ablated flies display normal immediate recall after a one hour training session. During a training session of five to eight hours duration, a male will engage in multiple bouts of courtship with a mated female with breaks in between. These repeated attempts at mating are thought to represent the repetition that is required for consolidation of some types of LTM and result in formation of a robust LTM that has been shown to persist at least five to seven days after training. The methodology has been previously described in detail. Male flies to be tested were collected and housed in single vials for 4 – 6 days. For each experiment, control genotypes were tested at the same time as those expressing the knockdown or overexpression construct. In all experiments, 14981513 the scorer was blind to the genotype of the flies. All nave and trained groups contained males. All experiments were performed under ambient light. For experiments using the TARGET system, the temperature was modulated by placing flies at the permissive temperature of 19C or the restrictive temperature of 30C, as appropriate. For induction of transgene expression, flies were transferred to 30C three days prior to training to allow maximum GAL4-mediated expression of the UAS construct. Flies were trained at 30C in an incubator under white light and remained at 30C until 30 minutes before testing, at which time they were transferred to 25C for equilibration to the testing conditions. A courtship index is MedChemExpress TG-101348 calculated as the percentage of the ten-minute period spent in courtship behavior. In 
order to compare memory across genotypes, a memory index was calculated by dividing the courtship index of each test fly by the mean CI of the sham flies of that genotype . A score of 0 indicates the highest memory performance possible, and a score 1.0 indicates no memory. For statistical analyses, data was arcsine transformed in order to approximate a normal distribution and significance was assessed by one-way ANOVA with post-hoc Tukey’s HSD test. When comparing only two genotypes, the student’s t-test was used. The significance level was set at P<0.05. Immunohistochemistry Whole flies were fixed in PFAT/DMSO for one hour then washed in 1xPBS. Brains were microdissected in 1xPBS then post fixed in PFAT/DMSO for 20 mins and stored in MeOH at -20C. Following rehydration in PBT brains were blocked in immunobuffer for two hours at room temperature. They were then incubated overnight at room temperature with primary antibody, rabbit anti-FLAG 1:1000, mouse anti-Trio 1:200, rabbit anti-Drosophila Rpd3, rabbit anti-GFP, rabbit antiMEF2 then incubated overnight at 4C with secondary antibody and mounted with Antifade. The monoclonal antibody anti-Trio was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. For confocal microscopy, optical sections were taken with a Leica TCS SP5 DM6000B Confocal Microscope. Image 11 HDAC4 Regulates Long-Term Memory stacks were taken at intervals of 1 m or 0.5 m and processed with Leica Application Suite Advanced Fluorescence software. Western Blotting 50 - 100 flies were collected in 15 ml tubes and frozen in a dry ice/ethanol bath. The tubes were vortexed 3986806 to snap the heads from the bodies, and then the heads were separated and collected on a piece of acetate over dry ice. Cytoplasmic extracts were prepared by homogenizing heads in 50 l of Buffe