Glia transmit negative valence information during aversive learning in Drosophila

INTRODUCTION During Drosophila aversive olfactory conditioning, flies are exposed simultaneously to an odor and aversive electrical shocks. Flies learn to associate the odor with the aversive stimulus and subsequently avoid the odor. Learning and memory of this association requires a brain region known as the mushroom bodies (MBs). Odor information is conveyed to the MBs by cholinergic projection neurons, which originate in the antennal lobes and synapse onto the dendrites of MB neurons. However, the cells and pathways that convey aversive shock information to the MBs have been uncertain. Activation of dopaminergic (DA) pathways that innervate the axons of MB neurons can bypass the requirement for aversive stimulation during conditioning, suggesting that DA may convey this information. On the other hand, dopamine is required for memory reinforcement and synaptic plasticity, complex functions that should occur after the transmission of aversive shock information to the MBs. Recent ex vivo imaging studies have suggested that glutamate (Glu) binding to N-methyl-d-aspartate (NMDA)-type Glu receptors (NMDARs) on MB neurons conveys shock information to the MBs, while DA functions subsequently to induce plasticity and reinforce associative memories.RATIONALE NMDARs are expressed abundantly on the MBs and are required for forming associative memories. However, presynaptic Glu terminals-identified by expression of the vesicular Glu transporter (VGLUT), which transports Glu into synaptic vesicles-are extremely sparse in the MBs. This suggested that unidentified Glu terminals expressing an uncharacterized VGLUT may play a role in the transmission of shock information to the MBs and subsequent memory formation.RESULTS We identified a previously unknown DVGLUT, DVGLUT2, through sequence homology and verified that it functions as a Glu transporter. DVGLUT2 is not expressed in neurons but instead is associated with vesicle-like structures in ensheathing glia (EG), which surround neuropil structures, including the MBs. Aversive electrical stimulation induces rapid Ca2+ transients in EG, vesicular exocytosis, and Glu release. Although EG surround all lobes of the MBs similarly, Glu release only occurs in select MB compartments. Other aversive stimuli, including bitter taste and noxious heat stimuli, also induce Glu release from EG in similar patterns. Blocking exocytosis from EG during conditioning impairs aversive learning, whereas artificial activation of EG can replace aversive stimuli during conditioning. Glu released from EG binds to NMDARs on MB neurons and kainate receptors (KARs) on DA neurons. Because of the Mg2+ block mechanism of NMDARs, Glu binding on its own is unable to induce Ca2+ influx into MB neurons. However, coincidental activation of MB neurons by both odors and aversive stimuli during aversive conditioning is sufficient to remove Mg2+ block and induce NMDAR-mediated Ca2+ influx, resulting in synergistic activation of MB neurons by odors and shocks. Glu binding to KARs is sufficient to induce Ca2+ influx into DA neurons and DA release onto MB neurons, but this release is not required for aversive learning after normal odor-shock paired conditioning. Instead, KAR-dependent DA release is required to prevent aberrant learning when shock onset precedes odor onset during backward conditioning.CONCLUSION Our results are consistent with a model in which shock information transmitted from EG bifurcates into two pathways. One pathway consists of Glu release onto NMDARs on MB neurons, and the second pathway induces the release of DA from DA neurons upon aversive but not neutral or appetitive stimulation. Glu released onto MB neurons works synergistically with odor signals and is required for learning, whereas Glu released onto DA neurons prevents inappropriate learning in situations in which an odor cannot be used to predict shocks. Overall, our data suggest that vesicle release of Glu from EG serves to transmit aversive information to higher-order brain areas and also functions to separate aversive sensory information from reinforcement signals during the formation of associative memories. Ensheathing glia transmit aversive information. Aversive stimuli induce vesicular exocytosis from EG and release of Glu onto KAR on dopaminergic neurons (DANs) and NMDARs on MB neurons. (Left) When aversive stimuli are unpaired, Mg2+ block prevents Ca2+ influx. (Right) When paired with odors, odor-dependent depolarization removes Mg2+ block, allowing Ca2+ influx into appropriate MB neurons.