(J-366) Focus ultrasound-based non-invasive optogenetics for deep brain modulation

Introduction:: Optogenetics has revolutionized neuroscience understanding by allowing spatiotemporal control over cell-type specific neurons in neural circuits. However, the sluggish development of noninvasive photon delivery in the brain has limited the clinical application of optogenetics. Focused ultrasound (FUS) drived mechanoluminescence has emerged as a promising tool for in situ photons emission, but there is not yet a biocompatible liquid-phase mechanoluminescence system for spatiotemporal optogenetics. To achieve noninvasive optogenetics with high temporal resolution and desirable biocompatibility, we have developed liposome (Lipo@IR780/L012) nanoparticles for focused ultrasound triggered mechanoluminescence for brain photon delivery. Synchronized and stable blue light emission was generated in solution under FUS irradiation due to the cascade reactions in liposomes . In vitro tests revealed that Lipo@IR780/L012 could be triggered by FUS for light emission at different stimulation frequencies and hence activate opsin-expressing spiking HEK cells under the FUS irradiation. In vivo optogenetic stimulation further demonstrated that motor cortex neurons could be noninvasively and reversibly activated under the repetitive FUS stimulation after i.v. injection of lipid nanoparticles to achieve limb motion control.


Materials and Methods:: Thy1-ChR2-YFP transgenic mice were used in our study.
The mice were anesthetized with 2.5% isoflurane, and cut the hair on the head of mice by hair clipper. After that, the mice were fixed in a stereotaxic frame, and placed in a 37 ℃ heating pad to maintain body temperature. The eyes of the mouse were protected via coating vet ointment. 0.2 mL Lipo@IR780/L012 was administered through the tail vein.After injection of Lipo@IR780/L012 nanoparticles, ultrasound gel was placed on the mouse head at first, and then the FUS transducer (1.5 MHz, Image Guided Therapy, France) with 5 mm water balloon was placed on the top of the gel. To achieve effective FUS stimulation, please make sure there are no gaps and bubbles between the transducer and brain, and the gel was totally filled. The stereotaxic coordinates of the motor cortex were anteroposterior (AP) ±1.0 mm, mediolateral (ML) +0.5 mm, dorsoventral (DV) −0.5 mm.[8] Then, the isoflurane concentration was adjusted to 0.5%. To guarantee the mouse was in light anesthesia status.before conducting the FUS stimulation, we pinched the toes of the mouse to check the response. The FUS stimulation could be started until we observe the body movement when pinching the toes of the mouse. FUS was conducted with repetition frequency of 1 Hz (100 ms FUS on, 900 ms FUS off) and 2.5 MPa, while the motions of the limbs were recorded through video camera during the FUS stimulation



Results, Conclusions, and Discussions:: We developed a biocompatible mechanoluminescence system based on organic lipid vehicles, sonosensitizer IR780 and chemiluminescence L012 to achieve noninvasive optogenetic stimulation of neural activity under focused ultrasound, without the need for charging of the nanoparticles. Ultrasound energy is noninvasively transmitted to brain tissue and sensed via the sonosensitizer IR780, generating ROS that triggers the nearby chemiluminescence L012, resulting in blue light emission. We revealed that these Lipo@IR780/L012 nanoparticles were able to emit blue light under FUS for activating the CheRiff expressed spiking HEK cells. Furthermore, in vivo experiments demonstrated that motor cortex neurons in Thy1-ChR2-YFP transgenic mice can be temporarily and reversibly activated under the repetitive FUS irradiation after i.v. injection of Lipo@IR780/L012, thus achieving noninvasive sono-optogenetic brain stimulation. Notably, the axial resolution of present FUS is around millimeter, and the development of FUS with submillimeter resolution is imperative for more delicate neural modulation.


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