Single cell RNA-sequencing reveals GINIP-expressing neurons as the main targets of focused ultrasound
Elena Brunet, Thibaud Parpaite, Sungjae Yoo, Eric Debieu, Khaled Metwally, Serge Mensah, Pascale Malapert, Andrew Saurin, Olivier Macherey, Emilie Franceschini, Aziz Moqrich
2024 · 10.1101/2024.07.17.604026
Abstract
Dorsal root ganglion (DRG) neurons have a wide range of functions, including touch, pain and itch. These neurons have emerged as promising targets for non-invasive focused ultrasound (FUS) neuromodulation. However, our knowledge of the molecular and physical mechanisms underlying FUS-evoked responses in DRG neurons is limited. Here, we investigate the neuromodulatory capabilities of FUS in cultured DRG neurons in combination with calcium imaging. We find that a 20-MHz FUS burst of 1-ms duration at an acoustic pressure of 5 MPa elicited calcium responses in 52% of DRG neurons. Single-cell RNA sequencing reveals that the majority of FUS-sensitive neurons belong to three subsets of DRG neurons; C-LTMRs, the MRGPRD-expressing C-HTMRs and A6-LTMRs. FUS excites all these neuronal subtypes by membrane deformation, suggesting a mechanism mediated by mechanosensitive ion channels. Our results identify FUS parameters that activate distinct subsets of DRG neurons and open new avenues for using FUS stimulation to modulate DRG neuron function.
Abstract via europepmc.
Exposures
Exposure 1: optimal FUS stimulus to cultured DRG neurons
Target: dorsal root ganglion culture — “cultured DRG neurons”
Device: Olympus / Panametrics · Olympus · V317-SM ✓
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 20,000 | ✓✓✓ |
| Pulse duration (ms) | not applicable | ⚑ |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | not applicable | |
| Sonication duration (s) | 0.0001, 0.001swept | ✓? |
| Free-field pressure (kPa) | 4,000, 5,000swept | ✓?⚑ |
|---|---|---|
| Free-field Isppa (W/cm²) | 346, 536swept | ✓? |
| 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 |
The FUS stimulus consisted of a 20-MHz sinusoidal signal with peak positive pressure of 4 to 5 MPa and stimulus duration of 0.1 to 1 ms. Each cell was subjected to a single FUS stimulus to avoid potential cumulative effects (the 'optimal FUS stimulus' was 5 MPa for 1 ms). For the scRNA-seq experiment, neurons instead received two consecutive optimal FUS stimulations 2 minutes apart; for the deformation experiments, neurons received up to 6-7 consecutive stimuli sweeping pressure or duration.
Flags from extraction
n_sessions_per_subject— Most cells received a single FUS stimulus (used for the pressure/duration dose-response and n=192 count), but the scRNA-seq and deformation sub-experiments used repeated stimulations (2 to 7 consecutive stimuli); n_sessions_per_subject=1 reflects the main dose-response experiment only.exposures[0].free_field.pressure_kpa— Cells were in a dish/culture setup (no skull); paper describes the beam as 'free-field' (measured by hydrophone) so these values were placed in free_field, though this is a bath-type in vitro exposure rather than a whole-water free-field beam characterization for a transcranial study.exposures[0].timing.pulse_duration_ms— Classified as continuous (single uninterrupted tone burst per trial) per the burst rule; sonication_duration_s carries the 0.1-1 ms burst length instead.