Semi-Automated Quantitative Evaluation of Neuron Developmental Morphology In Vitro Using the Change-Point Test

Abstract

Neuron morphology gives rise to distinct axons and dendrites and plays an essential role in neuronal functionality and circuit dynamics. In rat hippocampal neurons, morphological development occurs over roughly one week in vitro. This development has been qualitatively described as occurring in 5 stages. Still, there is a need to quantify cell growth to monitor cell culture health, understand cell responses to sensory cues, and compare experimental results and computational growth model predictions. To address this need, embryonic rat hippocampal neurons were observed in vitro over six days, and their processes were quantified using both standard morphometrics (degree, number of neurites, total length, and tortuosity) and new metrics (distance between change points, relative turning angle, and the number of change points) based on the Change-Point Test to track changes in path trajectories. Of the standard morphometrics, the total length of neurites per cell and the number of endpoints were significantly different between 0.5, 1.5, and 4 days in vitro, which are typically associated with Stages 2-4. Using the Change-Point Test, the number of change points and the average distance between change points per cell were also significantly different between those key time points. This work highlights key quantitative characteristics, both among common and novel morphometrics, that can describe neuron development in vitro and provides a foundation for analyzing directional changes in neurite growth for future studies.

Appendix: Distributions and Analyses on All Morphometrics for All Observed Time Points

A summary of the Dunn tests along with each feature’s distributions are showcased in Fig. 8. The Dunn tests with a Bonferroni correction indicated significant differences between several time points, as outlined in the corresponding Tables below, for segment length (Fig. 8a, Table 10), number of change points (Fig. 8c, Table 12), total length (Fig. 8d, Table 13), number of neurites (Fig. 8e, Table 14), and degree (Fig. 8g, Table 16). No significant differences between time points were detected for turning angle (Fig. 8b, Table 11), and significant differences were only detected between DIV 1.5 and 3 for tortuosity (Fig. 8f, Table 15).

Fig. 8

The distributions and results of the Dunn test with a Bonferroni correction used to assess each morphometric, a average segment length, b average relative turning angle, c number of change points; d total length, e number of neurites, f average tortuosity, g degree, for every time point pair are symbolically represented, as defined in h

Additionally, the sample sizes of the data set are reported in Table 1. The summary statistics and Anderson-Darling results for all of the morphometrics are detailed in Tables 2, 3, 4, 5, 6, 7 and 8. The $\chi ^2$ and $p$-values from the Kruskal-Wallis tests for each feature are in Table 9. Lastly, the post-hoc Dunn tests with a Bonferroni correction $p$-values are in Tables 10, 11, 12, 13, 14, 15 and 16.

Table 1 Overall Data Set Size

Table 2 Summary Statistics and Anderson-Darling Results for Average Segment Length Per Cell ($\mu$m)

Table 3 Summary Statistics Anderson-Darling Results for Average Relative Turning Angle Per Cell ($^\circ$)

Table 4 Summary Statistics Anderson-Darling Results for Number of Change Points Per Cell

Table 5 Summary Statistics Anderson-Darling Results for Total Length of All Neurites Per Cell ($\mu$m)

Table 6 Summary Statistics Anderson-Darling Results for Total Number of Neurites Per Cell

Table 7 Summary Statistics Anderson-Darling Results for Average Tortuosity Per Cell

Table 8 Summary Statistics Anderson-Darling Results for Degree Per Cell

Table 9 Kruskal-Wallis Test Results For All Features

Table 10 Dunn Test with Bonferroni Correction p-values for the Average Segment Length Per Cell ($\mu$m)

Table 11 Dunn Test with Bonferroni Correction p-values for the Average Relative Turning Angle Per Cell ($^\circ$)

Table 12 Dunn Test with Bonferroni Correction p-values for the Number of Change Points Per Cell

Table 13 Dunn Test with Bonferroni Correction p-values for the Total Length of All Neurites Per Cell ($\mu$m)

Table 14 Dunn Test with Bonferroni Correction p-values for the Number of Neurites Per Cell

Table 15 Dunn Test with Bonferroni Correction p-values for the Average Tortuosity Per Cell

Table 16 Dunn Test with Bonferroni Correction p-values for the Degree Per Cell