Ultrasonocoverslip: In-vitro platform for high-throughput assay of cell type-specific neuromodulation with ultra-low-intensity ultrasound stimulation
Keunhyung Lee, Jung Moo Lee, Tien Thuy Phan, C. Justin Lee, Joo Min Park, Jinhyoung Park
Brain Stimulation 2023 · 10.1016/j.brs.2023.08.002
Abstract
Brain stimulation with ultra-low-intensity ultrasound has rarely been investigated due to the lack of a reliable device to measure small neuronal signal changes made by the ultra-low intensity range. We propose Ultrasonocoverslip, an ultrasound-transducer-integrated-glass-coverslip that determines the minimum intensity for brain cell activation. Brain cells can be cultured directly on Ultrasonocoverslip to simultaneously deliver uniform ultrasonic pressure to hundreds of cells with real-time monitoring of cellular responses using fluorescence microscopy and single-cell electrophysiology. The sensitivity for detecting small responses to ultra-low-intensity ultrasound can be improved by averaging simultaneously obtained responses. Acoustic absorbers can be placed under Ultrasonocoverslip, and stimuli distortions are substantially reduced to precisely deliver user-intended acoustic stimulations. With the proposed device, we discover the lowest acoustic threshold to induce reliable neuronal excitation releasing glutamate. Furthermore, mechanistic studies on the device show that the ultra-low-intensity ultrasound stimulation induces cell type-specific neuromodulation by activating astrocyte-mediated neuronal excitation without direct neuronal involvement. The performance of ultra-low-intensity stimulation is validated by in vivo experiments demonstrating improved safety and specificity in motor modulation of tail movement compared to that with supra-watt-intensity.
Abstract via europepmc.
Exposures
Exposure 1: Ultrasonocoverslip in vitro stimulation of cultured astrocytes and neuron-astrocyte coculture
Target: cultured neurons, cultured glia — “cultured cortical astrocytes and neuron-astrocyte coculture”
Device: custom-built · Ultrasonocoverslip (PVDF-TrFE transducer integrated on a glass coverslip)
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 6,000 | ✓✓✓⚑ |
| Pulse duration (ms) | 0.333 | ✓✓✓ |
| Pulse repetition frequency (Hz) | 1,500 | ✓✓✓ |
| Duty cycle (%) | not reportedpulse duration × PRF gives 49.95% | ⚑ |
| Sonication duration (s) | 0.05, 0.5swept | ✓✓✓⚑ |
| Free-field pressure (kPa) | 50, 150swept | ✓✓✓ |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | 0.04, 0.372swept | ✓✓✓ |
| 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 |
The fundamental pulse sequence used a train of 333 μs long 6 MHz bursts with PRF of 1.5 kHz; 'pulse length' (total sonication) was varied by changing the number of bursts, e.g. 750 bursts for a 500 ms transmission. Frequency was also varied from 5 to 10 MHz at 0.15 MPa and 0.5 s.
Exposure 2: 750 kHz transcranial stimulation of mouse motor cortex (tail movement)
Target: motor cortex — “a motor cortical region ... to the best position where the tail movement was mostly elicited”
Device: custom-built · custom-made 750 kHz PZT concave transducer (DL-61HD), 31 mm diameter, 23 mm focal distance
| Waveform | not reported | |
|---|---|---|
| Fundamental frequency (kHz) | 750 | ✓✓✓ |
| Pulse duration (ms) | not reported | |
| Pulse repetition frequency (Hz) | not reported | |
| Duty cycle (%) | not reported | |
| Sonication duration (s) | 0.5 | ✓✓✓⚑ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | measurementsingle value | |
| In-situ pressure (kPa) | 70, 620swept | ✓✓✓⚑ |
| In-situ Isppa (W/cm²) | 12.32 | ✓✓✓ |
| In-situ Ispta (W/cm²) | 0.08, 6.16swept | ✓✓✓ |
Pulse length of 0.5 s at pressures between 0.07 and 0.62 MPa measured under a mouse skull piece; the acoustic parameters are given only in Fig. 6b, so PRF and duty cycle are not available in the text.
Flags from extraction
exposures[0].fundamental_frequency_khz— Frequency was swept from 5 to 10 MHz at 0.15 MPa and 0.5 s; 6 MHz recorded as the main frequency.exposures[0].unspecified_domain.pressure_kpa— Hydrophone-measured pressure 1 mm above the transducer (0.05-0.15 MPa across experiments, 0.11 MPa also used); medium not stated, so recorded as unspecified. Range read from a figure caption.exposures[0].timing.duty_cycle_pct— Duty cycle not stated; only 333 us bursts at 1.5 kHz PRF.exposures[0].timing.sonication_duration_s— Pulse length varied 0.05-0.5 s (0.1, 0.25, 0.5 s in figures); range from the patch-clamp methods sentence.exposures[1].in_situ.pressure_kpa— Pressure range is across a sweep and is the stated 'measured under a mouse skull piece' range.exposures[1].unspecified_domain.ispta_w_cm2— Ispta values 0.08 and 6.16 W/cm2 taken from a figure caption linked to 0.07 and 0.62 MPa; domain not stated. Isppa 12.32 W/cm2 given only for the highest pressure.exposures[1].timing.sonication_duration_s— 'Pulse length' of 0.5 s read as the sonication duration; PRF and duty cycle only in Fig. 6b.n_subjects— Group sizes for the behaviour experiment (wild type 4, TRPA1 KO 5); c-Fos and TUNEL groups had n = 3. In vitro cultures not counted.