Layered reward signalling through octopamine and dopamine in Drosophila

Abstract

Dopamine is synonymous with reward and motivation in mammals. However, only recently has dopamine been linked to motivated behaviour and rewarding reinforcement in fruitflies. Instead, octopamine has historically been considered to be the signal for reward in insects. Here we show, using temporal control of neural function in Drosophila, that only short-term appetitive memory is reinforced by octopamine. Moreover, octopamine-dependent memory formation requires signalling through dopamine neurons. Part of the octopamine signal requires the α-adrenergic-like OAMB receptor in an identified subset of mushroom-body-targeted dopamine neurons. Octopamine triggers an increase in intracellular calcium in these dopamine neurons, and their direct activation can substitute for sugar to form appetitive memory, even in flies lacking octopamine. Analysis of the β-adrenergic-like OCTβ2R receptor reveals that octopamine-dependent reinforcement also requires an interaction with dopamine neurons that control appetitive motivation. These data indicate that sweet taste engages a distributed octopamine signal that reinforces memory through discrete subsets of mushroom-body-targeted dopamine neurons. In addition, they reconcile previous findings with octopamine and dopamine and suggest that reinforcement systems in flies are more similar to mammals than previously thought.

Figure 1. Octopamine mediates the short-term reinforcing effects of sweet taste

a, Blocking OA neurons with Tdc2-GAL4/ uas-shi^ts1 during conditioning with sucrose lacks consequence (all p>0.4, n≥6). b, Blocking OA neurons during conditioning with arabinose significantly impairs appetitive learning, (p<0.05, n≥10). c, No significant defect is observed when flies are conditioned with arabinose at 23 °C, (all p>0.1, n≥8). d, Blocking OA neurons during conditioning with sorbitol supplemented arabinose has no significant effect (all p>0.4, n=8). Data are mean ± standard error of the mean (s.e.m.). Asterisks denote significant difference between marked groups and all others (all p<0.05, ANOVA).

Figure 2. OA neuron stimulation can replace sugar presentation during conditioning to form short-term appetitive memory

a, Conditioning protocol pairing a 2 min odour presentation with heat-activation (red) of uas-dTrpA1 expressing neurons. b, Tdc2-GAL4/uas-dTrpA1 driven OA neuron activation contingent with odour presentation forms appetitive olfactory memory in satiated flies (p<0.001, n≥14). c, Implanted memory remains significant 30 min after training in satiated flies (p<0.05, n≥8). d, No memory is observed at 3 h, even in hungry flies (p>0.5, n=6). e, Implanting memory with Tdc2-GAL4 neuron stimulation requires OA. Artificial conditioning does not form significant memory in hungry Tβh flies (p>0.05, n≥8). f, Schematic of all four OA neurons that innervate the MB calyx (OA-VUMa2, OA-VPM5, plus the antennal lobe, AL), heel (OA-VPM4, plus the MB γ lobe), or calyx and heel (OA-VPM3). Somata reside in the maxillary (Mx), mandibulary (Md), or labial (Lb) neuromere. g, NP7088-GAL4 expresses in many Tdc2 positive OA neurons. Projection of OA neurons common to Tdc2-GAL4/Tdc2-LexA and NP7088-GAL4 revealed by genetic intersection. MB-OA cell types in each GAL4 are colour-coded. h, 0665-GAL4 labels MB-innervating OA-VPM3 and OA-VPM4 neurons; 247-RFP labeled MB (red). i, 0891-GAL4 specifically labels MB-innervating OA-VPM4 neurons. Scale bar 50 μm. j, Stimulating OA neuron subsets cannot replace sugar presentation in appetitive conditioning. (p>0.05, n≥6).

Figure 3. Reinforcing DA neurons are functionally downstream of OA-dependent reinforcement

a, Memory cannot be implanted with Tdc2 neuron stimulation in dumb¹ (DopR) flies (all p>0.05, except Tdc2-GAL4/uas-dTrpA1 control p<0.001, n≥8). b, 0273-GAL4 labels PAM-DA neurons (dashed box) that innervate the MB (red). c, 0273-GAL4 labels all ~130 TH-positive PAM-DA neurons. d, 0104-GAL4 labels PAM-DA neurons (dashed box). e, 0104-GAL4 labels ~40 TH-positive PAM-DA neurons. Scale bar 50 μm (b and d), 20 μm (c and e). f, Robust appetitive memory implanted with 0104-GAL4 and 0273-GAL4 neuron activation contingent with odour presentation. Memory of 0104-GAL4;uas-dTrpA1 and 0273-GAL4;uas-dTrpA1 flies is significantly different from all others (p<0.01, n≥4). g, Blocking DA neurons with 0104-GAL4/uas-shi^ts1 during arabinose conditioning abolishes appetitive learning (p<0.001, n≥8). h, Blocking 0104-GAL4 DA neurons during sucrose conditioning significantly impairs learning, (p<0.05, n≥6). i, 0104-GAL4 neuron stimulation forms appetitive memory in satiated Tβh flies (p<0.001, n≥6).

Figure 4. OA-dependent reinforcement functions through discrete groups of DA neurons

a, Memory cannot be implanted with Tdc2 neuron stimulation in hungry oamb flies. Only Tdc2-GAL4/uas-dTrpA1 flies display significant learning (p<0.01, n≥8). b, Memory formation with arabinose requires oamb in 0104-GAL4 neurons. Memory of 0104-GAL4;uas-oamb^RNAi flies is significantly different to control groups (p<0.05, n≥18). Uas-oamb^RNAi causes ~40% decrease in oamb transcript (Supplementary Fig. 5A). c, Applying 5 mM OA to the exposed fly brain drives an increase in intracellular Ca²⁺, measured using GCaMP3.0, in 0104-DA neurons. OA-evoked response (red trace) is significantly decreased in brains treated with 2.45 mM mianserin (blue trace, see also Supplementary Fig. 6). First dotted arrow; time of mianserin or vehicle application. Solid arrow; OA or OA with mianserin application. Traces averaged (each n = 11 flies); solid line represents mean and shaded areas s.e.m. Panels, representative pseudocoloured images of fluorescence intensity 3 s before (left) and 3 s after (right) OA application. Dotted circle, analysed region of interest. Scale bar 10 μm. d, Memory cannot be implanted with Tdc2 neuron stimulation in satiated octβ2R/+ heterozygous flies (p>0.05 all groups, except Tdc2-GAL4/uas-dTrpA1, p<0.05, n=8). e Memory implantation is restored in hungry octβ2R/+ flies. Tdc2-GAL4/uas-dTrpA1 flies and Tdc2-GAL4/uas-dTrpA1; octβ2R/+ flies are significantly different to all other groups (p<0.05, n≥6). f, Memory cannot be formed with Tdc2 neuron stimulation in flies that express octβ2R^RNAi in MB-MP1 neurons (all p>0.05 except Tdc2-GAL4/uas-dTrpA1 control, p<0.001, n≥8). Uas-octβ2R^RNAi efficacy has been reported⁴². g, 0104-GAL4 co-expression of mCherry (magenta) and Bruchpilot::GFP (Brp::GFP, green) reveals presynaptic label in the horizontal MB lobe tips. Brp::GFP negative processes in ampr are presumed PAM-DA dendrites. h, Y-Z section (at the level of dashed line in b) reveals Brp::GFP expression only in β′ and γ lobe tips. Scale bar 20 μm. i, GRASP indicates contact between OA and 0104-DA neurons in the ampr (dashed circles). Scale bar 50 μm.