DPM neuron ITM trace across age. (*A* and *B*) Time course for the percent change in fluorescence (ΔF) relative to baseline (F_{o}) of G-CaMP in response to Oct (*A*) or Ben (*B*), measured in the distal portion of the vertical lobes of the MBs in flies 10 d or 30 d of age. The horizontal axis represents the time after odor presentation. (*C*) Diagram illustrating two conditioning protocols used for imaging experiments. Flies received paired or unpaired conditioning with 1 min of odor CS+ along with 12 electric shock pulses. After conditioning, each fly was mounted in a pipette tip and prepared for imaging. The calcium response to Oct was assayed first by imaging across a 3-s odor exposure. After a 3-min interval, the calcium response to Ben or Mch was assayed in an identical way. The imaging was performed across 25 to 99 min after conditioning in three time interval groups: 25, 25 to 49 min; 50, 50 to 74 min; 75, 75 to 99 min after conditioning. (*D*) Calcium responses in the vertical lobes at various times after conditioning using 10-d-old flies trained with Oct. As a control, imaging was performed using flies without conditioning (i.e., naive). A significant increase in %*ΔF/F*_{o} in response to CS+ odor (Oct) was detected in DPM neurons at 25 to 49 min after conditioning compared with naive flies [Fisher least significant difference (LSD) following ANOVA, *P* = 0.044]. The CS+ response for all other time intervals was not significantly different from the naive response (Fisher LSD following ANOVA, *P* ≥ 0.825). No significant difference was observed in the response magnitudes to the CS− odor across time (Fisher LSD following ANOVA, *P* ≥ 0.499). Given the constancy in CS− response, we also compared the CS+ response vs. the CS− response within each time interval group. The CS+ response was significantly higher than the CS− response at 25 to 49 min after conditioning; however, no significant difference was observed at 50 min after training and thereafter (Tukey post hoc following significant differences after one-way ANOVA: 25–49 min, *P* = 0.003; 50–74 min, *P* = 0.955; 75–99 min, *P* = 0.955; naive, *P* = 0.994). Asterisks indicate a statistically significant difference (*n* = 9–14 for all groups). (*E*) The ratio of the response to the CS+ odor (Oct) relative to the CS− odor (Ben) in flies receiving paired vs. unpaired conditioning. As the CS− responses were constant across time intervals (*D*), this allows the CS+ response to be normalized within each fly to the CS− response. The 25- to 49-min time interval showed a marked difference in CS+/CS− response in the paired group vs. unpaired group (Tukey post hoc following significant differences after one-way ANOVA, *P* = 0.003). No significant difference was observed at 50 min after training and thereafter (Tukey post hoc following significant differences after one-way ANOVA, *P* ≥ 0.929). Asterisks indicate a statistically significant difference (*n* = 9–14 for all groups). (*F*) Calcium responses in the vertical lobes of the MBs at various times after conditioning with 30-d-old flies trained with Oct. As a control, imaging was performed with flies without conditioning (i.e., naive). The CS+ response for all time intervals was not significantly different from the naive response (Tukey post hoc following significant differences after one-way ANOVA, *P* ≥ 0.817). No significant difference was observed in the response magnitudes to the CS− odor across time (Tukey post hoc following significant differences after one-way ANOVA, *P* ≥ 0.958). Given the constancy in CS− response, we also compared the CS+ response to the CS− response within each time interval group. No significant difference was observed across time (Tukey post hoc following significant differences after one-way ANOVA, *P* ≥ 0.998; *n* = 9–19 for all groups). (*G*) The ratio of the response to the CS+ odor (Oct) relative to the CS− odor (Ben) in flies receiving paired vs. unpaired conditioning. Because the CS− responses were constant across time intervals (*F*), this allows the CS+ response to be normalized within each fly to the CS− response. No significant difference in CS+/CS− response was observed with pairing compared with unpaired group across time intervals (Tukey post hoc following significant differences after one-way ANOVA, *P* ≥ 0.996). Asterisks indicate a statistically significant difference (*n* = 6–19 for all groups). (*H*) The ratio of CS+/CS− responses across time interval groups for 10- and 30-d-old flies. The 25- to 49-min time interval showed a robust difference in CS+/CS− response between in 10- and 30-d-old flies (Tukey post hoc following significant differences after one-way ANOVA, *P* = 0.014). (*I*) Flies 10 d or 30 d of age were trained with Mch. The ratio in the response to the CS+ odor (Mch) relative to the CS− odor (Oct) is plotted. The 25- to 49-min time interval after conditioning showed a marked increase in CS+/CS− response compared with naive flies in 10-d-old flies (Fisher LSD following ANOVA, *P* = 0.020), but not in 30-d-old flies (Fisher LSD following ANOVA, *P* = 0.788). The difference in CS+/CS− response between in 10- and 30-d-old flies was observed at 25 to 49 min (Fisher LSD following ANOVA, *P* = 0.002), 50 to 74 min (Fisher LSD following ANOVA, *P* = 0.001), and 75 to 99 min (Fisher LSD following ANOVA, *P* = 0.020; *n* = 10–12 for all groups). All error bars indicate SEM.

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