Published: August 7th, 2014
The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.
Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes8-10 and in flies to measure the contribution of adult plasticity genes1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment5,11-13. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal3,4,14,16.
Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory15-17. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory3-6,11-13,15-21.
Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS+) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal1,16. A second conditioned stimulus (CS-) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.
The method presented here is that described by Tully and Quinn with some small modifications1. The experiment is performed in two phases: the flies are trained in the first phase, and the trained flies are tested in the second phase. During training, a group of flies are simultaneously exposed to odor 1 (CS+) and an electric shock (US) in a training tube. The flies then receive odor 2 (CS-) without an electric shock. This single pairing of a particular odor with a shock is called 1-cycle training, and the odors that are most frequently used are 4-methylcyclohexanol (MCH) and 3-octanol (OCT).
One-cycle training leads to the formation of a labile phase of memory that can be detected for up to 7 hr; however, memory is typically tested immediately to determine what is termed learning, acquisition or 2 min memory. Memory measured at 30 min or 1 hr is referred to as short-term memory, whereas 3 hr memory is referred to as mid-term memory. The exposure of flies to repetitive training cycles with gaps between the training cycles (spaced training) leads to a consolidated form of long-term memory that is CREB transcription dependent and lasts up to a week. Training without gaps (massed training) leads to the formation of anesthesia-resistant memory (ARM), which similar to long-term memory, is typically measured 24 hr after 5 cycles of training7,13,15-17,20,21.
With this approach, the effect of various gene mutations on these different phases of memory can be determined. The promoter-driven expression of light- or temperature-sensitive transgenes to activate or block the neural activity of specific neurons allows one to investigate which neurons are required for memory acquisition, consolidation and retrieval3,4,11,15,16,20,22-24. Memory at 1 hr is typically measured when studying age-related memory impairment because this form of memory appears particularly vulnerable to the effects of ageing11-13. A full range of behavioral and genetic controls are performed with memory experiments, for example, to determine whether a performance defect is because of a central memory defect or a peripheral sensory defect that prevents the fly from sensing the shock or olfactory cue5-7,17,25,26.
1. Fly Preparation
2. Preparation Before the Experiment
3. Odor Dilutions
4. Training Protocol (Figures 1 and 2)
5. Calculation of the Performance Index: a Measure of the Flies’ Memory
6. Sensorimotor Controls
The performance index (PI) serves as a measure of memory. Table 1 illustrates a representative calculation of PI.
|MCH paired with shock
|3-OCT paired with shock
|Flies avoiding MCH (in OCT tube) = 80
Flies preferring MCH (in MCH tube) = 20
|Flies avoiding OCT (in MCH tube) = 75
Flies preferring OCT (in OCT tube) = 25
PI2 = (75-25)/(75+25)
|PI of the experiment= (0.6+0.5)/2=0.55
Table 1. A representative calculation of performance index using illustrative data. Performance indices for different experiments can be compared to elucidate memory effects. Once such comparison is shown in Figure 3, which contains the results from a series of experiments performed with Canton S wildtype adult flies (WT) and dunce learning mutant adult flies1. The mean of 10 PIs is provided, with error bars representing the standard error of mean (SEM). These results demonstrate that dunce flies show a reduction in learning compared to wildtype.
Figure 1. The adult experimental set-up. The flies are trained and tested in a T maze. The training involves presenting an odor A with electric shock followed by a second odor B without electric shock. After a rest period in the middle chamber the flies are presented with both the odors simultaneously. The flies are trapped in the two tubes and collected and counted to obtain learning/memory scores.
Figure 2. The adult training protocol. The flies are training in two steps. The first step where flies receive an odor (CS+) paired with electric shock (US) for 60 sec. In the next step flies receive a second odor (CS-) without electric shock. Flies are then allowed to rest for 90 sec after which they are tested for their choice between CS+ and CS-.
Figure 3. A representative graph showing dunce and wildtype learning in adult Drosophila. WT and dunce flies were tested following one session of training. dunce flies show a reduction in learning compared to the WT (n = 10).
The Drosophila adult olfactory shock learning assay presented here allows analysis of the molecular mechanisms underlying different phases of memory, including long-term memory15-17. As well as determination of the effect of circadian rhythms18, sleep19, diet20,21, senescence11-13, neurodegenerative disease5 and drug treatments5,6,19 on memory.
Many powerful approaches have been recently developed for the functional imaging of the neural circuits that mediate olfactory memory in flies3,4,7,11,16,27. These optogenetic techniques use the vast repertoire of the different promoters available in Drosophila14,16. These promoters are used to express genetically encoded calcium and cAMP reporters in the memory neurons16,27 to study the effect of specific gene mutations on memory traces.
The use of conditional promoters and mutations in adults allows the study of the post-developmental role of a gene product in memory3,4,6,7,13,14. Imaging and behavioral approaches can be combined with light- and heat-activated channels to stimulate or inhibit different neurons in the memory circuit11,14,16,22-24 to further elucidate their function. Furthermore, mushroom body memory neurons are accessible to whole-cell patch clamp recordings28, and mathematical and computational techniques are being used to model Drosophila olfactory memory29.
These experimental advances, combined with the different forms of associative memory protocols introduced here, allow Drosophila to be used to model the molecular- and circuit-level changes in associative memory that occur in response to reward, punishment, motivation, addiction, aging and disease5,6,11-13,16,30-31.
The authors have nothing to disclose.
We acknowledge Bloomington stock centers for the fly strain. This work was supported by research grant from BBSRC (BB/G008973/1).
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