To enable sophisticated optogenetic manipulation of neural circuits throughout the nervous system with limited disruption of animal behavior, light-delivery systems beyond fiber optic tethering and large, head-mounted wireless receivers are desirable. We report the development of an easy-to-construct, implantable wireless optogenetic device. Our smallest version (20 mg, 10 mm3) is two orders of magnitude smaller than previously reported wireless optogenetic systems, allowing the entire device to be implanted subcutaneously. With a radio-frequency (RF) power source and controller, this implant produces sufficient light power for optogenetic stimulation with minimal tissue heating (<1 °C). We show how three adaptations of the implant allow for untethered optogenetic control throughout the nervous system (brain, spinal cord and peripheral nerve endings) of behaving mice. This technology opens the door for optogenetic experiments in which animals are able to behave naturally with optogenetic manipulation of both central and peripheral targets.
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We acknowledge support from grants from the US National Institutes of Health (National Institute of Neurological Disorders and Stroke grant R01-NS080954), the US National Science Foundation (ECCS-1351687), the Stanford Bio-X NeuroVentures program and the Stanford Bio-X Interdisciplinary Initiatives Program. K.L.M. was supported by a Stanford Interdisciplinary Graduate Fellowship through the Bio-X program. We thank E. Tanabe for his advice about constructing the resonant cavity; A. Yee for assistance with photographing experimental setups; the Stanford Behavioral and Functional Neuroscience Laboratory for assistance with Rotarod experiments; and the members of the Deisseroth, Delp and Poon labs for useful discussions and assistance with experiments.
K.L.M., A.J.Y., J.S.H., V.T., S.M.I., L.G., E.A.F., Y.T., K.D., S.L.D. and A.S.Y.P. have disclosed this technology to the Stanford Office of Technology Licensing for potential therapy applications.
Integrated supplementary information
(a) Resonant cavity powers a wireless device in a mouse on the surface of the cavity. (b) Total setup: the mouse enclosure above the resonant cavity, resonant cavity, phase shifter, signal generator, amplifier, and fan. (c) Drawing for use with assembling the components (Supplementary Data) of the cavity.
Devices reported here have the same approximate mass of a black peppercorn and are much smaller than previously reported wireless optogenetic systems (A–E); reported ranges of mass represented by rectangles. A: Wentz, C.T., et al. A wirelessly powered and controlled device for optical neural control of freely-behaving animals. J Neural Eng 8, 046021 (2011). B: Iwai, Y., Honda, S., Ozeki, H., Hashimoto, M. & Hirase, H. A simple head-mountable LED device for chronic stimulation of optogenetic molecules in freely moving mice. Neurosci Res 70, 124–127 (2011). C: Hashimoto, M., Hata, A., Miyata, T. & Hirase, H. Programmable wireless light-emitting diode stimulator for chronic stimulation of optogenetic molecules in freely moving mice. Neurophotonics 1, 011002 (2014). D: Kim, T.I., et al. Injectable, cellular-scale optoelectronics with applications for wireless optogenetics. Science 340, 211–216 (2013). E: Lee, S., et al. A miniature, fiber-coupled, wireless, deep-brain optogenetic stimulator. IEEE Trans Neural Syst Rehabil Eng (2015).
Similar construction is used for all implants, but small design variations are used to customize the implants to different nervous system targets. In brain and peripheral implants, current is delivered to a 250-μm diameter extension made of a pair of magnet wires with a micro-LED attached at the tip. The extension can be inserted in the brain or routed to peripheral nerves. The micro-LED of the brain implant points parallel to the extension axis, whereas that of the peripheral nerve ending implant points perpendicular to the extension axis. Such an extension was not included in the spinal implant to avoid damage to the cord; instead, the LED was mounted directly onto the PCB. A “spinal cord” device could equivalently be used for superficial brain targets. Peripheral implants change spatial orientation relative to the cavity more than central implants during the course of locomotion. Due to this variability in orientation of the peripheral implant, the individual turns of the coil were set to be non-parallel, thus minimizing orientation-related power fluctuations.
(a) Circuit diagram of the rectifier. This circuit converts RF energy received from the implant coil to direct current for the LED. It is a two-stage voltage doubling circuit using Schottky diodes. (b) The printed circuit board (PCB) was cut to size, and solder paste was applied to the metal traces on the PCB. (c) The surface-mount devices (SMD) were bonded with reflow soldering. (d) The power receiving coil was soldered to the PCB. (e) The coil and SMD components were stabilized with acrylic. (f) The extension was formed from a pair of twisted 36 G wires. (g) The ends of the twisted wires were separated by 70 μm. (h) Solder paste was applied to the tips of the bared wires. (i) The LED was placed on the ends of the wires. (j) The extension was positioned for reflow with a butane torch (post-reflow not shown). (k) The extension was cut to desired length and tested for polarity. (l) The extension was soldered to the bottom of the PCB. (m) The extension was bent to the desired angle and a final coat of acrylic was applied. Scale bars: black, 1 mm; white, 0.5 mm.
Supplementary Figure 5 Power transferred to the wireless implant at different positions and input power levels.
(a) Measurement probe consisting of an LED and the same coil and rectifier circuitry as the brain and spinal cord implants. Light from the LED is directed to a photodiode through a silica optical fiber. (b) Power transferred to the implant at the output of the rectifier circuitry as a function of input power and position on the resonant cavity. Typical operating region on the cavity surface is shaded. The implant is operated at a 20% duty cycle; instantaneous transferred power and time-averaged input power are shown (n = 4 technical trials at each location).
(a) Experimental setup describing system for closed-loop adjustment of delivered power based on measurements on reflected power (Supplementary Note 1). Solid lines: power flow; dashed lines: control signals. (b) Real-time tracking with linear motion: output power increases when reflected power decreases. c) Real-time tracking with angular motion: output power increases when reflected power decreases. (d-e) Closed loop monitoring reduces variability in delivered power with changes in mouse position (d), and mouse angle (e) to within ± 10%. Center values: means, error bars: S.E.M. (n = 3 technical replicates). (f) Measurements of 'normal' stochastic variation in laser output. Variation magnitude in commonly used laser systems is similar to that achieved with closed-loop wireless system.
Supplementary Figure 7 Simulated power density as a function of the height, h, of the mouse above the surface of the cavity.
Thickness of the floor of the behavior chamber or bedding could alter height and therefore decrease delivered power, and should be accounted for (Supplementary Note 2).
Supplementary Figure 8 Local heating due to LED compared to heating due to light from a fiber-coupled laser (20 mW/mm2).
Local temperature of brain tissue of mice was measured with inserted probe (Supplementary Note 3). Center values: means, error bars: S.E.M. (n = 3 trials).
Supplementary Figure 9 Surface mouse temperature due to radio-frequency heating (4-W average input power).
(a) Temperatures of mice were measured with thermal imaging with either the cavity power off or on without an implant over 8 minutes (n = 3 mice). (b) Final reading values for both cavity power off and cavity power on. The mice exhibited a temperature rise of 0.76 °C with the power off, perhaps due to increased behavior in the enclosure tube. The temperature rise attributed to the radio-frequency energy alone is 0.52 °C.
(a) Craniotomy is drilled in the skull and the implant is positioned at the specified x-y-z coordinates of the target brain structure, in this case just superior to M2 (AP: +1.0, ML: 0.5, DV: –0.5; see reference below) using the disposable glass implantation tool attached to the stereotactic positioner. (b) The implant is bonded in place using blue light curable composite (Pentron, N11WA). (c) The stereotactic positioner is unscrewed from the implantation tool and moved out of the way. (d) The implantation tool is gently twisted off, and the skin is then sutured over the implant. Reference: Franklin, K.B.J. & Paxinos, G. The mouse brain in stereotaxic coordinates, (Academic Press, San Diego, 1997).
Supplementary Figure 11 Representative traces of mouse movement during on-off cycles (1 continuous session, 1 mouse).
Motor stimulation of the right motor cortex with 5 ms pulses at 20 Hz elicited circling behavior. Stimulation was wirelessly controlled in 20-s on-off cycles. Traces here representative of three out of five mice; the remaining two mice moved very little or not at all when cavity power was off.
Supplementary Figure 12 Implantation of spinal cord device to stimulate superficial laminae of the dorsal spinal cord.
(a) The spinal column is exposed and secured in stereotactic adaptors for the spinal cord. (b) Superficial soft tissue is removed from the vertebrae. The dorsal process of the vertebra is broken off and then drilled flat. A hole is drilled into the now flat surface of the vertebra directly dorsal to the region to be stimulated. (c) LED is positioned over the hole in the vertebra, either by hand or with a stereotax, using the glass implantation tool. The implant is cemented in place, and the implantation tool is broken off. (d) A second coat of cement is applied before the muscle is sutured in place around the implant. (e) The implant is checked for functionality using an alternate antenna. (f) Skin is sutured over the implant.
Supplementary Figure 13 Implantation of peripheral device to stimulate peripheral nerve endings in the heel of the mouse hind paw.
(a) Incision is made superficial to triceps surae muscles. (b) Size of implant compared to the incision. (c) LED at the tip of the extension is routed to the heel, and the power receiving coil is then placed under the skin, adjacent to the incision (not directly under it). (d) Incision is sutured.
LED was not powered on at any time. (a) Mice were evaluated for Rotarod performance before and after implantation of wireless device in lower hindlimb. Control mice did not undergo surgery. Rotarod latency was not significantly different between implanted and not implanted mice at any time point (n = 9 implanted mice, 10 not implanted mice, P [effect size (Hedge’s g)] - Pre: 0.49 [0.31], Day two: 0.18 [0.62], Day fourteen: 0.86 [0.078]). (b) Mechanical (von Frey) threshold of mice before and after implantation of the wireless device in lower hind limb. Thresholds are not significantly different between the implanted limb and the contralateral limb at any time point (n = 9 mice, P [effect size (Hedge’s g)] - Pre: 0.36 [0.30], Day one: 0.58 [0.23], Day two: 0.91 [0.0.085]).
(a) Withdrawal latency test (cohort means): ChR2+ mice withdrew their paws within a 20 s stimulation window significantly more often than EYFP+ control mice (n = 4 ChR2+ mice, 4 EYFP+ mice, unpaired t-test, P = 0.03, effect size (Hedge’s g) = 1.71). (b) Place aversion test (individual mice): All ChR2+ mice implanted with the peripheral device spent less time in the resonant cavity chamber when the cavity was powered on (n = 5 ChR2+ mice).
Supplementary Figures 1–15 and Supplementary Notes 1–6 (PDF 2268 kb)
Computer aided design file of resonant cavity. Precise specifications for machining the custom resonant cavity. (ZIP 524 kb)
Closed-loop reflection monitoring LabView program. Program to control and limit fluctuations of light power density by monitoring reflected power at the cavity ports. (ZIP 524 kb)
Representative video of circling behavior of a mouse during wireless optogenetic stimulation of premotor cortex (20 Hz frequency, 5 ms pulse width, 10 mW/mm2 light power density). (MP4 15947 kb)
Demonstration of wireless light delivery in mice while wheel running, walking through tube, and engaging in social behavior. (MP4 12452 kb)
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Montgomery, K., Yeh, A., Ho, J. et al. Wirelessly powered, fully internal optogenetics for brain, spinal and peripheral circuits in mice. Nat Methods 12, 969–974 (2015). https://doi.org/10.1038/nmeth.3536
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