By changing the conditioned discrimination paradigm of Quinn et al. (1974) from an instrumental procedure to a classical (Pavlovian) one, we have demonstrated strong learning in wildtype flies. About 150 flies were sequestered in a closed chamber and trained by exposing them sequentially to two odors in air currents. Flies received twelve electric shock pulses in the presence of the first odor (CS+) but not in the presence of the second odor (CS-). To test for conditioned avoidance responses, flies were transported to a T-maze choice point, between converging currents of the two odors. Typically, 95% of trained flies avoided the shock-associated odor (CS+). Acquisition of learning was a function of the number of shock pulses received during CS+ presentation and was asymptotic within one training cycle. Conditioned avoidance increased with increasing shock intensity or odor concentration and was very resistant to extinction. Learning was best when CS+ presentations overlap shock (delay conditioning) and then decreased with increasing CS-US interstimulus intervals. Shocking flies immediately before CS+ presentation (backward conditioning) produced no learning. Nonassociative control procedures (CS Alone, US Alone and Explicitly Unpaired) produced slight decreases in avoidance responses, but these affected both odors equally and did not alter our associative learning index (A). Memory in wild-type flies decayed gradually over the first seven hours after training and still was present 24 h later. The mutants amnesiac, rutabaga and dunce showed appreciable learning acquisition, but their memories decayed very rapidly during the first 30 min. After this, the rates of decay slowed sharply; conditioned avoidance still was measureable at least three hours after training.

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Convergent findings from our behavioral screen for memory mutants and DNA microarray analysis of transcriptional responses during memory formation in normal animals suggest the involvement of the pumilio/staufen pathway in memory. Behavioral experiments confirm a role for this pathway and suggest a molecular mechanism for synapse-specific modification.

Upon exposure to ethanol, Drosophila display behaviors that are similar to ethanol intoxication in rodents and humans. Using an inebriometer to measure ethanol-induced loss of postural control, we identified cheapdate, a mutant with enhanced sensitivity to ethanol. Genetic and molecular analyses revealed that cheapdate is an allele of the memory mutant amnesiac. amnesiac has been postulated to encode a neuropeptide that activates the cAMP pathway. Consistent with this, we find that enhanced ethanol sensitivity of cheapdate can be reversed by treatment with agents that increase cAMP levels or PKA activity. Conversely, genetic or pharmacological reduction in PKA activity results in increased sensitivity to ethanol. Taken together, our results provide functional evidence for the involvement of the cAMP signal transduction pathway in the behavioral response to intoxicating levels of ethanol.

Disruptions in mushroom body (MB) or central complex (CC) brain structures impair Drosophila associative olfactory learning. Perturbations in adenosine 3Ј,5Ј monophosphate signaling also disrupt learning. To integrate these observations, expression of a constitutively activated stimulatory heterotrimeric guanosine triphosphate-binding protein ␣ subunit (G␣ s *) was targeted to these brain structures. The ability to associate odors with electroshock was abolished when G␣ s * was targeted to MB, but not CC, structures, whereas sensorimotor responses to these stimuli remained normal. Expression of G␣ s * did not affect gross MB morphology, and wild-type G␣ s expression did not affect learning. Thus, olfactory learning depends on regulated G s signaling in Drosophila MBs.

Surgical, pharmacological and genetic lesion studies have revealed distinct anatomical sites involved with different forms of learning. Studies of patients with localized brain damage and work in rodent model systems, for example, have shown that the hippocampal formation participates in acquisition of declarative tasks but is not the site of their long-term storage. Such lesions are usually irreversible, however, which has limited their use for dissecting the temporal processes of acquisition, storage and retrieval of memories. Studies in bees and flies have similarly revealed a distinct anatomical region of the insect brain, the mushroom body, that is involved specifically in olfactory associative learning. We have used a temperature-sensitive dynamin transgene, which disrupts synaptic transmission reversibly and on the time-scale of minutes, to investigate the temporal requirements for ongoing neural activity during memory formation. Here we show that synaptic transmission from mushroom body neurons is required during memory retrieval but not during acquisition or storage. We propose that the hebbian processes underlying olfactory associative learning reside in mushroom body dendrites or upstream of the mushroom body and that the resulting alterations in synaptic strength modulate mushroom body output during memory retrieval.

Unlike most organ systems, which have evolved to maintain homeostasis, the brain has been selected to sense and adapt to environmental stimuli by constantly altering interactions in a gene network that functions within a larger neural network. This unique feature of the central nervous system provides a remarkable plasticity of behavior, but also makes experimental investigations challenging. Each experimental intervention ramifies through both gene and neural networks, resulting in unpredicted and sometimes confusing phenotypic adaptations. Experimental dissection of mechanisms underlying behavioral plasticity ultimately must accomplish an integration across many levels of biological organization, including genetic pathways acting within individual neurons, neural network interactions which feed back to gene function, and phenotypic observations at the behavioral level. This dissection will be more easily accomplished for model systems such as Drosophila, which, compared with mammals, have relatively simple and manipulable nervous systems and genomes. The evolutionary conservation of behavioral phenotype and the underlying gene function ensures that much of what we learn in such model systems will be relevant to human cognition. In this essay, we have not attempted to review the entire Drosophila memory field. Instead, we have tried to discuss particular findings that provide some level of intellectual synthesis across three levels of biological organization: behavior, neural circuitry and biochemical pathways. We have attempted to use this integrative approach to evaluate distinct mechanistic hypotheses, and to propose critical experiments that will advance this field.