Classical reward conditioning in Drosophila melanogaster

Negatively reinforced olfactory conditioning has been widely employed to identify learning and memory genes, signal transduction pathways and neural circuitry in Drosophila. To delineate the molecular and cellular processes underlying reward-mediated learning and memory, we developed a novel assay system for positively reinforced olfactory conditioning. In this assay, flies were involuntarily exposed to the appetitive unconditioned stimulus sucrose along with a conditioned stimulus odour during training and their preference for the odour previously associated with sucrose was measured to assess learning and memory capacities. After one training session, wild-type Canton S flies displayed reliable performance, which was enhanced after two training cycles with 1-min or 15-min inter-training intervals. Higher performance scores were also obtained with increasing sucrose concentration. Memory in Canton S flies decayed slowly when measured at 30 min, 1 h and 3 h after training; whereas, it had declined significantly at 6 h and 12 h post-training. When learning mutant t beta h flies, which are deficient in octopamine, were challenged, they exhibited poor performance, validating the utility of this assay. As the Drosophila model offers vast genetic and transgenic resources, the new appetitive conditioning described here provides a useful tool with which to elucidate the molecular and cellular underpinnings of reward learning and memory. Similar to negatively reinforced conditioning, this reward conditioning represents classical olfactory conditioning. Thus, comparative analyses of learning and memory mutants in two assays may help identify the molecular and cellular components that are specific to the unconditioned stimulus information used in conditioning.     

Olfactory conditioning of the proboscis extension in bumble bees

The foraging behaviour of bumble bees is well documented for nectar and/or pollen gathering, but little is known about the learning processes underlying such behaviour. We report olfactory conditioning in worker bumble bees Bombus terrestris L. (Hymenoptera: Apidae) obtained under laboratory conditions on restrained individuals. The protocol was adapted from the proboscis extension conditioning previously described in the honey bee Apis mellifera L. Bumble bees were found to be able to learn a pure odorant when it was presented in paired association with a sugar reward, but not when odour and reward were presented in an explicitly unpaired procedure. This suggests an associative basis for this olfactory learning. Bumble bees showed similar conditioning abilities when stimulated with two different floral odours. An effect of the sugar reward concentration on the learning performances was found.     

Exposure to cold: aversive Pavlovian conditioning in individual Drosophila melanogaster

Abstract. Temperature changes can be especially threatening for ectotherms, such as Drosophila melanogaster (Diptera: Drosophilidea Meigen, 1830 ), and in this study we tested whether flies can associate olfactory stimuli with a sudden drop in temperature. Such Pavlovian conditioning would allow them to make appropriate behavioural and/or physiological responses in the future. We found that exposing individual flies to one of two odours in the presence of a sudden drop in temperature resulted in Pavlovian conditioning with flies subsequently avoiding the odour paired with cold. The characteristics of Pavlovian conditioning in flies were comparable to those observed for mammalian species. Specifically, the strength of conditioning increased with increasing intensity of the cold and decreased as the time interval between the olfactory stimulus (CS) and cold (US) was lengthened. Finally, the order in which CS and US were presented affected the strength of conditioning. Learning was observed when the CS preceded US and when the US immediately preceded the CS, but not when the CS preceded the US by 30 s or more. These results provide further evidence for learning in individual flies, and confirm that Pavlovian conditioning is a general mechanism used by organisms to obtain information about their environment.     

Odor discrimination in classical conditioning of proboscis extension in two stingless bee species in comparison to Africanized honeybees

Learning in insects has been extensively studied using different experimental approaches. One of them, the proboscis extension response (PER) paradigm, is particularly well suited for quantitative studies of cognitive abilities of honeybees under controlled conditions. The goal of this study was to analyze the capability of three eusocial bee species to be olfactory conditioned in the PER paradigm. We worked with two Brazilian stingless bees species, Melipona quadrifasciata and Scaptotrigona aff. depilis, and with the invasive Africanized honeybee, Apis mellifera. These three species present very different recruitment strategies, which could be related with different odor-learning abilities. We evaluated their gustatory responsiveness and learning capability to discriminate floral odors. Gustatory responsiveness was similar for the three species, although S. aff. depilis workers showed fluctuations along the experimental period. Results for the learning assays revealed that M. quadrifasciata workers can be conditioned to discriminate floral odors in a classical differential conditioning protocol and that this discrimination is maintained 15 min after training. During conditioning, Africanized honeybees presented the highest discrimination, for M. quadrifasciata it was intermediate, and S. aff. depilis bees presented no discrimination. The differences found are discussed considering the putative different learning abilities and procedure effect for each species.     

A new paradigm for operant conditioning of Drosophila melanogaster

A freely walking single fly (Drosophila melanogaster) can be conditioned to avoid one side of a small test chamber if the chamber is heated whenever the fly enters this side. In a subsequent memory test without heat it keeps avoiding the heat-associated side. The memory mutants dunce ¹ and rutabaga ¹ successfully avoid the heated side but show no avoidance in the memory test. Wildtype flies can be trained to successively avoid alternating sides in a reversal conditioning experiment. Every single fly shows strong avoidance and a positive memory score. The new conditioning apparatus has several advantages: (1) Statistically significant learning scores can be obtained for individual flies. (2) Learning scores are obtained fully automatically without interference of the experimenter. (3) The procedure is fast, robust and requires little handling. Therefore the apparatus is suitable for largescale mutant screening. (4) Animals are not attached to a hook and thus can easily be used for breeding.Abbreviations dnc dunce gene - PI performance Index - rut rutabaga gene - S.E.M. standard error of mean     

Spatial serial conditioning maintained with minimal temporal contingency

Two experiments used a spatial serial conditioning paradigm to assess the effectiveness of spatially informative conditioned stimuli in eliciting tracking behavior in pigeons. The experimental paradigm consisted of the simultaneous presentation of 2 key lights (CS2 and CTRL), followed by another key light (CS1), followed by food (the unconditioned stimulus or US). CS2 and CTRL were presented in 2 of 3 possible locations, randomly assigned; CS1 was always presented in the same location as CS2. CS2 was designed to signal the spatial, but not the temporal locus of CS1; CS1 signaled the temporal locus of the US. In experiment 1, differential pecking on CS2 was observed even when CS2 was present throughout the interval between consecutive presentations of CS1, but only in a minority of pigeons; prevalence of differential pecking was enhanced when CS2 duration was halved. A control condition verified that pecking on CS2 was not due to temporal proximity between CS2 and US. Experiment 2 demonstrated the reversibility of spatial conditioning between CS2 and CTRL. Asymptotic performance never involved tracking CTRL more than CS2 for any of 16 pigeons. It is inferred that pigeons learned the spatial association between CS2 and CS1, and that temporal contingency facilitated its expression as tracking behavior.     

Identification of a new contingency-based response in honey bees (Apis mellifera) through revision of the proboscis extension conditioning paradigm

The two experiments reported present new information in the area of classical conditioning experiments with honey bees. Experiment 1 establishes a single unconditioned stimulus (US) technique as a preferred technique for conditioning of the proboscis extension response. Experiment 1 further identifies a new head turn response which occurs when the standard compound US technique is used. Experiment 2 demonstrates that the newly identified head turn response is contingency-based and provides important new response to the repertoire of honey bee learning experiments.     

Drosophila Adult Olfactory Shock Learning

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 diseases^1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes^8-10 and in flies to measure the contribution of adult plasticity genes^1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment^5,11-13. 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 signal^3,4,14,16.Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory^15-17. 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 memory^{3-6,11-13,15-21}.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 animal^1,16. 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.     

Odor information transfer in the stingless bee<Emphasis Type="Italic"> Melipona quadrifasciata</Emphasis>: effect of in-hive experiences on classical conditioning of proboscis extension

A recent study showed that the stingless bee Melipona quadrifasciata could learn to discriminate odors in a classical conditioning of proboscis extension response (PER). Here we used this protocol to investigate the ability of these bees to use olfactory information obtained within the colony in an experimental context: the PER paradigm. We compared their success in solving a classical differential conditioning depending on the previous olfactory experiences received inside the nest. We found that M. quadrifasciata bees are capable of transferring the food-odor information acquired in the colony to a differential conditioning in the PER paradigm. Bees attained higher discrimination levels when they had previously encountered the rewarded odor associated to food inside the hive. The increase in the discrimination levels, however, was in some cases unspecific to the odor used indicating a certain degree of generalization. The influence of the food scent offered at a field feeder 24 h before the classical conditioning could also be seen in the discrimination attained by the foragers in the PER setup, detecting the presence of long-term memory. Moreover, the improved performance of recruited bees in the PER paradigm suggests the occurrence of social learning of nectar scents inside the stingless bees’ hives.     

Consequences of food-attraction conditioning in Helix: a behavioral and electrophysiological study

Food-attraction conditioning is a learning phenomenon by which adult Helix pomatia acquire the ability to locate food through exposure to that particular food. Food-conditioned snails can be distinguished from naive snails during their approach to food. Naive snails keep their tentacles upright — whereas food-conditioned animals bend the tentacles down-ward, in a horizontal orientation, pointed in the direction of the food.Tentacle musculature is innervated by two peritentacular nerves (PTn), each projecting to approximately one hemi-section of the tentacle wall. Stimulating the peritentacular nerves caused the tentacles to bend downward in a manner reflecting the full complement of tentacle movements performed by conditioned snails.The neural correlate of tentacle movements was investigated in isolated ganglion preparations with the posterior tentacles attached. PT nerve activity was recorded while the olfactory epithelia were stimulated with natural food odors. Preparations obtained from conditioned animals responded with a substantial increase in unit activity (mean increase 280%) to stimulation with odor of the conditioned food but not to other odors. Preparations from naive animals did not respond to food odor stimulation. The electrophysiological results demonstrated that plasticity due to conditioning the snails in vivo survived dissection and could be monitored in vitro.Abbreviations ext PTn external peritentacular nerve - int PTn internal peritentacular nerve     

A Quantitative Study of the Ca2+/Calmodulin Sensitivity of Adenylyl Cyclase in Aplysia, Drosophila, and Rat

Studies in Aplysia and Drosophila have suggested that Ca2+/calmodulin-sensitive adenylyl cyclase may act as a site of convergence for the cellular representations of the conditioned stimulus (Ca2+ influx) and unconditioned stimulus (facilitatory transmitter) during elementary associative learning. This hypothesis predicts that the rise in intracellular free Ca2+ concentration produced by spike activity during the conditioned stimulus will cause an increase in the activity of adenylyl cyclase. However, published values for the Ca2+ sensitivity of Ca2+/calmodulin-sensitive adenylyl cyclase in mammals and in Drosophila vary widely. The difficulty in evaluating whether adenylyl cyclase would be activated by physiological elevations in intracellular Ca2+ levels is in part a consequence of the use of Ca2+/EGTA buffers, which are prone to several types of errors. Using a procedure that minimizes these errors, we have quantified the Ca2+ sensitivity of adenylyl cyclase in membranes from Aplysia, Drosophila, and rat brain with purified species-specific calmodulins. In all three species, adenylyl cyclase was activated by an increase in free Ca2+ concentration in the range caused by spike activity. Ca2+ sensitivity was dependent on both calmodulin concentration and Mg2+ concentration. Mg2+ raised the threshold for adenylyl cyclase activation by Ca2+ but also acted synergistically with Ca2+ to activate maximally adenylyl cyclase.     

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces^1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates^5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)^7-10.Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar^11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement ⁹. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups⁹.     

Anopheles gambiae females readily learn to associate complex visual cues with the quality of sugar sources

The ability to learn plays a key role in tuning and adapting the behaviours of animals for their unpredictable biotopes. This also applies to insect vectors of disease. Anautogenous mosquitoes need to find both sugar and blood for survival and reproduction. Learning processes are expected to contribute to a mosquito’s ability to undertake repeated feeding behaviours more efficiently with time, serving to decrease energy demands and avoid hazards. Here we report how visual learning by the Afrotropical malaria mosquito Anopheles gambiae allows it to readily associate visual cues with the quality of a sugar source. Circular black and white patterns were used as visual cues. An. gambiae females were conditioned in cages with a chequered pattern paired with sucrose and a concentric pattern paired with non-palatable sucrose-NaCl and with reverse combinations. Hours later, significantly higher numbers of feeding attempts were counted on sucrose paired with the chequered pattern following conditioning with the same combination. This was also the case on the concentric pattern paired with sucrose following conditioning with this combination. However, the effect was weaker than with sucrose paired with the chequered pattern. These findings indicate a differential capacity of visual stimuli to induce learning, explained in our experiments by a significantly higher mosquito appetence on sucrose paired with a chequered pattern that mimics floral shape. Training that induced a higher propensity for feeding attempts was found to allow the females to display a fast learning curve (<4 min) on the less suitable concentric pattern paired with sucrose, several hours after conditioning on the chequered pattern paired with sucrose. This has important implications for mosquito behavioural ecology, suggesting that An. gambiae shows plasticity in its learning capacities that would allow it to readily turn to alternative sources for a sugar meal once initiated in the process by an appropriate stimulus combination.     

Choline acetyltransferase-like immunoreactivity in the brain of Drosophila melanogaster

Summary Using a monoclonal antibody selective for the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT) of Drosophila melanogaster we find ChAT-like immunoreactivity in specific synaptic regions throughout the brain of Drosophila melanogaster apart from the lobes and the peduncle of the mushroom body and most of the first visual neuropile (lamina). Several anatomically well-defined central brain structures exhibit particularly strong binding. Characteristic differential staining patterns are observed for each of the four neuromeres of the optic lobes. Cell bodies appear not to bind this antibody. The prominent features of the distribution of ChAT-like immunoreactivity are paralleled by the distribution of acetylcholine hydrolyzing enzymatic activity as revealed by histochemical staining for acetylcholine esterase (AChE). These results are discussed in comparison with published data on enzyme distribution, choline uptake and ACh receptor binding in the nervous system of Drosophila melanogaster.     

Lateral axonal modulation is required for stimulus-specific olfactory conditioning in Drosophila

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Activation phase ensures kinematic efficacy in flight-steering muscles of Drosophila melanogaster

During tethered flight in Drosophila melanogaster, spike activity of the second basalar flight-control muscle (M.b2) is correlated with an increase in both the ipsilateral wing beat amplitude and the ipsilateral flight force. The frequency of muscle spikes within a burst is about 100 Hz, or 1 spike for every two wing beat cycles. When M.b2 is active, its spikes tend to occur within a comparatively narrow phase band of the wing beat cycle. To understand the functional role of this phase-lock of firing in the control of flight forces, we stimulated M.b2 in selected phases of the wing beat cycle and recorded the effect on the ipsilateral wing beat amplitude. Varying the phase timing of the stimulus had a significant effect on the wing beat amplitude. A maximum increase of wing beat amplitude was obtained by stimulating M.b2 at the beginning of the upstroke or about 1 ms prior to the narrow phase band in which the muscle spikes typically occur during flight. Assuming a delay of 1 ms between the stimulation of the motor nerve and muscle activation, these results indicate that M.b2 is activated at an instant of the stroke cycle that produces the greatest effect on wing beat amplitude.     

The genetic basis for mating-induced sex differences in starvation resistance in Drosophila melanogaster

Multiple genetic and environmental factors interact to influence starvation resistance, which is an important determinant of fitness in many organisms, including Drosophila melanogaster. Recent studies have revealed that mating can alter starvation resistance in female D. melanogaster, but little is known about the behavioral and physiological mechanisms underlying such mating-mediated changes in starvation resistance. In the present study, we first investigated whether the effect of mating on starvation resistance is sex-specific in D. melanogaster. As indicated by a significant sex × mating status interaction, mating increased starvation resistance in females but not in males. In female D. melanogaster, post-mating increase in starvation resistance was mainly attributed to increases in food intake and in the level of lipid storage relative to lean body weight. We then performed quantitative genetic analysis to estimate the proportion of the total phenotypic variance attributable to genetic differences (i.e., heritability) for starvation resistance in mated male and female D. melanogaster. The narrow-sense heritability (h²) of starvation resistance was 0.235 and 0.155 for males and females, respectively. Mated females were more resistant to starvation than males in all genotypes, but the degree of such sexual dimorphism varied substantially among genotypes, as indicated by a significant sex × genotype interaction for starvation resistance. Cross-sex genetic correlation was greater than 0 but less than l for starvation resistance, implying that the genetic architecture of this trait was partially shared between the two sexes. For both sexes, starvation resistance was positively correlated with longevity and lipid storage at genetic level. The present study suggests that sex differences in starvation resistance depend on mating status and have a genetic basis in D. melanogaster.     

Reassessment of the effect of biological rhythm mutations on learning in Drosophila melanogaster

A link between learning deficits and circadian period-lengthening mutations in Drosophila melanogaster previously has been reported. Mutant long-period males performed poorly in two learning assays involving experience-dependent courtship inhibition. In one, normal males that have courted fertilized females subsequently show courtship inhibition with virgin females. In the other, normal males that have courted sexually immature males subsequently fail to court other immature males. Those results have been reassessed in an extended study of genetic variants involving the period gene. 1. Long-period perL1 males demonstrated poor conditioned courtship inhibition when exposed to fertilized females; they showed normal courtship conditioning when exposed to immature males. This could be due to a perL1-associated olfactory deficit with fertilized females, since perL1 males were unable to discriminate behaviorally between fertilized and virgin females. 2. Other long-period males, including perL2 males and transgenic perL1 males bearing a truncated form of the per+ gene, were conditioned normally by fertilized females. Thus, the courtship inhibition defect is specific to the perL1 mutant strain. 3. perL1 (and other per mutant) flies showed normal acquisition and retention of a classically conditioned olfactory avoidance response. 4. Results from a new conditioned courtship inhibition experiment are presented; males exposed to fertilized females during training showed further courtship inhibition during subsequent exposure to fertilized females. From the perspective of learning theory, this can be viewed as a savings experiment.