Email this article 
Three-Dimensional Arenas for the Assessment of Caenorhabditis elegans Behavior
Vol 8, Issue 4, 2022, Article identifier:610
VIEWS - 303 (Abstract) 228 (PDF) 151 (Supp.File)
Abstract
Caenorhabditis elegans nematode is a well-established model organism in numerous fields of experimental biology. In nature, C. elegans live in a rich three-dimensional (3D) environment. However, their behavior has been assessed almost exclusively on the open, flat surface of nematode growth medium (NGM) plates, the golden standard for C. elegans culture in the laboratory. We present two methods to build 3D behavioral arenas for C. elegans, by casting and by directly 3D-printing NGM hydrogel. The latter is achieved using a highly customized fused deposition modeling (FDM) 3D printer, modified to employ NGM hydrogel as ink. The result is the advancement of 3D complexity of behavioral assays. To demonstrate the potential of our method, we use the 3D-printed arenas to assess C. elegans physical barriers crossing. C. elegans decision to cross physical obstacles is affected by aging, physiological status (i.e., starvation), and prior experience. The 3D-printed structures can be used to spatially confine C. elegans behaviors, that is, egg laying. We consider these findings a decisive step toward characterizing C. elegans 3D behavior, an area long overlooked due to technical constrains. We envision our method of 3D-printing NGM arenas as a powerful tool in behavioral neurogenetics, neuroethology, and invertebrate model organisms’ neurobiology.
Keywords
References
Tissenbaum HA, 2015, Using C. elegans for Aging Research. Invertebr Reprod Dev, 59:59–63. https://doi.org/10.1080/07924259.2014.940470
Corsi AK, Wightman B, Chalfie M, 2015, A Transparent Window into Biology: A Primer on Caenorhabditis elegans. Genetics, 200:387–407. https://doi.org/10.1534/genetics.115.176099
Kaletta T, Hengartner MO, 2006, Finding Function in Novel Targets: C. elegans as a Model Organism. Nat Rev Drug Discov, 5:387–99. https://doi.org/10.1038/nrd2031
Rankin CH, Beck CD, Chiba CM, 1990, Caenorhabditis elegans: A New Model System for the Study of Learning and Memory. Behav Brain Res, 37:89–92. https://doi.org/10.1016/0166-4328(90)90074-o
Frézal L, Félix MA, 2015, C. elegans Outside the Petri Dish. Elife, 4:e05849. https://doi.org/10.7554/eLife.05849
Stiernagle T, 2006, Maintenance of C. elegans. Pasadena, CA: WormBook In: WormBook.
Gourgou E, Adiga K, Goettemoeller A, et al., 2021, Caenorhabditis elegans Learning in a Structured Maze is a Multisensory Behavior. iScience, 24:102284. https://doi.org/10.1016/j.isci.2021.102284
Gourgou E, Hsu AL, 2021, A Maze Platform for the Assessment of Caenorhabditis elegans Behavior and Learning. STAR Protoc, 2:100829. https://doi.org/10.1016/j.xpro.2021.100829
Han B, Dong Y, Zhang L, et al., 2017, Dopamine Signaling Tunes Spatial Pattern Selectivity in C. elegans. Elife, 6:e22896. https://doi.org/10.7554/eLife.22896
Bilbao A, Patel AK, Rahman M, et al., 2018, Roll Maneuvers are Essential for Active Reorientation of Caenorhabditis elegans in 3D Media. Proc Natl Acad Sci, 115:E3616–25 https://doi.org/10.1073/pnas.170675411
Shaw M, Zhan H, Elmi M, et al., 2018, Three-dimensional Behavioural Phenotyping of Freely Moving C. elegans using Quantitative Light Field Microscopy. PLoS One, 13:e0200108. https://doi.org/10.1371/journal.pone.0200108
Kwon N, Hwang AB, You YJ, et al., 2015, Dissection of C. elegans Behavioral Genetics in 3-D Environments. Sci Rep, 5:9564.
Kwon N, Pyo J, Lee SJ, et al., 2013, 3-D Worm Tracker for Freely Moving C. elegans. PloS One, 8:e57484. https://doi.org/10.1371/journal.pone.0057484
Li H, Tan C, Li L, 2018, Review of 3D Printable Hydrogels and Constructs. Mater Des, 159:20–38. https://doi.org/10.1016/j.matdes.2018.08.023
Li J, Wu C, Chu PK, et al., 2020, 3D Printing of Hydrogels: Rational Design Strategies and Emerging Biomedical Applications. Mater Sci Eng R Rep, 140:100543. https://doi.org/10.1016/j.mser.2020.100543
Spicer CD, 2020, Hydrogel Scaffolds for Tissue Engineering: The Importance of Polymer Choice. Polym Chem, 11:184–219. https://doi.org/10.1039/C9PY01021A
Unagolla JM, Jayasuriya AC, 2020, Hydrogel-based 3D Bioprinting: A Comprehensive Review on Cell-Laden Hydrogels, Bioink Formulations, and Future Perspectives. Appl Mater Today, 18:100479. https://doi.org/10.1016/j.apmt.2019.100479
Stein GM, Murphy CT, 2012, The Intersection of Aging, Longevity Pathways, and Learning and Memory in C.elegans. Front Genet, 3:259. https://doi.org/10.3389/fgene.2012.00259
Glenn CF, Chow DK, David L, et al., 2004, Behavioral Deficits During Early Stages of Aging in Caenorhabditis elegans Result From Locomotory Deficits Possibly Linked to Muscle Frailty. J Gerontol A Biol Sci Med Sci, 59:1251–60. https://doi.org/10.1093/gerona/59.12.1251
Golden TR, Hubbard A, Dando C, et al., 2008, Agerelated Behaviors have Distinct Transcriptional Profiles in Caenorhabditis elegans. Aging Cell, 7:850–65. https://doi.org/10.1111/j.1474-9726.2008.00433.x
Tahernia M, Mohammadifar M, Choi S, 2020, Paper-supported High-throughput 3D Culturing, Trapping, and Monitoring of Caenorhabditis elegans. Micromachines (Basel), 11:99. https://doi.org/10.3390/mi11010099
Lockery SR, Lawton KJ, Doll JC, et al., 2008, Artificial Dirt: Microfluidic Substrates for Nematode Neurobiology and Behavior. J Neurophysiol, 99:3136–43. https://doi.org/10.1152/jn.91327.2007
Lee TY, Yoon KH, Lee JI, 2016, Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory. J Vis Exp, 118:55048. https://doi.org/10.3791/55048
Jiang T, Munguia-Lopez JG, Flores-Torres S, et al., 2019, Extrusion Bioprinting of Soft Materials: An Emerging Technique for Biological Model Fabrication. Appl Phys Rev, 6:011310. https://doi.org/10.1063/1.5059393
Ng WL, Huang X, Shkolnikov V, et al., 2021, Controlling Droplet Impact Velocity and Droplet Volume: Key Factors to Achieving High Cell Viability in Sub-nanoliter Droplet-based Bioprinting. Int J Bioprint, 8:424. https://doi.org/10.18063/ijb.v8i1.424
Li X, Liu B, Pei B, et al., 2020, Inkjet Bioprinting of Biomaterials. Chem Rev, 120:10793–833. https://doi.org/10.1021/acs.chemrev.0c00008
Zhuang P, Ng WL, An J, et al., 2019, Layer-by-layer Ultraviolet Assisted Extrusion-based (UAE) Bioprinting of Hydrogel Constructs with High Aspect Ratio for Soft Tissue Engineering Applications. PLoS One, 14:e0216776. https://doi.org/10.1371/journal.pone.0216776
Li W, Mille LS, Robledo JA, et al., 2020, Recent Advances in Formulating and Processing Biomaterial Inks for Vat Polymerization-based 3D Printing. Adv Healthc Mater, 9:2000156. https://doi.org/10.1002/adhm.202000156
Tavana H, Mosadegh B, Takayama S, 2010, Polymeric Aqueous Biphasic Systems for Non-contact Cell Printing on Cells: Engineering Heterocellular Embryonic Stem Cell Niches. Adv Mater, 22:2628–31. https://doi.org/10.1002/adma.200904271
Chung JHY, Naficy S, Yue Z, et al., 2013, Bio-ink Properties and Printability for Extrusion Printing Living Cells. Biomater Sci, 1:763–73. https://doi.org/10.1039/C3BM00012E
He Y, Yang F, Zhao H, et al., 2016, Research on the Printability of Hydrogels in 3D Bioprinting. Sci Rep, 6:29977.
Yan Y, Wang X, Xiong Z, et al., 2005, Direct Construction of a Three-dimensional Structure with Cells and Hydrogel. J Bioact Compat Polym, 20:259–69. https://doi.org/10.1177/0883911505053658
Ozbolat IT, Hospodiuk M, 2016, Current Advances and Future Perspectives in Extrusion-based Bioprinting. Biomaterials, 76:321–43. https://doi.org/10.1016/j.biomaterials.2015.10.076
Kessel B, Lee M, Bonato A, et al., 2020, 3D Bioprinting of Macroporous Materials Based on Entangled Hydrogel Microstrands. Adv Sci, 7:2001419.
Stanton MM, Samitier J, Sánchez S, 2015, Bioprinting of 3D Hydrogels. Lab Chip, 15:3111–5. https://doi.org/10.1039/c5lc90069g
Fan R, Piou M, Darling E, et al., 2016, Bio-printing Cell-laden Matrigel–agarose Constructs. J Biomater Appl, 31:684–92. https://doi.org/10.1177/0885328216669238
Fan D, Staufer U, Accardo A, 2019, Engineered 3D Polymer and Hydrogel Microenvironments for Cell Culture Applications. Bioengineering (Basel), 6:113. https://doi.org/10.3390/bioengineering6040113
Gross BC, Erkal JL, Lockwood SY, et al., 2014, Evaluation of 3D Printing and its Potential Impact on Biotechnology and the Chemical Sciences. Anal Chem, 86:3240–53. https://doi.org/10.1021/ac403397r
Landers R, Hübner U, Schmelzeisen R, et al., 2002, Rapid Prototyping of Scaffolds Derived from Thermoreversible Hydrogels and Tailored for Applications in Tissue Engineering. Biomaterials, 23:4437–47. https://doi.org/10.1016/s0142-9612(02)00139-4
White JG, Southgate E, Thomson JN, et al., 1986, The Structure of the Nervous System of the Nematode Caenorhabditis elegans. Philos Trans R Soc Lond B Biol Sci, 314:1–340.
Schulenburg H, Félix MA, 2017, The Natural Biotic Environment of Caenorhabditis elegans. Genetics, 206:55-86. https://doi.org/10.1534/genetics.116.195511
Kauffman AL, Ashraf JM, Corces-Zimmerman MR, et al., 2010, Insulin Signaling and Dietary Restriction Differentially Influence the Decline of Learning and Memory with Age. PLoS Biol, 8:e1000372. https://doi.org/10.1371/journal.pbio.1000372
Hsu AL, Feng Z, Hsieh MY, et al., 2009, Identification by Machine Vision of the Rate of Motor Activity Decline as a Lifespan Predictor in C. elegans. Neurobiol Aging, 30:1498–503.chttps://doi.org/10.1016/j.neurobiolaging.2007.12.007
Newell Stamper BL, Cypser JR, Kechris K, et al., 2018, cMovement Decline Across Lifespan of Caenorhabditis elegans Mutants in the Insulin/Insulin-like Signaling Aging Cell, 17:e12704. https://doi.org/10.1111/acel.12704
Stern S, Kirst C, Bargmann CI, 2017, Neuromodulatory Control of Long-term Behavioral Patterns and Individuality Across Development. Cell, 171:1649–62.e10. https://doi.org/10.1016/j.cell.2017.10.041
Ahadi S, Zhou W, Schüssler-Fiorenza Rose SM, et al., 2020, Personal Aging Markers and Ageotypes Revealed by Deep Longitudinal Profiling. Nat Med, 26:83–90. https://doi.org/10.1038/s41591-019-0719-5
Schreiber MA, Pierce-Shimomura JT, Chan S, et al., 2010, Manipulation of Behavioral Decline in Caenorhabditis elegans with the Rag GTPase raga-1. PLoS Genet, 6:e1000972. https://doi.org/10.1371/journal.pgen.1000972
Hills T, Brockie PJ, Maricq AV, 2004, Dopamine and Glutamate Control Area-restricted Search Behavior in Caenorhabditis elegans. J Neurosci, 24:1217–25. https://doi.org/10.1523/JNEUROSCI.1569-03.2004
Tsalik EL, Hobert O, 2003, Functional Mapping of Neurons that Control Locomotory Behavior in Caenorhabditis elegans. J Neurobiol, 56:178–97. https://doi.org/10.1002/neu.10245
Wakabayashi T, Kitagawa I, Shingai R, 2004, Neurons Regulating the Duration of Forward Locomotion in Caenorhabditis elegans. Neurosci Res, 50:103–11. https://doi.org/10.1016/j.neures.2004.06.005
Gray JM, Hill JJ, Bargmann CI, 2005, A Circuit for Navigation in Caenorhabditis elegans. Proc Natl Acad Sci U S A, 102:3184–91. https://doi.org/10.1073/pnas.0409009101
López-Cruz A, Sordillo A, Pokala N, et al., 2019, Parallel Multimodal Circuits Control an Innate Foraging Behavior. Neuron, 102:407–19.e8. https://doi.org/10.1016/j.neuron.2019.01.053
Ghosh DD, Sanders T, Hong S, et al., 2016, Neural Architecture of Hunger-dependent Multisensory Decision Making in C. elegans. Neuron, 92:1049–62.
Schafer WR, 2005, Egg-laying. In: WormBook: The Online Review of C. elegans Biology. Pasadena, CA: WormBook.
Trent C, 1983, Genetic and Behavioral Studies of the Egg laying System in Caenorhabditis elegans. Thesis (Ph. D.). Cambridge, MA: Massachusetts Institute of Technology.
Brenner S, 1974, The Genetics of Caenorhabditis elegans. Genetics, 77:71–94.
Duran C, Subbian V, Giovanetti MT, et al., 2015, Experimental Desktop 3D Printing using Dual Extrusion and Water-soluble Polyvinyl Alcohol. Rapid Prototyp J, 21:528–34.
Tagami T, Fukushige K, Ogawa E, et al., 2017, 3D Printing Factors Important for the Fabrication of Polyvinylalcohol Filament-based Tablets. Biol Pharm Bull. 40:357–64. https://doi.org/10.1248/bpb.b16-00878
Wei J, Wang J, Su S, et al., 2015, 3D Printing of an Extremely Tough Hydrogel. RSC Adv, 5:81324–9.
Hinton TJ, Jallerat Q, Palchesko RN, et al., 2015, Three-dimensional Printing of Complex Biological Structures by Freeform Reversible Embedding of Suspended Hydrogels. Sci Adv, 1:e1500758. https://doi.org/10.1126/sciadv.1500758
DOI: http://dx.doi.org/10.18063/ijb.v8i4.610
Refbacks
- There are currently no refbacks.
Copyright (c) 2022 Author(s).
This work is licensed under a Creative Commons Attribution 4.0 International License.