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 in vivo 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_OA1.0. This sensor is highly selective for OA and exhibits a robust and rapid increase in fluorescence in response to extracellular OA. Using GRAB_OA1.0, we monitored OA release in the Drosophila 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_OA1.0 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.

Directional hearing is crucial for animals depending on acoustic signals to locate a mate. We focused on crickets to explore the reliability of directional information forwarded to the brain by the ascending auditory interneuron AN1, which is crucial for phonotactic behavior. We presented calling song from −45° to +45° in steps of 3° and compared the phonotactic steering of females walking on a trackball with the directional responses of AN1. Forty percent of females showed good steering behavior and changed their walking direction when the speaker passed the body’s longitudinal axis. The bilateral latency difference between right and left AN1 responses was small and may not be reliable for auditory steering. In respect to spike count, all AN1 recordings presented significant bilateral differences for angles larger than ±18°, yet 35% showed a mean significant difference of 1–3 action potentials per chirp when the frontal stimulus deviated by 3° from their length axis. For small angles, some females had a very similar AN1 activity forwarded to the brain, but the accuracy of their steering behavior was substantially different. Our results indicate a correlation between directional steering and the response strength of AN1, especially for large angles. The reliable steering of animals at small angles would have to be based on small bilateral differences of AN1 activity, if AN1 is the only source providing directional information. We discuss whether such bilateral response difference at small angles can provide a reliable measure to generate auditory steering commands descending from the brain, as pattern recognition is intensity independent.NEW & NOTEWORTHY The ascending auditory interneuron AN1 has been implicated in cricket auditory steering, but at small acoustic stimulation angles, it does not provide reliable directional information. We conclude that either the small bilateral auditory activity differences of the AN1 neurons are enhanced to generate reliable descending steering commands or, more likely, directional auditory steering is mediated via a thoracic pathway, as indicated by the reactive steering hypothesis.