Automated Multichamber Time-lapse Videography for Long-term In Vivo Observation of Migrating Cells

Cerebral function and viability are critically dependent on efficient delivery of oxygen and glucose through the microvasculature. Here, we studied individual microvessels in the intact brain using high-resolution confocal imaging and long-term time-lapse two-photon microscopy across the lifetime of a mouse. In the first postnatal month, we found large-scale sprouting but to our surprise the majority of sprouts underwent pruning and only a small fraction became perfused capillaries. After the first month, microvessel formation and elimination decreased and the net number of vessels stabilized. Although vascular stability was the hallmark of the adult brain, some vessel formation and elimination continued throughout life. In young adult mice, vessel formation was markedly increased after exposure to hypoxia; however, upon return to normoxia, no vessel elimination was observed, suggesting that new vessels constitute a long-term adaptive response to metabolic challenges. This plasticity was markedly reduced in older adults and aging where hypoxia-induced angiogenesis was absent. Our study describes, for the first time in vivo patterns of cerebral microvascular remodeling throughout life. Disruption of the observed balance between baseline turnover and vascular stability may underlie a variety of developmental and age-related degenerative neurological disorders.

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

Journal of Neurophysiology ◽

10.1152/jn.00588.2013 ◽

2014 ◽

Vol 111 (1) ◽

pp. 208-216 ◽

Cited By ~ 14

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.

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The dynamic interplay between ATP/ADP levels and autophagy sustain neuronal migration in vivo

10.1101/2020.04.02.022228 ◽

2020 ◽

Author(s):

Bressan Cedric ◽

Pecora Alessandra ◽

Gagnon Dave ◽

Snapyan Marina ◽

Labrecque Simon ◽

...

Keyword(s):

Cell Migration ◽

Stationary Phase ◽

Neuronal Migration ◽

Stationary Phases ◽

Focal Adhesions ◽

Time Lapse ◽

List Type ◽

Atp Levels ◽

Migrating Cells

AbstractCell migration is a dynamic process that entails extensive protein synthesis and recycling, structural remodeling, and a considerable bioenergetic demand. Autophagy is one of the pathways that maintain cellular homeostasis. Time-lapse imaging of autophagosomes and ATP/ADP levels in migrating cells in the rostral migratory stream of mice revealed that decrease in ATP levels force cells into the stationary phase and induce autophagy. Genetic impairment of autophagy in neuroblasts using either inducible conditional mice or CRISPR/Cas9 gene editing decreased cell migration due to the longer duration of the stationary phase. Autophagy is modulated in response to migration-promoting and inhibiting molecular cues and is required for the recycling of focal adhesions. Our results show that autophagy and energy consumption act in concert in migrating cells to dynamically regulate the pace and periodicity of the migratory and stationary phases in order to sustain neuronal migration.HighlightsADP levels dynamically change during cell migrationA decrease in ATP levels leads to cell pausing and autophagy induction via AMPKAutophagy is required to sustain neuronal migration by recycling focal adhesionsAutophagy level is dynamically modulated by migration-promoting and inhibiting cues

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Rapid disruption of the cortical microcirculation after mild traumatic brain injury

10.1101/788455 ◽

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.

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Regulation of leading edge microtubule and actin dynamics downstream of Rac1

The Journal of Cell Biology ◽

10.1083/jcb.200303082 ◽

2003 ◽

Vol 161 (5) ◽

pp. 845-851 ◽

Cited By ~ 182

Author(s):

Torsten Wittmann ◽

Gary M. Bokoch ◽

Clare M. Waterman-Storer

Keyword(s):

Rho Gtpases ◽

Actin Polymerization ◽

Leading Edge ◽

Time Lapse ◽

Dominant Negative ◽

Actin Dynamics ◽

Retrograde Flow ◽

Rho Proteins ◽

Migrating Cells

Actin in migrating cells is regulated by Rho GTPases. However, Rho proteins might also affect microtubules (MTs). Here, we used time-lapse microscopy of PtK1 cells to examine MT regulation downstream of Rac1. In these cells, “pioneer” MTs growing into leading-edge protrusions exhibited a decreased catastrophe frequency and an increased time in growth as compared with MTs further from the leading edge. Constitutively active Rac1(Q61L) promoted pioneer behavior in most MTs, whereas dominant-negative Rac1(T17N) eliminated pioneer MTs, indicating that Rac1 is a regulator of MT dynamics in vivo. Rac1(Q61L) also enhanced MT turnover through stimulation of MT retrograde flow and breakage. Inhibition of p21-activated kinases (Paks), downstream effectors of Rac1, inhibited Rac1(Q61L)-induced MT growth and retrograde flow. In addition, Rac1(Q61L) promoted lamellipodial actin polymerization and Pak-dependent retrograde flow. Together, these results indicate coordinated regulation of the two cytoskeletal systems in the leading edge of migrating cells.

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The dynamic interplay between ATP/ADP levels and autophagy sustain neuronal migration in vivo

eLife ◽

10.7554/elife.56006 ◽

2020 ◽

Vol 9 ◽

Author(s):

Cedric Bressan ◽

Alessandra Pecora ◽

Dave Gagnon ◽

Marina Snapyan ◽

Simon Labrecque ◽

...

Keyword(s):

Cell Migration ◽

Stationary Phase ◽

Neuronal Migration ◽

Stationary Phases ◽

Focal Adhesions ◽

Time Lapse ◽

Time Lapse Imaging ◽

Migratory Stream ◽

Migrating Cells

Cell migration is a dynamic process that entails extensive protein synthesis and recycling, structural remodeling, and considerable bioenergetic demand. Autophagy is one of the pathways that maintain cellular homeostasis. Time-lapse imaging of autophagosomes and ATP/ADP levels in migrating cells in the rostral migratory stream of mouse revealed that decreases in ATP levels force cells into the stationary phase and induce autophagy. Pharmacological or genetic impairments of autophagy in neuroblasts using either bafilomycin, inducible conditional mice, or CRISPR/Cas9 gene editing decreased cell migration due to the longer duration of the stationary phase. Autophagy is modulated in response to migration-promoting and inhibiting molecular cues and is required for the recycling of focal adhesions. Our results show that autophagy and energy consumption act in concert in migrating cells to dynamically regulate the pace and periodicity of the migratory and stationary phases to sustain neuronal migration.

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Computer assisted detection of axonal bouton structural plasticity in in vivo time-lapse images

eLife ◽

10.7554/elife.29315 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 9

Author(s):

Rohan Gala ◽

Daniel Lebrecht ◽

Daniela A Sahlender ◽

Anne Jorstad ◽

Graham Knott ◽

...

Keyword(s):

Laser Scanning ◽

Structural Changes ◽

Barrel Cortex ◽

Time Lapse ◽

Laser Scanning Microscopy ◽

Computer Assisted ◽

3D Electron Microscopy ◽

Computer Assisted Detection

The ability to measure minute structural changes in neural circuits is essential for long-term in vivo imaging studies. Here, we propose a methodology for detection and measurement of structural changes in axonal boutons imaged with time-lapse two-photon laser scanning microscopy (2PLSM). Correlative 2PLSM and 3D electron microscopy (EM) analysis, performed in mouse barrel cortex, showed that the proposed method has low fractions of false positive/negative bouton detections (2/0 out of 18), and that 2PLSM-based bouton weights are correlated with their volumes measured in EM (r = 0.93). Next, the method was applied to a set of axons imaged in quick succession to characterize measurement uncertainty. The results were used to construct a statistical model in which bouton addition, elimination, and size changes are described probabilistically, rather than being treated as deterministic events. Finally, we demonstrate that the model can be used to quantify significant structural changes in boutons in long-term imaging experiments.

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In-vivo third-harmonic generation microscopy at 1550nm three-dimensional long-term time-lapse studies in living C. elegans embryos

10.1117/12.874931 ◽

2011 ◽

Author(s):

Rodrigo Aviles-Espinosa ◽

Susana I. C. O. Santos ◽

Andreas Brodschelm ◽

Wilhelm G. Kaenders ◽

Cesar Alonso-Ortega ◽

...

Keyword(s):

Harmonic Generation ◽

Three Dimensional ◽

Time Lapse ◽

Third Harmonic Generation ◽

Third Harmonic ◽

C Elegans ◽

Third Harmonic Generation Microscopy

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ORIGIN, DEVELOPMENT, AND NATURE OF INTRANUCLEAR RODLETS AND ASSOCIATED BODIES IN CHICKEN SYMPATHETIC NEURONS

The Journal of Cell Biology ◽

10.1083/jcb.44.1.172 ◽

1970 ◽

Vol 44 (1) ◽

pp. 172-191 ◽

Cited By ~ 67

Author(s):

Edmund B. Masurovsky ◽

Helena H. Benitez ◽

Seung U. Kim ◽

Margaret R. Murray

Keyword(s):

Sympathetic Neurons ◽

Time Lapse ◽

Structural Features ◽

Intrinsic Factors ◽

Intranuclear Rodlets ◽

Functional Dynamics ◽

Small Clusters ◽

Derivatives Of

Correlative data are presented here on the developmental history, dynamics, histochemistry, and fine structure of intranuclear rodlets in chicken sympathetic neurons from in vivo material and long-term organized tissue cultures. The rodlets consist of bundles of ∼70 ± 10 A proteinaceous filaments closely associated with ∼0.4–0.8 µ spheroidal, granulofibrillar (gf) bodies of a related nature. These bodies are already present in the developing embryo a week or more in advance of the rodlets. In early formative stages rodlets consist of small clusters of aligned filaments contiguous with the gf-bodies. As neuronal differentiation progresses these filaments increase in number and become organized into well-ordered polyhedral arrays. Time-lapse cinemicrography reveals transient changes in rodlet contour associated with intrinsic factors, changes in form and position of the nucleolus with respect to the rodlet, and activity of the gf-bodies. With the electron microscope filaments may be seen extending between the nucleolus, gf-bodies, and rodlets; nucleoli display circumscribed regions with fine structural features and staining reactions reminiscent of those of gf-bodies, We suggest that the latter may be derivatives of the nucleolus and that the two may act together in the assemblage and functional dynamics of the rodlet. The egress of rodlet filaments into the cytoplasm raises the possibility that these might represent a source of the cell's filamentous constituents.

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Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells

The Journal of Cell Biology ◽

10.1083/jcb.200203022 ◽

2002 ◽

Vol 158 (1) ◽

pp. 31-37 ◽

Cited By ~ 156

Author(s):

Wendy C. Salmon ◽

Michael C. Adams ◽

Clare M. Waterman-Storer

Keyword(s):

Cell Body ◽

Time Lapse ◽

Lung Epithelial Cells ◽

Dual Wavelength ◽

Convergence Zone ◽

Retrograde Flow ◽

Filamentous Actin ◽

Actin Bundles ◽

Migrating Cells

Interactions between microtubules (MTs) and filamentous actin (f-actin) are involved in directed cell locomotion, but are poorly understood. To test the hypothesis that MTs and f-actin associate with one another and affect each other's organization and dynamics, we performed time-lapse dual-wavelength spinning-disk confocal fluorescent speckle microscopy (FSM) of MTs and f-actin in migrating newt lung epithelial cells. F-actin exhibited four zones of dynamic behavior: rapid retrograde flow in the lamellipodium, slow retrograde flow in the lamellum, anterograde flow in the cell body, and no movement in the convergence zone between the lamellum and cell body. Speckle analysis showed that MTs moved at the same trajectory and velocity as f-actin in the cell body and lamellum, but not in the lamellipodium or convergence zone. MTs grew along f-actin bundles, and quiescent MT ends moved in association with f-actin bundles. These results show that the movement and organization of f-actin has a profound effect on the dynamic organization of MTs in migrating cells, and suggest that MTs and f-actin bind to one another in vivo.

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