This stimulation caused a robust and significant decrease in memory. To further ensure that this decrement was due to a loss of consolidated memory rather than any remaining labile memory, we imposed a cold shock at 2 hr to eliminate labile memory, followed by a 20 min stimulation with PS-341 in vitro trpA1 just prior to a 3 hr memory test ( Figure 4B). Remarkably, we found that this stimulation of MBgal80/+; c150-gal4/+ neurons led to a complete loss of consolidated memory. These data, along with Figure 1D, indicate that, while

early labile memories are more sensitive than consolidated memories to endogenous dopamine activity after learning ( Figures 3A–3B), excessive stimulation of these neurons with TrpA1 is sufficient to weaken both forms of memory. Appetitive olfactory memories are consolidated within the first few hours

after training to form a stable memory that lasts for days (Tempel et al., 1983 and Krashes and Waddell, 2008). Although the formation of appetitive memory has been shown to be independent of synaptic activity of DANs during acquisition (Schwaerzel et al., 2003), we wondered whether this form of memory is vulnerable to DAN-mediated forgetting. Interestingly, AZD6738 stimulating TH-gal4 neurons for 20 or 80 min after appetitive memory training led to a robust and significant decrease in memory expression measured at 3 hr ( Figures 4C

and 4C′), an effect we mapped to the MBgal80/+; c150-gal4/+ neurons ( Figure 4D). To eliminate the possibility that stimulation of c150-gal4 DANs was interfering with the consolidation of appetitive memory, we performed an 80 min stimulation of MBgal80/+; c150-gal4/+ neurons just prior to a 6 hr retrieval test ( Figure 4E). Once again, we observed a significant decrease in memory performance when stimulating just prior to testing at 6 hr. Together, these data indicate that found stimulated activity of c150-gal4 DANs can also induce the forgetting of consolidated appetitive memories. Our blocking experiments of synaptic activity strongly indicate that some of the c150-gal4 PPL1 DANs (MP1, heel/peduncle; MV1, junction/lower stalk; V1, upper stalk; Figure 5A) that innervate the mushroom bodies have continued synaptic activity after conditioning. To verify and measure this activity, we expressed UAS-GCaMP3.0 ( Tian et al., 2009), which encodes a Ca2+-sensitive enhanced green fluorescent protein (GFP), within the DANs via TH-gal4. In order to isolate the Ca2+-based increases in fluorescence from motion-based changes in fluorescence, we included a UAS-RFP ( Pramatarova et al., 2003), which encodes a Ca2+-insensitive red fluorescent protein (RFP) with an emission spectrum largely separate and distinct from the GCaMP3.0.