Stretch-Induced Injury Affects Cortical Neuronal Networks in a Time- and Severity-Dependent Manner.

Traumatic brain injury (TBI) is the leading cause of accident-related death and disability in the world and can lead to long-term neuropsychiatric symptoms, such as a decline in cognitive function and neurodegeneration. TBI includes primary and secondary injury, with head trauma and deformation of the brain caused by the physical force of the impact as primary injury, and cellular and molecular cascades that lead to cell death as secondary injury. Currently, there is no treatment for TBI-induced cell damage and neural circuit dysfunction in the brain, and thus, it is important to understand the underlying cellular mechanisms that lead to cell damage. In the current study, we use stretchable microelectrode arrays (sMEAs) to model the primary injury of TBI to study the electrophysiological effects of physically injuring cortical cells. We recorded electrophysiological activity before injury and then stretched the flexible membrane of the sMEAs to injure the cells to varying degrees. At 1, 24, and 72 h post-stretch, we recorded activity to analyze differences in spike rate, Fano factor, burstlet rate, burstlet width, synchrony of firing, local network efficiency, and Q statistic. Our results demonstrate that mechanical injury changes the firing properties of cortical neuron networks in culture in a time- and severity-dependent manner. Our results suggest that changes to electrophysiological properties after stretch are dependent on the strength of synchronization between neurons prior to injury.

Förster resonance energy transfer efficiency of the vinculin tension sensor in cultured primary cortical neuronal growth cones.

Significance: Interaction of neurons with their extracellular environment and the mechanical forces at focal adhesions and synaptic junctions play important roles in neuronal development. Aim: To advance studies of mechanotransduction, we demonstrate the use of the vinculin tension sensor (VinTS) in primary cultures of cortical neurons. VinTS consists of TS module (TSMod), a Förster resonance energy transfer (FRET)-based tension sensor, inserted between vinculin's head and tail. FRET efficiency decreases with increased tension across vinculin. Approach: Primary cortical neurons cultured on glass coverslips coated with poly-d-lysine and laminin were transfected with plasmids encoding untargeted TSMod, VinTS, or tail-less vinculinTS (VinTL) lacking the actin-binding domain. The neurons were imaged between day in vitro (DIV) 5 to 8. We detail the image processing steps for calculation of FRET efficiency and use this system to investigate the expression and FRET efficiency of VinTS in growth cones. Results: The distribution of fluorescent constructs was similar within growth cones at DIV 5 to 8. The mean FRET efficiency of TSMod ( 28.5 ± 3.6 % ) in growth cones was higher than the mean FRET efficiency of VinTS ( 24.6 ± 2 % ) and VinTL ( 25.8 ± 1.8 % ) ( p < 10 - 6 ). While small, the difference between the FRET efficiency of VinTS and VinTL was statistically significant ( p < 10 - 3 ), suggesting that vinculin is under low tension in growth cones. Two-hour treatment with the Rho-associated kinase inhibitor Y-27632 did not affect the mean FRET efficiency. Growth cones exhibited dynamic changes in morphology as observed by time-lapse imaging. VinTS FRET efficiency showed greater variance than TSMod FRET efficiency as a function of time, suggesting a greater dependence of VinTS FRET efficiency on growth cone dynamics compared with TSMod. Conclusions: The results demonstrate the feasibility of using VinTS to probe the function of vinculin in neuronal growth cones and provide a foundation for studies of mechanotransduction in neurons using this tension probe.