Dendritic Spines

Brains were harvested whole from P14 or P25 mice on a 129 background and stained using the FD Rapid GolgiStain kit (FD NeuroTechnologies). Brains were rinsed with double distilled water and then immersed in a 1∶1 mixture of FD Solution A∶B for 2 weeks at room temperature in the dark. Brains were then transferred to FD Solution C and kept in the dark at 4°C for 48 hours. Solution C was replaced after the first 24 hours. In preparation for freezing, individual brains were placed in Peel-A-Way disposable embedding molds (VWR) and immersed in Tissue Freezing Medium (Triangle Biomedical Sciences). Dry ice was used to line the bottom of an ice bucket, which was then filled with 190-proof ethanol (Koptec). Using forceps, the molds were lowered into the ethanol (being careful not to allow the ethanol to spill into the top of the mold) and held until the TFM froze. Brains were kept at −80°C until sectioning. Cryosectioning was performed on a Leica CM 3050 S at −22°C. Coronal sections of 100 µm thickness were cut and transferred to gelatin coated slides (LabScientific) onto small drops of FD Solution C. This thickness enabled optimal staining and preservation of spines on the secondary and tertiary dendritic segments used for analysis in the examples presented here. However, any thickness between 80 to 240 µm (recommended in the FD Rapid GolgiStain instructions) can be used in order to satisfy the user’s unique requirements, providing that individual dendritic spines can still be differentiated and measured. After allowing sections to dry at room temperature in the dark for at least 4 hours (or overnight), slides were then stained exactly as described in the FD Rapid GolgiStain instructions (under “Part VI. Staining Procedure”). Permount (Fisher) was used for coverslipping.

All images relating to DF motility and immunofluorescence were collected on a Nikon Ti-E Total Internal Reflection Fluorescence (TIRF) microscope (Nikon Instruments) equipped with an ORCA-Flash 4.0 V2 sCMOS camera (Hamamatsu), motorized stage, perfect focus system, and environmental chamber (InVivo Scientific) to maintain humidity, 37°C and 5% CO₂ ambient conditions. Neuron cultures were imaged in standard Neurobasal Media containing phenol red. For widefield fluorescence imaging, microscope is equipped with a SOLA solid-state LED white light source (Lumencor, 100% power), and a DAPI/FITC/CY3/CY5 excitation, emission, and dichroic filter set (89000 Sedat Quad ET, Chroma. Excitation Filters: D350/50x, ET402/15x, ET490/20x, ET555/25x, ET645/30x. Emission Filters: ET455/50 m, ET525/36 m, ET605/52 m, ET705/72 m). For TIRF imaging, microscope is equipped with a 405/488/561/640 nm laser launch (LUN4; Nikon, 15 mW, 100% power except for optogenetic activation at 10% power), Ti-TIRF-E Motorized Illuminator Unit, and utilized with C-FL TIRF Ultra Hi S/N 405/488/561/638 Quad Cube, Z Quad HC Cleanup, and HC TIRF Quad Dichroic. All images observing synapse formation and dendritic spine density were performed on a Nikon Ti2-E (Nikon) equipped with a CSU-W1 SoRa spinning disk confocal (Yokogawa), Kinetix sCMOS (Photometrics), motorized stage, perfect focus system, and an environmental chamber (Tokai). Microscope is further equipped with a 405/488/561/640 nm laser launch (Nikon, LUNF-XL, 50/60/50/40 mW respectively, 25–50% power), dichroics (100199, Chroma, ZT405/488/561/640RPCV2 MTD TIRF cube), and emission filters (ET455/50 m, ET535/30 m, ET605/52 m, ET705/72 m). Acquisition software was NIS Elements (Nikon).

Tip tracking was performed using Manual Tracking in NIH ImageJ (FIJI build [Schindelin et al., 2012 (link)]). DF were only tracked if they met the following conditions: no contact with axons, neighboring DF, or debris during the time course; emanated from dendrites at least 50 µm away from the center of the soma; clearly visible by brightfield during time course; if they initiated or retracted during imaging, non-existent timepoints were removed from further analysis; buckling and wagging DF were included in tracking. Using the manually tracked positions of the DF base and tip, the image files were then further analyzed with a custom MATLAB script to determine the centerline path along each DF (Mendeley data hyperlink). This script used the fluorescent intensity in either the LifeAct or GFP space-filler channel in the vicinity of the tip and base coordinates to define the average tangent direction of the long axis of the DF by computing the tangent angle q at pixel i using ${\theta }_i=\frac{1}{2}{\tan }^{-1}\left(\frac{\langle y-y_i\rangle \langle x-x_i\rangle }{\langle x^2-x_i^2\rangle \langle y^2-y_i^2\rangle }\right)\text{,}$ where brackets denote the intensity-weighted average over a 15 × 15 pixel domain centered on the ith pixel. The centerline curve (x(s),y(s)) was then determined by solving $\frac{{\partial }^{2}x}{\partial s^2}=-\sin \theta \frac{\partial \theta }{\partial s};\frac{{\partial }^{2}y}{\partial s^2}=\cos \theta \frac{\partial \theta }{\partial s}$
subject to the constraint that the starting and ending positions were the tracked positions of the base and tip of the DF. Using the centerline curves for each DF at each time point, we then calculated the absolute tip displacement, DF length, and mean tip fluorescence intensity and were able to extract the following metrics: average filopodial tip speed calculated as the average of the instantaneous speeds (absolute tip displacement per 5 s interval) between successive timepoints; percent motile, percent of total DF population with average tip speeds greater than 0.0128 µm/s (motile; one pixel displacement or greater per 5 s interval) or less than 0.0128 µm/s (non-motile); percent time motile, the percent of time per DF in which instantaneous speed was greater than 0.0128 µm/s; average length, the distance from base to tip along the centerline curve, median protrusion or retraction rate, the positive or negative change in length between successive timepoints, when instantaneous change in length was greater than ±0.0128 µm/s (motile); mean fluorescence intensity for a circular area of 384 nm radius surrounding the distal DF tip with non-cell background omitted; fluorescence intensity variance, a measure of the spread of intensity values compared to the mean. Fluorescence intensity values were normalized for expression by the minimum local intensity during the duration of imaging. For defining motile versus non-motile filopodia, or substantiative protrusion/retraction rates, a threshold of 0.0128 µm/s was chosen as it represents one pixel (effective size at 100X = 0.064 µm) displacement per 5-s interval and undistinguishable from tracking error. Neurite morphology was measured using the ImageJ plug-in Simple Neurite Tracer (Longair et al., 2011 (link)). Tracings were used to determine the number and length of primary and higher order neurites, and length of the axon (the longest Tau-positive process). Protrusion and spine density was determined by counting proturbences or dendritic spines along a length of dendrite. PSD95 foci analysis was performed by generating a binary mask of foci, and using the automated 2D tracking module in NIS-Elements (Nikon) to follow their trajectories.

Fig. S1 shows characterization of synapse precursor DF in cultured mouse primary cortical neurons. Fig. S2 shows that EVL is the predominant Ena/VASP paralog regulating DF. Fig. S3 shows that MENA and EVL overexpression enhance DF motility. Fig. S4 shows tip enrichment of EVL precedes DF protrusion. Fig. S5 shows that EVL is required for DF morphogenesis, and influences dendritic spine plasticity. Fig. S6 shows that EVL tip localization is necessary and sufficient for DF motility. Fig. S7 shows that MIM/MTSS1 cooperates with EVL to promote DF initiation and motility.