The staufen/pumilo pathway is involved in Drosophila long-term

memoryJosh Dubnau et al.

• 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.

• 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.

• 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.

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.

• 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.

• 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.

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)

• 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.

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.

• 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.

• 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.

• 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.

• 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.

• 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

• 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