ASIC1a is required for neuronal activation via low-intensity ultrasound stimulation in mouse brain
Jormay Lim, Hsiao-Hsin Tai, Wei-Hao Liao, Ya-Cherng Chu, Chen-Ming Hao, Yueh-Chun Huang, Cheng-Han Lee, Shao-Shien Lin, Sherry Hsu, Ya-Chih Chien, Dar-Ming Lai, Wen-Shiang Chen, Chih-Cheng Chen, Jaw-Lin Wang
eLife 2021, 10 · 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.
Abstract via europepmc.
Exposures
Exposure 1: transcranial ultrasound to mouse brain (p-ERK activation and DCX/neurogenesis experiments)
Target: whole brain or unfocused — “mouse brain, transducer positioned between the nasal process of maxilla and the axis of the ear”
Device: Olympus / Panametrics · Olympus · C539-SM ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 1,000 | ✓✓✓ |
| Pulse duration (ms) | not reportedimplied by duty cycle ÷ PRF: 0.01 ms (not stated by the paper) | ⚑ |
| Pulse repetition frequency (Hz) | 1,000 | ✓✓✓ |
| Duty cycle (%) | 1 | ✓✓✓ |
| Sonication duration (s) | 60, 300swept | ✓✓✓⚑ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | 0.005 | ✓✓✓ |
| In-situ estimate | not reported | |
| In-situ pressure (kPa) | not reported | |
| In-situ Isppa (W/cm²) | not reported | |
| In-situ Ispta (W/cm²) | not reported |
Mice received either a single 1-min ultrasound exposure (p-ERK experiments) or three consecutive daily treatments (DCX/neurogenesis experiments, stated as either 5-min sessions at 5 mW/cm2 in the main text, or 1-min sessions at 5 mW/cm2 for the Asic1-/- comparison); Figure 5's legend instead describes the first neurogenesis experiment as 4 mW/cm2, 1% for 5 min. Mice were randomly assigned to sham (transducer on head, not powered) or real ultrasound treatment under isoflurane anaesthesia.
Exposure 2: micropipette-guided ultrasound to cultured cortical neurons (calcium imaging mechanotransduction experiments)
Target: cultured neurons — “primary cultured cortical neurons”
Device: custom-built
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 1,000 | ✓✓✓ |
| Pulse duration (ms) | not reported | |
| Pulse repetition frequency (Hz) | not reported | |
| Duty cycle (%) | 0.05, 20, 100swept | ✓✓✓⚑ |
| Sonication duration (s) | 3 | ✓✓✓⚑ |
| Free-field pressure (kPa) | 8, 8.72, 12, 15.3swept | ✓✓✓ |
|---|---|---|
| Free-field Isppa (W/cm²) | 0.0074 | ✓✓✓ |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not applicable | |
| In-situ pressure (kPa) | not applicable | |
| In-situ Isppa (W/cm²) | not applicable | |
| In-situ Ispta (W/cm²) | not applicable |
A micropipette-guided 1-MHz transducer (15 mm diameter) delivered ultrasound at varying input voltage and duty factor (DF): an ultrasound-predominant condition (up to 2000 mVpp, DF 0.05%), an acoustic-streaming-predominant condition (up to 100 mVpp, DF 100%, i.e. continuous), and a mixed condition (700 mVpp, DF 20%) that most effectively elevated calcium. Most dose-response figures used a 3 s stimulation; some supplementary comparisons extended stimulation to 10 s (no response, ultrasound-predominant) or reduced it to 1.5 s (response present, mixed condition).
Flags from extraction
exposures[0].timing.sonication_duration_s— the in vivo sonication duration differs across experiments and across text vs. figure legend: 1 min (p-ERK experiment) and 3x1 min (Asic1 DCX experiment) at 5 mW/cm2, but the first DCX/neurogenesis experiment is stated as 3x5 min at 5 mW/cm2 in text while Figure 5's own legend states 4 mW/cm2, 1% for 5 min for the same experiment -- text and figure disagree on intensity.exposures[0].timing.pulse_duration_ms— paper states duty factor (1%) and pulse rate (1 kHz) for in vivo stimulation but never states pulse duration directly; not computed per instructions.exposures[0].free_field— domain of the 5 mW/cm2 (ISPTA) in vivo intensity is not stated explicitly as free-field or in-brain; it is described in the same sentence as a hydrophone-in-water characterization, so placed in free_field, but this is transcranial delivery and the value could represent an unspecified/derated domain.n_subjects— the paper reports many different small group sizes across separate p-ERK, calcium-imaging, and DCX/neurogenesis sub-experiments (e.g. n=5, n=11, n=10, n=4, n=9...) with no single aggregate total exposed to ultrasound stated.n_sessions_per_subject— sessions per subject differ by experiment: single 1-min session for p-ERK; three consecutive daily sessions for the DCX/neurogenesis experiments.exposures[1].timing.duty_cycle_pct— micropipette ultrasound duty factor was swept across several values (0.05%, 20%, 100%) to test different physical mechanisms (compression vs. streaming vs. mixed); recording the sweep as a list.exposures[1].timing.sonication_duration_s— most micropipette dose-response figures use a 3 s stimulation, but the same section also reports responses with stimulation extended to 10 s and reduced to 1.5 s for other conditions; 3 s recorded as the primary/most common value.