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Parametric and genetic analysis of Drosophila appetitive
long-term memory and sugar motivation
J. Colomb, Laetitia Kaiser, M.-A. Chabaud, T. Preat
To cite this version:
J. Colomb, Laetitia Kaiser, M.-A. Chabaud, T. Preat. Parametric and genetic analysis of Drosophila
appetitive long-term memory and sugar motivation. Genes, Brain and Behavior, Wiley, 2009, 8 (4),
pp.407-415. �10.1111/j.1601-183X.2009.00482.x�. �hal-02667883�
Parametric and genetic analysis of Drosophila
appetitive long-term memory and sugar motivation
J. Colomb
†, L. Kaiser
‡,§,¶, M.-A. Chabaud
†,‡and
T. Preat*
,††
Ge`nes et Dynamique des Syste`mes de Me´moire, CNRS UMR 7637, ESPCI, Paris,‡De´veloppement Evolution et Plasticite´ du Syste`me Nerveux, CNRS UPR 2197, Institut de Neurobiologie Alfred Fessard, Gif-sur-Yvette, and§INRA Physiologie de l’Insecte Signalisation et Communication, UMR 1272, Versailles, France *Corresponding author: T. Preat, Ge`nes et Dynamique des Syste`mes de Me´moire, CNRS UMR 7637, ESPCI, 10 rue Vauquelin, 75005 Paris, France. E-mail: thomas.preat@espci.fr
Distinct forms of memory can be highlighted using different training protocols. In Drosophila olfactory aver-sive learning, one conditioning session triggers memory formation independently of protein synthesis, while five spaced conditioning sessions lead to the formation of long-term memory (LTM), a long-lasting memory depen-dent on de novo protein synthesis. In contrast, one session of odour–sugar association appeared sufficient for the fly to form LTM. We designed and tuned an apparatus that facilitates repeated discriminative condi-tioning by alternate presentations of two odours, one being associated with sugar, as well as a new paradigm to test sugar responsiveness (SR). Our results show that both SR and short-term memory (STM) scores increase with starvation length before conditioning. The protein dependency of appetitive LTM is independent of the repetition and the spacing of training sessions, on the starvation duration and on the strength of the uncondi-tioned stimulus. In contrast to a recent report, our test measures an abnormal SR of radish mutant flies, which might initiate their STM and LTM phenotypes. In addi-tion, our work shows that crammer and tequila mutants, which are deficient for aversive LTM, present both an SR and an appetitive STM defect. Using the MB247-P[switch] system, we further show that tequila is required in the adult mushroom bodies for normal sugar motivation.
Keywords: Appetitive learning, conditioning, crammer, mem-ory dynamics, motivation, protein synthesis, radish, starva-tion, sugar responsiveness, tequila
Received 17 October 2008, revised 23 December 2008, accepted for publication 5 January 2009
Appetitive learning plays a role in different psychiatric disor-ders, like substance abuse disordisor-ders, eating disordisor-ders, depression and schizophrenia (Martin-Soelch et al. 2007). Yet, this form of learning is rarely studied in Drosophila, with research generally focusing on aversive learning paradigms. Many behavioural, anatomical, cellular and molecular pro-cesses involved in Drosophila learning and memory have been discovered in the last 30 years (for a review, see Davis 2005), but, until recently, these processes were considered nearly exclusively in aversive learning paradigms.
The scarcity of appetitive learning studies can be explained by the difficulty in finding suitable appetitive stimuli (Martin-Soelch et al. 2007). Usually, food sources (for instance sucrose) are used as an unconditioned stimulus (US) on food-deprived animals. In honeybees, the starvation regime was shown to be correlated both with sugar responsiveness (SR) and appetitive memory performance (Scheiner et al. 1999). This correlation makes it difficult to interpret memory retention differences without a reliable control for the animal sugar motivation: the lack of such a test is a crucial caveat in the majority of studies on appetitive memory in Drosophila (see Discussion).
Despite the technical difficulties, analogies between appe-titive and aversive processes were found in Drosophila, such as the involvement of the adenylate cyclase rutabaga (rut; Schwa¨rzel et al. 2003; Thum et al. 2007; Zars et al. 2000) and the dDA1 dopamine receptor (Kim et al. 2007b) in the mushroom bodies for learning, and the requirement of normal synaptic output of these same neurons for short-term mem-ory (STM) retrieval (Dubnau et al. 2001; Krashes et al. 2007; McGuire et al. 2001; Schwa¨rzel et al. 2003). In contrast, the radish (rsh) gene was suggested to have different functions in the two learning situations (Krashes & Waddell 2008). More-over, the kinetics of aversive and appetitive memory retention are different (Tempel et al. 1983). Appetitive learning leads to the formation of protein-synthesis-dependent long-term memory (LTM) after only one training session of 2 min (Krashes & Waddell 2008), while aversive learning leads to protein-synthesis-independent memory, unless five spaced sessions are experienced (Dudai 2002; Isabel et al. 2004; Mery & Kawecki 2005; Tully et al. 1994).
This study presents a new apparatus designed and tuned for repeated and synchronized exposure of flies to odour and sugar as well as a new paradigm to test SR. We validated our SR test using a positive control, comparing the scores of flies differently starved before the test. We further confirmed in our set up that LTM was formed after one session (Krashes &
¶Present address: Institut de Recherche pour le
De´veloppe-ment, UR 072, c/o Laboratoire Evolution, Ge´nomes et Spe´ci-ation, UPR 9034, CNRS 91198, Gif-sur-Yvette cedex, France
Waddell 2008); and we additionally showed that this forma-tion is independent of the number and spacing of sessions as well as on the US strength and the starvation time. We then studied the genetics underlying this behaviour, questioning previous interpretations and results on rsh, tequila (teq) and crammer (cer) appetitive memory phenotypes (Krashes & Waddell 2008). Indeed, rsh, a gene known to be required during the formation of protein-synthesis-independent aver-sive memory (Folkers et al. 2006; Tully et al. 1994) and two genes involved in aversive LTM, teq (Didelot et al. 2006) and cer (Comas et al. 2004), appear to be involved in starvation-induced SR.
Materials and methods
Flies
Drosophila melanogaster, wild-type Canton Special (Canton S) and mutant flies were reared at 188C on a standard diet. For the experi-ments, 80–100 flies (1–2 days old) were transferred to fresh medium for 24 h at 188C before starvation. cerPand teqPlines, which were previously isolated in our laboratory, were outcrossed for five gen-erations before being used. rsh mutant flies were obtained from S. Waddell (UMASS, MA, USA).
Starvation protocols
Standard protocol
Before conditioning, groups of 40 flies were kept in plastic bottles (175 ml; Greiner Bio-one 960177, Courtaboeuf, France) with a cotton wool pad (local store) imbibed with 6.5 ml of mineral water (pH¼ 7.2; Evianâ, Danone, Paris, France) at 258C for 21.5, 14.5 or 7.5 0.5 h. Except when the test was performed more than 1 day after condi-tioning, flies were starved in the same bottle after training; they were kept at 258C if tested the day of the conditioning or at 188C if tested on the following day. For the 3-day tests, flies were transferred from the starvation bottle into a food bottle for 2 h at 22 h and 46 h after conditioning, and then starved again for 22 h before tests began. For the 7-day test, flies were starved for 22 h after conditioning, kept on food until day 6 and then starved again for 22 h before tests began. A short feeding period between conditioning and testing appears not to affect memory scores when flies are starved again (Fig. 2; Krashes & Waddell 2008). In contrast, the longer feeding period might partly explain the drop in the 7-day memory retention score (Fig. 2), possibly because feeding experience may interfere with memory for the sugar-reinforced odour.
CXM treatments
We modified a protocol from Yu et al. (2006). After 1 day on fresh medium, flies were transferred into 15 ml Falcon tubes with a What-man filter paper (1 2.5 cm) soaked with 125 ml of 35 mM cyclohex-imide (CXM) solution (94% purity; Sigma C7698, St Louis, MO, USA) diluted in mineral water (pH¼ 7; Volvicâ, Danone, Paris, France), or with mineral water alone (control), for 20 h. Flies were then trans-ferred into regular starvation bottles for 1 h before being conditioned, and transferred back into those bottles afterwards. In control experi-ments assessing sugar or odour responsiveness, the same protocol was used but flies were tested for their SR instead of being conditioned, or conditioned and then tested for their SR or their odour acuity instead of for their memory.
CXM for 7-h starvation
In this experiment, we first put flies on CXM without starving them during 13 h (in a Falcon tube as above, but with a solution of CXM containing ethanol and 5% sugar). Then, we transferred the flies to normal food bottles for 1 h to ensure that the flies were not food
deprived. Finally, we started the 7-h period of starvation in CXM (as above). We then transferred the flies to normal starvation bottles 1 h before conditioning and put them back into the same bottles afterwards. Control flies were treated similarly, except for the absence of CXM in the solutions.
RU486 treatment
While using the P[switch] system, we transferred the flies onto food with RU486 (40 mg/l; Sigma-Aldrich M8046, St Louis, MO, USA) for 1 day and then transferred them to 15-ml Falcon tubes with Whatman filter paper (1 2.5 cm) soaked with 125 ml of RU486 solution (100 mg/l, mixed in Evian water at about 508C) for 20 h. Flies were then transferred into regular starvation bottles for 1 h before being conditioned or tested for their SR and transferred back to those bottles after training.
Conditioning apparatus
The conditioning apparatus (Fig. 1) was based on the barrel designed by Pascual & Preat (2001) for electric shock conditioning, in which Drosophila are loaded into six compartments lined with an electrifiable copper grid. The appetitive barrel has three compartments, in which we place a removable plastic tube (internal diameter¼ 2.4 cm) covered with sugar on 2/5 of their surface (Fig. 1c). In each compart-ment, a removable brass hemicylinder (the rotating inserts; Fig. 1b) masks the sugar when flies are loaded (Fig. 1d). The hemicylinders are mounted on stainless steel ball bearings. Therefore, when the barrel is rotated by 1808, the hemicylinders stay in the inferior part of the tubes by the force of gravity and the sugar becomes accessible to the flies (Fig. 1e). Barrels are gently rotated as soon as the condi-tioned odour is flowing through the barrel. Sugar intake is almost immediate, ensuring synchrony of odour–sugar presentation (90% of flies extend their proboscis, touching the sugar within 5 seconds following rotation of the barrel). Computer-controlled electronic valves ensure fresh air and odour delivery at a flow rate of 2 l/min for each barrel.
Odour sources
3-octanol (>95% purity; Fluka 74878, Sigma-Aldrich, St Louis, MO, USA) and 4-methylcyclohexanol (99% purity; Fluka 66360) were diluted in paraffin oil (Prod. 24 679.360; VWR international, Sigma-Aldrich, St Louis, MO, USA) at 3.60 104
and 3.25 104 M respectively (Pascual & Preat 2001).
Sugar delivery
If not otherwise indicated, a 1.5Msucrose solution in mineral water (Evian) was applied on 2/5 of the inner surface of plastic tubes, using a piece of felt (2.7 4.5 cm, local store) imbibed with 1-ml sugar solution (three tubes were prepared with one felt). To let the sugar dry, tubes were left at room temperature for 18–28 h before conditioning. The sugar tubes were replaced for each reciprocal experiments for massed and spaced conditionings.
Conditioning session
Conditioning and testing were performed at 258C and 65–80% relative humidity.
After an initial period of 90 seconds of non-odourized airflow, one session consisted of 60 seconds of one of the odours, 52 seconds of non-odourized airflow, 60 seconds of the other odour and 52 seconds of non-odourized airflow. We rotated the barrels to allow for sugar association at the onset of odour delivery for 1 min. We associated sugar with either the first or the second odour. As expected from a previous report (Kim et al. 2007a), this had no effect on performance and data were pooled. In order to average out potential odour preference bias, one experiment consisted of two reciprocal con-ditionings with two groups of flies from the same rearing bottle, in
408
Genes, Brain and Behavior (2009) 8: 407–415which the order of the two odours was swapped. In the case of five massed conditioning sessions, there was no time delay between sessions. In the five spaced conditionings procedure, a 15-min period of nonodourized airflow was introduced between the end of a session and the beginning of the next one. For unpaired conditioning, sugar exposure was scheduled 7 min after the end of the session.
Memory tests
Tests were performed with a T-maze apparatus (Tully & Quinn 1985). Flies had to choose between two arms, delivering octanol vs. methylcyclohexanol odour. After 3 min (Figs 2–4) or 1 min, flies were trapped in either arm (the two durations had no effect on the memory scores). An index was calculated as the difference between the number of flies in each arm, divided by the sum of flies in both arms. Two indexes were calculated from two reciprocal experiments and were averaged to give a memory score, labelled performance index theoretically ranging from1 to 1 (all flies choosing the conditioned
odour for the two reciprocal conditionings), with naive flies giving on average a zero score.
Olfactory acuity
Tests were performed with a T-maze apparatus. One odour was tested against its solvent (paraffin oil), for 3 min. The response index (RI) was calculated as above and used as a score. The odour was delivered alternately through the right or left side of the maze. The index ranged theoretically from 1 (total repulsion) to 1 (total attraction).
SR tests
Tests were performed with a T-maze apparatus, in a protocol modified from Kim et al. (2007b). One arm with sugar was tested against one regular arm, in an odourless airflow. Flies were trapped in either arm after 1 min under red light. The sugar arm was placed alternately on the right or left. The RI was calculated as above and used as a score. We prepared the sugar arm as follows: a sucrose solution in mineral water (Evian) was applied on a band in the inner surface of plastic test tubes, using a piece of imitation felt (0.5 6 cm) imbibed with 0.4-ml sugar solution (three tubes prepared with one felt). If not noted otherwise, we used a concentration of 0.15M. For the sugar to dry, tubes were left at room temperature for 18–28 h before tests. Each tube was used for four consecutive tests.
Data analysis
Treatment effects on memory scores or RIs to sugar or odours were analysed using Student’s t-tests to compare two groups, or one-way ANOVAfollowed by the Newman–Keuls multiple comparisons test if significant at P < 0.05, to compare several groups. A paired t-test was used to compare scores of male and female flies that were condi-tioned and tested together. When the condition on variances was not fulfilled (Zar, 1999), the Kruskal–Wallis test was used. Scores indicated in the text are given as mean SEM.
Results
Long-lasting appetitive memory is formed after single
or multiple conditioning sessions
We investigated the time–course of memory on a long timescale. While unpaired conditioning leads to a null memory
Figure 1: Barrel-type apparatus for odour–sugar associative conditioning. (a) Side view of the closed barrel; the arrow indicates the direction of the airflow. Odour delivery is ensured with computer-controlled electronic valves (Pascual & Preat, 2001). (b) Close up view of a rotating insert. The base can be wedged in the barrel, while the inner part rotates freely, thanks to the stainless steel balls. A hole in the base terminates in an air inlet in order to let air through. (c) Open barrel, top view. Plastic cylinders with sugar applied on the 2/5 of the inner surface are inserted in the three holes, fitting to the rotating inserts. (d) Flies are loaded into three chambers delimitated by the plastic cylinders and the rotating inserts. During the loading phase, the rotating inserts hide the sugared area and the flies do not access the sugar. (e) When the conditioned odour is delivered, the barrel is rotated by 1808. The rotating insert stays in position because of gravity and flies are able to access the sugar. Note that the labels (‘no sugar’ and ‘sugar’) refer to the same chamber.
b b NS a NS 0.0 0.2 0.4 0.6 0.8 1 3 7
Testing time (days) Memory retention score
1session 5 massed 5 spaced
/ / PI
Figure 2: Time–course of memory over days, as a function of number and spacing of conditioning sessions. Mean memory performance indexes SEM compared after one or five (massed or spaced) conditioning sessions (respectively, one session, five massed and five spaced), from 1 to 7 days after conditioning with the usual protocol (21-h starvation, 1.5Msucrose solution left to
dry as US). Sample sizes: n¼ 11–12.ANOVAshowed a significant
group effect on day 3: F2,32¼ 3.61; P ¼ 0.038 (Newman–Keuls statistic, lowercase letters indicate significant differences at P < 0.05), but not on days 1 and 7 (NS, P > 0.05).
score independently of the number of sessions (memory retention score, after one session: 0.04 0.07, n ¼ 9, after five spaced sessions: 0.06 0.06, n ¼ 9), conditioning flies for odour–sugar association during one or five sessions induced similarly high scores at 22 h, regardless of the spacing of the conditioning sessions (Fig. 2). Importantly, data for a similar retention time were tested in parallel, which reduces the chance of a sampling error. Three days after conditioning, scores became significantly lower than the memory score induced by five spaced sessions, which was unchanged compared with the score at 1 day. Seven days after conditioning, scores dropped. The score obtained with five spaced sessions was the only one still significantly higher
than 0 (Student’s t-test: t¼ 2.99, df ¼ 11; P < 0.05), but
ANOVAfailed to show a significant group effect. In all cases,
mean mortality until the testing day was inferior to 5%, 0.0 0.2 0.4 0.6 0.15 1.5
Sugar response
14 h 21 hSucrose concentration on the felt (M)
*
NS1.5
-h memory
Starvation length: 0.0 0.2 0.4 76*
76 44 42 20 20 PI RI(a)
(b)
Figure 3: 1.5-h memory retention score and SR as a function of starvation length. Memory performance indexes for the conditioned odours 1.5 h after one-session conditioning (a) and RIs to sugar directly after starvation (b) were tested in indepen-dent groups. SR was tested with tubes prepared with either a 0.15 or a 1.5Msolution. The graph displays mean values
SEM; sample sizes are indicated in the bars. Student’s t-test indicates a significant effect of starvation duration on memory score, and on the SR while tested with 0.15Msugar on the felt, but not with 1.5M; NS, P > 0.05,wP < 0.05. PI, performance index. 21 0.0 0.2 0.4 1 session Water control CXM NS 21 21 17 19 13 14 0.0 0.2 0.4
**
**
**
10 0.0 0.2 0.4 Aversive 5 massed NS Treatment 21 101 session 5 massed 5 spaced 1.5 - h memory 22 - h memory 22 - h memory PI PI PI
(a)
(b)
(c)
Figure 4: Effect of protein synthesis inhibition on short-term and long-term memory scores following different condition-ing protocols. (a,b) Effect of CXM on memory performance indexes after different sugar-reward conditionings after 21-h starvation (1.5Msucrose solution let to dry as US), tested at
1.5 h (a) or 22 h (b). (c) Control with electroshock massed conditioning. CXM was added to the water for 20.5 h before conditioning. The graph displays mean values SEM; sample sizes are indicated in the bars. A Student’s t-test was used to compare memory scores between CXM-treated flies and control flies: NS, P > 0.05,wwP < 0.01. PI, performance index.
410
Genes, Brain and Behavior (2009) 8: 407–415indicating that the starvation protocols allow the formation of a long-lasting appetitive memory without affecting survival.
Accurate control for sugar motivation
Fly sugar motivation appears to be essential in determining memory scores: for instance, non-starved flies show no appetitive memory (1.5-h memory score: 0.03 0.04, n¼ 15). We therefore needed a control for the starvation effect, independent from the learning assay and sensitive enough to detect slight differences in motivation. A small but significant difference in STM scores can be seen between flies starved for 14 vs. 21 h (Fig. 3). We sought to implement an SR test that is able to distinguish between the responses of these two groups of flies. We achieved this by using a new paradigm based on the work of Kim et al. (2007b). It involves testing fly chemotaxis in a T-maze, where one arm is covered with dry sugar derived from a sucrose solution diluted at 0.15M (see Materials and methods section for further
details). In this assay, a differential SR was measured following the two starvation durations. Interestingly, if a large amount of sugar is present in the arm (when sugar is coming from a 1.5M solution), the difference vanishes (Fig. 3). In summary, we found that both SR and STM increased significantly with starvation length of 14–21 h, indicating that the starvation protocol is crucial and that our motivation control is accurate.
22-h memory is protein synthesis dependent after all
conditioning procedures
In order to confirm that the long-lasting memory induced in our protocol was dependent on de novo protein synthesis, we used an inhibitor of protein synthesis (CXM) that we admin-istered to the flies for 20.5 h and until 30 min before conditioning. Because the presence of ethanol during starva-tion lowered memory scores (data not shown), we simply diluted the drug in water. The treatment had no effect on the memory retention score tested shortly (1.5 h) after the one-session sugar conditioning (Fig. 4a; t¼ 0.17, df ¼ 40, P > 0.05). However, when tested 22 h after conditioning, the memory score was significantly lower in the CXM-treated groups, after one session as well as five sessions of appetitive conditioning, massed or spaced (Fig. 4a; t 2.88, df 23, P < 0.008). Importantly, the drug had no effect on SR, neither at the conditioning time nor at the retrieval time 22 h after conditioning (Table 1). Because the drug treatment is different from the one used in previous studies on aversive learning (Tully et al. 1994), we tested its effect on olfactory acuity. We found no effect of the drug on the response to the odour at the concentration used for conditioning, or diluted five times (Table 2). In contrast with previous studies (Kim et al. 2007b), both untreated and CXM-treated flies showed stronger aversion towards more diluted odours, especially in the case of octanol, a surprising result that remains inexpli-cable at this point.
We next wanted to make sure that the observed deficit of memory was not because of a combined effect of CXM and starvation, which might have affected the retrieval of any
form of olfactory memory at 22 h. Unlike LTM, the memory formed after massed aversive conditioning does not depend on de novo protein synthesis (Mery & Kawecki 2005; Tully et al. 1994) and serves as a control. We therefore looked at the memory formed after aversive massed conditioning of flies that had been prepared (and starved) similarly as in the appetitive protocol and found no effect of CXM (Fig. 4b). This indicates that the combination of CXM administration and starvation does not unspecifically affect memory retrieval. Taken together, the results confirm that appetitive LTM is formed after a single appetitive training session.
Finally, we examined the nature of long-lasting memories obtained after shorter starvation before conditioning (7 h instead of 21 h) and with a weaker US (by diluting 10 times the sugar solution applied to the plastic). Both protocols lead to weak performance 22 h after conditioning, and this per-formance is abolished by CXM application (Fig. 5). Flies therefore also formed LTM under these conditions.
Genetic analysis of appetitive memory and SR
We next analysed the role of different genes that show specific involvement in aversive memory, first focusing on the rsh gene. In contrast to a previous report (Krashes & Waddell 2008) and using the same mutant strain, we observed a difference between the SR of rsh and Canton S flies (Fig. 6). However, because the rsh strain used was not freshly outcrossed, this difference in SR may be explained by genetic background effects.
We then examined the phenotype of teqPand cerP, two mutant strains showing a specific LTM defect in the aversive paradigm and allowing easy outcross because the mutations correspond to the insertion of a traceable transposable P-element. teqPand cerPwere previously shown to display
an LTM defect after appetitive conditioning, with a normal STM and SR (Krashes & Waddell 2008). In contrast, we found that both teqP and cerP display an STM defect (Fig. 7b).
Moreover, both mutants present a defect in their SR after 21 h of starvation (Fig. 7a). Interestingly, these defects in SR were not observable with a less sensitive test: using 1.5M
sucrose solution instead of a 0.15 M to prepare the
Table 1: Control for sugar responsiveness after CXM treatment Sugar responsiveness Water control CXM 35 mM Student’s t-test Conditioning time 0.39 0.06 (27) 0.27 0.07 (27) t¼ 1.27; P¼ 0.21 22-h post-conditioning 0.61 0.04 (35) 0.54 0.04 (35) t¼ 1.10; P¼ 0.27 Values are given as mean SEM of RIs to sugar and numbers in parentheses are the sample sizes. SR was tested at a time corre-sponding to that of conditioning, that is 30 min after the end of the 20.5-h treatment, or 22 h later, that is at time of test. There is no significant difference between the water control and CXM-treated groups (Student’s t-test, P > 0.05).
sugar-coated arm of the T-maze, the responses of cerPand
teqP flies were indistinguishable from Canton S (cerP: 0.48 0.07, n ¼ 18; teqP
: 0.44 0.10, n ¼ 10; Canton S: 0.56 0.05, n ¼ 23; Kruskal–Wallis, P > 0.05).
Because the SR defect displayed by these mutants pre-vented us from assessing their potential memory deficien-cies, we tried to decorrelate the two effects. We reasoned that a defect in teq expression only in adult mushroom bodies might not affect SR, but might affect memory retention. We therefore made use of the P[switch] system (Roman et al. 2001) combined with a UAS-teq-RNAi (Didelot et al. 2006), which allows us to decrease teq expression specifically during adulthood and specifically in the cells labelled by the MB247 Gal4 line (principally mushroom body intrinsic neu-rons; Mao et al. 2004). The expression of the RNAi induced both a defect in SR and STM (Fig. 7c,d), a phenotype similar to the teqPflies (Fig. 7b). When the expression is not induced
by the drug, no phenotype is present (data not shown), which refutes a role of the genetic background in this effect.
Discussion
Using a newly designed barrel for precisely scheduled odour– sugar association in Drosophila, we obtained high memory scores that lasted for days following one or several sessions of conditioning. The scores are comparable with those currently observed for electroshock conditioning (Pascual & Preat 2001) and are the result of associative learning because unpaired exposure to odour and sugar yields null scores. This may have been favoured by limited manipulation of flies during the conditioning (Kim et al. 2007a) and by the starva-tion protocols that kept flies vigorous, showing less than 5% mortality within 3 days after conditioning.
The present work also provides for the first time a validated control for SR in the study of appetitive memory in Drosophila. Learning scores following sugar associative conditioning are correlated to the feeding status of individuals both in Dro-sophila (Fig. 3; Tempel et al. 1983) and in the honeybee, where this correlation has been studied in more detail (Friedrich et al. 2004; Scheiner et al. 2001, 2004, 2005). Any apparent memory retention defect (obtained with a pharma-cological treatment or a genetic alteration) may thus be because of an effect on sugar motivation. In order to be confident in the SR test, we set up a positive control. We reasoned that different starvation times would lead to differ-ent sugar motivations, and consequdiffer-ently differdiffer-ent memory
scores. We measured memory of such flies and found that 14 h of starvation induces a lower memory retention score than 21 h (Fig. 3). An accurate SR test must therefore be able to measure this difference, and ours can (Fig. 3); we conclude that the SR test is sensitive enough: an absence of SR phenotype proves that both sugar motivation and sugar detection are normal. Of course, if an SR deficit is observed, we cannot determine whether motivation or taste processing is affected, but we cannot rule out that an associated memory deficit is because of a lower motivation. Indeed, the correlation between SR and STM defects appears robust (Figs 3, 6, 7).
Interestingly, we needed to use a sugar solution 10 times more diluted for the preparation of SR test tubes than for the preparation of conditioning tubes in order to observe differ-ences in the SR score of differently starved flies (Fig. 3). Similarly, only the sensitive test was able to detect teqPand cerPdefect (Fig. 7 and data in text). This result suggests that
previous SR paradigms, whose sensitivity was not evaluated, might have been inaccurate. Actually, the high SR scores (above 70% of the time passed on a filter soaked with sugar) reported with the paradigm used in Schwa¨rzel et al. (2003) and Thum et al. (2007) hint for a low sensitivity of this test. On the other hand, the paradigm used by Krashes and collabo-rators (chemotaxis test between sugared and pure agar; Keene et al. 2006; Krashes et al. 2007; Krashes & Waddell 2008) appeared to be incapable of detecting the SR deficiencies of the rsh mutant strain (Fig. 6).
Using the new conditioning apparatus and the validated SR test, we showed that a single appetitive conditioning session (as well as massed and spaced conditioning sessions) leads to the formation of memory depending on de novo protein synthesis, in accordance with a previous report (Krashes & Waddell 2008). Oral administration of the protein synthesis inhibitor CXM impairs memory at 22 h for all three condition-ing protocols, without affectcondition-ing the memory tested after 1.5 h following one conditioning session. Because CXM applied during starvation may have different and stronger effects than with the usual protocol, we also tested olfactory acuity. We found no effect of the drug, while starvation itself appeared to affect olfactory responses. Importantly, the SR of these flies, both at the time of conditioning and 22 h after a conditioning session, was normal. This indicates that CXM application does not affect sugar motivation or detection, neither at the time of conditioning nor at the time of the test. In addition, the drug treatment has no effect on the protein-synthesis-independent memory generated after an aversive
Table 2: Control for odour responsiveness after CXM treatment
Odour response Water control CXM 35 mM Student’s t-test
Methylcyclohexanol 1/1 0.29 0.07 (16) 0.27 0.07 (16) t¼ 0.13; P ¼ 0.90
Octanol 1/1 0.03 0.09 (25) 0.02 0.08 (25) t¼ 0.40; P ¼ 0.69
Methylcyclohexanol 1/5 0.64 0.07 (11) 0.60 0.08 (11) t¼ 0.40; P ¼ 0.70
Octanol 1/5 0.61 0.08 (9) 0.67 0.06 (10) t¼ 0.60; P ¼ 0.56
Values are given as mean SEM of RIs to the odours at the time of test, at the concentration used for memory assays or diluted 5 times (1/5) and numbers in parentheses are the sample sizes. There is no significant difference between the water control and CXM-treated groups (Student’s t-test, P > 0.05).
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Genes, Brain and Behavior (2009) 8: 407–415massed conditioning of starved flies. We can thus exclude unspecific effects of CXM as a result of a prolonged protein deprivation at 22 h. All three training procedures induce a long-lasting memory dependent on de novo protein synthesis and is therefore defined as LTM. Because CXM inhibits only 50% of protein synthesis at the concentration we used (Tully et al. 1994), the residual memory may or may not be LTM.
Although generating similar 1-day memory levels, the three conditioning protocols are not equivalent because memory scores tested 3 days after conditioning are higher after five spaced sessions (Fig. 2). This evokes the situation in the honeybee, where scores obtained after massed proboscis extension reflex conditioning sessions are generally lower than after spaced ones, although memories formed after both
procedures rely on de novo protein synthesis (Menzel et al., 2001). Whether this difference is quantitative or whether qualitatively different forms of LTM are formed still needs to be examined.
Several different factors may underlie LTM formation after one session of conditioning. For instance, studies in the honeybee suggest the involvement of motivation during the formation of appetitive LTM via elevation of cyclic AMP-dependent protein kinase (Friedrich et al. 2004; Mu¨ller 2000). But our data suggest that this is not the case here: while starvation before aversive conditioning had no facilitating effect on aversive LTM formation (Fig. 4 and data not shown), a shorter starvation regime did not prevent appetitive LTM formation (Fig. 5). We then reasoned that a very strong US might lead to LTM while a weaker may not. In order to test this hypothesis, we reduced the amount of sugar available to the fly during conditioning by diluting the solution applied on the plastic tubes. In these conditions, the memory scores are much lower, but memory remained CXM sensitive (Fig. 5). Despite our efforts, we failed to develop a protocol generating exclusively protein-synthesis-independent memory 22 h after appetitive conditioning.
We next examined the role of different genes in appetitive memory and SR. The rsh mutation was reported to have a specific defect in appetitive 3-h and 24-h memory (Krashes & Waddell 2008). In contrast, the same mutant stock showed an SR defect in our hands (Fig. 6). A difference in genetic background of the Canton S and rsh strains prevents us from drawing definite conclusions about the role of rsh in SR. Nevertheless, our results suggest that the memory retention phenotypes seen in rsh mutant flies (Krashes & Waddell 2008) may be, at least in part, because of a deficit in motivation or sugar taste processing.
We next examined teq and cer function, two genes whose mutation was reported to display an LTM-specific defect in CXM Control 0.0 0.2 0.4
*
22 - h memory, 7 - h starvation only CXM Control 0.0 0.1 0.2*
22 - h memory, weak US 26 26 19 20 PI PI (a) (b)Figure 5: Effect of protein-synthesis inhibition on long-term memory formed following weaker conditioning. Effect of CXM on memory performance indexes while flies are starved for only 7 h before normal conditioning (a), or while normally starved flies are conditioned with less sugar on the tubes (b). In this second experiment, the conditioning tubes were prepared with a sugar solution ten times more diluted than in the normal protocol. The graph displays mean values SEM; sample sizes are indicated in the bars. A Student’s t-test was used to compare memory scores between CXM-treated flies and control flies: w
P < 0.05. PI, performance index.
0.0 0.2 0.4 38 26
*
Canton S rsh 0.6 Sugar response RIFigure 6: Sugar responsiveness deficit of radish mutant flies. SR indexes of Canton S and rsh mutant flies tested after 21 h of starvation. The graph displays mean values SEM; sample sizes are indicated in the bars: A Student’s t-test was used to compare the two groups:w
aversive memory (Comas et al. 2004; Didelot et al. 2006) as well as in appetitive LTM (Krashes & Waddell 2008). In contrast to Krashes & Waddell (2008), we found strong STM defects, which were correlated with SR shortcom-ings. These discrepancies may be because of different factors. First, the conditioning protocols are slightly differ-ent: their tests were performed at 3-h post-conditioning (compared with 1.5 h in our protocol) and their conditioning time is 2 min (as opposed to 1 min in our protocol). Second, the starvation protocols differ. Finally, the genetic back-ground of the flies may have been different and may have concealed the early memory and SR phenotypes in their case.
Interestingly, both genes were involved in starvation-induced response to sugar. Using the P[switch] system, we further showed that the teq phenotype was not because of a developmental effect (Fig. 7), but that the gene acts during the starvation or the test, in certain cells labelled by the MB247-switch line, most likely in the mushroom bodies (Didelot et al. 2006). Because taste perception is thought not to require this brain area (Vosshall & Stocker 2007), this result suggests that teq may be involved in sugar motivation. Indeed, both learning and starvation induce a change in behaviour because of past experience and might therefore share molecular pathways.
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Acknowledgments
The authors are grateful to Jean-Yves Tiercelin for contributing to the design, realization and set up of the sugar barrel prototype, to Patrick Para for his help in the maintenance of the apparatus and to Jean Paul Bouillot for pictures. The authors thank Philippe Vernier and Ge´rard Baux for their interest and general support during the experiments at the INAF, Elsa Bonnard for her help with setting up the new training apparatus, Guillaume Isabel and Fre´de´ric Mery for helpful discussions, Michel Chaminade for his logistic help during the experiments as well as Niki Scaplehorn for helpful comments on the manuscript. This work was sup-ported by the Swiss National Fund (grant for young researcher PBFRA-116951, to J.C.), by the Universite´ Paris 13 Ecole Doctor-ale Sciences du Vivant et Socie´te´ (to M.A.C.), and by grants from the Agence Nationale pour la Recherche, the Fondation Bettencourt-Schueller and the Fondation pour la Recherche Me´dicale (to T.P.).
