2024 AIChE Annual Meeting
Mechanosensory Constraints in Habituation Learning of Single-Celled Spirostomum Ambiguum
Our research targets the fundamental mechanisms underlying non-associative learning in the unicellular organism Spirostomum ambiguum. Spirostomum is a ciliated protozoa that can contract its body length by 75% when exposed to mechanical stress using a 5 millisecond calcium driven ultrafast contraction. Previous studies have concluded that the protozoa demonstrates a decreasing probability of contraction when exposed to a repetitive vibratory stimulus. These prior examinations of the protozoans' habituation behavior have induced mechanical stimulation through an incident voltage or a mechanical agitation of the organisms. These methods heavily favor binary "high" or "low" force measures, which result in a repetitive stimulation that has been correlated to affect behaviors beyond the probability of contraction. Using such a system we employed a low-cost, Arduino-controlled solenoid-based mechanical stimulus, and successfully induced population-level habituation through a contractile-response decrement during the training regime. Building on this, we have designed a microfluidic device that applies precise radially continuous hydrodynamic stresses to probe relationships between stimulus force and habituation—an area previously overlooked by binary stimuli. In order to effectively examine the protozoans' contractions over time we utilize a high speed imaging system capable of capturing high resolution videos over the entire volume of the apparatus. In order to preserve the fidelity of measurements the time and position of each contraction in the apparatus volume will be measured using a machine learning-based neural network for image analysis, leveraging its ability to extract intricate patterns and features from complex datasets. We also utilize a similar computer vision software to analyze video data from a custom-designed apparatus, which induces low Reynolds number flow for real-time cell counting. The system accurately tracks the number of cells passing through the apparatus, ensuring a constant sample size. This cost-effective method, which can be manufactured and implemented for only pennies, provides a scalable solution for cell counting in resource-constrained settings. The hydrodynamic stimuli creates a rigorously controlled and easily adjusted stimulus force. This apparatus is designed to induce a controlled Jeffery-Hammel flow, creating a polar gradient of shear stress experienced by the organism. As the organism progresses radially, it encounters continuously increasing shear forces on its membrane, eventually reaching a threshold that triggers contraction when a critical shear level is attained. To investigate S. ambiguum habituation learning under these conditions, we will carefully recycle the cells through equivalent flow fields repeatedly and observe if the critical radial distance decreases with each repetition. Our device's continuous stimulus gradient allows us to explore how S. ambiguum integrates environmental information, questioning whether habituation occurs in a continuous or discrete manner. Ultimately, we'll compare learning rates under different mechanical stimuli to reveal any preference in habituation behavior, shedding light on cellular-level learning and its broader biological implications. A thorough understanding of Spirostomum ambiguum’s response to hydrodynamic stimuli presents the opportunity to learn more about the mechanism responsible for the organism's contraction. This would yield more insight into basic biophysical concepts and present a new understanding of how single cells induce extreme ultrafast shape changes. Such an understanding presents the opportunity to inspire new engineering applications and could help identify the specific molecules responsible for the protozoans' contractions. Once identified the molecules responsible for habituation can be transplanted into unhabituated organisms in order to better examine the habituation process at the cellular level, laying the groundwork for future studies.