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The small ventrolateral neurons (sLNvs) are key components of the central clock in the Drosophila brain. They signal via the neuropeptide pigment-dispersing factor (PDF) to align the molecular clockwork of different central clock neurons and to modulate downstream circuits. The dorsal terminals of the sLNvs undergo daily morphological changes that affect presynaptic sites organised by the active zone protein Bruchpilot (BRP), a homolog of mammalian ELKS proteins. However, the role of these presynaptic sites for PDF release is ill-defined. Here, we combined expansion microscopy with labelling of active zones by endogenously tagged BRP to examine the spatial correlation between PDF-containing dense-core vesicles and BRP-labelled active zones. We found that the number of BRP-labelled puncta in the sLNv terminals was similar while their density differed between Zeitgeber time (ZT) 2 and 14. The relative distance between BRP- and PDF-labelled puncta was increased in the morning, around the reported time of PDF release. Spontaneous dense-core vesicle release profiles of sLNvs in a publicly available ssTEM dataset (FAFB) consistently lacked spatial correlation to BRP-organised active zones. RNAi-mediated downregulation of brp and other active zone proteins expressed by the sLNvs did not affect PDF-dependent locomotor rhythmicity. In contrast, down-regulation of genes encoding proteins of the canonical vesicle release machinery, the dense-core vesicle-related protein CADPS, as well as PDF impaired locomotor rhythmicity. Taken together, our study suggests that PDF release from the sLNvs is independent of BRP-organised active zones, while BRP may be redistributed to active zones in a time-dependent manner., (© 2024 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Phloem loading and transport are fundamental processes for allocating carbon from source organs to sink tissues. Cotton (Gossypium spp.) has a high sink demand for the cellulosic fibers that grow on the seed coat and for the storage reserves in the developing embryo, along with the demands of new growth in the shoots and roots. Addressing how cotton mobilizes resources from source leaves to sink organs provides insight into processes contributing to fiber and seed yield. Plasmodesmata frequencies between companion cells and flanking parenchyma in minor veins are higher than expected for an apoplastic loader, and cotton's close relatedness to Tilia spp. hints at passive loading. Suc was the only canonical transport sugar in leaves and constituted 87% of 14C-labeled photoassimilate being actively transported. [14C]Suc uptake coupled with autoradiography indicated active [14C]Suc accumulation in minor veins, suggesting Suc loading from the apoplast; esculin, a fluorescent Suc analog, did not accumulate in minor veins. Of the nine sucrose transporter (SUT) genes identified per diploid genome, only GhSUT1-L2 showed appreciable expression in mature leaves, and silencing GhSUT1-L2 yielded phenotypes characteristic of blocked phloem transport. Furthermore, only GhSUT1-L2 cDNA stimulated esculin and [14C]Suc uptake into yeast, and only the GhSUT1-L2 promoter caused uidA (β-glucuronidase) reporter gene expression in minor vein phloem of Arabidopsis thaliana. Collectively, these results argue that apoplastic phloem loading mediated by GhSUT1-L2 is the dominant mode of phloem loading in cotton., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Neurons utilize plasticity of dendritic arbors as part of a larger suite of adaptive plasticity mechanisms. This explicitly manifests with motoneurons in the Drosophila embryo and larva, where dendritic arbors are exclusively postsynaptic and are used as homeostatic devices, compensating for changes in synaptic input through adapting their growth and connectivity. We recently identified reactive oxygen species (ROS) as novel plasticity signals instrumental in this form of dendritic adjustment. ROS correlate with levels of neuronal activity and negatively regulate dendritic arbor size. Here, we investigated NADPH oxidases as potential sources of such activity-regulated ROS and implicate Dual Oxidase (but not Nox), which generates hydrogen peroxide extracellularly. We further show that the aquaporins Bib and Drip, but not Prip, are required for activity-regulated ROS-mediated adjustments of dendritic arbor size in motoneurons. These results suggest a model whereby neuronal activity leads to activation of the NADPH oxidase Dual Oxidase, which generates hydrogen peroxide at the extracellular face; aquaporins might then act as conduits that are necessary for these extracellular ROS to be channeled back into the cell where they negatively regulate dendritic arbor size., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dhawan, Myers, Bailey, Ostrovsky, Evers and Landgraf.)

Root hairs are tubular protrusions of the root epidermis that significantly enlarge the exploitable soil volume in the rhizosphere. Trichoblasts, the cell type responsible for root hair formation, switch from cell elongation to tip growth through polarization of the growth machinery to a predefined root hair initiation domain (RHID) at the plasma membrane. The emergence of this polar domain resembles the establishment of cell polarity in other eukaryotic systems [1-3]. Rho-type GTPases of plants (ROPs) are among the first molecular determinants of the RHID [4, 5], and later play a central role in polar growth [6]. Numerous studies have elucidated mechanisms that position the RHID in the cell [7-9] or regulate ROP activity [10-18]. The molecular players that target ROPs to the RHID and initiate outgrowth, however, have not been identified. We dissected the timing of the growth machinery assembly in polarizing hair cells and found that positioning of molecular players and outgrowth are temporally separate processes that are each controlled by specific ROP guanine nucleotide exchange factors (GEFs). A functional analysis of trichoblast-specific GEFs revealed GEF3 to be required for normal ROP polarization and thus efficient root hair emergence, whereas GEF4 predominantly regulates subsequent tip growth. Ectopic expression of GEF3 induced the formation of spatially confined, ROP-recruiting domains in other cell types, demonstrating the role of GEF3 to serve as a membrane landmark during cell polarization., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

[This corrects the article DOI: 10.1371/journal.pgen.1003452.].

Developing neuronal networks display spontaneous bursts of action potentials that are necessary for circuit organization and tuning. While spontaneous activity has been shown to instruct map formation in sensory circuits, it is unknown whether it plays a role in the organization of motor networks that produce rhythmic output. Using computational modeling, we investigate how recurrent networks of excitatory and inhibitory neuronal populations assemble to produce robust patterns of unidirectional and precisely timed propagating activity during organism locomotion. One example is provided by the motor network inDrosophilalarvae, which generates propagating peristaltic waves of muscle contractions during crawling. We examine two activity-dependent models, which tune weak network connectivity based on spontaneous activity patterns: a Hebbian model, where coincident activity in neighboring populations strengthens connections between them; and a homeostatic model, where connections are homeostatically regulated to maintain a constant level of excitatory activity based on spontaneous input. The homeostatic model successfully tunes network connectivity to generate robust activity patterns with appropriate timing relationships between neighboring populations. These timing relationships can be modulated by the properties of spontaneous activity, suggesting its instructive role for generating functional variability in network output. In contrast, the Hebbian model fails to produce the tight timing relationships between neighboring populations required for unidirectional activity propagation, even when additional assumptions are imposed to constrain synaptic growth. These results argue that homeostatic mechanisms are more likely than Hebbian mechanisms to tune weak connectivity based on spontaneous input in a recurrent network for rhythm generation and robust activity propagation., Significance Statement: How are neural circuits organized and tuned to maintain stable function and produce robust output? This task is especially difficult during development, when circuit properties change in response to variable environments and internal states. Many developing circuits exhibit spontaneous activity, but its role in the synaptic organization of motor networks that produce rhythmic output is unknown. We studied a model motor network, that when appropriately tuned, generates propagating activity as during crawling inDrosophilalarvae. Based on experimental evidence of activity-dependent tuning of connectivity, we examined plausible mechanisms by which appropriate connectivity emerges. Our results suggest that activity-dependent homeostatic mechanisms are better suited than Hebbian mechanisms for organizing motor network connectivity, and highlight an important difference from sensory areas., (Copyright © 2016 Gjorgjieva et al.)

Light-sheet microscopy is an increasingly popular technique in the life sciences due to its fast 3D imaging capability of fluorescent samples with low photo toxicity compared to confocal methods. In this work we present a new, fast, flexible and simple to implement method to optimize the illumination light-sheet to the requirement at hand. A telescope composed of two electrically tuneable lenses enables us to define thickness and position of the light-sheet independently but accurately within milliseconds, and therefore optimize image quality of the features of interest interactively. We demonstrated the practical benefit of this technique by 1) assembling large field of views from tiled single exposure each with individually optimized illumination settings; 2) sculpting the light-sheet to trace complex sample shapes within single exposures. This technique proved compatible with confocal line scanning detection, further improving image contrast and resolution. Finally, we determined the effect of light-sheet optimization in the context of scattering tissue, devising procedures for balancing image quality, field of view and acquisition speed.

Background: Much of our understanding of how neural networks develop is based on studies of sensory systems, revealing often highly stereotyped patterns of connections, particularly as these diverge from the presynaptic terminals of sensory neurons. We know considerably less about the wiring strategies of motor networks, where connections converge onto the dendrites of motoneurons. Here, we investigated patterns of synaptic connections between identified motoneurons with sensory neurons and interneurons in the motor network of the Drosophila larva and how these change as it develops., Results: We find that as animals grow, motoneurons increase the number of synapses with existing presynaptic partners. Different motoneurons form characteristic cell-type-specific patterns of connections. At the same time, there is considerable variability in the number of synapses formed on motoneuron dendrites, which contrasts with the stereotypy reported for presynaptic terminals of sensory neurons. Where two motoneurons of the same cell type contact a common interneuron partner, each postsynaptic cell can arrive at a different connectivity outcome. Experimentally changing the positioning of motoneuron dendrites shows that the geography of dendritic arbors in relation to presynaptic partner terminals is an important determinant in shaping patterns of connectivity., Conclusions: In the Drosophila larval motor network, the sets of connections that form between identified neurons manifest an unexpected level of variability. Synapse number and the likelihood of forming connections appear to be regulated on a cell-by-cell basis, determined primarily by the postsynaptic dendrites of motoneuron terminals., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

As animals grow, their nervous systems also increase in size. How growth in the central nervous system is regulated and its functional consequences are incompletely understood. We explored these questions, using the larval Drosophila locomotor system as a model. In the periphery, at neuromuscular junctions, motoneurons are known to enlarge their presynaptic axon terminals in size and strength, thereby compensating for reductions in muscle excitability that are associated with increases in muscle size. Here, we studied how motoneurons change in the central nervous system during periods of animal growth. We find that within the central nervous system motoneurons also enlarge their postsynaptic dendritic arbors, by the net addition of branches, and that these scale with overall animal size. This dendritic growth is gated on a cell-by-cell basis by a specific isoform of the steroid hormone receptor ecdysone receptor-B2, for which functions have thus far remained elusive. The dendritic growth is accompanied by synaptic strengthening and results in increased neuronal activity. Electrical properties of these neurons, however, are independent of ecdysone receptor-B2 regulation. We propose that these structural dendritic changes in the central nervous system, which regulate neuronal activity, constitute an additional part of the adaptive response of the locomotor system to increases in body and muscle size as the animal grows.

Drosophila larvae crawl by peristaltic waves of muscle contractions, which propagate along the animal body and involve the simultaneous contraction of the left and right side of each segment. Coordinated propagation of contraction does not require sensory input, suggesting that movement is generated by a central pattern generator (CPG). We characterized crawling behavior of newly hatched Drosophila larvae by quantifying timing and duration of segmental boundary contractions. We developed a CPG network model that recapitulates these patterns based on segmentally repeated units of excitatory and inhibitory (EI) neuronal populations coupled with immediate neighboring segments. A single network with symmetric coupling between neighboring segments succeeded in generating both forward and backward propagation of activity. The CPG network was robust to changes in amplitude and variability of connectivity strength. Introducing sensory feedback via "stretch-sensitive" neurons improved wave propagation properties such as speed of propagation and segmental contraction duration as observed experimentally. Sensory feedback also restored propagating activity patterns when an inappropriately tuned CPG network failed to generate waves. Finally, in a two-sided CPG model we demonstrated that two types of connectivity could synchronize the activity of two independent networks: connections from excitatory neurons on one side to excitatory contralateral neurons (E to E), and connections from inhibitory neurons on one side to excitatory contralateral neurons (I to E). To our knowledge, such I to E connectivity has not yet been found in any experimental system; however, it provides the most robust mechanism to synchronize activity between contralateral CPGs in our model. Our model provides a general framework for studying the conditions under which a single locally coupled network generates bilaterally synchronized and longitudinally propagating waves in either direction.

Olfactory sensory neurons connect to the antennal lobe of the fly to create the primary units for processing odor cues, the glomeruli. Unique amongst antennal-lobe neurons is an identified wide-field serotonergic neuron, the contralaterally-projecting, serotonin-immunoreactive deutocerebral neuron (CSDn). The CSDn spreads its termini all over the contralateral antennal lobe, suggesting a diffuse neuromodulatory role. A closer examination, however, reveals a restricted pattern of the CSDn arborization in some glomeruli. We show that sensory neuron-derived Eph interacts with Ephrin in the CSDn, to regulate these arborizations. Behavioural analysis of animals with altered Eph-ephrin signaling and with consequent arborization defects suggests that neuromodulation requires local glomerular-specific patterning of the CSDn termini. Our results show the importance of developmental regulation of terminal arborization of even the diffuse modulatory neurons to allow them to route sensory-inputs according to the behavioural contexts., Competing Interests: The authors have declared that no competing interests exist.

Drosophila have emerged as a model system to study mammalian neurodegenerative diseases. In the present study we have generated Drosophila transgenic for ovine PrP (prion protein) to begin to establish an invertebrate model of ovine prion disease. We generated Drosophila transgenic for polymorphic variants of ovine PrP by PhiC31 site-specific germ-line transformation under expression control by the bi-partite GAL4/UAS (upstream activating sequence) system. Site-specific transgene insertion in the fly genome allowed us to test the hypothesis that single amino acid codon changes in ovine PrP modulate prion protein levels and the phenotype of the fly when expressed in the Drosophila nervous system. The Arg(154) ovine PrP variants showed higher levels of PrP expression in neuronal cell bodies and insoluble PrP conformer than did His(154) variants. High levels of ovine PrP expression in Drosophila were associated with phenotypic effects, including reduced locomotor activity and decreased survival. Significantly, the present study highlights a critical role for helix-1 in the formation of distinct conformers of ovine PrP, since expression of His(154) variants were associated with decreased survival in the absence of high levels of PrP accumulation. Collectively, the present study shows that variants of the ovine PrP are associated with different spontaneous detrimental effects in ovine PrP transgenic Drosophila.

Many parts of the nervous system become active before development is complete, including the embryonic spinal cord. Remarkably, although the subject has been debated for over a century (Harrison, 1904), it is still unclear whether such activity is required for normal development of motor circuitry. In Drosophila, embryonic motor output is initially poorly organized, and coordinated crawling-like behavior gradually emerges over the subsequent phase of development. We show that reversibly blocking synaptic transmission during this phase severely delays the first appearance of coordinated movements. When we interfere with the pattern of neuronal firing during this period, coordination is also delayed or blocked. We conclude that there is a period during which endogenous patterns of neuronal activity are required for the normal development of motor circuits in Drosophila.

A fundamental strategy for organising connections in the nervous system is the formation of neural maps. Map formation has been most intensively studied in sensory systems where the central arrangement of axon terminals reflects the distribution of sensory neuron cell bodies in the periphery or the sensory modality. This straightforward link between anatomy and function has facilitated tremendous progress in identifying cellular and molecular mechanisms that underpin map development. Much less is known about the way in which networks that underlie locomotion are organised. We recently showed that in the Drosophila embryo, dendrites of motorneurons form a neural map, being arranged topographically in the antero-posterior axis to represent the distribution of their target muscles in the periphery. However, the way in which a dendritic myotopic map forms has not been resolved and whether postsynaptic dendrites are involved in establishing sets of connections has been relatively little explored. In this study, we show that motorneurons also form a myotopic map in a second neuropile axis, with respect to the ventral midline, and they achieve this by targeting their dendrites to distinct medio-lateral territories. We demonstrate that this map is "hard-wired"; that is, it forms in the absence of excitatory synaptic inputs or when presynaptic terminals have been displaced. We show that the midline signalling systems Slit/Robo and Netrin/Frazzled are the main molecular mechanisms that underlie dendritic targeting with respect to the midline. Robo and Frazzled are required cell-autonomously in motorneurons and the balance of their opposite actions determines the dendritic target territory. A quantitative analysis shows that dendritic morphology emerges as guidance cue receptors determine the distribution of the available dendrites, whose total length and branching frequency are specified by other cell intrinsic programmes. Our results suggest that the formation of dendritic myotopic maps in response to midline guidance cues may be a conserved strategy for organising connections in motor systems. We further propose that sets of connections may be specified, at least to a degree, by global patterning systems that deliver pre- and postsynaptic partner terminals to common "meeting regions.", Competing Interests: The authors have declared that no competing interests exist.

During nervous system development, different classes of neurons obtain different dendritic architectures, each of which receives a large number of input synapses. However, it is not clear whether synaptic inputs are targeted to specific regions within a dendritic tree and whether dendritic tree geometry and subdendritic synapse distributions might be optimized to support proper neuronal input-output computations. This study uses an insect model where structure and function of an individually identifiable neuron, motoneuron 5 (MN5), are changed while it develops from a slow larval crawling into a fast adult flight motoneuron during metamorphosis. This allows for relating postembryonic dendritic remodeling of an individual motoneuron to developmental changes in behavioral function. Dendritic architecture of MN5 is analyzed by three-dimensional geometric reconstructions and quantitative co-localization analysis to address the distribution of synaptic terminals. Postembryonic development of MN5 comprises distinct changes in dendritic shape and in the subdendritic distribution of GABAergic input synapses onto MN5. Subdendritic synapse targeting is not a consequence of neuropil structure but must rely on specific subdendritic recognition mechanisms. Passive multicompartment simulations indicate that postembryonic changes in dendritic architecture and in subdendritic input synapse distributions may tune the passive computational properties of MN5 toward stage-specific behavioral requirements.

During the metamorphosis of the holometabolous insect, Manduca sexta, the postembryonic acquisition of adult specific motor behaviors is accompanied by changes in dendritic architecture, membrane currents, and input synapses of identified motoneurons. This study aims to test whether increased activity affects dendritic architecture and sub-dendritic input synapse distribution of the identified flight motoneuron 5 (MN5). Systemic injections of the chloride channel blocker, picrotoxin (PTX), during early pupal stages increase pupal reflex responsiveness, but overall development is not impaired. MN5 input resistance, resting membrane potential, and spiking threshold are not affected. Bath application of PTX to isolated ventral nerve cords evokes spiking in pupal and adult flight motoneurons. Quantitative three-dimensional reconstructions of the dendritic tree of the adult MN5 show that systemic PTX injections into early pupae cause dendritic overgrowth and reduce the density of GABAergic inputs. In contrast, the distribution patterns of GABAergic terminals throughout the dendritic tree remain unaltered. This indicates that increased overall excitability might cause dendritic overgrowth and decreased inhibitory input during postembryonic motoneuron remodeling, whereas sub-dendritic synapse targeting might be controlled by activity-independent signals. Behavioral testing reveals that these neuronal changes do not impede the animal's ability to fly, but impair maximum flight performance.

We used non-invasive muscle imaging to study the onset of motor activity and emergence of coordinated movement in Drosophila embryos. Earliest movements are myogenic, and neurally controlled muscle contractions first appear with the onset of bursting activity 17 hours after egg laying. Initial episodes of activity are poorly organised and coordinated crawling sequences only begin to appear after a further hour of bursting. Thus, network performance improves during this first period of activity. The embryo continues to exhibit bursts of crawling-like sequences until shortly before hatching, while other reflexes also mature. Bursting does not begin as a reflex response to sensory input but appears to reflect the onset of spontaneous activity in the motor network. It does not require GABA-mediated transmission, and, by using a light-activated channel to excite the network, we demonstrate activity-dependent depression that may cause burst termination.

As the nervous system develops, there is an inherent variability in the connections formed between differentiating neurons. Despite this variability, neural circuits form that are functional and remarkably robust. One way in which neurons deal with variability in their inputs is through compensatory, homeostatic changes in their electrical properties. Here, we show that neurons also make compensatory adjustments to their structure. We analysed the development of dendrites on an identified central neuron (aCC) in the late Drosophila embryo at the stage when it receives its first connections and first becomes electrically active. At the same time, we charted the distribution of presynaptic sites on the developing postsynaptic arbor. Genetic manipulations of the presynaptic partners demonstrate that the postsynaptic dendritic arbor adjusts its growth to compensate for changes in the activity and density of synaptic sites. Blocking the synthesis or evoked release of presynaptic neurotransmitter results in greater dendritic extension. Conversely, an increase in the density of presynaptic release sites induces a reduction in the extent of the dendritic arbor. These growth adjustments occur locally in the arbor and are the result of the promotion or inhibition of growth of neurites in the proximity of presynaptic sites. We provide evidence that suggest a role for the postsynaptic activity state of protein kinase A in mediating this structural adjustment, which modifies dendritic growth in response to synaptic activity. These findings suggest that the dendritic arbor, at least during early stages of connectivity, behaves as a homeostatic device that adjusts its size and geometry to the level and the distribution of input received. The growing arbor thus counterbalances naturally occurring variations in synaptic density and activity so as to ensure that an appropriate level of input is achieved.

Dendritic filopodia are long thin protrusions occurring predominantly on developing neurons. Data from different systems suggest a range of crucial functions for filopodia in central circuit formation, including steering of dendritic growth, branch formation, synaptogenesis, and spinogenesis. Are the same filopodia competent to mediate all these processes, do filopodia acquire different functions through development, or do different filopodial types with distinct functions exist? In this study, 3-dimensional reconstructions from confocal image stacks demonstrate the existence of two morphologically and functionally distinct types of filopodia located on the dendritic tips versus the dendritic shafts of the same developing motoneuron. During dendritic growth, both filopodial types undergo a process of stage-specific morphogenesis. Using novel quantification strategies of 3-dimensional co-localization analysis for immunocytochemically labeled presynaptic specializations along postsynaptic filopodia, we find that presynaptic terminals accumulate along filopodia towards the dendrites at both stable dendritic shafts and on growing dendritic tips. On tips, this is likely to reflect synaptotrophic growth of the dendrite. At stable shafts, however, presynaptic sites become relocated along filopodia towards dendritic branches. This indicates the interactive growth of both pre- and postsynaptic partner towards one another during synaptogenesis, using filopodia as guides.

The dendritic trees of different neuronal types display an astonishing diversity in structure and function. How this diversity is generated remains incompletely understood. However, recent studies have revealed some of the underlying mechanisms by which intrinsic programs of cell-type specification and extrinsic factors exert their effects on the dendritic cytoskeleton to regulate patterns of growth and branching.

Dendritic architecture provides the structural substrate for myriads of input and output synapses in the brain and for the integration of presynaptic inputs. Understanding mechanisms of evolution and development of neuronal shape and its respective function is thus a formidable problem in neuroscience. A fundamental prerequisite for finding answers is a precise quantitative analysis of neuronal structure in situ and in vivo. Therefore we have developed a tool set for automatic geometric reconstruction of neuronal architecture from stacks of confocal images. It provides exact midlines, diameters, surfaces, volumes, and branch point locations and allows analysis of labeled molecule distribution along neuronal surfaces as well as direct export into modeling software. We show the high accuracy of geometric reconstruction and the analysis of putative input synapse distribution throughout entire dendritic trees from in situ light microscopy preparations as a possible application. The binary version of the reconstruction module is downloadable at no cost.

Exact geometrical reconstructions of neuronal architecture are indispensable for the investigation of neuronal function. Neuronal shape is important for the wiring of networks, and dendritic architecture strongly affects neuronal integration and firing properties as demonstrated by modeling approaches. Confocal microscopy allows to scan neurons with submicron resolution. However, it is still a tedious task to reconstruct complex dendritic trees with fine structures just above voxel resolution. We present a framework assisting the reconstruction. User time investment is strongly reduced by automatic methods, which fit a skeleton and a surface to the data, while the user can interact and thus keeps full control to ensure a high quality reconstruction. The reconstruction process composes a successive gain of metric parameters. First, a structural description of the neuron is built, including the topology and the exact dendritic lengths and diameters. We use generalized cylinders with circular cross sections. The user provides a rough initialization by marking the branching points. The axes and radii are fitted to the data by minimizing an energy functional, which is regularized by a smoothness constraint. The investigation of proximity to other structures throughout dendritic trees requires a precise surface reconstruction. In order to achieve accuracy of 0.1 microm and below, we additionally implemented a segmentation algorithm based on geodesic active contours that allow for arbitrary cross sections and uses locally adapted thresholds. In summary, this new reconstruction tool saves time and increases quality as compared to other methods, which have previously been applied to real neurons.

The brain's link between perception and action involves several steps, which include stimulus transduction, neuronal coding of the stimulus, comparison to a memory template and choice of an appropriate behavioral response. All of these need time, and many studies report that the time needed to compare two stimuli correlates inversely with the perceived distance between them. We developed a behavioral assay in which we tested the time that a honeybee needs to discriminate between odors consisting of mixtures of two components, and included both very similar and very different stimuli spanning four log-concentration ranges. Bees learned to discriminate all odors, including very similar odors and the same odor at different concentrations. Even though discriminating two very similar odors appears to be a more difficult task than discriminating two very distinct substances, we found that the time needed to make a choice for or against an odor was independent of odor similarity. Our data suggest that, irrespective of the nature of the olfactory code, the bee olfactory system evaluates odor quality after a constant interval. This may ensure that odors are only assessed after the olfactory network has optimized its representation.

The orcokinins are a highly conserved family of crustacean peptides that enhance hindgut contractions in the crayfish Orconectes limosus (Stangier et al. [1992] Peptides 13:859-864). By combining immunocytochemical and mass spectrometrical analysis of the stomatogastric nervous system (STNS) in the crayfish Cherax destructor, we show that multiple orcokinins are synthesized in single neurons. Immunocytochemistry demonstrated orcokinin-like immunoreactivity in all four ganglia of the STNS and in the pericardial organs, a major neurohaemal organ. Identified neurons in the STNS were stained, including a pair of modulatory interneurons (inferior ventricular nerve neuron, IVN), a neuron with its cell body in the stomatogastric ganglion that innervates cardiac muscle c6 via the anterior median nerves (AM-c6), and a sensory neuron (anterior gastric receptor neuron). Five orcokinin-related peptides were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) post source decay fragmentation in samples of either the stomatogastric ganglion or the pericardial organs. Four of these peptides are identical to peptides derived from the cloned Procambarus clarkii precursor (Yasuda-Kamatani and Yasuda [2000] Gen. Comp. Endocrinol. 118:161-172), including the original [Asn(13)]-orcokinin (NFDEIDRSGFGFN, [M+H](+) = 1,517.7 Da), [Val(13)]-orcokinin ([M+H](+) = 1,502.7 Da), [Thr(8)-His(13)]-orcokinin ([M+H](+) = 1,554.8 Da), and FDAFTTGFGHS ([M+H](+) = 1,186.5 Da). The fifth peptide is a hitherto unknown orcokinin variant: [Ala(8)-Ala(13)]-orcokinin ([M+H](+) = 1,458.7 Da). The masses of all five peptides were also detected in the inferior ventricular nerve of C. destructor, which contains the cell bodies and axons of the IVNs as well as the axons of two other orcokinin-like immunoreactive neurons. In the oesophageal nerve, in which all the orcokinin-like immunoreactivity derives from the IVNs, at least two of the orcokinins were detected, indicating that multiple orcokinins are synthesized in these neurons. Similarly, all four orcokinin masses were detected in the anterior median nerves, in which all the orcokinin-like immunoreactivity derives from the AM-c6 neuron. This study therefore lays the groundwork to investigate the function of the orcokinin peptide family using single identified neurons in a well-studied system., (Copyright 2002 Wiley-Liss, Inc.)