For decades, scientists and physicians have electrically stimulated neurons deep in the brain with implanted electrodes connected through wires to a pacemaker-like device under the skin of the chest. This approach, known as deep brain stimulation (DBS), can treat patients with various neurological symptoms, such as Parkinson’s disease and major depression. However, it requires the implantation of electrodes in the brain, a costly and highly risky procedure. Potential candidates of brain tissue-penetrating stimuli include electric [1-2], magnetic [1,3,4], acoustic [5,6], and optical  signals. For example, recent work has demonstrated activated magnetic nanoparticles can trigger the firing of deep brain neurons expressing heat-sensitive TRPV1 receptors 3 . On the other hand, optical approaches, such as optogenetics, harness genetically encoded light-gated ion channels, the so-called rhodopsins, to optically stimulate target neurons . For deep brain applications, optogenetics has hitherto required the insertion of invasive optical fibers because the activating blue-green wavelengths are strongly scattered and absorbed by endogenous chromophore in brain tissue . To make optogenetics non-invasive, one option is to use near- infrared light (NIR, 650-1350 nm), which can well penetrate the biological tissue and reach deep brain regions. However, NIR optogenetics for deep brain stimulation has yet to be demonstrated due to fails in the development of NIR-shifted rhodopsin variants [9-15].
Recently we developed a novel approach for transcranial NIR stimulation of specifically labelled neurons in deep brain regions by taking advantage of molecularly tailored nanomaterials – the upconversion nanoparticles (UCNPs) . These lanthanide-doped nanocrystals are optically unique in their ability to absorb tissue-penetrating NIR and convert it to wavelength-specific visible emission [17,18]. Once injected into target brain regions, they could serve as optogenetic actuators to stimulate rhodopsin-expressing neurons upon transcranial NIR irradiation. The emission of UCNPs can be precisely tuned to a particular wavelength by control of energy transfer via selective lanthanide-ion doping. Incorporation of Tm 3+ into Yb 3+ -doped host lattices leads to blue emission that matches the maximum absorption of channelrhodopsin-2 (ChR2) for neuronal activation, while the Yb 3+ -Er 3+ couple emits green light compatible with activation of halorhodopsin (NpHR) or archaerhodopsin (Arch) for neuronal inhibition.
We first synthesized blue-emitting NaYF 4 nanocrystals co-doped with Yb 3+ /Tm 3+. The UCNPs exhibited an emission spectrum peaking at 450 and 475 nm upon excitation at 980 nm. We then performed in vivo fiber photometry to examine NIR upconversion by UCNPs in the ventral tegmental area (VTA) of mouse brain, a region located ~4.2 mm below the skull. Upon transcranial delivery of 980 nm laser pulses at a peak power of 2.0 W, an upconverted emission with a power density sufficient for channelrhodopsin activation was detected. To validate the feasibility of UCNP-mediated NIR optogenetics, we performed in vitro electrophysiology to examine NIR activation of dopamine (DA) neurons in acute slices of the mouse VTA. 980 nm NIR pulses triggered membrane depolarization sufficient to generate photocurrents and evoke spikes in VTA DA neurons. The photocurrent amplitudes of ChR2 increased in response to elevated intensity of the incident NIR light under voltage clamp. NIR irradiation could also evoke action potentials of DA neurons as shown in current-clamp traces.
We next tested the in vivo utility of UCNP-mediated NIR upconversion optogenetics. We sensitized VTA DA neurons of TH-Cre mice to transcranial NIR stimulation through viral delivery of ChR2 followed by UCNP injection. Neuronal excitation was successfully triggered by NIR light in ChR2-transfected mice in the presence of UCNPs, as indicated by significantly higher proportion of c-Fos-positive cells in areas where UCNP injection and ChR2 expression overlapped. We further evaluated the real-time efficacy of NIR-evoked excitation of VTA DA neurons with fast-scan cyclic voltammetry. In nomifensine-pretreated mice with both UCNP injection and ChR2 expression in VTA, we detected DA release that was temporally locked to transcranial NIR stimulation.
We further expanded the in vivo application of NIR upconversion optogenetics to neuronal inhibition, as well as to different brain regions such as the hippocampus. First we developed green-emitting UCNPs to match the maximum absorption of rhodopsins that hyperpolarize neurons, such as NpHR and Arch, to achieve neuronal inhibition. With the help of the NaYF 4 :Yb/Er@SiO 2 UCNPs, transcranial low-intensity chronic pulsed NIR irradiation successfully silenced hippocampal neurons during chemically induced hyperexcitability. We further used NIR optogenetics to alter behavior of an awake animal by targeting NIR to granule cells in the hippocampus involved in memory recall. We injected blue-emitting UCNPs into the DG of c-fos-tTA transgenic mice and labeled active c-Fos-expressing DG granule cells with ChR2 during the encoding of fear memory in the absence of doxycycline. We then applied transcranial NIR stimulation to reactivate the labeled granule cells and successfully recalled the fear memory, reflected by increased freezing behavior of the mice during NIR illumination in a
safe context, indicating that our technique works on the behavioral level for transcranial neuronal manipulation.
Our study shows that UCNPs could work as actuators to compile with the current toolbox of light activated channels for functional activation and inhibition of deep brain structures. In the future, refinements of the nanoparticles to establish precise cell-type or intracellular targeting [17,18], as well as improved delivery methods that would further reduce invasiveness , will advance the utility of the approach. The UCNP technology we developed may provide an alternative approach to less invasive deep brain stimulation and neurological disorder therapies.
Fig.1. Principle of UCNP-medicated NIR optogenetics. (A) Schematic mechanism of upconversion optogenetic stimulation. (B,C) Representative TEM images (B) and emission (C) of UCNPs.
Fig.2. In vitro and in vivo applications of UCNP-mediated NIR optogenetics. (A) Validation of the technique by in vivo electrophysiological recordings. (B) Electron micrographs of UCNPs distributed in the VTA tissue. (C) Voltage-clamp traces of neurons from VTA slice preparations in response to 100-ms NIR stimulation at various intensities. (D) Scheme of in vivo cyclic voltammetry to measure DA transients in ventral striatum during NIR stimulation of the VTA. (E) NIR-driven c-Fos expression in the VTA. (F) A trace of background subtracted current measured by cyclic voltammetry in the ventral striatum of a nomifensine-pretreated mouse in response to transcranial NIR stimulation of the VTA (15-ms pulses at 20 Hz, 700-mW peak power).
- E. Dayan, N. Censor, E. R. Buch, M. Sandrini, L. G. Cohen, Noninvasive brain stimulation: from physiology to network dynamics and back. Nat Neurosci 16, 838-844 (2013).
- N. Grossman et al., Noninvasive Deep Brain Stimulation via Temporally Interfering Electric Fields. Cell 169, 1029-1041 e1016 (2017).
- R. Chen, G. Romero, M. G. Christiansen, A. Mohr, P. Anikeeva, Wireless magnetothermal deep brain stimulation. Science 347, 1477-1480 (2015).
- S. A. Stanley et al., Bidirectional electromagnetic control of the hypothalamus regulates feeding and metabolism. Nature 531, 647-650 (2016).
- W. Legon et al., Transcranial focused ultrasound modulates the activity of primary somatosensory cortex in humans. Nat Neurosci 17, 322-329 (2014).
- R. Airan, Neuromodulation with nanoparticles. Science 357, 465 (2017).
- M. R. Hamblin, Shining light on the head: Photobiomodulation for brain disorders. BBA Clin 6, 113-124 (2016).
- L. Fenno, O. Yizhar, K. Deisseroth, The development and application of optogenetics. Annu Rev Neurosci 34, 389-412 (2011).
- J. Y. Lin, P. M. Knutsen, A. Muller, D. Kleinfeld, R. Y. Tsien, ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation. Nat Neurosci 16, 1499-1508 (2013).
- F. Zhang et al., Red-shifted optogenetic excitation: a tool for fast neural control derived from Volvox carteri. Nat Neurosci 11, 631-633 (2008).
- O. Yizhar et al., Neocortical excitation/inhibition balance in information processing and social dysfunction. Nature 477, 171-178 (2011).
- A. S. Chuong et al., Noninvasive optical inhibition with a red-shifted microbial rhodopsin. Nat Neurosci 17, 1123-1129 (2014).
- N. C. Klapoetke et al., Independent optical excitation of distinct neural populations. Nat Methods 11, 338-346 (2014).
- P. Rajasethupathy et al., Projections from neocortex mediate top-down control of memory retrieval. Nature 526, 653-659 (2015).
- R. Prakash et al., Two-photon optogenetic toolbox for fast inhibition, excitation and bistable modulation. Nat Methods 9, 1171-1179 (2012).
- Chen et al., Near-infrared deep brain stimulation via upconversion nanoparticle–mediated optogenetics. Science 359, 679-684 (2018).
- G. Chen, H. Qiu, P. N. Prasad, X. Chen, Upconversion nanoparticles: design, nanochemistry, and applications in theranostics. Chem Rev 114, 5161-5214 (2014).
- B. Zhou, B. Shi, D. Jin, X. Liu, Controlling upconversion nanocrystals for emerging applications. Nat Nanotechnol 10, 924-936 (2015).
- J. Shi, A. R. Votruba, O. C. Farokhzad, R. Langer, Nanotechnology in drug delivery and tissue engineering: from discovery to applications. Nano Lett 10, 3223-3230 (2010).
About the Author
Shuo Chen is a Human Frontier Postdoctoral Fellow at University of California, Berkeley, working with Drs. Na Ji and David Foster. He received his bachelor's degree in chemical engineering at Tsinghua University, and PhD in organic and supramolecular chemistry with Dr. Takuzo Aida at The University of Tokyo. Strong interest in neuroscience led him to embark a journey in neuroscience.Before he joined UC Berkeley, he had been working as a JSPS Postdoctoral Fellow at RIKEN with Dr. Thomas McHugh where he developed NIR upconversion optogenetics. He is interested in developing chemical tools for neuroscience as well as studying the neural mechanism of hippocampal circuitry and memory.