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The ability to directly measure acetylcholine (ACh) release is an essential step toward understanding its physiological function. Here we optimized the GRAB ACh (GPCR-activation-based ACh) sensor to achieve substantially improved sensitivity in ACh detection, as well as reduced downstream coupling to intracellular pathways. The improved version of the ACh sensor retains the subsecond response kinetics, physiologically relevant affinity and precise molecular specificity for ACh of its predecessor. Using this sensor, we revealed compartmental ACh signals in the olfactory center of transgenic flies in response to external stimuli including odor and body shock. Using fiber photometry recording and two-photon imaging, our ACh sensor also enabled sensitive detection of single-trial ACh dynamics in multiple brain regions in mice performing a variety of behaviors. A genetically encoded acetylcholine sensor with improved sensitivity allows detection of cholinergic neurotransmission in vivo in the Drosophila and mouse brain.

Recent studies in mammals have documented the neural expression and mobility of retrotransposons and have suggested that neural genomes are diverse mosaics. We found that transposition occurs among memory-relevant neurons in the Drosophila brain. Cell type-specific gene expression profiling revealed that transposon expression is more abundant in mushroom body (MB) αβ neurons than in neighboring MB neurons. The Piwi-interacting RNA (piRNA) proteins Aubergine and Argonaute 3, known to suppress transposons in the fly germline, are expressed in the brain and appear less abundant in αβ MB neurons. Loss of piRNA proteins correlates with elevated transposon expression in the brain. Paired-end deep sequencing identified more than 200 de novo transposon insertions in áâ neurons, including insertions into memory-relevant loci. Our observations indicate that genomic heterogeneity is a conserved feature of the brain.

Dopamine is synonymous with reward in mammals but associated with aversive reinforcement in insects, where reward seems to be signalled by octopamine; here it is shown that flies have discrete populations of dopamine neurons representing positive or negative values that are coordinately regulated by octopamine. Shared neuronal reward signals The neurotransmitter dopamine has been synonymous with reward in mammals, but is associated with aversive reinforcement in insects. In insects, it was thought, reward was signalled by octopamine. Now Scott Waddell and colleagues show that flies have discrete 'negative' and 'positive' populations of dopamine neurons, which are coordinately regulated by octopamine. This work reconciles previous findings with octopamine and dopamine, and suggests that reinforcement systems in flies are more like those in mammals than previously thought. Dopamine is synonymous with reward and motivation in mammals 1 , 2 . However, only recently has dopamine been linked to motivated behaviour and rewarding reinforcement in fruitflies 3 , 4 . Instead, octopamine has historically been considered to be the signal for reward in insects 5 , 6 , 7 . Here we show, using temporal control of neural function in Drosophila , that only short-term appetitive memory is reinforced by octopamine. Moreover, octopamine-dependent memory formation requires signalling through dopamine neurons. Part of the octopamine signal requires the α-adrenergic-like OAMB receptor in an identified subset of mushroom-body-targeted dopamine neurons. Octopamine triggers an increase in intracellular calcium in these dopamine neurons, and their direct activation can substitute for sugar to form appetitive memory, even in flies lacking octopamine. Analysis of the β-adrenergic-like OCTβ2R receptor reveals that octopamine-dependent reinforcement also requires an interaction with dopamine neurons that control appetitive motivation. These data indicate that sweet taste engages a distributed octopamine signal that reinforces memory through discrete subsets of mushroom-body-targeted dopamine neurons. In addition, they reconcile previous findings with octopamine and dopamine and suggest that reinforcement systems in flies are more similar to mammals than previously thought.

Dopamine plays a central role in motivating and modifying behavior, serving to invigorate current behavioral performance and guide future actions through learning. Here we examine how this single neuromodulator can contribute to such diverse forms of behavioral modulation. By recording from the dopaminergic reinforcement pathways of the Drosophila mushroom body during active odor navigation, we reveal how their ongoing motor-associated activity relates to goal-directed behavior. We found that dopaminergic neurons correlate with different behavioral variables depending on the specific navigational strategy of an animal, such that the activity of these neurons preferentially reflects the actions most relevant to odor pursuit. Furthermore, we show that these motor correlates are translated to ongoing dopamine release, and acutely perturbing dopaminergic signaling alters the strength of odor tracking. Context-dependent representations of movement and reinforcement cues are thus multiplexed within the mushroom body dopaminergic pathways, enabling them to coordinately influence both ongoing and future behavior. By recording from the Drosophila mushroom body during active odor navigation, Zolin et al. reveal that dopaminergic reinforcement pathways encode rewards and goal-directed actions through similar patterns of neural activity and dopamine release.

The fruit fly can evaluate its energy state and decide whether to pursue food-related cues. Here, we reveal that the mushroom body (MB) integrates hunger and satiety signals to control food-seeking behavior. We have discovered five pathways in the MB essential for hungry flies to locate and approach food. Blocking the MB-intrinsic Kenyon cells (KCs) and the MB output neurons (MBONs) in these pathways impairs food-seeking behavior. Starvation bi-directionally modulates MBON responses to a food odor, suggesting that hunger and satiety controls occur at the KC-to-MBON synapses. These controls are mediated by six types of dopaminergic neurons (DANs). By manipulating these DANs, we could inhibit food-seeking behavior in hungry flies or promote food seeking in fed flies. Finally, we show that the DANs potentially receive multiple inputs of hunger and satiety signals. This work demonstrates an information-rich central circuit in the fly brain that controls hunger-driven food-seeking behavior.

Neuronal communication across synapses relies on neurotransmitter release from presynaptic active zones (AZs) followed by postsynaptic transmitter detection. Synaptic plasticity homeostatically maintains functionality during perturbations and enables memory formation. Postsynaptic plasticity targets neurotransmitter receptors, but presynaptic mechanisms regulating the neurotransmitter release apparatus remain largely enigmatic. By studying Drosophila neuromuscular junctions (NMJs) we show that AZs consist of nano-modular release sites and identify a molecular sequence that adds modules within minutes of inducing homeostatic plasticity. This requires cognate transport machinery and specific AZ-scaffolding proteins. Structural remodeling is not required for immediate potentiation of neurotransmitter release, but necessary to sustain potentiation over longer timescales. Finally, mutations in Unc13 disrupting homeostatic plasticity at the NMJ also impair short-term memory when central neurons are targeted, suggesting that both plasticity mechanisms utilize Unc13. Together, while immediate synaptic potentiation capitalizes on available material, it triggers the coincident incorporation of modular release sites to consolidate synaptic potentiation. Synaptic plasticity ensures functionality during perturbations and enables memory formation. Here, the authors describe homeostatic functional and nano-modular active zone modifications for immediate and long-lasting enhancement of neurotransmitter release, and identify Unc13 as a presynaptic molecular target for homeostatic potentiation and learning.

In dynamic environments, animals make behavioural decisions on the basis of the innate valences of sensory cues and information learnt about these cues across multiple timescales 1 – 3 . However, it remains unclear how the innate valence of a sensory stimulus affects the acquisition of learnt valence information and subsequent memory dynamics. Here we show that in the Drosophila brain, interconnected short- and long-term memory units of the mushroom body jointly regulate memory through dopamine signals that encode innate and learnt sensory valences. By performing time-lapse in vivo voltage-imaging studies of neural spiking in more than 500 flies undergoing olfactory associative conditioning, we found that protocerebral posterior lateral 1 dopamine neurons (PPL1-DANs) 4 heterogeneously and bidirectionally encode innate and learnt valences of punishment, reward and odour cues. During learning, these valence signals regulate memory storage and extinction in mushroom body output neurons (MBONs) 5 . During initial conditioning bouts, PPL1-γ1pedc and PPL1-γ2α′1 neurons control short-term memory formation, which weakens inhibitory feedback from MBON-γ1pedc>α/β to PPL1-α′2α2 and PPL1-α3. During further conditioning, this diminished feedback allows these two PPL1-DANs to encode the net innate plus learnt valence of the conditioned odour cue, which gates long-term memory formation. A computational model constrained by the fly connectome 6 , 7 and our spiking data explains how dopamine signals mediate the circuit interactions between short- and long-term memory traces, yielding predictions that our experiments confirmed. Overall, the mushroom body achieves flexible learning through the integration of innate and learnt valences in parallel learning units sharing feedback interconnections. This hybrid physiological–anatomical mechanism may be a general means by which dopamine regulates memory dynamics in other species and brain structures, including the vertebrate basal ganglia. In the brain of fruit flies, the mushroom body achieves flexible learning by using interconnected short- and long-term memory units for dopamine-mediated integration of innate valences with learnt valences obtained through experience.

Long-term memory (LTM) requires transcription and translation of new proteins, yet the transcriptional control of memory remains poorly understood. Here, we performed a transcriptome time-course during LTM formation in Drosophila melanogaster exposed to courtship conditioning. We identified a mushroom body-specific transcriptional memory trace that becomes activated during memory consolidation. Using scRNAseq of CREB-activated cells we were able to detect a persistent transcriptional response in MB neurons after LTM consolidation and retrieval. As a proof of causality, we conducted a loss-of-function screen for genes comprising the transcriptional memory trace, finding 16 positive hits whose disruption impaired LTM. Among them, we identified two neuron activity-regulated genes, Hr38 and sr , which encode transcription factors that are activated by courtship LTM training, required for LTM, and bind to many genes comprising the transcriptional memory trace. Overall, we further define the transcriptional response to LTM and identify transcription factors that may help shape it. Gene expression programs underlying memory are not well understood in Drosophila. Here, authors use mushroom body-specific RNA sequencing to characterize the transcriptional trace of long-term memory and key TFs that may regulate it.

Efficient energy use has constrained the evolution of nervous systems. However, it is unresolved whether energy metabolism may resultantly regulate major brain functions. Our observation that Drosophila flies double their sucrose intake at an early stage of long-term memory formation initiated the investigation of how energy metabolism intervenes in this process. Cellular-resolution imaging of energy metabolism reveals a concurrent elevation of energy consumption in neurons of the mushroom body, the fly’s major memory centre. Strikingly, upregulation of mushroom body energy flux is both necessary and sufficient to drive long-term memory formation. This effect is triggered by a specific pair of dopaminergic neurons afferent to the mushroom bodies, via the D5-like DAMB dopamine receptor. Hence, dopamine signalling mediates an energy switch in the mushroom body that controls long-term memory encoding. Our data thus point to an instructional role for energy flux in the execution of demanding higher brain functions. Energy consumption in the brain is thought to respond to changes in neuronal activity, without informational role. Here the authors show that increased energy flux in the mushroom body, driven by a pair of input dopaminergic neurons, is a command for the formation of long-term memory in Drosophila.

Neurons can adjust synaptic output according to the postsynaptic partners. However, the target-specific regulation of synaptic structures within individual neurons in the central nervous system remains unresolved. Applying the CRISPR/Cas9-mediated split-GFP tagging, we visualized the endogenous active zone scaffold protein, Bruchpilot (Brp), in specific cells. This technology enabled the spatial characterization of the presynaptic scaffolds only within the Kenyon cells (KCs) of the Drosophila mushroom bodies. We found the patterned accumulation of Brp among the compartments of axon terminals, where a KC synapses onto different postsynaptic neurons. Mechanistically, the localized octopaminergic projections along γ KC terminals regulate this compartmental Brp heterogeneity via Octβ2R and cAMP signaling. We further found that physiological stress, such as food or sleep deprivation reorganizes this intracellular pattern in an octopamine-dependent manner. Such concurrent regulation of local active zone assemblies thus suggests how the mushroom bodies integrate changing physiological states.