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Multiplexed Optogenetic Stimulation of Neurons with Spectrum-Selective Upconversion Nanoparticles Xudong Lin, Ying Wang, Xian Chen, Runhuai Yang, Zixun Wang, Jingyu Feng, Haitao Wang, King W. C. Lai, Jufang He, Feng Wang,* and Peng Shi* the membrane of neuron cells.[8,9] Since the first use of channelrhodopsin (ChR) in rodent animals, a wide range of ChR variants have been deve loped to control different aspects of neural function.[10–12] Most of these proteins respond to visible lights ranging from blue to green spectrum, such as ChR2 and C1V1.[13] ChR2 is one of the most common light-sensitive proteins in use today, which can be activated with the blue light;[14] and C1V1 is a ChR variant with red-shifted peak absorption at 539 nm but relatively slower channel kinetics.[14,15] Recently, there have been great efforts for developing stronger red-shifted photo sensitive proteins, aiming to provide direct optogenetic modulation with light of better tissue penetration capability,[10,13] so that tetherless delivery of light into animal’s brain could be implemented. Even though implantation of wirelessly powered light-emitting diode (LED)-devices provides an electronic method to perform tetherless optogenetic experiments,[16–18] an all-optical optogenetic strategy for deep brain stimulation in live animal is yet to be achieved. Alternatively, the use of nanomaterials that are responsive to tissue-penetrating signals, such as near-infrared (NIR) light or ultrasounds, holds great promise for instrumentation of novel ways to control neural activity.[19–22] For example, nanoparticles or nanorods have been utilized to modulate neural activity by photothermal effects;[20,22,23] wireless neural stimulation has also been demonstrated with piezoelectric nanoparticles that
Optical modulation of nervous system becomes increasingly popular as the wide adoption of optogenetics. For these applications, upconversion materials hold great promise as novel photonic elements. This study describes an upconversion based strategy for combinatorial neural stimulation both in vitro and in vivo by using spectrum-selective upconversion nanoparticles (UCNPs). NaYF4 based UCNPs are used to absorb near-infrared (NIR) energy and to emit visible light for stimulating neurons expressing different channelrhodopsin (ChR) proteins. The emission spectrum of the UCNPs is selectively tuned by different doping strategy (Tm3+ or Er3+) to match the responsive wavelength of ChR2 or C1V1. When the UCNPs are packaged into a glass microoptrode, and placed close to or in direct contact with neurons expressing ChR2 or C1V1, the cells can be reliably activated by NIR illumination at single cell level as well as network level, which is characterized by patch-clamping and multielectrode-array recording in culture primary neurons. Furthermore, the UCNP-based optrode is implanted into the brain of live rodents to achieve all-optical remote activation of brain tissues in mammalian animals. It is believed that this proof-of-concept study opens up completely new applications of upconversion materials for regulating physio logical functions, especially in neuroscience research.
1. Introduction Selective activation or inhibition of targeted neurons is essential for modulating of brain functions.[1,2] In the past decade, optogenetics has evolved to be a versatile tool for precise manipu lation of brain circuits for various neuroscience studies.[3–7] This technique is based on optical stimulation of light-sensitive ion channel proteins that are genetically expressed on X. Lin, Y. Wang, R. Yang, Z. Wang, Dr. K. W. C. Lai, Dr. P. Shi Department of Mechanical and Biomedical Engineering City University of Hong Kong Kowloon, Hong Kong SAR 999077, China E-mail:
[email protected] X. Lin, Dr. F. Wang, Dr. P. Shi Shenzhen Research Institute City University of Hong Kong Shenzhen, Guangdong 518000, China E-mail:
[email protected] X. Chen, Dr. F. Wang Department of Physics and Material Science City University of Hong Kong Kowloon, Hong Kong SAR 999077, China
J. Feng, Prof. J. He Department of Biomedical Science City University of Hong Kong Kowloon, Hong Kong SAR 999077, China Dr. H. Wang Chinese Academy of Science Key Laboratory of Brain Function and Disease Department of Neurobiology and Biophysics University of Science and Technology of China Hefei, Anhui 230000, China
DOI: 10.1002/adhm.201700446
Adv. Healthcare Mater. 2017, 1700446
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were actuated by ultrasound signals.[21] In this regard, upconversion nanoparticles (UCNPs) have also been explored for different biological applications, as they can absorb NIR irradiation and reliably emit visible lights.[24,25] UCNPs have been used in different in vivo applications, including noninvasive imaging of deep tissues,[26] photodynamic therapy,[27] and neural activities activation.[28] Particularly, upconversion strategy has recently been suggested to activate ChR proteins in vitro,[28–30] however, UCNP’s practical implementation has not yet been demonstrated in living rodent animals to enable optogenetic control of targeted neural circuits. Here, we systematically characterized an upconversion based strategy for stimulating ChR proteins, and showed multiplexed optogenetic stimulation of neuron cells with spectrum-selective UCNPs. We also further demonstrated tetherless optogenetic brain stimulation in living rodent animals by using UCNP-based microdevices. Specifically, NaYF4 based nanoparticles were used as optical transducers to convert NIR energy to visible light for stimulating neurons expressing opsin proteins. To render the UCNP-based microdevices biocompatibility, the nanoparticles were packaged into a glass micro optrode, which was excited by NIR laser (980 nm) to emit green or blue light, depending on different dopants. When the UCNP-optrodes were placed close to or in direct contact with neurons expressing ChR2 or C1V1, the cells can be reliably activated by NIR illumination at single cell level as well as network level, which was thoroughly characterized by patch-clamp and multielectrode-array (MEA) recording in culture primary neurons. Importantly, for the first time, we also implanted the UCNP-based microoptrode into the brain of live rodent animals, and achieved all-optical tetherless optogenetic activation brain tissues in vivo.
2. Results 2.1. Design of the UCNP-Based Microdevice for Optogenetic Experiments The unique optical advantage of UCNPs is their capability to emit higher-energy light in the visible spectrum after sequential absorption of two or more lower-energy photons, thus providing the opportunity of using NIR to stimulate commonly used opsin proteins without the need of intriguing molecular engineering to change the protein’s responsive wavelength (Figure 1). Given proper instrumentation development, this
configuration can further enable an all-optical tetherless strategy for optogenetic neural activation/inhibition, in virtue of the superior tissue-penetrating capability of NIR. In addition, the highly tunable optical property of UCNPs can potentially be tailored to match different ChR variants for multiplexed neural modulation.
2.2. Synthesis of Upconversion Nanoparticles We fabricated core–shell UCNPs (Figure 2a) composed of lanthanide-doped NaYF4 with emission spectrum (emission of Tm3+ doped UCNPs peaked at 470 nm, while the Er3+ doped UCNPs showed a peak emission at 540 nm) that well matches the excitation wavelengths of ChR2 or C1V1, respectively (Figure 2b). NaYF4 is widely accepted to render the most efficient upconversion emission and the core–shell structure is believed to eliminate surface quenching that usually impedes the upconversion process.[31–33] In this study, core–shell particles were fabricated through a layer-by-layer growth process.[25,34] As shown in Figure 2c and Figure S1a (Supporting Information), transmission electron microscopy (TEM) micrographs revealed a spherical shape of the core with an average size of ≈20 nm. The corresponding core–shell UCNPs showed an increased size with slight variation in morphology (Figure 2d; Figure S1b, Supporting Information). Moreover, dynamic light scattering analysis confirmed that the particle size increased from ≈20 to ≈25 nm upon epitaxial coating of NaYF4 shells (Figure S1c,d, Supporting Information). To make UCNPs biocompatible both in vitro and in vivo, we loaded dry UCNPs in a glass micropipette to form a microoptrode, so that UCNPs were sealed and could be placed in close proximity to neurons in a very concentrated format without direct contact with neuronal cells. The tip of an optrode was excited to emit either blue or green light by remotely applied NIR pulses (980 nm, Movie S1, Supporting Information). Generally, the power of upconverted emission from UCNP-optrodes positively correlated with NIR irradiation power (Figure 2e,f). The Er3+ doped UCNPs showed slightly higher upconversion efficiency than Tm3+ doped UCNPs, especially under high intensity of NIR irradiation, which should not affect the function of neural tissues.[35] Still, we measured local temperature fluctuation within brain tissues around an implanted optrode during the NIR irradiation (Figure S2, Supporting Information). Pulsed illumination by 980 nm laser (8 mW mm−2) induced almost no temperature change on the brain tissue, or at the tip of the UCNP-optrode.
Figure 1. Schematic of near-infrared optogenetic stimulation of neurons with spectrum-selective upconversion nanoparticles.
Adv. Healthcare Mater. 2017, 1700446
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Figure 2. Characterization of upconversion nanoparticles (UCNPs) for neuronal stimulation. a) Schematic illustration of the UCNPs with a core–shell structure. b) The luminescence emission spectrum of UCNPs doped with either Tm3+ (blue line) or Er3+ (green line). c) Transmission electron microscopy (TEM) images of the NaYF4:Yb/Er core. Scale bar, 50 nm. d) TEM image of the NaYF4:Yb/Er@NaYF4 core–shell particles. Scale bar, 50 nm. e) The power of upconverted blue emission from an optrode containing UCNPs doped with Tm3+ at various input power of 980 nm laser. f) The power of upconverted green emission from an optrode containing UCNPs doped with Er3+ at various input power of 980 nm laser.
2.3. Photoactivation of Ion Channels To test whether the upconversion emission from UCNPoptrode is sufficient to induce photocurrents in cultured neurons expressing different opsin proteins, we then performed whole-cell patch-clamping to characterize the upconversion based neural stimulation method in cultured primary neurons. In this experiment, the recording electrode was held by a micromanipulator, and a second micromanipulator was used to position the UCNP-optrode close (
