Ultrastructural differences impact cilia shape and external exposure across cell classes in the visual cortex.

A primary cilium is a membrane-bound extension from the cell surface that contains receptors for perceiving and transmitting signals that modulate cell state and activity. Primary cilia in the brain are less accessible than cilia on cultured cells or epithelial tissues because in the brain they protrude into a deep, dense network of glial and neuronal processes. Here, we investigated cilia frequency, internal structure, shape, and position in large, high-resolution transmission electron microscopy volumes of mouse primary visual cortex. Cilia extended from the cell bodies of nearly all excitatory and inhibitory neurons, astrocytes, and oligodendrocyte precursor cells (OPCs) but were absent from oligodendrocytes and microglia. Ultrastructural comparisons revealed that the base of the cilium and the microtubule organization differed between neurons and glia. Investigating cilia-proximal features revealed that many cilia were directly adjacent to synapses, suggesting that cilia are poised to encounter locally released signaling molecules. Our analysis indicated that synapse proximity is likely due to random encounters in the neuropil, with no evidence that cilia modulate synapse activity as would be expected in tetrapartite synapses. The observed cell class differences in proximity to synapses were largely due to differences in external cilia length. Many key structural features that differed between neuronal and glial cilia influenced both cilium placement and shape and, thus, exposure to processes and synapses outside the cilium. Together, the ultrastructure both within and around neuronal and glial cilia suggest differences in cilia formation and function across cell types in the brain.

Motor neurons generate pose-targeted movements via proprioceptive sculpting.

Motor neurons are the final common pathway1 through which the brain controls movement of the body, forming the basic elements from which all movement is composed. Yet how a single motor neuron contributes to control during natural movement remains unclear. Here we anatomically and functionally characterize the individual roles of the motor neurons that control head movement in the fly, Drosophila melanogaster. Counterintuitively, we find that activity in a single motor neuron rotates the head in different directions, depending on the starting posture of the head, such that the head converges towards a pose determined by the identity of the stimulated motor neuron. A feedback model predicts that this convergent behaviour results from motor neuron drive interacting with proprioceptive feedback. We identify and genetically2 suppress a single class of proprioceptive neuron3 that changes the motor neuron-induced convergence as predicted by the feedback model. These data suggest a framework for how the brain controls movements: instead of directly generating movement in a given direction by activating a fixed set of motor neurons, the brain controls movements by adding bias to a continuing proprioceptive-motor loop.

Nanometer-scale views of visual cortex reveal anatomical features of primary cilia poised to detect synaptic spillover.

A primary cilium is a thin membrane-bound extension off a cell surface that contains receptors for perceiving and transmitting signals that modulate cell state and activity. While many cell types have a primary cilium, little is known about primary cilia in the brain, where they are less accessible than cilia on cultured cells or epithelial tissues and protrude from cell bodies into a deep, dense network of glial and neuronal processes. Here, we investigated cilia frequency, internal structure, shape, and position in large, high-resolution transmission electron microscopy volumes of mouse primary visual cortex. Cilia extended from the cell bodies of nearly all excitatory and inhibitory neurons, astrocytes, and oligodendrocyte precursor cells (OPCs), but were absent from oligodendrocytes and microglia. Structural comparisons revealed that the membrane structure at the base of the cilium and the microtubule organization differed between neurons and glia. OPC cilia were distinct in that they were the shortest and contained pervasive internal vesicles only occasionally observed in neuron and astrocyte cilia. Investigating cilia-proximal features revealed that many cilia were directly adjacent to synapses, suggesting cilia are well poised to encounter locally released signaling molecules. Cilia proximity to synapses was random, not enriched, in the synapse-rich neuropil. The internal anatomy, including microtubule changes and centriole location, defined key structural features including cilium placement and shape. Together, the anatomical insights both within and around neuron and glia cilia provide new insights into cilia formation and function across cell types in the brain.

A comprehensive neuroanatomical survey of the Drosophila Lobula Plate Tangential Neurons with predictions for their optic flow sensitivity.

Flying insects exhibit remarkable navigational abilities controlled by their compact nervous systems. Optic flow, the pattern of changes in the visual scene induced by locomotion, is a crucial sensory cue for robust self-motion estimation, especially during rapid flight. Neurons that respond to specific, large-field optic flow patterns have been studied for decades, primarily in large flies, such as houseflies, blowflies, and hover flies. The best-known optic-flow sensitive neurons are the large tangential cells of the dipteran lobula plate, whose visual-motion responses, and to a lesser extent, their morphology, have been explored using single-neuron neurophysiology. Most of these studies have focused on the large, Horizontal and Vertical System neurons, yet the lobula plate houses a much larger set of 'optic-flow' sensitive neurons, many of which have been challenging to unambiguously identify or to reliably target for functional studies. Here we report the comprehensive reconstruction and identification of the Lobula Plate Tangential Neurons in an Electron Microscopy (EM) volume of a whole Drosophila brain. This catalog of 58 LPT neurons (per brain hemisphere) contains many neurons that are described here for the first time and provides a basis for systematic investigation of the circuitry linking self-motion to locomotion control. Leveraging computational anatomy methods, we estimated the visual motion receptive fields of these neurons and compared their tuning to the visual consequence of body rotations and translational movements. We also matched these neurons, in most cases on a one-for-one basis, to stochastically labeled cells in genetic driver lines, to the mirror-symmetric neurons in the same EM brain volume, and to neurons in an additional EM data set. Using cell matches across data sets, we analyzed the integration of optic flow patterns by neurons downstream of the LPTs and find that most central brain neurons establish sharper selectivity for global optic flow patterns than their input neurons. Furthermore, we found that self-motion information extracted from optic flow is processed in distinct regions of the central brain, pointing to diverse foci for the generation of visual behaviors.

Whole-brain annotation and multi-connectome cell typing quantifies circuit stereotypy in Drosophila.

The fruit fly Drosophila melanogaster combines surprisingly sophisticated behaviour with a highly tractable nervous system. A large part of the fly's success as a model organism in modern neuroscience stems from the concentration of collaboratively generated molecular genetic and digital resources. As presented in our FlyWire companion paper 1 , this now includes the first full brain connectome of an adult animal. Here we report the systematic and hierarchical annotation of this ~130,000-neuron connectome including neuronal classes, cell types and developmental units (hemilineages). This enables any researcher to navigate this huge dataset and find systems and neurons of interest, linked to the literature through the Virtual Fly Brain database 2 . Crucially, this resource includes 4,552 cell types. 3,094 are rigorous consensus validations of cell types previously proposed in the hemibrain connectome 3 . In addition, we propose 1,458 new cell types, arising mostly from the fact that the FlyWire connectome spans the whole brain, whereas the hemibrain derives from a subvolume. Comparison of FlyWire and the hemibrain showed that cell type counts and strong connections were largely stable, but connection weights were surprisingly variable within and across animals. Further analysis defined simple heuristics for connectome interpretation: connections stronger than 10 unitary synapses or providing >1% of the input to a target cell are highly conserved. Some cell types showed increased variability across connectomes: the most common cell type in the mushroom body, required for learning and memory, is almost twice as numerous in FlyWire as the hemibrain. We find evidence for functional homeostasis through adjustments of the absolute amount of excitatory input while maintaining the excitation-inhibition ratio. Finally, and surprisingly, about one third of the cell types proposed in the hemibrain connectome could not yet be reliably identified in the FlyWire connectome. We therefore suggest that cell types should be defined to be robust to inter-individual variation, namely as groups of cells that are quantitatively more similar to cells in a different brain than to any other cell in the same brain. Joint analysis of the FlyWire and hemibrain connectomes demonstrates the viability and utility of this new definition. Our work defines a consensus cell type atlas for the fly brain and provides both an intellectual framework and open source toolchain for brain-scale comparative connectomics.

Neuronal wiring diagram of an adult brain.

Connections between neurons can be mapped by acquiring and analyzing electron microscopic (EM) brain images. In recent years, this approach has been applied to chunks of brains to reconstruct local connectivity maps that are highly informative, yet inadequate for understanding brain function more globally. Here, we present the first neuronal wiring diagram of a whole adult brain, containing 5×107 chemical synapses between ~130,000 neurons reconstructed from a female Drosophila melanogaster. The resource also incorporates annotations of cell classes and types, nerves, hemilineages, and predictions of neurotransmitter identities. Data products are available by download, programmatic access, and interactive browsing and made interoperable with other fly data resources. We show how to derive a projectome, a map of projections between regions, from the connectome. We demonstrate the tracing of synaptic pathways and the analysis of information flow from inputs (sensory and ascending neurons) to outputs (motor, endocrine, and descending neurons), across both hemispheres, and between the central brain and the optic lobes. Tracing from a subset of photoreceptors all the way to descending motor pathways illustrates how structure can uncover putative circuit mechanisms underlying sensorimotor behaviors. The technologies and open ecosystem of the FlyWire Consortium set the stage for future large-scale connectome projects in other species.

Structured sampling of olfactory input by the fly mushroom body.

Associative memory formation and recall in the fruit fly Drosophila melanogaster is subserved by the mushroom body (MB). Upon arrival in the MB, sensory information undergoes a profound transformation from broadly tuned and stereotyped odorant responses in the olfactory projection neuron (PN) layer to narrowly tuned and nonstereotyped responses in the Kenyon cells (KCs). Theory and experiment suggest that this transformation is implemented by random connectivity between KCs and PNs. However, this hypothesis has been challenging to test, given the difficulty of mapping synaptic connections between large numbers of brain-spanning neurons. Here, we used a recent whole-brain electron microscopy volume of the adult fruit fly to map PN-to-KC connectivity at synaptic resolution. The PN-KC connectome revealed unexpected structure, with preponderantly food-responsive PN types converging at above-chance levels on downstream KCs. Axons of the overconvergent PN types tended to arborize near one another in the MB main calyx, making local KC dendrites more likely to receive input from those types. Overconvergent PN types preferentially co-arborize and connect with dendrites of αß and α'ß' KC subtypes. Computational simulation of the observed network showed degraded discrimination performance compared with a random network, except when all signal flowed through the overconvergent, primarily food-responsive PN types. Additional theory and experiment will be needed to fully characterize the impact of the observed non-random network structure on associative memory formation and recall.

Drosophila gustatory projections are segregated by taste modality and connectivity.

Gustatory sensory neurons detect caloric and harmful compounds in potential food and convey this information to the brain to inform feeding decisions. To examine the signals that gustatory neurons transmit and receive, we reconstructed gustatory axons and their synaptic sites in the adult Drosophila melanogaster brain, utilizing a whole-brain electron microscopy volume. We reconstructed 87 gustatory projections from the proboscis labellum in the right hemisphere and 57 from the left, representing the majority of labellar gustatory axons. Gustatory neurons contain a nearly equal number of interspersed pre- and postsynaptic sites, with extensive synaptic connectivity among gustatory axons. Morphology- and connectivity-based clustering revealed six distinct groups, likely representing neurons recognizing different taste modalities. The vast majority of synaptic connections are between neurons of the same group. This study resolves the anatomy of labellar gustatory projections, reveals that gustatory projections are segregated based on taste modality, and uncovers synaptic connections that may alter the transmission of gustatory signals.

Synaptic targets of photoreceptors specialized to detect color and skylight polarization in Drosophila.

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.

Automatic detection of synaptic partners in a whole-brain Drosophila electron microscopy data set.

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.

Circuit reorganization in the Drosophila mushroom body calyx accompanies memory consolidation.

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.

Spatial readout of visual looming in the central brain of Drosophila.

Visual systems can exploit spatial correlations in the visual scene by using retinotopy, the organizing principle by which neighboring cells encode neighboring spatial locations. However, retinotopy is often lost, such as when visual pathways are integrated with other sensory modalities. How is spatial information processed outside of strictly visual brain areas? Here, we focused on visual looming responsive LC6 cells in Drosophila, a population whose dendrites collectively cover the visual field, but whose axons form a single glomerulus-a structure without obvious retinotopic organization-in the central brain. We identified multiple cell types downstream of LC6 in the glomerulus and found that they more strongly respond to looming in different portions of the visual field, unexpectedly preserving spatial information. Through EM reconstruction of all LC6 synaptic inputs to the glomerulus, we found that LC6 and downstream cell types form circuits within the glomerulus that enable spatial readout of visual features and contralateral suppression-mechanisms that transform visual information for behavioral control.

Distinct subpopulations of mechanosensory chordotonal organ neurons elicit grooming of the fruit fly antennae.

Diverse mechanosensory neurons detect different mechanical forces that can impact animal behavior. Yet our understanding of the anatomical and physiological diversity of these neurons and the behaviors that they influence is limited. We previously discovered that grooming of the Drosophila melanogaster antennae is elicited by an antennal mechanosensory chordotonal organ, the Johnston's organ (JO) (Hampel et al., 2015). Here, we describe anatomically and physiologically distinct JO mechanosensory neuron subpopulations that each elicit antennal grooming. We show that the subpopulations project to different, discrete zones in the brain and differ in their responses to mechanical stimulation of the antennae. Although activation of each subpopulation elicits antennal grooming, distinct subpopulations also elicit the additional behaviors of wing flapping or backward locomotion. Our results provide a comprehensive description of the diversity of mechanosensory neurons in the JO, and reveal that distinct JO subpopulations can elicit both common and distinct behavioral responses.

The Neuroanatomical Ultrastructure and Function of a Biological Ring Attractor.

Neural representations of head direction (HD) have been discovered in many species. Theoretical work has proposed that the dynamics associated with these representations are generated, maintained, and updated by recurrent network structures called ring attractors. We evaluated this theorized structure-function relationship by performing electron-microscopy-based circuit reconstruction and RNA profiling of identified cell types in the HD system of Drosophila melanogaster. We identified motifs that have been hypothesized to maintain the HD representation in darkness, update it when the animal turns, and tether it to visual cues. Functional studies provided support for the proposed roles of individual excitatory or inhibitory circuit elements in shaping activity. We also discovered recurrent connections between neuronal arbors with mixed pre- and postsynaptic specializations. Our results confirm that the Drosophila HD network contains the core components of a ring attractor while also revealing unpredicted structural features that might enhance the network's computational power.

The Mind of a Mouse.

Large scientific projects in genomics and astronomy are influential not because they answer any single question but because they enable investigation of continuously arising new questions from the same data-rich sources. Advances in automated mapping of the brain's synaptic connections (connectomics) suggest that the complicated circuits underlying brain function are ripe for analysis. We discuss benefits of mapping a mouse brain at the level of synapses.

Connectomics Analysis Reveals First-, Second-, and Third-Order Thermosensory and Hygrosensory Neurons in the Adult Drosophila Brain.

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.

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.

The Wiring Logic of an Identified Serotonergic Neuron That Spans Sensory Networks.

Serotonergic neurons project widely throughout the brain to modulate diverse physiological and behavioral processes. However, a single-cell resolution understanding of the connectivity of serotonergic neurons is currently lacking. Using a whole-brain EM dataset of a female Drosophila, we comprehensively determine the wiring logic of a broadly projecting serotonergic neuron (the CSDn) that spans several olfactory regions. Within the antennal lobe, the CSDn differentially innervates each glomerulus, yet surprisingly, this variability reflects a diverse set of presynaptic partners, rather than glomerulus-specific differences in synaptic output, which is predominately to local interneurons. Moreover, the CSDn has distinct connectivity relationships with specific local interneuron subtypes, suggesting that the CSDn influences distinct aspects of local network processing. Across olfactory regions, the CSDn has different patterns of connectivity, even having different connectivity with individual projection neurons that also span these regions. Whereas the CSDn targets inhibitory local neurons in the antennal lobe, the CSDn has more distributed connectivity in the LH, preferentially synapsing with principal neuron types based on transmitter content. Last, we identify individual novel synaptic partners associated with other sensory domains that provide strong, top-down input to the CSDn. Together, our study reveals the complex connectivity of serotonergic neurons, which combine the integration of local and extrinsic synaptic input in a nuanced, region-specific manner.SIGNIFICANCE STATEMENT All sensory systems receive serotonergic modulatory input. However, a comprehensive understanding of the synaptic connectivity of individual serotonergic neurons is lacking. In this study, we use a whole-brain EM microscopy dataset to comprehensively determine the wiring logic of a broadly projecting serotonergic neuron in the olfactory system of Drosophila Collectively, our study demonstrates, at a single-cell level, the complex connectivity of serotonergic neurons within their target networks, identifies specific cell classes heavily targeted for serotonergic modulation in the olfactory system, and reveals novel extrinsic neurons that provide strong input to this serotonergic system outside of the context of olfaction. Elucidating the connectivity logic of individual modulatory neurons provides a ground plan for the seemingly heterogeneous effects of modulatory systems.