Firing rate of in-vitro hippocampal neuronal network depends on the mechanical tension

Introduction:: Introduction: The firing pattern of neurons plays a critical role in learning, memory, and computation. Loss of firing is associated with several neurodegenerative diseases, making it essential to understand the factors that regulate neuron firing. While biological and chemical processes are traditionally considered the regulators of neuron firing, recent evidence suggests that mechanical tension may also play a role. We hypothesize that mechanical tension in hippocampal neurons regulates neuron firing. However, there is currently no technique available to measure the force produced by a network of neurons in 3D. We have developed an ultra-sensitive force sensor with 1 nN resolution capable of measuring the force of a neuronal network. Using this novel force sensor, we measured the force generated by a network of rat hippocampal neurons and explored the role of mechanical tension in regulating neuron firing. Our results demonstrate that disrupting mechanical tension reduces the firing rate and presynaptic vesicle clustering while restoring tension recovers firing.


Materials and Methods::
Materials and Methods: Primary neurons are harvested from day 18 embryonic (E18) rat hippocampi using the standard protocol. Briefly, hippocampi were dissected in slice dissection solution (SLDS) and then digested in 3mg/ml protease 23 in SLDS for 10 minutes to get dissociated single cells. The sensor consists of a force-sensing spring and two grips that support a neural tissue (Fig. 1a,b). One grip is attached to the spring, the other to a rigid anchor. It was made by casting PDMS into a microfabricated silicon mold. ECM solution was made with collagen I and Matrigel, 1 mg/ml of each. The ECM-cell mixture solution was dispensed onto the sensor grips, which fills the two grips and forms a capillary bridge between them (Fig. 1b). Neurites originating from the neurons extend into the 3D ECM and form synapses resulting in a neural network. After synaptogenesis, the neurites contract and the network applies tensile force on the grids, deforming the sensor spring. Neuron network force was calculated from the spring displacement of the sensor with subpixel resolution and its spring constant (Fig. 1b).




Results, Conclusions, and Discussions:: Results and Discussion: The force of neuronal tissue from several samples was measured using the force sensor. It was observed that neurons gradually increased the force at approximately 0.5 nN/hr. (Fig. 1d), reaching saturation after 6 to 8 days. Maximum force varied from 20 nN to 65 nN across samples (Fig. 1e). The Multi-Electrode Array (MEA) system was used to measure the firing rate of rat hippocampal neuron networks. We found that a 1-hour treatment with Blebbistatin to disrupt the tension in the neuron decreased the spike rate by more than 60% and the burst rate by around 90%. However, the neural network recovered both spike and burst rates after 48 hours of washout (Fig. 1f). To assess the effect of force disruption on neuronal firing at the synaptic scale, iGluSnFR, a genetically engineered fluorescent protein, was used. Results indicated that Blebbistatin caused a significant (~90%) decrease in the spike rate of individual synapses (Fig. 1g). Synaptic vesicle intensity was also found to drop (~20%) with Blebbistatin treatment (Fig. 1h). These findings suggest that neuronal tension regulates the firing of neuronal networks.

Conclusions: To summarize, our study provides insights into the role of neuronal tension in the functionality of neuronal networks of the central nervous system. Our self-assembled 3D neuronal tissue platform allowed us to investigate the evolution of force during the growth and development of the neuronal network. Our results indicate that neurons generate and maintain tension after the post-synaptogenesis stage. We have also shown that blebbistatin, a drug that disrupts force generation in neurons, can significantly decrease the firing rates of individual synapses, highlighting the importance of tension in neuronal activity. Our findings have implications for understanding the fundamental mechanisms underlying neuronal function and could inform the development of new treatments for neurological disorders.


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