Continuous, long-term crawling behavior characterized by a robotic transport system.

Detailed descriptions of behavior provide critical insight into the structure and function of nervous systems. In Drosophila larvae and many other systems, short behavioral experiments have been successful in characterizing rapid responses to a range of stimuli at the population level. However, the lack of long-term continuous observation makes it difficult to dissect comprehensive behavioral dynamics of individual animals and how behavior (and therefore the nervous system) develops over time. To allow for long-term continuous observations in individual fly larvae, we have engineered a robotic instrument that automatically tracks and transports larvae throughout an arena. The flexibility and reliability of its design enables controlled stimulus delivery and continuous measurement over developmental time scales, yielding an unprecedented level of detailed locomotion data. We utilize the new system's capabilities to perform continuous observation of exploratory search behavior over a duration of 6 hr with and without a thermal gradient present, and in a single larva for over 30 hr. Long-term free-roaming behavior and analogous short-term experiments show similar dynamics that take place at the beginning of each experiment. Finally, characterization of larval thermotaxis in individuals reveals a bimodal distribution in navigation efficiency, identifying distinct phenotypes that are obfuscated when only analyzing population averages.

Continuous, long-term crawling behavior characterized by a robotic transport system.

Detailed descriptions of behavior provide critical insight into the structure and function of nervous systems. In Drosophila larvae and many other systems, short behavioral experiments have been successful in characterizing rapid responses to a range of stimuli at the population level. However, the lack of long-term continuous observation makes it difficult to dissect comprehensive behavioral dynamics of individual animals and how behavior (and therefore the nervous system) develops over time. To allow for long-term continuous observations in individual fly larvae, we have engineered a robotic instrument that automatically tracks and transports larvae throughout an arena. The flexibility and reliability of its design enables controlled stimulus delivery and continuous measurement over developmental time scales, yielding an unprecedented level of detailed locomotion data. We utilize the new system’s capabilities to perform continuous observation of exploratory behavior over a duration of six hours with and without a thermal gradient present, and in a single larva for over 30 hours. Long-term free-roaming behavior and analogous short-term experiments show similar dynamics that take place at the beginning of each experiment. Finally, characterization of larval thermotaxis in individuals reveals a bimodal distribution in navigation efficiency, identifying distinct phenotypes that are obfuscated when only analyzing population averages.

Assessment of Transport of Lipid Metabolites Within Trabecular Meshwork Cells.

Lipids from trabecular meshwork (TM) cells are of particular interest to ophthalmological researchers as a therapeutic target for lowering intraocular pressure (IOP) in glaucomatous eyes. Fluorescence-based lipid transport assays (FBLTA) and immunocytochemistry (ICC) are dynamic fluorescence analysis techniques that allow for quantitative and qualitative comparisons, respectively, between multiple samples. Here we describe methods for FBLTA, ICC, and mass spectroscopy designed to measure the kinetics and localization of lipid metabolites within the trabecular meshwork.