Focal Neurostimulation of Calcium Signaling and Dopamine Release in Human Dopaminergic Neurons Using Megahertz-range Single-Pulse Focused Ultrasound

Sarvenaz Khodayari, Ivan Suarez-Castellanos, Magali Perier, Tom Aubier, Parastoo Hashemi, Alexandre Carpentier, Stephane Marinesco, W. Apoutou N’djin

2026 · 10.64898/2026.02.14.705923

in vitro cellhealthycellular imagingother

Abstract

Focused Ultrasound (FUS) neurostimulation has increasingly attracted attention given its ability for localized targeting and non- to minimally-invasive capacity. However, the understanding of the biological and neurochemical mechanisms triggered by this neurostimulation modality remains limited. Indeed, further progress of this technology could benefit from spatiotemporal evaluation of neurotransmitter secretory activity resulting from FUS stimulation. Recently, we demonstrated in-vitro the ability of FUS to evoke calcium ( Ca 2+ ) waves, in a mixture of human neuronal and glial cells (standard cells), with single-pulse megahertz-range FUS compatible with a transdural approach relying on an intracranial FUS implant. In the present work, we investigated the ability to evoke Ca 2+ signaling and dopamine (DA) release by single-pulse megahertz-range FUS stimulation of cultured human dopaminergic neurons in-vitro . A hybrid-platform integrating a custom-made FUS implant prototype (concave spherical transducer, Ø and focal distance: 15 mm) with real-time Ca 2+ fluorescence microscopy imaging (FMI), and fast scan cyclic voltammetry (FSCV) was constructed to evaluate FUS-evoked Ca 2+ signaling and concurrent DA release. Ca 2+ and DA releases evoked by FUS on dopaminergic neurons (DA neurons) were compared to those evoked in standard cells. Application of single-pulse FUS (frequency: 5.11 MHz, pulse duration: 700 µs, spatial average pulse average intensity, I sapa : 19.59 ± 4.12 W.cm −2 ) was shown to causally mobilize Ca 2+ dynamics in both cell types. Immediate (< 1 s) responses were focally evoked in cell clusters of 290 µm in diameters; corresponding to the −3 dB FUS focal diameter. However, while standard cells exhibited continuous and omnidirectional delayed propagating dynamics following FUS stimulation, DA neurons showed more spatially sparse responses. The FUS-induced Ca 2+ activity in DA neurons was accompanied by DA release detected by FSCV, but not in standard cells. This study demonstrates that single-pulse megahertz-range FUS induced with an intracranial implant prototype smaller than conventionally used FUS devices (transcranial applications) can evoke in-vitro intracellular Ca 2+ activity while stimulating DA release from dopaminergic neurons, underscoring its potential as a neuro-stimulation/-modulation tool for targeting dopaminergic circuits in various pathologies.

Abstract via europepmc.

Specieshuman (ReNCell VM neural progenitor-derived dopaminergic neuron and standard glial/neuronal cultures)

Subjects6, 6swept cultures

Sessions per subjectnot applicable

Randomisednot applicable

Blindingnot applicable

Sham / controlnone

Auditory controlnot reported

Readout timingonline

Anaesthesianot applicable

Readoutscellular imaging, other — Ca2+ fluorescence microscopy imaging (Fluo-4); fast-scan cyclic voltammetry (FSCV) with carbon-fiber microelectrode for dopamine release; immunocytochemistry (tyrosine hydroxylase, Nestin, GFAP)

Direction of effectexcitatorySingle-pulse FUS evoked immediate (<1 s) Ca2+ transients in both dopaminergic neurons and standard cells, and evoked dopamine release detected by FSCV specifically in dopaminergic neurons but not in standard cells.

Adverse eventsnot applicable

Exposures

Exposure 1: Single-pulse FUS stimulation of cultured human dopaminergic neurons and standard (mixed glial/neuronal) cultures

Target: cultured neurons, cultured glia — “cultured dopaminergic (DA) neurons and standard differentiated ReNCell VM cultures (glia + neurons)”
Device: custom-built · Fuji Ceramics Corp. · C-21 ✓

Pulse timing
Waveform	pulsed
Fundamental frequency (kHz)	5,110	✓✓✓
Pulse duration (ms)	0.7	✓✓✓
Pulse repetition frequency (Hz)	not applicable
Duty cycle (%)	not applicable
Sonication duration (s)	0.0007	✓✓✓⚑

Pressure and intensity, by domain
Free-field pressure (kPa)	24,080	✓✓✓
Free-field Isppa (W/cm²)	19,940	✓✓✓⚑
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

Protocol, in the paper’s words

Single 700 µs FUS pulses applied at pulse average acoustic powers (Pac) ranging from 12 W to 24 W were experimentally identified as capable of reliably triggering Ca2+ responses. A single-pulse protocol was chosen because low-energy FUS sequences can modulate neuronal activity without causing irreversible damage, and single, higher-intensity pulses provide a simpler framework for characterizing fundamental neuromodulatory effects before progressing to more complex repetitive-pulse protocols.

Flags from extraction

exposures[0].free_field.isppa_w_cm2 — Isppa value taken from Table 1 (Free-field column); the corresponding in-text paragraph reports Isapa (spatial-average pulse-average), a different quantity, for this condition.
n_subjects — Study reports several sub-analyses each with n=6 independent trials (calcium imaging, FSCV) but never states one overall total number of culture preparations/dishes used.
auditory_control — Not applicable: in vitro cell culture study, no auditory system present.
exposures[0].timing.sonication_duration_s — Single 700 microsecond pulse treated as one uninterrupted 'continuous' burst per the burst-duration convention for single-tone-burst exposures; duty cycle within the pulse explicitly stated as 100%.
exposures[0].timing.waveform — set to pulsed by the tie-break: the ruled timing has a pulse repetition or duty cycle