Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRPC6 is a mechanosensitive channel essential for ultrasound neuromodulation in the mammalian brain

doi: 10.1073/pnas.2404877121

Figure Lengend Snippet: A hypothetical model for the mechanism of ultrasound neuromodulation via TRPC6 activation proposed in the present study. Ultrasound irradiation potentially distorts the cell membrane by acoustic pressure, which triggers the opening of a mechanosensitive TRPC6 channel followed by membrane depolarization in cultured cortical neurons. The generation of action potentials may be induced by the opening of VGSC following the TRPC6-mediated depolarization, which may further elicit extracellular Ca 2+ influx via voltage-gated calcium channels, resulting in excitation of the neuronal network.

Article Snippet: TRPC6 deficient mouse strain (TRPC6-KO; B6;129S- Trpc6 tm1Lbi / Mmjax, Stock # 37345-JAX) was purchased from the Jackson Laboratory (Bar Harbor, ME) ( ).

Techniques: Activation Assay, Irradiation, Membrane, Cell Culture

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRPC6 is a mechanosensitive channel essential for ultrasound neuromodulation in the mammalian brain

doi: 10.1073/pnas.2404877121

Figure Lengend Snippet: TRPC6 mediates ultrasound-induced neuronal activation in vivo. ( A ) An ultrasound burst pulse sequence used for in vivo experiments. ( B ) Pressure distribution of stimulating ultrasound measured inside free water. ( C and D ) Schematic diagrams of in vivo multiunit recordings with a TRPC6 blocker, BI-749327. ( C ) Ultrasound was transcranially irradiated to the cerebral cortex of mice, and a tungsten microelectrode for recordings was inserted through a cranial window. A white arrowhead indicates a representative recording site labeled with an electrical lesion after recordings. ( D ) BI-749327 was intracerebroventricularly administered 30 min before post recordings. ( E and F ) Representative in vivo multiunit recordings around US irradiations before ( E ) and after ( F ) BI-749327 administration. Top : high-pass filtered extracellular voltage traces; Bottom : peristimulus time histograms (PSTHs). Orange patches represent ultrasound irradiation. Dashed pink, red, and blue lines in each PSTH represent global significance levels, local significance levels, and mean counts calculated with 1,000 randomly generated datasets of timestamps of population neural activities with time jittering, respectively. The significance of each recording was determined by whether PSTH goes above or down across the global significant levels. ( G ) Fraction of significantly activated population responses by US irradiations of several intensities. Significance on each stimulus intensity was determined by the Χ 2 test on a two-by-two (significance × control or BI-749327) frequency table. n = 73, 80 sessions from three mice for pre- and postrecordings, respectively. * P < 0.05.

Article Snippet: TRPC6 deficient mouse strain (TRPC6-KO; B6;129S- Trpc6 tm1Lbi / Mmjax, Stock # 37345-JAX) was purchased from the Jackson Laboratory (Bar Harbor, ME) ( ).

Techniques: Activation Assay, In Vivo, Sequencing, Irradiation, Labeling, Generated, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRPC6 is a mechanosensitive channel essential for ultrasound neuromodulation in the mammalian brain

doi: 10.1073/pnas.2404877121

Figure Lengend Snippet: Pharmacological investigation of the involvement of mechanosensitive channels in neuronal responses to ultrasound stimulation. ( A ) A schematic diagram of mechanosensitive receptors and effects of antagonists. RuR (RuR, 5 µM) was used to block TRPVs channel and GsMT×4 (10 µM) was used to prevent gating Piezo1/2, TRPC1, and TRPC6. ( B ) Averaged traces of neuronal Ca 2+ transients against ultrasound irradiation under the condition of control (black), RuR-treated (red), or GsMT×4-treated (blue) neurons. Data from five, six, and six independent experiments for control, RuR, and GsMTx4 are described as mean ± SEM, respectively. The arrowhead indicates the time of ultrasound stimulation. ( C ) Heatmap demonstration of normalized fluorescence intensity (ΔF/F 0 ) of GCaMP6s in neurons under the condition of control, or the presence of RuR or GsMT×4. Arrowheads indicate the time of ultrasound stimulation. Data from 60 cells in an experiment are plotted. Arrowheads indicate the time of ultrasound stimulation. ( D ) The amplitude of Ca 2+ transients in responses to ultrasound stimulation is quantified as area under the curve (AU). Bar graph values show mean ± SEM. * P < 0.05 (one-way ANOVA followed by the Dunnett post hoc test). ( E ) A scheme of the effects of selective antagonists on Piezo1 or TRPC6. Dooku1 (10 µM) and BI-749327 (1 µM) were used to inhibit Piezo1 and TRPC6 activation, respectively. ( F ) Averaged traces of neuronal Ca 2+ transients against ultrasound irradiation under the condition of control (black), or the presence of Dooku1 (purple) or BI-749327 (light blue). Fourteen, thirteen, and twelve independent experiments for control, Dooku1, and BI-749327, respectively, were performed. Traces show mean ± SEM. ( G ) Heatmap analysis of neuronal responses under the condition of control, Dooku1 treated, or BI-749327 treated neurons. ( H ) Bar graph values of area under the curve in each condition show mean ± SEM. ** P < 0.01 (one-way ANOVA followed by the Dunnett post hoc test).

Article Snippet: TRPC6 deficient mouse strain (TRPC6-KO; B6;129S- Trpc6 tm1Lbi / Mmjax, Stock # 37345-JAX) was purchased from the Jackson Laboratory (Bar Harbor, ME) ( ).

Techniques: Blocking Assay, Irradiation, Control, Fluorescence, Activation Assay