Direct activation of zebrafish neurons by ultrasonic stimulation revealed by whole CNS calcium imaging
N Meneghetti, F Dedola, V Gavryusev, G Sancataldo, L Turrini, G de Vito, N Tiso, F Vanzi, J Carpaneto, A Cutrone, F Saverio Pavone, S Micera, A Mazzoni
Journal of Neural Engineering 2020, 17, 056033 · 10.1088/1741-2552/abae8b
Abstract
Objective Ultrasounds (US) use in neural engineering is so far mainly limited to ablation through high intensity focused ultrasound, but interesting preliminary results show that low intensity low frequency ultrasound could be used instead to modulate neural activity. However, the extent of this modulatory ability of US is still unclear, as in in vivo studies it is hard to disentangle the contribution to neural responses of direct activation of the neuron by US stimulation and indirect activation due either to sensory response to mechanical stimulation associated to US, or to propagation of activity from neighboring areas. Here, we aim to show how to separate the three effects and assess the presence of direct response to US stimulation in zebrafish. Approach We observed in zebrafish larvae brain-wide US-induced activity patterns through calcium imaging microscopy. Sensory response to mechanical stimulation was assessed with a US shield. Activity propagation was assessed with inter-area latency evaluation. Main results We prove that in selected brain regions the zebrafish's neural response is mainly due to direct activation, later spreading to the other regions. Shielding the neurons from direct US stimulation resulted in a significantly attenuated response, showing that sensory stimulation does not play a prominent role. Significance US non-invasive neuromodulatory approach might lead to novel ways to test and control neural activity, and hence to novel neuromodulatory therapies. Future studies will focus on the biophysical structure of directly responsive neurons to capture the mechanisms of US induced activity.
Abstract via europepmc.
Exposures
Exposure 1: Whole-CNS unfocused ultrasound stimulation of zebrafish encephalon
Target: whole brain or unfocused — “whole zebrafish encephalon (CNS-wide, unfocused)”
Device: other named manufacturer · Precision Acoustics LTD, Dorchester, UK ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 490 | ✓✓✓ |
| Pulse duration (ms) | 25, 50, 75, 100swept | ✓✓✓ |
| Pulse repetition frequency (Hz) | 10 | ✓✓✓ |
| Duty cycle (%) | 25, 50, 75, 100swept | ✓✓✓ |
| Sonication duration (s) | 0.3 | ✓✓✓ |
| Free-field pressure (kPa) | 8, 40swept | ✓✓✓⚑ |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | 0.0015, 0.12swept | ✓✓✓ |
| 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 |
Pulsed ultrasound stimulations of 490 kHz were delivered in blocks of three stimuli of 100 ms each, with a duty cycle of 25%, 50%, 75% or 100%. A stimulus duration of 300 ms with a pulse repetition frequency of 10 Hz was chosen. Each recording session consisted of three identical US stimulation windows delivered every 15 s, with the overall acquisition time for every recording session fixed at 60 s. Pressure amplitude varied from 8 to 40 kPa in nine discrete steps; each larva underwent two sets of nine recording sessions, one for each pressure amplitude, and for each pressure amplitude the duty cycle was first set at either 25% or 50% and then increased to 100% or 75%. In a separate 'US-shielded' control condition, a 10 mm thick soundproofing element (transmitting 15.85% of incident acoustic pressure) was placed between the transducer and the Petri dish to block direct US stimulation of the brain while preserving water-movement stimulation.
Flags from extraction
exposures[0].free_field.pressure_kpa— Pressure/Ispta calibration was performed both at the experimental distance in free field and, separately, inside the Petri dish to account for reflections/attenuation ('additional intensity measurements were performed by positioning the hydrophone tip inside the Petri dish...These measurements yielded the Ispta calibration reported in figure 1'); it is unclear whether the 8-40 kPa / 1.5-120 mW/cm2 range used for the actual protocol reflects the free-field or dish-corrected calibration.n_subjects— Paper gives separate group sizes for the US-unshielded (12, 10, 8 by dpf) and US-shielded (8, 8, 6 by dpf) cohorts rather than a single total; listed as separate group sizes rather than summed.auditory_control— Paper discusses possible low-frequency (~10 Hz envelope) auditory confound and argues zebrafish larvae have low sensitivity at this frequency, but no active auditory masking/control was implemented; classified as 'other' for this reasoning-based argument rather than an explicit control manipulation.sham_type— The 'US-shielded' condition uses a soundproofing element to block/attenuate (not fully eliminate, 15.85% pressure transmitted) direct ultrasound to the brain while preserving water movement; classified as inactive_transducer ('blocked') though it is a partial block rather than a fully inactive transducer.