBSwitch/+;UAS-Tauwt were placed onto food containing RU486 on days 3–5. No learning impairments were seen in 10- to 12-day-old MBSwitch/+; UAS-Tauwt flies (data not shown). Thus, flies were maintained on food containing RU until they were 23–25 days old; food was changed every 5 days. As seen in Fig. 5f, age-matched Cs flies learn normally at this age, while RU+-treated flies are learning impaired. Age-matched siblings fed vehicle (RU−) also displayed normal learning. Both RU+ and RU− treated MBSwitch/+;UAS-Tauwt flies displayed normal PI and QSI, indicating that the change in performance was not because of changes in sensory thresholds. Together, these data indicate that the APS is sensitive enough to measure deficits in fly models of human conditions associated with cognitive deficits.

Discussion

Aversive phototaxic suppression is a simple, inexpensive and reliable assay that can be used to assess how genetic manipulations or environmental perturbations alter short-term memory and response inhibition. The assay does not require special equipment and does not require dedicated space, and the apparatus can be easily stored in a drawer in between uses. A reliable assessment of learning can be achieved by evaluating behavior in as few as 8–10 individual flies in 2 h. Moreover, the protocol does not harm the fly, which can then be retrieved and used for other purposes, including evaluating learning at subsequent ages, etc.

Olfactory conditioning has proven to be a powerful tool to identify genes involved in the formation, stabilization and retrieval of memory. One reason olfactory conditioning has been so powerful has been that it is possible to exclude nonassociative effects using differential conditioning protocols. Such protocols are not easily applied to the APS, the heat box or to courtship conditioning thereby requiring additional characterization of each of these assays (Le Bourg 2005; Mehren et al. 2004; Perisse et al. 2007; Putz & Heisenberg 2002). Our data extend the results of Le Bourg and provide evidence that the increased visits to the dark vial in the APS cannot be attributed to either sensitization or habituation. Importantly, learning is only observed under a narrow set of conditions defined by a precise association between light and quinine/humidity. Indeed, a 10-fold increase in the duration of exposure to quinine does not alter preference for the dark vial, indicating that habituation does not influence behavior under the conditions used in these experiments. Similarly, no evidence for learning is observed in flies for which the contingency between the light vial and quinine/humidity has been removed but that receive the same amount of sensory stimulation as during training (unpaired test).

In operant learning, an animal must learn the relationship between the unconditioned stimulus and its own behavior (Brembs & Heisenberg 2000; Putz & Heisenberg 2002; Rescorla 1988). In the APS, the fly can avoid the quinine/ humidity by selecting the dark vial. If the fly cannot accurately avoid the quinine based upon its own behavior, as is the case in our yoked control experiments, learning is disrupted. In these experiments, the two flies received the same stimulus protocol, but only the experimental fly has operant control. Thus, the difference in the final performance of the two flies can be attributed to operant learning. Because the APS does not allow precise temporal control over the administration of either the CS or the US, as is the case in the flight arena (Brembs & Wiener 2006), we conducted three independent experiments where the exposure of a fly to quinine/humidity and either dark or light was determined by the behavior of an experimental fly. Even when the fly’s choice resulted in an incongruent outcome in only 6 of 16 trials, acquisition was significantly disrupted. Interestingly, the scores obtained with the partially modified training protocols shown in Fig. 2e,f were higher than those obtained by naive flies, suggesting that the trials that remained consistent with the standard test were still able to elicit some association. Thus, together with the work conducted by Le Bourg, these data indicate the APS is an operant task.

We conducted a series of experiments to determine the extent to which results obtained using olfactory conditioning would be observed in the APS. To begin, we evaluated five classic learning/memory mutants that were identified using olfactory conditioning (Heisenberg 2003; Margulies et al. 2005); all were significantly impaired. Moreover, we rescued learning deficits in male dnc1 and rut2080 by expressing their respective wild-type transgenes in the MBs. We proceeded to block synaptic release from the MB αβγ lobes and found that this was sufficient to prevent memory retrieval. This result indicates that MBs are playing a role in memory recall in APS as has been reported for olfactory conditioning (Dubnau et al. 2001; McGuire et al. 2001). MBs are sensitive to context change in visual learning (Brembs & Wiener 2006; Liu et al. 1999). Thus, impaired memory retrieval may also originate from an inability to recall an association after a temporary context disruption. Note that as the MBSwitch driver uses the 247 promoter, it does not induce expression in either the α′ or β′ lobes and thus should not affect acquisition (Krashes et al. 2007). We have previously shown that other manipulations that disrupt performance in olfactory conditioning, including ablating the MBs with hydroxylurea, and pharmacological and genetically disrupting dopamine also disrupt performance in the APS (Seugnet et al. 2008). We have also shown that, like olfactory conditioning, flies mutant for the Drosophila dopamine 1 receptor (dDA1) are learning impaired in the APS and that this deficit could be rescued when the dDA1 receptor was expressed in the MBs. Altogether, we have conducted 12–13 independent experiments in the APS that produce outcomes that are similar to that obtained with olfactory conditioning. Thus, when taken in their entirety, our data strongly indicate that the APS is an effective assay that can be used to evaluate learning in flies.

A major difference between the APS and other assays including olfactory conditioning, the heat box, courtship conditioning and the flight arena is that in the APS it is not possible to evaluate performance in the absence of the unconditioned stimulus. In the case of the APS, removing quinine during the test also removes a contextual cue, humidity, that is required for recall. Thus, every test requires quinine/humidity and is also training. This concern applies strongly to our retention experiments. That is, after a delay, the difference in learning between two groups could reflect either poor retention or a different rate of relearning (acquisition) in one of the groups. The rut2080 females exhibit the same rate of acquisition as Cs females (Fig. 3f), thus favoring the interpretation the poor learning scores in rut2080 are because of poor retention (Fig. 4a). The effects of relearning are reduced by evaluating behavior over four trials (the minimal unit that can reliably evaluate performance). Nonetheless, the issue of relearning may very well limit the use of the APS for evaluating retention.

Although our data show that we can identify mutations that disrupt short-term memory, the APS may be best suited to investigate how environmental perturbations alter learning. For example, we have shown that the APS is sensitive for detecting learning deficits following sleep loss (Seugnet et al. 2008). When used in this context, the learning score of a fly that has had a full nights’ sleep is compared with a sleep-deprived sibling that shares the same genetic background and thus similar photosensitivities, quinine sensitivity and motoric abilities. A wild-type response to sleep loss is defined as a learning impairment after sleep deprivation when compared with its genetically identical, untreated sibling. Thus, if a genotype can obtain a learning score that can get worse (≈0.375 or above), then potential differences in baseline-learning scores between genotypes are not directly relevant to the question under investigation. That is, our primary question is: does a mutation prevent sleep-loss-induced learning impairments? If an experimental line does not show a learning deficit after sleep loss but the parental lines are impaired when compared with their respective baseline learning scores, it is not meaningful to make additional and unnecessary comparisons of the sleep-deprived experimental line to the learning score achieved by the parental lines after sleep loss. A relevant example from this manuscript is shown in Fig. 5a,b. While both baseline and sleep-deprived learning scores differ in flies tested at high- and low-light intensities, each group shows similar reductions in performance after sleep loss. Thus, we conclude that both groups display a wild-type response to sleep loss.

Examination of Fig. 5 reveals that sleep deprivation disrupts learning by 18–25% compared with baseline. Is a difference of this magnitude meaningful? Such a question is difficult to address. From a statistical perspective, the effect size is quite large and the results are highly reproducible. Power analysis using G*Power (http://www.psycho.uni-duesseldorf.de/aap/projects/gpower/) calculates a Cohen’s d of 1.8 and that only eight flies/group are needed to obtain statistical differences. By way of comparison, olfactory conditioning uses five to eight batches of 50–100 flies, the heat box uses 50–200 flies and courtship conditioning uses 16–25 flies. Is a difference of 18–25% functionally relevant to the organism? Again, this is a difficult question to assess. Interestingly, the difference in learning between flies mutant for genes that are known to effect learning and memory is also ≈18–30% as is also true for flies that have had the MBs ablated. Thus, performance of a fly after sleep deprivation is roughly comparable with being mutant for a gene that is important for memory formation or having no MBs. These data emphasize that performance in the APS is constrained by both ceiling and floor effects. While the range of scores is narrow in the APS, the effect size following sleep deprivation is not small. Importantly, the magnitude of the deficit observed following sleep deprivation in flies is well within the range of effect sizes observed following sleep loss in humans and rodents across a number of cognitive domains (Frey et al. 2004; Fu et al. 2007; Graves et al. 2003; Pierard et al. 2007). Clearly what may appear to us to be small differences in short-term memory may have important consequences in ecological settings including finding food or a mate and avoiding danger.

For APS to be a useful assay, it should be able to detect impairments in fly models of human conditions associated with cognitive deficits. Expression of Aβ40 and Aβ42 peptides results in impairments in olfactory conditioning (Iijima et al. 2004, 2008). Similarly, expression of Tau in the MBs also induces deficits in memory using olfactory conditioning (Mershin et al. 2004). We observed a substantial impairment in young flies expressing Tauwt using a pan-neuronal driver. Moreover, learning impairments were also detected when Tauwt was expressed in the MBs using the GeneSwitch system, suggesting that the expression of Tau during development is not required to induce deficits. Importantly, these data indicate that the APS is a simple and reliable assay that can be effectively used to identify impairments in short-term memory in fly models of human disease states.

Supplementary Material

Supp Table S1

Acknowledgments

We thank Thomas Préat, Matthew Thimgan, Jeff Donlea and William Vanderheyden for helpful comments This study was funded in part by 1 R01 NS051305-01A1, 5 K07 AG21164-02 and the McDonnell Center for Cellular and Molecular Neurobiology.

Footnotes

Supporting Information

The following supporting information are available for this article.

Table S1: Photosensitivity and quinine sensitivity

Additional supporting information may be found in the online version of this article.

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author.

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Supplementary Materials

Supp Table S1
NIHMS564348-supplement-Supp_Table_S1.pdf (58.3KB, pdf)

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