Poster D8 - High-Throughput Biophysical Sensors for Quantification of Neuronal Tensile Force and Its Effects on Synaptic Plasticity in 3-D Neural Tissues

Friday, October 25, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Aja A. Phan (she/they)

Undergraduate Researcher
University of Illinois at Urbana-Champaign
Morton, Illinois, United States

Co-Author(s)

Md Saddam Hossain Joy (he/him/his)

Graduate Research Assistant
University of Illinois at Urbana Champaign
champaign, Illinois, United States
BE

Bashar Emon

Postdoctoral Research Associate
University of Illinois at Urbana-Champaign
Champaign, Illinois, United States
William Drennan (he/him/his)

PhD student
University of Illinois at Urbana Champaign
Champaign, Illinois, United States

Primary Investigator(s)

MS

M. Taher A. Saif

Professor
University of Illinois at Urbana-Champaign
Urbana, Illinois, United States

Introduction: In the quest to build computational systems out of living neurons that achieve the basic hallmarks of cognition—such as learning, attention, and curiosity—understanding the mechanical forces that neurons exert and experience offers valuable insights into how these properties influence neuronal function and information processing. Previous studies have shown that synapses without mechanical tension fail to fire in an in vitro network of neurons. However, the impact of mechanical forces on neuronal plasticity remains unknown. To address this challenge, a high-throughput biophysical sensor array was developed to sense and apply tension forces to light-trained 3-D in vitro optogenetic neuron tissue cultures. This system enables the quantitative analysis of neuronal behavior and memory recall under varying tension environments with a 1 nN resolution, while simultaneously capturing neuronal firing patterns within 3-D extracellular matrices.

Materials and

Methods: A high-throughput biophysical sensor array was developed to measure the forces exerted by neurons at the nano-newton scale. Each sensor in the array includes a force-sensing spring, a stiff spring, and two, pronged grips to support neural tissues. An additional grip on the inner edge holds a gelatin droplet to resist external capillary forces during neural tissue construction (Fig. 1,2,3). The sensor array mold was microfabricated using a CNC-milled acrylic sheet. A 10:1 concentration of PDMS (Slygard 184) was cast to form the sensor positive. The positive mold was subsequently treated with O2 plasma and chemically etched with silane gas allowing more PDMS to be applied and removed to create the sensor (Fig. 4).

The sensor surfaces were surface-treated using APTES-glutaraldehyde to enhance cell attachment. The extracellular matrix (ECM) solution was prepared with collagen I and Matrigel (Corning), each at 1 mg/mL concentration. Primary E-18 optogenetic hippocampal rat neurons are transfected with adeno-associated virus, combined with this ECM solution, and dispensed onto the sensor grips, forming a capillary bridge between them. The mixture is polymerized for 60 minutes and inundated with neuron culture media, allowing neurites to grow within the ECM and form synapses.

Substrate forces were determined by measuring the displacement of the soft, force-sensing grip and applied with a needle mounted on a 3-axis stage under a microscope. Neural firing patterns were recorded using GCaMP calcium imaging. Displacement data were analyzed using Fiji ImageJ, while calcium imaging data were processed with the open-source pipeline, suite2P (Fig. 5).

Results, Conclusions, and Discussions: This study presents a novel, high-throughput approach to modulate and characterize the mechanical forces experienced by neurons and their subsequent influence on synaptic plasticity. We developed biocompatible sensors with soft spring beams, ranging from 50-80μm in width and having spring constants between 1-4.2 nN/μm (Fig. 1,2,3,4). These sensors, integrated with advanced imaging and transfection techniques, enable the study of 3D optogenetic neural tissue cultures that more closely mimic their in vivo microenvironment (Fig. 5). The ability to apply, modulate, and measure forces with high precision allows for a deeper understanding of mechanobiological influences on neural behavior. These high-resolution force measurements lay the groundwork for establishing foundational knowledge for more stable and functional integration of living neurons into engineered systems and more sophisticated neural network models. Future research is focused on optimizing sensor-culture integration, refining optogenetic light-training sequences, and elucidating the impact of tension on neuronal network dynamics and cognitive processes through mechanical manipulation and chemical modulation.

Acknowledgements (Optional): [1] M. S. H. Joy et al., Synapses without tension fail to fire in an in vitro network of hippocampal neurons. Proc Natl Acad Sci USA, Vol. 120, no. 52, p. e2311995120 (2023). DOI: 10.1073/pnas.2311995120.
[2] Bashar Emon et al., A novel method for sensor-based quantification of single/multicellular force dynamics and stiffening in 3D matrices. Sci. Adv. 7, eabf2629 (2021). DOI: 10.1126/sciadv.abf2629
[3] Pachitariu, M., Stringer, C., Schröder, S., Dipoppa, M., Rossi, L. F., Carandini, M., & Harris, K. D. (2016). Suite2p: beyond 10,000 neurons with standard two-photon microscopy. BioRxiv, 061507.