Background: neuron development and the role of neurite growth dynamics
During development, neurons build the processes (neurites) required for cell-cell communication. This requires several complex morphological changes including growth cone formation, axon specification and branching. Subtle abnormalities in neuron morphology have been implicated in the development of several neurodegenerative diseases [1,2]. Teasing apart abnormalities from the normal heterogeneous behaviour of neurons requires a detailed understanding of neurite growth dynamics . In this case study, we show that Nanolive cell imaging offers a perfect solution for monitoring neuron development.
Experimental approach: how to image cortical neurons label-free using Nanolive live cell imaging solutions?
Unstimulated human cortical neuron 2 cells (hCN2, ATCC, USA) were grown as a monolayer in Dulbecco‘s Minimum Essential Medium (DMEM) supplemented with 10% of heat-inactivated foetal bovine serum, penicillin (20 units/ml) and streptomycin (20 mg/ml). Temperature (37°C) and humidity (5% CO2) were kept constant throughout the experiment. Unstimulated cells were imaged. Neurite stimulation media (25 ng/ml β nerve growth factor, 0.5 mM dbcAMP and 0.5 mM IBMX) was then added and cells were re-imaged 20 h, 3 d, 9 d and 20 d post-media addition. All images were acquired at a rate of one image every 6 seconds using Nanolive’s 3D Cell Explorer. The resulting images were subjected to a
maximum intensity z-projection, segmented (with cell body shown in green and neurites shown in orange, separated) and quantified, using tools developed in-house.
Morphological evolution of cortical neuron cells over time. Observations made in unstimulated cells, hCN2 cells 20h after neurite growth stimulation media addition, hCN2 cells 3 days after neurite growth stimulation media addition, hCN2 cells 9 days after neurite growth stimulation media addition, and hCN2 cells 20 days after neurite growth stimulation media addition.
Experimental setting and processing of imaging and quantitative data
1. A timeline of the morphological changes unstimulated primary cortical neuron undergo after exposure to neurite stimulation media
In the figure below, one frame from each experimental timepoint is shown (top row), alongside its corresponding segmentation mask (bottom row). In the segmentation mask, the position of the cell body is shown in green, and the neurites are shown in orange. To see the videos in full, please watch the webinar.
Unstimulated cells have a typical undifferentiated morphology; flat, triangular-shaped, and are well spread across the surface of the dish. Bright, round vesicles, which likely contain neuronal material, fill the cell cytoplasm.
• At 20 h post-stimulation, cells remain spread across the surface of the dish, but have developed their first neurite (orange in segmented image).
• At 3 d post-stimulation, cells have already formed advanced growth cones, with complex branching morphology.
• At 9 d post-stimulation, cells start to take on classic neuron morphology; the cell body has contracted, and five neurites have developed.
• At 20 d post-stimulation, cells had adopted a multipolar physiology with four well-defined, elongated neurites.
*Note the high precision of the segmentation masks; no detail is missed. Fine protrusions, spines, and varicosities are all captured with high accuracy, confirming that Nanolive imaging can be used to monitor fine scale morphological changes in neurons.
2. Visualize and quantify fine scale dynamic behaviours with high precision
In the video on the right, we quantify the dynamic behaviour of the primary neurite, which grew after 20 h after the stimulation media was added.
Quantification of the dynamic behaviour of the primary neurite, 20h after stimulation media addition
• The high spatio-temporal resolution of Nanolive imaging permits to detect changes in neurite volume and shape with high precision. The large fluctuations in neurite area (A), perimeter (B) and dry mass (C) reflect the speed and dynamism of these changes.
• Bubble plots (e.g. D where bubble size represents dry mass) can be used to investigate the relationship between the three variables A, B and C.
3. Investigate potential reasons for novel behavioural observations
Two cells were imaged 3 days after the neurite stimulation media was added. The high temporal resolution of Nanolive imaging enabled us to capture a novel cell pulsing behaviour. In the video on the right, we examine the cell metrics in one pulsing cell.
Cell metrics on a pulsing cell, 3 days after stimulation media addition
• To our knowledge, this is the first-time pulsing has been observed in primary cortical neurons. The phenomenon is clearer in the video but even in the still images above changes in shape of the cell body as the cell contracts are evident.
• The cell metrics accurately reflect the behaviour captured in the video. The cell body for example, accumulates dry mass as it expands and then shows a sharp decrease in dry mass during contraction, before stabilizing (A), whereas the inverse pattern is observed in the dry mass of the neurites (B).
• The peaks in the ratio of the cell body and the area of the neurites (C) suggest this could be how cells inject material (and thus grow) their neurites, although this hypothesis would need to be confirmed with additional experiments.
• In future, it may be interesting to monitor neurons over longer periods of time to determine whether these pulsing events are cyclical and to calculate the time that passes between contractions.
4. Examine neurite morphological plasticity and determine how cellular material is distributed between neurites
In the video on the right, we quantify the dynamic behaviour of individual growth cones, which grew 3 days after the stimulation media was added.
• We segmented the growth cones as before, but this time we took the analysis one step further by adding a unique object identifier to each neurite and tracking how dry mass changed over time.
• The graph on the right shows that dry mass fluctuates more in some neurites than in others, and that the magnitude of the fluctuations are not necessarily linked to the size of the neurite.
• This type of analysis could be used to investigate the morphological plasticity of neurites over time and determine a) how cellular material is distributed between neurites and b) which neurites are growing/shrinking.
5. Quantify complex changes in shape over time
The cell imaged 9 days after the neurite stimulation was added, underwent a complex change in shape during the video. In the video on the right, we show it is possible to accurately quantify these complex changes in shape.
Quantification of complex changes in shape on a cortical neuron 9 days after stimulation media addition
• Over the course of the video, we see a clear reduction in shape complexity and neurites appear to fuse back into one big growth cone. The loss of shape complexity is clearly reflected in the metrics. There is a sharp decrease in neurite perimeter as the branching structure is lost (A). The simultaneous increase of the ratio of cell body dry mass to neurite dry mass (B) shows that this cellular material is reabsorbed by the cell body. It is not possible to conclude why the cell does this, but this would be an interesting topic for future research.
6. Examine vesicular trafficking in high-resolution, at every step in the timeline
Vesicles (likely containing neuronal material) observed in unstimulated cortical neurons (left) and 20 days after stimulation media addition (right)
• From unstimulated cells (left), to 20 days after neurite stimulation was added (right) vesicles (that likely contain neuronal material) are present in the cell cytoplasm. In the future, it might be interesting to quantify the content or dynamics of intracellular trafficking within or between neurons.
Conclusions: could my research in neuroscience benefit from non-invasive, label-free Nanolive live cell imaging?
• Many of the observations we made during this case study are novel because our microscopes offer the only non-invasive, label-free option for studying delicate cells such as neurons, unperturbed.
• These observations open several interesting new directions for research, some of which we have highlighted in this case study. These suggestions are by no means exhaustive; but they do showcase the wealth of quantitative information that is just waiting to be exploited.
 Knowles, R. B. et al. Plaque-induced neurite abnormalities: implications for disruption of neural networks in Alzheimer’s disease. Proc. Natl. Acad. Sci. 96(9), 5274-5279. (1999).
 Nagy, J. et al. Altered neurite morphology and cholinergic function of induced pluripotent stem cell-derived neurons from a patient with Kleefstra syndrome and autism. Transl. Psychiatry. 7(7), e1179-e1179. (2017).
 Wissner-Gross, Z. D. et al. Large-scale analysis of neurite growth dynamics on micropatterned substrates. Integr. Biol. 3(1), 65-74 (2011).
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