We wish to acknowledge Ian T. Ryde, for contributions to the development of the scoring scale and for his support during the creation of this manuscript. This work was supported by the National Institutes of Health (T32ES021432 supported KSM, and P42ES010356 to JNM).

1. Prepare Worms for Imaging
NOTE: See related JoVE video article31:&#160;https://www.jove.com/v/835/&#160;
<ol>
	<li>For each experimental group, pipette or pick 20 to 30 worms to an imaging platform compatible with the imaging microscope. Most common platforms include 2% agarose gel pads mounted on glass slides with a coverslip31 and 96-well plates containing well volumes at or less than 100 &#181;L of liquid medium.</li>
	<li>Paralyze the worms by adding 30-90 mM sodium azide (NaN3), 2.5-8.5 mM levamisole HCl, or other paralyzing agent to the worms. If paralyzing in liquid, use a higher concentration of paralyzing agent than if paralyzing on agarose pads. Tap imaging platform gently to mix.</li>
	<li>Allow worms to paralyze completely. 
	NOTE: This may take several minutes.</li>
</ol>
2. Image Dopaminergic Neurons
<ol>
	<li>Locate worms&#39; head regions under single-color GFP fluorescence using imaging microscope capable of taking z-stacks.
	<ol>
		<li>Be mindful of exposure and aperture settings; avoid overexposure of dendrites and keep settings consistent across trials. Make the dendrites as bright as needed for clear visualization; this typically results in overexposure of the soma. 
		NOTE: Images included in this protocol were captured using 400x magnification.</li>
	</ol>
	</li>
	<li>Scroll through the focus to find upper and lower bounds where the dendrites are clear. Set these as upper and lower bounds for a z-stack image capture.</li>
	<li>Click to capture z-stack images for each worm. 
	&#8203;NOTE: All following steps may be performed at any time.</li>
</ol>
3. Prepare Dopaminergic Neuron Images for Scoring
<ol>
	<li>For each z-stack, open the image file using either the microscope software or an external image analysis software, load the stack in the software, and compress the stack into a single flattened image.</li>
	<li>Blind images between and within treatment groups manually or using automatic blinding software.</li>
</ol>
4. Score Dopaminergic Neuron Dendrites
<ol>
	<li>Work with one neuron image at a time. Choose one of the four CEP dendrites to assess for blebs, breaks, and irregularities including bends, kinks, and curves. Scoring from the left to right when the nose is at the top of the image is recommended to ensure repeatability in scoring.</li>
	<li>Using the following guidelines, assign one score value to the dendrite. See Figure 1 for representative scoring image examples. 
	0- no damage, &#34;perfect&#34; neurons 
	1- irregular (kinks, curves, etc.) 
	2- &#60;5 blebs 
	3- 5-10 blebs 
	4- &#62;10 blebs and/or breakage removing &#60;25% of total dendrite 
	5- breakage, 25-75% of dendrite removed 
	6- &#8203;breakage, &#62;75% dendrite removed
	<ol>
		<li>If multiple criteria are met within a single dendrite (i.e., kinks and blebs), assign the highest applicable score.</li>
		<li>Do not score dendrites that are not clearly visible, due to issues with image capture, overlapping with other dendrites, etc. If zooming in on a flattened z stack image, be mindful of enlarged pixels resembling false blebs.</li>
	</ol>
	</li>
	<li>Repeat for each dendrite. Repeat for all images.</li>
	<li>Record all scores. Scores may be un-blinded at this time.</li>
</ol>
5. Prepare and Present Data
<ol>
	<li>Calculate the total number of dendrites in each treatment group assigned to each neurodegeneration score. Calculate the total number of scored dendrites in each treatment group.</li>
	<li>Divide the neurodegeneration score tallies by the total number of dendrites scored in the treatment group. Present data as a proportion of dendrites within a treatment group at each neurodegeneration score.</li>
</ol>
6. Perform Statistical Analysis
<ol>
	<li>Using a programming software or manually, run a chi-squared test for independence between all treatment group pairs to be compared. When appropriate, apply a Bonferroni correction of the p-value according to the number of compared experimental groups to account for multiple comparisons. 
	NOTE: This test will determine significant differences between two groups, but details of the type of difference must be qualified by eye.
	<ol>
		<li>Select comparisons between experimental groups. This will vary based on experimental design. 
		&#8203;NOTE: In our experiments, typically, controls are compared to their respective treatment groups, all controls are compared, and all treatments are compared.</li>
	</ol>
	</li>
</ol>
7. Practice Scoring with Practice Set of Dopaminergic Neuron Images
<ol>
	<li>See Supplementary File 1 for a set of neuron images presenting across the full range of our scoring system with commentary and score key. This practice set is intended to train researchers new to this method and ensure inter-rater reliability.</li>
</ol>
8. Consider Alternate Protocol Options
<ol>
	<li>Instead of capturing z-stacks, complete scoring at the microscope, without saving or stacking images. 
	NOTE: This option reduces requirements for technology capabilities, but removes the option for creating an archive of neuron images to return to at a later time, requires manual blinding, and permits blinding only between and not within treatment groups.</li>
	<li>Instead of creating a single compressed image per stack, complete scoring by scrolling through the images of each z-stack. 
	NOTE: This option may be easier for some scorers and it reduces the risk for seeing false blebs on worms that moved during imaging and allows for scoring overlapping dendrites, but requires manual blinding and permits blinding only between and not within treatment groups.</li>
</ol>

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The authors have no disclosures.

This protocol demonstrates how to use the seven-point scale developed in our laboratory to quantify levels of dopaminergic neuron morphologic alteration and degeneration in C. elegans. We created and shared this scale as a tool to standardize analysis of dopaminergic neurodegeneration work in worms. Recognizing the importance of studying pathways involved in highly prevalent neurodegenerative diseases, many investigators take advantage of the C. elegans model&#39;s suitability for neurobiology visualization to study neurodegeneration29,31,32,33. However, there has yet to be an effort to reduce the large variation in how neuron damage is quantified across neurodegeneration research in worms. The scoring system presented here is thus intended to promote consistency in analyses and allow for comparison between studies.
Our scoring system may be used to analyze data derived from C. elegans experiments that use cell-specific fluorescent reporters that allow for visualization of dopaminergic neurons - specifically the CEP dendrites. Specifically, strains tagged at the dat-1 gene for GFP visualization of the dopaminergic neurons are compatible with this scoring protocol, though many other related transgenic models of PD do exist. It is possible that this scoring system would also be useful with those models; however, this should be validated prior to using them. In particular, it is possible (but not tested to our knowledge) strains with mCherry may not be well suited for this protocol as mCherry aggregation may be indistinguishable from blebs or lead to cell stress. Rather than providing a commentary on all specific models of PD and related neurodegenerative disorders, we focus on the scoring of neurodegeneration data itself. Additionally, this protocol focuses only on neuronal morphology and does not consider fluorescence levels of the soma. Neurodegeneration assays may be performed alongside behavioral assays relevant to neurodegenerative diseases, such as locomotion, lifespan, and health-span experiments. Levels of degeneration in established chemical, age-based, and genetic models of PD can also be confirmed and detailed using this scoring system. Measuring models, contributors, and pathways associated with PD and other neurodegenerative diseases can add to the scientific knowledge about these disorders and point towards how to manage the growing population of affected individuals. Having comparable neurodegeneration results across literature is key in supporting this goal.
To interpret the results derived from this scoring system, we propose considering each dendrite scored as n=1, because different neurons within the same worm often respond differently to treatment. This may be driven by the fact that only the CEPD neurons are directly exposed to the pseudocoelomic body fluid. As such, this allows the score spread of experimental groups to be displayed as proportions of the total number of dendrites scored in each group. This method, used for the representative results shown here, allows for easy comparison across treatment groups, accounts for differential responses within the same worm, and is easily analyzed with a Chi-squared test complimented by a Bonferroni correction for multiple comparisons. An example template for recording neuron scores and calculating percentages can be found in Supplementary File 2. We have considered two alternate methods for data analysis and identify flaws in each. The first option is averaging the scores of the four CEP neurons for each worm. This parametrizes the data; however, it assumes a linear relationship with increasing score and loses information about any variations in response to treatment within the same worm. The second option is to sum the scores of all four CEP neurons for each worm, which also parametrizes the data. This still assumes a linear relationship between scores, however it more capably accounts for differences within each worm than averaged scores by expanding the parameters of possible scores. However individual researchers decide to display their data, the results should be considered alongside experimental variables such as strain and worm age; for example, older worms have a higher expected baseline level of degeneration.
As these neurodegeneration score results are interpreted, researchers should also be aware of a few caveats and limitations of the scoring method. First, certain technological requirements are necessary to capture images suitable for scoring. The imaging microscope must support fluorescence channels and magnification and exposure settings that allow for clear visualization of CEP dendrites. As noted in the protocol, technological requirements may be reduced by protocol adjustments like scoring live images through the microscopic field rather than capturing images to be archived and scored at a later time. Second, possible statistical analysis methods for this data are limited as the data is non-parametric. The scoring scale is presumed to be progressive, but cannot be considered numeric since there are discrete score options and score increases are not necessarily proportional to each other with respect to biological function. For these reasons, chi-squared tests for independence are best suited for this type of data, meaning the statistical analysis depends on the observer to determine the direction of any statistical significance. Notably, the chi-squared test also only analyzes for differences in score distribution and is unable to provide evidence of differences in specific scoring categories. Finally, the functional significance of the morphological changes quantified by this scoring system have yet to be studied.
The future directions prompted by the development of this scoring system involve determining biological bases and correlations with individual neuron scores. Studying the functional significance (e.g. neuronal signaling, worm behavior) of all points on the scoring scale will inform how to better translate results to conclusions applicable to understanding the causes and consequences of neurodegenerative diseases and developing prevention and treatment options. Future research on neurodegeneration in worms should aim to discover connections to other morphology, such as worm shape and size. Additionally, neurodegeneration research can be supported by studying other reporter C. elegans strains to measure endpoints such as bioenergetics, reactive oxygen species production, and mitochondrial morphology.

Parkinson&#39;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 age1,2. 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 PD1,3.
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 dysfunction6,7,8,9,10,11,12,13,14,15,16,17,18,19,20. 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 diseases1,4. Combinations of genetic and environmental factors, or &#34;gene-environment interactions,&#34; which likely play a major role in PD2,17,23,24,25,26,27,28, have been examined by several groups using C. elegans. Finally, age-related dopaminergic neurodegeneration has also been observed29,30. 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&#39;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 &#945;-syn]], BZ555 [egIs1 [dat-1p::GFP]], and several others available from the Caenorhabditis Genetics Center (CGC) or upon request from specific laboratories1,21,22,29. 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 &#60;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 damage29,31,32,33,34. 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 neurons31,32. 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 lab29,33. 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&#39;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well plate</td>
			<td>VWR</td>
			<td>29442-056</td>
			<td>For imaging in wells</td>
		</tr>
		<tr>
			<td>Blinder</td>
			<td>Solibyte Solutions LLC</td>
			<td></td>
			<td>Free software that blinds between and within uploaded sets of image files</td>
		</tr>
		<tr>
			<td>BY200 [vtIs1 (dat-1p::GFP, rol-6)]</td>
			<td>Aschner Lab</td>
			<td></td>
			<td>C. elegans strain suitable for dopaminergic neuron fluorescent imaging. May be subsituted by other strains with a fluorescent reporter driven by cell-specific promotors 
			Available upon request from the Meyer lab</td>
		</tr>
		<tr>
			<td>complete K-medium</td>
			<td></td>
			<td></td>
			<td>51 mM sodium chloride, 32 mM potassium chloride, 3 mM calcium chloride, 3 mM magnesium sulfate, 13 mM cholesterol</td>
		</tr>
		<tr>
			<td>Coverslips 22x22mm, No.1 glass</td>
			<td>VWR VistaVision</td>
			<td>48366-067</td>
			<td>For imaging on slides</td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>472301</td>
			<td>Solvent for rotenone exposures</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td>ImageJ 1.5e or newer. Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https:// imagej.nih.gov/ij/, 1997-2016.</td>
			<td>Sotware for image manipulation</td>
		</tr>
		<tr>
			<td>Keyence BZ-X All-in-one Fluoresence Microscope</td>
			<td>Keyence</td>
			<td></td>
			<td>Used for fluorescent dopaminergic neuron image capture. May be substituted by other microscopes stuitable for fluorescent, high-resolution imaging</td>
		</tr>
		<tr>
			<td>Microscope Slides 3x1&#34;</td>
			<td>VWR VistaVision</td>
			<td>16004-420</td>
			<td>For imaging on slides</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875</td>
			<td>Electron transport chain complex I inhibitor</td>
		</tr>
		<tr>
			<td>Sodium Azide (NaN_3)</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td>Paralytic</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td>For bleach lysis</td>
		</tr>
		<tr>
			<td>Sodium Hypochlorite</td>
			<td>VWR</td>
			<td>RC7495.5-32</td>
			<td>For bleach lysis</td>
		</tr>
		<tr>
			<td>Tetramisole (Levamisole) Hydrochloride (HCl)</td>
			<td>Sigma-Aldrich</td>
			<td>L9756</td>
			<td>Paralytic</td>
		</tr>
	</tbody>
</table>

quantifying levels of dopaminergic neuron morphological alteration and degeneration in caenorhabditis elegans

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million people worldwide. Since many details about PD etiology remain unknown, studies investigating genetic and environmental contributors to PD are needed to discover methods of prevention, management, and treatment. Proper characterization of dopaminergic neuronal loss may be relevant not only to PD research, but to other increasingly prevalent neurodegenerative disorders.
There are established genetic and chemical models of dopaminergic neurodegeneration in the Caenorhabditis elegans model system, with easy visualization of neurobiology supported by the nematodes&#39; transparency and invariant neuronal architecture. In particular, hermaphroditic C. elegans&#39; dopaminergic neuron morphological changes can be visualized using strains with fluorescent reporters driven by cell-specific promotors such as the dat-1 dopamine transporter gene, which is expressed exclusively in their eight dopaminergic neurons.
With the capabilities of this model system and the appropriate technology, many laboratories have studied dopaminergic neurodegeneration. However, there is little consistency in the way the data is analyzed and much of the present literature uses binary scoring analyses that capture the presence of degeneration but not the full details of the progression of neuron loss. Here, we introduce a universal scoring system to assess morphological changes and degeneration in C. elegans&#39; cephalic neuron dendrites. This seven-point scale allows for analysis across a full range of dendrite morphology, ranging from healthy neurons to complete dendrite loss, and considering morphological details including kinks, branching, blebs, and breaks. With this scoring system, researchers can quantify subtle age-related changes as well as more dramatic chemical-induced changes. Finally, we provide a practice set of images with commentary that can be used to train, calibrate, and assess the scoring consistency of researchers new to this method. This should improve within- and between- laboratory consistency, increasing rigor and reproducibility.

The scoring system described here was used to assess neurodegeneration in L4 larval stage BY200 [vtIs1 (dat-1p::GFP, rol-6)] C. elegans after rotenone exposure. Results of this experiment are shown in Figure 2 and represent our scoring system&#39;s ability to detect and quantify variable levels of dopaminergic neuron damage. Rotenone is a naturally occurring electron transport chain complex I inhibitor used in some pesticides, piscicides, and insecticides36,37. Note that working with toxic chemicals such as rotenone is inherently dangerous, and all labs should comply with all use and disposal regulations set by their institutions. In this experiment, liquid rotenone exposures at two doses, 0.03 &#181;M and 0.5 &#181;M, along with a control group, were begun immediately following a 0.5 M sodium hydroxide/1% sodium hypochlorite lysis to harvest eggs38. Eggs hatched in complete K-medium33,39 with 0.25% dimethyl sulfoxide (DMSO), and worms remained in liquid for ~48 hours until mid-L4 larval stage, at which point they were removed from chemical exposure, and prepared, imaged, and, using Figure 1 as a reference, scored according to the protocol steps above. For the higher dose of 0.5 &#181;M rotenone, eggs were harvested 24 hours in advance to account for a rotenone-induced developmental delay and ensure all worms were stage synchronized at the time of imaging.
Figure 2&#160;further demonstrates how our laboratory visualizes data collected using this scoring system. In this figure, a dose-dependent neurodegeneration response can be appreciated, and the specific breakdown of the score distribution is displayed clearly. These particular results showcase how neuronal damage can present in different ways. For example, the 0.03 &#181;M rotenone-exposed group has a decreased proportion of healthy neurons with a score of 0, as compared to the control group, yet also has a decreased proportion of 5 scores. Detecting this detail about the score distributions between experimental groups highlights the sensitivity of our seven-point scoring system. This data was analyzed for statistical significance according to the protocol, using a chi-squared test for independence with a Bonferroni correction.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62894/62894fig01.jpg" /> 
Figure 1. Dopamine neuron morphological alteration and degeneration scoring system representative images. This consolidated chart contains examples of neurons at each score and is intended to be used as a reference for scoring. Here, each labelled score corresponds to the most damaged dendrite in each worm, as indicated by the arrow in each panel. These images were taken using the protocol described in this paper with BY200 C. elegans. Please see Supplementary File 1 for a set of scored images with commentary to be used for training those new to this scoring method. <a href="https://www.jove.com/files/ftp_upload/62894/62894fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62894/62894fig02.jpg" /> 
Figure 2. BY200 L4 dopamine neuron morphology and degeneration scores after rotenone exposure. This figure shows representative results analyzed using the scoring methods described here. The visualized greater proportions of damaged dopaminergic neurons with higher rotenone exposure concentrations were statistically analyzed using chi-squared tests for independence. Both rotenone treatment groups yielded statistically significant p-values when compared to the control group. Different letters indicate statistical difference. <a href="https://www.jove.com/files/ftp_upload/62894/62894fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1. <a href="https://www.jove.com/files/ftp_upload/62894/Supplemental_Training_Set_KM_V5.docx" target="_blank">Please click here to download this file.</a>
Supplementary File 2. <a href="https://www.jove.com/files/ftp_upload/62894/SupplementaryFile2.xlsx" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans at JoVE.com

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

In this article, we showcase how to use a seven-point scoring system to consistently quantify changes to dopaminergic neuron dendrite morphology in C. elegans. This system is intended for analyses of dopaminergic neurodegeneration assays utilizing genetic, chemical, and age-based models of neurodegenerative disorders.

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

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million ...

quantifying-levels-dopaminergic-neuron-morphological-alteration

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JoVE Journal

Neuroscience

4.0K Views.  Duke University. In this article, we showcase how to use a seven-point scoring system to consistently quantify changes to dopaminergic neuron dendrite morphology in C. elegans. This system is intended for analyses of dopaminergic neurodegeneration assays utilizing genetic, chemical, and age-based models of neurodegenerative disorders. 

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

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the substantia nigra, is essential. To estimate the total dopaminergic neuron number, unbiased stereology using the optical fractionator method is currently considered the gold standard. Because the theory behind the optical fractionator method is complex and because stereology is difficult to achieve without specialized equipment, several commercially available complete stereology systems that include the necessary software do exist, purely for cell counting reasons. Since purchasing a specialized stereology setup is not always feasible, for many reasons, this report describes a method for the stereological estimation of dopaminergic neuronal cell counts using standard microscopy equipment, including a light microscope, a motorized object table (x, y, z plane) with imaging software, and a computer for analysis. A step-by-step explanation is given on how to perform stereological quantification using the optical fractionator method, and pre-programmed files for the calculation of estimated cell counts are provided. To assess the accuracy of this method, a comparison to data obtained from a commercially available stereology apparatus was performed. Comparable cell numbers were found using this protocol and the stereology device, thus demonstrating the precision of this protocol for unbiased stereology.

This work presents a step-by-step protocol for the unbiased stereological estimation of dopaminergic neuronal cell numbers in the mouse substantia nigra using standard microscopy equipment (i.e., a light microscope, a motorized object table (x, y, z plane), and public domain software for digital image analysis.

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the ...

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal degeneration are remarkably similar in humans and invertebrates as are the molecular mechanisms that drive these processes. The fruit fly, Drosophila melanogaster, provides a powerful yet simple genetic model organism to study the cellular complexities of neurodegenerative diseases. In fact, approximately 70% of disease-associated human genes have a Drosophila homolog and a plethora of tools and assays have been described using flies to study human neurodegenerative diseases. More specifically the neuromuscular junction (NMJ) in Drosophila has proven to be an effective system to study neuromuscular diseases because of the ability to analyze the structural connections between the neuron and the muscle. Here, we report on an in vivo motor neuron injury assay in Drosophila, which reproducibly induces neurodegeneration at the NMJ by 24 h. Using this methodology, we have described a temporal sequence of cellular events resulting in motor neuron degeneration. The injury method has diverse applications and has also been utilized to identify specific genes required for neurodegeneration and to dissect transcriptional responses to neuronal injury.

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

Here we describe a simple and widely accessible method to injure segmental nerves in Drosophila larvae to visualize and quantify neurodegeneration of motor neurons at the neuromuscular junction (NMJ) of third instar larvae.

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...