Rapid consolidation to a radish and protein synthesis-dependent long-term memory after single-session appetitive olfactory conditioning in Drosophila

Abstract

In Drosophila, formation of aversive olfactory long-term memory (LTM) requires multiple training sessions pairing odor and electric shock punishment with rest intervals. In contrast, here we show that a single 2 min training session pairing odor with a more ethologically relevant sugar reinforcement forms long-term appetitive memory that lasts for days. Appetitive LTM has some mechanistic similarity to aversive LTM in that it can be disrupted by cycloheximide, the dCreb2-b transcriptional repressor, and the crammer and tequila LTM-specific mutations. However, appetitive LTM is completely disrupted by the radish mutation that apparently represents a distinct mechanistic phase of consolidated aversive memory. Furthermore, appetitive LTM requires activity in the dorsal paired medial neuron and mushroom body alpha'beta' neuron circuit during the first hour after training and mushroom body alphabeta neuron output during retrieval, suggesting that appetitive middle-term memory and LTM are mechanistically linked. Last, experiments feeding and/or starving flies after training reveals a critical motivational drive that enables appetitive LTM retrieval.

Figure 1.

Persistent memory after a single session of appetitive conditioning. Wild-type flies were food deprived for 16–20 h and were conditioned with odor and sucrose reinforcement (as described in Materials and Methods). After training, they were housed in empty vials with water-dampened filter paper until they were tested. Different populations were tested once for odor memory 1, 3, 6, 9, 12, 24, 27, 30, 33, and 36 h after training. Beyond 36 h, a significant number of animals perished, presumably of starvation. However, those that survived displayed robust memory performance. Error bars are SEM. All n ≥ 8.

Figure 2.

A single appetitive training session forms memory that requires new protein synthesis. Flies were either fed 35 mm CXM in 3% ethanol solution (open squares) or 3% ethanol alone (filled diamonds) during a 16 h starvation period before training. All flies were then trained, and different populations were tested once for odor memory 1, 3, 6, 12, or 24 h later. Error bars are SEM. Asterisks denote a significant difference (p < 0.05) at that time point from the performance of the other group. All n ≥ 8.

Figure 3.

Inducible or region-restricted expression of dCREB2b disrupts appetitive LTM but not MTM. Wild-type flies and flies harboring an hs-dCreb2-b transgene were either heat shocked (+hs) for 30 min 2 h before training or were untreated (−hs). A, B, All groups were then trained and tested for 24 h memory (A) or 3 h memory (B). Flies harboring hs-dCreb2-b that were heat shocked displayed defective LTM compared with all other groups, but MTM was unaffected. C, Expressing a uas-dCreb2-b transgene in the MBs with c772, MB247, and c739 GAL4 drivers disrupts LTM. Appetitive LTM performance of c772/uas-dCreb2-b, uas-dCreb2-b;MB247, and c739/uas-dCreb2-b flies was statistically different from all other groups. D, Appetitive MTM was not affected by expressing a uas-dCreb2-b transgene in the MBs. Error bars are SEM. Asterisks denote a significant difference (p < 0.05) from all other unmarked groups.

Figure 4.

Appetitive memory is quickly consolidated and is disrupted by crammer, tequila, and radish mutation. A, Appetitive memory becomes resistant to disruption by cold-shock anesthesia within 2 h after training. Different populations of wild-type flies were subjected to a 2 min cold-shock anesthesia 1 h before, immediately after, or 2 or 12 h after training (open diamonds). They were then allowed to recover and were tested for 24 h appetitive memory. Only the performance of flies that were anesthetized immediately after training differed from that of the other groups and from flies that had not been anesthetized (filled square). All n ≥ 8. B, Twenty-four-hour appetitive memory is disrupted in crammer, tequila, and radish mutant flies. Twenty-four-hour memory performance of cer, teq, and rsh mutant flies was statistically different from wild-type flies. All n ≥ 12. C, Three-hour appetitive MTM is unaffected by cer and teq mutation but is significantly disrupted by rsh mutation. Three-hour memory performance of rsh flies was statistically different from that of all other groups. All n ≥ 11. Error bars are SEM. Asterisks denote a significant difference (p < 0.05) from all other unmarked groups.

Figure 5.

Neurotransmission from MB α′β′ neurons and DPM neurons is required for consolidation of appetitive LTM, whereas transmission from MB αβ neurons is only required for retrieval. The temperature shift protocols are shown pictographically above each graph. A, The permissive temperature of 25°C does not affect 24 h appetitive LTM of any of the lines used in this study. All genotypes were trained and tested for 24 h memory at 25°C. All n ≥ 8. B, Blocking DPM neuron or MB α′β′ neuron output, but not MB αβ neuron output, for 1 h immediately after training severely impairs 24 h appetitive LTM. Flies were trained at 25°C, and immediately after training, they were shifted to 31°C for 60 min. Flies were then returned to 25°C and stored in empty vials until they were tested for 24 h appetitive LTM at 25°C. All n ≥ 11. C, Blocking MB αβ neuron output, but not DPM neuron or MB α′β′ neuron output, during testing abolishes 24 h appetitive LTM. Flies were trained at 25°C and stored in empty vials for 24 h. They were then shifted to 31°C for 15 min before they were tested for appetitive LTM. All n ≥ 8. Error bars are SEM. Asterisks denote a significant difference (p < 0.05) from all other unmarked groups. All flies harbor one copy of the uas-shi ^ts1 transgene.

Figure 6.

Evidence against a role for EB ring neurons in LTM retrieval. A–C, Projections of the entire midbrain of flies driving a uas-CD8::GFP transgene with the c547 (A), Ruslan (B), and Feb170 (C) enhancer trap lines. All lines show clear expression in R2/R4 ring neurons in the outer layers of the EB compared with R1/R3 neurons in c739 (data not shown). The driver name is listed in the lower left-hand corner of each panel. D, Blocking c547 or Feb170 neurons, but not Ruslan neuron output, for 15 min before and during testing impairs 24 h appetitive LTM performance. The temperature shift protocol is shown pictographically. All genotypes were trained at 25°C and tested for 24 h memory at 31°C. All flies with uas-shi ^ts1, n ≥ 8; heterozygous GAL4/+ flies, n ≥ 4. E, Blocking c547 or Feb170 neurons, but not Ruslan neurons, impairs phototaxis performance. All flies were tested at 31°C. F, Blocking c547 neurons or Feb170 neurons impairs negative geotaxis performance. All flies were tested at 31°C. All n ≥ 6. Error bars are SEM. Asterisks denote a significant difference (p < 0.05) from all other unmarked groups. All flies harbor one copy of the uas-shi ^ts1 transgene.

Figure 7.

Experiment to test whether satiation reversibly suppresses memory retrieval. Wild-type flies were starved for 16–20 h, trained with a single appetitive conditioning session, and were either returned to food vials for 48 h (group A) or for 24 h and then subsequently food deprived for the next 24 h (group B). Both groups were trained, stored, and tested for 48 h appetitive LTM at 25°C.