scholarly journals From whole organism to ultrastructure: progress in axonal imaging for decoding circuit development

Development ◽  
2021 ◽  
Vol 148 (18) ◽  
Author(s):  
Cory J. Weaver ◽  
Fabienne E. Poulain
Keyword(s):  
Neural Circuit ◽  
Super Resolution ◽  
Axon Growth ◽  
Light Sheet ◽  
Growth Cones ◽  
Novel Technologies ◽  
Future Technology ◽  
Axon Development ◽  
Circuit Formation ◽  
Super Resolution Microscopy

ABSTRACT Since the pioneering work of Ramón y Cajal, scientists have sought to unravel the complexities of axon development underlying neural circuit formation. Micrometer-scale axonal growth cones navigate to targets that are often centimeters away. To reach their targets, growth cones react to dynamic environmental cues that change in the order of seconds to days. Proper axon growth and guidance are essential to circuit formation, and progress in imaging has been integral to studying these processes. In particular, advances in high- and super-resolution microscopy provide the spatial and temporal resolution required for studying developing axons. In this Review, we describe how improved microscopy has revolutionized our understanding of axonal development. We discuss how novel technologies, specifically light-sheet and super-resolution microscopy, led to new discoveries at the cellular scale by imaging axon outgrowth and circuit wiring with extreme precision. We next examine how advanced microscopy broadened our understanding of the subcellular dynamics driving axon growth and guidance. We finally assess the current challenges that the field of axonal biology still faces for imaging axons, and examine how future technology could meet these needs.

A Multi-Functional Microfluidic Device Compatible with Widefield and Light Sheet Imaging

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Regan P Moore ◽  
Ellen C O’Shaughnessy ◽  
Yu Shi ◽  
Ana T Nogueira ◽  
Katelyn M Heath ◽  
...  
Keyword(s):  
High Resolution ◽  
Microfluidic Device ◽  
Super Resolution ◽  
Light Sheet ◽  
Ethylene Propylene ◽  
Super Resolution Microscopy ◽  
Fluorinated Ethylene Propylene

We present a microfluidic device compatible with high resolution light sheet and super-resolution microscopy. Our device is a 150 μm thick chamber with a transparent fluorinated ethylene propylene (FEP) cover...

Cubic spline-based depth-dependent localization of mitochondria–endoplasmic reticulum contacts by three-dimensional light-sheet super-resolution microscopy

The Analyst ◽  
2021 ◽  
Author(s):  
Yucheng Sun ◽  
Seungah Lee ◽  
Seong Ho Kang
Keyword(s):  
Endoplasmic Reticulum ◽  
Calcium Homeostasis ◽  
Three Dimensional ◽  
Super Resolution ◽  
Light Sheet ◽  
Cubic Spline ◽  
Contact Distance ◽  
Super Resolution Microscopy

The contact distance between mitochondria (Mito) and endoplasmic reticulum (ER) has received considerable attention owing to their crucial function in maintaining lipid and calcium homeostasis. Herein, cubic spline algorithm-based depth-dependent...

Emerging roles for HOX proteins in synaptogenesis

2018 ◽  
Vol 62 (11-12) ◽  
pp. 807-818 ◽  
Author(s):  
Françoise Gofflot ◽  
Benoit Lizen
Keyword(s):  
Cell Fate ◽  
Synapse Formation ◽  
Neural Circuit ◽  
Target Selection ◽  
Axon Growth ◽  
Current Data ◽  
Cell Fate Specification ◽  
Hox Proteins ◽  
Circuit Formation ◽  
Vertebrate Central Nervous System

Neural circuit formation requires the intricate orchestration of multiple developmental events including cell fate specification, cell migration, axon guidance, dendritic growth, synaptic target selection, and synaptogenesis. The HOX proteins are well-known transcriptional regulators that control embryonic development. Investigations into their action in the vertebrate central nervous system have demonstrated pivotal roles in specifying neural subpopulations, but also in several successive steps required for the assembly of neuronal circuitry, such as neuron migration, axon growth and pathfinding and synaptic target selection. Several lines of evidence suggest that the HOX transcription factors could also regulate synaptogenesis processes even after the process of axonal and dendritic guidance has concluded. Here we will review the current data on HOX proteins in neural circuit formation in order to evaluate their potential roles in establishing neuronal connectivity with specific emphasis on synapse formation and maturation.

scholarly journals 3D Single-Molecule Super-Resolution Microscopy in Mammalian Cells Using a Tilted Light Sheet

2018 ◽  
Vol 114 (3) ◽  
pp. 14a
Author(s):  
Anna-Karin Gustavsson ◽  
Petar N. Petrov ◽  
Maurice Y. Lee ◽  
Yoav Shechtman W.E. Moerner
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
Super Resolution ◽  
Light Sheet ◽  
Super Resolution Microscopy

scholarly journals Analysis of growth cone extension in standardized coordinates highlights self-organization rules during wiring of the Drosophila visual system

PLoS Genetics ◽  
2021 ◽  
Vol 17 (11) ◽  
pp. e1009857
Author(s):  
Weiyue Ji ◽  
Lani F. Wu ◽  
Steven J. Altschuler
Keyword(s):  
Visual System ◽  
Neural Circuit ◽  
Growth Cones ◽  
Local Regularity ◽  
Tissue Organization ◽  
Cellular Behaviors ◽  
Neural Superposition ◽  
The Right ◽  
Circuit Formation

A fascinating question in neuroscience is how ensembles of neurons, originating from different locations, extend to the proper place and by the right time to create precise circuits. Here, we investigate this question in the Drosophila visual system, where photoreceptors re-sort in the lamina to form the crystalline-like neural superposition circuit. The repeated nature of this circuit allowed us to establish a data-driven, standardized coordinate system for quantitative comparison of sparsely perturbed growth cones within and across specimens. Using this common frame of reference, we investigated the extension of the R3 and R4 photoreceptors, which is the only pair of symmetrically arranged photoreceptors with asymmetric target choices. Specifically, we found that extension speeds of the R3 and R4 growth cones are inherent to their cell identities. The ability to parameterize local regularity in tissue organization facilitated the characterization of ensemble cellular behaviors and dissection of mechanisms governing neural circuit formation.

scholarly journals A dynamic formin-dependent deep F-actin network in axons

2015 ◽  
Vol 210 (3) ◽  
pp. 401-417 ◽  
Author(s):  
Archan Ganguly ◽  
Yong Tang ◽  
Lina Wang ◽  
Kelsey Ladt ◽  
Jonathan Loi ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Super Resolution ◽  
Growth Cones ◽  
Filamentous Actin ◽  
Actin Organization ◽  
Cytoskeletal Network ◽  
Presynaptic Boutons ◽  
Super Resolution Microscopy ◽  
Neuronal Growth Cones ◽  
Actin Rings

Although actin at neuronal growth cones is well-studied, much less is known about actin organization and dynamics along axon shafts and presynaptic boutons. Using probes that selectively label filamentous-actin (F-actin), we found focal “actin hotspots” along axons—spaced ∼3–4 µm apart—where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin polymerization, generating long filaments spurting bidirectionally along axons—a phenomenon we call “actin trails.” Super-resolution microscopy reveals intra-axonal deep actin filaments in addition to the subplasmalemmal “actin rings” described recently. F-actin hotspots colocalize with stationary axonal endosomes, and blocking vesicle transport diminishes the actin trails, suggesting mechanistic links between vesicles and F-actin kinetics. Actin trails are formin—but not Arp2/3—dependent and help enrich actin at presynaptic boutons. Finally, formin inhibition dramatically disrupts synaptic recycling. Collectively, available data suggest a two-tier F-actin organization in axons, with stable “actin rings” providing mechanical support to the plasma membrane and dynamic "actin trails" generating a flexible cytoskeletal network with putative physiological roles.

scholarly journals Filopodial dynamics and growth cone stabilization in Drosophila visual circuit development

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Mehmet Neset Özel ◽  
Marion Langen ◽  
Bassem A Hassan ◽  
P Robin Hiesinger
Keyword(s):  
Growth Cone ◽  
Synapse Formation ◽  
Neural Circuit ◽  
Stochastic Dynamics ◽  
Axon Growth ◽  
Growth Cones ◽  
Direct Role ◽  
Control Growth ◽  
Photoreceptor Neurons ◽  
Circuit Assembly

Filopodial dynamics are thought to control growth cone guidance, but the types and roles of growth cone dynamics underlying neural circuit assembly in a living brain are largely unknown. To address this issue, we have developed long-term, continuous, fast and high-resolution imaging of growth cone dynamics from axon growth to synapse formation in cultured Drosophila brains. Using R7 photoreceptor neurons as a model we show that >90% of the growth cone filopodia exhibit fast, stochastic dynamics that persist despite ongoing stepwise layer formation. Correspondingly, R7 growth cones stabilize early and change their final position by passive dislocation. N-Cadherin controls both fast filopodial dynamics and growth cone stabilization. Surprisingly, loss of N-Cadherin causes no primary targeting defects, but destabilizes R7 growth cones to jump between correct and incorrect layers. Hence, growth cone dynamics can influence wiring specificity without a direct role in target recognition and implement simple rules during circuit assembly.

scholarly journals Meeting experiments at the diffraction barrier: an in-silico widefield fluorescence microscopy

2021 ◽  
Author(s):  
Subhamoy Mahajan ◽  
Tian Tang
Keyword(s):  
Fluorescence Microscopy ◽  
In Silico ◽  
Super Resolution ◽  
Light Sheet ◽  
Atomic Resolution ◽  
Live Cells ◽  
Two Photon ◽  
Color Combinations ◽  
Super Resolution Microscopy ◽  
Diffraction Barrier

AbstractFluorescence microscopy allows the visualization of live cells and their components, but even with advances in super- resolution microscopy, atomic resolution remains unattainable. On the other hand, molecular simulations (MS) can easily access atomic resolution, but comparison with experimental microscopy images has not been possible. In this work, a novel in-silico widefield fluorescence microscopy is proposed, which reduces the resolution of MS to generate images comparable to experiments. This technique will allow cross-validation and compound the knowledge gained from experiments and MS. We demonstrate that in-silico images can be produced with different optical axis, object focal planes, exposure time, color combinations, resolution, brightness and amount of out-of-focus fluorescence. This allows the generation of images that resemble those obtained from widefield, confocal, light-sheet, two-photon and super-resolution microscopy. This technique not only can be used as a standalone visualization tool for MS, but also lays the foundation for other in-silico microscopy methods.

scholarly journals Autophagy is required for midbrain dopaminergic axon development and their responsiveness to guidance cues

2020 ◽  
Author(s):  
M.S. Profes ◽  
A. Saghatelyan ◽  
M. Lévesque
Keyword(s):  
Molecular Mechanisms ◽  
Axon Growth ◽  
Brain Functions ◽  
Guidance Cues ◽  
Axon Development ◽  
Wide Range ◽  
Growth Guidance ◽  
Circuit Formation

AbstractMesodiencephalic dopamine (mDA) neurons play a wide range of brain functions. Distinct subtypes of mDA neurons regulate these functions but the molecular mechanisms that drive the mDA circuit formation are largely unknown. Here we show that autophagy, the main recycling cellular pathway, is present in the growth cones of developing mDA neurons and its level changes dynamically in response to guidance cues. To characterize the role of autophagy in mDA axon growth/guidance, we knocked-out (KO) essential autophagy genes (Atg12, Atg5) in mice mDA neurons. Autophagy deficient mDA axons exhibit axonal swellings and decreased branching both in vitro and in vivo, likely due to aberrant microtubule looping. Strikingly, deletion of autophagy-related genes, blunted completely the response of mDA neurons to chemo-repulsive and chemo-attractive guidance cues. Our data demonstrate that autophagy plays a central role in regulating mDA neurons development, orchestrating axonal growth and guidance.

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