Connectomic reconstruction predicts visual features used for navigation.

Many animals use visual information to navigate1-4, but how such information is encoded and integrated by the navigation system remains incompletely understood. In Drosophila melanogaster, EPG neurons in the central complex compute the heading direction5 by integrating visual input from ER neurons6-12, which are part of the anterior visual pathway (AVP)10,13-16. Here we densely reconstruct all neurons in the AVP using electron-microscopy data17. The AVP comprises four neuropils, sequentially linked by three major classes of neurons: MeTu neurons10,14,15, which connect the medulla in the optic lobe to the small unit of the anterior optic tubercle (AOTUsu) in the central brain; TuBu neurons9,16, which connect the AOTUsu to the bulb neuropil; and ER neurons6-12, which connect the bulb to the EPG neurons. On the basis of morphologies, connectivity between neural classes and the locations of synapses, we identify distinct information channels that originate from four types of MeTu neurons, and we further divide these into ten subtypes according to the presynaptic connections in the medulla and the postsynaptic connections in the AOTUsu. Using the connectivity of the entire AVP and the dendritic fields of the MeTu neurons in the optic lobes, we infer potential visual features and the visual area from which any ER neuron receives input. We confirm some of these predictions physiologically. These results provide a strong foundation for understanding how distinct sensory features can be extracted and transformed across multiple processing stages to construct higher-order cognitive representations.

Network statistics of the whole-brain connectome of Drosophila.

Brains comprise complex networks of neurons and connections, similar to the nodes and edges of artificial networks. Network analysis applied to the wiring diagrams of brains can offer insights into how they support computations and regulate the flow of information underlying perception and behaviour. The completion of the first whole-brain connectome of an adult fly, containing over 130,000 neurons and millions of synaptic connections1-3, offers an opportunity to analyse the statistical properties and topological features of a complete brain. Here we computed the prevalence of two- and three-node motifs, examined their strengths, related this information to both neurotransmitter composition and cell type annotations4,5, and compared these metrics with wiring diagrams of other animals. We found that the network of the fly brain displays rich-club organization, with a large population (30% of the connectome) of highly connected neurons. We identified subsets of rich-club neurons that may serve as integrators or broadcasters of signals. Finally, we examined subnetworks based on 78 anatomically defined brain regions or neuropils. These data products are shared within the FlyWire Codex ( https://codex.flywire.ai ) and should serve as a foundation for models and experiments exploring the relationship between neural activity and anatomical structure.

Neuronal parts list and wiring diagram for a visual system.

A catalogue of neuronal cell types has often been called a 'parts list' of the brain1, and regarded as a prerequisite for understanding brain function2,3. In the optic lobe of Drosophila, rules of connectivity between cell types have already proven to be essential for understanding fly vision4,5. Here we analyse the fly connectome to complete the list of cell types intrinsic to the optic lobe, as well as the rules governing their connectivity. Most new cell types contain 10 to 100 cells, and integrate information over medium distances in the visual field. Some existing type families (Tm, Li, and LPi)6-10 at least double in number of types. A new serpentine medulla (Sm) interneuron family contains more types than any other. Three families of cross-neuropil types are revealed. The consistency of types is demonstrated by analysing the distances in high-dimensional feature space, and is further validated by algorithms that select small subsets of discriminative features. We use connectivity to hypothesize about the functional roles of cell types in motion, object and colour vision. Connectivity with 'boundary types' that straddle the optic lobe and central brain is also quantified. We showcase the advantages of connectomic cell typing: complete and unbiased sampling, a rich array of features based on connectivity and reduction of the connectome to a substantially simpler wiring diagram of cell types, with immediate relevance for brain function and development.

Developmental biology: Wait a bit and then you'll smell it.

Animals change how they respond to the world around them as they age, giving rise to developmental stage and status appropriate behaviours. New work finds that changes in the primary olfactory neuropil are correlated with the natural developmental shift in alarm pheromone-specific responses of an ant.

Diversity of visual inputs to Kenyon cells of the Drosophila mushroom body.

The arthropod mushroom body is well-studied as an expansion layer representing olfactory stimuli and linking them to contingent events. However, 8% of mushroom body Kenyon cells in Drosophila melanogaster receive predominantly visual input, and their function remains unclear. Here, we identify inputs to visual Kenyon cells using the FlyWire adult whole-brain connectome. Input repertoires are similar across hemispheres and connectomes with certain inputs highly overrepresented. Many visual neurons presynaptic to Kenyon cells have large receptive fields, while interneuron inputs receive spatially restricted signals that may be tuned to specific visual features. Individual visual Kenyon cells randomly sample sparse inputs from combinations of visual channels, including multiple optic lobe neuropils. These connectivity patterns suggest that visual coding in the mushroom body, like olfactory coding, is sparse, distributed, and combinatorial. However, the specific input repertoire to the smaller population of visual Kenyon cells suggests a constrained encoding of visual stimuli.

3D atlas of cerebral neuropils with previously unknown demarcations in the honey bee brain.

Honey bees (Apis mellifera) express remarkable social interactions and cognitive capabilities that have been studied extensively. In many cases, behavioral studies were accompanied by neurophysiological and neuroanatomical investigations. While most studies have focused on primary sensory neuropils, such as the optic lobes or antennal lobes, and major integration centers, such as the mushroom bodies or the central complex, many regions of the cerebrum (the central brain without the optic lobes) of the honey bee are only poorly explored so far, both anatomically and physiologically. To promote studies of these brain regions, we used anti-synapsin immunolabeling and neuronal tract tracings followed by confocal imaging and 3D reconstructions to demarcate all neuropils in the honey bee cerebrum and close this gap at the anatomical level. We demarcated 35 neuropils and 25 fiber tracts in the honey bee cerebrum, most of which have counterparts in the fly (Drosophila melanogaster) and other insect species that have been investigated so far at this level of detail. We discuss the role of cerebral neuropils in multisensory integration in the insect brain, emphasize the importance of this brain atlas for comparative studies, and highlight specific architectural features of the honey bee cerebrum.

Extraction of bouton-like structures from neuropil calcium imaging data.

The neuropil, the plexus of axons and dendrites, plays a critical role in operating the circuit processing of the nervous system. Revealing the spatiotemporal activity pattern within the neuropil would clarify how the information flows throughout the nervous system. However, calcium imaging to examine the circuit dynamics has mainly focused on the soma population due to their discrete distribution. The development of a methodology to analyze the calcium imaging data of a densely packed neuropil would provide us with new insights into the circuit dynamics. Here, we propose a new method to decompose calcium imaging data of the neuropil into populations of bouton-like synaptic structures with a standard desktop computer. To extract bouton-like structures from calcium imaging data, we introduced a new type of modularity, a widely used quality measure in graph theory, and optimized the clustering configuration by a simulated annealing algorithm, which is established in statistical physics. To assess this method's performance, we conducted calcium imaging of the neuropil of Drosophila larvae. Based on the obtained data, we established artificial neuropil imaging datasets. We applied the decomposition procedure to the artificial and experimental calcium imaging data and extracted individual bouton-like structures successfully. Based on the extracted spatiotemporal data, we analyzed the network structure of the central nervous system of fly larvae and found it was scale-free. These results demonstrate that neuropil calcium imaging and its decomposition could provide new insight into our understanding of neural processing.

Matched function of the neuropil processing optic flow in flies and crabs: the lobula plate mediates optomotor responses in Neohelice granulata.

When an animal rotates (whether it is an arthropod, a fish, a bird or a human) a drift of the visual panorama occurs over its retina, termed optic flow. The image is stabilized by compensatory behaviours (driven by the movement of the eyes, head or the whole body depending on the animal) collectively termed optomotor responses. The dipteran lobula plate has been consistently linked with optic flow processing and the control of optomotor responses. Crabs have a neuropil similarly located and interconnected in the optic lobes, therefore referred to as a lobula plate too. Here we show that the crabs' lobula plate is required for normal optomotor responses since the response was lost or severely impaired in animals whose lobula plate had been lesioned. The effect was behaviour-specific, since avoidance responses to approaching visual stimuli were not affected. Crabs require simpler optic flow processing than flies (because they move slower and in two-dimensional instead of three-dimensional space), consequently their lobula plates are relatively smaller. Nonetheless, they perform the same essential role in the visual control of behaviour. Our findings add a fundamental piece to the current debate on the evolutionary relationship between the lobula plates of insects and crustaceans.

The neuronal logic of how internal states control food choice.

When deciding what to eat, animals evaluate sensory information about food quality alongside multiple ongoing internal states1-10. How internal states interact to alter sensorimotor processing and shape decisions such as food choice remains poorly understood. Here we use pan-neuronal volumetric activity imaging in the brain of Drosophila melanogaster to investigate the neuronal basis of internal state-dependent nutrient appetites. We created a functional atlas of the ventral fly brain and find that metabolic state shapes sensorimotor processing across large sections of the neuropil. By contrast, reproductive state acts locally to define how sensory information is translated into feeding motor output. These two states thus synergistically modulate protein-specific food intake and food choice. Finally, using a novel computational strategy, we identify driver lines that label neurons innervating state-modulated brain regions and show that the newly identified 'borboleta' region is sufficient to direct food choice towards protein-rich food. We thus identify a generalizable principle by which distinct internal states are integrated to shape decision making and propose a strategy to uncover and functionally validate how internal states shape behaviour.

Neural organization of the third optic neuropil, the lobula, in the highly visual semiterrestrial crab Neohelice granulata.

The visual neuropils (lamina, medulla, and lobula complex) of malacostracan crustaceans and hexapods have many organizational principles, cell types, and functional properties in common. Information about the cellular elements that compose the crustacean lobula is scarce especially when focusing on small columnar cells. Semiterrestrial crabs possess a highly developed visual system and display conspicuous visually guided behaviors. In particular, Neohelice granulata has been previously used to describe the cellular components of the first two optic neuropils using Golgi impregnation technique. Here, we present a comprehensive description of individual elements composing the third optic neuropil, the lobula, of that same species. We characterized a wide variety of elements (140 types) including input terminals and lobula columnar, centrifugal, and input columnar elements. Results reveal a very dense and complex neuropil. We found a frequently impregnated input element (suggesting a supernumerary cartridge representation) that arborizes in the third layer of the lobula and that presents four variants each with ramifications organized following one of the four cardinal axes suggesting a role in directional processing. We also describe input elements with two neurites branching in the third layer, probably connecting with the medulla and lobula plate. These facts suggest that this layer is involved in the directional motion detection pathway in crabs. We analyze and discuss our findings considering the similarities and differences found between the layered organization and components of this crustacean lobula and the lobula of insects.

A projectome of the bumblebee central complex.

Insects have evolved diverse and remarkable strategies for navigating in various ecologies all over the world. Regardless of species, insects share the presence of a group of morphologically conserved neuropils known collectively as the central complex (CX). The CX is a navigational center, involved in sensory integration and coordinated motor activity. Despite the fact that our understanding of navigational behavior comes predominantly from ants and bees, most of what we know about the underlying neural circuitry of such behavior comes from work in fruit flies. Here, we aim to close this gap, by providing the first comprehensive map of all major columnar neurons and their projection patterns in the CX of a bee. We find numerous components of the circuit that appear to be highly conserved between the fly and the bee, but also highlight several key differences which are likely to have important functional ramifications.

Bumblebees forage widely for pollen and nectar from flowers, sometimes travelling kilometers away from their nest, but they can somehow always find their way home in a nearly straight line. These insects have been known to return to their nest from new locations almost 10 kilometers away. This homing ability is a complex neurological feat and requires the brain to combine several processes, including observing the external world, controlling bodily movements and drawing on memory. While the navigational behavior of bees has been well-studied, the neuronal circuitry behind it has not. Unfortunately, most of what is known about insects' brain activity comes from studies in species such as locusts or fruit flies. In these species, a region of the brain known as the central complex has been shown to have an essential role in homing behaviors. However, it is unknown how similar the central complex of bumblebees might be to fruit flies' or locusts', or how these differences may affect navigational abilities. Sayre et al. obtained images of thin slices of the bumblebee central complex using a technique called block-face electron microscopy, which produces high-resolution image volumes. These images were used to obtain a three-dimensional map of over 1300 neurons. This cellular atlas showed that key aspects of the central complex are nearly identical between flies and bumblebees, including the internal compass that monitors what direction the insect is travelling in. However, hundreds of millions of years of independent evolution have resulted in some differences. These were found in neurons possibly involved in forming memories of the directions and lengths of travelled paths, and in the circuits that use such vector memories to steer the insects towards their targets. Sayre et al. propose that these changes underlie bees' impressive ability to navigate. These results help explain how the structure of insects' brains can determine homing abilities. The insights gained could be used to develop efficient autonomous navigation systems, which are challenging to build and require a lot more processing power than offered by a small part of an insect brain.

Distinct protocerebral neuropils associated with attractive and aversive female-produced odorants in the male moth brain.

The pheromone system of heliothine moths is an optimal model for studying principles underlying higher-order olfactory processing. In Helicoverpa armigera, three male-specific glomeruli receive input about three female-produced signals, the primary pheromone component, serving as an attractant, and two minor constituents, serving a dual function, that is, attraction versus inhibition of attraction. From the antennal-lobe glomeruli, the information is conveyed to higher olfactory centers, including the lateral protocerebrum, via three main paths - of which the medial tract is the most prominent. In this study, we traced physiologically identified medial-tract projection neurons from each of the three male-specific glomeruli with the aim of mapping their terminal branches in the lateral protocerebrum. Our data suggest that the neurons' widespread projections are organized according to behavioral significance, including a spatial separation of signals representing attraction versus inhibition - however, with a unique capacity of switching behavioral consequence based on the amount of the minor components.

Astrocytes close a motor circuit critical period.

Critical periods-brief intervals during which neural circuits can be modified by activity-are necessary for proper neural circuit assembly. Extended critical periods are associated with neurodevelopmental disorders; however, the mechanisms that ensure timely critical period closure remain poorly understood1,2. Here we define a critical period in a developing Drosophila motor circuit and identify astrocytes as essential for proper critical period termination. During the critical period, changes in activity regulate dendrite length, complexity and connectivity of motor neurons. Astrocytes invaded the neuropil just before critical period closure3, and astrocyte ablation prolonged the critical period. Finally, we used a genetic screen to identify astrocyte-motor neuron signalling pathways that close the critical period, including Neuroligin-Neurexin signalling. Reduced signalling destabilized dendritic microtubules, increased dendrite dynamicity and impaired locomotor behaviour, underscoring the importance of critical period closure. Previous work defined astroglia as regulators of plasticity at individual synapses4; we show here that astrocytes also regulate motor circuit critical period closure to ensure proper locomotor behaviour.

A multi-scale brain map derived from whole-brain volumetric reconstructions.

Animal nervous system organization is crucial for all body functions and its disruption can lead to severe cognitive and behavioural impairment1. This organization relies on features across scales-from the localization of synapses at the nanoscale, through neurons, which possess intricate neuronal morphologies that underpin circuit organization, to stereotyped connections between different regions of the brain2. The sheer complexity of this organ means that the feat of reconstructing and modelling the structure of a complete nervous system that is integrated across all of these scales has yet to be achieved. Here we present a complete structure-function model of the main neuropil in the nematode Caenorhabditis elegans-the nerve ring-which we derive by integrating the volumetric reconstructions from two animals with corresponding3 synaptic and gap-junctional connectomes. Whereas previously the nerve ring was considered to be a densely packed tract of neural processes, we uncover internal organization and show how local neighbourhoods spatially constrain and support the synaptic connectome. We find that the C. elegans connectome is not invariant, but that a precisely wired core circuit is embedded in a background of variable connectivity, and identify a candidate reference connectome for the core circuit. Using this reference, we propose a modular network architecture of the C. elegans brain that supports sensory computation and integration, sensorimotor convergence and brain-wide coordination. These findings reveal scalable and robust features of brain organization that may be universal across phyla.

Thermoresponsive motor behavior is mediated by ring neuron circuits in the central complex of Drosophila.

Insects are ectothermal animals that are constrained in their survival and reproduction by external temperature fluctuations which require either active avoidance of or movement towards a given heat source. In Drosophila, different thermoreceptors and neurons have been identified that mediate temperature sensation to maintain the animal's thermal preference. However, less is known how thermosensory information is integrated to gate thermoresponsive motor behavior. Here we use transsynaptic tracing together with calcium imaging, electrophysiology and thermogenetic manipulations in freely moving Drosophila exposed to elevated temperature and identify different functions of ellipsoid body ring neurons, R1-R4, in thermoresponsive motor behavior. Our results show that warming of the external surroundings elicits calcium influx specifically in R2-R4 but not in R1, which evokes threshold-dependent neural activity in the outer layer ring neurons. In contrast to R2, R3 and R4d neurons, thermogenetic inactivation of R4m and R1 neurons expressing the temperature-sensitive mutant allele of dynamin, shibireTS, results in impaired thermoresponsive motor behavior at elevated 31 °C. trans-Tango mediated transsynaptic tracing together with physiological and behavioral analyses indicate that integrated sensory information of warming is registered by neural activity of R4m as input layer of the ellipsoid body ring neuropil and relayed on to R1 output neurons that gate an adaptive motor response. Together these findings imply that segregated activities of central complex ring neurons mediate sensory-motor transformation of external temperature changes and gate thermoresponsive motor behavior in Drosophila.

In vivo microstimulation with cathodic and anodic asymmetric waveforms modulates spatiotemporal calcium dynamics in cortical neuropil and pyramidal neurons of male mice.

Electrical stimulation has been critical in the development of an understanding of brain function and disease. Despite its widespread use and obvious clinical potential, the mechanisms governing stimulation in the cortex remain largely unexplored in the context of pulse parameters. Modeling studies have suggested that modulation of stimulation pulse waveform may be able to control the probability of neuronal activation to selectively stimulate either cell bodies or passing fibers depending on the leading polarity. Thus, asymmetric waveforms with equal charge per phase (i.e., increasing the leading phase duration and proportionately decreasing the amplitude) may be able to activate a more spatially localized or distributed population of neurons if the leading phase is cathodic or anodic, respectively. Here, we use two-photon and mesoscale calcium imaging of GCaMP6s expressed in excitatory pyramidal neurons of male mice to investigate the role of pulse polarity and waveform asymmetry on the spatiotemporal properties of direct neuronal activation with 10-Hz electrical stimulation. We demonstrate that increasing cathodic asymmetry effectively reduces neuronal activation and results in a more spatially localized subpopulation of activated neurons without sacrificing the density of activated neurons around the electrode. Conversely, increasing anodic asymmetry increases the spatial spread of activation and highly resembles spatiotemporal calcium activity induced by conventional symmetric cathodic stimulation. These results suggest that stimulation polarity and asymmetry can be used to modulate the spatiotemporal dynamics of neuronal activity thus increasing the effective parameter space of electrical stimulation to restore sensation and study circuit dynamics.

Nomen est omen, cognitive dissonance, and homology of memory centers in crustaceans and insects.

In 1882, the Italian embryologist Giuseppe Bellonci introduced a nomenclature for structures in the stomatopod crustacean Squilla mantis that he claimed correspond to insect mushroom bodies, today recognized as cardinal centers that in insects mediate associative memory. The use of Bellonci's terminology has, through a series of misunderstandings and entrenched opinions, led to contesting views regarding whether centers in crustacean and insect brains that occupy corresponding locations and receive comparable multisensory inputs are homologous or homoplasic. The following describes the fate of terms used to denote sensory association neuropils in crustacean species and relates how those terms were deployed in the 1920s and 1930s by the Swedish neuroanatomist Bertil Hanström to claim homology in insects and crustaceans. Yet the same terminology has been repurposed by subsequent researchers to promote the very opposite view: that mushroom bodies are a derived trait of hexapods and that equivalent centers in crustaceans evolved independently.

The brain of Cataglyphis ants: Neuronal organization and visual projections.

Cataglyphis ants are known for their outstanding navigational abilities. They return to their inconspicuous nest after far-reaching foraging trips using path integration, and whenever available, learn and memorize visual features of panoramic sceneries. To achieve this, the ants combine directional visual information from celestial cues and panoramic scenes with distance information from an intrinsic odometer. The largely vision-based navigation in Cataglyphis requires sophisticated neuronal networks to process the broad repertoire of visual stimuli. Although Cataglyphis ants have been subjected to many neuroethological studies, little is known about the general neuronal organization of their central brain and the visual pathways beyond major circuits. Here, we provide a comprehensive, three-dimensional neuronal map of synapse-rich neuropils in the brain of Cataglyphis nodus including major connecting fiber systems. In addition, we examined neuronal tracts underlying the processing of visual information in more detail. This study revealed a total of 33 brain neuropils and 30 neuronal fiber tracts including six distinct tracts between the optic lobes and the cerebrum. We also discuss the importance of comparative studies on insect brain architecture for a profound understanding of neuronal networks and their function.

Visual pathways in the brain of the jumping spider Marpissa muscosa.

Some animals have evolved task differentiation among their eyes. A particular example is spiders, where most species have eight eyes, of which two (the principal eyes) are used for object discrimination, whereas the other three pairs (secondary eyes) detect movement. In the ctenid spider Cupiennius salei, these two eye types correspond to two visual pathways in the brain. Each eye is associated with its own first- and second-order visual neuropil. The second-order neuropils of the principal eyes are connected to the arcuate body, whereas the second-order neuropils of the secondary eyes are linked to the mushroom body. We explored the principal- and secondary eye visual pathways of the jumping spider Marpissa muscosa, in which size and visual fields of the two eye types are considerably different. We found that the connectivity of the principal eye pathway is the same as in C. salei, while there are differences in the secondary eye pathways. In M. muscosa, all secondary eyes are connected to their own first-order visual neuropils. The first-order visual neuropils of the anterior lateral and posterior lateral eyes are connected with a second-order visual neuropil each and an additional shared one (L2). In the posterior median eyes, the axons of their first-order visual neuropils project directly to the arcuate body, suggesting that the posterior median eyes do not detect movement. The L2 might function as an upstream integration center enabling faster movement decisions.

The brain of a nocturnal migratory insect, the Australian Bogong moth.

Every year, millions of Australian Bogong moths (Agrotis infusa) complete an astonishing journey: In Spring, they migrate over 1,000 km from their breeding grounds to the alpine regions of the Snowy Mountains, where they endure the hot summer in the cool climate of alpine caves. In autumn, the moths return to their breeding grounds, where they mate, lay eggs and die. These moths can use visual cues in combination with the geomagnetic field to guide their flight, but how these cues are processed and integrated into the brain to drive migratory behavior is unknown. To generate an access point for functional studies, we provide a detailed description of the Bogong moth's brain. Based on immunohistochemical stainings against synapsin and serotonin (5HT), we describe the overall layout as well as the fine structure of all major neuropils, including the regions that have previously been implicated in compass-based navigation. The resulting average brain atlas consists of 3D reconstructions of 25 separate neuropils, comprising the most detailed account of a moth brain to date. Our results show that the Bogong moth brain follows the typical lepidopteran ground pattern, with no major specializations that can be attributed to their spectacular migratory lifestyle. These findings suggest that migratory behavior does not require widespread modifications of brain structure, but might be achievable via small adjustments of neural circuitry in key brain areas. Locating these subtle changes will be a challenging task for the future, for which our study provides an essential anatomical framework.