1. The staufen/pumilo pathway is involved in Drosophila long-term memory Josh Dubnau et al.

2. Memory formation after olfactory learning in Drosophila is similar in both behavioral and molecular aspects to memory formation in other species, including humans. Studies in Drosophila have shown that long-term memory requires cAMP signaling and CREB-dependent transcription.

3. At the cellular level, long-lasting synaptic plasticity appears to depend on CREB- mediated gene transcription and subsequent structural and functional changes to relevant synapses. At present, little is known about the molecular mechanisms that contribute to this process.

4. To identify genes that function “downstream” of CREB, Dubnau et al performed two complementary searches. 1. Microarray experiments to find genes with altered expression during memory formation. 2. Large-scale behavioral screens for mutants defective in long-term memory.

5. Pavlovian olfactory conditioning Flies can distinguish between chemical with different smells (octanol vs. methylcyclohexanol). If one smell (such as octanol) is associated with an unpleasant experience (mild electrical foot shock) the flies will learn to avoid that smell.

6. On the first training day, a group of approximately 100 flies was exposed consecutively to octanol (positive conditioned stimulus, CS+) paired with foot shock (unconditioned stimulus) and then to methylcyclohexanol (negative conditioned stimulus, CS-) without foot shock, piped through the training chamber in an air current.

7. Flies were subjected to 10 training sessions with a 15 minute rest between sessions. The next day, conditioned odor avoidance responses were assessed in a T-maze, where the CS+ and CS- were each delivered to one of the two arms of the T-maze. The experimenters counted the number of flies in each arm of the maze at the end of 2 minutes.

8. Microarray experiments Used a prototype Affymetrix array containing 1542 fly genes. Gave flies to olfactory memory tests Collected RNA Identified genes with significant changes in expression at various time points after the memory task (t = 0 hour, 6 hour, and 24 hour)

9. Found 129 differentially expressed candidate memory genes Because of the high rate of false positive results from microarrays, Dubnau et al. sought to confirm their results using other measurement methods, including Quantitative PCR and a later commercial Affymetrix fly array. These confirmatory studies showed that differential expression of 42 candidate memory genes could be reproducibly measured.

10. Candidate memory genes from the behavioral (mutation) screen Introduced mutations in genes using a P-element transposon. P-element transposons can be inserted in a single gene, so as to disrupt its function, and the experimenter can determine the specific gene affected. Dubnau et al. created 6681 viable strains of flies containing P-element transposons, and screened them for performance deficits in the olfactory memory task. Found 60 mutants with defective one-day memory but normal sensorimotor response to the odors and foot shocks used in training.

11. Both the microarray experiment and the mutation experiment identified the pumilio gene. Prior research had indicated that a pumilio mutation altered neuron excitability, but had not identified a role in memory. Pumilio was best known as part of the cellular machinery for sub-cellular targeting and localized translation of mRNA in oocytes.

12. Sub-cellular targeting and localized translation of mRNA provides a way for the cell to produce proteins at specific locations where they are required within the cell (targeted RNA) In particular, neurons perform localized mRNA translation in nerve synapses, presumably as part of the mechanism to strengthen or alter the synaptic connections.

13. Other genes known to be involved in mRNA transcription or translation were identified in the microarray experiments, including staufen, orb, moesin, and elF- 2G. Needed to test if altering these genes affected memory. Focus on staufen.

14. The staufen gene, has a temperature- sensitive mutant. At the restrictive temperature (29 C), the staufen mutant stauC8/stauD3 shows loss-of-function. At the permissive temperature (18 C) only slight defects are seen.

15. Raised staufen mutants at permissive temperature. Trained adults staufen and wild type. Split trained flies into 2 groups: 1. Kept at permissive temp 2. Kept at restrictive temp Tested for memory retention 1 day later

16. Learning was normal for staufen flies kept at permissive temp. Learning was abolished for staufen flies kept at restrictive temp Staufen is essential for learning