1. Olfactory based learning and memory in the Fruit Fly Drosophila girl boy

2. What’s so great about Drosophila? Small: you can have large populations of flies in a small area Reproduce rapidly and in great quantity Easy to maintain in culture Small genome

3. Seymore Benzer: father of fly genetics and one of the pioneers of the molecular revolution Established a method of mutating DNA Apply a mutagen which breaks up the DNA DNA reassembles with errors Most errors are lethal Rarely a mutation is not but it will affect the animal in some nominal to major way. Those which live can be established as highly inbred (homogenous) colonies and studied.

4. Limitations of the approach: What if the mutation disrupts its proteins function not only as an adult but during development and/or in systems other that the one being studied? Effect of interest can be compounded with other effects and/or secondary effects. Solution: mutations can now be triggered in adults at will. Non affected genes/proteins can compensate: Functional protein isoforms can compensate for mutated proteins. Assumptions of the approach Genes -> proteins -> structural and functional basis of behavior basis of behavior Mutating genes one by one should cause specific defects in behavior Identification of gene reveals molecules necessary for that behavior

5. Odor-shock learning in the fruit fly (the early days) Groups of flies are placed in a start tube. Start tube shuttles them to the grid and rest tubes. Grid tubes contain an odor (A or B) Grid tubes contain shock grids Flourescent light used to attract flies into and out of tubes. One odor is paired with shock Between training trials flies are shuttled to rest tube. What kind of conditioning paradigm is this?

6. Differential conditioning yields a measure of associative processes. Comparative differences in this measure as a function of mutations provides an assay of learning and memory. Key learning mutants: Dunce Turnip Cabbage Rutabaga DCO Amnesiac CS+ CS- CS+ CS-

7. Different learning and memory mutants show subtly different deficits Normal learning in amnesiac flies Abnormal retention

8. Tim Tully and the new and improved odor-shock learning method No light: Flies are put into training chamber and odor is shuttled in and out. More Pavlovian: The paradigm is imposed on the flies (they cannot escape odor-shock pairing by not going into the grid tubes. Flies can’t avoid the test (forced choice)

9. Results: up to 90 percent as compared to 33 percent change in behavior Old results

10. Inter-stimulus interval is important Best learning occurs when CS and US are overlapped

11. Comparison of learning and retention across different mutants reveals multiple phases Acquisition in wildtype Index of learning Index of STM Index of LTM Summary of mutation effects In all cases mutants had learned something and lost it but: Dunce: poor learning no STM LTM Rutabaga/turnip: moderate learning little STM little LTM Amnesiac: good learning little STM LTM Based on what we know of Aplysia, what can we predict about the molecules involved in these mutants?

12. Rutabaga: disrupts the adenylyl cyclase binding site for calmodulin: blocks coincidence detection (of CS and US) DCO: Disrupts the PKA catalytic subunit thus blocks the phosphoralation of the K channels (no spike broadening) Genetic lesioning: Specific ablation of a single gene/protein Dunce: disrupted PDE increases ambient cAMP levels: possibly blocks cAMP pathway by masking learning-dependent changes in cAMP levels amnesiac Amnesiac: disrupted PACAP-like protein which acts like serotonin leaving the second messenger pathway active Other potential mutants that fit the cAMP model: Turnip: gene regulates PKC Ddc: gene regulates 5HT and Dopamine Shaker: A potassium channel gene

13. So where are these genes expressed in the fruit fly brain?

14. Antibody staining indicates that the mushroom bodies contain the molecular components that are key for learning: Adenylyl cyclase PKA cAMP phosphodiesterase

15. Proposed CS-US and behavior pathways Genetic dissection of MB function MB deranged: normal MB as larvae but deformed MBs as adults Learning is fine in larvae but abolished in adults (similar results in bees). Martin Heisenberg