Conclusion
In this work, we established the FVIO-mediated magnetothermal neurostimulation technique by systematically investigating the effect of FVIO dose on stimulus-response time and the in vitro and in vivo biosafety, as well as exploring the effectiveness of using this system for long-term, repeated stimulation and magnetothermal regulation in the deep brains of transgene-free mice. Benefiting from the superior heat-generating performance of the FVIOs, anti-His antibody-coated FVIOs triggered Ca2+ transients in both transfected 293T cells and cortical neurons at a minimum Fe concentration of 54μg/mL, which was 20.27-fold lower than the Fe concentration in the SPIOs. In vivo magnetothermal stimulation of the CeA in freely moving mice demonstrated that the mice treated with FVIOs at the optimal dose of 0.05 μg had a short latency to fear behavior response of approximately 2.51 s , which was 2.3 times faster than that of the SPIO-treated mice under the same AMF conditions. Furthermore, FVIO-mediated magnetothermal stimulation was safer and allowed long-term control of the freezing behaviors of mice for more than 60 days. Even in nongenetically modified mice, 0.28 μg of FVIOs was able to activate the endogenous TRPV1 in the CeA and elicit fear behaviors. Overall, we believe that the FVIO-mediated efficient and safe neuromodulation technique established in this study has potential for future neuroscience and therapeutic applications.