Fig. 4: Influence of the indentation speed and anchoring between neuronal membrane and actin cytoskeleton on mechanically evoked action potentials.
a, Bar plots depict the percentage of cortical neurons showing a response (grey) and no response (white) to mechanical stimuli of a bead indenting the soma at various indentation speeds. The neurons were indented with a 5-μm-diameter silica bead, glued to the AFM cantilever, by applying a force of 200 nN (∼5 kPa). The response of the neurons expressing the genetically encoded calcium sensor GcaMP6s was monitored by confocal microscopy. b, Differential neuronal responses to indentations at different speeds. Time series of extracellular potentials of a cortical neuron, in which soma was indented with a 5 µm bead. The solid line at the top represents the vertical movement of AFM cantilever and bead at 10 µm s–1 (black) and 0.1 µm s–1 (blue). Number of action potentials evoked through 10 µm s–1 and 0.1 µm s–1 indentations were compared using a two-tailed Mann–Whitney U-test. The P value of 0.004 (n = 8 individual neurons) suggests that neurons can differentiate indentation speeds. c, Coupling between the neuronal membrane and the actin cytoskeleton is required for cortical neurons to respond to mechanical stimuli. Bar plots give the percentage of cortical neurons showing a response (grey) and no response (white) to mechanical stimulation of untreated (control) neurons and of neurons treated with 0.1 μM latrunculin A (LatA) or 50 μM NSC668394C. The neurons were indented with a 5-μm-diameter silica bead by applying a force of 200 nN (∼5 kPa) at 10 μm s–1. The neurons expressed the genetically encoded calcium sensor GCaMP6s. The response of the neurons expressing GCaMP6s was monitored by confocal microscopy. In all experiments, spontaneous activity of the neurons was blocked with glutamate receptor antagonists (10 μM DNQX and 40 μM D-AP5) to isolate mechanically evoked responses. n represents the number of characterized independent neurons. P values of the χ2 analysis for different conditions are given above the bars in a and c.
