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Strengths and limitations of morphological and behavioral analyses in detecting dopaminergic deficiency in Caenorhabditis elegans.

ABSTRACT: In order to develop a better understanding of the role environmental toxicants may play in the onset and progression of neurodegenerative diseases, it has become increasingly important to optimize sensitive methods for quickly screening toxicants to determine their ability to disrupt neuronal function. The nematode Caenorhabditis elegans can help with this effort. This species has an integrated nervous system producing behavioral function, provides easy access for molecular studies, has a rapid lifespan, and is an inexpensive model. This study focuses on methods of measuring neurodegeneration involving the dopaminergic system and the identification of compounds with actions that disrupt dopamine function in the model organism C. elegans. Several dopamine-mediated locomotory behaviors, Area Exploration, Body Bends, and Reversals, as well as Swimming-Induced Paralysis and Learned 2-Nonanone Avoidance, were compared to determine the best behavioral method for screening purposes. These behavioral endpoints were also compared to morphological scoring of neurodegeneration in the dopamine neurons. We found that in adult worms, Area Exploration is more advantageous than the other behavioral methods for identifying DA-deficient locomotion and is comparable to neuromorphological scoring outputs. For larval stage worms, locomotion was an unreliable endpoint, and neuronal scoring appeared to be the best method. We compared the wild-type N2 strain to the commonly used dat-1p::GFP reporter strains BY200 and BZ555, and we further characterized the dopamine-deficient strains, cat-2 e1112 and cat-2 n4547. In contrast to published results, we found that the cat-2 strains slowed on food almost as much as N2s. Both showed decreased levels of cat-2 mRNA and DA content, rather than none, with cat-2 e1112 having the greatest reduction in DA content in comparison to N2. Finally, we compared and contrasted strengths, limitations, cost, and equipment needs for all primary methods for analysis of the dopamine system in C. elegans.

SUBMITTER: Smith LL

PROVIDER: S-EPMC6751008 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Strengths and limitations of morphological and behavioral analyses in detecting dopaminergic deficiency in Caenorhabditis elegans.

Smith Latasha L LL Ryde Ian T IT Hartman Jessica H JH Romersi Riccardo F RF Markovich Zachary Z Meyer Joel N JN

Neurotoxicology 20190716

In order to develop a better understanding of the role environmental toxicants may play in the onset and progression of neurodegenerative diseases, it has become increasingly important to optimize sensitive methods for quickly screening toxicants to determine their ability to disrupt neuronal function. The nematode Caenorhabditis elegans can help with this effort. This species has an integrated nervous system producing behavioral function, provides easy access for molecular studies, has a rapid ...[more]

PMID: 31323240

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Project description:Dopamine neuron loss is involved in the pathology of Parkinson'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' transparency and invariant neuronal architecture. In particular, hermaphroditic C. elegans' 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' 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.

Project description:Perfluorooctane sulfonate (PFOS) has been widely utilized in numerous industries. Due to long environmental and biological half-lives, PFOS is a major public health concern. Although the literature suggests that PFOS may induce neurotoxicity, neurotoxic mechanisms, and neuropathology are poorly understood. Thus, the primary goal of this study was to determine if PFOS is selectively neurotoxic and potentially relevant to specific neurological diseases. Nematodes (Caenorhabditis elegans) were exposed to PFOS or related per- and polyfluoroalkyl substances (PFAS) for 72 h and tested for evidence of neuropathology through examination of cholinergic, dopaminergic, gamma-amino butyric acid (GABA)ergic, and serotoninergic neuronal morphologies. Dopaminergic and cholinergic functional analyses were assessed through 1-nonanol and Aldicarb assay. Mechanistic studies assessed total reactive oxygen species, superoxide ions, and mitochondrial content. Finally, therapeutic approaches were utilized to further examine pathogenic mechanisms. Dopaminergic neuropathology occurred at lower exposure levels (25 ppm, approximately 50 µM) than required to produce neuropathology in GABAergic, serotonergic, and cholinergic neurons (100 ppm, approximately 200 µM). Further, PFOS exposure led to dopamine-dependent functional deficits, without altering acetylcholine-dependent paralysis. Mitochondrial content was affected by PFOS at far lower exposure level than required to induce pathology (≥1 ppm, approximately 2 µM). Perfluorooctane sulfonate exposure also enhanced oxidative stress. Further, mutation in mitochondrial superoxide dismutase rendered animals more vulnerable. Neuroprotective approaches such as antioxidants, PFAS-protein dissociation, and targeted (mitochondrial) radical and electron scavenging were neuroprotective, suggesting specific mechanisms of action. In general, other tested PFAS were less neurotoxic. The primary impact is to prompt research into potential adverse outcomes related to PFAS-induced dopaminergic neurotoxicity in humans.

Project description:BackgroundNociception evokes a rapid withdrawal behavior designed to protect the animal from potential danger. C. elegans performs a reflexive reversal or forward locomotory response when presented with noxious stimuli at the head or tail, respectively. Here, we have developed an assay with precise spatial and temporal control of an infrared laser stimulus that targets one-fifth of the worm's body and quantifies multiple aspects of the worm's escape response.ResultsWhen stimulated at the head, we found that the escape response can be elicited by changes in temperature as small as a fraction of a degree Celsius, and that aspects of the escape behavior such as the response latency and the escape direction change advantageously as the amplitude of the noxious stimulus increases. We have mapped the behavioral receptive field of thermal nociception along the entire body of the worm, and show a midbody avoidance behavior distinct from the head and tail responses. At the midbody, the worm is sensitive to a change in the stimulus location as small as 80 ?m. This midbody response is probabilistic, producing either a backward, forward or pause state after the stimulus. The distribution of these states shifts from reverse-biased to forward-biased as the location of the stimulus moves from the middle towards the anterior or posterior of the worm, respectively. We identified PVD as the thermal nociceptor for the midbody response using calcium imaging, genetic ablation and laser ablation. Analyses of mutants suggest the possibility that TRPV channels and glutamate are involved in facilitating the midbody noxious response.ConclusionThrough high resolution quantitative behavioral analysis, we have comprehensively characterized the C. elegans escape response to noxious thermal stimuli applied along its body, and found a novel midbody response. We further identified the nociceptor PVD as required to sense noxious heat at the midbody and can spatially differentiate localized thermal stimuli.

Dataset Information

Strengths and limitations of morphological and behavioral analyses in detecting dopaminergic deficiency in Caenorhabditis elegans.

Publications

Strengths and limitations of morphological and behavioral analyses in detecting dopaminergic deficiency in Caenorhabditis elegans.

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