Three-Dimensional Arenas for the Assessment of Caenorhabditis elegans Behavior

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.