The geometry of photopolymerized topography influences neurite pathfinding by directing growth cone morphology and migration

Abstract

Cochlear implants (CIs) provide auditory perception to those with profound sensorineural hearing loss: however, the quality of sound perceived by a CI user does not approximate natural hearing. This limitation is due in part to the large physical gap between the stimulating electrodes and their target neurons. Therefore, directing the controlled outgrowth of processes from spiral ganglion neurons (SGNs) into close proximity to the electrode array could provide significantly increased hearing function. For this objective to be properly designed and implemented, the ability and limits of SGN neurites to be guided must first be determined. In this work, we engineered precise topographical microfeatures with angle turn challenges of various geometries to study SGN pathfinding. Additionally, we analyze sensory neurite growth in response to topographically patterned substrates and use live imaging to better understand how neurite growth is guided by these cues. In assessing the ability of neurites to sense and turn in response to topographical cues, we find that the geometry of the angled microfeatures determines the ability of neurites to navigate the angled microfeature turns. SGN neurite pathfinding fidelity can be increased by 20-70% through minor increases in microfeature amplitude (depth) and by 25% if the angle of the patterned turn is made more obtuse. Further, by using engineered topographies and live imaging of dorsal root ganglion neurons (DRGNs), we see that DRGN growth cones change their morphology and migration to become more elongated within microfeatures. However, our observations also indicate complexities in studying neurite turning. First, as the growth cone pathfinds in response to the various cues, the associated neurite often reorients across the angle topographical microfeatures. This reorientation is likely related to the tension the neurite shaft experiences when the growth cone elongates in the microfeature around a turn. Additionally, neurite branching is observed in response to topographical guidance cues, most frequently when turning decisions are most uncertain. Overall, the multi-angle channel micropatterned substrate is a versatile and efficient system to assess SGN neurite turning and pathfinding in response to topographical cues. These findings represent fundamental principles of neurite pathfinding that will be essential to consider for the design of 3D systems aiming to guide neurite growth in vivo.

Keywords: growth cone; neurite guidance; pathfinding; photopolymerization; spiral ganglion neurons; topography.

Figure 1:. Schematic of Photopatterning Process and Pattern Characterization.

A. Schematic of photopolymerizing micropatterned substrates. A monomer solution (yellow) is added to a silane coupled glass cover slip (grey). Then the photomask (black outlined pattern) is placed on top of the solution before exposing the system to UV light. The monomer polymerizes to form a solid substrate and the photomask is removed. B. The photomask (black). This pattern repeats over the photomask to create the topographically micropatterned substrate. C. A depth coded confocal microscopy image of multi-angled microfeatures patterned in the substrate surface. Scale bars = 100 μm.

Figure 2:. Feature Geometry Determines the Ability of SGNs to Navigate Complex Angle Microfeature Cues.

A, B, C. Representative images of SGN neurite encountering a 120° angle microfeatures across the amplitude conditions A. 2 μm (green), B. 4 μm (white), and C. 8 μm (purple). D. 95% confidence interval of the distance that SGN neurites follow a microfeature once encountering that feature. n for each sub-condition ranges from 30 to 123. Unpatterned represents the shape of an angled microfeature superimposed onto a flat substrate. Two-way ANOVA shows that both microfeature amplitude and angle of turn affect SGN length in microfeatures with length increasing with deeper microfeatures and lower magnitude turns. p < 0.01. Scale bar = 50 μm

Figure 3:. Microfeature Amplitude Promotes the Ability of SGN Neurites to Turn.

A, B, C. Representative images of SGN neurite encountering a 90° angle microfeature across the amplitude conditions A. 2 μm (green) failing to turn, B. 4 μm (white) aligning across the microfeature, and C. 8μm (purple) making a turn. D. Proportion of SGN neurites that successfully navigate a turn in a microfeature when they encountered. n for each sub-condition ranges from 20 to 64. Two-way ANOVA shows that increasing microfeature amplitude improves the ability of SGN neurites to navigate turns. Error bars represent +/− SEM. p < 0.05. Scale bar = 50 μm

Figure 4:. Growth Cones More Faithfully Remain in Deeper Microfeatures.

A, B. Representative images from Supplemental Video 1a in which a rDRGN growth cone encounters and remains in an 8 μm amplitude, 120° angle microfeature. C, D. Representative images from Supplemental Video 1b in which a rDRGN growth cone encounters and exits a 4 μm amplitude, 120° angle microfeature. E. Percent of rDRGN growth cones that remained in the microfeature over the duration of 1.5 h recording. Z-test shows greater proportion remain in the microfeature in the 8 μm amplitude condition. Error bars represent +/− SEM. p < 0.01. Scale bar = 20 μm

Figure 5:. Growth Cones Exhibit Distinct Morphology and Behavior on Topographically Micropatterned Substrate.

A. Depth coded confocal image of repeating rows of ridges and grooves substrate used for this experiment (3 μm amplitude and 10 μm periodicity). B. Representative image of a rDRGN growth cone grown on an unpatterned substrate. C. Representative image of rDRGN growth cone grown on the micropatterned substrate in A. D. Growth cone shape was approximated as a spheroid and its prolaticity was calculated as per Equation 1. T-test shows growth cones on the patterned substrate were more prolate. Error bars represent +/−SEM, p < 0.05. E. The major axis of this spheroid was found, and the angle difference between this axis and the neurite shaft was measured. Mann-Whitney shows that this angle was smaller for the neurons on the patterned substrate. Error bars represent 95% confidence interval. p < 0.001. Scale bars = 5 μm

Figure 6:. Neurite Shafts Remain in Microfeatures at Similar Rates when Navigating Turns Regardless of Feature Amplitude.

A. Representative image of DRGN neurite unable to hold position around a turn through a 4 μm amplitude, 60° angle microfeature. B. Representative image of DRGN neurite holding position around 2 turns through an 8 μm amplitude, 60° angle microfeature. Red circles indicate neurite shafts not holding the turn while blue arrows show neurites that do hold the turn. C. Percent of neurites that successfully navigate the angle turn challenge and with shafts holding that position. One-way ANOVA shows no difference in treatment groups (p = 0.24). n = 49, 45, and 39. Error bars represent +/− SEM. Scale bar = 50 μm

Figure 7:. Neurite Branching is Most Likely When Neurite Turning Result is Uncertain.

A. SGN neurite turning in response to an 8 μm amplitude, 120° angle microfeature, representing an “easy turn” toward the far right of the graph. B. SGN neurite exiting the microfeature in a 2 μm amplitude, represents a “challenging turn” towards the left of the graph. C. SGN neurite branching to both follow and exit the microfeature in a 4 μm amplitude, 60° angle feature, representing an intermediate challenge where branching was seen to be most likely. D. Neurite branching plotted as a function of neurites successfully turning. A second order polynomial fits the data with r² = 0.54 which equates to p = 0.036.