Second-order conditioning in Drosophila

Abstract

Associative conditioning in Drosophila melanogaster has been well documented for several decades. However, most studies report only simple associations of conditioned stimuli (CS, e.g., odor) with unconditioned stimuli (US, e.g., electric shock) to measure learning or establish memory. Here we describe a straightforward second-order conditioning (SOC) protocol that further demonstrates the flexibility of fly behavior. In SOC, a previously conditioned stimulus (CS1) is used as reinforcement for a second conditioned stimulus (CS2) in associative learning. This higher-order context presents an opportunity for reassessing the roles of known learning and memory genes and neuronal networks in a new behavioral paradigm.

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.

Figure 2.

Odor discrimination. (A) Timeline representations of training and testing. Squares represent odor—solid indicate electric shock (90 V dc) reinforcement, open indicate the absence of reinforcement. We assessed discrimination of odors from odor mixtures during both training (protocols 1 and 2) and testing (protocols 3 and 4). (B) Flies demonstrated significant avoidance of all conditioned stimuli (t-test, t₍₁₃₎ = 8.64, P< 0.0001; t₍₁₃₎ = 9.13, P< 0.0001; t₍₁₃₎ = 11.50, P< 0.0001; and t₍₁₃₎ = 14.24, P< 0.0001). Differences between groups were not significant (ANOVA, F_(3,52) = 1.779, P= 0.1626). Bars indicate mean ± SEM; n= 14/bar.

Figure 3.

Extinction. (A) Timeline representations of training and testing. Squares represent odor presentation—solid indicate reinforcement, open indicate the absence of reinforcement. We assessed the effect of CS1 extinction during SOC (protocol 1), relative to the control group (protocol 2). (B) Presenting CS1 in the absence of the US did not accelerate extinction of the conditioned response (t-test, t₍₁₀₎ = 1.083, P= 0.3042). Bars indicate mean ± SEM; n= 6/bar.