An octopamine-specific GRAB sensor reveals a monoamine relay circuitry that boosts aversive learning.

Octopamine (OA), analogous to norepinephrine in vertebrates, is an essential monoamine neurotransmitter in invertebrates that plays a significant role in various biological functions, including olfactory associative learning. However, the spatial and temporal dynamics of OA remain poorly understood due to limitations associated with the currently available methods used to detect it. To overcome these limitations, we developed a genetically encoded GPCR  activation-based (GRAB) OA sensor called GRAB. This sensor is highly selective for OA and exhibits a robust and rapid increase in fluorescence in response to extracellular OA. Using GRAB, we monitored OA release in the mushroom body (MB), the fly's learning center, and found that OA is released in response to both odor and shock stimuli in an aversive learning model. This OA release requires acetylcholine (ACh) released from Kenyon cells, signaling via nicotinic ACh receptors. Finally, we discovered that OA amplifies aversive learning behavior by augmenting dopamine-mediated punishment signals via Octβ1R in dopaminergic neurons, leading to alterations in synaptic plasticity within the MB. Thus, our new GRAB sensor can be used to monitor OA release in real time under physiological conditions, providing valuable insights into the cellular and circuit mechanisms that underlie OA signaling.