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

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 (Ca2+) 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 Ca2+ 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, O and focal distance: 15 mm) with real-time Ca2+ fluorescence microscopy imaging (FMI), and fast scan cyclic voltammetry (FSCV) was constructed to evaluate FUS-evoked Ca2+ signaling and concurrent DA release. Ca2+ 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 {micro}s, spatial average pulse average intensity, Isapa: 19.59 {+/-} 4.12 W.cm-2) was shown to causally mobilize Ca2+ dynamics in both cell types. Immediate (< 1 s) responses were focally evoked in cell clusters of 290 {micro}m 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 Ca2+ 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 Ca2+ activity while stimulating DA release from dopaminergic neurons, underscoring its potential as a neuro-stimulation/-modulation tool for targeting dopaminergic circuits in various pathologies.