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Color and polarization provide complementary information about the world and are detected by specialized photoreceptors. However, the downstream neural circuits that process these distinct modalities are incompletely understood in any animal. Using electron microscopy, we have systematically reconstructed the synaptic targets of the photoreceptors specialized to detect color and skylight polarization in Drosophila, and we have used light microscopy to confirm many of our findings. We identified known and novel downstream targets that are selective for different wavelengths or polarized light, and followed their projections to other areas in the optic lobes and the central brain. Our results revealed many synapses along the photoreceptor axons between brain regions, new pathways in the optic lobes, and spatially segregated projections to central brain regions. Strikingly, photoreceptors in the polarization-sensitive dorsal rim area target fewer cell types, and lack strong connections to the lobula, a neuropil involved in color processing. Our reconstruction identifies shared wiring and modality-specific specializations for color and polarization vision, and provides a comprehensive view of the first steps of the pathways processing color and polarized light inputs.

The formation and consolidation of memories are complex phenomena involving synaptic plasticity, microcircuit reorganization, and the formation of multiple representations within distinct circuits. To gain insight into the structural aspects of memory consolidation, we focus on the calyx of the Drosophila mushroom body. In this essential center, essential for olfactory learning, second- and third-order neurons connect through large synaptic microglomeruli, which we dissect at the electron microscopy level. Focusing on microglomeruli that respond to a specific odor, we reveal that appetitive long-term memory results in increased numbers of precisely those functional microglomeruli responding to the conditioned odor. Hindering memory consolidation by non-coincident presentation of odor and reward, by blocking protein synthesis, or by including memory mutants suppress these structural changes, revealing their tight correlation with the process of memory consolidation. Thus, olfactory long-term memory is associated with input-specific structural modifications in a high-order center of the fly brain.

Serotonergic neurons modulate diverse physiological and behavioral processes in a context-dependent manner, based on their complex connectivity. However, their connectivity has not been comprehensively explored at a single-cell resolution. Using a whole-brain EM dataset we determined the wiring logic of a broadly projecting serotonergic neuron (the “CSDn”) inDrosophila. Within the antennal lobe (AL; first-order olfactory region), the CSDn receives glomerulus-specific input and preferentially targets distinct local interneuron subtypes. Furthermore, the wiring logic of the CSDn differs between olfactory regions. The CSDn innervates the AL and lateral horn (LH), yet does not maintain the same synaptic relationship with individual projection neurons that also span both regions. Consistent with this, the CSDn has more distributed connectivity in the LH relative to the AL, preferentially synapsing with principal neuron types based on presumptive transmitter content. Lastly, we identify protocerebral neurons that provide abundant synaptic input to the CSDn. Our study demonstrates how an individual modulatory neuron can interact with local networks and integrate input from non-olfactory sources.

Summary 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 γ5β′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, β′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., Highlights • Nanoscale anatomy reveals additional subtypes of rewarding dopaminergic neurons • Connectomics reveals input specificity to subtypes of dopaminergic neurons • Axon morphology implies dopaminergic neurons provide subcompartment-level function • Unique dopaminergic subtypes serve aversive memory extinction and sugar learning, Otto et al. use electron microscope resolution connectomics to describe the structure and organization of neurons providing synaptic input to functionally discrete subtypes of dopaminergic neurons. The nanoscale anatomy reveals further anatomical and functional specialization of dopaminergic neurons, which is confirmed with behavioral experiments.

SUMMARYAnimals exhibit innate and learned preferences for temperature and humidity – conditions critical for their survival and reproduction. Here, we leveraged a whole adult brain electron microscopy volume to study the circuitry associated with antennal thermosensory and hygrosensory neurons, which target specific ventroposterior (VP) glomeruli in theDrosophila melanogasterantennal lobe. We have identified two new VP glomeruli, in addition to the five known ones, and the projection neurons (VP PNs) that relay VP information to higher brain centres, including the mushroom body and lateral horn, seats of learned and innate olfactory behaviours, respectively. Focussing on the mushroom body lateral accessory calyx (lACA), a known thermosensory neuropil, we present a comprehensive connectome by reconstructing neurons downstream of heating- and cooling-responsive VP PNs. We find that a few lACA-associated mushroom body intrinsic neurons (Kenyon cells) solely receive thermosensory inputs, while most receive additional olfactory and thermo- or hygrosensory PN inputs in the main calyx. Unexpectedly, we find several classes of lACA-associated neurons that form a local network with outputs to other brain neuropils, suggesting that the lACA serves as a general hub for thermosensory circuitry. For example, we find DN1 pacemaker neurons that link the lACA to the accessory medulla, likely mediating temperature-based entrainment of the circadian clock. Finally, we survey strongly connected downstream partners of VP PNs across the protocerebrum; these include a descending neuron that receives input mainly from dry-responsive VP PNs, meaning that just two synapses might separate hygrosensory inputs from motor neurons in the nerve cord. (249)HIGHLIGHTSTwo novel thermo/hygrosensory glomeruli in the fly antennal lobeFirst complete set of thermosensory and hygrosensory projection neuronsFirst connectome for a thermosensory centre, the lateral accessory calyxNovel third order neurons, including a link to the circadian clock

The study of neural circuits requires the reconstruction of neurons and the identification of synaptic connections between them. To scale the reconstruction to the size of whole-brain datasets, semi-automatic methods are needed to solve those tasks. Here, we present an automatic method for synaptic partner identification in insect brains, which uses convolutional neural networks to identify post-synaptic sites and their pre-synaptic partners. The networks can be trained from human generated point annotations alone and require only simple post-processing to obtain final predictions. We used our method to extract 244 million putative synaptic partners in the fifty-teravoxel full adult fly brain (FAFB) electron microscopy (EM) dataset and evaluated its accuracy on 146,643 synapses from 702 neurons with a total cable length of 312 mm in four different brain regions. The predicted synaptic connections can be used together with a neuron segmentation to infer a connectivity graph with high accuracy: between 92% and 96% of edges linking connected neurons are correctly classified as weakly connected (less than five synapses) and strongly connected (at least five synapses). Our synaptic partner predictions for the FAFB dataset are publicly available, together with a query library allowing automatic retrieval of up- and downstream neurons.

Neural representations of head direction have been discovered in many different species. A large body of theoretical work has proposed that the dynamics associated with these representations might be generated, maintained, and updated by recurrent network structures called ring attractors. Ring attractor models rely on specific assumptions about the structure of excitatory and inhibitory connectivity. These assumptions have been difficult to test directly. We therefore performed electron-microscopy-based circuit reconstruction and RNA profiling of identified cell types in the heading direction system of Drosophila melanogaster to directly examine excitatory and inhibitory synaptic connectivity, generating a dataset that should serve as a reference for future functional studies of the network. Consistent with several theoretical models, we identified network motifs that have been hypothesized to maintain the heading representation in darkness, update it when the animal turns, and tether it to visual cues. Genetically targeted two-photon calcium imaging and thermogenetic perturbation of the constituent neuron types during behavior provided additional support for these functional roles. However, we also discovered network motifs absent in current models, including a surprising degree of recurrence between arbors of different neurons with mixed pre- and post-synaptic specializations. Overall, our results confirm that the Drosophila heading direction network contains the core components of a ring attractor while also revealing unpredicted structural features that might enable the heading system to accurately track the animal9s heading with a small number of neurons.

Serotonin plays different roles across networks within the same sensory modality. Previously, we used whole-cell electrophysiology in Drosophila to show that serotonergic neurons innervating the first olfactory relay are inhibited by odorants (Zhang and Gaudry, 2016). Here we show that network-spanning serotonergic neurons segregate information about stimulus features, odor intensity and identity, by using opposing coding schemes in different olfactory neuropil. A pair of serotonergic neurons (the CSDns) innervate the antennal lobe and lateral horn, which are first and second order neuropils. CSDn processes in the antennal lobe are inhibited by odors in an identity independent manner. In the lateral horn, CSDn processes are excited in an odor identity dependent manner. Using functional imaging, modeling, and EM reconstruction, we demonstrate that antennal lobe derived inhibition arises from local GABAergic inputs and acts as a means of gain control on branch-specific inputs that the CSDns receive within the lateral horn.

All centralized nervous systems possess modulatory neurons that arborize broadly across multiple brain regions. Such modulatory systems are critical for proper sensory, motor, and cognitive processing. How single modulatory neurons integrate into circuits within their target destination remains largely unexplored due to difficulties in both labeling individual cells and imaging across distal parts of the CNS. Here, we take advantage of an identified modulatory neuron in Drosophila that arborizes in multiple olfactory neuropils. We demonstrate that this serotonergic neuron has opposing odor responses in its neurites of the antennal lobe and lateral horn, first and second order olfactory neuropils respectively. Specifically, processes of this neuron in the antennal lobe have responses that are inhibitory and odor-independent, while lateral horn responses are excitatory and odor-specific. The results show that widespread modulatory neurons may not function purely as integrate-and-fire cells, but rather their transmitter release is locally regulated based on neuropil. As nearly all vertebrate and invertebrate neurons are subject to synaptic inputs along their dendro-axonic axis, it is likely that our findings generalize across phylogeny and other broadly-projecting modulatory systems.SignificanceThe centrifugal innervation of neuronal circuits is ubiquitous across centralized nervous systems. Such inputs often arise from modulatory neurons that arborize broadly throughout the brain. How information is integrated in such cells and how release from their distant terminals is regulated remains largely unknown. We show that a serotonergic neuron that innervates multiple stages of odor processing in Drosophila has distinct activity throughout its neurites, including opposite polarity responses in first and second order olfactory neuropils. Disparate activity arises from local interactions within each target region. Our results show that such neurons exhibit dendritic computation rather than somatic integration alone, and that examining local interactions at release sites is critical for understanding centrifugal innervation.

Animals exhibit innate behaviours in response to a variety of sensory stimuli such as olfactory cues. In Drosophila, a higher olfactory centre called the lateral horn (LH) is implicated in innate behaviour. However, our knowledge of the structure and function of the LH is scant, due to the lack of sparse neurogenetic tools for this brain region. Here we generate a collection of split-GAL4 driver lines providing genetic access to 82 LH cell-types. We identify the neurotransmitter and axo-dendritic polarity for each cell-type. Using these lines were create an anatomical map of the LH. We found that ∼30% of LH projections converge with outputs from the mushroom body, the site of olfactory learning and memory. Finally, using optogenetic activation of small groups of LH neurons. We identify cell-types that drive changes in either valence or specific motor programs, such as turning and locomotion. In summary we have generated a resource for manipulating and mapping LH neurons in both light and electron microscopy and generated insights into the anatomy and function of the LH.

Summary In pursuit of food, hungry animals mobilize significant energy resources and overcome exhaustion and fear. How need and motivation control the decision to continue or change behavior is not understood. Using a single fly treadmill, we show that hungry flies persistently track a food odor and increase their effort over repeated trials in the absence of reward suggesting that need dominates negative experience. We further show that odor tracking is regulated by two mushroom body output neurons (MBONs) connecting the MB to the lateral horn. These MBONs, together with dopaminergic neurons and Dop1R2 signaling, control behavioral persistence. Conversely, an octopaminergic neuron, VPM4, which directly innervates one of the MBONs, acts as a brake on odor tracking by connecting feeding and olfaction. Together, our data suggest a function for the MB in internal state-dependent expression of behavior that can be suppressed by external inputs conveying a competing behavioral drive., Graphical Abstract, Highlights • Hunger motivates persistent food odor tracking even without reward • Two synaptically connected MBONs, -γ1pedc>αβ and -α2sc, regulate odor tracking • Octopamine neurons connect feeding and counteract MBON and odor tracking • Dopaminergic neurons and Dop1R2 signaling promote persistent tracking, What drives behavioral persistence versus quitting? Sayin et al. propose that circuit modules in the fly’s learning center and dopamine drive gradually increasing food odor tracking, which can be efficiently suppressed by extrinsic, but directly innervating, feeding-related neuromodulatory neurons.

We demonstrate a meaningful prospective power analysis for an (admittedly idealized) illustrative connectome inference task. Modeling neurons as vertices and synapses as edges in a simple random graph model, we optimize the trade-off between the number of (putative) edges identified and the accuracy of the edge identification procedure. We conclude that explicit analysis of the quantity/quality trade-off is imperative for optimal neuroscientific experimental design. In particular, more though more errorful edge identification can yield superior inferential performance., 8 pages, 1 figure

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 entireDrosophila melanogasterbrain, we reconstruct the first complete inventory of olfactory projections connecting the antennal lobe, the insect analogue 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 analogue of the mammalian cortical amygdala, is the main target for this olfactory information and has been shown to guide innate behaviour. Here, we find new connectivity motifs, including: axo-axonic connectivity between projection neurons; feedback and lateral inhibition of these axons by local neurons; and the convergence of different inputs, including non-olfactory inputs and memory-related feedback onto lateral horn neurons. This differs from the configuration of the second most prominent target for olfactory projection neurons: the mushroom body calyx, the insect analogue of the mammalian piriform cortex and a centre for associative memory. Our work provides a complete neuroanatomical platform for future studies of the adultDrosophilaolfactory system.HighlightsFirst complete parts list for second-order neurons of an adult olfactory systemQuantification of left-right stereotypy in cell and synapse numberAxo-axonic connections form hierarchical communities in the lateral hornLocal neurons and memory-related feedback target projection neuron axons