Focused ultrasound excites cortical neurons via mechanosensitive calcium accumulation and ion channel amplification

Non-invasive neuromodulation is crucial for neuroscience research and developing neurological and psychiatric disease therapies. Existing techniques like transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS) have limitations in spatial targeting and penetration depth. Focused ultrasound (FUS), however, offers the potential for precise, deep-brain stimulation without genetic or chemical modifications. While FUS has shown promise in eliciting neural and behavioral responses in various animal models and humans, the underlying cellular and molecular mechanisms remain poorly understood. The challenge lies in FUS's multiple physical effects: mechanical force, heating, and cavitation. This study aims to comprehensively elucidate the molecular and cellular mechanisms of FUS-induced neuromodulation in primary cortical neurons, addressing the gap in understanding how ultrasound translates into neuronal excitation.

Previous research has explored the potential of FUS for neuromodulation, demonstrating its ability to elicit neural and behavioral responses in various organisms. However, the precise mechanisms remain unclear, with hypotheses ranging from thermal effects and cavitation to direct mechanical forces on cell membranes. Studies have suggested a role for various ion channels and potential involvement of synaptic transmission. These prior studies often used in vivo models, which complicate the identification of specific underlying mechanisms due to potential confounds like indirect auditory stimulation. This research aimed to overcome those limitations by using in vitro models.

Primary murine cortical neurons were cultured on an acoustically transparent mylar film, allowing for optical recording of calcium and voltage responses to FUS. A focused ultrasound transducer delivered 300 kHz continuous-wave stimulation at varying intensities and durations. Genetically encoded fluorescent indicators (GCaMP6f and Ace2N) were used to monitor calcium and voltage changes, respectively. To determine the role of various physical effects, experiments were conducted under different conditions: varying ultrasound parameters (intensity, duration, frequency), degassed vs. gassed media (to assess cavitation), and cytochalasin D treatment (to alter cellular mechanics). Pharmacological blockers (TTX, Gd³⁺, ruthenium red, suramin, GsMTx4, TTA-P2, AP5, CNQX) and CRISPR/Cas9 gene editing were used to investigate the roles of specific ion channels (TRPV1, TRPV2, TRPV4, Piezo1, TRPC1, TRPM7, TRPP1/2, TRPM4). Ultra-high-speed imaging was employed to examine for bubble formation or cell deformation during ultrasound application. Temperature changes were monitored using a fiber optic thermometer and mCherry fluorescence. The experiments included controls for each condition and treatment. Data analysis involved calculating area under the curve (AUC) of responses and statistical analysis using ANOVA and t-tests.

The study demonstrated that FUS robustly activates cortical neurons in a dose-dependent manner. Temperature elevation and cavitation were ruled out as primary mechanisms. Depolymerizing actin cytoskeleton with cytochalasin D reduced the response, suggesting a role for mechanical stress. Blocking voltage-gated sodium channels with TTX only partially reduced the response, indicating direct calcium influx. Voltage imaging in calcium-free media eliminated the ultrasound-induced depolarization, confirming extracellular calcium as the essential ionic initiator. Pharmacological inhibition and CRISPR/Cas9 knockdown studies implicated specific mechanosensitive ion channels (TRPP1/2, TRPC1) and amplifier channels (TRPM4) in the response pathway. The T-type calcium channels also appear to be involved in the sustained response. Overexpression of TRPC1, TRPP2, and TRPM4 significantly enhanced the ultrasound response, confirming their roles and providing potential avenues for sonogenetic applications. These findings propose that the mechanical effects of ultrasound cause opening of calcium-permeable mechanosensitive channels which then trigger a cascade of events amplifying the calcium response and resulting in neuronal firing.

This study provides a detailed mechanistic understanding of FUS-induced neuronal excitation, resolving conflicting findings from previous research. The findings highlight a primarily mechanical mechanism mediated by specific mechanosensitive and voltage-gated ion channels, ruling out significant contributions from thermal effects and cavitation within the tested parameter range. The identification of key ion channels involved provides crucial information for developing more effective and targeted sonogenetic strategies. The in vitro approach using primary cortical neurons minimizes confounding factors present in in vivo studies, allowing for precise identification of the cellular and molecular events mediating the response. The study's findings are consistent with in vivo observations, suggesting broad applicability of the identified mechanisms.

This research provides a comprehensive mechanistic explanation for FUS-induced neuronal excitation, revealing a pathway involving mechanosensitive channels (TRPP1/2, TRPC1), calcium influx, and calcium-gated amplifier channels (TRPM4) and T-type calcium channels. This detailed understanding opens up new avenues for optimizing FUS-based neuromodulation and for the development of improved sonogenetic tools. Future research could explore the precise mechanical interactions at the membrane level and expand on the potential therapeutic applications.

The study focused on primary murine cortical neurons in vitro, which may not perfectly replicate the complex environment of the in vivo brain. While the selected parameters were chosen to be relevant to in vivo studies, variations in tissue properties and acoustic conditions in vivo could influence the exact mechanisms and efficiency of stimulation. The use of pharmacological and genetic manipulations may have off-target effects or incomplete inhibition/knockdown, potentially influencing the results. Further research using in vivo models is needed to validate the findings and to fully understand the translation of these in vitro findings to clinical applications.