In The Spotlight
We read an interesting paper! Here is what it says…about Sono-Optogenetics
Written by Chiara
Last year, researchers at Stanford University worked on a novel approach to perform optogenetics. The study published in late 2020 by Wu and colleagues  (PNAS, 116 (52); 26332-26342) describes sono-optogenetics, which couples the use of ultrasound with optogenetics in order to specifically target deeper brain areas without the use of invasive techniques. Optogenetics is a tool widely used by neuroscientists to control neural activity via expression of light-sensitive proteins, e.g Channelrhodopsins. Thanks to optogenetics, it is possible to excite or inhibit different subtypes of neurons or neuronal populations in a specific neuronal layer. However, optogenetics normally depends on invasive procedures, such as the implantation of optic fibers in the brain, which in turn can lead to gliosis in the site of implantation . This is where sono-optogenetics makes things better. So how does it work and what are its advantages?
The method developed by the team at Stanford exploits mechanoluminescent nanoparticles that are injected in the bloodstream via intravenous injection. With a 400 nm light positioned close to the skin at the level of the jugular vein and facial artery, it is possible to activate the nanoparticles delivered to that area of the body by the bloodstream. These activated particles continue to travel and reach all tissues, including deep brain areas, via the circulatory system. Using Focused Ultrasound (FUS), the nanoparticles are excited by sound waves when they reach the target area. Upon excitation, they emit light at 470nm, which in turn activates ChR2 expressed by cells of the nervous system in the target area.
Here are the major features of this new system:
Excitation light A 400nm excitation light source is placed near the skin, located in close proximity to the blood vessels leading to the brain area to be activated. Light at 400nm activates the mechanoluminescent nanoparticles.
Mechanoluminescent nanoparticles (ZnS:Ag,Co@ZnS particles) The core of the particles is composed of a matrix of ZnS co-doped with Ag+ and Co2+ ions (ZnS:Ag,Co). This core is coated with an un-doped ZnS particle (ZnS:Ag,Co@ZnS). What is the role of these nanoparticles? When ZnS is excited by 400nm light, it is photoactivated. At this point, the Co2+ dopant ions present in the matrix trap the excited electrons. This allows the storage of the photoexcitation of the matrix, without leading to emission. Insteadm,the Ag+ dopant ions receive the energy released by the electrons that were trapped, thus leading to emission of light at 470nm.
Focused Ultrasound (FUS) Sound waves at a specific frequency are transmitted to a very focused area, thus spatially limiting the effect only to the target site. Such a technique is also being used in the medical field, e.g. to treat some types of cancer. The pressure determined by the sound waves reaching the tissue acts as a mechanical stimulus that excites the nanoparticles. The experiments from this paper were performed using a 1.5 MHz frequency, which successfully activated the nanoparticles in vitro and in vivo. While the use of FUS can induce an increase of temperature in the target tissue, ultrasound at this low frequency induced only an increase of less than 0.2°C, meaning rendering the possible alteration of the neural physiology due to local heating effect negligible.
Intravenous injections (i.v.) of the nanoparticles The ZnS:Ag,Co@ZnS nanoparticles have been administered via i.v. injection, thus avoiding the use of invasive techniques. The presence of the nanoparticles in the bloodstream means that they are constantly flowing through the entire body via the circulatory system. This constant supply of nanoparticles leads to renewable sound-induced emission in the target area. Moreover, if the system is continuously excited via FUS stimulation, it reaches a steady-state of excitation which means that the system is rechargeable. The particles are also able to reach deeper brain regions via capillary tracts.
Sono-optogenetics successfully worked in a transgenic mouse line expressing ChR2 for the control of motor behaviour, thus proving that this new method has potential to become widely used in neuroscience research. The main advantages being: it does not require invasive surgeries to implant fibers in the mouse brain, thus avoiding gliosis in response to the implant; it reaches deeper brain regions than normal optogenetic approaches; and that it enables long term experiments (up to hours or days) without renewing the i.v. injection of nanoparticles. Next, it will be important to test the compatibility of sono-optogenetics with in vivo electrophysiology or more complex behavioural tests, that are commonly used to study neuronal activity in various physiological and pathophysiological contests. Seems like sono-optogenetics could help shine some light on more secrets of the brain!
Written by Chiara Galante; Edited by Radhika Menon. Featured image: NGC/Design.
 Wu X, Zhu X, Chong P, Liu J, Andre LN, Ong KS, Brinson K, Mahdi AI, Li J, Fenno LE, Wang H, Hong H. Sono-optogenetics facilitated by a circulation-delivered rechargeable light source for minimally invasive optogenetics. Proceedings of the National Academy of Sciences 2019;116(52):26332-26342. doi: 10.1073/pnas.1914387116
 Fenno L, Yizhar O, Deisseroth K. The development and application of optogenetics. Annu Rev Neurosci. 2011;34:389–412. doi:10.1146/annurev-neuro-061010-113817