Multisensory learning binds neurons into a cross-modal memory engram.

Associating multiple sensory cues with objects and experience is a fundamental brain 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 both visual and olfactory memory after multisensory training. Voltage imaging in head-fixed flies showed that multisensory learning binds activity between streams of modality-specific KCs so that unimodal sensory input generates a multimodal neuronal response. Binding occurs between regions of the olfactory and visual KC axons, which receive valence-relevant dopaminergic reinforcement, and is propagated downstream. Dopamine locally releases GABAergic inhibition to permit specific microcircuits within KC-spanning serotonergic neurons to function as an excitatory bridge between the previously 'modality-selective' KC streams. Cross-modal binding thereby expands the KCs representing the memory engram for each modality into those representing the other. This broadening of the engram improves memory performance after multisensory learning and permits a single sensory feature to retrieve the memory of the multimodal experience.

Developmental and evolutionary constraints on olfactory circuit selection.

SignificanceIn this work, we explore the hypothesis that biological neural networks optimize their architecture, through evolution, for learning. We study early olfactory circuits of mammals and insects, which have relatively similar structure but a huge diversity in size. We approximate these circuits as three-layer networks and estimate, analytically, the scaling of the optimal hidden-layer size with input-layer size. We find that both longevity and information in the genome constrain the hidden-layer size, so a range of allometric scalings is possible. However, the experimentally observed allometric scalings in mammals and insects are consistent with biologically plausible values. This analysis should pave the way for a deeper understanding of both biological and artificial networks.

Loss of Bicra impairs Drosophila learning and choice abilities.

The Drosophila Bicra (CG11873) gene encodes the sole ortholog of mammalian GLTSCR1 and GLTSCR1L, which are components of a chromatin remodeling complex involved in neoplasia and metastasis of cancer cells. Bicra is highly expressed in Drosophila larval CNS and adult brain, yet its physiological functions in the nervous system remain elusive. Here we report that Bicra is expressed in both neurons and glia of adult brains, and is required for courtship learning and choice ability of male flies. The function of Bicra in the mushroom body, and in particular, Bicra expression in neurons but not glia, is responsible for the male courtship learning and choice performance. This study unravels a novel function of Bicra in cognition-related courtship behaviors in Drosophila, and may provide insight into the neuronal functions of its mammalian orthologs.

Compensatory variability in network parameters enhances memory performance in the Drosophila mushroom body.

Neural circuits use homeostatic compensation to achieve consistent behavior despite variability in underlying intrinsic and network parameters. However, it remains unclear how compensation regulates variability across a population of the same type of neurons within an individual and what computational benefits might result from such compensation. We address these questions in the Drosophila mushroom body, the fly's olfactory memory center. In a computational model, we show that under sparse coding conditions, memory performance is degraded when the mushroom body's principal neurons, Kenyon cells (KCs), vary realistically in key parameters governing their excitability. However, memory performance is rescued while maintaining realistic variability if parameters compensate for each other to equalize KC average activity. Such compensation can be achieved through both activity-dependent and activity-independent mechanisms. Finally, we show that correlations predicted by our model's compensatory mechanisms appear in the Drosophila hemibrain connectome. These findings reveal compensatory variability in the mushroom body and describe its computational benefits for associative memory.

Neurofibromin 1 in mushroom body neurons mediates circadian wake drive through activating cAMP-PKA signaling.

Various behavioral and cognitive states exhibit circadian variations in animals across phyla including Drosophila melanogaster, in which only ~0.1% of the brain's neurons contain circadian clocks. Clock neurons transmit the timing information to a plethora of non-clock neurons via poorly understood mechanisms. Here, we address the molecular underpinning of this phenomenon by profiling circadian gene expression in non-clock neurons that constitute the mushroom body, the center of associative learning and sleep regulation. We show that circadian clocks drive rhythmic expression of hundreds of genes in mushroom body neurons, including the Neurofibromin 1 (Nf1) tumor suppressor gene and Pka-C1. Circadian clocks also drive calcium rhythms in mushroom body neurons via NF1-cAMP/PKA-C1 signaling, eliciting higher mushroom body activity during the day than at night, thereby promoting daytime wakefulness. These findings reveal the pervasive, non-cell-autonomous circadian regulation of gene expression in the brain and its role in sleep.

The amyloid precursor protein is a conserved Wnt receptor.

The Amyloid Precursor Protein (APP) and its homologues are transmembrane proteins required for various aspects of neuronal development and activity, whose molecular function is unknown. Specifically, it is unclear whether APP acts as a receptor, and if so what its ligand(s) may be. We show that APP binds the Wnt ligands Wnt3a and Wnt5a and that this binding regulates APP protein levels. Wnt3a binding promotes full-length APP (flAPP) recycling and stability. In contrast, Wnt5a promotes APP targeting to lysosomal compartments and reduces flAPP levels. A conserved Cysteine-Rich Domain (CRD) in the extracellular portion of APP is required for Wnt binding, and deletion of the CRD abrogates the effects of Wnts on flAPP levels and trafficking. Finally, loss of APP results in increased axonal and reduced dendritic growth of mouse embryonic primary cortical neurons. This phenotype can be cell-autonomously rescued by full length, but not CRD-deleted, APP and regulated by Wnt ligands in a CRD-dependent manner.

Models of heterogeneous dopamine signaling in an insect learning and memory center.

The Drosophila mushroom body exhibits dopamine dependent synaptic plasticity that underlies the acquisition of associative memories. Recordings of dopamine neurons in this system have identified signals related to external reinforcement such as reward and punishment. However, other factors including locomotion, novelty, reward expectation, and internal state have also recently been shown to modulate dopamine neurons. This heterogeneity is at odds with typical modeling approaches in which these neurons are assumed to encode a global, scalar error signal. How is dopamine dependent plasticity coordinated in the presence of such heterogeneity? We develop a modeling approach that infers a pattern of dopamine activity sufficient to solve defined behavioral tasks, given architectural constraints informed by knowledge of mushroom body circuitry. Model dopamine neurons exhibit diverse tuning to task parameters while nonetheless producing coherent learned behaviors. Notably, reward prediction error emerges as a mode of population activity distributed across these neurons. Our results provide a mechanistic framework that accounts for the heterogeneity of dopamine activity during learning and behavior.

Juvenile hormone drives the maturation of spontaneous mushroom body neural activity and learned behavior.

Mature behaviors emerge from neural circuits sculpted by genetic programs and spontaneous and evoked neural activity. However, how neural activity is refined to drive maturation of learned behavior remains poorly understood. Here, we explore how transient hormonal signaling coordinates a neural activity state transition and maturation of associative learning. We identify spontaneous, asynchronous activity in a Drosophila learning and memory brain region, the mushroom body. This activity declines significantly over the first week of adulthood. Moreover, this activity is generated cell-autonomously via Cacophony voltage-gated calcium channels in a single cell type, α'/ß' Kenyon cells. Juvenile hormone, a crucial developmental regulator, acts transiently in α'/ß' Kenyon cells during a young adult sensitive period to downregulate spontaneous activity and enable subsequent enhanced learning. Hormone signaling in young animals therefore controls a neural activity state transition and is required for improved associative learning, providing insight into the maturation of circuits and behavior.

Social facilitation of long-lasting memory is mediated by CO2 in Drosophila.

How social interactions influence cognition is a fundamental question, yet rarely addressed at the neurobiological level. It is well established that the presence of conspecifics affects learning and memory performance, but the neural basis of this process has only recently begun to be investigated. In the fruit fly Drosophila melanogaster, the presence of other flies improves retrieval of a long-lasting olfactory memory. Here, we demonstrate that this is a composite memory composed of two distinct elements. One is an individual memory that depends on outputs from the α'ß' Kenyon cells (KCs) of the mushroom bodies (MBs), the memory center in the insect brain. The other is a group memory requiring output from the αß KCs, a distinct sub-part of the MBs. We show that social facilitation of memory increases with group size and is triggered by CO2 released by group members. Among the different known neurons carrying CO2 information in the brain, we establish that the bilateral ventral projection neuron (biVPN), which projects onto the MBs, is necessary for social facilitation. Moreover, we demonstrate that CO2-evoked memory engages a serotoninergic pathway involving the dorsal-paired medial (DPM) neurons, revealing a new role for this pair of serotonergic neurons. Overall, we identified both the sensorial cue and the neural circuit (biVPN>αß>DPM>αß) governing social facilitation of memory in flies. This study provides demonstration that being in a group recruits the expression of a cryptic memory and that variations in CO2 concentration can affect cognitive processes in insects.

Dopamine-based mechanism for transient forgetting.

Active forgetting is an essential component of the memory management system of the brain1. Forgetting can be permanent, in which prior memory is lost completely, or transient, in which memory exists in a temporary state of impaired retrieval. Temporary blocks on memory seem to be universal, and can disrupt an individual's plans, social interactions and ability to make rapid, flexible and appropriate choices. However, the neurobiological mechanisms that cause transient forgetting are unknown. Here we identify a single dopamine neuron in Drosophila that mediates the memory suppression that results in transient forgetting. Artificially activating this neuron did not abolish the expression of long-term memory. Instead, it briefly suppressed memory retrieval, with the memory becoming accessible again over time. The dopamine neuron modulates memory retrieval by stimulating a unique dopamine receptor that is expressed in a restricted physical compartment of the axons of mushroom body neurons. This mechanism for transient forgetting is triggered by the presentation of interfering stimuli immediately before retrieval.

The central nervous system of whip spiders (Amblypygi): Large mushroom bodies receive olfactory and visual input.

Whip spiders (Amblypygi) are known for their nocturnal navigational abilities, which rely on chemosensory and tactile cues and, to a lesser degree, on vision. Unlike true spiders, the first pair of legs in whip spiders is modified into extraordinarily long sensory organs (antenniform legs) covered with thousands of mechanosensory, olfactory, and gustatory sensilla. Olfactory neurons send their axons through the leg nerve into the corresponding neuromere of the central nervous system, where they terminate on a particularly large number (about 460) of primary olfactory glomeruli, suggesting an advanced sense of smell. From the primary glomeruli, olfactory projection neurons ascend to the brain and terminate in the mushroom body calyx on a set of secondary olfactory glomeruli, a feature that is not known from olfactory pathways of other animals. Another part of the calyx receives visual input from the secondary visual neuropil (the medulla). This calyx region is composed of much smaller glomeruli ("microglomeruli"). The bimodal input and the exceptional size of their mushroom bodies may support the navigational capabilities of whip spiders. In addition to input to the mushroom body, we describe other general anatomical features of the whip spiders' central nervous system.

Availability of food determines the need for sleep in memory consolidation.

Sleep remains a major mystery of biology, with little understood about its basic function. One of the most commonly proposed functions of sleep is the consolidation of memory1-3. However, as conditions such as starvation require the organism to be awake and active4, the ability to switch to a memory consolidation mechanism that is not contingent on sleep may confer an evolutionary advantage. Here we identify an adaptive circuit-based mechanism that enables Drosophila to form sleep-dependent and sleep-independent memory. Flies fed after appetitive conditioning needed increased sleep for memory consolidation, but flies starved after training did not require sleep to form memories. Memory in fed flies is mediated by the anterior-posterior α'/ß' neurons of the mushroom body, while memory under starvation is mediated by medial α'/ß' neurons. Sleep-dependent and sleep-independent memory rely on distinct dopaminergic neurons and corresponding mushroom body output neurons. However, sleep and memory are coupled such that mushroom body neurons required for sleep-dependent memory also promote sleep. Flies lacking Neuropeptide F display sleep-dependent memory even when starved, suggesting that circuit selection is determined by hunger. This plasticity in memory circuits enables flies to retain essential information in changing environments.

Visualization of naive and learned odor representations using in vivo calcium imaging and immunohistochemical bouton mapping of single Drosophila mushroom body neurons.

This protocol enables the quantification of odor-evoked calcium activity in mushroom body Kenyon cells of the Drosophila melanogaster brain at the single bouton level. We also present subsequent characterization of naive and learned odor representations in the context of olfactory coding. This approach to analyzing the neuronal basis of associative learning provides a substrate for similar studies, perhaps in other animals, to probe the attributes of a neuronal memory trace at the level of synapses distributed across neurons. For complete details on the use and execution of this protocol, please refer to Bilz et al. (2020).

Serotonin receptor 5-HT7 in Drosophila mushroom body neurons mediates larval appetitive olfactory learning.

Serotonin (5-HT) and dopamine are critical neuromodulators known to regulate a range of behaviors in invertebrates and mammals, such as learning and memory. Effects of both serotonin and dopamine are mediated largely through their downstream G-protein coupled receptors through cAMP-PKA signaling. While the role of dopamine in olfactory learning in Drosophila is well described, the function of serotonin and its downstream receptors on Drosophila olfactory learning remain largely unexplored. In this study we show that the output of serotonergic neurons, possibly through points of synaptic contacts on the mushroom body (MB), is essential for training during olfactory associative learning in Drosophila larvae. Additionally, we demonstrate that the regulation of olfactory associative learning by serotonin is mediated by its downstream receptor (d5-HT7) in a cAMP-dependent manner. We show that d5-HT7 expression specifically in the MB, an anatomical structure essential for olfactory learning in Drosophila, is critical for olfactory associative learning. Importantly our work shows that spatio-temporal restriction of d5-HT7 expression to the MB is sufficient to rescue olfactory learning deficits in a d5-HT7 null larvae. In summary, our results establish a critical, and previously unknown, role of d5-HT7 in olfactory learning.

BAcTrace, a tool for retrograde tracing of neuronal circuits in Drosophila.

Animal behavior is encoded in neuronal circuits in the brain. To elucidate the function of these circuits, it is necessary to identify, record from and manipulate networks of connected neurons. Here we present BAcTrace (Botulinum-Activated Tracer), a genetically encoded, retrograde, transsynaptic labeling system. BAcTrace is based on Clostridium botulinum neurotoxin A, Botox, which we engineered to travel retrogradely between neurons to activate an otherwise silent transcription factor. We validated BAcTrace at three neuronal connections in the Drosophila olfactory system. We show that BAcTrace-mediated labeling allows electrophysiological recording of connected neurons. Finally, in a challenging circuit with highly divergent connections, BAcTrace correctly identified 12 of 16 connections that were previously observed by electron microscopy.

Immune Receptor Signaling and the Mushroom Body Mediate Post-ingestion Pathogen Avoidance.

In spite of the positive effects of bacteria on health, certain species are harmful, and therefore, animals must weigh nutritional benefits against negative post-ingestion consequences and adapt their behavior accordingly. Here, we use Drosophila to unravel how the immune system communicates with the brain, enabling avoidance of harmful foods. Using two different known fly pathogens, mildly pathogenic Erwinia carotovora (Ecc15) and highly virulent Pseudomonas entomophila (Pe), we analyzed preference behavior in naive flies and after ingestion of either of these pathogens. Although survival assays confirmed the harmful effect of pathogen ingestion, naive flies preferred the odor of either pathogen to air and also to harmless mutant bacteria, suggesting that flies are not innately repelled by these microbes. By contrast, feeding assays showed that, when given a choice between pathogenic and harmless bacteria, flies-after an initial period of indifference-shifted to a preference for the harmless strain, a behavior that lasted for several hours. Flies lacking synaptic output of the mushroom body (MB), the fly's brain center for associative memory formation, lost the ability to distinguish between pathogenic and harmless bacteria, suggesting this to be an adaptive behavior. Interestingly, this behavior relied on the immune receptors PGRP-LC and -LE and their presence in octopaminergic neurons. We postulate a model wherein pathogen ingestion triggers PGRP signaling in octopaminergic neurons, which in turn relay the information about the harmful food source directly or indirectly to the MB, where an appropriate behavioral output is generated.

Visual Input into the Drosophila melanogaster Mushroom Body.

The patterns of neuronal connectivity underlying multisensory integration, a fundamental property of many brains, remain poorly characterized. The Drosophila melanogaster mushroom body-an associative center-is an ideal system to investigate how different sensory channels converge in higher order brain centers. The neurons connecting the mushroom body to the olfactory system have been described in great detail, but input from other sensory systems remains poorly defined. Here, we use a range of anatomical and genetic techniques to identify two types of input neuron that connect visual processing centers-the lobula and the posterior lateral protocerebrum-to the dorsal accessory calyx of the mushroom body. Together with previous work that described a pathway conveying visual information from the medulla to the ventral accessory calyx of the mushroom body, our study defines a second, parallel pathway that is anatomically poised to convey information from the visual system to the dorsal accessory calyx.

Flexible neural connectivity under constraints on total connection strength.

Neural computation is determined by neurons' dynamics and circuit connectivity. Uncertain and dynamic environments may require neural hardware to adapt to different computational tasks, each requiring different connectivity configurations. At the same time, connectivity is subject to a variety of constraints, placing limits on the possible computations a given neural circuit can perform. Here we examine the hypothesis that the organization of neural circuitry favors computational flexibility: that it makes many computational solutions available, given physiological constraints. From this hypothesis, we develop models of connectivity degree distributions based on constraints on a neuron's total synaptic weight. To test these models, we examine reconstructions of the mushroom bodies from the first instar larva and adult Drosophila melanogaster. We perform a Bayesian model comparison for two constraint models and a random wiring null model. Overall, we find that flexibility under a homeostatically fixed total synaptic weight describes Kenyon cell connectivity better than other models, suggesting a principle shaping the apparently random structure of Kenyon cell wiring. Furthermore, we find evidence that larval Kenyon cells are more flexible earlier in development, suggesting a mechanism whereby neural circuits begin as flexible systems that develop into specialized computational circuits.

Input Connectivity Reveals Additional Heterogeneity of Dopaminergic Reinforcement in Drosophila.

Different types of Drosophila dopaminergic neurons (DANs) reinforce memories of unique valence and provide state-dependent motivational control [1]. Prior studies suggest that the compartment architecture of the mushroom body (MB) is the relevant resolution for distinct DAN functions [2, 3]. Here we used a recent electron microscope volume of the fly brain [4] to reconstruct the fine anatomy of individual DANs within three MB compartments. We find the 20 DANs of the γ5 compartment, at least some of which provide reward teaching signals, can be clustered into 5 anatomical subtypes that innervate different regions within γ5. Reconstructing 821 upstream neurons reveals input selectivity, supporting the functional relevance of DAN sub-classification. Only one PAM-γ5 DAN subtype γ5(fb) receives direct recurrent feedback from γ5ß'2a mushroom body output neurons (MBONs) and behavioral experiments distinguish a role for these DANs in memory revaluation from those reinforcing sugar memory. Other DAN subtypes receive major, and potentially reinforcing, inputs from putative gustatory interneurons or lateral horn neurons, which can also relay indirect feedback from MBONs. We similarly reconstructed the single aversively reinforcing PPL1-γ1pedc DAN. The γ1pedc DAN inputs mostly differ from those of γ5 DANs and they cluster onto distinct dendritic branches, presumably separating its established roles in aversive reinforcement and appetitive motivation [5, 6]. Tracing also identified neurons that provide broad input to γ5, ß'2a, and γ1pedc DANs, suggesting that distributed DAN populations can be coordinately regulated. These connectomic and behavioral analyses therefore reveal further complexity of dopaminergic reinforcement circuits between and within MB compartments.

Complete Connectomic Reconstruction of Olfactory Projection Neurons in the Fly Brain.

Nervous systems contain sensory neurons, local neurons, projection neurons, and motor neurons. To understand how these building blocks form whole circuits, we must distil these broad classes into neuronal cell types and describe their network connectivity. Using an electron micrograph dataset for an entire Drosophila melanogaster brain, we reconstruct the first complete inventory of olfactory projections connecting the antennal lobe, the insect analog of the mammalian olfactory bulb, to higher-order brain regions in an adult animal brain. We then connect this inventory to extant data in the literature, providing synaptic-resolution "holotypes" both for heavily investigated and previously unknown cell types. Projection neurons are approximately twice as numerous as reported by light level studies; cell types are stereotyped, but not identical, in cell and synapse numbers between brain hemispheres. The lateral horn, the insect analog of the mammalian cortical amygdala, is the main target for this olfactory information and has been shown to guide innate behavior. Here, we find new connectivity motifs, including axo-axonic connectivity between projection neurons, feedback, and lateral inhibition of these axons by a large population of neurons, and the convergence of different inputs, including non-olfactory inputs and memory-related feedback onto third-order olfactory neurons. These features are less prominent in the mushroom body calyx, the insect analog of the mammalian piriform cortex and a center for associative memory. Our work provides a complete neuroanatomical platform for future studies of the adult Drosophila olfactory system.