Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

Parkinson's disease (PD) is an increasingly common neurodegenerative disease affecting up to 10 million individuals worldwide¹. Males and older individuals are at a higher risk for developing PD; the average age of onset for the disease is 60 years, and PD incidence climbs from a 0.3% incidence in the general population to 3% in individuals over 80 years of age^1,2. Although the details of PD pathology are not fully understood, this progressive disorder involves the loss of dopaminergic neurons in the substantia nigra region of the midbrain. Hypothesized mechanisms of this neuronal loss involve mitochondrial dysfunction, oxidative stress, and inflammation². The causes and risk factors for the disease are still being explored, but involve a combination of environmental and genetic factors¹. For example, studies have found positive associations between lifelong pesticide use and PD, as well as genetic susceptibility to familial PD^1,3.

The C. elegans model system, originally developed in part for neurobiology research⁴, is well suited for evaluating dopaminergic neuron loss in vivo. Nass and colleagues pioneered the use of C. elegans for dopaminergic neurodegeneration⁵, and many groups have since adopted the worm as a successful model for PD and dopaminergic dysfunction^{6,7,8,9,10,11,12,13,14,15,16,17,18,19,20}. C. elegans are good neurodegenerative disease models for many of the same reasons that they are such a popular model organism for other areas of biology; their transparency allows for in vivo study of cellular processes, genetic manipulation in worms is relatively quick and easy, they have a short generation time of about three days, and they are easy to maintain²¹. Most PD worm models fall into one of three categories: age-based models, chemical models, and genetic models. The ability to synchronize a population of worms allows for the study of age-related neurodegeneration for an age based-model of neurodegenerative diseases associated with aging, such as PD²². Chemical exposures inducing PD-like neuronal defects have been established using a variety of chemicals including 6-hydroxydopamine (6-OHDA), rotenone, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)²². Worms are also successfully used as genetic models of PD; strains with select neural gene knockouts can model various neurodegenerative diseases^1,4. Combinations of genetic and environmental factors, or "gene-environment interactions," which likely play a major role in PD^{2,17,23,24,25,26,27,28}, have been examined by several groups using C. elegans. Finally, age-related dopaminergic neurodegeneration has also been observed^29,30. If using an appropriate neural transgenic strain in fluorescent imaging, any of these PD worm models may be used to study dopaminergic neurodegeneration.

Quantifying changes to neuronal morphology is a critical component of neurodegenerative research. In C. elegans, many fluorescent reporter strains have been used to visualize morphological changes and loss of neurons. Strains suitable for neuronal imaging feature a fluorescent protein associated with cell-specific promoters. For dopaminergic neurodegeneration assays, our laboratory has used the BY200 [vtIs1 (dat-1p::GFP, rol-6)] strain, which has a green fluorescent protein (GFP) tag in the dat-1 gene, expressed in the dopaminergic neurons. Note that the BY200's roller phenotype has a very low penetrance and is rarely observed. Other common strains used for this type of imaging include BY250 [dat-1p::GFP], BY273 [baEx18[dat-1p::GFP+dat-1p::WT α-syn]], BZ555 [egIs1 [dat-1p::GFP]], and several others available from the Caenorhabditis Genetics Center (CGC) or upon request from specific laboratories^1,21,22,29. These strains typically allow for visualization of all three classes of dopaminergic neurons: cephalic (CEP), anterior deirid (ADE), and postdeirid (PDE) neurons. C. elegans does not naturally express the alpha synuclein protein, but strains such as BY273 have been engineered to express it. However, we note that the scoring system we present was developed using BY200, which does not express alpha synuclein, and would need to be validated with that strain (or any other new strain) prior to use. Additional dopaminergic neurons are present in males but are rarely considered because males normally comprise <1% of a C. elegans population. Here, we focus on the four CEP dopaminergic neurons found in the head region of C. elegans. This set of neurons is easily located under fluorescent microscopy, is present in both hermaphrodite and male worms, does not typically overlap with other areas of auto-fluorescence, and is commonly reported on in worm studies. Notably, though these neurons are not myelinated, the CEP dorsal (CEPD) neurons are directly exposed to the pseudocoelomic body fluid where as the CEP ventral neurons are not. A healthy set of CEP dendrites typically displays as relatively straight, uninterrupted lines. Degenerated dendrites may show any combination of irregularities and signs of damage, including pronounced dots called blebs along the line of the dendrite and breaks in the line of the dendrite. Examples of CEP neurons at varying levels of degeneration can be seen in Figure 1.

Although dopaminergic neurodegeneration is being studied by a growing number of C. elegans laboratories, there has been a large variation in analytical methods of quantifying dopaminergic neuron damage^{29,31,32,33,34}. Many published studies have reported on the presence or absence of CEP soma with a binary scoring system of degenerative versus typical or wild type neurons^31,32. These scoring methods can identify certain stressors that induce neurodegeneration but cannot quantify the details of the progression of more subtle neuronal damage, or easily detect differences between neurodegeneration as induced by unique chemicals or other variables. Additionally, scoring systems focused on the cell bodies may not be sensitive to less severe levels of damage or to neuronal damage affecting only part of the cell, such as the dendrite. Since the dendrite appears to have the largest range of consistently detectable morphologic changes in response to chemical stressors, we have selected them as the basis for our analysis. The scoring system we present here is modified from dendrite morphology based multi-point scales that have been previously used in our lab^29,33. This system expands these five- and six-point scales into a seven-point scale to account for age-related morphological changes, such as higher expected numbers of kinks in older adult dendrites, and to differentiate between severe damage and complete dendrite loss. The purpose of introducing this scoring system is to provide the ability to capture a comprehensive picture of neurodegeneration at all levels of neuronal damage and provide a universal system to support consistency across C. elegan dopaminergic neurodegeneration research. Because scoring is inherently subjective, it is critical to maximize consistency between individuals scoring, and to blind the scorer to the identity of the images using manual blinding or an automatic blinding program³⁵. To improve consistency, we present a series of training images and utilize JoVE's video capabilities to demonstrate our scoring system in detail. We recommend using a system that both permits automated blinded scoring and allows the scorer to quantify her or his scoring consistency by re-scoring a subset of images. This is particularly important when combining or comparing data from multiple scientists, or training scientists new to scoring.