3-dimensional arenas for the assessment of C. elegans behavior

C. elegans nematodes are a well-established model organism in numerous fields of experimental biology. In nature, C. elegans live in a rich 3-dimensional environment. However, their behavior has been assessed almost exclusively on the open, flat surface of NGM (Nematode Growth Medium) plates, the golden standard for C. elegans culture in the lab. We present two methods to build 3-dimensional behavioral arenas for C. elegans , by casting, and by directly 3D printing NGM hydrogel. The latter is achieved by using a highly customized fused deposition modeling (FDM) 3D-printer, modified to employ NGM hydrogel as ink. The result is the advancement of 3-dimensional 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, i.e., egg laying. We consider these findings a decisive step toward characterizing C. elegans 3-dimensional 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.