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Abstract Neurons in the brain communicate with each other at their synapses. It has long been understood that this communication occurs through biochemical processes. Here, we reveal a previously unrecognized paradigm wherein mechanical tension in neurons is essential for communication. Usingin vitrorat hippocampal neurons, we find that (1) neurons become tout/tensed after forming synapses resulting in a contractile neural network, and (2) without this contractility, neurons fail to fire. To measure time evolution of network contractility in 3D (not2D) extracellular matrix, we developed an ultra-sensitive force sensor with 1 nN resolution. We employed Multi-Electrode Array (MEA) and iGluSnFR, a glutamate sensor, to quantify neuronal firing at the network and at the single synapse scale, respectively. When neuron contractility is relaxed, both techniques show significantly reduced firing. Firing resumes when contractility is restored. Neural contractility may play a crucial role in memory, learning, cognition, and various neuropathologies.
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Vinculin tension probe in neurons
Vinculin is a known key regulator of focal adhesions; it undergoes tension in the locations of attachment to the extracellular matrix. In this study, we explore the use of a vinculin tension FRET probe to investigate vinculin tension within neurons. A critical component of neuronal growth is migration, which is dependent on the mechanical cues between the cells and the extracellular matrix. An understanding of tension variation within the neuron may help us understand mechanisms of neurogenesis. To study these forces, we use a previously developed molecular tension sensor, which consists of an elastic linker, TSMod, a 40-amino-acid-long peptide inserted between teal fluorescent protein (mTFP1) and mVenus. The vinculin tension sensor, VinTS, consists of TSMod embedded between the Vinculin head and tail. When under tension, VinTS will exhibit a lower fluorescence resonance energy transfer (FRET) efficiency between mTFP1 and mVenus. Cortical neurons were isolated from embryonic rat brains and cultured on glass coverslips coated with poly-D-lysine and laminin. The neurons were transfected with TSMod (the unloaded tension sensor) or VinTS. Neurons expressing TSMod are used as the experiment’s control group since TSMod on its own is not affected by vinculin tension. The mean FRET efficiency of 171 TSMod and 127 VinTS expressing neurons was 27.08 ± 4.98%, and 22.86 ± 3.98%, respectively. The FRET efficiency of VinTS was significantly lower than that of TSMod (p = 6.6e15 by Welch’s t-test). These results support the feasibility of using the VinTS probe in neurons and provide a first assessment of VinTS FRET efficiency in neurons. The lower FRET efficiency of VinTS compared with TSMod suggests that VinTS may be under tension in neurons. However, additional studies are required to further characterize these results.
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- Award ID(s):
- 1825433
- PAR ID:
- 10285504
- Editor(s):
- Yang, Victor X.; Kainerstorfer, Jana M.; Luo, Qingming; Ding, Jun; Fu, Ling; Mohanty, Samarendra K.; Roe, Anna W.; Shoham, Shy
- Date Published:
- Journal Name:
- Optical Techniques in Neurosurgery, Neurophotonics, and Optogenetics
- Volume:
- 11629
- Page Range / eLocation ID:
- 1162921
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Neurons in the brain communicate with each other at their synapses. It has long been understood that this communication occurs through biochemical processes. Here, we reveal that mechanical tension in neurons is essential for communication. Using in vitro rat hippocampal neurons, we find that 1) neurons become tout/tensed after forming synapses resulting in a contractile neural network, and 2) without this contractility, neurons fail to fire. To measure time evolution of network contractility in 3D (not 2D) extracellular matrix, we developed an ultrasensitive force sensor with 1 nN resolution. We employed Multi-Electrode Array and iGluSnFR, a glutamate sensor, to quantify neuronal firing at the network and at the single synapse scale, respectively. When neuron contractility is relaxed, both techniques show significantly reduced firing. Firing resumes when contractility is restored. This finding highlights the essential contribution of neural contractility in fundamental brain functions and has implications for our understanding of neural physiology.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Conference Paper:
- https://doi.org/10.1117/12.2578501
