Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

1. Neuronal Culture

Note: Fibroblast reprogramming in pluripotent stem cells, commitment to the dorsal telencephalon lineage, derivation, amplification, and banking of late cortical progenitors (LCP) were described in Boissart et al⁴. Neuronal differentiation of LCP-like cells was also performed according to Boissart et al⁴ with slight modifications. Other procedures have been developed for direct reprogramming of fibroblasts into induced pluripotent stem cells followed by their differentiation into neurons. This protocol was retained since it allows the selective production of pyramidal glutamatergic neurons.

1. Treat 6-well culture plates with glass coverslips with poly-ornithine (diluted to 1/6 in DPBS, stock concentration 0.01%) O/N, followed by three washes in DPBS. Then add laminin (stock concentration 1 mg/ml, diluted 500 times in DPBS) for at least 10 hr under the flow hood.
2. Plate and dispatch NSC at low density (50,000 cells/cm²) in 6-well culture plates with glass coverslips in 3 ml of culture medium consisting of DMEM/F12 (500 ml), 2 vials (5 ml each) of N2 supplement, 2 vials (10 ml each) of B27 supplement, 10 ml of Pen-Streptomycin (Penicillin = 10,000 units/ml and Streptomycin = 10,000 units/ml), 1 ml of 2-mercaptoethanol (Stock solution: 50 mM) and laminin (1/500), without growth factors. CRITICAL STEP: Carry out this step carefully by adding cells with slow rotating movements in order to reduce cell clustering.
3. Remove the culture medium. Add fresh N2B27 medium containing 2 µg/ml of fresh laminin solution to keep the neuron attached on the glass coverslips and avoid clumping. Change the medium every 3 days. Keep some of the remaining medium (200 µl) before adding fresh medium in order to prevent the cell from drying. Alternatively, proceed rapidly and change the total volume (3 ml).

2. Lentiviral Transduction

Note: GFP lentiviral vectors were kindly provided by Dr. Uwe Maskos Laboratory at Institut Pasteur (Paris) and prepared according to the published protocol ⁵. GFP expression is driven by the mouse phosphoglycerate kinase (PGK) promoter. For this study, viral titer was of 400 ng/µl (stock solution in PBS 1x).

1. Transduce human iPSC-derived neurons at any step of maturation by viral particles by adding 1 µl of the stock solution containing 40 ng of GFP lentiviral vector per culture well (6-well plates) and incubate for 48 hr in fresh culture medium. Note: For this protocol, the duration of incubation allows a good labeling of spine structures.

3. Immunofluorescence

Note: In order to improve the labeling of the whole spine morphology, immunofluorescence labeling was performed using an anti-GFP antibody in permeabilized conditions.

1. Remove culture medium and fix transduced cells on coverslips in 4% paraformaldehyde for 10 min at RT, then wash 3 times in 1x PBS (10 min each).
2. Immerge coverslips in PBS supplemented with 0.05% Triton (100x) and 10% horse serum for 1 hr at RT, then wash 3 times in 1x PBS.
3. Add 100 µl of 1x PBS supplemented with 4% horse serum and primary antibody (1/1,000) raised against GFP and diluted by a factor of 1,000 on each coverslip. Incubate in a dark box O/N at 4°C, then wash 3 times with 1x PBS.
4. Dilute Alexa Fluor 488-conjugated antibody (1/200) in PBS supplemented with 0.5% of Tween 20 and incubate for 1 hr at RT. Then wash 3 times in 1x PBS and mount coverslips on glass slides with mounting medium for fluorescence microscopy.

4. Dendritic Spine Imaging

1. Perform confocal Imaging through a confocal laser-scanning microscope.
2. Select healthy neurons with pyramidal morphology and a full dendritic arborization, and quantify at least 10 neurons per condition from separate experiments. Quantify 60 – 100 µm per dendrite.
3. Acquire images using a 40X oil NA = 1.3 objective and a 488 nm laser line for GFP excitation, with a typical peak power at sample level around 20 µW. Set pixel size around 80 nm to properly sample dendritic spines.
   Note: The subsequent analysis call for an image in which the noise does not compromise the proper segmentation of dendrites and spines. With the spatial sampling and power settings mentioned above, we observed that a pixel dwell-time of 3.15 µs is sufficient to collect enough photons to build such an image. For dim samples, image quality can be improved by averaging 2 to 4 scans. The acquisition of several XY tiles may be needed to cover the region of interest, which then must be stitched together before processing.
4. To sample the whole neuron volume, acquire a Z-stack, with a Z spacing ranging from 150 nm to 300 nm, yielding 20 to 30 Z slices.
   Note: The lateral spatial resolution achieved with these settings is 234 nm and the axial resolution is 591 nm. The sampling chosen here is adequate, but as the axial resolution is larger than the smallest size of the spines to be imaged, the analysis favors the spines that extend laterally from the dendrites.
    

5. 3D Quantification of Dendritic Spines

Note: The following sections specifically describe the use of the Imaris software for analysis. Alternative implementations exist, including NeuronStudio¹⁵ or Metamorph⁸, which can provide similar results.

Use the following key settings:

1. As a pre-processing stage, use Gaussian filtering through the image processing facilities offered by the software. Perform Gaussian filtering via the Image Processing > Smoothing > Gaussian Filter. Set the filter width to be equal to the pixel size in XY.
2. Perform a Semi-automatic tracing of dendrites, using the Filament Tracer module of the software.
   1. First, estimate the dendrite diameter, by using the distance tool in the Slice tab of the software. 
   2. In the Surpass tab, click on the Filaments tool. For a better robustness, this protocol relies on semi-automated tracking; click on Skip automatic creation. The module interface is now showing the Draw tab. Here, select AutoPath as a method, Dendrite as a type, and input the estimated dendrite diameter.
   3. Use the Select mode of the pointer, turning the cursor into a box. Shift-right-click on the dendrite starting point. Note: the software performs initial calculations.
   4. Move the pointer along the dendrite. From the starting point (represented as a blue sphere), a yellow line representing the most likely dendrite path is shown. Shift-left-click on the dendrite endpoint.
3. Perform the automated spines segmentation. Note: The spines on the traced dendrite are to be found automatically by the module interface. 
   1. In the module interface, click on the Creation tab. In the Rebuild drop-list, pick Rebuild Dendrite Diameter and check the Keep data box. Then click Rebuild.
   2. Set the Threshold so that the segmented volume corresponds to the actual dendrite volume. As an Algorithm, select Shortest Distance from Distance Map. Click the Next button.
   3. Determine the smallest spine head diameter and the maximal length, again by using the distance tool in the Slice tab of the software, then come back to the Surpass tab and enter the parameters. For this protocol, values around 200 – 300 nm for minimal diameter and 4 µm for maximal length are good starting points. Do not check the Allow Branch Spines box. Click the Next button.
   4. Adjust the Seed Points Threshold so that the blue points representing spines localize to actual spine heads. Click the Next button. Note: The core calculation is done now and can be lengthy.
4. Classify spines by going to the Tools tab of the module interface. Click on Classify Spines. In the prompted user interface, make sure that there are four classes, defined by their morphology as follows: Stubby: length <1 µm; Mushroom: Length (spine) >3 and Max width (head) >mean width (neck) x 2; Long thin: Mean width (head) ≥ Mean width (neck); Filopodia-like: Length ≤ 4 µm (no head).  
   Note: The module interface generates four new Filament objects containing the results of the classification. 
5. Export Statistical data: on any of these four object interfaces, go to the Statistics tab. 
   Click on the Export All Statistics to File button.
   Note: Other statistical values can be exported for dendrites (e.g., length, area, mean diameter, branch depth, branching angle, volume, etc.), and for spines (e.g., straightness, area of attachment, length and volume of distinct spine parts, spine diameter, densities, etc.)