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We are now in the era of millimeter-scale electron microscopy (EM) volumes collected at nanometer resolution (Shapson-Coe et al., 2021; Consortium et al., 2021). Dense reconstruction of cellular compartments in these EM volumes has been enabled by recent advances in Machine Learning (ML) (Lee et al., 2017; Wu et al., 2021; Lu et al., 2021; Macrina et al., 2021). Automated segmentation methods can now yield exceptionally accurate reconstructions of cells, but despite this accuracy, laborious post-hoc proofreading is still required to generate large connectomes free of merge and split errors. The elaborate 3-D meshes of neurons produced by these segmentations contain detailed morphological information, from the diameter, shape, and branching patterns of axons and dendrites, down to the fine-scale structure of dendritic spines. However, extracting information about these features can require substantial effort to piece together existing tools into custom workflows. Building on existing open-source software for mesh manipulation, here we present “NEURD”, a software package that decomposes each meshed neuron into a compact and extensively-annotated graph representation. With these feature-rich graphs, we implement workflows for state of the art automated post-hoc proofreading of merge errors, cell classification, spine detection, axon-dendritic proximities, and other features that can enable many downstream analyses of neural morphology and connectivity. NEURD can make these new massive and complex datasets more accessible to neuroscience researchers focused on a variety of scientific questions.

Deriving the detailed synaptic connections of an entire nervous system is the unrealized goal of the nascent field of connectomics. For the fruit fly Drosophila, in particular, we need to dissect the brain, connectives, and ventral nerve cord as a single continuous unit, fix and stain it, and undertake automated segmentation of neuron membranes. To achieve this, we designed a protocol using progressive lowering of temperature dehydration (PLT), a technique routinely used to preserve cellular structure and antigenicity. We combined PLT with low temperature en bloc staining (LTS) and recover fixed neurons as round profiles with darkly stained synapses, suitable for machine segmentation and automatic synapse detection. Here we report three different PLT-LTS methods designed to meet the requirements for FIB-SEM imaging of the Drosophila brain. These requirements include: good preservation of ultrastructural detail, high level of en bloc staining, artifact-free microdissection, and smooth hot-knife cutting to reduce the brain to dimensions suited to FIB-SEM. In addition to PLT-LTS, we designed a jig to microdissect and pre-fix the fly’s delicate brain and central nervous system. Collectively these methods optimize morphological preservation, allow us to image the brain usually at 8 nm per voxel, and simultaneously speed the formerly slow rate of FIB-SEM imaging.

1.AbstractNeuVue is a software platform created for large-scale proofreading of machine segmentation and neural circuit reconstruction in high-resolution electron microscopy connectomics datasets. The NeuVue platform provides a robust web-based interface for proofreaders to collaboratively view, annotate, and edit segmentation and connectivity data. A backend queuing service organizes proofreader tasks into purpose-driven task types and increases proofreader throughput by limiting proofreader actions to simple, atomic operations. A collection of analytical dashboards, data visualization tools, and Application Program Interface (API) capabilities provide stakeholders real-time access to proofreading progress at an individual proofreader level as well as insights on task generation priorities. NeuVue is agnostic to the underlying data being proofread and improves upon the traditional proofreader experience through quality-of-life features that streamline complex editing operations such as splitting and merging objects in dense nanoscale segmentation.NeuVue heavily leverages cloud resources to enable proofreaders to simultaneously access and edit data on the platform. Production-quality features such as load-balancing, auto-scaling, and pre-deployment testing are all integrated into the platform’s cloud architecture. Additionally, NeuVue is powered by well-supported open-source connectomics tools from the community such as Neuroglancer, PyChunkedGraph, and Connectomics Annotation Versioning Engine (CAVE). The modular design of NeuVue facilitates easy integration and adoption of useful community tools to allow proofreaders to take advantage of the latest improvements in data visualization, processing, and analysis.We demonstrate our framework through proofreading of the mouse visual cortex data generated on the IARPA MICrONS Project. This effort has yielded over 40,000 proofreader edits across the 2 petavoxels of “Minnie” neuroimaging data. 44 unique proofreaders of various skill levels have logged a cumulative 3,740 proofreading hours, and we have been able to validate the improved connectivity of thousands of neurons in the volume. With sustained development on the platform, new integrated error detection and error correction capabilities, and continuous improvements to the proofreader model, we believe that the NeuVue framework can enable high-throughput proofreading for large-scale connectomics datasets of the future.

Insect neural systems are a promising source of inspiration for new navigation algorithms, especially on low size, weight, and power platforms. There have been unprecedented recent neuroscience breakthroughs with Drosophila in behavioral and neural imaging experiments as well as the mapping of detailed connectivity of neural structures. General mechanisms for learning orientation in the central complex (CX) of Drosophila have been investigated previously; however, it is unclear how these underlying mechanisms extend to cases where there is translation through an environment (beyond only rotation), which is critical for navigation in robotic systems. Here, we develop a CX neural connectivity-constrained model that performs sensor fusion, as well as unsupervised learning of visual features for path integration; we demonstrate the viability of this circuit for use in robotic systems in simulated and physical environments. Furthermore, we propose a theoretical understanding of how distributed online unsupervised network weight modification can be leveraged for learning in a trajectory through an environment by minimizing orientation estimation error. Overall, our results may enable a new class of CX-derived low power robotic navigation algorithms and lead to testable predictions to inform future neuroscience experiments.

Insect neural systems are a promising source of inspiration for new algorithms for navigation, especially on low size, weight, and power platforms. There have been unprecedented recent neuroscience breakthroughs with Drosophila in behavioral and neural imaging experiments as well as the mapping of detailed connectivity of neural structures. General mechanisms for learning orientation in the central complex (CX) of Drosophila have been investigated previously; however, it is unclear how these underlying mechanisms extend to cases where there is translation through an environment (beyond only rotation), which is critical for navigation in robotic systems. Here, we develop a CX neural connectivity-constrained model that performs sensor fusion, as well as unsupervised learning of visual features for path integration; we demonstrate the viability of this circuit for use in robotic systems in simulated and physical environments. Furthermore, we propose a theoretical understanding of how distributed online unsupervised network weight modification can be leveraged for learning in a trajectory through an environment by minimizing of orientation estimation error. Overall, our results here may enable a new class of CX-derived low power robotic navigation algorithms and lead to testable predictions to inform future neuroscience experiments.SummaryAn insect neural connectivity-constrained model performs sensor fusion and online learning for orientation estimation.

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

The neural circuits responsible for animal behavior remain largely unknown. We summarize new methods and present the circuitry of a large fraction of the brain of the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. We define cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. We provide detailed circuits consisting of neurons and their chemical synapses for most of the central brain. We make the data public and simplify access, reducing the effort needed to answer circuit questions, and provide procedures linking the neurons defined by our analysis with genetic reagents. Biologically, we examine distributions of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence that maximizing packing density is an important criterion in the evolution of the fly’s brain.

The neural circuits responsible for behavior remain largely unknown. Previous efforts have reconstructed the complete circuits of small animals, with hundreds of neurons, and selected circuits for larger animals. Here we (the FlyEM project at Janelia and collaborators at Google) summarize new methods and present the complete circuitry of a large fraction of the brain of a much more complex animal, the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses, and proofread such large data sets; new methods that define cell types based on connectivity in addition to morphology; and new methods to simplify access to a large and evolving data set. From the resulting data we derive a better definition of computational compartments and their connections; an exhaustive atlas of cell examples and types, many of them novel; detailed circuits for most of the central brain; and exploration of the statistics and structure of different brain compartments, and the brain as a whole. We make the data public, with a web site and resources specifically designed to make it easy to explore, for all levels of expertise from the expert to the merely curious. The public availability of these data, and the simplified means to access it, dramatically reduces the effort needed to answer typical circuit questions, such as the identity of upstream and downstream neural partners, the circuitry of brain regions, and to link the neurons defined by our analysis with genetic reagents that can be used to study their functions.Note: In the next few weeks, we will release a series of papers with more involved discussions. One paper will detail the hemibrain reconstruction with more extensive analysis and interpretation made possible by this dense connectome. Another paper will explore the central complex, a brain region involved in navigation, motor control, and sleep. A final paper will present insights from the mushroom body, a center of multimodal associative learning in the fly brain.

Deriving the detailed synaptic connections of the entire nervous system has been a long term but unrealized goal of the nascent field of connectomics. For Drosophila, in particular, three sample preparation problems must be solved before the requisite imaging and analysis can even begin. The first is dissecting the brain, connectives, and ventral nerve cord (roughly comparable to the brain, neck, and spinal cord of vertebrates) as a single contiguous unit. Second is fixing and staining the resulting specimen, too large for previous techniques such as flash freezing, so as to permit the necessary automated segmentation of neuron membranes. Finally the contrast must be sufficient to support synapse detection at imaging speeds that enable the entire connectome to be collected. To address these issues, we report three major novel methods to dissect, fix, dehydrate and stain this tiny but complex nervous system in its entirety; together they enable us to uncover a Focused Ion-Beam Scanning Electron Microscopy (FIB-SEM) connectome of the entire Drosophila brain. They reliably recover fixed neurons as round profiles with darkly stained synapses, suitable for machine segmentation and automatic synapse detection, for which only minimal human intervention is required. Our advanced procedures use: a custom-made jig to microdissect both regions of the central nervous system, dorsal and ventral, with their connectives; fixation and Durcupan embedment, followed by a special hot-knife slicing protocol to reduce the brain to dimensions suited to FIB; contrast enhancement by heavy metals; together with a progressive lowering of temperature protocol for dehydration. Collectively these optimize the brain’s morphological preservation, imaging it at a usual resolution of 8nm per voxel while simultaneously speeding the formerly slow rate of FIB-SEM. With these methods we could recently obtain a FIB-SEM image stack of the Drosophila brain eight times faster than hitherto, at approximately the same rate as, but without the requirement to cut, nor imperfections in, EM serial sections.

Understanding the circuit mechanisms behind motion detection is a long-standing question in visual neuroscience. In Drosophila melanogaster, recently discovered synapse-level connectomes in the optic lobe, particularly in ON-pathway (T4) receptive-field circuits, in concert with physiological studies, suggest a motion model that is increasingly intricate when compared with the ubiquitous Hassenstein-Reichardt model. By contrast, our knowledge of OFF-pathway (T5) has been incomplete. Here, we present a conclusive and comprehensive connectome that, for the first time, integrates detailed connectivity information for inputs to both the T4 and T5 pathways in a single EM dataset covering the entire optic lobe. With novel reconstruction methods using automated synapse prediction suited to such a large connectome, we successfully corroborate previous findings in the T4 pathway and comprehensively identify inputs and receptive fields for T5. Although the two pathways are probably evolutionarily linked and exhibit many similarities, we uncover interesting differences and interactions that may underlie their distinct functional properties.

Programs that focus on student outreach are often disjoint from sponsored research efforts, despite the mutually beneficial opportunities that are possible with a combined approach. We designed and piloted a program to simultaneously meet the needs of underserved students and a large-scale sponsored research goal. Our program trained undergraduates to produce neuron maps for a major connectomics effort (i.e., single synapse brain maps), while providing these students with the resources and mentors to conduct novel research. Students were recruited from Johns Hopkins University to participate in a ten-week summer program. These students were trained in computational research, scientific communication skills and methods to map electron microscopy volumes. The students also had regular exposure to mentors and opportunities for guided, small group, independent discovery. A Learning-for-Use model was leveraged to provide the students with the tools, skills, and knowledge to pursue their research questions, while an Affinity Research Group model was adapted to provide students with mentorship in conducting cutting-edge research. A focus was placed on recruiting students who had limited opportunities and access to similar experiences. Program metrics demonstrated a substantial increase in knowledge (e.g., neuroscience, graph theory, machine learning, and scientific communication), while students also showed an overall increase in awareness and responsiveness to computational research after the program. Ultimately, the program positively impacted students' career choices and research readiness, and successfully achieved sponsor goals in a compact timeframe. This framework for combining outreach with sponsored research can be broadly leveraged for other programs across domains.

Understanding memory formation, storage and retrieval requires knowledge of the underlying neuronal circuits. In Drosophila, the mushroom body (MB) is the major site of associative learning. We reconstructed the morphologies and synaptic connections of all 983 neurons within the three functional units, or compartments, that compose the adult MB’s α lobe, using a dataset of isotropic 8 nm voxels collected by focused ion-beam milling scanning electron microscopy. We found that Kenyon cells (KCs), whose sparse activity encodes sensory information, each make multiple en passant synapses to MB output neurons (MBONs) in each compartment. Some MBONs have inputs from all KCs, while others differentially sample sensory modalities. Only 6% of KC>MBON synapses receive a direct synapse from a dopaminergic neuron (DAN). We identified two unanticipated classes of synapses, KC>DAN and DAN>MBON. DAN activation produces a slow depolarization of the MBON in these DAN>MBON synapses and can weaken memory recall. DOI: http://dx.doi.org/10.7554/eLife.26975.001

Animal behaviour arises from computations in neuronal circuits, but our understanding of these computations has been frustrated by the lack of detailed synaptic connection maps, or connectomes. For example, despite intensive investigations over half a century, the neuronal implementation of local motion detection in the insect visual system remains elusive. Here we develop a semi-automated pipeline using electron microscopy to reconstruct a connectome, containing 379 neurons and 8,637 chemical synaptic contacts, within the Drosophila optic medulla. By matching reconstructed neurons to examples from light microscopy, we assigned neurons to cell types and assembled a connectome of the repeating module of the medulla. Within this module, we identified cell types constituting a motion detection circuit, and showed that the connections onto individual motion-sensitive neurons in this circuit were consistent with their direction selectivity. Our results identify cellular targets for future functional investigations, and demonstrate that connectomes can provide key insights into neuronal computations. Reconstruction of a connectome within the fruitfly visual medulla, containing more than 300 neurons and over 8,000 chemical synapses, reveals a candidate motion detection circuit; such a circuit operates by combining displaced visual inputs, an operation consistent with correlation based motion detection. Three papers in this issue of Nature use the retina as a model for mapping neuronal circuits from the level of individual synaptic contacts to the long-range scale of dendritic interactions. Helmstaedter et al. used electron microscopy to map a mammalian retinal circuit of close to a thousand neurons. The work reveals a new type of retinal bipolar neuron and suggests functional mechanisms for known visual computations. The other two groups study the detection of visual motion in the Drosophila visual system — a classic neural computation model. Takemura et al. used semi-automated electron microscopy to reconstruct the basic connectome (8,637 chemical synapses among 379 neurons) of Drosophila's optic medulla. Their results reveal a candidate motion detection circuit with a wiring plan consistent with direction selectivity. Maisak et al. used calcium imaging to show that T4 and T5 neurons are divided into specific subpopulations responding to motion in four cardinal directions, and are specific to 'ON' versus 'OFF' edges, respectively.

SUMMARY The role of sound in Drosophila melanogaster courtship, along with its perception via the antennae, is well established, as is the ability of this fly to learn in classical conditioning protocols. Here, we demonstrate that a neutral acoustic stimulus paired with a sucrose reward can be used to condition the proboscis-extension reflex, part of normal feeding behavior. This appetitive conditioning produces results comparable to those obtained with chemical stimuli in aversive conditioning protocols. We applied a logistic model with general estimating equations to predict the dynamics of learning, which successfully predicts the outcome of training and provides a quantitative estimate of the rate of learning. Use of acoustic stimuli with appetitive conditioning provides both an alternative to models most commonly used in studies of learning and memory in Drosophila and a means of testing hearing in both sexes, independently of courtship responsiveness.

Despite the growing research investigating the sex-specific organization of courtship behavior in Drosophila melanogaster, much remains to be understood about the sex-specific organization of the motor circuit that drives this behavior. To investigate the sex-specification of a tightly patterned component of courtship behavior, courtship song, the authors used the GAL4/UAS targeted gene expression system to feminize the ventral ganglia in male Drosophila and analyzed the acoustic properties of courtship song. More specifically, the authors used the thoracic-specifying teashirt promoter (tsh(GAL4)) to express feminizing transgenes specifically in the ventral ganglia. When tsh(GAL4) drove expression of transformer (tra), males were unable to produce prolonged wing extensions. Transgenic expression of an RNAi construct directed against male-specific fruitless (fru(M)) transcripts resulted in normal wing extension, but highly defective courtship song, with 58% of males failing to generate detectable courtship song. Of those that did sing, widths of individual pulses were significantly broader than controls, suggesting thoracic fru(M) function serves to mediate proprioceptive-dependent wing vibration damping during pulse song. However, the most critical signal in the song, the interpulse interval, remained intact. The inability to phenocopy this effect by reducing fru(M) expression in motor neurons and proprioceptive neurons suggests thoracic interneurons require fru(M) for proper pulse song execution and patterning of pulse structure, but not for pulse timing. This provides evidence that genes establishing sex-specific activation of complex behaviors may also be used in establishing pattern-generating motor networks underlying these sex-specific behaviors.

We reconstructed the synaptic circuits of seven columns in the second neuropil or medulla behind the fly's compound eye. These neurons embody some of the most stereotyped circuits in one of the most miniaturized of animal brains. The reconstructions allow us, for the first time to our knowledge, to study variations between circuits in the medulla's neighboring columns. This variation in the number of synapses and the types of their synaptic partners has previously been little addressed because methods that visualize multiple circuits have not resolved detailed connections, and existing connectomic studies, which can see such connections, have not so far examined multiple reconstructions of the same circuit. Here, we address the omission by comparing the circuits common to all seven columns to assess variation in their connection strengths and the resultant rates of several different and distinct types of connection error. Error rates reveal that, overall

Synaptic circuits for identified behaviors in the Drosophila brain have typically been considered from either a developmental or functional perspective without reference to how the circuits might have been inherited from ancestral forms. For example, two candidate pathways for ON- and OFF-edge motion detection in the visual system act via circuits that use respectively either T4 or T5, two cell types of the fourth neuropil, or lobula plate, that exhibit narrow-field direction-selective responses and provide input to wide-field tangential neurons. T4 or T5 both have four subtypes that terminate one each in the four strata of the lobula plate. Representatives are reported in a wide range of Diptera, and both cell types exhibit various similarities in: 1) the morphology of their dendritic arbors; 2) their four morphological and functional subtypes; 3) their cholinergic profile in Drosophila; 4) their input from the pathways of L3 cells in the first neuropil, or lamina, and by one of a pair of lamina cells, L1 (to the T4 pathway) and L2 (to the T5 pathway); and 5) their innervation by a single, wide-field contralateral tangential neuron from the central brain. Progenitors of both also express the gene atonal early in their proliferation from the inner anlage of the developing optic lobe, being alone among many other cell type progeny to do so. Yet T4 receives input in the second neuropil, or medulla, and T5 in the third neuropil or lobula. Here we suggest that these two cell types were originally one, that their ancestral cell population duplicated and split to innervate separate medulla and lobula neuropils, and that a fiber crossing – the internal chiasma – arose between the two neuropils. The split most plausibly occurred, we suggest, with the formation of the lobula as a new neuropil that formed when it separated from its ancestral neuropil to leave the medulla, suggesting additionally that medulla input neurons to T4 and T5 may also have had a common origin.

N-ethylmaleimide sensitive fusion protein (NSF) is an ATPase necessary for vesicle trafficking, including exocytosis. Current models hold that NSF is required in a step that readies vesicles for fusion by disassembling postfusion SNARE protein complexes allowing them to participate in further rounds of vesicle cycling. Whereas most organisms have only one NSF isoform, Drosophila has two. dNSF1 is the predominant functional isoform in the adult nervous system. Conditional mutations in the dNSF1 gene, comatose, are paralytic and lead to disruption of synaptic transmission and the rapid accumulation of SNARE complexes in adult flies. This isoform is not required for synaptic transmission in larvae. In contrast, dNSF2 is important at earlier developmental stages, and its broad expression indicates its importance in neural and non-neural tissues alike. To study dNSF2, and to circumvent the lethality of dNSF2 null mutants, we have constructed transgenic flies carrying a dominant negative form of dNSF2. When this construct was expressed in neurons we observed suppression of synaptic transmission, activity-dependent fatigue of transmitter release, and a reduction in the number of releasable vesicles. However, we unexpectedly found that there was no accumulation of SNARE complexes accompanying these physiological phenotypes. Intriguingly, we also found that expression of mutant dNSF2 induced pronounced overgrowth of the neuromuscular junction and some misrouting of axons. These results support the idea that dNSF2 has multiple roles in cellular function and adds that not all of its functions require disassembly of the SNARE complex. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 261–271, 2002

Mutations in the homeotic gene, Ultrabithorax (Ubx), result in the transformation of the third thoracic (T3) segment into the second thoracic (T2) segment. Although it has been well established that these mutations have striking effects on adult epidermal structures in T3, the effect of these mutations on the adult musculature has been controversial. In this study, a series of Ubx regulatory mutations, anterobithorax, bithorax, postbithorax, and bithoraxoid, as well as combinations of these alleles were used to reevaluate the role of Ubx in the patterning of the T3 musculature. Homeotic indirect and direct flight muscles (IFMs and DFMs) were identified in the transformed T3 segment of all alleles and allelic combinations with the exception of postbithorax. We critically evaluated the pattern and amount of these muscles and found that while the amount and/or quality of homeotic IFMs increased, the amount of homeotic DFMs did not vary significantly as the severity of the ectodermal transformation increased. Because Ubx is not expressed in the adult mesoderm of T3, these results suggest that inductive cues play a major role in the patterning of adult thoracic muscles. We provide a model that illustrates the central role of inductive cues in generating the final adult muscle pattern in the thorax.

The resting membrane potential (RMP) of most cells is not greatly influenced by the transmembrane calcium gradient because at rest, the membrane has very low permeability to calcium. We have observed, however, that the resting membrane potential of muscle cells in the larval bodywall of Drosophila melanogaster varies widely as the external calcium concentration is modified. The RMP depolarized as much as 21.8 mV/mM calcium at low concentrations, and on average, about 10 mV/mM across a range typical of neurophysiological investigations. The extent to which muscle RMP varies has important implications for the measurement of synaptic potentials as well. Two parameters of excitatory junctional potential (EJP) voltage were compared across a range of RMPs. EJP amplitude (DeltaV) and peak voltage (maxima) change as a function of RMP; on average, a 10 mV change in RMP elicits a 4-5 mV change in EJP amplitude and peak voltage. The influence of the calcium gradient on resting and synaptic membrane potentials led us to investigate the endogenous ion concentrations of larval hemolymph. In addition to the major monovalent ions and calcium, we report the first voltammetric analysis of magnesium concentration in larval fruit fly hemolymph.

Background In both vertebrates and invertebrates, the oviduct is an epithelial tube surrounded by visceral muscles that serves as a conduit for gamete transport between the ovary and uterus. While Drosophila is a model system for tubular organ development, few studies have addressed the development of the fly's oviduct. Recent studies in Drosophila have identified mating-responsive genes and proteins whose levels in the oviduct are altered by mating. Since many of these molecules (e.g. Muscle LIM protein 84B, Coracle, Neuroglian) have known roles in the differentiation of muscle and epithelia of other organs, mating may trigger similar differentiation events in the oviduct. This led us to hypothesize that mating mediates the last stages of oviduct differentiation in which organ-specific specializations arise. Results Using electron- and confocal-microscopy we identified tissue-wide post-mating changes in the oviduct including differentiation of cellular junctions, remodeling of extracellular matrix, increased myofibril formation, and increased innervation. Analysis of once- and twice-mated females reveals that some mating-responsive proteins respond only to the first mating, while others respond to both matings. Conclusion We uncovered ultrastructural changes in the mated oviduct that are consistent with the roles that mating-responsive proteins play in muscle and epithelial differentiation elsewhere. This suggests that mating triggers the late differentiation of the oviduct. Furthermore, we suggest that mating-responsive proteins that respond only to the first mating are involved in the final maturation of the oviduct while proteins that remain responsive to later matings are also involved in maintenance and ongoing function of the oviduct. Taken together, our results establish the oviduct as an attractive system to address mechanisms that regulate the late stages of differentiation and maintenance of a tubular organ.

We describe exercises that illustrate the temperature sensitivity of synaptic transmission. The temperature dependence of synaptic transmission is demonstrated by cooling the larval Drosophila melanogaster preparation and recording excitatory junction potentials. Vesicle recycling is explored by utilizing a mutation of the shibire gene. This shibire mutant shows a robust reduction in synaptic vesicle recycling when temperature exceeds a known threshold (∼29° C). Students gain proficiency with the Drosophila larval neuromuscular junction preparation while investigating principles of vesicle release, vesicle recycling, synaptic facilitation and synaptic depression. We show that the viability of the larval preparation is prolonged in vitro with moderate cooling, which is particularly important when introducing the preparation as a novel exercise.

While the larval neuromuscular junction (NMJ) of Drosophila has emerged as a model system to study synaptic function and development, little attention has been given to the study of the adult NMJ. Here we report an immunocytochemical and morphological characterization of an adult NMJ preparation of the prothorax. All muscles examined were innervated by small, uniform type II terminals (0.5-1.5 microm), a subset of which contained octopamine. Terminals classified as type I varied in their morphology across different muscles, ranging from strings or clusters of boutons (0.8-5.5 microm) to an elongate terminal (80-100 microm long) with few branches and contiguous swellings (3-15 microm) along its length. Analysis of the molecular composition of the NMJs during the first 5 days after eclosion revealed four major findings: 1) type I boutons increase in size during early adulthood; 2) Fasciclin II-immunoreactivity is not detectable at type I terminals, while DLG-immunoreactivity is observed at the synapse; 3) a Shaker-GFP fusion protein that localizes to all type I boutons in the larva is differentially localized at adult prothoracic NMJs; and 4) while all type I terminals contain glutamate, the glutamate receptor subunits, DGluRIIA and DGluRIIB, are expressed and clustered in only a subset of muscles. These findings suggest that maturation of the adult NMJ occurs during early adulthood and that muscle-specific properties may play a role in organizing synaptic components in the adult. Furthermore, these results demonstrate that there are major differences in the molecular organization of the adult and larval NMJs.

Vesicle fusion in eukaryotic cells is mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). In neurons, the t-SNARE SNAP-25 is essential for synaptic vesicle fusion but its exact role in this process is unknown. We have isolated a SNAP-25 temperature-sensitive paralytic mutant in Drosophila, SNAP-25(ts). The mutation causes a Gly50 to Glu change in SNAP-25's first amphipathic helix. A similar mutation in the yeast homologue SEC9 also results in temperature sensitivity, implying a conserved role for this domain in secretion. In vitro-generated 70 kDa SNARE complexes containing SNAP-25(ts) are thermally stable but the mutant SNARE multimers (of approximately 120 kDa) rapidly dissociate at 37 degrees C. The SNAP-25(ts) mutant has two effects on neurotransmitter release depending upon temperature. At 22 degrees C, evoked release of neurotransmitter in SNAP-25(ts) larvae is greatly increased, and at 37 degrees C, the release of neurotransmitter is reduced as compared with controls. Our data suggest that at 22 degrees C the mutation causes the SNARE complex to be more fusion competent but, at 37 degrees C the same mutation leads to SNARE multimer instability and fusion incompetence.

Pyramidal neurons within the cerebral cortex are known to make long-range horizontal connections via an extensive axonal collateral system. The synaptic characteristics and specificities of these connections were studied at the ultrastructural level. Two superficial layer pyramidal cells in the primate striate cortex were labeled by intracellular injections with horseradish peroxidase (HRP) and their axon terminals were subsequently examined with the technique of electron microscopic (EM) serial reconstruction. At the light microscopic level both cells showed the characteristic pattern of widespread, clustered axon collaterals. We examined collateral clusters located near the dendritic field (proximal) and approximately 0.5 mm away (distal). The synapses were of the asymmetric/round vesicle variety (type I), and were therefore presumably excitatory. Three-quarters of the postsynaptic targets were the dendritic spines of other pyramidal cells. A few of the axodendritic synapses were with the shafts of pyramidal cells, bringing the proportion of pyramidal cell targets to 80%. The remaining labeled endings were made with the dendritic shafts of smooth stellate cells, which are presumed to be (GABA)ergic inhibitory cells. On the basis of serial reconstruction of a few of these cells and their dendrites, a likely candidate for one target inhibitory cell is the small-medium basket cell. Taken together, this pattern of outputs suggests a mixture of postsynaptic effects mediated by consequence the horizontal connections may well be the substrate for the variety of influences observed between the receptive field center and its surround.

Dividing cells and their progeny in retinae of young goldfish were labeled with [3H]thymidine, and selected cells were reconstructed from serial sections processed for electron microscopic autoradiography. Our goals were to characterize the cells that were identified as rod precursors in previous light microscopic autoradiographical studies and to determine their origin and fate. (In fish the population of rods increases several-fold postembryonically by proliferation of rod precursor cells scattered across the retina.) Over 200 labeled cells taken from 11 retinas were examined, and 20 of these were reconstructed in their entirety. Some retinas were examined at short intervals (1 to 48 hr) after [3H]thymidine injection in order to study mitotically active cells, and others were examined after longer intervals (9 or 14 days) to discover the nature of the progeny of labeled dividing cells. Previous evidence from thymidine studies in larval goldfish suggested that proliferating cells destined to produce rods appear first in the inner nuclear layer and later in the outer nuclear layer, where they continue to divide and generate new rods (P. R. Johns, (1982) J. Neurosci. 2, 179). The present results provide morphological evidence in support of the suggestion that rod precursors migrate from inner to outer nuclear layer and, furthermore, show that the precursors are closely associated with, and perhaps guided by, the radial processes of Muller glial cells. Examination of EM autoradiographs of labeled cells at 9 and 14 days after a pulse label with thymidine confirms that the differentiated progeny of dividing precursor cells are exclusively rods. To our knowledge, rod precursors are the first example of a neuronal germinal cell in the vertebrate central nervous system that under normal conditions produces only one type of neuron.

We describe a technique, modified from Langford and Coggeshall [Anat. Rec., 197 (1980) 297-303; J. Comp. Neurol., 203 (1981) 745-750], for enhancing membrane contrast and defining cellular boundaries, that is useful for reconstructing individual cells from ultrathin sections. The cells of interest in our study were neuronal germinal cells and their differentiated progeny in the retinas of young goldfish. These cells were labeled by pulse injections of [3H]thymidine, and they were subsequently identified in EM autoradiographs by the presence of silver grains overlying their nuclei. In tissue prepared by traditional procedures (fixation in mixed aldehydes, postfixation in osmium tetroxide) it was difficult to follow the processes of these cells through the complex, dense network of cells in the differentiated retina. However, in tissue postfixed with a mixture of osmium tetroxide and potassium ferricyanide, the contrast of the cell membranes was improved and, in favorable preparations, a dense precipitate was formed in the extracellular spaces, serving to outline individual cells. This greatly faciliated the preparation of reconstructions from serial ultrathin sections.

In insects, specialized mesodermal cells serve as templates to organize myoblasts into distinct muscle fibers during embryogenesis. In the grasshopper embryo, large mesodermal cells called muscle pioneers extend between the epidermal attachment points of future muscle fibers and serve as foci for myoblast fusion. In the Drosophila embryo, muscle founder cells serve a similar function, organizing large numbers of myoblasts into larval muscles. During the metamorphosis of Drosophila, nearly all larval muscles degenerate and are replaced by a set of de novo adult muscles. The extent to which specialized mesodermal cells homologous to the founders and pioneers of the insect embryo are involved in the development of adult-specific muscles has yet to be established. In the larval thorax, the majority of imaginal myoblasts are associated with the imaginal discs. We report here the identification of a morphologically distinct class of disc-associated myoblasts, which we call imaginal pioneers, that prefigures the formation of at least three adult-specific muscles, the tergal depressor of the trochanter and dorsoventral muscles I and II. Like the muscle pioneers of the grasshopper, the imaginal pioneers attach to the epidermis at sites where the future muscle insertions will arise and erect a scaffold for developing adult muscles. These findings suggest that a prior segregation of imaginal myoblasts into at least two populations, one of which may act as pioneers or founders, must occur during development.

This study describes regeneration of the neural retina in juvenile goldfish. The retina was destroyed with an intraocular injection of ouabain, a technique introduced by Wolburg and colleagues (Maier and Wolburg, 1979; Kurz-Isler and Wolburg, 1982). We confirmed their observation that the level of damage produced by the toxin was graded, in that neurons in the inner retinal layers were preferentially destroyed, and only in the more severely affected retinas were cells in the outer nuclear layer (i.e., photoreceptor cells) damaged. Evidence of retinal regeneration could be seen beginning about 2 weeks after the injection of ouabain. In contrast to previous studies (Maier and Wolburg, 1979), we found that regeneration took place only in those retinas in which photoreceptors had been destroyed. In cases in which the outer nuclear layer was spared, no regeneration of inner layers occurred, even after 6 months. Thymidine autoradiography was used to document the regeneration of new retinal neurons and to show that rod precursors, like other dividing cells, were not destroyed by the ouabain, but in contrast showed an increased mitotic activity. Regeneration did not proceed uniformly, but was initiated at neurogenic foci scattered across the retina. These foci consisted of clusters of dividing neuroepithelial-like cells. The evidence is consistent with the proposal that these cells were derived from rod precursors. These results imply that rod precursors are capable of a wider range of developmental fates than they normally express.

When larval goldfish hatch, their retinas contain postmitotic, undifferentiated cones but no rods. Cones rapidly differentiate during the first few days of larval life. Meanwhile, rods begin to appear; they are produced by dividing, specialized progenitors - rod precursors - scattered across the retina. Rod precursors continue to divide throughout larval and into adult life, adding new rods to the growing retina. These dividing cells, intercalated among differentiated retinal neurons, are unique and have not been described previously. The purpose of this study was to identify rod precursors in the larval retina, to characterize them ultrastructurally and to reconstruct their morphology.Dividing rod precursors were labeled by 3H-thymidine injections in late larval/early juvenile goldfish (26-51 days posthatch). Survival times ranged from 1 to 48 hours; fish were decapitated, the corneas were punctured with an insect pin and the eyes (or whole heads) were fixed by immersion in a mixture of 3% glutaraldehyde, 3% paraformaldehyde, 0.1% picric acid, 1 mM calcium chloride, 0.1 M phosphate buffer (pH 7.4) with 5% sucrose.