An unbiased, automated platform for scoring dopaminergic neurodegeneration inC. elegans

Abstract

Caenorhabditis elegans (C. elegans) has served as a simple model organism to study dopaminergic neurodegeneration, as it enables quantitative analysis of cellular and sub-cellular morphologies in live animals. These isogenic nematodes have a rapid life cycle and transparent body, making high-throughput imaging and evaluation of fluorescently tagged neurons possible. However, the current state-of-the-art method for quantifying dopaminergic degeneration requires researchers to manually examine images and score dendrites into groups of varying levels of neurodegeneration severity, which is time consuming, subject to bias, and limited in data sensitivity. We aim to overcome the pitfalls of manual neuron scoring by developing an automated, unbiased image processing algorithm to quantify dopaminergic neurodegeneration in C. elegans. The algorithm can be used on images acquired with different microscopy setups and only requires two inputs: a maximum projection image of the four cephalic neurons in the C. elegans head and the pixel size of the user’s camera. We validate the platform by detecting and quantifying neurodegeneration in nematodes exposed to rotenone, cold shock, and 6-hydroxydopamine using 63x epifluorescence, 63x confocal, and 40x epifluorescence microscopy, respectively. Analysis of tubby mutant worms with altered fat storage showed that, contrary to our hypothesis, increased adiposity did not sensitize to stressor-induced neurodegeneration. We further verify the accuracy of the algorithm by comparing code-generated, categorical degeneration results with manually scored dendrites of the same experiments. The platform, which detects 19 different metrics of neurodegeneration, can provide comparative insight into how each exposure affects dopaminergic neurodegeneration patterns.

Morphological hallmarks of dopaminergic neurodegeneration are associated with altered neuron function inCaenorhabditis elegans

“…Subsequently, an adapted version of our previously reported image processing algorithm, AUDDIT (Clark et al, 2023), would analyze and report the median break percent of each dendrite and the total number of blebs. Worms were then either sent to the collection chamber or trash chamber depending on the group being collected at the time.…”

“…Our approach to accomplish this goal integrated two technologies we have previously: a microfluidic device capable of imaging and sorting worms (D. F. Midkiff et al, 2022;San-Miguel et al, 2016) and an automated image processing algorithm used to detect CEP dendrite morphologies (Fig. 2A) (Clark et al, 2023). This semi-automated platform enabled worm imaging, automated dopaminergic dendrite analysis, and worm sorting at a throughput of up to 75 worms per hour.…”

“…The CEP dendrites in healthy nematodes are nearly straight lines with no apparent irregularities, particularly early in life. However, the CEP dendrites in worms exposed to 6-OHDA often exhibit clear breakage (the disappearance of dendrite) or blebbing (protrusions from the string-like dendrites) (Bijwadia et al, 2021;Clark et al, 2023;González-Hunt et al, 2014). To gain a better understanding of chemical-induced morphological changes in DA neurons, levels of dendritic degeneration have been categorized into a variety of classifications, from presence/absence to more nuanced scales such as 5-or 7-point systems (Bijwadia et al, 2021;Cothren et al, 2018).…”

“…We then tested whether worms with specific dendritic morphologies exhibited differences in functional behavior. In our previous study, we developed an automated image processing algorithm capable of rapidly detecting and quantifying levels of dopaminergic neurodegeneration from maximum intensity projections (MIP) (Clark et al, 2023). Thus, we integrated our previously developed algorithm, fluorescent microscopy, and a microfluidic device to create a semi-automated sorting platform.…”

The effect of common paralytic agents used for fluorescence imaging on redox tone and ATP levels in Caenorhabditis elegans

“…Cold shock at 2 C was only lethal after 12-hour exposures or longer, though cold shocked worms show decreased gonad size, loss of pigmentation, and increased vulval abnormalities after recovery [23]. Cold shock at 4 o C for 16 hours is capable of inducing blebbing of dopaminergic neurons, suggesting it may induce dopaminergic neurodegeneration [27]. As most paralytic uses of cold shock are far shorter in duration, it is unclear whether these effects occur in this timeframe.…”