Abstract
Formation of long term memory (LTM) formation requires delicate regulation of numerous molecules in order to change the plasticity of neurons. To elucidate the expression of memory genes and neural activity, we are dedicated to providing molecular tools for investigating the temporal and spatial expression of memory regulated genes during the formation of learning and memory at single cell resolution. We first generated a series of promoter driven lines for the purpose of reporting spatial expressions of specific memory genes in response to neuronal activity. After the expression pattern is confirmed either via in situ hybridization or antibody staining, Gal4 sequence will subsequently be replaced by photoconvertible fluorescent protein to visualize the transcriptional activity real time. Thus far, we have generated several constructs, including a 5.5 kb and a 3 kb regions of N-methyl-D-aspartate receptor 2 (NR2) promoter in random insertion vector, pPTGAL, and a modified version replacing GAL4 with fluorescent protein EOS. And constructs of NR1, NR2, cAMP response element-binding protein A (CREBA), cAMP response element-binding protein 2 (CREB2), dopamine receptor (DopR1), DopR2, 5-hydroxytryptamine receptor (5-HT1A), 5-HT7 serotonin transporter (SERT), dopa-decarboxylase (Ddc), slow border cells (Slbo), ephrin genes with different length of regulatory elements on specific insertion vector pBPGAL4 have also been generated and sequenced. These constructs were used to generate promoter driven fly lines. Thus far, we have obtained several promoter lines expressing pattern of GFP (ex. CREBA 8k, Slbo 5.4k) that needed further confirmation and one confirmed construct of 5.2 kb 5-HT1A on pBPGAL4 that expresses GFP fluorescence in and lobes of the mushroom body, AMMC and the optic lobe of fly brain. The expression pattern of 5-HT1A promoter line correlates well with published in situ hybridization result and its known anatomical function. To better monitor the activity of 5-HT1A in response to learning within short period of time, promoter constructs with photoconvertible fluorescent protein-PSmOrange and Kaede have also been generated. Two different pH-sensitive 5-HT1A constructs were made for evaluating the protein trafficking of 5-HT1A during memory formation. These two constructs have been made and are in process of generating transgenic fly lines.