Olfactory associative learning in Drosophila is mediated by synaptic plasticity between the Kenyon cells of the mushroom body and their output neurons. Both Kenyon cells and their inputs from projection neurons are cholinergic, yet little is known about the physiological function of muscarinic acetylcholine receptors in learning in adult flies. Here, we show that aversive olfactory learning in adult flies requires type A muscarinic acetylcholine receptors (mAChR-A), particularly in the gamma subtype of Kenyon cells. mAChR-A inhibits odor responses and is localized in Kenyon cell dendrites. Moreover, mAChR-A knockdown impairs the learning-associated depression of odor responses in a mushroom body output neuron. Our results suggest that mAChR-A function in Kenyon cell dendrites is required for synaptic plasticity between Kenyon cells and their output neurons.

Highlights d mAChR-A affects GABAergic local neurons in the fly's antennal lobe d mAChR-A activity changes the antennal lobe odor coding and odor valence d mAChR-A protects the GABAergic local neurons from shortterm depression d mAChR-A activation increases the excitability of GABAergic local neurons

Value coding of external stimuli in general, and odor valence in particular, is crucial for survival. In flies, odor valence is thought to be coded by two types of neurons: mushroom body output neurons (MBONs) and lateral horn (LH) neurons. MBONs are classified as neurons that promote either attraction or aversion, but not both, and they are dynamically activated by upstream neurons. This dynamic activation updates the valence values. In contrast, LH neurons receive scaled, but non-dynamic, input from their upstream neurons. It remains unclear how such a non-dynamic system generates differential valence values. Recently, PD2a1/b1 LH neurons were demonstrated to promote approach behavior at low odor concentration in starved flies. Here, we demonstrate that at high odor concentrations, these same neurons contribute to avoidance in satiated flies. The contribution of PD2a1/b1 LH neurons to aversion is context dependent. It is diminished in starved flies, although PD2a1/b1 neural activity remains unchanged, and at lower odor concentration. In addition, PD2a1/b1 aversive effect develops over time. Thus, our results indicate that, even though PD2a1/b1 LH neurons transmit hardwired output, their effect on valence can change. Taken together, we suggest that the valence model described for MBONs does not hold for LH neurons.In order to survive, animals must attach value to external stimuli, be it due to innate or learned behavior. In particular, the ability to accurately evaluate potential food resources is a critical trait for survival. To make such an assessment, animals use many senses, with olfaction often serving as a primary cue. The Drosophila olfactory system resembles that of mammals, including our own, and uses similar principles to decode olfactory information 1,2 . Odors bind to olfactory receptor neurons (ORNs), which are located in the antennae and maxillary palps, where each ORN expresses a single type of odorant receptor (OR) 3-5 . All ORNs expressing the same OR converge onto the same region in the antennal lobe termed the glomerulus 6-8 . Second-order excitatory cholinergic projection neurons (ePNs) have dendrites that are restricted to a single glomerulus, whereas inhibitory GABAergic projection neurons (iPNs) are mostly multiglomerular 9 . Both PN types project to the lateral horn (LH), whereas only ePNs project to the calyx of the mushroom body (MB) 9 . Until recently, associative learning and memory processes were generally believed to occur in the MB, with innate behavior driven by the LH 10,11 . However, although the LH is still believed to contribute greatly to innate behavior, it has become apparent that the rigid functional distinction between the two neuropils cannot be upheld. There is now evidence that the MB also plays a role in some innate olfactory behaviors, mostly attractive 12-14 , while the LH is involved in some forms of associative memory 15 .The LH compartment contains over 1300 cells that are categorized into over 150 types, each with individual morphology 16 . Cells that share mor...

G-protein coupled receptors (GPCRs) play a paramount role in diverse brain functions. Almost 20 years ago, GPCR activity was shown to be regulated by membrane potential in vitro, but whether the voltage dependence of GPCRs contributes to neuronal coding and behavioral output under physiological conditions in vivo has never been demonstrated. Here we show that muscarinic GPCR mediated neuronal potentiation in vivo is voltage dependent. This voltage dependent potentiation is abolished in mutant animals expressing a voltage independent receptor. Depolarization alone, without a muscarinic agonist, results in a nicotinic ionotropic receptor potentiation that is mediated by muscarinic receptor voltage dependency. Finally, muscarinic receptor voltage independence causes a strong behavioral effect of increased odor habituation. Together, this study identifies a physiological role for the voltage dependency of GPCRs by demonstrating crucial involvement of GPCR voltage dependence in neuronal plasticity and behavior. Thus, this study suggests that GPCR voltage dependency plays a role in many diverse neuronal functions including learning and memory.

Highlights d MooSEZs elicit backward locomotion via MDN-dependent and MDN-independent pathways d MooSEZs are connected to MDNs and other descending neurons d MooSEZs and MDNs both respond to olfactory input d MooSEZs can trigger rotational backward locomotion