Design and implementation of multi-signal and time-varying neural reconstructions

Abstract

Several efficient procedures exist to digitally trace neuronal structure from light microscopy, and mature community resources have emerged to store, share, and analyze these datasets. In contrast, the quantification of intracellular distributions and morphological dynamics is not yet standardized. Current widespread descriptions of neuron morphology are static and inadequate for subcellular characterizations. We introduce a new file format to represent multichannel information as well as an open-source Vaa3D plugin to acquire this type of data. Next we define a novel data structure to capture morphological dynamics, and demonstrate its application to different time-lapse experiments. Importantly, we designed both innovations as judicious extensions of the classic SWC format, thus ensuring full back-compatibility with popular visualization and modeling tools. We then deploy the combined multichannel/time-varying reconstruction system on developing neurons in live Drosophila larvae by digitally tracing fluorescently labeled cytoskeletal components along with overall dendritic morphology as they changed over time. This same design is also suitable for quantifying dendritic calcium dynamics and tracking arbor-wide movement of any subcellular substrate of interest.

Distinct relations of microtubules and actin filaments with dendritic architecture

“…We previously confirmed that expression of the F-actin and microtubule transgene reporters did not themselves exert any effects on dendritic development (Das et al, 2017a). Live confocal imaging of agematched third instar larval mature da neuron Class I and Class IV arbors was performed as previously described (Das et al, 2017a;Nanda et al, 2018aNanda et al, , 2018b. Briefly, larvae were immersed in a 1:5 (v/v) diethyl ether to halocarbon oil on a cover slipped glass slide and neurons expressing fluorescent protein cytoskeletal reporter transgenes were visualized on a Zeiss LSM 780 confocal microscope.…”

“…Microtubules and F-actin have long been implicated in neurostructural plasticity; however, their exact roles have not been fully elucidated due to the lack of their simultaneous quantification across the entirety of the neural arbor. With the use of the newly developed multi-signal reconstruction system (Nanda et al, 2018a), we quantified the arbor-wide microtubule and F-actin distributions from live images of fruit fly sensory neurons. Analysis of cytoskeletal distribution revealed the association of local microtubule with dendritic elongation (arbor size) and of local F-actin with dendritic branching (arbor complexity).…”

“…Time-lapse imaging of dendritic morphology with multiple fluorescent proteins tagging the orientation of microtubule and F-actin polymerization will be informative for future studies to investigate further arbor plasticity and the underlying subcellular changes that drive it. We have recently developed an enhanced reconstruction system to annotate temporal changes in neuronal structure simultaneously with subcellular changes (Nanda et al, 2018a). Time varying reconstructions with local polymerization information (rate and direction) may foster a deeper understanding of potential developmental mechanisms.…”

“…Here we deploy the recently developed multi-modal confocal image stack tracing of multi-fluorescent neurons (Nanda et al, 2018a) to reconstruct a representative sample of mature da neuron morphologies containing arbor-wide local quantifications of microtubule and F-actin concentrations. We test whether the distributions of microtubule and F-actin in mature arbors can provide topological and developmental insights on the emergent dendritic structure.…”

An open-source framework for neuroscience metadata management applied to digital reconstructions of neuronal morphology

“…To annotate the reconstructions with proper metadata, the repository administrators have also been inviting data contributors to provide suitable information through a semi-structured Excel spreadsheet [33]. While the ecosystem of neuronal reconstructions has coalesced around a simple data standard for over two decades [30], selection and interpretation of metadata concepts remain highly variable and inconsistent. Thus, for every new dataset, a team of trained curators must validate or reconcile the author-provided information, complemented as needed by the associated publication, with the metadata schema and preferred nomenclature of the database.…”