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ASIC1a is required for neuronal activation via low-intensity ultrasound stimulation in mouse brain

  1. Jormay Lim
  2. Hsiao-Hsin Tai
  3. Wei-Hao Liao
  4. Ya-Cherng Chu
  5. Chen-Ming Hao
  6. Yueh-Chun Huang
  7. Cheng-Han Lee
  8. Shao-Shien Lin
  9. Sherry Hsu
  10. Ya-Chih Chien
  11. Dar-Ming Lai
  12. Wen-Shiang Chen
  13. Chih-Cheng Chen  Is a corresponding author
  14. Jaw-Lin Wang  Is a corresponding author
  1. Department of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taiwan
  2. Department of Physical Medicine and Rehabilitation, National Taiwan Hospital University, Taiwan
  3. Institute of Biomedical Sciences, Academia Sinica, Taiwan
  4. Department of Surgery, National Taiwan Hospital University, Taiwan
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Cite this article as: eLife 2021;10:e61660 doi: 10.7554/eLife.61660

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
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).

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
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.

Video 1
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
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
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
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
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.

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 24). 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
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.

Video 5
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.

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.

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 quantification and student t-test analysis indicated that the increase was partially compromised in Asic1-/- (Figure 6A–I). We tested whether there was an interaction between the two factors, that is the two-way ANOVA analysis results showed that the ultrasound treatment (F = 9.4; p = 0.0098) and the Asic1-/- (F = 26.35; p = 0.0002) in regulating the DCX-positive cell counts (Supplementary file 1). In addition, there was no significant interaction (F = 0.22; p = 0.65) of these two factors while both factors contributed significantly and independently to the DCX cell counts (Supplementary file 1).

DCX staining-positive cells are increased but partially compromised by Asic1-/- after consecutive 3 days of ultrasound treatments.

(A) Micrographs stitched to show the representative DCX staining pattern (green fluorescence) of the dentate gyrus (DG) in the 5 weeks old mice of wildtype sham treated controls. (B) Magnified DCX positive cells in wildtype control DG. (C, D) DCX-positive cells increased significantly upon three continuous days of ultrasound treatments. (E, F) Representative stitched micrographs of sham treated Asic1-/- dentate gyrus. (G, H) The increase of DCX staining upon ultrasound stimulation partially compromised by Asic1-/-. (I) Quantitative analysis of DCX cell counts/mm in the 100 μm brain slices with clear DCX and DAPI staining using confocal microscopy scanning stacks of 8–10 z-planes. Scale bar 100 μm. There were two batches of mice of 5 weeks and 7 weeks old and the cells counts from all the z-stacks were normalized based on the mean value of wildtype controls to include both batches of mice. Each data point represents quantification of one animal; wildtype control n = 4, wildtype ultrasound treated n = 5, Asic1-/- control n = 3, Asic1-/- ultrasound stimulated n = 4. Refer to Supplementary file 1 for the two-way ANOVA analysis of this graph.

Asic1-/- suppressed transcranial ultrasound induced P-ERK in mouse brain

To study whether ASIC1a is also responsible for the responses of p-ERK in mouse brain, we employed Asic1-/- and Asic3-/- in the vibratome brain slices p-ERK staining experiments. The inclusion of Asic3-/- is to elucidate the role of peripheral nerves in brain activation, as ASIC3 is highly expressed in in somatosensory neurons, trigeminal ganglion neurons, and spiral ganglion neurons. Comparing to the mock controls (Figure 7A, H and O), the p-ERK cell counts in wildtype mice upon transcranial ultrasound stimulations (Figure 7B, I and P) were significantly increased (Figure 7). Comparing to the mock controls (Figure 7C, J and Q), the cell counts increase of p-ERK staining in Asic1-/- was partially decreased in hippocampal region while greatly reduced in cortical and amygdala regions (Figure 7D, K and R). The reduction of p-ERK cell counts caused the difference between mock control and ultrasound stimulation to be statistically insignificant in Asic1-/- mice (Figure 7G, N and U). On the other hand, Asic3-/- mice showed a more consistent lower background of p-ERK in mock controls (Figure 7E, L and S) and exhibited a significant increase of p-ERK cell counts (Figure 7F, M and T) to the ultrasound treatment. Quantification of p-ERK responses in these three genotypes of mice led us to conclude that ASIC1a plays an important role in mediating transcranial ultrasound stimulation in mouse brain. The two-way ANOVA analysis of p-ERK cell counts showed that there was only an interaction of two factors, namely genotype and ultrasound treatment in the cortex (F = 6.45, p = 0.0037) but not in hippocampus and amygdala (Supplementary file 1). To test whether there is a role of peripheral nerves in mediating ultrasound stimulation, we included the Asic3-/- in our p-ERK response phenotypes studies and indeed the genotype did not reduce the activation of p-ERK as Asic1-/- did. These results indicated that the p-ERK response in mouse brain is likely directly caused by the transcranial ultrasound instead of caused by the secondary effects due to the neurons wired with auditory circuits or other sensory circuits, as ASIC3 being mainly expressed in somatosensory neurons and spiral ganglion neurons (Lin et al., 2016; Wu et al., 2009).

Figure 7 with 1 supplement see all
Asic1-/- suppressed the p-ERK cell count increases in cortex, hippocampus, and amygdala of mouse brain.

The IHC stained brain slices of wildtype mice, Asic1-/- mice and Asic3-/- mice. Mice of all genotypes were randomly assigned to sham treatment group and ultrasound treatment group. The quantification of p-ERK-positive cells were performed using ImageJ with setting of threshold and particle sizes that representing the actual staining pattern. (A–F) Micrographs depicting p-ERK IHC staining in the cortex of the vibratome brain slices. (G) Quantification comparing cortical p-ERK-positive cells in three different genotypes of mice either mock treated or ultrasound stimulated. (H–M) IHC micrograph depicting p-ERK staining in hippocampus. (N) Quantification comparing hippocampal p-ERK-positive cells in mice with indicated the genotypes and treatments. (O–T) IHC micrographs depicting p-ERK staining in amygdala. (U) Quantification comparing p-ERK-positive cells in amygdala. Scale bar 100 μm. Each data point represents the total cell count of one mouse brain; wildtype control n = 11, wildtype ultrasound treated n = 10, Asic1-/- control n = 5, Asic1-/- ultrasound stimulated n = 9, Asic3-/- control n = 5, Asic3-/- ultrasound stimulated n = 5. Refer to Supplementary file 1 for the two-way ANOVA analysis of this graph.

To further address the cell types showing p-ERK response upon ultrasound stimulation, we performed immunofluorescent co-staining of several markers, such as NeuN, NMDAR, GAD67, and PV. There was an obvious p-ERK co-staining with NeuN (94% or 197/209) however these cells were not NMDAR positive (Figure 7—figure supplement 1). On the other hand, there was a very small population co-staining with interneuron marker GAD67 (4.5% or 10/223) and even smaller population co-staining PV (0.9% or 2/211). An ASIC1a-specific antibody for IHC or IF staining will shed light on whether the p-ERK-responsive cells are ASIC1a-positive neurons. Nevertheless, further study is needed to have a comprehensive picture of the cell types that are specific to ultrasound-activated response.

Discussion

Accumulating evidence has shown ASICs are involved in different types of mechanotransduction in the sensory nervous system, including nociception, baroreception, proprioception, and hearing (Cheng et al., 2018; Lin et al., 2016; Chen and Wong, 2013). However, the mechanosensitive role of ASICs in the brain is still not known, although ASIC1a is a predominant acid sensor modulating neural activity in physiological and pathological conditions (Baron and Lingueglia, 2015; Wemmie et al., 2013). Here, we demonstrated low-intensity ultrasound could modulate neural activity in mouse brain and directly activate neurons (Figure 1) via an ASIC1a-depenent manner (Figures 23). While the current view of transcranial ultrasound activation of neurons in brain is through the auditory nerves (Sato et al., 2018), our results from Asic3-/- mice suggest that the peripheral nerves may not play a role in the activation of p-ERK in mouse brain by low intensity ultrasound. Alternatively, the low-intensity ultrasound-mediated mechanotransduction may act via a channel subtype-dependent manner specific for ASIC1a but not for other ASIC subtypes as shown in dextrose prolotherapy (Han et al., 2021). Moreover, repeated transcranial low-intensity ultrasound stimulations are safe and able to elicit adult neurogenesis in mouse brains (Figure 4).

Although ASIC1a was determined as the molecular determinant involved in low-intensity ultrasound mechanotransduction, non-selective mechanosensor inhibitors such as gadolinium and ruthenium red were partially suppressing the calcium response triggered by micropipette ultrasound (Figure 2G and H). Of note gadolinium also blocked ASICs in μM to mM ranges. However, we cannot rule out a role of TRP channels in the micropipette ultrasound mechanotransduction, because there is no evidence showing ruthenium red can also inhibit ASICs. Previous studies have proposed a role of TRP for ultrasound-mediated mechanotransduction while high-intensity ultrasound was applied. More studies are required to validate the role of TRP in low-intensity ultrasound mechanotransduction or the unexpected role of ruthenium red in neuronal ASIC1a signaling pathways.

ASIC1a is widely expressed in the brain and could form as homotrimeric and heterotrimeric channels with different sensitivity to PcTx1 inhibition (Joeres et al., 2016; Sherwood et al., 2011). Specifically, heterotrimeric ASIC2b/ASIC1a can be inhibited 50 % by approximately 3 nM (Sherwood et al., 2011) PcTx1 and ASIC1a/ASIC2a heterotrimeric can be inhibited by 50 nM (Joeres et al., 2016), whereas 0.5 nM to 1 nM can inhibit the homotrimeric ASIC1a (Escoubas et al., 2003; Saez et al., 2011). Therefore, since PcTx1 in low-doses effectively inhibited ASIC1a-mediated calcium signal by micropipette ultrasound, homotrimeric ASIC1a channels may be the predominant subtype involved in ultrasound mechanotransduction in cortical neurons (Figure 3A and Figure 3—figure supplement 1).

To explain the mode of ultrasound induced ASIC1a mechanotransduction, we hypothesized a physical effect of micropipette ultrasound at cell level; which the acoustic streaming imposes shear stress on cell apical surfaces while ultrasound exerts compressional stresses throughout the cells. Considering the combinatorial forces mode in vitro, we argue, in a mixed loading condition (Figures 2B and 3 F), the extracellular domains of ASIC1a are under shear force pulling the protein to the flow direction while the intracellular domains of the ASIC1a are connected to cortical actin or other cytoskeleton, which experiences dynamic reorganization coupling with membrane withdrawals in response to ultrasound (Chu et al., 2019; Lim et al., 2020). As such, mechano-signal triggered ASIC1a, essentially a sodium channel, results in the intracellular calcium elevation possibly by activating voltage-gated calcium channels (Boillat et al., 2014).

The condition in vivo on the other hand (Figure 3—figure supplement 1), is accomplished differently. Neurons are embedded in extracellular matrixes (earthy yellow color) such as laminin, poly-lysine, or poly-ornithine in the brain. ASIC1a is N-glycosylated at N366 and N393, both residues extracellular located (Jing et al., 2012). While N-glycosylation is reported to be involved in the surface trafficking and dendritic spine trafficking of ASIC1 (Jing et al., 2012; Kadurin et al., 2008), the N-glycosylation of many proteins has been known to be important for adhesion and migration (Gu, 2012; Medina-Cano et al., 2018; Stevens and Spang, 2017), implicating the extracellular matrix interacting nature of N-glycans. When ultrasound is applied to the brain, the acoustic pressure exerted through extracellular matrixes, can possibly activate ASIC1a via a cytoskeletal-dependent manner (Figure 3—figure supplement 1), in addition to other mechanosensitive machineries such as PIEZO and TRPV4 (Poole et al., 2014; Servin-Vences et al., 2017), since these mechanoreceptors have all been proven to be triggered by indentation of substrates. Consequently, this leads to an activation of the cells manifesting as ERK phosphorylation.

Since DCX has been accepted as a surrogate mark for neurogenesis in dentate gyrus (Jin et al., 2010; Salvi et al., 2016), increased DCX-positive cells in dentate gyrus with a consecutive 3 day of ultrasound treatment suggests a therapeutic implication. Of note, DCX plays multiple roles in brain development including hippocampal pyramidal neuronal lamination, cortical neuronal migration, and axonal wiring (Germain et al., 2013; Koizumi et al., 2006). The use of ultrasound in developing brains should be extremely cautious.

In conclusion, here we provide evidence that a clinically safely low-intensity transcranial ultrasound could modulate neuronal activity in mouse brain. The low-intensity ultrasound can directly activate neurons via ASIC1a, which provides a molecular basis for future development of ultrasound neuromodulation.

Materials and methods

Ultrasound devices and stimulation parameters

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Two different ultrasonic setups were used in our study. We used a commercial 1MHz transducer (C539-SM, Olympus, Tokyo, Japan) for mouse brain stimulation in the in vivo experiment (Figure 1A). Simulation for calcium imaging was done with micropipette ultrasound (Chu et al., 2021) attaching a 1MHz transducer (15 mm in diameter). Schematics of experimental setup for ultrasound calcium imaging is shown in Figure 2A. All the transducers were controlled by a function generator (Tektronix AFG1022, Beaverton, OR, USA) through a power amplifier (E&I 210 L, Rochester, NY, USA). The input voltage was 900 mVpp with a duty factor of 1% at 1 kHz pulse rate for the in vivo stimulation. We characterized cellular exposure to ultrasound using a hydrophone (HGL-1000, Onda, Sunnyvale, CA, US) immersed in water. The intensity used for in vivo animal experiments was 5 mW/cm2 (ISPTA), and 7.4 mW/cm2 (ISPPA, at 700 mVpp) for micropipette in vitro experiment. These intensity values are within the range of not causing any side effects from ultrasound, like heat and cavitation.

Exploring upstream mechanoreceptors requires a calcium imaging assay that can respond to ultrasound stimulation repeatedly and reliably so that the effect of inhibitors can be demonstrated clearly. Micropipette ultrasound offers a wide range controllability. To select appropriate parameters for calcium experiments, we tested two extreme conditions: one with high input voltage and low duty factor (1500 mVpp, 0.05% duty factor) for a predominant ultrasound stimulus and the other with low input voltage (100 mVpp) and continuous waves for a predominant acoustic streaming stimulus. As a predominant ultrasound stimulus, micropipette ultrasound exhibits a point source characteristic (Figure 2—figure supplement 1A). As a predominant streaming stimulus, micropipette ultrasound yields an inward flow pattern (Figure 2—figure supplement 1B).

The position of the micropipette is adjusted to stimulate the neurons as shown in (Figure 2—figure supplement 1C). The distance from the tip of micropipette to the apical membrane of the cells is approximately controlled to be 20 μm (Figure 2—figure supplement 1D).

Animals

All animal procedures complied with the guidelines of the Institutional Animal Care and Use Committee in Academia Sinica, Taipei, Taiwan. Asic1−/− mice were a gift from Dr. CC Lien of NYCU of Taiwan and generated by crossing Asic1 conditional KO (Asic1f/f) mice (Wu, 2013) with protamine-Cre mice (Lin et al., 2015). Asic3-knockout/eGFP-f-knockin mice (Asic3-/-) were generated based on the Accn3 gene; in brief the design was mainly the targeting allele with a 6 kb long arm (HincII∼HincII DNA fragment located 82 bp downstream the transcription start site) and a 240 bp short arm upstream of the ATG translation start site of Accn3 was used for homologous recombination (Lin et al., 2016). Either wildtype, or Asic1-/- or Asic3-/- C57B6/J mice of 6–8 weeks were shaved under isoflurane anesthesia the day previous to ultrasound stimulation. The mice were randomly assigned to be either sham treated by placing ultrasound transducer on top of head or really exposed to ultrasound for 1 min to evaluate neuronal activities in the mouse brain after the ultrasound stimulation under isoflurane anesthesia. Immediately after the treatment, mice were first anesthetized with urethane (1.5 g/kg; intraperitoneal) and perfused transcardially with 25 ml 0.02 M Tris buffer saline (1 x TBS, pH7.4, at 4°C) and then 25 ml cold fixative (4%[w/v] formaldehyde, 0.02 M TBS (pH7.4, at 4°C)).

Brain histology and immunohistochemistry

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Mouse brain was dissected and post-fixed with 4% formaldehyde at 4°C for 16 hr; tissues were sectioned with Vibratome 1000 Plus (Rankin Biomedical, Holly, MI) at 100 μm thickness and incubated with antibody in free-floating method. For ABC-DAB-Nickel staining, tissue sections were first bleached in 1 x TBS containing 0.03% H2O2 for 30 min, and then blocked in TBST (TBS +0.05% Triton X-100) containing 5% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) and 5% normal goat serum (NGS from Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at room temperature for 60 min, and incubated with Rabbit polyclonal Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) primary antibody [(1:500) #9101, Cell Signaling Technology, Danvers, MA, USA] diluted in blocking solution overnight at 4°C. Sections were then washed three times with TBST and incubated with secondary biotinylated goat-anti-rabbit antibodies (1:1000, Vector Laboratories, Burlingame, CA, USA) for 1 hr at room temperature. After three TBST washes, sections were incubated in the Avidin-Biotin pre-mix solution (1:200, Vector Laboratories, Burlingame, CA, USA). After 3 1xTBS washes, positive immunoreactivity signals were visualized using a Nickel-DAB method [DAB Peroxidase (HRP) Substrate Kit (with Nickel), 3,3’-diaminobenzidine SK-4100, Vector Laboratories, Burlingame, CA, USA or Sigma-Aldrich, St. Louis, MO, USA].

Primary cell culture

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In order to ensure the detection of neuron specific p-ERK, we set up primary culture from neonatal mouse brain. Briefly, cortex isolated from neonatal mouse brain were mechanically minced by Castroviejo scissor and trypsinized by Trypsin (SI-T4174-100ml, Thermo Fisher Scientific, Waltham, MA, USA) diluted in Hanks Buffered Salt Solution (HBSS) (SI-H6648-500ml, Thermo Fisher Scientific, Waltham, MA, USA) with L-glutamine (2 mM/ml) (SI-G7513-100ml, Thermo Fisher Scientific, Waltham, MA, USA) for 15 min at 37°C with three subsequent HBSS washes before treated by deoxyribosenuclease I (SI-D4513-1vl, Thermo Fisher Scientific, Waltham, MA, USA). The treated tissues were then triturated with fire polished glass pipette and strained through 40 μm strainer (431750, Corning Inc, Corning, NY, USA) and seeded on plasma treated and poly-D-Lysine (SI-P7405 Thermo Fisher Scientific, Waltham, MA, USA) coated glass cover slips at a density of 105/ml in B27+ supplemented (Gibco A3582801, Thermo Fisher Scientific, Waltham, MA, USA) neurobasal media (Gibco A3582901, Thermo Fisher Scientific, Waltham, MA, USA) with 10% horse serum (Gibco 26050070, Thermo Fisher Scientific, Waltham, MA, USA) and penicillin/streptomycin (100 U/ml) (Life Technologies, Carlsbad, CA, USA). Culture was gradually replaced with serum free B27+ neurobasal media until day seven for either immunofluorescence or for live cell calcium signal detection.

Live cell calcium signal imaging

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In order to visualize calcium signal in the neurites and in the cell bodies of neuron, we treated the primary cultures with three different green fluorescent dyes, that is Invitrogen Oregon Green 488 BAPTA-1, AM cell permeant (O6807, Thermo Fisher Scientific, Waltham, MA, USA), Invitrogen Fluo-4, AM, FluoroPure grade (F23917, Thermo Fisher Scientific Waltham, MA, USA), or Invitrogen Fura-2, AM, cell permeant (1 mM Solution in Anhydrous DMSO) (F1225, Thermo Fisher Scientific Waltham, MA, USA). Living primary culture on cover slip was immersed in HHBS (20 mM Hepes pH7.4, 1 mM CaCl2, 0.5 mM MgCl2, 0.4 mM MgSO4-7H2O, 5 mM KCl, 0.4 mM KH2PO4, 4 mM NaHCO3, 138 mM NaCl, 0.3 mM Na2HPO4, 6 mM D-Glucose) with 2–5 μM of fluorescent dye and incubate in incubator for 90 min. Subsequently, calcium staining solution was replaced with HHBS with 17% neurobasal media. Cover glass was mounted on an imaging chamber and placed under the fluorescent microscope and micropipette ultrasound was set up to the proximity of targeted cells.

Images were recorded using Olympus IX71 fluorescent microscope (Olympus Corporation, Shinjuku, Tokyo, Japan) with digital camera for microscope Camera attachment with 0.63 x lens (DP80, Olympus Corporation, Shinjuku, Tokyo, Japan). Stacked images were analyzed in ImageJ. ROI of neurites or cell bodies were determined for stacks resliced to obtain data of fluorescence intensities plotted against time points.

Molecular signaling protein inhibitors

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To determine whether PIEZO receptor or TRPC1 was responsible for the signal, we applied the GsMTx-4 (500 nM) (Bae et al., 2011; Bowman et al., 2007) (ab141871, Abcam Inc, Cambridge, MA, USA) isolated from tarantula venom and Gadolinium (10 μM) (Coste et al., 2010) (G7532, Sigma-Aldrich, St. Louis, MO, USA) to the tissues or cells before ultrasound treatment. To investigate the potential role of ASIC channels in ultrasound signal transduction, we utilized the inhibitors such as Amiloride (100 μM) (Leng et al., 2016) (A7410-1G Sigma-Aldrich, St. Louis, MO, USA) and PcTx1 (0.1–50 nM) (Cristofori-Armstrong et al., 2019) (Tocris #5042, Bio-Techne Corporation, Minneapolis, MN, USA). To test whether endoplasmic reticular stored calcium was involved in the calcium signal detected, RyR inhibitor, JTV519 fumarate (10 μM) (Hunt et al., 2007) (Tocris #4564, Bio-Techne Corporation, Minneapolis, MN, USA) and Thapsigargin (T9033, Sigma-Aldrich, St. Louis, MO, USA) was tested.

Immunofluorescence staining of DCX

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After the VLIUS stimulation, the mice were sacrificed and perfused with 10 % formaldehyde/PBS. The brain was then harvested and fixed with 10% formaldehyde/PBS at room temperature. Samples were embedded in paraffin and serial 7 μm transverse sections were mounted on slides. The samples were deparaffinized, rehydrated, antigen retrieved (100℃, 20 min) and washed in PBST. Slices were blocked with 10% newborn calf serum (NCS) and 1% BSA in PBST for 1 hr, incubated with primary antibody overnight at 4℃. After washing with PBST, the samples were incubated with the secondary antibody for 1 hr at room temperature, washed with PBST and mounted with EverBrite Hardset Mounting Medium containing DAPI to label the nuclei (Biotium). Slides were viewed, and images were captured with LSM780 confocal microscope (Zeiss, Jena, Germany). The primary antibodies used for immunostaining and their dilutions were as follows: rabbit anti-DCX (1:200, Cell signaling), mouse anti-MAP2 (1:200, Thermo). The secondary antibodies used were Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:100, Thermo) and Alexa Fluor 555-conjugated goat anti-mouse IgG (1:100, Thermo).

Data and statistical analyses

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Cells were counted using ImageJ. Cells were identified using a global threshold with watershed segmentation. The number of pixel groups was evaluated as the number of cells. Cells were also manually counted from bright-field images. Measurements were compared between control and ultrasound groups using student t-test. A p value ≤ 0.05 was considered to indicate statistical significance. All statistical analyses of animal studies were performed using GraphPad Prism 8.

Appendix 1

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
OtherWildtype C57BL/6 J (Mus musculus)The Jackson LaboratoryStock number: 000664Experimental Animal Facility, Institute of Biomedical Sciences, Academia Sinica IACUC 12-03-332, 20-06-1492
OtherAsic1a-/-(specific targeting alternative spliced isoform Asic1a-/-) C57BL6/J (Mus musculus)Eur J Neurosci 2015 Jun; 41(12):1553–68. doi:10.1111/ejn.12905.Asic1-/-Experimental Animal Facility, Institute of Biomedical Sciences, Academia Sinica IACUC 12-03-332, 20-06-1492
OtherAsic3-/- C57BL6/J (Mus musculus)Nat Commun 2016 May; 7:11,460. doi:10.1038/ncomms11460.Asic3-/-Experimental Animal Facility, Institute of Biomedical Sciences, Academia Sinica IACUC 12-03-332, 20-06-1492
cell line (Cricetulus griseus)Epithelial like Chinese Hamster ovary cellsATCCCCL-61 (RRID:CVCL_0214)
Transfected construct (Discosoma sp.)pCMV - mCherryGift from Dr. Huang, Yi-Shuian, IBMS, Academia Sinica, TaiwanVector control1 μg DNA: 3 μl Lipofectamin 2000 in 1 ml opti-MEM medium
Transfected construct (Mus musculus)pT7-RFP-N2- T▲T - P2A - mASIC1a (alternative spliced isoform Asic1a)The cDNA of mouse ASIC1a was subcloned into the plasmid (pT7-RFP-N2)Asic11 μg DNA: 3 μl Lipofectamin 2000 in 1 ml opti-MEM medium
AntibodypERK; Rabbit polyclonal [polyclonal Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204)]Cell Signaling Technology#9,101 (RRID:AB_331646)IHC (1:500)
AntibodyDoublecortin; (Rabbit polyclonal)Cell Signaling#4,604 (RRID:AB_561007)ICC (1:200)
AntibodyMAP2; (Mouse monoclonal)Thermo Fisher ScientificMA5-12823 (RRID:AB_10982160)ICC (1:200)
AntibodySecondary biotinylated goat-anti-rabbit antibodies; (Goat monoclonal)Vector LaboratoriesBA-1000–1.5 (RRID:AB_2313606)(1:1000)
AntibodyAlexa Fluor 488-conjugated goat anti-rabbit IgG; (Goat monoclonal)Thermo Fisher Scientific16–237 (RRID:AB_436053)(1:100)
AntibodyAlexa Fluor 555-conjugated goat anti-mouse IgG; (Goat monoclonal)Thermo Fisher ScientificA-21422 (RRID:AB_141822)(1:100)
Commercial assay or kitIHC Reagent; Avidin-Biotin pre-mix solutionVector LaboratoriesA-2004–5 (RRID:AB_2336507)(1:200)
Commercial assay or kitIHC staining kit; DAB Peroxidase Substrate Kit with Nickel, 3,3’-diaminobenzidineVector LaboratoriesSK-4100 (RRID:AB_2336382)According to instruction manual
Commercial assay or kitLive cell imaging staining reagent for calcium; Invitrogen Oregon Green 488 BAPTA-1, AM, cell permeantThermo Fisher ScientificO68075 μM
Commercial assay or kitLive cell imaging staining reagent for calcium; Invitrogen Fluo-4, AM, FluoroPureThermo Fisher ScientificF239175 μM
Commercial assay or kitLive cell imaging staining reagent for calcium; Fura-2, AM, cell permeantThermo Fisher ScientificF12215 μM
Chemical compound, drugGsMTx-4Abcam Incab141871500 nM
Chemical compound, drugAmilorideSigma-AldrichA7410-1G100 μM
Chemical compound, drugPcTx1Tocris#5,0420.1–50 nM
Chemical compound, drugRuthenium RedTocris#1,4395–10 μM
Chemical compound, drugRyR inhibitor, JTV519 fumarateTocris#4,56410 μM
Chemical compound, drugThapsigarginSigma-AldrichT9033100 nM
Chemical compound, drugCytochalasin DTocris#1,2335 μg- 10 μg/ml
Chemical compound, drugNocodazoleTocris#1,2285 μg- 10 μg/ml
Software, algorithmImage JAbràmoff, M. D., Magalhães, P. J., & Ram, S. J. (2004). Image processing with ImageJ. Biophotonics international, 11(7), 36–42.Particle analysisSet threshold and define particle sizes
Software, algorithmGraphPad Prism 8Swift, M. L. (1997). GraphPad prism, data analysis, and scientific graphing. Journal of chemical information and computer sciences, 37(2), 411–412.Student t-test

Data availability

All data generated relevant to this study are included in the manuscript are presented in the manuscript either as main figures or as figure supplements. Source data files will be provided when there is a need.

References

Decision letter

  1. Rohini Kuner
    Reviewing Editor; Universität Heidelberg, Germany
  2. Andrew J King
    Senior Editor; University of Oxford, United Kingdom

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This study reports a novel role for the mechanosensitive trimeric cation channel ASIC1 in activation of neurons of the mouse brain by ultrasound stimulation.

Decision letter after peer review:

Thank you for submitting your article "ASIC1a is required for neuronal activation via low-intensity ultrasound stimulation in mouse brain" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Andrew King as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it can be considered further for publication, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Summary:

This is an interesting manuscript suggesting that ultrasound stimuli induce movements of the extracellular matrix and the cytoskeleton to cause mechanical activation of ASIC1a in cortical neurons. This is a novel finding.

Essential revisions:

The reviewers find the results of the study to be intriguing, but have also voiced major concerns about open questions and methodological issues. Most importantly, the reviewers felt that there is insufficient evidence to support a key role for ASIC1a in the responses to ultrasound stimulation.

A revised version would need to address and include the following:

1. It is critically important to back up the claim that ASIC1a mediates the effects described in both in vivo and in vitro experiments. For in vivo experiments pertaining to pERK activation by ultrasound stimulation, appropriate ASIC knockout mice should be used. This experiment is critical for the manuscript to be considered further for a re-review. For in vitro experiments in HEK cells, the reviewers wish to see transfection with ASIC1a and tests performed to determine whether this is sufficient to confer sensitivity to ultrasound stimulation.

2. Calcium imagine experiments: Number of cells tested in calcium imaging experiments must be specified in legends, it should be clarified whether data points refer to individual cells or to mean values, and are sufficient enough for robust interpretations. Data on internal calibration and integrity of cellular responses, e.g. using an ionophore, should be shown. You should clarify if the inhibitors were applied to the control cells from the same panel or to different cells. Please specify how many control cells actually responded to the ultrasound stimulation.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "ASIC1a is required for neuronal activation via low-intensity ultrasound stimulation in mouse brain" for consideration by eLife. Your article has been reviewed by 1 peer reviewer, and the evaluation has been overseen by a Reviewing Editor and Andrew King as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Gary R Lewin (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1. Revise the text to avoid any claim that activation of ASIC1 channel is sufficient for neuronal activation by ultrasound stimulation and that this is necessarily linked to mechanical activation of the channel. Please discuss the alternative scenarios via which ASIC3 could be contributing to this effect.

2. Correct the assertion that ASIC1 is gated via tethers.

3. Ensure that all the images and photomicrographs have scale bars.

Reviewer #3:

The authors have made a very significant effort to add new data to the manuscript. Most noteworthy is the new analysis of ultrasound induced neuron activation (pERK as a surrogate) in both ASIC1 and ASIC3 knockout mouse models. The authors noted a higher baseline number of pERK positive cells in ASIC1-/- mice but no increase was seen after ultrasound exposure. This data together with the data from ASIC3 mutant mice where no attenuation of the ultrasound effect was seen, better support a role for ASIC1 in the ultrasound effect. The authors should still note that this data implicates a role for ASIC1 in being necessary for the effect, but does not show that the mechanical activation of this trimeric channel is the mechanism accounting for the in vivo effect. For example the presence of ASIC1 could be necessary for the trafficking or functionality of other proteins necessary for the effect. Indeed the authors should at least comment in the discussion on the fact that ASIC1a is highly selective for sodium ions cannot directly account for calcium influx measured in the in vitro experiments.

One other general point that the authors should take care of is their assertion that ASIC1a is gated via tethers. As far as I am aware there is no direct evidence of a molecular tether that binds to this protein to gate the channel. Rather, this assertion is based on the observation that indentation of the substrate can activate a such channels (work from the Chen lab). However, this has also been shown for PIEZO and TRPV4 channels PMID: 28135189,PMID: 24662763. These data were reviewed in a short review here PMID: 24981693.

https://doi.org/10.7554/eLife.61660.sa1

Author response

Reviewer #1:

In the manuscript entitled “ASIC1a is required for neuronal activation via low-intensity ultrasound stimulation in mouse brain", Lim et al. investigate the mechanism underlying the activation of brain neurons by transcranial low-intensity ultrasound stimulation. The authors propose that ultrasound stimuli-induced movements of the extracellular matrix and the cytoskeleton cause mechanical activation of ASIC1a in cortical neurons, which leads to ca2+ influx and subsequent expression of pERK, which the authors used as a surrogate marker for neuronal activation.

While I agree that the finding that ultrasound activates neurons via activation of a mechanosensitive ion channel is per se very interesting, I have to say that in my opinion most of the conclusions and claims are not supported by the actual data.

1. The entire study is purely correlative. Thus, the authors made two independent experiments; on the one hand they show that in-vivo transcranial ultrasound stimulation induces pERK in various brain regions and on the other hand they shown that ultrasound-evoked ca2+ influx in cultures of cortical neurons is probably mediated by ASIC1a. From this data they conclude that pERK activation is also mediated by ASIC1a activation. This is, however, pure speculation. The authors must provide additional evidence to support their claim. In my opinion the sole use of PcTx1 is not sufficient to prove that the ca2+ signals are mediated by ASIC1a. Hence, firstly the authors should demonstrate that ASIC1a is indeed activated by ultrasound. This is a very simple experiment. All they would have to do is express ASIC1a in a cell line (e.g. HEK293, CHO, etc) and show that this expression renders the cells sensitive to ultrasound. Second, I would appreciate it if the authors would show that cortical neurons, especially those that show pERK activation, express ASIC1a in the first place. This would also be quite simple – just co-stain the brain sections with an anti-ASIC1a antibody. Third, if the authors want to keep up their claim (see title) that ASIC1a is required for ultrasound activation of brain neurons they should examine ultrasound-induced pERK activation in ASIC1a-knockout mice.

We have performed the Asic1a overexpression experiment suggested by the reviewer using CHO cells. The CHO cells are in favour instead of HEK293 because of the endogenous ASIC1a expression levels in HEK293. The results are organized into Manuscript Figure 4. (Unless otherwise indicated, all the figures are referred to according to the manuscript figure number system). Basically, our results confirm the notion that ASIC1a is mediating the neuronal calcium response. The CHO cells calcium response to pipette delivered ultrasound with a time lag of about 10-15 seconds (Figure 4A,B). We think this may be due to acoustic flow however the delay response is not within our scope of study. The CHO cells overexpressing Asic1a show an immediate calcium response to ultrasound (Figure 4 A, B) comparable to that was detected in primary cultured neurons (Manuscript Figure 2). In order to make sure that the difference is not due to the mere effect to an introduction of the ectopic DNA into the cells, same experiments were conducted using a vector transfected control to compare with Asic1a transfected cells. We have observed and confirmed that the shortened response time to the ultrasound stimulation is due to ectopic ASIC1a (Figure 4C). The responsive time was significantly reduced in Asic1a overexpressed CHO cells (Figure 4 C). After the stimulations, intracellular calcium can be elevated again by 0.1% saponin treatment to cause cell permeation (Figure 4D). The calcium response calibrated by 0.01% Saponin cell perforation could surge to the maximal Fura-2 ratio (F340/380nm) (Figure 4—figure supplement 1 C, D). The 2-way ANOVA analysis of this set of data showed that ASIC1a overexpression did not contribute to the difference of with an insignificant p value (F=0.11; p=0.74) (Figure 4-table supplement 2) while cell perforation contributed significantly to the calcium responses (F=10.52; p<0.0001) (Figure 4-table supplement 2). Note that the calcium response upon ultrasound treatment remained effective under the PcTx1 treatments 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 interaction (F=1.44; p=0.15) (Figure 4-table supplement 3) of drug treatments with ultrasound stimulations (Figure 4-table supplement 3). In contrast, when CHO cells were overexpressing ASIC1a, ultrasound stimulations had no significant effects on the calcium response under the PcTx1 inhibition (Figure 4F) (F=1.26; p=0.29) (Figure 4-table supplement 4), while the two factors interact significantly (F=4.46, p<0.0001) (Figure 4-table supplement 4). In short, different from the failed PcTx1 inhibition of mock control (Figure 4 E), the ASIC1a mediated calcium response upon ultrasound was significantly blocked by PcTx1 starting from as diluted as 2nM (Figure 4F).

The reviewer ask for the immunostaining of ASIC1a expression in the cortical neurons. However, the immunostaining of ASIC1a is currently not possible due to the lack of reliable antibody. We have tried many commercially available antibodies using Asic1a-/- to validate the specificity however we have found no antibody that detect specific signal that is absent in knockout samples.

Despite the co-staining of ASIC1a is not possible because there is no good and specific antibody available that can meet the standard of knockout validation. Nevertheless, we have obtained three batches of Asic1a-/- mice and performed pERK immunohistochemical staining. The results confirmed that the deletion of Asic1a in mice can sufficiently suppressed pERK cell count in cortex, hippocampus and amygdala Figure 7, demonstrating that ASIC1a is required for ultrasound activation of cells in mouse brain. The two-way ANOVA analysis of p-ERK cell counts showed that there was only an interaction of two factors, namely genotype and ultrasound treatment in the cortex (F=6.45, p=0.0037) but not in hippocampus and amygdala (Figure 7-table supplement 1). To further confirm the specific function of ASIC1a in mediating ultrasound stimulation, we included the Asic3-/- in our p-ERK response phenotypes studies and indeed the genotype did not reduce the activation of p-ERK as Asic1a-/- did. These results indicated that the p-ERK response in mouse brain is likely directly caused by the transcranial ultrasound instead of caused by the secondary effects due to the neurons wired with auditory circuits or other sensory circuits, as ASIC3 being mainly expressed in somatosensory neurons and spiral ganglion neurons (1, 2).

2. It is difficult to evaluate the ca2+ imaging experiments, because the method – especially the ultrasound stimulation – is not very well described. Hence it is unclear to me how close to the cell the ultrasound stimulator was placed. Moreover, the N-numbers of the ca2+ imaging experiments are rather small (by the way, it would make reading much easier if the N-numbers were indicated in the figure). Most importantly, it is unclear if the inhibitors (Gadolinium, GsMTx4 etc – Figure 2B-H) were applied to the control cells from the same panel or to different cells. In this context it would be important to know how many control cells actually responded to the ultrasound stimulation. Considering the low N-number, I was wondering if the authors may have had a hard time finding cells that responded and that this is the reason why the N-numbers are so small? I suggest examining many more control neurons and provide information about the proportion of cells that respond. If only for the controls as well as for the cells treated the various channel inhibitors.

To address the concern of the validity of the ca2+ imaging experiments and to allow better evaluation of calcium response, we have performed the experiment again using Fura-2 staining reagent and setting up the imaging system recording F340/380nm images. The results were shown in (Figure 2—figure supplement 2 and Video ).

The same cells were stimulated for the second time and still show a good response within the duration of ultrasound stimulation (Figure 2—figure supplement 2). It is probably due to the imaging system in our laboratory, the photo-decay problem is quite overwhelming, and it will be difficult for us to keep recording the same visual field for subsequent treatments. Thus, although the inhibitors were applied to the same dish, a different visual field is recorded for the quantification of the calcium response. The N-numbers presented in our manuscript is representing a subset of what we have tested, mainly from the experiments using Invitrogen Oregon Green 488 BAPTA-1, AM, cell permeant. When this project was funded year 2019, the Fura-2 staining method and imaging system was not available in our lab yet. When we used Oregon Green 488 BAPTA-1, almost all of the tested visual fields (>90%) were responsive to ultrasound stimulation. Thus, this allowed a better consistency in quantification. On the other hand, if Invitrogen Fluo-4, AM, FluoroPure was used, only 28.6%-38.5% of the tested visual fields were showing the calcium response upon ultrasound stimulation (Video 4). When this staining method is applied, each treatment is performed using a fresh dish of cells. After the image recording, we selected the best responding cells from each experimental group for comparison to the sham-treated control dish of cells. The N-numbers of every graph and chart presented are added to the manuscript figure legends.

Figure 2—figure supplement 1C shows the position of micropipette in the visual field of video 3.

Reviewer #2:

In this study the authors claim that short lasting low intensity ultrasound stimulation activates many neurons in the whole brain. They further claim that the activation mechanism is via the ASIC1a channel. There are some intriguing results in this paper, but there are also many open questions and methodological issues that should be addressed. The authors use pERK as a surrogate for neuronal activation by a global ultrasound stimulus. Some but not all neurons in cortex seem to show activation (it seems only large pyramidal cells, why not interneurons? More analysis needed here).

We have tried to identify the p-ERK cells by co-staining them with several markers, including NeuN, NMDAR, GAD67 and PV. The results in shown in the following micrographs Figure 7—figure supplement 1. We observed that there were visibly a large number of p-ERK co-stained with NeuN (94% or 197/209). However, we have not found any NMDAR co-stained cells in our experiment. We cautioned that this could be a false negative result that was probably due to the limitation of antibody specificities and staining procedures. We found that there is very small population of p-ERK positive cells co-stained with GAD67 (4.5% or 10/223) while even smaller percentage co-stained cells of p-ERK with Parvalbumin (PV) (0.9% or 2/211).

This experiment is followed by an in vitro experiment with cultured cortical neurons from neonates (no ages given for the animals used in this experiment as far as I can see). These are also not equivalent to the adult cells tested in the in vivo experiment. In the bulk of the experiments calcium imaging is used as a surrogate to measure neuronal activation. Unfortunately, in none of the graphs displayed of the Δ F/Fo is there any indication of the number of cells selected and measured. This is critical to evaluate the robustness of the results. In addition, it is normal at the end of the experiment to permeabilize the neurons to calcium by using an ionophore. This allows the assessment of the maximum fluorescence signal when calcium outside concentration equilibrates with the intracellular concentration. This was not done which means the experiments have no internal calibration.

The neuronal cultures were prepared using postnatal day 3 – postnatal day 5 pups. The usage of neonatal primary culture is to have a proof-of-concept that ultrasound can directly activate neurons in a simplified in vitro system. Of note, it is almost impossible to culture adult cortical neurons for calcium imaging studies. To ensure that the results are reproducible and robust, we have applied four different staining reagents; namely (1) Invitrogen Oregon Green 488 BAPTA-1, AM, cell permeant, (2) Invitrogen Fluo-4, AM, FluoroPure, (3) Rhod-2 AM, fluorescent ca2+ indicator (ab142780) and (4) Fura-2, AM, cell permeant. We found that Rhod-2 AM is not compatible with our system and we cannot obtain any good signal from the staining. Thus, this staining method was ruled out. Our observation can be reproduced by using the other three reagents. Of note only that when Fluo-4 is applied, the responding neurons are about 28.6%-38.5% among tested (6/21 and 10/26, in total 46 cells were tested).

We have added 0.1% saponin to show the assessment of the maximum fluorescence signal. Representative results are shown in Figure 4—figure supplement 1.

Likewise, similar cell perforation experiments have been performed using Oregon Green 488 BAPTA-1 for primary neurons to show the maximal calcium response presented in DF/F0 (Figure 2—figure supplement 3). We have also measured the repeated stimulation of the same cells using this staining method and the comparisons of first and second stimulation is shown in Figure 2—figure supplement 3D.

It is for me impossible to assess the robustness of the calcium imaging experiment when I do not know what each data point corresponds to, take Figure 2I as an example. Are these individual cells or means values from many cells from individual cultures? Many critical methodological details are indeed missing from the paper.

Yes. Every data point corresponds to individual cell. Although we have performed the experiments for either two or three times for each treatment, we presented the data from a representative experiment because the staining reagents and imaging settings are sometimes changed, and it is quite impossible to merge all the measurements into one graph. To demonstrate that the calcium response is indeed reproducibly stimulated by the micropipette guided ultrasound and not caused by cell damage, we have set up a new imaging system in the lab to perform the experiment again using Fura-2 staining reagent and the quantification is presented by a ratio of F340nm/380nm. The results were shown in Figure 2—figure supplement 2 and Video 5.

The same cells were stimulated for the second time and still show a good response within the duration of ultrasound stimulation (Figure 2—figure supplement 2). The photo-decay problem causes a difficulty in keeping the same visual field for different treatments of inhibitors. Thus, although the inhibitors were applied to the same dish, a different visual field is recorded for the quantification of the calcium response. The N-numbers presented in our manuscript is representing a subset of what we have tested, mainly from the experiments using Invitrogen Oregon Green 488 BAPTA-1, AM, cell permeant. When we use this reagent, almost every visual field of the tested cells (>90%) were responsive to ultrasound stimulation (Video 3). We place the pipette right on top of the imaged neuron or its neurites under 40X objective lens, the calcium response can be observed at the time points of 1.5 to 5 seconds after the turning on of ultrasound. This can be because of the limitation of our recording setting, which is usually capturing 1 or 2 images per second. In most of the cases, about 1or 2 neuronal cells per visual field can be quantified plotting fluorescence (DF/F0) against time (s). On the other hand, if Invitrogen Fluo-4, AM, FluoroPure is used, only 28.6%-38.5% of the visual fields were showing calcium response upon ultrasound stimulation (Video 4). When this staining method is applied, each treatment is performed using a fresh dish of cells. After the image recording, we select the top responding visual fields from each experimental group for comparison to the control dish of cells.

The idea that ASIC1a is THE critical mediator of this effect is quite surprising and the more dramatic and implausible the conclusion may seem, the more solid the evidence needed. The authors should use ASIC1a mutant mice both in vivo and in vitro to prove that ASIC1a really is critical. The same applies to the apparent effect on neurogenesis.

We have prepared Asic1a gene deletion mice (Asic1a-/-) and Asic3 gene deletion mice (Asic3-/-) for IHC experiments measuring p-ERK response to address the reviewer’s concern for whether Asic1a really play a major role mediating transcranial ultrasound stimulation in mice. Transcranial ultrasound induces p-ERK response in various regions of the mice including cortex, hippocampus and amygdala (representative micrographs Figure 7 A, B, H, I, O, P), while the activation is partially abolished in the Asic1a-/- (Figure 7 C, D, J, K Q, R). Therefore, the Asic1a-/- causes the p-ERK response upon transcranial ultrasound stimulation in the three regions of the brain to become not statistically significant (Figure 7 G, N, U). On the other hand, the effects of Asic3-/- on the p-ERK response to ultrasound are less noticeable (Figure 7 E, F, L, M, S, T). The ultrasound induced p-ERK response is therefore remaining statistically significant (Figure 7 G, N, U). The two-way ANOVA analysis of p-ERK cell counts showed that there was only an interaction of two factors, namely genotype and ultrasound treatment in the cortex (F=6.45, p=0.0037) but not in hippocampus and amygdala (Figure 7-table supplement 1). To further confirm the specific function of ASIC1a in mediating ultrasound stimulation, we included the Asic3-/- in our p-ERK response phenotypes studies and indeed the genotype did not reduce the activation of p-ERK as Asic1a-/- did. These results indicated that the p-ERK response in mouse brain is likely directly caused by the transcranial ultrasound instead of caused by the secondary effects due to the neurons wired with auditory circuits or other sensory circuits, as ASIC3 being mainly expressed in somatosensory neurons and spiral ganglion neurons (1, 2).

We have shown the increase levels of DCX caused by consecutive ultrasound stimulations. This phenomenon has been confirmed independently by two other labs of our collaborators using 4-weeks old mice although we have only reported data produced by the initial batch of mice of 6-weeks. And to address the reviewer’s concern, we have performed the same experiment again using mice with Asic1a-/- (Figure 6).

The videos show quite large physical effects of the ultrasound on the cultures (cells moving around). This is problematic as it may be that what the calcium signals are purely indicative of cell damage. Controls should be provided to ensure this was not the case.

Previously our data were collected by staining reagent Invitrogen Oregon Green 488 BAPTA-1, AM, cell permeant. To ensure that the calcium response is not due to cell damage, we have performed an experiment with the ultrasound of input voltage of 400mVpp and Duty Factor 10% (Video 5). The cells tested under this condition are not moved while showing clear local calcium response with Fura-2 staining, which requires imaging system recording F340nm/380nm. We have stimulated the cells consecutively as shown in Figure 2—figure supplement 2.

Although there is obvious photo-decay after the first stimulation, the cells can recover after 5-10 minutes and be stimulated for the second time showing decent calcium response (Figure 2—figure supplement 2B). Similarly, the repeat stimulation of the same cells was evident in the Oregon Green 488 BAPTA-1-stained cells as shown in Figure 2—figure supplement 3D. In addition to the second round of stimulation despite overwhelming photo-bleaching effects, the calcium response of cell perforation due to the 0.01% Saponin treatment showed a different profile as presented in Figure 2—figure supplement 3C.

References

1. S. H. Lin et al., Evidence for the involvement of ASIC3 in sensory mechanotransduction in proprioceptors. Nat Commun 7, 11460 (2016).

2. W. L. Wu, C. H. Wang, E. Y. Huang, C. C. Chen, Asic3(-/-) female mice with hearing deficit affects social development of pups. Plos One 4, e6508 (2009).

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Reviewer #3:

The authors have made a very significant effort to add new data to the manuscript. Most noteworthy is the new analysis of ultrasound induced neuron activation (pERK as a surrogate) in both ASIC1 and ASIC3 knockout mouse models. The authors noted a higher baseline number of pERK positive cells in ASIC1-/- mice but no increase was seen after ultrasound exposure. This data together with the data from ASIC3 mutant mice where no attenuation of the ultrasound effect was seen, better support a role for ASIC1 in the ultrasound effect. The authors should still note that this data implicates a role for ASIC1 in being necessary for the effect, but does not show that the mechanical activation of this trimeric channel is the mechanism accounting for the in vivo effect. For example the presence of ASIC1 could be necessary for the trafficking or functionality of other proteins necessary for the effect. Indeed the authors should at least comment in the discussion on the fact that ASIC1a is highly selective for sodium ions cannot directly account for calcium influx measured in the in vitro experiments.

One other general point that the authors should take care of is their assertion that ASIC1a is gated via tethers. As far as I am aware there is no direct evidence of a molecular tether that binds to this protein to gate the channel. Rather, this assertion is based on the observation that indentation of the substrate can activate a such channels (work from the Chen lab). However, this has also been shown for PIEZO and TRPV4 channels PMID: 28135189,PMID: 24662763. These data were reviewed in a short review here PMID: 24981693.

We thank the reviewer Prof Gary R Lewin and the editor Prof Rohini Kuner for your thorough examination of our works and your effort to help us refining our data interpretation and statements.

We have taken all the advices and altered our manuscript as listed below.

1. Original text line 47:

“ASIC1a and the tether-mode mechanotransduction were involved in the low-intensity ultrasound-mediated mechanotransduction and cultured neuron activation, which was inhibited by ASIC1a blockade and cytoskeleton-modified agents.”

Sentence altered to line 47:

“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.”

2. Original text line 75-79:

Mechanosensitve ion channels are mainly grouped into bilayer model, such as PIEZO and TRP channels gated by membrane tension change, and extracellular matrix tethered model such as acid sensing ion channels (ASICs) (8-10). Here we aim to identify possible mechanical sensors in mouse brain that can directly respond to low-intensity ultrasound.

Sentences altered to line 77-80:

“Mechanosensitve ion channels such as PIEZO and TRP channels and acid sensing ion channels (ASICs) (8-10) 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.”

3. Original text line 174-176:

Previous studies have shown ASICs are involved in tether mode mechanotransduction, which relies on intact cytoskeletal structures (8, 13).

Sentence altered to 184-186:

“Previous studies have suggested ASICs are involved in tether-mode mechanotransduction, which relies on intact cytoskeletal structures (8, 13).”

4. Original text line 180-183:

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 via tether mode mechanotransduction (Figure 3 F).

Sentence altered to line 190-192:

“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 3 F).”

5. Addressing the issue of whether ASIC3 could be contributing to ultrasound induced changes.

Adding sentence to line 261-263:

“The inclusion of Asic3-/- is to elucidate the role of peripheral nerves in brain activation, as ASIC3 is highly expressed in in somatosensory neurons, trigeminal ganglion neurons, and spiral ganglion neurons.”

6. Original text line 259:

To further confirm the specific function of ASIC1a in mediating ultrasound stimulation,

Sentence altered to line 278:

“To test whether there is a role of peripheral nerves in mediating ultrasound stimulation,”

7. Addressing the issue of whether ASIC3 could be contributing to ultrasound induced changes.

Adding sentences to line 308-319:

“While the current view of transcranial ultrasound activation of neurons in brain is through the auditory nerves (22), our results from Asic3-/- mice suggest that the peripheral nerves may not play a role in the activation of p-ERK in mouse brain by low intensity ultrasound. Alternatively, the low intensity ultrasound-mediated mechanotransduction may act via a channel subtype-dependent manner specific for ASIC1a but not for other ASIC subtypes as shown in dextrose prolotherapy (23).”

8. Original text line 306:

To explain how ultrasound could activate ASIC1a via the tether-mode mechanotransduction,

Sentence altered to line 341:

“To explain the mode of ultrasound induced ASIC1a mechanotransduction,”

9. Original text line 309-310:

Considering the tether model in vitro,

Sentence altered to line 348-349:

“Considering the combinatorial forces mode in vitro,”

10. Addressing the issue of ASIC1a is functioning as a sodium channel, not calcium channel.

Adding sentence to line 353-355:

“As such, mechano-signal triggered ASIC1a, essentially a sodium channel, results in the intracellular calcium elevation possibly by activating voltage-gated calcium channels (30).”

11. Original text line 314-320:

The anchor-pulling condition in vivo on the other hand (Figure 3—figure supplement 1), is accomplished differently. Neurons are embedded in extracellular matrixes (earthy yellow color) such as laminin, poly-lysine, or poly-ornithine in the brain. When ultrasound is applied to the brain, extracellular matrixes anchor the ASIC1a (earthy arrow) while cytoskeletal changes pull it in a different direction (purple arrow), causing an activation of the cells manifesting as ERK phosphorylation.

Sentences altered to line 356-369:

“The condition in vivo on the other hand (Figure 3—figure supplement 1), is accomplished differently. Neurons are embedded in extracellular matrixes (earthy yellow color) such as laminin, poly-lysine, or poly-ornithine in the brain. ASIC1a is N-glycosylated at N366 and N393, both residues extracellular located (31). While N-glycosylation is reported to be involved in the surface trafficking and dendritic spine trafficking of ASIC1 (31, 32), the N-glycosylation of many proteins has been known to be important for adhesion and migration (33-35), implicating the extracellular matrix interacting nature of N-glycans. When ultrasound is applied to the brain, the acoustic pressure exerted through extracellular matrixes, can possibly activate ASIC1a via a cytoskeletal dependent manner (Figure 3—figure supplement 1), in addition to other mechanosensitive machineries such as PIEZO and TRPV4 (36, 37), since these mechanoreceptors have all been proven to be triggered by indentation of substrates. Consequently, this leads to an activation of the cells manifesting as ERK phosphorylation.”

12. Original text line 328-331:

The low-intensity ultrasound can directly activate neurons via tether-mode mechanotransduction and ASIC1a, which provides a molecular basis for future development of ultrasound neuromodulation.

Sentence altered to line 384-386:

“The low-intensity ultrasound can directly activate neurons via ASIC1a, which provides a molecular basis for future development of ultrasound neuromodulation.”

13. Original text line 695-696:

A schematic tether mode mechanotransduction model for in vivo circumstance.

Sentence altered to line 785-786:

“A schematic mechanotransduction model for in vivo circumstance.”

14. We have added scale bar in the micrographs for the following figures:

Figure 2 figure supplement 2

Figure 5

Figure 6

Figure 7 figure supplement 1

https://doi.org/10.7554/eLife.61660.sa2

Article and author information

Author details

  1. Jormay Lim

    Department of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei, Taiwan
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Supervision, Validation, Writing - original draft, Writing – review and editing
    Contributed equally with
    Hsiao-Hsin Tai, Wei-Hao Liao and Ya-Cherng Chu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7191-545X
  2. Hsiao-Hsin Tai

    Department of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei, Taiwan
    Contribution
    Data curation, Formal analysis, Investigation, Methodology, Validation
    Contributed equally with
    Jormay Lim, Wei-Hao Liao and Ya-Cherng Chu
    Competing interests
    No competing interests declared
  3. Wei-Hao Liao

    Department of Physical Medicine and Rehabilitation, National Taiwan Hospital University, Taipei, Taiwan
    Contribution
    Data curation, Formal analysis, Investigation, Methodology
    Contributed equally with
    Jormay Lim, Hsiao-Hsin Tai and Ya-Cherng Chu
    Competing interests
    No competing interests declared
  4. Ya-Cherng Chu

    Department of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei, Taiwan
    Contribution
    Conceptualization, Data curation, Validation, Writing – review and editing
    Contributed equally with
    Jormay Lim, Hsiao-Hsin Tai and Wei-Hao Liao
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6408-2813
  5. Chen-Ming Hao

    Department of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei, Taiwan
    Contribution
    Data curation, Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared
  6. Yueh-Chun Huang

    Department of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei, Taiwan
    Contribution
    Data curation, Formal analysis, Methodology, Validation
    Competing interests
    No competing interests declared
  7. Cheng-Han Lee

    Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
    Contribution
    Animal experiments, Methodology
    Competing interests
    No competing interests declared
  8. Shao-Shien Lin

    Department of Surgery, National Taiwan Hospital University, Taipei, Taiwan
    Contribution
    Data curation, Formal analysis, Methodology, Validation
    Competing interests
    No competing interests declared
  9. Sherry Hsu

    Department of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei, Taiwan
    Contribution
    Data curation, Formal analysis, Investigation
    Competing interests
    No competing interests declared
  10. Ya-Chih Chien

    Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  11. Dar-Ming Lai

    Department of Surgery, National Taiwan Hospital University, Taipei, Taiwan
    Contribution
    Funding acquisition, Resources, Supervision
    Competing interests
    No competing interests declared
  12. Wen-Shiang Chen

    Department of Physical Medicine and Rehabilitation, National Taiwan Hospital University, Taipei, Taiwan
    Contribution
    Funding acquisition, Resources, Supervision
    Competing interests
    No competing interests declared
  13. Chih-Cheng Chen

    Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
    Contribution
    Conceptualization, Methodology, Supervision, Visualization, Writing – review and editing
    For correspondence
    chih@ibms.sinica.edu.tw
    Competing interests
    No competing interests declared
  14. Jaw-Lin Wang

    Department of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei, Taiwan
    Contribution
    Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review and editing
    For correspondence
    jlwang@ntu.edu.tw
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5734-9276

Funding

Ministry of Science and Technology, Taiwan (MOST 107-2221-E-002-068-MY3)

  • Jaw-Lin Wang

Ministry of Science and Technology, Taiwan (MOST108-2321-B-002-047)

  • Jaw-Lin Wang

Ministry of Science and Technology, Taiwan (MOST 108-2321-B-002-061-MY2)

  • Jaw-Lin Wang

National Health Research Institutes (NHRI-EX109-10924EI)

  • Jaw-Lin Wang

Ministry of Science and Technology, Taiwan (MOST 108-2321-B-001-028-MY2)

  • Chih-Cheng Chen

Ministry of Science and Technology, Taiwan (MOST 110-2321-B001-010)

  • Chih-Cheng Chen

National Taiwan University (NTU-CC-107L891105)

  • Jaw-Lin Wang

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This study was supported by Ministry of Science and Technology, Taiwan (MOST 107-2221-E-002-068-MY3, MOST108-2321-B-002-047, MOST 108-2321-B-002-061-MY2), National Health Research Institute, Taiwan (NHRI-EX109-10924EI), National Taiwan University (NTU-CC-107L891105); and grants from MOST, Taiwan (MOST 108-2321-B-001-028-MY2, MOST 110-2321-B-001-010) to CCC.

Ethics

The animal work has been performed following the recommendations in the Guide for the Care and Use of Laboratory Animals of National Taiwan University. All of the animals handling complies to the IACUC protocol #20190055 of National Taiwan University Hospital.

Senior Editor

  1. Andrew J King, University of Oxford, United Kingdom

Reviewing Editor

  1. Rohini Kuner, Universität Heidelberg, Germany

Publication history

  1. Preprint posted: July 10, 2020 (view preprint)
  2. Received: July 31, 2020
  3. Accepted: September 22, 2021
  4. Accepted Manuscript published: September 27, 2021 (version 1)
  5. Version of Record published: October 12, 2021 (version 2)

Copyright

© 2021, Lim et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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