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Many animals use visual information to navigate 1-4 , 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 direction 5 by integrating visual input from ER neurons 6-12 , which are part of the anterior visual pathway (AVP) 10,13-16 . Here we densely reconstruct all neurons in the AVP using electron-microscopy data 17 . The AVP comprises four neuropils, sequentially linked by three major classes of neurons: MeTu neurons 10,14,15 , which connect the medulla in the optic lobe to the small unit of the anterior optic tubercle (AOTUsu) in the central brain; TuBu neurons 9,16 , which connect the AOTUsu to the bulb neuropil; and ER neurons 6-12 , 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., (© 2024. The Author(s).)

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 connections 1-3 , 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 annotations 4,5 , 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., (© 2024. The Author(s).)

A catalogue of neuronal cell types has often been called a 'parts list' of the brain 1 , and regarded as a prerequisite for understanding brain function 2,3 . In the optic lobe of Drosophila, rules of connectivity between cell types have already proven to be essential for understanding fly vision 4,5 . 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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

The lateral complexes (LXs) are bilaterally paired neuropils in the insect brain that mediate communication between the central complex (CX), a brain center controlling spatial orientation, various sensory processing areas, and thoracic motor centers that execute locomotion. The LX of the desert locust consists of the lateral accessory lobe (LAL), and the medial and lateral bulb. We have analyzed the anatomical organization and the neuronal connections of the LX in the locust, to provide a basis for future functional studies. Reanalyzing the morphology of neurons connecting the CX and the LX revealed likely feedback loops in the sky compass network of the CX via connections in the gall of the LAL and a newly identified neuropil termed ovoid body. In addition, we characterized 16 different types of neuron that connect the LAL with other areas in the brain. Eight types of neuron provide information flow between both LALs, five types are LAL input neurons, and three types are LAL output neurons. Among these are neurons providing input from sensory brain areas such as the lobula and antennal neuropils. Brain regions most often targeted by LAL neurons are the posterior slope, the wedge, and the crepine. Two descending neurons with dendrites in the LAL were identified. Our data support and complement existing knowledge about how the LAL is embedded in the neuronal network involved in processing of sensory information and generation of appropriate behavioral output for goal-directed locomotion., (© 2021 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

Neuropil is a fundamental form of tissue organization within the brain 1 , in which densely packed neurons synaptically interconnect into precise circuit architecture 2,3 . However, the structural and developmental principles that govern this nanoscale precision remain largely unknown 4,5 . Here we use an iterative data coarse-graining algorithm termed 'diffusion condensation' 6 to identify nested circuit structures within the Caenorhabditis elegans neuropil, which is known as the nerve ring. We show that the nerve ring neuropil is largely organized into four strata that are composed of related behavioural circuits. The stratified architecture of the neuropil is a geometrical representation of the functional segregation of sensory information and motor outputs, with specific sensory organs and muscle quadrants mapping onto particular neuropil strata. We identify groups of neurons with unique morphologies that integrate information across strata and that create neural structures that cage the strata within the nerve ring. We use high resolution light-sheet microscopy 7,8 coupled with lineage-tracing and cell-tracking algorithms 9,10 to resolve the developmental sequence and reveal principles of cell position, migration and outgrowth that guide stratified neuropil organization. Our results uncover conserved structural design principles that underlie the architecture and function of the nerve ring neuropil, and reveal a temporal progression of outgrowth-based on pioneer neurons-that guides the hierarchical development of the layered neuropil. Our findings provide a systematic blueprint for using structural and developmental approaches to understand neuropil organization within the brain.

Animal nervous system organization is crucial for all body functions and its disruption can lead to severe cognitive and behavioural impairment 1 . 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 brain 2 . 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 corresponding 3 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.

Neural organization of mushroom bodies is largely consistent across insects, whereas the ancestral ground pattern diverges broadly across crustacean lineages resulting in successive loss of columns and the acquisition of domed centers retaining ancestral Hebbian-like networks and aminergic connections. We demonstrate here a major departure from this evolutionary trend in Brachyura, the most recent malacostracan lineage. In the shore crab Hemigrapsus nudus , instead of occupying the rostral surface of the lateral protocerebrum, mushroom body calyces are buried deep within it with their columns extending outwards to an expansive system of gyri on the brain's surface. The organization amongst mushroom body neurons reaches extreme elaboration throughout its constituent neuropils. The calyces, columns, and especially the gyri show DC0 immunoreactivity, an indicator of extensive circuits involved in learning and memory., Competing Interests: NS, MS No competing interests declared, (© 2021, Strausfeld and Sayre.)

Only a few studies have examined the central visual system of Solifugae until now. To get new insights suitable for phylogenetic analysis we studied the R-cell (or retinula cell) projections and visual neuropils of Galeodes granti using various methods. G. granti possesses large median eyes and rudimentary lateral eyes. In this study, only the R-cells and neuropils of the median eyes were successfully stained. The R-cells terminate in two distinct visual neuropils. The first neuropil is located externally to the protocerebrum directly below the retina, the second neuropil lies in the cell body rind of the protocerebrum, and immediately adjacent is the arcuate body. This layout of the median eye visual system differs from Arachnopulmonata (Scorpiones + Tetrapulmonata). However, there are several similarities with Opiliones. In both, (1) the R-cells are connected to a first and second visual neuropil and not to any other region of the brain, (2) the first neuropil is not embedded in the cell body rind of the protocerebrum, it is rather external to the protocerebrum, (3) the second visual neuropil is embedded in the cell body rind, and (4) the second neuropil abuts the arcuate body. These findings may provide important new characters for the discussion on arachnid phylogeny., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (© 2020 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals, Inc.)

The pectines of scorpions are comb-like structures, located ventrally behind the fourth walking legs and consisting of variable numbers of teeth, or pegs, which contain thousands of bimodal peg sensillae. The associated neuropils are situated ventrally in the synganglion, extending between the second and fourth walking leg neuromeres. While the general morphology is consistent among scorpions, taxon-specific differences in pecten and neuropil structure remain elusive but are crucial for a better understanding of chemosensory processing. We analysed two scorpion species (Mesobuthus eupeus and Heterometrus petersii) regarding their pecten neuropil anatomy and the respective peg afferent innervation with anterograde and lipophilic tracing experiments, combined with immunohistochemistry and confocal laser-scanning microscopy. The pecten neuropils consisted of three subcompartments: a posterior pecten neuropil, an anterior pecten neuropil and a hitherto unknown accessory pecten neuropil. These subregions exhibited taxon-specific variations with regard to compartmentalisation and structure. Most notable were structural differences in the anterior pecten neuropils that ranged from ovoid shape and strong fragmentation in Heterometrus petersii to elongated shape with little compartmentalisation in Mesobuthus eupeus. Labelling the afferents of distinct pegs revealed a topographic organisation of the bimodal projections along a medio-lateral axis. At the same time, all subregions along the posterior-anterior axis were innervated by a single peg's afferents. The somatotopic projection pattern of bimodal sensillae appears to be common among arachnids, including scorpions. This includes the structure and organisation of the respective neuropils and the somatotopic projection patterns of chemosensory afferents. Nonetheless, the scorpion pecten pathway exhibits unique features, e.g. glomerular compartmentalisation superimposed on somatotopy, that are assumed to allow high resolution of substrate-borne chemical gradients., Competing Interests: The authors have declared that no competing interests exist.

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., (© 2020 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals, Inc.)

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., (© 2020 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals, Inc.)

The compound eye of cockroaches is obligatory for entrainment of the Madeira cockroach's circadian clock, but the cellular nature of its entrainment pathways is enigmatic. Employing multiple-label immunocytochemistry, histochemistry, and backfills, we searched for photic entrainment pathways to the accessory medulla (AME), the circadian clock of the Madeira cockroach. We wanted to know whether photoreceptor terminals could directly contact pigment-dispersing factor-immunoreactive (PDF-ir) circadian pacemaker neurons with somata in the lamina (PDFLAs) or somata next to the AME (PDFMEs). Short green-sensitive photoreceptor neurons of the compound eye terminated in lamina layers LA1 and LA2, adjacent to PDFLAs and PDFMEs that branched in LA3. Long UV-sensitive compound eye photoreceptor neurons terminated in medulla layer ME2 without direct contact to ipsilateral PDFMEs that arborized in ME4. Multiple neuropeptide-ir interneurons branched in ME4, connecting the AME to ME2. Before, extraocular photoreceptors of the lamina organ were suggested to send terminals to accessory laminae. There, they overlapped with PDFLAs that mostly colocalized PDF, FMRFamide, and 5-HT immunoreactivities, and with terminals of ipsi- and contralateral PDFMEs. We hypothesize that during the day cholinergic activation of the largest PDFME via lamina organ photoreceptors maintains PDF release orchestrating phases of sleep-wake cycles. As ipsilateral PDFMEs express excitatory and contralateral PDFMEs inhibitory PDF autoreceptors, diurnal PDF release keeps both PDF-dependent clock circuits in antiphase. Future experiments will test whether ipsilateral PDFMEs are sleep-promoting morning cells, while contralateral PDFMEs are activity-promoting evening cells, maintaining stable antiphase via the largest PDFME entrained by extraocular photoreceptors of the lamina organ., (© 2019 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals, Inc.)

Understanding neuronal function at the local and circuit level requires understanding astrocyte function. We have provided a detailed analysis of astrocyte morphology and territory in the Drosophila third-instar ventral nerve cord where there already exists considerable understanding of the neuronal network. Astrocyte shape varies more than previously reported; many have bilaterally symmetrical partners, many have a high percentage of their arborization in adjacent segments, and many have branches that follow structural features. Taken together, our data are consistent with, but not fully explained by, a model of a developmental growth process dominated by competitive or repulsive interactions between astrocytes. Our data suggest that the model should also include cell-autonomous aspects, as well as the use of structural features for growth. Variation in location of arborization territory for identified astrocytes was great enough that a standardized scheme of neuropil division among the six astrocytes that populate each hemi-segment is not possible at the third instar. The arborizations of the astrocytes can extend across neuronal functional domains. The ventral astrocyte in particular, whose territory can extend well into the proprioceptive region of the neuropil, has no obvious branching pattern that correlates with domains of particular sensory modalities, suggesting that the astrocyte would respond to neuronal activity in any of the sensory modalities, perhaps integrating across them. This study sets the stage for future studies that will generate a robust, functionally oriented connectome that includes both partners in neuronal circuits-the neurons and the glial cells, providing the foundation necessary for studies to elucidate neuron-glia interactions in this neuropil., (© 2020 Wiley Periodicals, Inc.)

Mantis shrimps (Stomatopoda) possess in common with other crustaceans, and with Hexapoda, specific neuroanatomical attributes of the protocerebrum, the most anterior part of the arthropod brain. These attributes include assemblages of interconnected centers called the central body complex and in the lateral protocerebra, situated in the eyestalks, paired mushroom bodies. The phenotypic homologues of these centers across Panarthropoda support the view that ancestral integrative circuits crucial to action selection and memory have persisted since the early Cambrian or late Ediacaran. However, the discovery of another prominent integrative neuropil in the stomatopod lateral protocerebrum raises the question whether it is unique to Stomatopoda or at least most developed in this lineage, which may have originated in the upper Ordovician or early Devonian. Here, we describe the neuroanatomical structure of this center, called the reniform body. Using confocal microscopy and classical silver staining, we demonstrate that the reniform body receives inputs from multiple sources, including the optic lobe's lobula. Although the mushroom body also receives projections from the lobula, it is entirely distinct from the reniform body, albeit connected to it by discrete tracts. We discuss the implications of their coexistence in Stomatopoda, the occurrence of the reniform body in another eumalacostracan lineage and what this may mean for our understanding of brain functionality in Pancrustacea., (© 2019 Wiley Periodicals, Inc.)

The central complex (CX) comprises a group of midline neuropils in the insect brain, consisting of the protocerebral bridge (PB), the upper (CBU) and lower division (CBL) of the central body and a pair of globular noduli. It receives prominent input from the visual system and plays a major role in spatial orientation of the animals. Vertical slices and horizontal layers of the CX are formed by columnar, tangential, and pontine neurons. While pontine and columnar neurons have been analyzed in detail, especially in the fruit fly and desert locust, understanding of the organization of tangential cells is still rudimentary. As a basis for future functional studies, we have studied the morphologies of tangential neurons of the CX of the desert locust Schistocerca gregaria. Intracellular dye injections revealed 43 different types of tangential neuron, 8 of the PB, 5 of the CBL, 24 of the CBU, 2 of the noduli, and 4 innervating multiple substructures. Cell bodies of these neurons were located in 11 different clusters in the cell body rind. Judging from the presence of fine versus beaded terminals, the vast majority of these neurons provide input into the CX, especially from the lateral complex (LX), the superior protocerebrum, the posterior slope, and other surrounding brain areas, but not directly from the mushroom bodies. Connections are largely subunit- and partly layer-specific. No direct connections were found between the CBU and the CBL. Instead, both subdivisions are connected in parallel with the PB and distinct layers of the noduli., (© 2019 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals, Inc.)

Neural remodeling is essential for the development of a functional nervous system and has been extensively studied in the metamorphosis of Drosophila Despite the crucial roles of glial cells in brain functions, including learning and behavior, little is known of how adult glial cells develop in the context of neural remodeling. Here, we show that the architecture of neuropil-glia in the adult Drosophila brain, which is composed of astrocyte-like glia (ALG) and ensheathing glia (EG), robustly develops from two different populations in the larva: the larval EG and glial cell missing -positive ( gcm + ) cells. Whereas gcm + cells proliferate and generate adult ALG and EG, larval EG dedifferentiate, proliferate and redifferentiate into the same glial subtypes. Each glial lineage occupies a certain brain area complementary to the other, and together they form the adult neuropil-glia architecture. Both lineages require the FGF receptor Heartless to proliferate, and the homeoprotein Prospero to differentiate into ALG. Lineage-specific inhibition of gliogenesis revealed that each lineage compensates for deficiency in the proliferation of the other. Together, the lineages ensure the robust development of adult neuropil-glia, thereby ensuring a functional brain., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The complexity of the nervous system requires the coordination of multiple cellular processes during development. Among them, we find boundary formation, axon guidance, cell migration and cell segregation. Understanding how different cell populations such as glial cells, developing neurons and neural stem cells contribute to the formation of boundaries and morphogenesis in the nervous system is a critical question in neurobiology. Slit is an evolutionary conserved protein essential for the development of the nervous system. For signaling, Slit has to bind to its cognate receptor Robo, a single-pass transmembrane protein. Although the Slit/Robo signaling pathway is well known for its involvement in axon guidance, it has also been associated to boundary formation in the Drosophila visual system. In the optic lobe, Slit is expressed in glial cells, positioned at the boundaries between developing neuropils, and in neurons of the medulla ganglia. Although it has been assumed that glial cells provide Slit to the system, the contribution of the neuronal expression has not been tested. Here, we show that, contrary to what was previously thought, Slit protein provided by medulla neurons is also required for boundary formation and morphogenesis of the optic lobe. Furthermore, tissue specific rescue using modified versions of Slit demonstrates that this protein acts at long range and does not require processing by extracellular proteases. Our data shed new light on our understanding of the cellular mechanisms involved in Slit function in the fly visual system morphogenesis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Copepoda is one of the crustacean taxa with still unresolved phylogenetic relationships within Tetraconata. Recent phylogenomic studies place them close to Malacostraca and Cirripedia. Little is known about the morphological details of the copepod nervous system, and the available data are sometimes contradictory. We investigated several representatives of the subgroup Calanoida using immunohistochemical labeling against alpha-tubulin and various neuroactive substances, combining this with confocal laser scanning analysis and 3D reconstruction. Our results show that the studied copepods exhibit only a single anterior protocerebral neuropil which is connected to the nerves of two protocerebral sense organs: the frontal filament organ and a photoreceptor known as the Gicklhorn's organ. We suggest, on the basis of its position and the innervation it provides, that Gicklhorn's organ is homologous to the compound eye in arthropods. With regard to the frontal filament organ, we reveal detailed innervation to the lateral protocerebrum and the appearance of spherical bodies that stain intensely against alpha tubulin. A potential homology of these bodies to the onion bodies in malacostacan crustaceans and in Mystacocarida is suggested. The nauplius eye in all the examined calanoids shows the same basic pattern of innervation with the middle cup sending its neurites into the median nerve, while the axons of the lateral cups proceed into both the median and the lateral nerves. The early development of the axonal scaffold of the nauplius eye neuropil from the proximal parts of the nauplius eye nerves follows the same pattern as in other crustaceans. In our view, this specific innervation pattern is a further feature supporting the homology of the nauplius eye in crustaceans., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The fan-shaped body (FB) neuropil in the Drosophila brain central complex (CX) controls a variety of adult behaviors, including navigation and sleep. How neuronal processes are organized into precise layers and columns in the FB and how alterations in FB neural-circuit wiring affect animal behaviors are unknown. We report here that secreted semaphorin 2b (Sema-2b) acts through its transmembrane receptor Plexin B (PlexB) to locally attract neural processes to specific FB laminae. Aberrant Sema-2b/PlexB signaling leads to select disruptions in neural lamination, and these disruptions result in the formation of ectopic inhibitory connections between subsets of FB neurons. These structural alternations and connectivity defects are associated with changes in fly sleep and arousal, emphasizing the importance of lamination-mediated neural wiring in a central brain region critical for normal sleep behavior., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Only a few studies have examined the central visual system of Thelyphonida (whip scorpions) until now. To obtain new insights suitable for phylogenetic analysis we studied the axonal trajectories and neuropil architecture of the central visual systems of two whip scorpion species (Mastigoproctus giganteus and Typopeltis dalyi) with different neuroanatomical techniques (Cobalt fills, Wigglesworth stains, and μCT). The central visual system of whip scorpion comprises one pair of median eyes and one pair of lateral eye triplets. The R-cells (or retinula cells) of both eye types each terminate in a first and a second visual neuropil. Furthermore, a few R-cell fibres from the median eyes leave the second median eye visual neuropil and terminate in the second and the first lateral eye neuropil. This means R-cell terminals from the lateral eyes and the median eyes overlap here. Additionally, the arcuate body and mushroom bodies are described. A detailed comparison of our findings with previously studied chelicerate central visual systems seems to support a monophyly of Arachnopulmonata, i.e. a clade comprising Tetrapulmonata (Thelyphonida, Schizomida, Amblypygi, and Araneae) and Scorpions. Furthermore, the architecture of the central visual systems hints at a close evolutionary relationship of Arachnopulmonata and Xiphosura., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Nasonia, a genus of parasitoid wasps, is a promising model system in the study of developmental and evolutionary genetics, as well as complex traits such as learning. Of these "jewel wasps", the species Nasonia vitripennis is widely spread and widely studied. To accelerate neuroscientific research in this model species, fundamental knowledge of its nervous system is needed. To this end, we present an average standard brain of recently eclosed naïve female N. vitripennis wasps obtained by the iterative shape averaging method. This "Jewel Wasp Standard Brain" includes the optic lobe (excluding the lamina), the anterior optic tubercle, the antennal lobe, the lateral horn, the mushroom body, the central complex, and the remaining unclassified neuropils in the central brain. Furthermore, we briefly describe these well-defined neuropils and their subregions in the N. vitripennis brain. A volumetric analysis of these neuropils is discussed in the context of brains of other insect species. The Jewel Wasp Standard Brain will provide a framework to integrate and consolidate the results of future neurobiological studies in N. vitripennis. In addition, the volumetric analysis provides a baseline for future work on age- and experience-dependent brain plasticity., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Miniaturized microscopes are lightweight imaging devices that allow optical recordings from neurons in freely moving animals over the course of weeks. Despite their ubiquitous use, individual neuronal responses measured with these microscopes have not been directly compared to those obtained with established in vivo imaging techniques such as bench-top two-photon microscopes. To achieve this, we performed calcium imaging in mouse primary visual cortex while presenting animals with drifting gratings. We identified the same neurons in image stacks acquired with both microscopy methods and quantified orientation tuning of individual neurons. The response amplitude and signal-to-noise ratio of calcium transients recorded upon visual stimulation were highly correlated between both microscopy methods, although influenced by neuropil contamination in miniaturized microscopy. Tuning properties, calculated for individual orientation tuned neurons, were strongly correlated between imaging techniques. Thus, neuronal tuning features measured with a miniaturized microscope are quantitatively similar to those obtained with a two-photon microscope., Competing Interests: The authors have declared that no competing interests exist.

The pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in anterior pituitary hormone secretion, neurotransmission, and the control of breathing. Mice lacking PACAP die suddenly mainly in the 2 nd postnatal week, coinciding temporally with a critical period of respiratory development uncovered by our laboratory in the rat. The goal of the current study was to test our hypothesis that PACAP expression is reduced during the critical period in normal rats. We undertook immunohistochemistry and optical densitometry of PACAP (specifically PACAP38) in several brain stem respiratory-related nuclei of postnatal days P2-21 rats, and found that PACAP immunoreactivity was significantly reduced at P12 in the pre-Bötzinger complex, nucleus ambiguus, hypoglossal nucleus, and the ventrolateral subnucleus of the nucleus tractus solitarius. No changes were observed in the control, non-respiratory cuneate nucleus at P12. Results imply that the down-regulation of PACAP during normal postnatal development may contribute to the critical period of vulnerability, when the animals' response to hypoxia is at its weakest., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Relatively little neuroscience research has been focused on artiodactyls. Recent observations of complex social interactions in domestic and wild species suggest that analyses of artiodactyl brain anatomy would be of comparative value. In this study, we examined how the distribution of cortical neuropil space (a proxy for connectivity) varies across representative members of this diverse clade. Using image analysis techniques, we quantified the neuropil space in the anterior cingulate cortex (ACC) and the occipital (putative primary visual) cortex (OC) of 12 artiodactyl species from adult specimens. Additionally, we conducted a preliminary investigation of variation in ACC neuropil space in a developmental series of five white-tailed deer (Odocoileus virginianus). Results indicate a consistent pattern of greater neuropil space in the ACC in comparison to the OC among all species, and a gradual increase in ACC neuropil space toward maturity in the white-tailed deer. Given the taxa that have the greatest cortical neuropil space, we hypothesize that such enhanced connectivity might be needed to support behaviors such as group foraging and attentiveness to conspecifics. These results help advance a broader understanding of diversity in neural circuitry in artiodactyls and point to the need for more in-depth comparisons of cortical neuron morphology and organization in this relatively understudied taxonomic group. Anat Rec, 301:1871-1881, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

The central complex, a set of neuropils in the center of the insect brain, plays a crucial role in spatial aspects of sensory integration and motor control. Stereotyped neurons interconnect these neuropils with one another and with accessory structures. We screened over 5,000 Drosophila melanogaster GAL4 lines for expression in two neuropils, the noduli (NO) of the central complex and the asymmetrical body (AB), and used multicolor stochastic labeling to analyze the morphology, polarity, and organization of individual cells in a subset of the GAL4 lines that showed expression in these neuropils. We identified nine NO and three AB cell types and describe them here. The morphology of the NO neurons suggests that they receive input primarily in the lateral accessory lobe and send output to each of the six paired noduli. We demonstrate that the AB is a bilateral structure which exhibits asymmetry in size between the left and right bodies. We show that the AB neurons directly connect the AB to the central complex and accessory neuropils, that they target both the left and right ABs, and that one cell type preferentially innervates the right AB. We propose that the AB be considered a central complex neuropil in Drosophila. Finally, we present highly restricted GAL4 lines for most identified protocerebral bridge, NO, and AB cell types. These lines, generated using the split-GAL4 method, will facilitate anatomical studies, behavioral assays, and physiological experiments., (© 2018 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals, Inc.)

Precise control of neurite guidance during development is essential to ensure proper formation of neuronal networks and correct function of the central nervous system (CNS). How neuronal projections find their targets to generate appropriate synapses is not entirely understood. Although transcription factors are key molecules during neurogenesis, we do not know their entire function during the formation of networks in the CNS. Here, we used the Drosophila melanogaster optic lobe as a model for understanding neurite guidance during development. We assessed the function of Sox102F/SoxD, the unique Drosophila orthologue of the vertebrate SoxD family of transcription factors. SoxD is expressed in immature and mature neurons in the larval and adult lobula plate ganglia (one of the optic lobe neuropils), but is absent from glial cells, neural stem cells and progenitors of the lobula plate. SoxD RNAi knockdown in all neurons results in a reduction of the lobula plate neuropil, without affecting neuronal fate. This morphological defect is associated with an impaired optomotor response of adult flies. Moreover, knocking down SoxD only in T4/T5 neuronal types, which control motion vision, affects proper neurite guidance into the medulla and lobula. Our findings suggest that SoxD regulates neurite guidance, without affecting neuronal fate.

Ultrastructure of the neuropil of the brain oculomotor nuclei was studied in mice after 30-day exposure to microgravity on Bion-M1 biosatellite and after 13-h exposure to Earth gravity. The number of axo-dendritic synapses in the neuropil of the oculomotor nucleus significantly decreased after the flight. Degenerated axon terminals containing conglomerates of presynaptic vesicles appeared. The number of synapses with high functional activity increased and the length of active zones of the axo-dendritic synapses significantly increased. The observed ultrastructural changes of the neuropil of the oculomotor nuclei of mice exposed to microgravity reflect the development of long-term deafferentation of the analyzed brain structures. These changes in the neuropil ultrastructure can determine the disturbances in the oculomotor system, e.g. development of atypical nystagmus under conditions of microgravity.

In most animals, the brain controls the body via a set of descending neurons (DNs) that traverse the neck. DN activity activates, maintains or modulates locomotion and other behaviors. Individual DNs have been well-studied in species from insects to primates, but little is known about overall connectivity patterns across the DN population. We systematically investigated DN anatomy in Drosophila melanogaster and created over 100 transgenic lines targeting individual cell types. We identified roughly half of all Drosophila DNs and comprehensively map connectivity between sensory and motor neuropils in the brain and nerve cord, respectively. We find the nerve cord is a layered system of neuropils reflecting the fly's capability for two largely independent means of locomotion -- walking and flight -- using distinct sets of appendages. Our results reveal the basic functional map of descending pathways in flies and provide tools for systematic interrogation of neural circuits., Competing Interests: SN, MD, AW, WK, GC No competing interests declared, (© 2018, Namiki et al.)

Despite divergent evolutionary origins, the organization of olfactory systems is remarkably similar across phyla. In both insects and mammals, sensory input from receptor cells is initially processed in synaptically dense regions of neuropil called glomeruli, where neural activity is shaped by local inhibition and centrifugal neuromodulation prior to being sent to higher-order brain areas by projection neurons. Here we review both similarities and several key differences in the neuroanatomy of the olfactory system in honey bees, mice, and humans, using a combination of literature review and new primary data. We have focused on the chemical identity and the innervation patterns of neuromodulatory inputs in the primary olfactory system. Our findings show that serotonergic fibers are similarly distributed across glomeruli in all three species. Octopaminergic/tyraminergic fibers in the honey bee also have a similar distribution, and possibly a similar function, to noradrenergic fibers in the mammalian OBs. However, preliminary evidence suggests that human OB may be relatively less organized than its counterparts in honey bee and mouse.

The Caenorhabditis elegans cell lineage is nearly invariant. Whether this stereotyped cell-division pattern promotes reproducibility in cell shapes/positions is not generally known, as manual spatiotemporal cell-shape/position alignments are labor-intensive, and fully-automated methods are not described. Here, we report automated algorithms for spatiotemporal alignments of C. elegans embryos from pre-morphogenesis to motor-activity initiation. We use sparsely-labeled green-fluorescent nuclei and a pan-nuclear red-fluorescent reporter to register consecutive imaging time points and compare embryos. Using our method, we monitor early assembly of the nerve-ring (NR) brain neuropil. While NR pioneer neurons exhibit reproducible growth kinetics and axon positions, cell-body placements are variable. Thus, pioneer-neuron axon locations, but not cell-body positions, are under selection. We also show that anterior NR displacement in cam-1/ROR Wnt-receptor mutants is not an early NR assembly defect. Our results demonstrate the utility of automated spatiotemporal alignments of C. elegans embryos, and uncover key principles guiding nervous-system development in this animal.

Knowing the proportions of asymmetric (excitatory) and symmetric (inhibitory) synapses in the neuropil is critical for understanding the design of cortical circuits. We used focused ion beam milling and scanning electron microscopy (FIB/SEM) to obtain stacks of serial sections from the six layers of the juvenile rat (postnatal day 14) somatosensory cortex (hindlimb representation). We segmented in three-dimensions 6184 synaptic junctions and determined whether they were established on dendritic spines or dendritic shafts. Of all these synapses, 87-94% were asymmetric and 6-13% were symmetric. Asymmetric synapses were preferentially located on dendritic spines in all layers (80-91%) while symmetric synapses were mainly located on dendritic shafts (62-86%). Furthermore, we found that less than 6% of the dendritic spines establish more than one synapse. The vast majority of axospinous synapses were established on the spine head. Synapses on the spine neck were scarce, although they were more common when the dendritic spine established multiple synapses. This study provides a new large quantitative dataset that may contribute not only to the knowledge of the ultrastructure of the cortex, but also towards defining the connectivity patterns through all cortical layers.

The subesophageal zone (SEZ) of the Drosophila brain processes mechanosensory and gustatory sensory input from sensilla located on the head, mouth cavity and trunk. Motor output from the SEZ directly controls the movements involved in feeding behavior. In an accompanying paper (Hartenstein et al., ), we analyzed the systems of fiber tracts and secondary lineages to establish reliable criteria for defining boundaries between the four neuromeres of the SEZ, as well as discrete longitudinal neuropil domains within each SEZ neuromere. Here we use this anatomical framework to systematically map the sensory projections entering the SEZ throughout development. Our findings show continuity between larval and adult sensory neuropils. Gustatory axons from internal and external taste sensilla of the larva and adult form two closely related sensory projections, (a) the anterior central sensory center located deep in the ventromedial neuropil of the tritocerebrum and mandibular neuromere, and (b) the anterior ventral sensory center (AVSC), occupying a superficial layer within the ventromedial tritocerebrum. Additional, presumed mechanosensory terminal axons entering via the labial nerve define the ventromedial sensory center (VMSC) in the maxilla and labium. Mechanosensory afferents of the massive array of chordotonal organs (Johnston's organ) of the adult antenna project into the centrolateral neuropil column of the anterior SEZ, creating the antenno-mechanosensory and motor center (AMMC). Dendritic projections of dye back-filled motor neurons extend throughout a ventral layer of the SEZ, overlapping widely with the AVSC and VMSC. Our findings elucidate fundamental structural aspects of the developing sensory systems in Drosophila., (© 2017 Wiley Periodicals, Inc.)

The Drosophila visual system has become a premier model for probing how neural diversity is generated during development. Recent work has provided deeper insight into the elaborate mechanisms that control the range of types and numbers of neurons produced, which neurons survive, and how they interact. These processes drive visual function and influence behavioral preferences. Other studies are beginning to provide insight into how neuronal diversity evolved in insects by adding new cell types and modifying neural circuits. Some of the most powerful comparisons have been those made to the Drosophila visual system, where a deeper understanding of molecular mechanisms allows for the generation of hypotheses about the evolution of neural anatomy and function. The evolution of new neural types contributes additional complexity to the brain and poses intriguing questions about how new neurons interact with existing circuitry. We explore how such individual changes in a variety of species might play a role over evolutionary timescales. Lessons learned from the fly visual system apply to other neural systems, including the fly central brain, where decisions are made and memories are stored.

Crustaceans and insects share many similarities of brain organization suggesting that their common ancestor possessed some components of those shared features. Stomatopods (mantis shrimps) are basal eumalacostracan crustaceans famous for their elaborate visual system, the most complex of which possesses 12 types of color photoreceptors and the ability to detect both linearly and circularly polarized light. Here, using a palette of histological methods we describe neurons and their neuropils most immediately associated with the stomatopod retina. We first provide a general overview of the major neuropil structures in the eyestalks lateral protocerebrum, with respect to the optical pathways originating from the six rows of specialized ommatidia in the stomatopod's eye, termed the midband. We then focus on the structure and neuronal types of the lamina, the first optic neuropil in the stomatopod visual system. Using Golgi impregnations to resolve single neurons we identify cells in different parts of the lamina corresponding to the three different regions of the stomatopod eye (midband and the upper and lower eye halves). While the optic cartridges relating to the spectral and polarization sensitive midband ommatidia show some specializations not found in the lamina serving the upper and lower eye halves, the general morphology of the midband lamina reflects cell types elsewhere in the lamina and cell types described for other species of Eumalacostraca., (© 2017 Wiley Periodicals, Inc.)

Visual information processing in animals with large image forming eyes is carried out in highly structured retinotopically ordered neuropils. Visual neuropils in Drosophila form the optic lobe, which consists of four serially arranged major subdivisions; the lamina, medulla, lobula and lobula plate; the latter three of these are further subdivided into multiple layers. The visual neuropils are formed by more than 100 different cell types, distributed and interconnected in an invariant highly regular pattern. This pattern relies on a protracted sequence of developmental steps, whereby different cell types are born at specific time points and nerve connections are formed in a tightly controlled sequence that has to be coordinated among the different visual neuropils. The developing fly visual system has become a highly regarded and widely studied paradigm to investigate the genetic mechanisms that control the formation of neural circuits. However, these studies are often made difficult by the complex and shifting patterns in which different types of neurons and their connections are distributed throughout development. In the present paper we have reconstructed the three-dimensional architecture of the Drosophila optic lobe from the early larva to the adult. Based on specific markers, we were able to distinguish the populations of progenitors of the four optic neuropils and map the neurons and their connections. Our paper presents sets of annotated confocal z-projections and animated 3D digital models of these structures for representative stages. The data reveal the temporally coordinated growth of the optic neuropils, and clarify how the position and orientation of the neuropils and interconnecting tracts (inner and outer optic chiasm) changes over time. Finally, we have analyzed the emergence of the discrete layers of the medulla and lobula complex using the same markers (DN-cadherin, Brp) employed to systematically explore the structure and development of the central brain neuropil. Our work will facilitate experimental studies of the molecular mechanisms regulating neuronal fate and connectivity in the fly visual system, which bears many fundamental similarities with the retina of vertebrates., (Copyright © 2017. Published by Elsevier Inc.)

Cortical neuropil modulations recorded by calcium imaging reflect the activity of large aggregates of axo-dendritic processes and synaptic compartments from a large number of neurons. The organization of this activity impacts neuronal firing but is not well understood. Here we used in vivo 2-photon imaging with Oregon Green Bapta (OGB) and GCaMP6s to study neuropil visual responses to moving gratings in layer 2/3 of mouse area V1. We found neuropil responses to be strongly modulated and more reliable than neighboring somatic activity. Furthermore, stimulus independent modulations in neuropil activity, i.e., noise correlations, were highly coherent across the cortical surface, up to distances of at least 200 μm. Pairwise neuropil-to-neuropil-patch noise correlation strength was much higher than cell-to-cell noise correlation strength and depended strongly on brain state, decreasing in quiet wakefulness relative to light anesthesia. The profile of neuropil noise correlation strength decreased gently with distance, dropping by ~11% at a distance of 200 μm. This was comparatively slower than the profile of cell-to-cell noise correlations, which dropped by ~23% at 200 μm. Interestingly, in spite of the "salt & pepper" organization of orientation and direction encoding across mouse V1 neurons, populations of neuropil patches, even of moderately large size (radius ~100 μm), showed high accuracy for discriminating perpendicularly moving gratings. This was commensurate to the accuracy of corresponding cell populations. The dynamic, stimulus dependent, nature of neuropil activity further underscores the need to carefully separate neuropil from cell soma activity in contemporary imaging studies.

The praying mantis is an insect which relies on vision for capturing prey, avoiding being eaten and for spatial orientation. It is well known for its ability to use stereopsis for estimating the distance of objects. The neuronal substrate mediating visually driven behaviors, however, is not very well investigated. To provide a basis for future functional studies, we analyzed the anatomical organization of visual neuropils in the brain of the praying mantis Hierodula membranacea and provide supporting evidence from a second species, Rhombodera basalis, with particular focus on the lobula complex (LOX). Neuropils were three-dimensionally reconstructed from synapsin-immunostained whole mount brains. The neuropil organization and the pattern of γ-aminobutyric acid immunostaining of the medulla and LOX were compared between the praying mantis and two related polyneopteran species, the Madeira cockroach and the desert locust. The investigated visual neuropils of the praying mantis are highly structured. Unlike in most insects the LOX of the praying mantis consists of five nested neuropils with at least one neuropil not present in the cockroach or locust. Overall, the mantis LOX is more similar to the LOX of the locust than the more closely related cockroach suggesting that the sensory ecology plays a stronger role than the phylogenetic distance of the three species in structuring this center of visual information processing., (© 2017 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.)

The brain anatomy of Niphargus puteanus and Orchestia cavimana, two amphipod species with different lifestyles, has been studied using a variety of recent techniques. The general aspects of the brain anatomy of both species correspond to those of other malacostracans. However, both species lack hemiellipsoid bodies. Furthermore, related to their lifestyle certain differences have been observed. The aquatic subterranean species N. puteanus lacks eye structures, the optic nerve, and the two outer optic neuropils lamina and medulla. Only partial remains of the lobula have been detected. In contrast to this, the central complex in the protocerebrum and the olfactory glomeruli in the deutocerebrum are well differentiated. The terrestrial species Orchestia cavimana shows a reduced first antenna, the absence of olfactory neuropils in the deutocerebrum, and a reduction of the olfactory globular tract. The characteristics in defining the hemiellipsoid bodies are critically discussed. Contradictions about presence or absence of this neuropil are due to different conceptualizations. A comparison with other crustaceans that live in dark environments reveal similar patterns of optic system reduction, but to different degrees following a centripetal pattern. Retaining the olfactory system seems a general problem of terrestrialization in crustaceans with the notable exception of terrestrial hermit crabs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The central complex comprises an elaborate system of modular neuropils which mediate spatial orientation and sensory-motor integration in insects such as the grasshopper and Drosophila. The neuroarchitecture of the largest of these modules, the fan-shaped body, is characterized by its stereotypic set of decussating fiber bundles. These are generated during development by axons from four homologous protocerebral lineages which enter the commissural system and subsequently decussate at stereotypic locations across the brain midline. Since the commissural organization prior to fan-shaped body formation has not been previously analyzed in either species, it was not clear how the decussating bundles relate to individual lineages, or if the projection pattern is conserved across species. In this study, we trace the axonal projections from the homologous central complex lineages into the commissural system of the embryonic and larval brains of both the grasshopper and Drosophila. Projections into the primordial commissures of both species are found to be lineage-specific and allow putatively equivalent fascicles to be identified. Comparison of the projection pattern before and after the commencement of axon decussation in both species reveals that equivalent commissural fascicles are involved in generating the columnar neuroarchitecture of the fan-shaped body. Further, the tract-specific columns in both the grasshopper and Drosophila can be shown to contain axons from identical combinations of central complex lineages, suggesting that this columnar neuroarchitecture is also conserved.

To avoid the fierce competition for food, South African ball-rolling dung beetles carve a piece of dung off a dung-pile, shape it into a ball and roll it away along a straight line path. For this unidirectional exit from the busy dung pile, at night and day, the beetles use a wide repertoire of celestial compass cues. This robust and relatively easily measurable orientation behavior has made ball-rolling dung beetles an attractive model organism for the study of the neuroethology behind insect orientation and sensory ecology. Although there is already some knowledge emerging concerning how celestial cues are processed in the dung beetle brain, little is known about its general neural layout. Mapping the neuropils of the dung beetle brain is thus a prerequisite to understand the neuronal network that underlies celestial compass orientation. Here, we describe and compare the brains of a day-active and a night-active dung beetle species based on immunostainings against synapsin and serotonin. We also provide 3D reconstructions for all brain areas and many of the fiber bundles in the brain of the day-active dung beetle. Comparison of neuropil structures between the two dung beetle species revealed differences that reflect adaptations to different light conditions. Altogether, our results provide a reference framework for future studies on the neuroethology of insects in general and dung beetles in particular., (© 2017 Wiley Periodicals, Inc.)

To generate rhythmic motor behaviors, both single neurons and neural circuits require a balance between excitatory inputs that trigger action potentials and inhibitory inputs that promote a stable resting potential (E/I balance). Previous studies have focused on individual neurons and have shown that, over a short spatial scale, excitatory and inhibitory (E/I) synapses tend to form structured territories with inhibitory inputs enriched on cell bodies and proximal dendrites and excitatory inputs on distal dendrites. However, systems-level E/I patterns, at spatial scales larger than single neurons, are largely uncharted. We used immunostaining for PSD-95 and gephyrin postsynaptic scaffolding proteins as proxies for excitatory and inhibitory synapses, respectively, to quantify the numbers and map the distributions of E/I synapses in zebrafish spinal cord at both an embryonic stage and a larval stage. At the embryonic stage, we found that PSD-95 puncta outnumber gephyrin puncta, with the number of gephyrin puncta increasing to match that of PSD-95 puncta at the larval stage. At both stages, PSD-95 puncta are enriched in the most lateral neuropil corresponding to distal dendrites while gephyrin puncta are enriched on neuronal somata and in the medial neuropil. Significantly, similar to synaptic puncta, neuronal processes also exhibit medial-lateral territories at both developmental stages with enrichment of glutamatergic (excitatory) processes laterally and glycinergic (inhibitory) processes medially. This establishment of neuropil excitatory-inhibitory structure largely precedes dendritic arborization of primary motor neurons, suggesting that the structured neuropil could provide a framework for the development of E/I balance at the cellular level. J. Comp. Neurol. 525:1649-1667, 2017. © 2016 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

The Drosophila central brain consists of stereotyped neural lineages, developmental-structural units of macrocircuitry formed by the sibling neurons of single progenitors called neuroblasts. We demonstrate that the lineage principle guides the connectivity and function of neurons, providing input to the central complex, a collection of neuropil compartments important for visually guided behaviors. One of these compartments is the ellipsoid body (EB), a structure formed largely by the axons of ring (R) neurons, all of which are generated by a single lineage, DALv2. Two further lineages, DALcl1 and DALcl2, produce neurons that connect the anterior optic tubercle, a central brain visual center, with R neurons. Finally, DALcl1/2 receive input from visual projection neurons of the optic lobe medulla, completing a three-legged circuit that we call the anterior visual pathway (AVP). The AVP bears a fundamental resemblance to the sky-compass pathway, a visual navigation circuit described in other insects. Neuroanatomical analysis and two-photon calcium imaging demonstrate that DALcl1 and DALcl2 form two parallel channels, establishing connections with R neurons located in the peripheral and central domains of the EB, respectively. Although neurons of both lineages preferentially respond to bright objects, DALcl1 neurons have small ipsilateral, retinotopically ordered receptive fields, whereas DALcl2 neurons share a large excitatory receptive field in the contralateral hemifield. DALcl2 neurons become inhibited when the object enters the ipsilateral hemifield and display an additional excitation after the object leaves the field of view. Thus, the spatial position of a bright feature, such as a celestial body, may be encoded within this pathway., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Jumping spiders are known for their extraordinary cognitive abilities. The underlying nervous system structures, however, are largely unknown. Here, we explore and describe the anatomy of the brain in the jumping spider Marpissa muscosa (Clerck, 1757) by means of paraffin histology, X-ray microCT analysis and immunohistochemistry as well as three-dimensional reconstruction. In the prosoma, the CNS is a clearly demarcated mass that surrounds the esophagus. The anteriormost neuromere, the protocerebrum, comprises nine bilaterally paired neuropils, including the mushroom bodies and one unpaired midline neuropil, the arcuate body. Further ventrally, the synganglion comprises the cheliceral (deutocerebrum) and pedipalpal neuropils (tritocerebrum). Synapsin-immunoreactivity in all neuropils is generally strong, while allatostatin-immunoreactivity is mostly present in association with the arcuate body and the stomodeal bridge. The most prominent neuropils in the spider brain, the mushroom bodies and the arcuate body, were suggested to be higher integrating centers of the arthropod brain. The mushroom body in M. muscosa is connected to first and second order visual neuropils of the lateral eyes, and the arcuate body to the second order neuropils of the anterior median eyes (primary eyes) through a visual tract. The connection of both, visual neuropils and eyes and arcuate body, as well as their large size corroborates the hypothesis that these neuropils play an important role in cognition and locomotion control of jumping spiders. In addition, we show that the architecture of the brain of M. muscosa and some previously investigated salticids differs significantly from that of the wandering spider Cupiennius salei, especially with regard to structure and arrangement of visual neuropils and mushroom body. Thus, we need to explore the anatomical conformities and specificities of the brains of different spider taxa in order to understand evolutionary transformations of the arthropod brain., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In the Drosophila optic lobes, 800 retinotopically organized columns in the medulla act as functional units for processing visual information. The medulla contains over 80 types of neuron, which belong to two classes: uni-columnar neurons have a stoichiometry of one per column, while multi-columnar neurons contact multiple columns. Here we show that combinatorial inputs from temporal and spatial axes generate this neuronal diversity: all neuroblasts switch fates over time to produce different neurons; the neuroepithelium that generates neuroblasts is also subdivided into six compartments by the expression of specific factors. Uni-columnar neurons are produced in all spatial compartments independently of spatial input; they innervate the neuropil where they are generated. Multi-columnar neurons are generated in smaller numbers in restricted compartments and require spatial input; the majority of their cell bodies subsequently move to cover the entire medulla. The selective integration of spatial inputs by a fixed temporal neuroblast cascade thus acts as a powerful mechanism for generating neural diversity, regulating stoichiometry and the formation of retinotopy.

In the vertebrate CNS, afferent sensory inputs are targeted to specific depths or layers of their target neuropil. This patterning exists ab initio, from the very beginning, and therefore has been considered an activity-independent process. However, here we report that, during circuit development, the subcellular segregation of the visual and mechanosensory inputs to specific regions of tectal neuron dendrites in the tadpole optic tectum requires NMDA receptor activity. Blocking NMDARs during the formation of these sensory circuits, or removing the visual set of inputs, leads to less defined segregation, and suggests a correlation-based mechanism in which correlated inputs wire to common regions of dendrites. This can account for how two sets of inputs form synapses onto different regions of the same dendrite. Blocking NMDA receptors during later stages of circuit development did not disrupt segregation, indicating a critical period for activity-dependent shaping of patterns of innervation., Competing Interests: The authors declare that no competing interests exist.

The adult insect brain is composed of neuropils present in most taxa. However, the relative size, shape, and developmental timing differ between species. This diversity of adult insect brain morphology has been extensively described while the genetic mechanisms of brain development are studied predominantly in Drosophila melanogaster. However, it has remained enigmatic what cellular and genetic mechanisms underlie the evolution of neuropil diversity or heterochronic development. In this perspective paper, we propose a novel approach to study these questions. We suggest using genome editing to mark homologous neural cells in the fly D. melanogaster, the beetle Tribolium castaneum, and the Mediterranean field cricket Gryllus bimaculatus to investigate developmental differences leading to brain diversification. One interesting aspect is the heterochrony observed in central complex development. Ancestrally, the central complex is formed during embryogenesis (as in Gryllus) but in Drosophila, it arises during late larval and metamorphic stages. In Tribolium, it forms partially during embryogenesis. Finally, we present tools for brain research in Tribolium including 3D reconstruction and immunohistochemistry data of first instar brains and the generation of transgenic brain imaging lines. Further, we characterize reporter lines labeling the mushroom bodies and reflecting the expression of the neuroblast marker gene Tc-asense, respectively.

Astrocytes are crucial in the formation, fine-tuning, function and plasticity of neural circuits in the central nervous system. However, important questions remain about the mechanisms instructing astrocyte cell fate. We have studied astrogenesis in the ventral nerve cord of Drosophila larvae, where astrocytes exhibit remarkable morphological and molecular similarities to those in mammals. We reveal the births of larval astrocytes from a multipotent glial lineage, their allocation to reproducible positions, and their deployment of ramified arbors to cover specific neuropil territories to form a stereotyped astroglial map. Finally, we unraveled a molecular pathway for astrocyte differentiation in which the Ets protein Pointed and the Notch signaling pathway are required for astrogenesis; however, only Notch is sufficient to direct non-astrocytic progenitors toward astrocytic fate. We found that Prospero is a key effector of Notch in this process. Our data identify an instructive astrogenic program that acts as a binary switch to distinguish astrocytes from other glial cells., (© 2016. Published by The Company of Biologists Ltd.)

The twin nerve tracts of the antenna of the grasshopper Schistocerca gregaria are established early in embryogenesis by sibling pairs of pioneers which delaminate from the epithelium into the lumen at the antennal tip. These cells can be uniquely identified via their co-expression of the neuronal labels horseradish peroxidase and the lipocalin Lazarillo. The apical pioneers direct axons toward the antennal base where they encounter guidepost-like cells called base pioneers which transiently express the same molecular labels as the apical pioneers. To what extent the pioneer growth cones then progress into the brain neuropil proper, and what their targets there might be, has remained unclear. In this study, we show that the apical antennal pioneers project centrally beyond the antennal base first into the deutocerebral, and then into the protocerebral brain neuropils. In the protocerebrum, we identify their target circuitry as being identified Lazarillo-positive cells which themselves pioneer the primary axon scaffold of the brain. The apical and base antennal pioneers therefore form part of a molecularly contiguous pathway from the periphery to an identified central circuit of the embryonic grasshopper brain.