A cartesian coordinate robot for dispensing fruit fly food.

The fruit fly, Drosophila melanogaster, continues to be one of the most widely used model organisms in biomedical research. Though chosen for its ease of husbandry, maintaining large numbers of stocks of fruit flies, as done by many laboratories, is labour-intensive. One task which lends itself to automation is the production of the vials of food in which the flies are reared. Fly facilities typically have to generate several thousand vials of fly food each week to sustain their fly stocks. The system presented here combines a cartesian coordinate robot with a peristaltic pump. The design of the robot is based on an open hardware CNC (computer numerical control) machine, and uses belt and pulley actuators for the X and Y axes, and a leadscrew actuator for the Z axis. CNC motion and operation of the peristaltic pump are controlled by grbl (gnea 2018), an open source, embedded, G-code parser. Grbl is written in optimized C and runs directly on an Arduino. A Raspberry Pi is used to generate and stream G-code instructions to Grbl. A touch screen on the Raspberry Pi provides a graphical user interface to the system. Whilst the robot was built for the express purpose of filling vials of fly food, it could potentially be used for other liquid handling tasks in the laboratory.Equipment grant from the School of Biological Sciences, University of Cambridg

Identification of genes influencing dendrite morphogenesis in developing peripheral sensory and central motor neurons.

BACKGROUND: Developing neurons form dendritic trees with cell type-specific patterns of growth, branching and targeting. Dendrites of Drosophila peripheral sensory neurons have emerged as a premier genetic model, though the molecular mechanisms that underlie and regulate their morphogenesis remain incompletely understood. Still less is known about this process in central neurons and the extent to which central and peripheral dendrites share common organisational principles and molecular features. To address these issues, we have carried out two comparable gain-of-function screens for genes that influence dendrite morphologies in peripheral dendritic arborisation (da) neurons and central RP2 motor neurons. RESULTS: We found 35 unique loci that influenced da neuron dendrites, including five previously shown as required for da dendrite patterning. Several phenotypes were class-specific and many resembled those of known mutants, suggesting that genes identified in this study may converge with and extend known molecular pathways for dendrite development in da neurons. The second screen used a novel technique for cell-autonomous gene misexpression in RP2 motor neurons. We found 51 unique loci affecting RP2 dendrite morphology, 84% expressed in the central nervous system. The phenotypic classes from both screens demonstrate that gene misexpression can affect specific aspects of dendritic development, such as growth, branching and targeting. We demonstrate that these processes are genetically separable. Targeting phenotypes were specific to the RP2 screen, and we propose that dendrites in the central nervous system are targeted to territories defined by Cartesian co-ordinates along the antero-posterior and the medio-lateral axes of the central neuropile. Comparisons between the screens suggest that the dendrites of peripheral da and central RP2 neurons are shaped by regulatory programs that only partially overlap. We focused on one common candidate pathway controlled by the ecdysone receptor, and found that it promotes branching and growth of developing da neuron dendrites, but a role in RP2 dendrite development during embryonic and early larval stages was not apparent. CONCLUSION: We identified commonalities (for example, growth and branching) and distinctions (for example, targeting and ecdysone response) in the molecular and organizational framework that underlies dendrite development of peripheral and central neurons.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Prion-induced neurotoxicity: Possible role for cell cycle activity and DNA damage response.

Protein misfolding neurodegenerative diseases arise through neurotoxicity induced by aggregation of host proteins. These conditions include Alzheimer's disease, Huntington's disease, Parkinson's disease, motor neuron disease, tauopathies and prion diseases. Collectively, these conditions are a challenge to society because of the increasing aged population and through the real threat to human food security by animal prion diseases. It is therefore important to understand the cellular and molecular mechanisms that underlie protein misfolding-induced neurotoxicity as this will form the basis for designing strategies to alleviate their burden. Prion diseases are an important paradigm for neurodegenerative conditions in general since several of these maladies have now been shown to display prion-like phenomena. Increasingly, cell cycle activity and the DNA damage response are recognised as cellular events that participate in the neurotoxic process of various neurodegenerative diseases, and their associated animal models, which suggests they are truly involved in the pathogenic process and are not merely epiphenomena. Here we review the role of cell cycle activity and the DNA damage response in neurodegeneration associated with protein misfolding diseases, and suggest that these events contribute towards prion-induced neurotoxicity. In doing so, we highlight PrP transgenic Drosophila as a tractable model for the genetic analysis of transmissible mammalian prion disease.Supported by The NC3Rs No. NC/K000462/1 (in part).This is the final published version. It first appeared at http://dx.doi.org/10.5501/wjv.v4.i3.188

Development of connectivity in a motoneuronal network in Drosophila larvae.

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.L.C. was supported by a Fyssen Foundation post-doctoral fellowship. This work was supported by a Biotechnology and Biological Sciences Research Council (UK) grant (BB/I022414/1) to M.L., a Wellcome Trust Programme Grant (WT075934) to Michael Bate and M.L., a Grass Foundation fellowship to A.S.M., and a Sir Isaac Newton Trust grant to A.S.M. and M.L. The work benefited from facilities supported by a Wellcome Trust Equipment Grant (WT079204) and contributions by the Sir Isaac Newton Trust in Cambridge.This paper was originally published in Current Biology (Couton L, Mauss AS, Yunusov T, Diegelmann S, Evers JF, Landgraf M, Current Biology 2015, 25, 568–576, doi:10.1016/j.cub.2014.12.056

MicroRNA-8 promotes robust motor axon targeting by coordinate regulation of cell adhesion molecules during synapse development

Neuronal connectivity and specificity rely upon precise coordinated deployment of multiple cell-surface and secreted molecules. MicroRNAs have tremendous potential for shaping neural circuitry by fine-tuning the spatio-temporal expression of key synaptic effector molecules. The highly conserved microRNA miR-8 is required during late stages of neuromuscular synapse development in Drosophila. However, its role in initial synapse formation was previously unknown. Detailed analysis of synaptogenesis in this system now reveals that miR-8 is required at the earliest stages of muscle target contact by RP3 motor axons. We find that the localization of multiple synaptic cell adhesion molecules (CAMs) is dependent on the expression of miR-8, suggesting that miR-8 regulates the initial assembly of synaptic sites. Using stable isotope labelling in vivo and comparative mass spectrometry, we find that miR-8 is required for normal expression of multiple proteins, including the CAMs Fasciclin III (FasIII) and Neuroglian (Nrg). Genetic analysis suggests that Nrg and FasIII collaborate downstream of miR-8 to promote accurate target recognition. Unlike the function of miR-8 at mature larval neuromuscular junctions, at the embryonic stage we find that miR-8 controls key effectors on both sides of the synapse. MiR-8 controls multiple stages of synapse formation through the coordinate regulation of both pre- and postsynaptic cell adhesion proteins

Drosophila astrocytes cover specific territories of the CNS neuropil and are instructed to differentiate by Prospero, a key effector of Notch.

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.This work was supported by grants from the Canadian Institutes of Health Research (CIHR) and the Canada Foundation for Innovation (CFI) to D.J.v.M.; by a Biotechnology and Biological Sciences Research Council (UK) grant [BB/I022414/1] to M.L.; and by studentship awards to S.D. from McGill University (Max Stern) and the Integrated Program in Neuroscience.This is the author accepted manuscript. The final version is available from the Company of Biologists via http://dx.doi.org/10.1242/dev.13316

Metamorphosis of an identified serotonergic neuron in the Drosophila olfactory system.

BACKGROUND: Odors are detected by sensory neurons that carry information to the olfactory lobe where they connect to projection neurons and local interneurons in glomeruli: anatomically well-characterized structures that collect, integrate and relay information to higher centers. Recent studies have revealed that the sensitivity of such networks can be modulated by wide-field feedback neurons. The connectivity and function of such feedback neurons are themselves subject to alteration by external cues, such as hormones, stress, or experience. Very little is known about how this class of central neurons changes its anatomical properties to perform functions in altered developmental contexts. A mechanistic understanding of how central neurons change their anatomy to meet new functional requirements will benefit greatly from the establishment of a model preparation where cellular and molecular changes can be examined in an identified central neuron. RESULTS: In this study, we examine a wide-field serotonergic neuron in the Drosophila olfactory pathway and map the dramatic changes that it undergoes from larva to adult. We show that expression of a dominant-negative form of the ecdysterone receptor prevents remodeling. We further use different transgenic constructs to silence neuronal activity and report defects in the morphology of the adult-specific dendritic trees. The branching of the presynaptic axonal arbors is regulated by mechanisms that affect axon growth and retrograde transport. The neuron develops its normal morphology in the absence of sensory input to the antennal lobe, or of the mushroom bodies. However, ablation of its presumptive postsynaptic partners, the projection neurons and/or local interneurons, affects the growth and branching of terminal arbors. CONCLUSION: Our studies establish a cellular system for studying remodeling of a central neuromodulatory feedback neuron and also identify key elements in this process. Understanding the morphogenesis of such neurons, which have been shown in other systems to modulate the sensitivity and directionality of response to odors, links anatomy to the development of olfactory behavior.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Selective Inhibition Mediates the Sequential Recruitment of Motor Pools.

Locomotor systems generate diverse motor patterns to produce the movements underlying behavior, requiring that motor neurons be recruited at various phases of the locomotor cycle. Reciprocal inhibition produces alternating motor patterns; however, the mechanisms that generate other phasic relationships between intrasegmental motor pools are unknown. Here, we investigate one such motor pattern in the Drosophila larva, using a multidisciplinary approach including electrophysiology and ssTEM-based circuit reconstruction. We find that two motor pools that are sequentially recruited during locomotion have identical excitable properties. In contrast, they receive input from divergent premotor circuits. We find that this motor pattern is not orchestrated by differential excitatory input but by a GABAergic interneuron acting as a delay line to the later-recruited motor pool. Our findings show how a motor pattern is generated as a function of the modular organization of locomotor networks through segregation of inhibition, a potentially general mechanism for sequential motor patterns.This work was supported by the Howard Hughes Medical Institute, the HHMI Janelia Visitor Program (MFZ and ML), an Isaac Newton Trust/ISSF Wellcome Trust and a Wellcome Trust grant (092986/Z) to ML.This is the author accepted manuscript. The final version is available from Cell Press via http://dx.doi.org/10.1016/j.neuron.2016.06.03

First data set of H<sub>2</sub>O/HDO columns from the Tropospheric Monitoring Instrument (TROPOMI)

Global measurements of atmospheric water vapour isotopologues aid to better understand the hydrological cycle and improve global circulation models. This paper presents a new data set of vertical column densities of H2O and HDO retrieved from short-wave infrared (2.3 µm) reflectance measurements by the Tropospheric Monitoring Instrument (TROPOMI) onboard the Sentinel-5 Precursor satellite. TROPOMI features daily global coverage with a spatial resolution of up to 7 km×7 km. The retrieval utilises a profile-scaling approach. The forward model neglects scattering, and strict cloud filtering is therefore necessary. For validation, recent ground-based water vapour isotopologue measurements by the Total Carbon Column Observing Network (TCCON) are employed. A comparison of TCCON δD with ground-based measurements by the Multi-platform remote Sensing of Isotopologues for investigating the Cycle of Atmospheric water (MUSICA) project for data prior to 2014 (where MUSICA data are available) shows a bias in TCCON δD estimates. As TCCON HDO is currently not validated, an overall correction of recent TCCON HDO data is derived based on this finding. The agreement between the corrected TCCON measurements and co-located TROPOMI observations is good with an average bias of (−0.2±3)×10$^{21}$ molec cm$^{-2}$ ((1.1±7.2) %) in H$_{2}$O and (−2±7)×10$^{17}$ molec cm$^{-2}$ ((−1.1±7.3) %) in HDO, which corresponds to a mean bias of (−14±17) ‰ in a posteriori δD. The bias is lower at low- and mid-latitude stations and higher at high-latitude stations. The use of the data set is demonstrated with a case study of a blocking anticyclone in northwestern Europe in July 2018 using single-overpass data

Nitric oxide mediates activity-dependent change to synaptic excitation during a critical period in Drosophila.

The emergence of coordinated network function during nervous system development is often associated with critical periods. These phases are sensitive to activity perturbations during, but not outside, of the critical period, that can lead to permanently altered network function for reasons that are not well understood. In particular, the mechanisms that transduce neuronal activity to regulating changes in neuronal physiology or structure are not known. Here, we take advantage of a recently identified invertebrate model for studying critical periods, the Drosophila larval locomotor system. Manipulation of neuronal activity during this critical period is sufficient to increase synaptic excitation and to permanently leave the locomotor network prone to induced seizures. Using genetics and pharmacological manipulations, we identify nitric oxide (NO)-signaling as a key mediator of activity. Transiently increasing or decreasing NO-signaling during the critical period mimics the effects of activity manipulations, causing the same lasting changes in synaptic transmission and susceptibility to seizure induction. Moreover, the effects of increased activity on the developing network are suppressed by concomitant reduction in NO-signaling and enhanced by additional NO-signaling. These data identify NO signaling as a downstream effector, providing new mechanistic insight into how activity during a critical period tunes a developing network