Vice President of Research The University of Texas at Dallas, United States
Introduction:
PIEZO1 is a mechanosensitive ion channel expressed in various organs including but not limited to brain, heart, lung, kidney, bone, and skin. In the mammalian cortex, PIEZO1 has been implicated in astrocyte, microglia, capillary, and oligodendrocyte signaling. Using murine embryonic frontal cortex tissue, we examined the protein expression and functionality of PIEZO1 channels in cultured networks leveraging substrate-integrated microelectrode arrays (MEAs). Our results demonstrate that PIEZO1 is expressed in cortical neurons, the PEIZO1 antagonist GsMTx4 inhibits spontaneous activity in cortical networks while the PIEZO1 agonist Yoda1 transiently enhances the mean firing rate (MFR) of single units. Our work suggests that murine cortical neurons express functional PIEZO1 channels and demonstrates the electrophysiological effects of channel activation in vitro.
Materials and Methods:
Female pregnant mice were used to create embryonic derived cortical cultures by dissociating cortical tissue from the frontal lobe of each embryo and seeding it onto 48 well microelectrode arrays and multi-well culture plates. After maturation to DIV21, cells were fixed for immunocytochemistry (ICC) using GFAP, NeuN, and Piezo1 antibodies. Electrophysiological activity of the embryonic derived neuronal networks was recorded using 48 well MEA plates, and Piezo1 channel antagonist, GsMTx4, and agonist, Yoda1, were introduced to determine the changes in mean firing rate from electrode sites. Results, Conclusions, and Discussions:
ICC results: As shown in Figure 1, cultures at DIV21 were labeled for NeuN, GFAP, and PIEZO1. Quantification of six regions of interest (ROI) with at least 110 neurons in each showed that 88.0 ± 0.1% (mean ± SEM) of the neurons identified by NeuN labeling co-express PIEZO1. In contrast, quantification of four ROIs with at least 45 cells in each ROI showed 45 ± 0.13% of astrocytes identified by GFAP labeling co-express PEIZO1. These observations suggest that PIEZO1 is predominately expressed in the neuronal population within these cultured cortical networks.
Electrophysiology results: Following a 5-minute basal recording of spontaneous activity at DIV22, we added 3 µM of GsMTx4 to six separate wells. Interestingly, in the absence of any exogenous mechanical stimuli, the recordings showed a significant decrease in MFR of the neuronal networks within 15 min of exposure to GsMTx4 (Figure 2). We also introduced concentrations of 1, 5, 10, 30, 50, 100 µM for Yoda1 and the largest concentration of DMSO used as a solvent for the agonist. For Yoda1 concentrations of 10, 30, 50, and 100 µM, we observed a transient increase in the MFR that typically lasted between 30-50 seconds. The results are shown in Figure 3.
In summary, mechanosensitive ion channels, including PIEZO1 channels, are expressed and contribute to the spontaneous firing activity in mouse embryonic-derived cortical networks. Inhibition of mechanosensitive channel activity can decrease network firing rate whereas the transient elevation in spike rate associated with the PIEZO1 agonist is similar to that which has been observed for focused ultrasound.
