Ultrasonic Neuromodulation via Astrocytic TRPA1
Soo-Jin Oh, Jung Moo Lee, Hyun-Bum Kim, Jungpyo Lee, Sungmin Han, Jin Young Bae, Gyu-Sang Hong, Wuhyun Koh, Jea Kwon, Eun-Sang Hwang, Dong Ho Woo, Inchan Youn, Il-Joo Cho, Yong Chul Bae, Sungon Lee, Jae Wan Shim, Ji-Ho Park, C. Justin Lee
Current Biology 2019, 29, 3386-3401.e8 · 10.1016/j.cub.2019.08.021
Abstract
Low-intensity, low-frequency ultrasound (LILFU) is the next-generation, non-invasive brain stimulation technology for treating various neurological and psychiatric disorders. However, the underlying cellular and molecular mechanism of LILFU-induced neuromodulation has remained unknown. Here, we report that LILFU-induced neuromodulation is initiated by opening of TRPA1 channels in astrocytes. The Ca 2+ entry through TRPA1 causes a release of gliotransmitters including glutamate through Best1 channels in astrocytes. The released glutamate activates NMDA receptors in neighboring neurons to elicit action potential firing. Our results reveal an unprecedented mechanism of LILFU-induced neuromodulation, involving TRPA1 as a unique sensor for LILFU and glutamate-releasing Best1 as a mediator of glia-neuron interaction. These discoveries should prove to be useful for optimization of human brain stimulation and ultrasonogenetic manipulations of TRPA1.
Abstract via europepmc.
Exposures
Exposure 1: Transcranial LILFU to motor cortex, in vivo tail movement test
Target: motor cortex — “motor cortex (transcranial, in vivo)”
Device: other named manufacturer · EofE Ultrasonics Co., Ltd ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 350 | ✓✓✓ |
| Pulse duration (ms) | 0.23 | ✓✓✓ |
| Pulse repetition frequency (Hz) | 1,500 | ✓✓✓ |
| Duty cycle (%) | not reportedpulse duration × PRF gives 34.5% | ⚑ |
| Sonication duration (s) | 0.0667 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| 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 |
Ultrasound was produced by two function generators in series, amplified by a linear power amplifier, and delivered through a single-element focused transducer (6.5 cm diameter, 7 cm geometrical focal length) via a water-filled cone housing. Tail movement was scored 1-3 depending on movement height, and was increased in an intensity-dependent manner in both WT and TRPA1 KO (or Best1 KO) mice, though less so in the KO lines.
Exposure 2: LILFU applied to organotypic rat hippocampal (CA1) slice culture during MEA recording
Target: CA1 — “CA1 region of organotypic rat hippocampal slice culture”
Device: custom-built · MKC Korea · ultrasound pulser MKPR-1025 with custom 10x10 mm water-immersion transducer ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 500 | ✓✓✓ |
| Pulse duration (ms) | 20.97 | ✓✓✓ |
| Pulse repetition frequency (Hz) | 1,160 | ✓✓✓ |
| Duty cycle (%) | not reportedpulse duration × PRF gives 2432.52% | |
| Sonication duration (s) | 180 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| 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 | |
| Pressure, domain unspecified (kPa) | 11.52 | ✓✓✓⚑ |
For all MEA recording, LILFU was applied for 3 min, following a 3 min baseline; the percent of neuronal firing from baseline was measured. For blocking experiments HC030031, niflumic acid, NPPB, AP-5 or kynurenic acid were added to the ACSF.
Consistency checks: pd exceeds period.
Exposure 3: LILFU applied via pMUT array to HEK293T cells (TRPA1/TRPC1/TRPV4/Piezo1) and cultured cortical astrocytes for Ca2+ imaging
Target: other — “TRPA1/TRPC1/TRPV4/Piezo1-expressing HEK293T cells and primary cultured cortical astrocytes”
Device: custom-built · not_reported (in-house fabrication) · custom piezoelectric micromachined ultrasonic transducer (pMUT) array, ~430 kHz resonance, 500 micron element diameter ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 430 | ✓✓✓ |
| Pulse duration (ms) | 0.25 | ✓✓✓ |
| Pulse repetition frequency (Hz) | 2,000 | ✓✓✓ |
| Duty cycle (%) | not reportedpulse duration × PRF gives 50% | |
| Sonication duration (s) | 0.1 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| 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 | |
| Pressure, domain unspecified (kPa) | 67.3 | ✓✓✓⚑ |
The acoustic power from a single transducer could be controlled from 10.2 kPa to 67.3 kPa by varying the applied actuation voltage from 11 V to 66 V. LEDs for Ca2+ imaging were triggered at 1 Hz and synchronized with sCMOS time-lapse imaging; LILFU-induced Ca2+ responses were compared with N-methylmaleimide (NMM)-induced responses.
Flags from extraction
n_subjects— Paper reports distinct group sizes per sub-experiment across three model systems (e.g., in vivo tail movement WT n=4 vs TRPA1 KO n=7 in Figure 1B; WT n=4 vs Best1 KO n=4 in Figure 6H; various cell/slice replicate counts n=3-15 in other figures) with no single combined total exposed to ultrasound; recorded as not_reported.auditory_control— Paper argues effects are not due to impaired hearing because TRPA1 KO and Best1 KO mice have normal hearing (cited from prior reports), but does not describe an explicit auditory-masking or sham-sound control paradigm; classified as 'other'.exposures[0].unspecified_domain— No pressure or intensity value is stated in text for the in vivo transcranial tail-movement exposure (only that effects were 'intensity-dependent'); left not_reported/null.exposures[1].unspecified_domain.pressure_kpa— Pressure measured at the MEA/slice interface in perfusing ACSF without skull; paper does not state whether this is a free-field or in-situ value, so placed in unspecified_domain.exposures[2].unspecified_domain.pressure_kpa— Acoustic power stated at the transducer surface for cultured cells with no domain terminology used by the paper; placed in unspecified_domain. A range (10.2-67.3 kPa) is achievable, but 67.3 kPa was the value explicitly used for the described Ca2+ imaging protocol.exposures[0].timing.duty_cycle_pct— Duty cycle is not stated as a percentage in the text for any of the three protocols; PRF and pulse duration are given separately and left unconverted.anaesthesia— No anaesthesia is mentioned for the in vivo tail-movement LILFU experiments (a separate, terminal EM procedure used sodium pentobarbital anaesthesia); left not_reported rather than inferring 'awake'.