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, 12 (10), 969-74

Wirelessly Powered, Fully Internal Optogenetics for Brain, Spinal and Peripheral Circuits in Mice

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Wirelessly Powered, Fully Internal Optogenetics for Brain, Spinal and Peripheral Circuits in Mice

Kate L Montgomery et al. Nat Methods.

Abstract

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 mm(3)) 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.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare competing financial interests: details are available in the online version of the paper.

Figures

Figure 1
Figure 1. Light delivery using wirelessly powered and fully internal implants
(a) Diagram of light-delivery system. (b) Schematic of wireless implant customized for the brain. (c) Size comparison of wireless implants (left to right: peripheral nerve endings, brain, spinal cord) with a US 1-cent coin.
Figure 2
Figure 2
The implant provides light power densities and pulse characteristics suited for optogenetic stimulation without generating excessive heat. (a) Light power density and efficiency of the LED are each a function of the power supplied to the micro-LED; here, we powered the LED with a wired circuit (not wirelessly). (b) Fidelity of light output for step-function pulses of various pulse widths. Relative transient intensities (arbitrary units) for 100-μs, 5-ms, 10-ms and 5-s pulses, as well as consecutive 5-ms pulses are shown. (c) Calculated light power density across the width of the behavioral area above the resonant cavity. (d,e) Local heating of tissue directly adjacent to the LED. A wired LED probe is inserted into brain with a light power density of 20 mW/mm2 at 5%, 10%, 20% and 40% duty cycles (5-ms pulse width; 10-Hz, 20-Hz, 40-Hz and 80-Hz frequencies, respectively; n = 3 technical trials). Dashed lines denote the temperature associated with neural damage. (d) Temperature versus time; each trace is an average of three trials. (e) Average of final 30 s of light delivery. Bar graphs show mean ± s.e.m.
Figure 3
Figure 3
Wireless optogenetic stimulation of premotor cortex (M2). (a) Positioning of the device: the circuit board and coil are above the skull and below the skin; the LED at the tip of the extension is inserted into the brain directly above motor cortex. (b) Freely moving mouse with the brain implant, shown at a light power of 60 mW/mm2 in order to be visible in the photograph. (c) Trace of mouse movement with device on for 20 s (5-ms pulse width, 20-Hz frequency); representative of 7 out of 10 such trials (mouse circling at 1 turn/min or faster). (d) Mean circling rate of ChR2+ mice, but not wild-type mice, significantly increased (0.40 turns/min to 2.5 turns/min; n = 5 ChR2+ mice, paired t-test, *P = 0.021, effect size (Hedge’s g) = 1.65; n = 3 wild-type mice, paired t-test, P = 0.57). (e,f) Mean speed, normalized by each mouse’s maximum speed, of ChR2+ but not wild-type mice was 40% greater with light stimulation than without. (e) Individual speeds of 5 ChR2+ mice and 3 wild-type mice. (f) Cohort means (n = 5 ChR2+ mice, paired t-test, **P = 0.0025, effect size (Hedge’s g) = 2.4; n = 3 wild-type mice, paired t-test, P = 0.18). Error bars, s.e.m. NS, not significant.
Figure 4
Figure 4
The wireless implant stimulates ChR2+, unmyelinated nociceptors at the spinal cord in freely moving mice. (a) The device is implanted on the right side of the dorsal surface of the vertebra; light is delivered through a drilled hole to L3/L4 of the spinal cord. (b) Freely moving mouse with the wireless spinal cord implant, shown at a light power of 60 mW/mm2 in order to be visible in the photograph. (c) Stimulation of the spinal cord was performed in awake mice (10-Hz frequency, 10-ms pulse width, 10-mW/mm2 light power density). ChR2+ mice showed increased unilateral c-Fos expression during light stimulation compared to EYFP+ control mice (n = 5 ChR2+ mice, 7 EYFP+ mice (two sections averaged per mouse), unpaired t-test, *P = 0.02, effect size (Hedge’s g) = 1.5). Data are shown as mean ± s.e.m. (d) Histology images. ChR2 images are representative of 4 out of 5 ChR2+ mice (15 or more c-Fos+ neurons); EYFP images are representative of 6 out of 7 EYFP+ mice (6 or fewer c-Fos+ neurons). Scale bars, 250 μm.
Figure 5
Figure 5
The implant allows for wireless optogenetic stimulation of peripheral nerve endings. (a) Positioning of the device: the circuit board and coil are subcutaneous and adjacent to the triceps surae muscles; the micro-LED extension is subcutaneously routed to the heel. (b) Freely moving mouse with the peripheral implant (10 mW/mm2). (c) Quantification of c-Fos expression after 10 min of stimulation (10 ms, 10 Hz, 10 mW/mm2). Unilateral c-Fos expression was greater, although not significantly so, in ChR2+ mice than in EYFP+ controls (n = 3 ChR2+ mice, 2 EYFP+ mice (two sections averaged per mouse), unpaired t-test, P = 0.08). (d) Histology images, representative of all ChR2+ mice (15 or more c-Fos+ neurons; left) and EYFP+ control mice (6 or fewer c-Fos+ neurons; right). Green, EYFP; magenta, c-Fos. Scale bars, 250 μm. (e) Schematic of the place-aversion experimental setup. Chambers are above the resonant cavity and support structure, with an open passageway between them. Implants produced light only when above the powered resonant cavity. (f) Change in the percentage of time spent in the resonant cavity chamber over the course of the experiment (cohort means of 5 ChR2+ and 6 EYFP+ mice). A ‘power off’ period (15 min) was followed by a ‘power on’ period (30 min; 10-Hz frequency, 10-ms pulse width, 10-mW/mm2 light power). (g) Overall change in percentage of time spent in the resonant cavity chamber, cohort mean. ChR2 mice spent 34.9% less time in the resonant cavity chamber than EYFP+ mice (n = 5 ChR2+, 6 EYFP+ mice, unpaired t-test, *P = 0.039, effect size (Hedge’s g) = 1.33). Data plotted in f,g as mean ± s.e.m.

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