Dopamine (DA) is synonymous with reward and motivation in mammals1,2. However, only recently has dopamine been linked to motivated behavior and rewarding reinforcement in fruit flies3,4. Instead octopamine (OA) has historically been considered the signal for reward in insects5–7. Here we show using temporal control of neural function in Drosophila that only short-term appetitive memory is reinforced by OA. Moreover, OA-dependent memory formation requires signaling through DA neurons. Part of the OA signal requires the α-adrenergic like OAMB receptor in an identified subset of mushroom body (MB)-targeted DA neurons. OA triggers an increase in intracellular calcium in these DA neurons and their direct activation can substitute for sugar to form appetitive memory, even in flies lacking OA. Analysis of the β-adrenergic like Octβ2R receptor reveals that OA-dependent reinforcement also requires an interaction with DA neurons that control appetitive motivation. These data suggest that sweet taste engages a distributed OA signal that reinforces memory through discrete subsets of MB-targeted DA neurons. In addition, they reconcile prior findings with OA and DA and suggest that reinforcement systems in flies are more similar to mammals than previously envisaged.

Synaptic vesicles fuse at active zone (AZ) membranes where Ca2+ channels are clustered and that are typically decorated by electron-dense projections. Recently, mutants of the Drosophila melanogaster ERC/CAST family protein Bruchpilot (BRP) were shown to lack dense projections (T-bars) and to suffer from Ca2+ channel–clustering defects. In this study, we used high resolution light microscopy, electron microscopy, and intravital imaging to analyze the function of BRP in AZ assembly. Consistent with truncated BRP variants forming shortened T-bars, we identify BRP as a direct T-bar component at the AZ center with its N terminus closer to the AZ membrane than its C terminus. In contrast, Drosophila Liprin-α, another AZ-organizing protein, precedes BRP during the assembly of newly forming AZs by several hours and surrounds the AZ center in few discrete punctae. BRP seems responsible for effectively clustering Ca2+ channels beneath the T-bar density late in a protracted AZ formation process, potentially through a direct molecular interaction with intracellular Ca2+ channel domains.

SummaryDuring olfactory learning in fruit flies, dopaminergic neurons assign value to odor representations in the mushroom body Kenyon cells. Here we identify a class of downstream glutamatergic mushroom body output neurons (MBONs) called M4/6, or MBON-β2β′2a, MBON-β′2mp, and MBON-γ5β′2a, whose dendritic fields overlap with dopaminergic neuron projections in the tips of the β, β′, and γ lobes. This anatomy and their odor tuning suggests that M4/6 neurons pool odor-driven Kenyon cell synaptic outputs. Like that of mushroom body neurons, M4/6 output is required for expression of appetitive and aversive memory performance. Moreover, appetitive and aversive olfactory conditioning bidirectionally alters the relative odor-drive of M4β′ neurons (MBON-β′2mp). Direct block of M4/6 neurons in naive flies mimics appetitive conditioning, being sufficient to convert odor-driven avoidance into approach, while optogenetically activating these neurons induces avoidance behavior. We therefore propose that drive to the M4/6 neurons reflects odor-directed behavioral choice.

SummaryIn Drosophila, negatively reinforcing dopaminergic neurons also provide the inhibitory control of satiety over appetitive memory expression. Here we show that aversive learning causes a persistent depression of the conditioned odor drive to two downstream feed-forward inhibitory GABAergic interneurons of the mushroom body, called MVP2, or mushroom body output neuron (MBON)-γ1pedc>α/β. However, MVP2 neuron output is only essential for expression of short-term aversive memory. Stimulating MVP2 neurons preferentially inhibits the odor-evoked activity of avoidance-directing MBONs and odor-driven avoidance behavior, whereas their inhibition enhances odor avoidance. In contrast, odor-evoked activity of MVP2 neurons is elevated in hungry flies, and their feed-forward inhibition is required for expression of appetitive memory at all times. Moreover, imposing MVP2 activity promotes inappropriate appetitive memory expression in food-satiated flies. Aversive learning and appetitive motivation therefore toggle alternate modes of a common feed-forward inhibitory MVP2 pathway to promote conditioned odor avoidance or approach.