High-resolution ultramicroscopy of the developing and adult nervous system in optically cleared Drosophila melanogaster (original) (raw)
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Three-dimensional reconstruction and segmentation of intact Drosophila by ultramicroscopy
Genetic mutants are invaluable for understanding the development, physiology and behaviour of Drosophila. Modern molecular genetic techniques enable the rapid generation of large numbers of different mutants. To phenotype these mutants sophisticated microscopy techniques are required, ideally allowing the 3D-reconstruction of the anatomy of an adult fly from a single scan. Ultramicroscopy enables up to cm fields of view, whilst providing micron resolution. In this paper, we present ultramicroscopy reconstructions of the flight musculature, the nervous system, and the digestive tract of entire, chemically cleared, drosophila in autofluorescent light. The 3D-reconstructions thus obtained verify that the anatomy of a whole fly, including the filigree spatial organization of the direct flight muscles, can be analysed from a single ultramicroscopy reconstruction.The recording procedure, including 3D-reconstruction using standard software, takes no longer than 30 min. Additionally, image segmentation, which would allow for further quantitative analysis, was performed.
PLoS Biology, 2010
The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile.
Long-term imaging of the ventral nerve cord in behaving adult Drosophila
2021
The dynamics and connectivity of neural circuits continuously change during an animal’s lifetime on timescales ranging from milliseconds to days. Therefore, to investigate how biological networks accomplish remarkable cognitive and behavioral tasks, minimally invasive methods are needed to perform repeated measurements, or perturbations of neural circuits in behaving animals across time. Such tools have been developed to investigate the brain but similar approaches are lacking for comprehensively and repeatedly recording motor circuits in behaving animals. Here we describe a suite of microfabricated technologies that enable long-term, minimally invasive optical recordings of the adult Drosophila melanogaster ventral nerve cord (VNC)—neural tissues that are functionally equivalent to the vertebrate spinal cord. These tools consist of (i) a manipulator arm that permits the insertion of (ii) a compliant implant into the thorax to expose the imaging region of interest; (iii) a numbered,...
Dynamic visualization of nervous system in live Drosophila
Proceedings of the National Academy of Sciences, 1999
We have constructed transgenic Drosophila melanogaster lines that express green f luorescent protein (GFP) exclusively in the nervous system. Expression is controlled with transcriptional regulatory elements present in the 5 f lanking DNA of the Drosophila Na ؉ ,K ؉-ATPase -subunit gene Nervana2 (Nrv2). This regulatory DNA is fused to the yeast transcriptional activator GAL4, which binds specifically to a sequence motif termed the UAS (upstream activating sequence). Drosophila lines carrying Nrv2-GAL4 transgenes have been genetically recombined with UAS-GFP (S65T) transgenes (Nrv2-GAL4؉UAS-GFP) inserted on the same chromosomes. We observe strong nervous system-specific f luorescence in embryos, larvae, pupae, and adults. The GFP f luorescence is sufficiently bright to allow dynamic imaging of the nervous system at all of these developmental stages directly through the cuticle of live Drosophila. These lines provide an unprecedented view of the nervous system in living animals and will be valuable tools for investigating a number of developmental, physiological, and genetic neurobiological problems.
A Complete Electron Microscopy Volume Of The Brain Of Adult Drosophila melanogaster
Drosophila melanogaster has a rich repertoire of innate and learned behaviors. Its 100,000-neuron brain is a large but tractable target for comprehensive neural circuit mapping. Only electron microscopy (EM) enables complete, unbiased mapping of synaptic connectivity; however, the fly brain is too large for conventional EM. We developed a custom high-throughput EM platform and imaged the entire brain of an adult female fly. We validated the dataset by tracing brain-spanning circuitry involving the mushroom body (MB), intensively studied for its role in learning. Here we describe the complete set of olfactory inputs to the MB; find a new cell type providing driving input to Kenyon cells (the intrinsic MB neurons); identify neurons postsynaptic to Kenyon cell dendrites; and find that axonal arbors providing input to the MB calyx are more tightly clustered than previously indicated by light-level data. This freely available EM dataset will significantly accelerate Drosophila neuroscience.
Optical dissection of experience-dependent pre- and postsynaptic plasticity in the Drosophila brain
Cell reports, 2015
Drosophila represents a key model organism for dissecting neuronal circuits that underlie innate and adaptive behavior. However, this task is limited by a lack of tools to monitor physiological parameters of spatially distributed, central synapses in identified neurons. We generated transgenic fly strains that express functional fluorescent reporters targeted to either pre- or postsynaptic compartments. Presynaptic Ca(2+) dynamics are monitored using synaptophysin-coupled GCaMP3, synaptic transmission is monitored using red fluorescent synaptophysin-pHTomato, and postsynaptic Ca(2+) dynamics are visualized using GCaMP3 fused with the postsynaptic matrix protein, dHomer. Using two-photon in vivo imaging of olfactory projection neurons, odor-evoked activity across populations of synapses is visualized in the antennal lobe and the mushroom body calyx. Prolonged odor exposure causes odor-specific and differential experience-dependent changes in pre- and postsynaptic activity at both lev...
BMC Neuroscience
Background: Synapse density is an essential indicator of development and functioning of the central nervous system. It is estimated indirectly through the accumulation of pre and postsynaptic proteins in tissue sections. 3D reconstruction of the electron microscopic images in serial sections is one of the most definitive means of estimating the formation of active synapses in the brain. It is tedious and highly skill-dependent. Confocal imaging of whole mounts or thick sections of the brain provides a natural alternative for rapid gross estimation of the synapse density in large areas. The optical resolution and other deep-tissue imaging aberrations limit the quantitative scope of this technique. Results: Here we demonstrate a simple sample preparation method that could enhance the clarity of the confocal images of the neuropil regions of the ventral nerve cord of Drosophila larvae, providing a clear view of synapse distributions. We estimated the gross volume occupied by the synaptic junctions using 3D object counter plug-in of Fiji/ ImageJ ®. It gave us a proportional estimate of the number of synaptic junctions in the neuropil region. The method is corroborated by correlated super-resolution imaging analysis and through genetic perturbation of synaptogenesis in the larval brain. Conclusions: The method provides a significant improvement in the relative estimate of region-specific synapse density in the central nervous system. Also, it reduced artifacts in the super-resolution images obtained using the stimulated emission depletion microscopy technique.