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W3207667095 abstract "Article Figures and data Abstract Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Accumulating evidence has shown transcranial low-intensity ultrasound can be potentially a non-invasive neural modulation tool to treat brain diseases. However, the underlying mechanism remains elusive and the majority of studies on animal models applying rather high-intensity ultrasound that cannot be safely used in humans. Here, we showed low-intensity ultrasound was able to activate neurons in the mouse brain and repeated ultrasound stimulation resulted in adult neurogenesis in specific brain regions. In vitro calcium imaging studies showed that a specific ultrasound stimulation mode, which combined with both ultrasound-induced pressure and acoustic streaming mechanotransduction, is required to activate cultured cortical neurons. ASIC1a and cytoskeletal proteins were involved in the low-intensity ultrasound-mediated mechanotransduction and cultured neuron activation, which was inhibited by ASIC1a blockade and cytoskeleton-modified agents. In contrast, the inhibition of mechanical-sensitive channels involved in bilayer-model mechanotransduction like Piezo or TRP proteins did not repress the ultrasound-mediated neuronal activation as efficiently. The ASIC1a-mediated ultrasound effects in mouse brain such as immediate response of ERK phosphorylation and DCX marked neurogenesis were statistically significantly compromised by ASIC1a gene deletion. Collated data suggest that ASIC1a is the molecular determinant involved in the mechano-signaling of low-intensity ultrasound that modulates neural activation in mouse brain. Introduction Transcranial ultrasound such as opening blood-brain barrier (BBB) (Cammalleri et al., 2020) for localized drug release and modulating neural activity (Nicodemus et al., 2019; Legon et al., 2014; David et al., 2014) has been used for therapeutic treatments of various brain diseases. Many in vivo animal experiments and human clinical trials (Supplementary file 1) proved the clinical potential of transcranial ultrasound stimulation. With the increased interest of this technique, the mechanisms underlying ultrasound-mediated neural modulation has also recently been learned. A study showed high-intensity transcranial ultrasound can elicit a startle-like motor response via an indirect auditory mechanism (Sato et al., 2018). Emerging sonogenetics in worm model also identified and engineered TRP-4 channels as a sensor for the ultrasound stimulus to activate neurons in living organisms at pressure level above 0.5 MPa (Ibsen et al., 2015), and ultrasound at 0.1 MPa acoustic pressure was found to activate neurons via Piezo one mechanosensitive ion channel (Qiu et al., 2019). Nonetheless, the energy intensity or acoustic pressure of most clinical trials or basic researches used for BBB opening or neuromodulation are both high, and safety issue of this technique in clinical application remains a concern. In this study, a much lower intensity ultrasound at the order lower than 10 mW/cm2 is proposed to activate neurons via mechanosensitive ion channels in mammals’ brain for potential clinical application. Mechanosensitve ion channels such as PIEZO and TRP channels and acid sensing ion channels (ASICs) (Cheng et al., 2018; Murthy et al., 2017; Nilius and Honoré, 2012) are considered the candidates likely responsive to ultrasound. Here, we aim to identify possible mechano-sensors in mouse brain that can respond to low-intensity ultrasound. Results Transcranial ultrasound-induced p-ERK in the cortex, hippocampus, and amygdala of mouse brain We kept ultrasound exposure to below 10 mW/cm2 (ISATA) in our experiments to ensure safe therapeutic applicability. The phosphorylation of extracellular-signal-regulated kinase (p-ERK), an established indicator of immediate neuronal activation (Gao and Ji, 2009), was used to evaluate whether transcranial low-intensity ultrasound can stimulate neuronal activity in mouse brain. Mice with 1 min ultrasound exposure (Figure 1A) had shown significant increase of p-ERK positive cells in certain brain regions, such as the cortex (Figure 1B–D), hippocampus (Figure 1E–G), and amygdala (Figure 1H–J) as compared with those received sham treatment (Supplementary file 1). More specifically, increased p-ERK expression generally occurred upon ultrasound stimulation in the visual, somatosensory, auditory, temporal associations, retrosplenial, piriform, and entorhinal areas of mouse cortex (Figure 1—figure supplements 1 and 2). In hippocampus, CA1 and CA2 were dramatically lightened up with p-ERK signals in response to ultrasound whereas CA3 and dentate gyrus showed sparsely stimulated (Figure 1—figure supplements 1 and 2). In amygdala, the central amygdala nucleus showed the strongest p-ERK signals, while medial and basolateral also obviously increased in p-ERK signals (Figure 1—figure supplements 1 and 2). We also observed a consistently unchanged p-ERK staining in the paraventricular nucleus of hypothalamus (PVH) (Figure 1—figure supplement 3), revealing an intriguing regional specificity of the ultrasound response. Figure 1 with 3 supplements see all Download asset Open asset Transcranial ultrasound induces p-ERK expression in neurons of the cortex, hippocampus and amygdala of mouse brain. (A) Illustration depicting mouse head stimulated by 1 MHz transducer which was positioned in between the mouse nasal process of maxilla and the axis of mouse ear of an anesthetized mouse. (B) Micrograph representing cortical region with basal level of p-ERK staining in sham control mice (n = 5), scale bar 100 μm. Sham control mice were handled with similar procedures of placing transducer on the head without turning on the ultrasound function generator. (C) Micrograph representing cortical region with p-ERK staining stimulated by ultrasound (ISPTA = 5 mW/cm2, 1 minute) (n = 5). (D) Quantitative bar graph of the number of p-ERK stained cells within comparable area of 1.224 mm2 (Length 1275 μm, Width 960 μm), showing significant difference (P = 0.0007) of cell count by ImageJ. (E) Micrograph representing hippocampal region with basal level of p-ERK staining in sham control mice. (F) Micrograph representing hippocampal region with p-ERK staining in mice stimulated by ultrasound. (G) Bar graph showing quantification of significantly p-ERK different cell count (1.224 mm2) (P = 0.0132) in hippocampal region. (H) Micrograph representing amygdala of sham controls. Scale bar 100 μm (I) Micrograph representing amygdala of ultrasound stimulated mice. (J) Quantification of amygdaloid significant difference (P = 0.0023) of p-ERK cell count (1.224 mm2). Figure 1—source data 1 Source data for Figure 1D, G, J. https://cdn.elifesciences.org/articles/61660/elife-61660-fig1-data1-v2.xlsx Download elife-61660-fig1-data1-v2.xlsx Micropipette Guided Ultrasound as the Mechanical Stimuli with Combined Ultrasound and Acoustic Streaming To determine whether low-intensity ultrasound can activate neurons mechanically, we next used in vitro calcium-imaging approach staining with the Oregon Green 488 BAPTA-1, AM to probe the possible ion channels responding to ultrasound mechanical stimulus in cultured cortical neurons. A micropipette was used to guide ultrasound to cultured cells (Figure 2A). The device can generate either an ultrasound induced pressure predominant condition (2000 mVpp, DF 0.05%, measured peak pressure versus the distance to the pipette tip shown in Figure 2B) or an acoustic streaming predominant condition (100 mVpp, DF 100%, flow pattern for the acoustic streaming shown in Figure 2—figure supplement 1); (Chu et al., 2021), or a mixed loading condition (700 mVpp, DF 20%) depending on duty cycle applied. Ultrasound pressure predominant conditions (up to 2000 mVpp, DF 0.05%) generated compressional stress on cells that were not able to elevate calcium responses (Figure 2B and Video 1), while acoustic streaming predominant conditions (up to 100 mVpp, DF 100%) invoked shear stress that could only activate little calcium responses in neurons (Figure 2B); whereas a mixed loading condition (700 mVpp, 20 % DF) effectively yielded much higher calcium responses (Figure 2B and Video 2). This response was also reproducible when we applied two additional live cell calcium staining reagents, including Fluo-4 (Video 3) and Fura-2 (Figure 2—figure supplement 2A, Video 4), and we observed similar responses upon micropipette guided ultrasound stimulations. To validate that the calcium response was not caused by a lipid bilayer damage, we repeated the stimulation on the same cell (Figure 2—figure supplement 2B), and found that despite an overwhelming photo-decay problem due to continuous light exposure, there was some repeated calcium elevations at certain sub-cellular sites (white arrows, Figure 2—figure supplement 2B). The quantification of the neuronal calcium responses (Figure 2—figure supplement 3A) using Oregon Green 488 BAPTA-1 AM was presented in an average value (Figure 2—figure supplement 3B) and the experiments were ended by a 0.01% Saponin cell perforation to calibrate the maximum response (Figure 2—figure supplement 3C). This method was going to be utilized for the tests for inhibitors and dosage studies. To ensure that this method is reliable, cells were repeatedly stimulated and clear responses were observed even though the magnitude of responses typically dropped to 30–60% during the second stimulation (Figure 2—figure supplement 3D). The ultrasound predominant mode with only compressional stress cannot induce a response even when the stimulation was extended to 10 s (Figure 2—figure supplement 4A). Similarly, the prolong stimulation of acoustic streaming invoking shear stresses also only elevated mildly the amplitude of calcium response (Figure 2—figure supplement 4B). On the other hand, the combined compression stress with acoustic streaming can reproducibly elevated calcium response even in 1.5 s stimulation (Figure 2B and Video 2, Figure 2—figure supplement 4C). The ultrasound-induced calcium responses were dose-dependent with a threshold of 400 mVpp (8 kPa) and EC50 of 700 mVpp (12 kPa) (Figure 2C and Figure 2—figure supplement 4D-K). The corresponding stress levels of ultrasound at 400 mVpp, 500 mVpp, 700 mVpp or 900 mVpp were 8 kPa, 8.72 kPa, 12 kPa, and 15.3 kPa, respectively. Figure 2 with 4 supplements see all Download asset Open asset Neuronal calcium signals induced by micropipette-guided ultrasound suppressed by ASIC1a inhibitors. (A) A micropipette positioned to the cortical neurons cultured on a 30 mm cover slips mounted to a chamber coupled to microscope platform. Calcium signals recorded from neurons stained by Invitrogen Oregon Green 488 BAPTA-1, AM cell permeant. (B) Line graphs of averaged calcium signals in four neurons stimulated by micropipette ultrasound for 3 s with an input voltage 2000mVpp, duty factor (DF) 0.05 % (n = 5); or 100mVpp, DF100% (n = 3) or 700mVpp, DF20% (n = 4). The red-dotted line denotes start of the stimulation while the yellow-dotted line denotes the end. (C) Calcium responses as a function of micropipette ultrasound in 20%DF. Dose-dependent (input voltages from 10mVpp, to 900mVpp, DF20%) responses of ultrasound with an EC50 of 700mVpp is shown (n = 5). (D) Effects of gadolinium (120 μM), a non-selective blocker of mechanically sensitive ion channels, on calcium signals in cortical neurons (n = 5). (E) Effects of GsMTx-4 (500 nM), a selective Piezo inhibitor, on calcium signals in cortical neurons (n = 4). Control n = 4. (F) Effects of Ruthenium red (10 μM), a non-selective TRP inhibitor, on calcium signals in cortical neurons (n = 4). Control n = 6. (G) Effects of amiloride (100 μm), an ASICs family inhibitor, on calcium signals in cortical neurons (n = 4). (H) Effects of PcTx1 treated (50 nM), a selective ASIC1a inhibitor, on calcium signals in cortical neurons (n = 5). (I) Statistical analyses of channel blockers on ultrasound-induced calcium signals in cortical neurons. Control n = 21, Gadolinium (50 μM) n = 5, Gadolinium (100 μM) n = 5, GsMTx-4 (500 nM) n = 5, Ruthenium red (5 μM) n = 5, Ruthenium red (10 μM) n = 5, Amiloride (100 μM) n = 5, PcTx1 (50 nM) n = 5. Figure 2—source data 1 Source data for Figure 2B and C. https://cdn.elifesciences.org/articles/61660/elife-61660-fig2-data1-v2.xlsx Download elife-61660-fig2-data1-v2.xlsx Figure 2—source data 2 Source data for Figure 2D. https://cdn.elifesciences.org/articles/61660/elife-61660-fig2-data2-v2.xlsx Download elife-61660-fig2-data2-v2.xlsx Figure 2—source data 3 Source data for Figure 2E and G. https://cdn.elifesciences.org/articles/61660/elife-61660-fig2-data3-v2.xlsx Download elife-61660-fig2-data3-v2.xlsx Figure 2—source data 4 Source data for Figure 2F. https://cdn.elifesciences.org/articles/61660/elife-61660-fig2-data4-v2.xlsx Download elife-61660-fig2-data4-v2.xlsx Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Neuronal calcium signal cannot be induced by micropipette guided ultrasound with 2000mVpp input voltage and duty cycle 0.05%. This setting induced produce predominantly ultrasound stimulation. When the dash line depicted micropipette tip appeared in the video, ultrasound function generator was turned on. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Micropipette-guided ultrasound stimulation of neuronal calcium elevation. The setting was 700mVpp input voltage and duty cycle 20% for 3 s. When the dash line depicted micropipette tip appeared in the video, ultrasound function generator was turned on. The setting generated both ultrasound and acoustic streaming effects. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Micropipette-guided ultrasound stimulation of neuronal calcium response. Ultrasound with 250mVpp input voltage and continuous waves stimulation. When the dash line depicted micropipette tip appeared in the video, ultrasound function generator was turned on. The setting generated both ultrasound and acoustic streaming effects. Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Micropipette-guided ultrasound with 400mVpp input voltage and duty factor 10% induced the neuronal calcium signals captured by Fura-2 imaging methods. Calcium elevations were measured by fluorescence ratios of Fura-2 emission at wavelengths 340 nm/380 nm (F340/380nm). Spectrum color coded fluorescence ratios in which the red color represents the highest ratio while purple color represents the lowest ratio of F340/380nm. When the dash line depicted micropipette tip appeared in the video, ultrasound function generator was turned on. The ultrasound setting generated both ultrasound and acoustic streaming effects. Neuronal calcium signal upon ultrasound simulation suppressed by ASIC channels inhibitors The micropipette ultrasound mechanotransduction was pharmacologically tested with selective or non-selective blockers of mechanosensitive ion channels for Piezo, TRP, and ASICs. First, the treatment with Gadolinium (10 μM), a non-selective blocker of mechanically sensitive ion channels, partially suppressed the ultrasound-induced calcium signals (Figure 2D). Treating the cells with a selective Piezo inhibitor, GsMTx-4, led to a marginal (not significant) inhibition of the ultrasound-induced calcium elevation as compared with the vehicle control (Figure 2E). Instead, the treatment with a TRP blocker ruthenium red (1–10 μM) partially suppressed the calcium signals (Figure 2F), whereas the non-selective ASIC inhibitor, amiloride, totally abolished the calcium signals by micropipette ultrasound (Figure 2G). Above results suggested ASICs might be the major channels involved in micropipette ultrasound mechanotransduction. To narrow down the specific candidate of ASICs, ASIC1a inhibitor, PcTx1 (50 nM) was tested. PcTx1 (50 nM) significantly inhibited the calcium response by micropipette ultrasound (Figure 2H and Figure 2—figure supplement 4C). The relative inhibitions of above channel blockers were summarized in Figure 2I. We further tested the dose-dependent inhibition of PcTx1 on micropipette ultrasound and determined an IC50 of 0.2 nM, suggesting a homotrimeric ASIC1a is the mechanoreceptor in action (Figure 3A and Figure 3—figure supplement 1 A-F). To validate how ASIC1a activation would lead to calcium responses to ultrasound, we treated the cells with calcium chelating agent, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) (1–5 mM) to block the extracellular calcium. The results showed that calcium influx was absolutely essential (Figure 3B and Figure 3—figure supplement 1G-H). To examine whether endoplasmic reticulum calcium was involved in calcium signaling, we found the calcium surge of cells treated with the RyR inhibitor, JTV519 fumarate (10 μM) (Figure 3—figure supplement 1G and I) was partially inhibited, while as the IP3R inhibitor, (-)-Xestonspongin C (1 μM) was most inhibited (Figure 3B). Figure 3 with 1 supplement see all Download asset Open asset ASIC1a as a mechanoreceptor responsive to mechanical stimuli with combined ultrasound and acoustic streaming. (A) PcTx1 dose-dependent inhibition curve of calcium responses induced by micropipette ultrasound of 700mVpp, DF20% for 3 s (n = 5). (B) Whisker plots showing comparison of peak ΔF/F0 within 3–5 s upon micropipette ultrasound (700mVpp, DF20%, 3 s) stimulation in the untreated control primary neurons (n = 16), 2- or 5 mM EGTA-treated neurons (n = 6 or 4, respectively), RyR inhibitor JTV519 fumarate (10 μM) treated neurons (n = 10), or IP3R inhibitor (-)-Xestonspongin C (1 μM)-treated neurons (n = 5). Student t-test with p value compared to control listed above the whisker plot. (C) Graph showing calcium response of actin polymerization inhibitor Cytochalasin D (5–10 μg/ml)-treated neurons (n = 7 and n = 9, respectively) compared to untreated control (n = 5). (D) Calcium signals showing the effect of the microtubule assembly inhibitor nocodazole (5–10 μg/ml) on neurons (n = 8 and n = 10, respectively). (E) Whisker plots showing comparison of peak ΔF/F0 within 3–5 s upon micropipette ultrasound (700mVpp, DF20%, 3 s) stimulation in the untreated control primary neurons (n = 11), cytochalasin D 5- or 10 μg/ml treated neurons (n = 7 or n = 9, respectively), nocodazole 5- or 10 μg/ml treated neurons (n = 8 or n = 10, respectively). Statistical p values of one-way ANOVA analysis were listed to show the significance of treatment. (F) Cartoon depicting ultrasound stimulating ASIC1a in the cell body of a neuron under the micropipette ultrasound stimulation. Green arrow represents the pulling force of acoustic stream and purple arrow represents the compression force of ultrasound that results in cytoskeletal rearrangement. Figure 3—source data 1 Source data for Figure 3A. https://cdn.elifesciences.org/articles/61660/elife-61660-fig3-data1-v2.xlsx Download elife-61660-fig3-data1-v2.xlsx Figure 3—source data 2 Source data for Figure 3B. https://cdn.elifesciences.org/articles/61660/elife-61660-fig3-data2-v2.xlsx Download elife-61660-fig3-data2-v2.xlsx ASIC1a mechano-response required cytoskeletal dynamics Previous studies have suggested ASICs are involved in tether-mode mechanotransduction, which relies on intact cytoskeletal structures (Cheng et al., 2018; Lin et al., 2009). We likewise treated the cells with either actin polymerization inhibitor, cytochalasin D (5–10 μg/ml) or microtubule assembly inhibitor, nocodazole (5–10 μg/ml). Indeed, inhibition of cytoskeletal dynamics could dose dependently and significantly suppress the calcium response stimulated by micropipette ultrasound (Figure 3C–E). The collated data revealed a novel mode of ultrasound mechanotransduction with a combination of compression force and shear force that activates ASIC1a channels in mouse neurons (Figure 3F). ASIC1a overexpression in CHO cells accelerated the calcium response upon ultrasound stimulation We next distinguished ASIC1a’s roles in causing the calcium response upon micropipette guided ultrasound stimulation in a heterologous expression system. We transfected Asic1 cDNA (the plasmid was constructed using specifically Asic1a alternative spliced isoform) in the Chinese hamster ovary (CHO) cells that contain no endogenous ASIC1a. Cells treated with mock transfection reagent served as a control for comparison. Interestingly, CHO cells might contain some endogenous mechanosensitive machinery that can manifest a delayed calcium response to micropipette-guided ultrasound (Figure 4A, Figure 4—figure supplement 1A). In contrast, Asic1-transfected cells showed an immediate calcium response to the ultrasound (Figure 4A–C, Video 5, Figure 4—figure supplement 1B), indicating a role for ASIC1a in the ultrasound-mediated mechanotransduction (Figure 4C). The ultrasound-induced calcium responses were analyzed based on the area under curve (AUC) in different time points. Two-way ANOVA analysis showed both ASIC1a overexpression and ultrasound treatments significantly regulated the calcium response of CHO cells (Supplementary file 1) and the p value was 0.086 for the interaction of the two factors. The immediate calcium response of the Asic1 -transfected CHO cells resembled that was detected in primary neurons (Videos 2–4). The calcium response calibrated by 0.01 % Saponin cell perforation could surge to the maximal Fura-2 ratio (F340/380nm) (Figure 4—figure supplement 1C, D). In the context of 0.01% Saponin, 2-way ANOVA analysis showed that ASIC1a overexpression did not contribute to the difference with an insignificant ppvalue (F = 0.11; p = 0.74) (Supplementary file 1) while cell perforation contributed significantly to the calcium responses (F = 10.52; p < 0.0001) (Supplementary file 1). Note that PcTx1 treatment did not affect the ultrasound-induced delayed calcium responses in vehicle transfected cells (Figure 4E), as ultrasound as a factor still contributed significantly (F = 17.2; p < 0.0001) to the changes of calcium while there was no significant (F = 1.44; p = 0.15) (Supplementary file 1) interaction of drug treatments with ultrasound stimulations (Supplementary file 1). In contrast, PcTx1 significantly inhibited ultrasound-induced calcium responses in Asic1-transfected cells (Figure 4F) (F = 1.26; p = 0.29) (Supplementary file 1), while the two factors interact significantly (F = 4.46, p < 0.0001) (Supplementary file 1). The overexpression of ASIC1a was validated by western analysis of CHO cell lysates (Figure 4G). Figure 4 with 1 supplement see all Download asset Open asset ASIC1a overexpression showed a fast calcium response upon micropipette-guided ultrasound stimulation in CHO cells. (A) Invitrogen Fura-2, AM, cell permeant (Fura-2) stained CHO cells. Fluorescence ratios of Fura-2 emission at wavelengths 340 nm/380 nm (F340/380nm) were recorded and averaged line graphs were shown. F340/380nm ratio values plotted against time were shown in Figure 4—figure supplement 1A, B. Ultrasound stimulation is indicated by the red dashed lines at time point 10 s for a duration of 3 s. The blue dashed line indicates the time of ultrasound termination. Control n = 18, ASIC1a overexpressed n = 15. (B) Area under curve (AUC) of F340/380nm were plotted in a 10 s bin manner. Each dot represents a single cell quantified. Three batches of experiments were represented by three different colors. Refer to Supplementary file 1 for the two-way ANOVA analysis of this graph. Control n = 14, ASIC1a overexpressed n = 15. (C) Calcium response time determined by the maximum F340/380nm was significantly shortened by ultrasound stimulation compared to the sham transfected controls. Control n = 18, ASIC1a overexpressed n = 15. (D) Cell perforation treatment with 0.1% saponin in HHBS after the experiments for internal calibration of maximal response. Refer to Supplementary file 1 for the two-way ANOVA analysis of this graph. Control n = 7, ASIC1a overexpressed n = 7. (E) CHO cells calcium response either with or without PcTx1 treatments. Refer to Supplementary file 1 for the two-way ANOVA analysis of this graph. Control n = 4, PcTx1 (2 nM) n = 5, (4 nM) n = 5, (10 nM) n = 5. (F) ASIC1a overexpressing CHO cells either with or without PcTx1 treatments. Refer to Supplementary file 1 for the two-way ANOVA analysis of this graph. Control n = 5, PcTx1 (2 nM) n = 3, (4 nM) n = 3, (10 nM) n = 3. (G) Western analysis of ASIC1a comparing the sham transfected control and Asic1-transfected cells. GAPDH detection serves as an internal control. Figure 4—source data 1 Source data for Figure 4A. https://cdn.elifesciences.org/articles/61660/elife-61660-fig4-data1-v2.xlsx Download elife-61660-fig4-data1-v2.xlsx Figure 4—source data 2 Source data for Figure 4B and C. https://cdn.elifesciences.org/articles/61660/elife-61660-fig4-data2-v2.xlsx Download elife-61660-fig4-data2-v2.xlsx Figure 4—source data 3 Source data for Figure 4D. https://cdn.elifesciences.org/articles/61660/elife-61660-fig4-data3-v2.xlsx Download elife-61660-fig4-data3-v2.xlsx Figure 4—source data 4 Source data for Figure 4E. https://cdn.elifesciences.org/articles/61660/elife-61660-fig4-data4-v2.xlsx Download elife-61660-fig4-data4-v2.xlsx Figure 4—source data 5 Source data for Figure 4F. https://cdn.elifesciences.org/articles/61660/elife-61660-fig4-data5-v2.xlsx Download elife-61660-fig4-data5-v2.xlsx Video 5 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg CHO cells calcium response induced by micropipette guided ultrasound. Right panel was CHO cells overexpressing ASIC1a while left panel was showing transfection sham control cells. Calcium elevations were measured by fluorescence ratios of Fura-2 emission at wavelengths 340 nm/380 nm (F340/380nm). Spectrum color coded fluorescence ratios in which the red color represents the highest ratio while purple color represents the lowest ratio of F340/380nm. When the dash line depicted micropipette tip appeared in the video, ultrasound function generator was turned on. The ultrasound setting was 600mVpp input voltage and duty cycle 10% for 3 s. This setting generated both ultrasound and acoustic streaming effects. Transcranial ultrasound treatments promoted neurogenesis in dentate gyrus We next investigated whether the low-intensity ultrasound stimulation in mouse brain could lead to a favorable outcome in terms of adult neurogenesis. We selected doublecortin (DCX) as a marker for neurogenesis in dentate gyrus (Germain et al., 2013; Jin et al., 2010; Rao and Shetty, 2004). Compared to the non-treated controls, after three consecutive days of 5 min ultrasound treatments (5 mW/cm2), DCX staining in dentate gyrus at day 4 and day seven showed a significant twofold increase (Figure 5). The results indicated that repeated stimulation of low-intensity ultrasound on mouse brain might achieve beneficial neural modulation and lead to neurogenesis in dentate gyrus. Figure 5 Download asset Open asset Neurogenesis in dentate gyrus induced by repeated transcranial ultrasound treatments. (A) The mice were treated three consecutive days by ultrasound of 4 mW/cm2, 1% for 5 min, subsequently perfused fixed at day 4 or day 7 and brains were dissected from the head and sectioned for immunofluorescence procedures. The DCX staining in dentate gyrus of treated mice were compared to control untreated one. (B) Cell count with clear DAPI stained nucleus surrounded by DCX markers compared in control, day 4 and day 7 post-ultrasound treatments. Statistical analysis: p = 0.0013 and F ratio = 15.18 in one-way ANOVA (n = 4). (C, D) Representative micrograph of untreated mice. The vibratome coronal brain sections (100 μm) of dentate gyrus region immunofluorescently stained for DCX (green) and MAP2 (red). Blue color indicates DAPI stained nuclei. Representative micrograph showing. (E, F) Representative micrograph from ultrasound treated mice fixed at day 4. (G, H) Representative micrograph from ultrasound treated mice fixed at day 7. Scale bar 200 μm. Figure 5—source data 1 Source data for Figure 5B. https://cdn.elifesciences.org/articles/61660/elife-61660-fig5-data1-v2.xlsx Download elife-61660-fig5-data1-v2.xlsx Neurogenesis marked by DCX induced by ultrasound was partially compromised by Asic1 knockout (specifically designed for Asic1a alternative spliced isoform) We performed the same experiments on either the wildtype mice or the Asic1 knockout (Asic1-/-) mice for 3 consecutive days of 1 min ultrasound treatments (5 mW/cm2) and sacrificed the mice at day seven to quantify the ultrasound effects on DCX staining in these mice. We observed a reproducibly significant increase of DCX staining in the ultrasound treated group while quantific" @default.
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W3207667095 title "Author response: ASIC1a is required for neuronal activation via low-intensity ultrasound stimulation in mouse brain" @default.
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