Scott Waddell

Dietary magnesium (Mg ²⁺ ) supplement enhances memory in mammals and Drosophila , but whether and how Mg ²⁺ changes molecular states of neurons, remains unknown. Here we used single-cell RNA sequencing to map cell-type specific transcriptional responses to memory-enhancing dietary Mg ²⁺ . These analyses revealed changes in Hippo-pathway gene expression in memory-relevant αβ Kenyon Cells (KCs) of the mushroom bodies. Targeted knock-down of Merlin , hippo ( hpo ) and warts demonstrated requirement in αβ KCs for Mg ²⁺ -enhanced, but not baseline memory. Further study of Hippo signaling identified Yorkie ( yki ) transcriptional coactivator and DNA-binding partner Scalloped ( sd ) to be required for baseline and Mg ²⁺ -enhanced memory. Moreover, releasing Yki::Sd of inhibition from Tondu-domain containing growth inhibitor ( Tgi ) specifically in αβ KCs enhanced memory, independent of diet. Analyses of Yki::Sd target genes identified several required in αβ KCs for baseline memory, including crb and Glut1 which are also Mg ²⁺ -induced in a Hippo-dependent manner in αβ KCs. These genes suggest that aiding the metabolic demands of long-term memory and facilitating structural neuronal plasticity are key mediators of Mg ²⁺ -induced memory enhancement. We propose pharmacological enhancement of Sd-dependent transcription, and activity of particular Sd-target genes may have therapeutic potential for memory disorders.

Animals navigate through dynamic environments in search of resources. Dopaminergic systems are known to control movement and assign motivational value to cues, actions and outcomes, but how they guide ongoing locomotion towards a goal remains unclear. Here, we demonstrate that Drosophila mushroom body innervating dopaminergic neurons encode steering signals in world-centred (allocentric) and body-centred (egocentric) coordinates during goal-directed navigation. Thirsty flies track water vapour through upwind directed turning and locomotion, that persists for several seconds after the vapour disappears. We identified a population of rewarding dopaminergic neurons to be specifically required for maintaining directed tracking following vapour offset. Recordings of these dopaminergic neurons during behaviour revealed delayed bilateral activity that followed upwind-directed turning in allocentric coordinates. Moreover, lateralized ipsilateral activity in a subset of these dopaminergic neurons encode whether the fly turns left or right in egocentric coordinates. Finally, these dopaminergic neurons also exhibited increased ipsilateral activity when turning in the direction where water vapour was previously encountered. Therefore, activity of mushroom body dopaminergic neurons represents steering and direction-specific actions that together maintain goal-directed tracking in turbulent environments.

Internal states such as hunger dynamically reshape activity across circuits to support resource seeking. Neuromodulation provides a means of controlling such physiological properties of neurons, but how this flexibility regulates memory networks remains unclear. Here, we describe how hunger reconfigures a dopaminergic food-reward circuit between two physiological modes that support memory formation, in Drosophila . Starvation suppresses baseline dopaminergic activity through peptidergic signalling, enabling reward-evoked, large-amplitude dopamine neuron spikes to reinforce learning. This spiking mode can be engaged by sugar consumption and persists beyond feeding, reflecting the fly’s satiety state.Persistent dopaminergic large-amplitude spiking reinforces learning and transitions the memory network into a mode that prioritizes consolidation of recently acquired memories. The transition from hunger to satiation is also reflected in the activity of postsynaptic output neurons that shift from a tonic, decorrelated mode that is responsive to dopamine into a bursting, correlated mode in which further dopaminergic modulation is occluded. Therefore, nutrient deprivation reconfigures the memory circuit into a learning competent dopamine-receptive mode, which persistent reinforcing dopamine then switches into a satiated mode driving memory consolidation. Together these processes mechanistically intertwine state-dependent reward-signalling with subsequent memory stabilization through transitions in physiological mode.

Associating multiple sensory cues with a single experience or object is a fundamental process that improves object recognition and memory performance. However, neural mechanisms that bind sensory features during learning and augment memory expression are unknown. Here we demonstrate multisensory appetitive and aversive memory in Drosophila. Combining colours and odours improved memory performance, even when each sensory modality was tested alone. Temporal control of neuronal function revealed visually-selective mushroom body Kenyon Cells (KCs) to be required for enhancement of visual and olfactory memory recall after multisensory training. Synapse-level connectomics suggests that valence-relevant dopaminergic reinforcement could permit the KC-spanning serotonergic DPM neurons to bridge between previously ‘modality-selective’ KC streams. Consistent with this model, DPM transmission is uniquely required during multisensory memory formation and for enhanced expression of olfactory memory afterwards. In addition, signalling via the Dop2R dopamine receptor is required in APL neurons, suggesting that reinforcing dopamine could locally release GABA-ergic inhibition to permit bridging microcircuits to function. Cross-modal binding thereby expands the KCs representing the olfactory memory engram into those representing the colour. We propose that broadening of the engram improves memory performance after multisensory learning and permits a single sensory feature to retrieve the memory of the multimodal experience.

Consolidation is a time when labile memories transition to a stable form. Malaise learning in Drosophila reveals consolidation to also permit memory updating. Flies taught to associate one of two odors with toxin-tainted sugar initially express conditioned odor approach, that following consolidation switches to avoidance. Behavioral reversal emerges from dopaminergic update of parallel memories for the two trained odors. Differential serotoninergic modulation of specific aversive and rewarding dopaminergic neuron subtypes permits post-ingestive intoxication to suppress consolidation of initial odor-sugar memory and simultaneously invert reward memory plasticity into “safety” memory for the odor experienced without food. Fat body release of the Toll-ligand activating protease modSP, and resilience factor Turandot A, instruct malaise updates by triggering autocrine Toll signaling in the same brain dopaminergic neurons that form and consolidate initial sugar memory. This neural mechanism overcomes the credit assignment problem of delayed post-ingestive reinforcement by updating earlier memories of the trained odors.

Sex differences in behaviour exist across all animals, typically under strong genetic regulation. In Drosophila , fruitless / doublesex transcription factors can identify dimorphic neurons but their organisation into functional circuits remains unclear. We present the connectome of the entire Drosophila male central nervous system. This contains 166,691 neurons spanning the brain and nerve cord, fully proofread and annotated including fruitless / doublesex expression and 11,691 types. We provide the first comprehensive comparison between male and female brain connectomes to synaptic resolution, finding 7,205 isomorphic, 114 dimorphic, 262 male-specific and 69 female-specific types. This resource enables analysis of full sensory-to-motor circuits underlying complex behaviours and the impact of dimorphic elements. Sex-specific/dimorphic neurons are concentrated in higher brain centres while the sensory and motor periphery are largely isomorphic. Within higher centres, male-specific connections are organised into hotspots defined by male-specific neurons or arbours. Numerous circuit switches reroute sensory information to form antagonistic circuits controlling opposing behaviours.

Nutrient state shapes not only what animals eat, but how they eat it. We lack circuit-level mechanisms explaining how gustatory and internal state information regulates feeding at the motor control level. In Drosophila , protein deprivation prolongs protein-specific feeding bursts, yet the detailed motor mechanisms underlying this change remain unknown. Using EM connectomics, we identified a feed-forward pathway from proteinsensitive gustatory receptor neurons to swallowing motor neurons. At its core is the Sustain neuron, which coordinates multiple swallowing motor neurons to move food efficiently through the cibarium and pharynx. This nutrient-dependent facilitation of swallowing sustains long feeding bursts, directly linking internal state to the temporal structure of the feeding motor program. Our findings reveal how a dedicated sensorimotor circuit translates physiological need into precise motor control to drive nutrient-specific feeding appetite and highlights the power of combining EM connectomics with experimental circuit dissection.

Using short-term memory (STM) requires that animals can distinguish memories of recent experience from those learned previously. In Drosophila the neuromodulator octopamine (OA) specifically affects STM through adrenergic-like receptors within neurons in the Mushroom Body (MB) network. However, why OA preferentially impacts STM remains unclear. A fly brain connectome reveals that two OA-VPM3 neurons provide most OA input to the MB network. Artificial OA-VPM3 activation during olfactory learning enhances STM and can divert odor salience. α-adrenergic-like receptor knock-down in MB β and γ lobe MB Output Neurons (MBONs) generally impaired STM, while β-adrenergic-like receptor loss from α lobe MBONs enhanced memory. The identity and connectivity of the relevant MBONs suggests OA modulates an interaction between sites of STM and long-term memory (LTM) storage. Indeed, synthetic, odor, and learning-driven, OA-VPM3 activation temporarily blocks LTM expression. Therefore, OA reconfigures the MBON network so the fly prioritizes expression of recent over remote memories.