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The forthcoming assembly of the adultDrosophila melanogastercentral brain connectome, containing over 125,000 neurons and 50 million synaptic connections, provides a template for examining sensory processing throughout the brain. Here, we create a leaky integrate-and-fire computational model of the entireDrosophilabrain, based on neural connectivity and neurotransmitter identity, to study circuit properties of feeding and grooming behaviors. We show that activation of sugar-sensing or water-sensing gustatory neurons in the computational model accurately predicts neurons that respond to tastes and are required for feeding initiation. Computational activation of neurons in the feeding region of theDrosophilabrain predicts those that elicit motor neuron firing, a testable hypothesis that we validate by optogenetic activation and behavioral studies. Moreover, computational activation of different classes of gustatory neurons makes accurate predictions of how multiple taste modalities interact, providing circuit-level insight into aversive and appetitive taste processing. Our computational model predicts that the sugar and water pathways form a partially shared appetitive feeding initiation pathway, which our calcium imaging and behavioral experiments confirm. Additionally, we applied this model to mechanosensory circuits and found that computational activation of mechanosensory neurons predicts activation of a small set of neurons comprising the antennal grooming circuit that do not overlap with gustatory circuits, and accurately describes the circuit response upon activation of different mechanosensory subtypes. Our results demonstrate that modeling brain circuits purely from connectivity and predicted neurotransmitter identity generates experimentally testable hypotheses and can accurately describe complete sensorimotor transformations.

SummarySex pheromones are key social signals in most animals. In Drosophila a dedicated olfactory channel senses a male pheromone, cis-vaccenyl acetate (cVA) that promotes female courtship while repelling males. Here we show that flies use separate cVA processing streams to extract qualitative and positional information. cVA olfactory neurons are sensitive to concentration differences in a 5 mm range around a male. Second-order projection neurons detect inter-antennal differences in cVA concentration, encoding the angular position of a male. We identify a circuit mechanism increasing left-right contrast through an interneuron which provides contralateral inhibition. At the third layer of the circuit we identify neurons with distinct response properties and sensory integration motifs. One population is selectively tuned to an approaching male with speed-dependent responses. A second population responds tonically to a male’s presence and controls female mating decisions. A third population integrates a male taste cue with cVA; only a simultaneous presentation of both signals promotes female mating via this pathway. Thus the olfactory system generates a range of complex percepts in discrete populations of central neurons that allow the expression of appropriate behaviors depending on context. Such separation of olfactory features resembles the mammalian what and where visual streams.HighlightscVA male pheromone has a 5 mm signaling range, activating two parallel central pathwaysPheromone-sensing neurons have spatial receptive fields sharpened by contralateral inhibitionPosition (where) and identity (what) are separated at the 3rd layer of cVA processingIntegrating taste and cVA in sexually dimorphic aSP-g controls female receptivity

We develop an automatic method for synaptic partner identification in insect brains and use it to predict synaptic partners in a whole-brain electron microscopy dataset of the fruit fly. The predictions can be used to infer a connectivity graph with high accuracy, thus allowing fast identification of neural pathways. To facilitate circuit reconstruction using our results, we develop CIRCUITMAP, a user interface add-on for the circuit annotation tool CATMAID. A deep-learning-based approach enables automatic identification of synaptically connected neurons in electron microscopy datasets of the fly brain.

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. BAcTrace is a predominantly retrograde tracing tool for connectomic analyses in Drosophila.

A new study reveals the effects of specific single cell type perturbations on the assembly and function of a mechanosensory circuit in the Drosophila melanogaster larva with synaptic resolution. This work paves the way for the use of comparative connectomics to understand general principles governing the development of wiring specificity.

Animal behavior is shaped both by evolution and by individual experience. Parallel brain pathways encode innate and learned valences of cues, but the way in which they are integrated during action-selection is not well understood. We used electron microscopy to comprehensively map with synaptic resolution all neurons downstream of all mushroom body (MB) output neurons (encoding learned valences) and characterized their patterns of interaction with lateral horn (LH) neurons (encoding innate valences) in Drosophila larva. The connectome revealed multiple convergence neuron types that receive convergent MB and LH inputs. A subset of these receives excitatory input from positive-valence MB and LH pathways and inhibitory input from negative-valence MB pathways. We confirmed functional connectivity from LH and MB pathways and behavioral roles of two of these neurons. These neurons encode integrated odor value and bidirectionally regulate turning. Based on this, we speculate that learning could potentially skew the balance of excitation and inhibition onto these neurons and thereby modulate turning. Together, our study provides insights into the circuits that integrate learned and innate valences to modify behavior.

The hemibrain connectome (Scheffer et al., 2020) provides large scale connectivity and morphology information for the majority of the central brain of Drosophila melanogaster. Using this data set, we provide a complete description of the most complex olfactory system studied at synaptic resolution to date, covering all first, second and third-order neurons of the olfactory system associated with the antennal lobe and lateral horn (mushroom body neurons are described in a parallel paper, Li et al., 2020). We develop a generally applicable strategy to extract information flow and layered organisation from synaptic resolution connectome graphs, mapping olfactory input to descending interneurons. This identifies a range of motifs including highly lateralised circuits in the antennal lobe and patterns of convergence downstream of the mushroom body and lateral horn. We also leverage a second data set (FAFB, Zheng et al., 2018) to provide a first quantitative assessment of inter- versus intra-individual stereotypy. Complete reconstruction of select developmental lineages in two brains (three brain hemispheres) reveals striking similarity in neuronal morphology across brains for >170 cell types. Within and across brains, connectivity correlates with morphology. Notably, neurons of the same morphological type show similar connection variability within one brain as across brains; this property should enable a rigorous quantitative approach to cell typing.

Reconstruction of neural circuitry at single-synapse resolution is a key target for improving understanding of the nervous system in health and disease. Serial section transmission electron microscopy (ssTEM) is among the most prolific imaging methods employed in pursuit of such reconstructions. We demonstrate how Flood-Filling Networks (FFNs) can be used to computationally segment a forty-teravoxel whole-brain Drosophila ssTEM volume. To compensate for data irregularities and imperfect global alignment, FFNs were combined with procedures that locally re-align serial sections as well as dynamically adjust and synthesize image content. The proposed approach produced a largely merger-free segmentation of the entire ssTEM Drosophila brain, which we make freely available. As compared to manual tracing using an efficient skeletonization strategy, the segmentation enabled circuit reconstruction and analysis workflows that were an order of magnitude faster.

Making inferences about the computations performed by neuronal circuits from synapse-level connectivity maps is an emerging opportunity in neuroscience. The mushroom body (MB) is well positioned for developing and testing such an approach due to its conserved neuronal architecture, recently completed dense connectome, and extensive prior experimental studies of its roles in learning, memory, and activity regulation. Here, we identify new components of the MB circuit inDrosophila, including extensive visual input and MB output neurons (MBONs) with direct connections to descending neurons. We find unexpected structure in sensory inputs, in the transfer of information about different sensory modalities to MBONs, and in the modulation of that transfer by dopaminergic neurons (DANs). We provide insights into the circuitry used to integrate MB outputs, connectivity between the MB and the central complex and inputs to DANs, including feedback from MBONs. Our results provide a foundation for further theoretical and experimental work.

Drosophila melanogaster olfactory neurons have long been thought to express only one chemosensory receptor gene family. There are two main olfactory receptor gene families in Drosophila, the Odorant Receptors (ORs) and the Ionotropic Receptors (IRs). The dozens of odorant binding receptors in each family require at least one co-receptor gene in order to function: Orco for ORs, and Ir25a, Ir8a, and Ir76b for IRs. Using a new genetic knock-in strategy, we targeted the four co-receptors representing the main chemosensory families in Drosophila (Orco, Ir8a, Ir76b, Ir25a). Co-receptor knock-in expression patterns were verified as accurate representations of endogenous expression. We find extensive overlap in expression among the different co-receptors. As defined by innervation into antennal lobe glomeruli, Ir25a is broadly expressed in 88% of all olfactory sensory neuron classes and is co-expressed in 82% of Orco+ neuron classes, including all neuron classes in the maxillary palp. Orco, Ir8a, and Ir76b expression patterns are also more expansive than previously assumed. Single sensillum recordings from Orco-expressing Ir25a mutant antennal and palpal neurons identify changes in olfactory responses. These results suggest co-expression of chemosensory receptors is common in olfactory neurons. Together, our data present the first comprehensive map of chemosensory co-receptor expression and reveal their unexpected widespread co-expression in the fly olfactory system.

The neural circuits responsible for animal behavior remain largely unknown. We summarize new methods and present the circuitry of a large fraction of the brain of the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. We define cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. We provide detailed circuits consisting of neurons and their chemical synapses for most of the central brain. We make the data public and simplify access, reducing the effort needed to answer circuit questions, and provide procedures linking the neurons defined by our analysis with genetic reagents. Biologically, we examine distributions of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence that maximizing packing density is an important criterion in the evolution of the fly’s brain., eLife digest Animal brains of all sizes, from the smallest to the largest, work in broadly similar ways. Studying the brain of any one animal in depth can thus reveal the general principles behind the workings of all brains. The fruit fly Drosophila is a popular choice for such research. With about 100,000 neurons – compared to some 86 billion in humans – the fly brain is small enough to study at the level of individual cells. But it nevertheless supports a range of complex behaviors, including navigation, courtship and learning. Thanks to decades of research, scientists now have a good understanding of which parts of the fruit fly brain support particular behaviors. But exactly how they do this is often unclear. This is because previous studies showing the connections between cells only covered small areas of the brain. This is like trying to understand a novel when all you can see is a few isolated paragraphs. To solve this problem, Scheffer, Xu, Januszewski, Lu, Takemura, Hayworth, Huang, Shinomiya et al. prepared the first complete map of the entire central region of the fruit fly brain. The central brain consists of approximately 25,000 neurons and around 20 million connections. To prepare the map – or connectome – the brain was cut into very thin 8nm slices and photographed with an electron microscope. A three-dimensional map of the neurons and connections in the brain was then reconstructed from these images using machine learning algorithms. Finally, Scheffer et al. used the new connectome to obtain further insights into the circuits that support specific fruit fly behaviors. The central brain connectome is freely available online for anyone to access. When used in combination with existing methods, the map will make it easier to understand how the fly brain works, and how and why it can fail to work correctly. Many of these findings will likely apply to larger brains, including our own. In the long run, studying the fly connectome may therefore lead to a better understanding of the human brain and its disorders. Performing a similar analysis on the brain of a small mammal, by scaling up the methods here, will be a likely next step along this path.

Summary Animals exhibit innate and learned preferences for temperature and humidity—conditions critical for their survival and reproduction. Leveraging a whole-brain electron microscopy volume, we studied the adult Drosophila melanogaster circuitry associated with antennal thermo- and hygrosensory neurons. We have identified two new target glomeruli in the antennal lobe, in addition to the five known ones, and the ventroposterior projection neurons (VP PNs) that relay thermo- and hygrosensory information to higher brain centers, including the mushroom body and lateral horn, seats of learned and innate behavior. We present the first connectome of a thermo- and hygrosensory neuropil, the lateral accessory calyx (lACA), by reconstructing neurons downstream of heating- and cooling-responsive VP PNs. A few mushroom body-intrinsic neurons solely receive thermosensory input from the lACA, while most receive additional olfactory and thermo- and/or hygrosensory PN inputs. Furthermore, several classes of lACA-associated neurons form a local network with outputs to other brain neuropils, suggesting that the lACA serves as a hub for thermo- and hygrosensory circuitry. For example, DN1a neurons link thermosensory PNs in the lACA to the circadian clock via the accessory medulla. Finally, we survey strongly connected downstream partners of VP PNs across the protocerebrum; these include a descending neuron targeted by dry-responsive VP PNs, meaning that just two synapses might separate hygrosensory inputs from motor circuits. These data provide a comprehensive first- and second-order layer analysis of Drosophila thermo- and hygrosensory systems and an initial survey of third-order neurons that could directly modulate behavior., Graphical Abstract, Highlights • Two novel thermo- and/or hygrosensory glomeruli in the fly antennal lobe • First complete set of thermosensory and hygrosensory projection neurons • First connectome for a thermo- and hygrosensory neuropil • Third-order thermo- and hygrosensory neurons, including link to circadian clock, Marin et al. use connectomics and genetics for comprehensive identification of temperature and humidity sensory neurons in the Drosophila brain. They reconstruct all projections to higher brain areas and select higher-order targets, including the mushroom body lateral accessory calyx, linking thermosensation to memory and the circadian clock.

Animal behavior is shaped both by evolution and by individual experience. In many species parallel brain pathways are thought to encode innate and learnt behavior drives and as a result may link the same sensory cue to different actions if innate and learnt drives are in opposition. How these opposing drives are integrated into a single coherent action is not well understood. In insects, the Mushroom Body Output Neurons (MBONs) and the Lateral Horn Neurons (LHNs) are thought to provide the learnt and innate drives, respectively. However their patterns of convergence and the mechanisms by which their outputs are used to select actions are not well understood. We used electron microscopy reconstruction to comprehensively map the downstream targets of all MBONs in Drosophila larva and characterise their patterns of convergence with LHNs. We discovered convergence neurons that receive direct input from MBONs and LHNs and compare opposite behaviour drives. Functional imaging and optogenetic manipulation suggest these convergence neurons compute the overall predicted value of approaching or avoiding an odor and mediate action selection. Our study describes the circuit mechanisms allowing integration of opposing drives from parallel olfactory pathways.

To analyse neuron data at scale, neuroscientists expend substantial effort reading documentation, installing dependencies and moving between analysis and visualisation environments. To facilitate this, we have developed a suite of interoperable open-source R packages called the natverse. The natverse allows users to read local and remote data, perform popular analyses including visualisation and clustering and graph-theoretic analysis of neuronal branching. Unlike most tools, the natverse enables comparison across many neurons of morphology and connectivity after imaging or co-registration within a common template space. The natverse also enables transformations between different template spaces and imaging modalities. We demonstrate tools that integrate the vast majority of Drosophila neuroanatomical light microscopy and electron microscopy connectomic datasets. The natverse is an easy-to-use environment for neuroscientists to solve complex, large-scale analysis challenges as well as an open platform to create new code and packages to share with the community.

The evolution of animal behaviour is poorly understood1,2. Despite numerous correlations between interspecific divergence in behaviour and nervous system structure and function, demonstrations of the genetic basis of these behavioural differences remain rare3–5. Here we develop a neurogenetic model, Drosophila sechellia, a species that displays marked differences in behaviour compared to its close cousin Drosophila melanogaster6,7, which are linked to its extreme specialization on noni fruit (Morinda citrifolia)8–16. Using calcium imaging, we identify olfactory pathways in D. sechellia that detect volatiles emitted by the noni host. Our mutational analysis indicates roles for different olfactory receptors in long- and short-range attraction to noni, and our cross-species allele-transfer experiments demonstrate that the tuning of one of these receptors is important for species-specific host-seeking. We identify the molecular determinants of this functional change, and characterize their evolutionary origin and behavioural importance. We perform circuit tracing in the D. sechellia brain, and find that receptor adaptations are accompanied by increased sensory pooling onto interneurons as well as species-specific central projection patterns. This work reveals an accumulation of molecular, physiological and anatomical traits that are linked to behavioural divergence between species, and defines a model for investigating speciation and the evolution of the nervous system. A neurogenetic model, Drosophila sechellia—a relative of Drosophila melanogaster that has developed an extreme specialization for a single host plant—sheds light on the evolution of interspecific differences in behaviour.

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.

New tools and techniques have enabled many key advances in our understanding of the brain. To elucidate circuit function, it is necessary to identify, record from and manipulate networks of connected neurons. Here we present BAcTrace (BotulinumActivatedTracer), the first fully genetically encoded, retrograde, transsynaptic labelling system. BAcTrace is based onC. botulinumneurotoxin A, Botox, which we have engineered to act as a Trojan horse that jumps retrogradely between neurons to activate an otherwise silent transcription factor. We validate BAcTrace at three connections in theDrosophilaolfactory system and show that it enables electrophysiological recordings of connected neurons. Finally, in a challenging circuit with highly divergent connections, we used Electron Microscopy connectomics to show that BAcTrace correctly identifies 12 out of 16 connections.

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

A new study investigates the distinct male courtship songs of two related Drosophila species and the neurons controlling this behavior, localizing a site of evolutionary divergence to the motor system, downstream of the central brain.

Parallels between invertebrates and vertebrates in nervous system development, organisation and circuits are powerful reasons to use insects to study the mechanistic basis of behaviour. The last few years have seen the generation in Drosophila melanogaster of very large light microscopy data sets, genetic driver lines and tools to report or manipulate neural activity. These resources in conjunction with computational tools are enabling large scale characterisation of neuronal types and their functional properties. These are complemented by 3D electron microscopy, providing synaptic resolution data. A whole brain connectome of the fly larva is approaching completion based on manual reconstruction of electron-microscopy data. An adult whole brain dataset is already publicly available and focussed reconstruction is under way, but its 40× greater volume would require ∼500-5000 person-years of manual labour. Nevertheless rapid technical improvements in imaging and especially automated segmentation will likely deliver a complete adult connectome in the next 5 years. To enhance our understanding of the circuit basis of behaviour, light and electron microscopy outputs must be integrated with functional and physiological information into comprehensive databases. We review presently available data, tools and opportunities in Drosophila. We then consider the limits and potential of future progress and how this may impact neuroscience in rich model systems provided by larger insects and vertebrates.

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.

Animals exhibit innate behaviours to a variety of sensory stimuli including olfactory cues. In Drosophila, one higher olfactory centre, the lateral horn (LH), is implicated in innate behaviour. However, our structural and functional understanding of the LH is scant, in large part due to a lack of sparse neurogenetic tools for this region. We generate a collection of split-GAL4 driver lines providing genetic access to 82 LH cell types. We use these to create an anatomical and neurotransmitter map of the LH and link this to EM connectomics data. We find ~30% of LH projections converge with outputs from the mushroom body, site of olfactory learning and memory. Using optogenetic activation, we identify LH cell types that drive changes in valence behavior or specific locomotor programs. In summary, we have generated a resource for manipulating and mapping LH neurons, providing new insights into the circuit basis of innate and learned olfactory behavior.

Reconstruction of neural circuitry at single-synapse resolution is a key target for improving understanding of the nervous system in health and disease. Serial section transmission electron microscopy (ssTEM) is among the most prolific imaging methods employed in pursuit of such reconstructions. We demonstrate how Flood-Filling Networks (FFNs) can be used to computationally segment a forty-teravoxel whole-brain Drosophila ssTEM volume. To compensate for data irregularities and imperfect global alignment, FFNs were combined with procedures that locally re-align serial sections as well as dynamically adjust and synthesize image content. The proposed approach produced a largely merger-free segmentation of the entire ssTEM Drosophila brain, which we make freely available. As compared to manual tracing using an efficient skeletonization strategy, the segmentation enabled circuit reconstruction and analysis workflows that were an order of magnitude faster.

The evolution of animal behaviour is poorly understood. Despite numerous correlations of behavioural and nervous system divergence, demonstration of the genetic basis of interspecific behavioural differences remains rare. Here we develop a novel neurogenetic model,Drosophila sechellia, a close cousin ofD. melanogasterthat displays profound behavioural changes linked to its extreme host fruit specialisation. Through calcium imaging, we identify olfactory pathways detecting host volatiles. Mutational analysis indicates roles for individual receptors in long- and short-range attraction. Cross-species allele transfer demonstrates that differential tuning of one receptor is important for species-specific behaviour. We identify the molecular determinants of this functional change, and characterise their behavioural significance and evolutionary origin. Circuit tracing reveals that receptor adaptations are accompanied by increased sensory pooling onto interneurons and novel central projection patterns. This work links molecular and neuronal changes to behavioural divergence and defines a powerful model for investigating nervous system evolution and speciation.

Summary 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., Graphical Abstract, Highlights • First complete parts list for second-order neurons of an adult olfactory system • Quantification of left-right stereotypy in cell and synapse number • Axo-axonic connections form hierarchical communities in the lateral horn • Local and memory-related feedback target projection neuron axons, Bates, Schlegel et al. report the first complete part list of second-order neurons in an adult arthropod olfactory system. They show that these neurons are stereotyped across hemispheres and reveal their hierarchical interconnectivity and how they connect to third-order targets and memory-related neurons.

At around 150 000 neurons, the adult Drosophila melanogaster central nervous system is one of the largest species, for which a complete cellular catalogue is imminent. While numerically much simpler than mammalian brains, its complexity is still difficult to parse without grouping neurons into consistent types, which can number 1-1000 cells per hemisphere. We review how neuroanatomical and gene expression data are being used to discover neuronal types at scale. The correlation among multiple co-varying neuronal properties, including lineage, gene expression, morphology, connectivity, response properties and shared behavioral significance is essential to the definition of neuronal cell type. Initial studies comparing morphological and transcriptomic definitions of neuronal type suggest that these are highly consistent, but there is much to do to match these approaches brain-wide. Matched single-cell transcriptomic and morphological data provide an effective reference point to integrate other data types, including connectomics data. This will significantly enhance our ability to make functional predictions from brain wiring diagrams as well facilitating molecular genetic manipulation of neuronal types. ispartof: Curr Opin Neurobiol vol:56 pages:125-134 ispartof: location:England

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.

Evolution has shaped nervous systems to produce stereotyped behavioural responses to ethologically relevant stimuli. For example when laying eggs, female Drosophila avoid geosmin, an odorant produced by toxic moulds. Here we identify second, third, and fourth order neurons required for this innate olfactory aversion. Connectomics data place these neurons in a complete synaptic circuit from sensory input to descending output. We find multiple levels of valence-specific convergence, including a novel form of axo-axonic input onto second order neurons conveying another danger signal, the pheromone of parasitoid wasps. However, we also observe extensive divergence: second order geosmin neurons connect with a diverse array of 80 third order cell types. We find a pattern of convergence of aversive odour channels at this level. Crossing one more synaptic layer, we identified descending neurons critical for egg-laying aversion. Our data suggest a transition from a labelled line organisation in the periphery to a highly distributed central brain representation that is then coupled to distinct descending pathways.

Summary Drosophila melanogaster has a rich repertoire of innate and learned behaviors. Its 100,000-neuron brain is a large but tractable target for comprehensive neural circuit mapping. Only electron microscopy (EM) enables complete, unbiased mapping of synaptic connectivity; however, the fly brain is too large for conventional EM. We developed a custom high-throughput EM platform and imaged the entire brain of an adult female fly at synaptic resolution. To validate the dataset, we traced brain-spanning circuitry involving the mushroom body (MB), which has been extensively studied for its role in learning. All inputs to Kenyon cells (KCs), the intrinsic neurons of the MB, were mapped, revealing a previously unknown cell type, postsynaptic partners of KC dendrites, and unexpected clustering of olfactory projection neurons. These reconstructions show that this freely available EM volume supports mapping of brain-spanning circuits, which will significantly accelerate Drosophila neuroscience. Video Abstract, Graphical Abstract, Highlights • A complete adult Drosophila brain was imaged with EM and has been made publicly available • The imaged volume enables brain-spanning mapping of circuits at synaptic resolution • All mushroom body (MB) calyx inputs were mapped, revealing a new cell type, MB-CP2 • Previously unidentified synaptic partners form recurrent microcircuits in MB calyx, Electron microscopy imaging of the entire adult fruit fly brain at synapse resolution reveals circuitry spanning multiple regions and connectivity between known and previously unknown cell types.

Most sensory systems are organized into parallel neuronal pathways that process distinct aspects of incoming stimuli. For example, in insects, second order olfactory projection neurons target both the mushroom body, which is required for learning, and the lateral horn (LH), which has been proposed to mediate innate olfactory behavior. Mushroom body neurons encode odors in a sparse population code, which does not appear stereotyped across animals. In contrast the functional principles of odor coding in the LH remain poorly understood. We have carried out a comprehensive anatomical analysis of the Drosophila LH, counting ~1400 neurons; combining genetic driver lines, anatomical and functional criteria, we identify 165 LHN cell types. We then show that genetically labeled LHNs have stereotyped odor responses across animals for 33 of these cell types. LHN tuning can be ultra-sparse (1/40 odors tested), but on average single LHNs respond to three times more odors than single projection neurons. This difference can be rationalized by our observation that LHNs are better odor categorizers, likely due to pooling of input projection neurons responding to different odors of the same category. Our results reveal some of the principles by which a higher sensory processing area can extract innate behavioral significance from sensory stimuli.

The use of genetically encoded ‘self-labeling tags’ with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.

The use of genetically encoded ‘self-labeling tags’ with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.

The use of genetically encoded ‘self-labeling tags’ with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.

The use of genetically encoded ‘self-labeling tags’ with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.

The use of genetically encoded ‘self-labeling tags’ with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.

The use of genetically encoded ‘self-labeling tags’ with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.

The use of genetically encoded ‘self-labeling tags’ with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.

In pursuit of palatable food, hungry animals mobilize significant energy resources and overcome obstacles, exhaustion and fear. Their perseverance depends on metabolic state, internal motivation and the expected benefit. Sustained commitment to a trying task is crucial, however, disengagement from one behavior to engage into another can be essential for optimal adaptation and survival. How neural circuits allow prioritizing perseverance over withdrawal based on the animal's need, however, is not understood. Using a single fly spherical treadmill, we show that hungry flies display increasing perseverance to track a food odor in the repeated absence of the predicted food reward. While this perseverance is mediated by a group of dopaminergic neurons, a subset of neurons expressing octopamine, the invertebrate noradrenaline, provide reward feedback and counteract dopamine-motivated food seeking. Our data and modeling suggest that two important neuromodulators tally internal and external signals to ultimately coordinate motivation-dependent antagonistic behavioral drives: perseverance vs. change of behavior.

SummaryThe same sensory signal can be interpreted differently according to context. A new study in Drosophila uses cell-type-specific tools to identify neural circuits that integrate context during olfactory processing and surprisingly implicates memory-recall neurons.

Summary The behavioral response to a sensory stimulus may depend on both learned and innate neuronal representations. How these circuits interact to produce appropriate behavior is unknown. In Drosophila, the lateral horn (LH) and mushroom body (MB) are thought to mediate innate and learned olfactory behavior, respectively, although LH function has not been tested directly. Here we identify two LH cell types (PD2a1 and PD2b1) that receive input from an MB output neuron required for recall of aversive olfactory memories. These neurons are required for aversive memory retrieval and modulated by training. Connectomics data demonstrate that PD2a1 and PD2b1 neurons also receive direct input from food odor-encoding neurons. Consistent with this, PD2a1 and PD2b1 are also necessary for unlearned attraction to some odors, indicating that these neurons have a dual behavioral role. This provides a circuit mechanism by which learned and innate olfactory information can interact in identified neurons to produce appropriate behavior. Video Abstract, Highlights • Specific Drosophila lateral horn neurons mediate innate attraction to food odors • The same neurons receive plastic odor information from the mushroom body • Recall after associative learning depends on reduced drive to lateral horn neurons • Connectomics circuit for integration of learned and innate odor representations, Sensory stimuli can engage both learned and innate behaviors. Dolan et al. identify neurons in Drosophila that directly integrate unlearned and plastic odor representations; they are required for innate approach to food odors but also learned aversive recall.

Thick tissue specimens present major challenges for labeling cells and subcellular structures in a rapid and reliable manner. Sutcliffe et al. present..., Labeling and visualizing cells and subcellular structures within thick tissues, whole organs, and even intact animals is key to studying biological processes. This is particularly true for studies of neural circuits where neurons form submicron synapses but have arbors that may span millimeters in length. Traditionally, labeling is achieved by immunofluorescence; however, diffusion of antibody molecules (>100 kDa) is slow and often results in uneven labeling with very poor penetration into the center of thick specimens; these limitations can be partially addressed by extending staining protocols to over a week (Drosophila brain) and months (mice). Recently, we developed an alternative approach using genetically encoded chemical tags CLIP, SNAP, Halo, and TMP for tissue labeling; this resulted in >100-fold increase in labeling speed in both mice and Drosophila, at the expense of a considerable drop in absolute sensitivity when compared to optimized immunofluorescence staining. We now present a second generation of UAS- and LexA-responsive CLIPf, SNAPf, and Halo chemical labeling reagents for flies. These multimerized tags, with translational enhancers, display up to 64-fold increase in sensitivity over first-generation reagents. In addition, we developed a suite of conditional reporters (4xSNAPf tag and CLIPf-SNAPf-Halo2) that are activated by the DNA recombinase Bxb1. Our new reporters can be used with weak and strong GAL4 and LexA drivers and enable stochastic, intersectional, and multicolor Brainbow labeling. These improvements in sensitivity and experimental versatility, while still retaining the substantial speed advantage that is a signature of chemical labeling, should significantly increase the scope of this technology.

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.

Summary The Drosophila sex pheromone cVA elicits different behaviors in males and females. First- and second-order olfactory neurons show identical pheromone responses, suggesting that sex genes differentially wire circuits deeper in the brain. Using in vivo whole-cell electrophysiology, we now show that two clusters of third-order olfactory neurons have dimorphic pheromone responses. One cluster responds in females; the other responds in males. These clusters are present in both sexes and share a common input pathway, but sex-specific wiring reroutes pheromone information. Regulating dendritic position, the fruitless transcription factor both connects the male-responsive cluster and disconnects the female-responsive cluster from pheromone input. Selective masculinization of third-order neurons transforms their morphology and pheromone responses, demonstrating that circuits can be functionally rewired by the cell-autonomous action of a switch gene. This bidirectional switch, analogous to an electrical changeover switch, provides a simple circuit logic to activate different behaviors in males and females., Graphical Abstract, Highlights • Two clusters of higher olfactory neurons show sex-specific responses to pheromone • Common sensory input is wired to a different cluster in each sex, rerouting information • Bidirectional circuit switch depends on dendritic location of third-order neurons • Circuit state determined by cell-autonomous action of a switch gene, fruitless, A circuitry change consisting of dendritic repositioning of third-order sensory neurons enables one pheromone to elicit differential responses in male and female flies. This bidirectional switch, analogous to an electrical changeover switch, provides a simple circuit logic to activate different behaviors in males and females.

Labeling and visualizing cells and sub-cellular structures within thick tissues, whole organs and even intact animals is key to studying biological processes. This is particularly true for studies of neural circuits where neurons form sub-micron synapses but have arbors that may span millimeters in length. Traditionally labeling is achieved by immunofluorescence; however diffusion of antibody molecules (>100 kDa) is slow and often results in uneven labeling with very poor penetration into the centre of thick specimens; these limitations can be partially addressed by extending staining protocols to over a week (Drosophilabrain) and months (mice). Recently we developed an alternative approach using genetically encoded chemical tags CLIP, SNAP, Halo and TMP for tissue labeling; this resulted in >100 fold increase in labeling speed in both mice andDrosophila, at the expense of a considerable drop in absolute sensitivity when compared to optimized immunofluorescence staining. We now present a second generation of UAS and LexA responsive CLIP, SNAPf and Halo chemical labeling reagents for flies. These multimerized tags with translational enhancers display up to 64 fold increase in sensitivity over first generation reagents. In addition we developed a suite of conditional reporters (4xSNAPf tag and CLIP-SNAP-Halo) that are activated by the DNA recombinase Bxb1. Our new reporters can be used with weak and strong GAL4 and LexA drivers and enable stochastic, intersectional and multicolor Brainbow labeling. These improvements in sensitivity and experimental versatility, while still retaining the substantial speed advantage that is a signature of chemical labeling, should significantly increase the scope of this technology.