ABSTRACT: Disruption of learning and memory can occur for a number of reasons including chronic alcohol abuse, head injury, anoxia, and various neurodegenerative disorders such as Alzheimer's disease. An understanding of the basic mechanisms underlying learning and memory is essential for prevention and treatment of such brain disorders. Recent studies unequivocally demonstrated the astonishing similarities in the basic mechanisms of learning and memory between divergent species, indicating that studies of experimentally tractable model organisms with relatively simpler nervous systems are likely to produce valuable information that will facilitate our understanding of learning and memory in higher organisms. We are studying the experience-dependent modification in Drosophila male courtship as a physiologically relevant model of learning and memory. The genome-scale gene-profiling analysis in this model system should provide us the basic information about learning-induced changes in gene expression, which will be the foundation of our future studies to identify genes and genetic pathways specifically involved in the formation of long-term memory. The proposed gene-profiling experiment is designed to identify candidate genes and genetic pathways involved in the formation of long-term memory in the Drosophila courtship conditioning. The specific aims of this project are to: 1) compare gene expression profiles between the naive and trained wild type males using the GeneChip Drosophila Genome 2.0 Arrays and identify genes that are up- or down-regulated in the fly head immediately or 24-hr after the male fly is exposed to the condition where the long-term courtship memory is established (i.e., paired with a mated female for 7 hrs), 2) carry out the same analysis for the per null mutant males, 3) compare the identified genes between the 0 and 24-hr after training as well as the wild type and per mutant males, 4) identify candidate genes which might be involved in the long-term memory formation either upstream or downstream of per, 5) classify the candidate genes according to their putative cellular function and discern the possible genetic pathways necessary for the long-term memory formation. When a male fly is presented with a previously mated female, he will initially court her vigorously but his courtship activity is reduced over time. Subsequently, when paired with a virgin female, the male will display a reduced amount of courtship compared with naïve males with no prior courtship experience. This modification of courtship is called âcourtship conditioningâ. We found that this type of courtship memory lasts at least 5 days when a male is trained with a mated female for 7 hrs. We have also found that the period gene (per) that is involved in circadian rhythm regulation plays a critical role in the formation of long-term, but not short-term, courtship memory. Our hypotheses are 1) the long-lasting memory in 7 h-trained males is accompanied with characteristic temporal changes in gene expression in the brain, 2) per mutant males are defective in regulation of critical genes involved in long-term courtship memory, 3) the memoryârelated changes in gene expression can be detected by comparing total head transcript levels between trained and naïve as well as the wild type and the per mutant males using the Affymetrix microarray. Two fly strains, a wild type Oregon R (OR) and a null mutant allele of per (per01) with the OR background for the second and third chromosomes, will be used. Virgin males and females will be collected without anesthesia within 6 hrs after eclosion and maintained individually in vials until experiments. Mated wild type females will be prepared by crossing to the wild type males the night before they are used for conditioning males. All flies will be raised at 25oC in a 12 h light/12 h dark cycle and the following behavioral experiments will be carried out during daytime. For courtship conditioning, a 3 day-old virgin male (either OR or per01) will be placed with a mated OR female in a conditioning chamber (15 mm x 5 mm in depth) containing fly food. As a control, a virgin male will be introduced into another conditioning chamber without a mated female. Seven hours after the male is introduced into the chamber, he will be anesthetized under CO2 gas or transferred to a new vial. For the anesthetized male, the head will be immediately excised and frozen with liquid nitrogen. The male fly transferred to a new vial will be decapitated 24 hrs after the end of the 7-hr training. Approximately thirty fly heads will be pooled for each of the 8-genotype/treatment groups, i.e., Oregon R 0-hr and 24-hr after conditioning, per01 0-hr and 24-hr after conditioning, in addition to the corresponding four naïve controls. Total RNA will be extracted from the pooled heads using the TRIzol Reagent (Life Technologies) followed by an RNeasy (Qiagen) cleanup step and a DNAase I digestion step. After purification, the RNA will be resuspended in DEPC water and the absorbance will be checked at 260 and 280 nm for determination of sample purity and concentration. The GeneChip Drosophila Genome 2.0 Arrays will be used to quantify the levels of transcripts in each sample.
