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

The neurexin genes (NRXN1/2/3) encode two families (α and β) of highly polymorphic presynaptic proteins that are involved in excitatory/inhibitory synaptic balance. Recent studies indicate that neuronal activation and memory formation affect NRXN1/2/3α expression and alternative splicing at splice sites 3 and 4 (SS#3/SS#4). Neurons in the biological clock residing in the suprachiasmatic nuclei of the hypothalamus (SCN) act as self-sustained oscillators, generating rhythms in gene expression and electrical activity, to entrain circadian bodily rhythms to the 24 hours day/night cycles. Cell autonomous oscillations in NRXN1/2/3α expression and SS#3/SS#4 exons splicing and their links to rhythms in excitatory/inhibitory synaptic balance in the circadian clock were explored. NRXN1/2/3α expression and SS#3/SS#4 splicing, levels of neurexin-2α and the synaptic scaffolding proteins PSD-95 and gephyrin (representing excitatory and inhibitory synapses, respectively) were studied in mRNA and protein extracts obtained from SCN of C3H/J mice at different times of the 24 hours day/night cycle. Further studies explored the circadian oscillations in these components and causality relationships in immortalized rat SCN2.2 cells. Diurnal rhythms in mNRXN1α and mNRXN2α transcription, SS#3/SS#4 exon-inclusion and PSD-95 gephyrin and neurexin-2α levels were found in the SCN in vivo. No such rhythms were found with mNRXN3α. SCN2.2 cells also exhibited autonomous circadian rhythms in rNRXN1/2 expression SS#3/SS#4 exon inclusion and PSD-95, gephyrin and neurexin-2α levels. rNRXN3α and rNRXN1/2β were not expressed. Causal relationships were demonstrated, by use of specific siRNAs, between rNRXN2α SS#3 exon included transcripts and gephyrin levels in the SCN2.2 cells. These results show for the first time dynamic, cell autonomous, diurnal rhythms in expression and splicing of NRXN1/2 and subsequent effects on the expression of neurexin-2α and postsynaptic scaffolding proteins in SCN across the 24-h cycle. NRXNs gene transcripts may have a role in coupling the circadian clock to diurnal rhythms in excitatory/inhibitory synaptic balance.

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...

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