, 1982; Kamyshev et al., 1999; McBride et al., 1999; Siegel and Hall, 1979; Tompkins et al., 1983). Memory in this assay is quantified as a learning index (LI), which measures the extent of the courtship suppression ( Experimental Procedures). Males homozygous for orb2+GFP had long-term, 24 hr memory comparable to that of control Canton-S males (2, orb2+GFP, LI = 31.7; 1, Canton-S, LI = 32.6), whereas

orb2ΔQGFP and orb2ΔQGFP/orb2attP males had no long-term memory (3, orb2ΔQGFP, LI = 0.63; 4, orb2ΔQGFP/orb2attP, LI = 3.03) ( Figure 1C; see Table S1 available online). These data are consistent with the lack of long-term memory previously reported for homozygous and hemizygous orb2ΔQ mutants ( Keleman et al., Dasatinib purchase 2007). Short-term, 1 hr memory of all tested genotypes was normal ( Figure 1C; Table S2), as previously reported also for orb2ΔQ mutants ( Keleman et al., 2007). These results validate our general strategy

for introducing targeted modifications at the orb2 locus and confirm that the C-terminal GFP tag does not impair Orb2 function. Accordingly, we also introduced the GFP tag for other modifications to the orb2 locus reported below, although for simplicity it is only indicated in allele or protein names when it is Lapatinib mouse exploited in immunolabeling or biochemistry experiments. We used antibodies against the GFP tag on the endogenous Orb2 protein encoded by orb2+GFP to determine its expression pattern and subcellular localization. At the level of light microscopy, Orb2 appeared to be broadly expressed throughout the nervous system of embryo, larvae, and adult, including the

ventral nerve cord (VNC) and the brain. In the adult brain Orb2 appeared to be widely expressed throughout various regions including the lobes, calyces, and soma of the mushroom bodies (MB), a center for olfactory memory formation in insect brains ( Heisenberg, 2003; Figure 2A). Previous studies in other species have variously placed CPEBs at either pre- or postsynaptic sites. Mouse CPEB3, for example, was reported to be present in postsynaptic densities, whereas Aplysia CPEB was shown to localize in presynaptic compartments ( Huang et al., 2003, 2006; Liu Sclareol and Schwartz, 2003; Wu et al., 1998). To examine the subcellular localization of Drosophila Orb2, we examined the calyx (input) region of the MB (see Experimental Procedures for details). Using immuno-electron microscopy, we detected Orb2 both in the presynaptic compartment of the extrinsic MB neurons, characterized by the presence of presynaptic specializations such as electron-dense active zones, synaptic vesicles and occasionally T bars, and the postsynaptic compartment likely to be the termini of the Kenyon cells (KCs) in the calyx, characterized by the presence of close membrane alignments with the presumptive presynaptic region ( Figure 2B). Furthermore, consistent with the reported role for Orb2 during development ( Hafer et al., 2011; Keleman et al.