A training duration of 60 s corresponds to normal single cycle training. Flies were exposed to OCT for 60 s with (OCT+) or without (OCT-) electrical shocks (1.5 s pulses every 5 s). Immediately after training, flies were placed in a T-maze, where flies were exposed to an odor (OCT, MCH, or Benzaldehyde) and air simultaneously to examine whether training to OCT affects responses to unrelated odors. Transcript levels were quantified using real-time PCR (model 7500, Applied Biosystems, Carlsbad, CA, USA) as described previously (Yamazaki et al., 2007). Heads were harvested on dry ice, and total

RNA was obtained using TRIzol reagent (Invitrogen, Carlsbad, Epigenetic assay CA, USA). cDNA was synthesized using a High-Capacity cDNA Archive Kit (Applied Biosystems), and qPCR was performed using sybr-green-based chemistry using specific primers (Table S1). Expression of each transcript was normalized to that of GAPDH1. To generate anti-dNR1 antibody, rabbit antiserum (αNM1) was raised against the most C-terminal amino

acid sequence of dNR1, CGKTRPQQSVLPPRYSPGYTSDVSHLVV. Affinity-purified antibody (1:1,000) was used for immunoblotting and immunohistochemistry as described previously (Xia et al., 2005). Fluorescence images of dNR1 distribution in fly brains were obtained using a confocal laser microscope (Fluoview FV500, Olympus, Shinjuku, Tokyo, Japan). Immunoblotting protocols for dCREB2-b (Xia et al., 2005) and for ERK (MAPK) and pERK (pMAPK) (Pagani et al., 2009) have been previously described. Anti-dCREB2-b monoclonal antibody (Yin et al., 1994) was used at a 1:10 dilution. Anti-phospho-p44/42 MAPK GDC-0068 datasheet and anti-total p44/42 MAPK primary antibodies were purchased from Cell Signaling Technology

(Danvers, MA, USA) and used at a 1:1,000 dilution. Signals were detected using HRP conjugated secondary antibodies and ECL blotting reagents (GE Healthcare, Waukesha, WI, USA). For analysis of dCREB2-b in single brains, adult heads were dissected and brains were placed in HL3 solution containing 100 μM TTX. After a first incubation in HL3 with TTX for 10 min in a 5% CO2 incubator, the HL3 solution was replaced with Mg2+-free HL3 solution MycoClean Mycoplasma Removal Kit (SrCl2 was used instead of MgCl2) and further incubated for 2 hr. To block dNMDAR activity in the Mg2+-free condition, 1 mM MK801 was used when indicated. Single brains were homogenized in 10 μl lysis buffer and processed for immunoblotting as described previously. Homer antibodies have been previously described (Diagana et al., 2002). For immunostaining, antibody was first preabsorbed overnight at 4°C with nitrocellulose strips blotted with protein from homerR102 flies and then used at a dilution of 1:1,000 ( Diagana et al., 2002). Immunostaining was performed as previously described ( Diagana et al., 2002). All data are expressed as means ± SEM. Graph Pad Prism version 4.01 was used for statistical analyses. Statistical significance between two groups was analyzed by Student’s t test.