scholarly journals Regulation of neurofilament length and transport by a dynamic cycle of polymer severing and annealing

2021 ◽  
Author(s):  
Atsuko Uchida ◽  
Anthony Brown
Keyword(s):  
Opposite Effect ◽  
Time Lapse ◽  
Site Directed Mutagenesis ◽  
Phosphorylation Sites ◽  
Neurofilament Protein ◽  
Enzymatic Mechanism ◽  
Time Lapse Imaging ◽  
Dynamic Cycle ◽  
Kinase A

ABSTRACTWe used long-term multi-field time-lapse imaging to analyze the movement, severing and annealing of single neurofilaments along axons of cultured neurons. All filaments were capable of rapid movement. However, long filaments paused and reversed more often, resulting in little net movement, whereas short filaments moved persistently for long distances, pausing and reversing less often. Long filaments severed more frequently, generating shorter filaments, and short filaments annealed more frequently, generating longer filaments. Site-directed mutagenesis to mimic phosphorylation at known phosphorylation sites in the neurofilament protein L head domain increased the severing rate, generating shorter neurofilaments that moved more frequently. A dephospho-mimic mutant had the opposite effect. Treatment with activators of protein kinase A increased filament severing, and this effect was blocked by the dephosphomimic. Thus, neurofilament length and transport are regulated by a dynamic cycle of severing and annealing. We propose that focal destabilization of intermediate filaments by N-terminal phosphorylation may be a general enzymatic mechanism for severing these cytoskeletal polymers.SUMMARYUchida & Brown demonstrate that neurofilament length and transport are regulated by a dynamic cycle of neurofilament polymer severing and end-to-end annealing and propose an enzymatic severing mechanism for neurofilaments involving N-terminal phosphorylation of their constituent polypeptides.

scholarly journals Rapid Intermittent Movement of Axonal Neurofilaments Observed by Fluorescence Photobleaching

2001 ◽  
Vol 12 (10) ◽  
pp. 3257-3267 ◽  
Author(s):  
Lei Wang ◽  
Anthony Brown
Keyword(s):  
Time Lapse ◽  
Cytoskeletal Proteins ◽  
Cultured Neurons ◽  
Neurofilament Proteins ◽  
Neurofilament Protein ◽  
Slow Axonal Transport ◽  
Fluorescent Intensity ◽  
Naturally Occurring ◽  
Time Lapse Imaging ◽  
Kinetics Of

Observations on naturally occurring gaps in the axonal neurofilament array of cultured neurons have demonstrated that neurofilament polymers move along axons in a rapid, intermittent, and highly asynchronous manner. In contrast, studies on axonal neurofilaments using laser photobleaching have not detected movement. Here, we describe a modified photobleaching strategy that does permit the direct observation of neurofilament movement. Axons of cultured neurons expressing GFP-tagged neurofilament protein were bleached by excitation with the mercury arc lamp of a conventional epifluorescence microscope for 12–60 s. The length of the bleached region ranged from 10 to 60 μm. By bleaching thin axons, which have relatively few neurofilaments, we were able to reduce the fluorescent intensity enough to allow the detection of neurofilaments that moved in from the surrounding unbleached regions. Time-lapse imaging at short intervals revealed rapid, intermittent, and highly asynchronous movement of fluorescent filaments through the bleached regions at peak rates of up to 2.8 μm/s. The kinetics of movement were very similar to our previous observations on neurofilaments moving through naturally occurring gaps, which indicates that the movement was not impaired by the photobleaching process. These results demonstrate that fluorescence photobleaching can be used to study the slow axonal transport of cytoskeletal polymers, but only if the experimental strategy is designed to ensure that rapid asynchronous movements can be detected. This may explain the failure of previous photobleaching studies to reveal the movement of neurofilament proteins and other cytoskeletal proteins in axons.

scholarly journals Long-Term Time-Lapse Imaging of Developing Hippocampal Neurons in Culture

2012 ◽  
Vol 2012 (3) ◽  
pp. pdb.prot068239-pdb.prot068239 ◽  
Author(s):  
S. Kaech ◽  
C.-F. Huang ◽  
G. Banker
Keyword(s):  
Hippocampal Neurons ◽  
Time Lapse ◽  
Time Lapse Imaging

scholarly journals Long-term in vivo time-lapse imaging of synapse development and plasticity in the cerebellum

2014 ◽  
Vol 111 (1) ◽  
pp. 208-216 ◽  
Author(s):  
Naoko Nishiyama ◽  
Jeremy Colonna ◽  
Elise Shen ◽  
Jennifer Carrillo ◽  
Hiroshi Nishiyama
Keyword(s):  
In Vivo Imaging ◽  
Time Window ◽  
Time Lapse ◽  
Mammalian Brain ◽  
Cerebellar Neurons ◽  
Brain Functions ◽  
Time Lapse Imaging ◽  
Synaptic Circuitry

Synapses are continuously formed and eliminated throughout life in the mammalian brain, and emerging evidence suggests that this structural plasticity underlies experience-dependent changes of brain functions such as learning and long-term memory formation. However, it is generally difficult to understand how the rewiring of synaptic circuitry observed in vivo eventually relates to changes in animal's behavior. This is because afferent/efferent connections and local synaptic circuitries are very complicated in most brain regions, hence it is largely unclear how sensorimotor information is conveyed, integrated, and processed through a brain region that is imaged. The cerebellar cortex provides a particularly useful model to challenge this problem because of its simple and well-defined synaptic circuitry. However, owing to the technical difficulty of chronic in vivo imaging in the cerebellum, it remains unclear how cerebellar neurons dynamically change their structures over a long period of time. Here, we showed that the commonly used method for neocortical in vivo imaging was not ideal for long-term imaging of cerebellar neurons, but simple optimization of the procedure significantly improved the success rate and the maximum time window of chronic imaging. The optimized method can be used in both neonatal and adult mice and allows time-lapse imaging of cerebellar neurons for more than 5 mo in ∼80% of animals. This method allows vital observation of dynamic cellular processes such as developmental refinement of synaptic circuitry as well as long-term changes of neuronal structures in adult cerebellum under longitudinal behavioral manipulations.

scholarly journals Rapid disruption of the cortical microcirculation after mild traumatic brain injury

2019 ◽  
Author(s):  
Ellen D. Witkowski ◽  
Şefik Evren Erdener ◽  
Kıvılcım Kılıç ◽  
Sreekanth Kura ◽  
Jianbo Tang ◽  
...  
Keyword(s):  
Traumatic Brain Injury ◽  
Brain Injury ◽  
Vascular Dysfunction ◽  
Metabolic Stress ◽  
Time Lapse ◽  
Post Injury ◽  
Injury Model ◽  
Time Lapse Imaging

AbstractTraumatic brain injury (TBI) is a major source of cognitive deficits affecting millions annually. The bulk of human injuries are mild, causing little or no macroscopic damage to neural tissue, yet can still lead to long-term neuropathology manifesting months or years later. Although the cellular stressors that ultimately lead to chronic pathology are poorly defined, one notable candidate is metabolic stress due to reduced cerebral blood flow (CBF), which is common to many forms of TBI. Here we used high-resolution in vivo intracranial imaging in a rodent injury model to characterize deficits in the cortical microcirculation during both acute and chronic phases after mild TBI. We found that CBF dropped precipitously during immediate post-injury periods, decreasing to less than half of baseline levels within minutes and remaining suppressed for 1.5-2 hours. Repeated time-lapse imaging of the cortical microvasculature revealed further striking flow deficits in the capillary network, where 18% of vessels were completely occluded for extended periods after injury, and an additional >50% showed substantial stoppages. Decreased CBF was paralleled by extensive vasoconstriction that is likely to contribute to loss of flow. Our data indicate a major role for vascular dysfunction in even mild forms of TBI, and suggest that acute post-injury periods may be key therapeutic windows for interventions that restore flow and mitigate metabolic stress.

scholarly journals Dendritic space-filling requires a neuronal type-specific extracellular permissive signal inDrosophila

2017 ◽  
Vol 114 (38) ◽  
pp. E8062-E8071 ◽  
Author(s):  
Amy R. Poe ◽  
Lingfeng Tang ◽  
Bei Wang ◽  
Yun Li ◽  
Maria L. Sapar ◽  
...  
Keyword(s):  
Dendritic Growth ◽  
Tyrosine Phosphatase ◽  
Receptive Fields ◽  
Time Lapse ◽  
Receptor Protein ◽  
Space Filling ◽  
Leukocyte Antigen ◽  
Somatosensory Neurons ◽  
Time Lapse Imaging

Neurons sometimes completely fill available space in their receptive fields with evenly spaced dendrites to uniformly sample sensory or synaptic information. The mechanisms that enable neurons to sense and innervate all space in their target tissues are poorly understood. UsingDrosophilasomatosensory neurons as a model, we show that heparan sulfate proteoglycans (HSPGs) Dally and Syndecan on the surface of epidermal cells act as local permissive signals for the dendritic growth and maintenance of space-filling nociceptive C4da neurons, allowing them to innervate the entire skin. Using long-term time-lapse imaging with intactDrosophilalarvae, we found that dendrites grow into HSPG-deficient areas but fail to stay there. HSPGs are necessary to stabilize microtubules in newly formed high-order dendrites. In contrast to C4da neurons, non–space-filling sensory neurons that develop in the same microenvironment do not rely on HSPGs for their dendritic growth. Furthermore, HSPGs do not act by transporting extracellular diffusible ligands or require leukocyte antigen-related (Lar), a receptor protein tyrosine phosphatase (RPTP) and the only knownDrosophilaHSPG receptor, for promoting dendritic growth of space-filling neurons. Interestingly, another RPTP, Ptp69D, promotes dendritic growth of C4da neurons in parallel to HSPGs. Together, our data reveal an HSPG-dependent pathway that specifically allows dendrites of space-filling neurons to innervate all target tissues inDrosophila.

scholarly journals Incubator-independent cell-culture perfusion platform for continuous long-term microelectrode array electrophysiology and time-lapse imaging

2015 ◽  
Vol 2 (6) ◽  
pp. 150031 ◽  
Author(s):  
Dirk Saalfrank ◽  
Anil Krishna Konduri ◽  
Shahrzad Latifi ◽  
Rouhollah Habibey ◽  
Asiyeh Golabchi ◽  
...  
Keyword(s):  
Cell Culture ◽  
Microelectrode Array ◽  
Low Cost ◽  
Time Lapse ◽  
Free Cell ◽  
Network Architectures ◽  
Hippocampal Networks ◽  
Time Lapse Imaging

Most in vitro electrophysiology studies extract information and draw conclusions from representative, temporally limited snapshot experiments. This approach bears the risk of missing decisive moments that may make a difference in our understanding of physiological events. This feasibility study presents a simple benchtop cell-culture perfusion system adapted to commercial microelectrode arrays (MEAs), multichannel electrophysiology equipment and common inverted microscopy stages for simultaneous and uninterrupted extracellular electrophysiology and time-lapse imaging at ambient CO 2 levels. The concept relies on a transparent, replica-casted polydimethylsiloxane perfusion cap, gravity- or syringe-pump-driven perfusion and preconditioning of pH-buffered serum-free cell-culture medium to ambient CO 2 levels at physiological temperatures. The low-cost microfluidic in vitro enabling platform, which allows us to image cultures immediately after cell plating, is easy to reproduce and is adaptable to the geometries of different cell-culture containers. It permits the continuous and simultaneous multimodal long-term acquisition or manipulation of optical and electrophysiological parameter sets, thereby considerably widening the range of experimental possibilities. Two exemplary proof-of-concept long-term MEA studies on hippocampal networks illustrate system performance. Continuous extracellular recordings over a period of up to 70 days revealed details on both sudden and gradual neural activity changes in maturing cell ensembles with large intra-day fluctuations. Correlated time-lapse imaging unveiled rather static macroscopic network architectures with previously unreported local morphological oscillations on the timescale of minutes.

scholarly journals An immobilization technique for long-term time-lapse imaging of explanted Drosophila tissues

2020 ◽  
Author(s):  
Matthew P. Bostock ◽  
Anadika R. Prasad ◽  
Rita Chaouni ◽  
Alice C. Yuen ◽  
Rita Sousa-Nunes ◽  
...  
Keyword(s):  
Time Lapse ◽  
Tissue Level ◽  
Anisotropic Pressure ◽  
Biological Processes ◽  
Effective Technique ◽  
Cell Dynamics ◽  
First Instar ◽  
Stress Damage ◽  
Time Lapse Imaging

AbstractTime-lapse imaging is an essential tool to study dynamic biological processes that cannot be discerned from fixed samples alone. However, imaging cell- and tissue-level processes in intact animals poses numerous challenges if the organism is opaque and/or motile. Explant cultures of intact tissues circumvent some of these challenges, but sample drift remains a considerable obstacle. We employed a simple yet effective technique to immobilize tissues in medium-bathed agarose. We applied this technique to study multiple Drosophila tissues from first-instar larvae to adult stages in various orientations and with no evidence of anisotropic pressure or stress damage. Using this method, we were able to image fine features for up to 18 hours and make novel observations. Specifically, we report that fibers characteristic of quiescent neuroblasts are inherited by their basal daughters during reactivation; that the lamina in the developing visual system is assembled roughly 2-3 columns at a time; that lamina glia positions are dynamic during development; and that the nuclear envelopes of adult testis cyst stem cells do not break down completely during mitosis. In all, we demonstrate that our protocol is well-suited for tissue immobilization and long-term live imaging, enabling new insights into tissue and cell dynamics in Drosophila.

scholarly journals Hippocampal ensemble dynamics timestamp events in long-term memory

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Alon Rubin ◽  
Nitzan Geva ◽  
Liron Sheintuch ◽  
Yaniv Ziv
Keyword(s):  
Activity Patterns ◽  
Time Lapse ◽  
Spatial Context ◽  
Long Term Memory ◽  
Neuronal Ensembles ◽  
Temporal Relationships ◽  
Hippocampal Ca1 ◽  
Underlying Mechanisms ◽  
Time Lapse Imaging

The capacity to remember temporal relationships between different events is essential to episodic memory, but little is currently known about its underlying mechanisms. We performed time-lapse imaging of thousands of neurons over weeks in the hippocampal CA1 of mice as they repeatedly visited two distinct environments. Longitudinal analysis exposed ongoing environment-independent evolution of episodic representations, despite stable place field locations and constant remapping between the two environments. These dynamics time-stamped experienced events via neuronal ensembles that had cellular composition and activity patterns unique to specific points in time. Temporally close episodes shared a common timestamp regardless of the spatial context in which they occurred. Temporally remote episodes had distinct timestamps, even if they occurred within the same spatial context. Our results suggest that days-scale hippocampal ensemble dynamics could support the formation of a mental timeline in which experienced events could be mnemonically associated or dissociated based on their temporal distance.

scholarly journals Versatile, Simple-to-Use Microfluidic Cell-Culturing Chip for Long-Term, High-Resolution, Time-Lapse Imaging

2015 ◽  
Vol 87 (8) ◽  
pp. 4144-4151 ◽  
Author(s):  
Olivier Frey ◽  
Fabian Rudolf ◽  
Gregor W. Schmidt ◽  
Andreas Hierlemann
Keyword(s):  
High Resolution ◽  
Time Lapse ◽  
Resolution Time ◽  
Cell Culturing ◽  
Time Lapse Imaging
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