Figure 1.

First- and second-order conditioning. (A) Schematic representation of our automated odor- and shock-delivery system. Bubblers contained odorants suspended in mineral oil [BEN (8 × 10⁻⁴), MCH (1.4 × 10⁻³), and OCT (2 × 10⁻³); CS1, CS2, and CS⁻] or mineral oil alone (O) and drew ambient room air using an in-house vacuum system (650 mL min⁻¹). Solenoids (Analytical Research Systems; white rectangles) directed airflow by opening or closing in response to computer-controlled relays. Air flowed through teflon-coated tubing (Tygon SE-200; solid lines; arrow indicates direction) from bubblers to solenoids, then into acrylic copper-coil-lined training tubes (gray rectangle). We presented mixed odors by opening two solenoids simultaneously. Electric shock (90 V dc; dotted line) was delivered from a dc-regulated power supply (Circuit Specialists) directly to the training tubes. (B) Timeline representations of training and testing. Squares represent stimuli—solid indicate reinforcement, open indicate the absence of reinforcement. All flies received both FOC and SOC and were tested for their responses to either CS1 (C) or CS2 (D) vs. the CS⁻. In the paired–paired protocol (P–P), both CS1 and CS2 were reinforced. In the paired–unpaired protocol (P–U), CS2 preceded CS1 by 45 sec, while in the unpaired–paired protocol (U–P), CS1 preceded the US by 45 sec. During the SOC phase of the P–P and U–P protocols, CS2 was presented alone for 7 sec, followed by simultaneous presentation of both CS1 and CS2. (C) Pairing stimuli during FOC was required to generate a conditioned response to CS1 vs. the CS⁻ (ANOVA, F_(2,21) = 152.0, P< 0.0001; Tukey, P≤ 0.05). Note that unpairing of stimuli during SOC did not reduce the first-order conditioned responses of flies using the P–U protocol. Bars indicate mean ± SEM; n= 8/bar. (D) Pairing of stimuli during both FOC and SOC was required to generate a conditioned response to CS2 vs. the CS⁻ (ANOVA, F_(2,21) = 14.68, P< 0.0001; Tukey, P≤ 0.05). Bars indicate mean ± SEM; n= 8/bar.