Abstract
Optogenetics, the selective excitation or inhibition of neural circuits by light, has become a transformative approach for dissecting functional brain microcircuits, particularly in in vivo rodent models, owing to the expanding libraries of opsins and promoters. Yet there is a lack of versatile devices that can deliver spatiotemporally patterned light while performing simultaneous sensing to map the dynamics of perturbed neural populations at the network level. We have created optoelectronic actuator and sensor microarrays that can be used as monolithic intracortical implants, fabricated from an optically transparent, electrically highly conducting semiconductor ZnO crystal. The devices can perform simultaneous light delivery and electrical readout in precise spatial registry across the microprobe array. We applied the device technology in transgenic mice to study light-perturbed cortical microcircuit dynamics and their effects on behavior. The functionality of this device can be further expanded to optical imaging and patterned electrical microstimulation.
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Acknowledgements
We thank W. Jiang and S. Kim for four-point probe measurement. This work was supported by the REPAIR Program (grant N66001-10-C-2010) from the US Defense Advanced Research Projects Agency, the US National Science Foundation (grants CBET-1402803 and CBET-1264816), and Emerging Frontiers in Research and Innovation from the US National Science Foundation (grant 0937848). The work was also partly supported by the Ministry of Science, ICT & Future Planning of Korea through the Center for Integrated Smart Sensors (grant CISS-2012366054204).
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A.V.N. and Y.-K.S. initiated the project. J.L. developed the ZnO MOA device and conducted benchtop characterizations. I.O. designed and led rodent experiments. I.O. and J.L. collected and analyzed neural recording data. I.O. performed Monte Carlo simulations. J.L., I.O. and A.V.N. wrote the manuscript. All authors contributed to the final version of the manuscript. A.V.N. contributed to and directed all aspects of this work.
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Integrated supplementary information
Supplementary Figure 1 Optical transmittance of materials used in MOA.
(a) An electrically conducting 2 mm-thick bulk ZnO substrate shows excellent optical transparency across the visible spectrum. The transmittance curves of other constituent materials used in MOA fabrication are also shown. Note that the ITO and polyimide are thin films; the oscillations in the transmittance of the polyimide film originate from optical interference, as expected in this thickness range for highly uniform films.
Supplementary Figure 2 Step-by-step fabrication process of ZnO MOA structure starting from bulk crystal.
Rounded square symbols identify key processes which are magnified in the figures: (a) Square array of Ti/Au electrical pads defined on O-face of ZnO substrate. (b) Dicing of grooves between electrical pads for electrical isolation. Pads are intentionally aligned off-center to preserve full optical access. (c) A stamping method is used to cover only the top surface of ZnO with the photoresist (PR) layer. (d) Application of polymer adhesive for electrical isolation and mechanical support. Excessive polymer was removed by a rubber blade. (e) Oxygen plasma etching step to disconnect adhesive layer and its removal in acetone. (f) Defining the square pillar array by dicing saw on the opposite side of ZnO in precise spatial registry with steps (a)-(c). (g) Transformation of the square pillars to a tapered, sharp needle array by two-step wet etch process in FeCl3 and diluted H2SO4 solutions, respectively. (h) Conformal deposition of an electrical insulation layer of parylene-C. (i) Spin coating the separation layer with PR and (j) application of PDMS etch mask layer. (k) Plasma etch process is used to expose the ZnO tips, followed by (l) thin ITO layer deposition for recording site definition. (m) PDMS mask removal by resolving PR layer in acetone.
Supplementary Figure 3 Photographs and scanning electron microscope (SEM) images showing additional details of MOA fabrication and process quality.
(a) Large area view of freshly diced and etched ZnO “needle array”, tips of the individual elements are here still covered by the PDMS etch mask and photoresist. (b) SEM image of the tapered ZnO tip geometry following the tip de-insulation process by precisely controlled plasma and wet etch steps, respectively. (c) An SEM view of the ZnO MOA array, with a protective ITO sputtered thin film covering the active tip areas. (d) Close-up cross-sectional image of the materials layers in the tip region, made here visible by intentional breaking of the MOA. The SEM image shows how sputtered ITO layer provides good semi-conformal coating even across the very end of the sharp ZnO tip. (e) Photograph of ZnO bulk single crystal substrate and microfabricated 4×4 optoelectrode array for rodent experiments. (f) Photograph of the multichannel electrical wiring arrangement, taken through semitransparent ribbon cable. Yellow dots (indicated by green circle) show the approximate size of the electrical contact pads; red square shows the unobstructed light accessible optical aperture. Optical apertures of optoelectrodes are spatially separated from gold traces to minimize optically-induced electrical artifacts. (g) The multichannel electrical connection from a 4×4 MOA to a printed circuit board and connector by a custom-made polyimide ribbon cable. (h) MOA inserted in the mouse brain. A transparent sapphire plate was placed above the ribbon cable for temporary mechanical support.
Supplementary Figure 4 Non-sequential ray tracing analysis of light propagation in the MOA structure.
(a) 3D computer model (left) and cross section of 1000 example traces from Gaussian source (w = 60 µm). Escaped rays are captured by detectors surrounding ZnO shank. Different colors of rays used to denote the segmented rays by reflection/refraction/transmission while tracing (b) Calculated intensity distribution of backreflected light measured at the back surface. Reflected rays are well confined in ZnO, away from metal contact and traces. (c) Analyzed irradiance distribution with various input beam sizes. Coupling efficiency is consistent with experimentally measured value; importantly any residual optical power leakage to brain tissue (through shank sidewalls) is negligible.
Supplementary Figure 5 Schematic diagram of laser scanning setup for spatio-temporal optical stimulation of neural activity.
(a) Time-modulated laser pulses were delivered through an optical fiber link onto a 2-axis MEMS mirror (diameter = 0.8 mm) and electronically directed to pre-programmed target positions at the optical input apertures of ZnO MOA elements. The optical alignment of the projection system was actively monitored and controlled by a CCD camera throughout the in vivo experiments. (b) Measured laser beam profile at the input aperture plane of an MOA element. Note that the beam diameter is somewhat smaller than the size of the optical aperture of the individual optoelectrodes to ensure high coupling efficiency and to avoid light scattering at the aperture edges.
Supplementary Figure 6 Demonstrating the capability for fluorescence detection by the transparent ZnO MOA.
(a) In a Thy1-ChR2 mouse, we collected fluorescence images at a variety of depths by a CCD camera while driving the MOA in to the motor cortex. Top row shows the location of those optoelectrode(s) which were targeted by the laser beam (473 nm, 225 µW). The same exposure time was used for all fluorescence images. When the MOA was outside the brain (second row from the top) only weak fluorescence was present, due to fluorescence emanating from the semitransparent polyimide cable. As the array was lowered into the brain (bottom three rows) fluorescence collected from the optoelectrodes intensified notably, now dominated by the yellow fluorescent protein (YFP) expression in the brain. (b) To confirm that these signals are not artifacts, we compared the results in a wild-type mouse (lacking YFP expression) where we only detected the weak fluorescence signals from the polyimide cable. (c) To quantify the practical influence of the fluorescence signals from the polyimide cable in performing fluorescence imaging in general, we performed fluorescence detection in fluorescein dye solutions across a range of concentrations. We inserted the optoelectrodes about 1 mm into the dye solutions and recorded the intensity of fluorescence signals. Even at dye concentrations of only about 100 nM we could clearly identify and separate the fluorescence signals originating from the dye solution from the background.
Supplementary Figure 7 Further examples of light modulated multichannel intracortical recording traces in Thy1-ChR2 mouse.
Raw sample traces (filtered between 0.3-10 kHz) of 16-channel ZnO MOA in anesthetized transgenic mouse, Blue shaded region indicates when optical stimuli are applied.
Supplementary Figure 8 LFP responses at different power regimes.
Peristimulus time plots of local field potential activity obtained in a transgenic mouse at varying levels of input optical power delivered onto optoelectrode 16, here as a light pulse of 1 s long in duration.
Supplementary Figure 9 Assessment of possible residual light-induced artifacts in electrophysiological recordings.
To quantify any light-induced artifacts, we performed simultaneous light delivery and electrophysiology using MOA in the motor cortex of a wild-type mouse lacking opsin expression. We delivered 1 s long laser pulses (n = 50) at the input aperture of the optoelectrodes and recorded multiunit spiking and LFP activity. The figure shows peristimulus time plots of average multiunit spiking rates in (a), and LFP traces in (b) when laser pulses were targeting only optoelectrode 16. Even at highest power levels laser stimulus did not trigger spiking activity in the recorded neurons in the wild type animal, and LFP band showed slight deflections only at optoelectrode 16. At levels beyond approximately 14 μW in optical power, the artifacts became detectable over the spontaneous activity in the LFP signals (only). At this power level, the artifacts had an average peak negative deflection of −0.043 ± 0.022 mV (p < 0.05) within 60 ms after the stimulus onset. In the high power regime (~1.4 mW), the average amplitude of negative deflections was higher (–0.17 ± 0.13 mV, p < 0.001).
The peak amplitude of the deflections in response to 1.36 mW light pulses (high power) was approximately 0.18 mV, some 20 fold smaller than the LFP deflections observed during high-power stimulation in the motor cortex of the transgenic mice. Thus the very small optically induced artifacts had no impact to the recorded activity observed in this work.
The noise levels with and without light in our recordings were also analyzed. To appreciate the individual contributions of the recording system and background neural activity to the overall noise level, we quantified the noise levels under three distinctly different conditions: (1) Thy1-ChR2 transgenic mouse (n = 3 animals); (2) wild type mouse (n = 1); and (3) a physiologically and anatomically dead Thy1-ChR2 transgenic mouse (n = 1, this data is collected about 1 hour after euthanizing the animal). The corresponding RMS noise levels without and with light were (1) 8.5 ± 2.9 µV and 10.1 ± 4.2 µV in transgenic mouse, (2) 7.0 ± 1.0 µV and 7.2 ± 1.5 µV in wild type mouse, and (3) 4.4 ± 0.3 µV and 4.3 ± 0.2 µV in the dead transgenic mouse, respectively.
Supplementary Figure 10 Medium power regime to explore local network connectivity.
(a) Peristimulus multiunit firing rate plots (red traces, 2 ms sliding window) and spike rasters (gray ticks) in the barrel cortex in response to 10 ms long light pulses at 5 Hz (n = 500 in 3 animals) delivered to optoelectrode 1. For each optoelectrode the response probability and latency is indicated if the neural activity at that optoelectrode showed statistically significant modulation in response to stimulation. Latencies were determined from PSTHs as the first time point after the stimulus onset where the PSTH amplitude reaches 5 standard deviations above the mean of PSTH before the stimulus onset. The response probabilities were determined as the probability of appearance of a spike within 20 ms after the stimulus (pstim), corrected against the spontaneous rate of firing pspon, i.e. (pstim – pspon)/(1 – pspon). The significance of the responses (p < 0.01) was determined by boot-straping, i.e. generating 10,000 data sets through random selections from the data. The response probability at optoelectrode 1 was 1.0 with a response latency of τ = 3.8 ms. Some neighboring optoelectrodes also responded to stimulus but with lower response probabilities and longer latencies. Assuming a spike transmission delay of about 1-5 ms during synaptic transmission in the cortex1,2, we infer that sites at optoelectrode 2, 5 and 16 might locate one synapse away from the illuminated site (τ = 7.3, 7.8 and 8.5 ms, respectively). (b) Same plot as in (a), but light pulses were delivered at optoelectrode 16 for comparison. In this case, only optoelectrodes 8, 11, 12 and 15 showed a response. An inference from these two cases is that while neurons at optoelectrode 1 have a strong modulatory influence on neurons at optoelectrode 16, the reverse is not the case.
1. Thomson, A.M. & Lamy, C. Functional maps of neocortical local circuitry. Front. Neurosci. 1, 19 (2007).
2. Fujisawa, S., Amarasingham, A., Harrison, M.T. & Buzsáki, G. Behavior-dependent short-term assembly dynamics in the medial prefrontal cortex. Nat. Neurosc. 11, 823-833 (2008).
Supplementary Figure 11 Comparison of functional motor cortical map obtained by different modalities of stimulation.
We performed joint mapping studies by deploying electrical stimulation (10 monopolar pulses of 50-125 µA at 333 Hz, 100 µs pulse width) and optogenetic stimulation (1 mW, 20-50 ms pulse width), sequentially in the motor cortex. The recorded maps overlapped significantly where the motor responses were identical, at 73 ± 4% (out of n = 48) of the optoelectrode sites. 21 ± 4% of the stimulation sites showed partial overlap in motor responses, i.e. one type of stimulus generated muscle twitches of two body area whereas muscle twitches at one these body areas was obtained in response to the other type of stimulus.
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Sequential control of forelimb movement of transgenic mouse: behavior generation by optical stimulus at approximately layer V. (MP4 6347 kb)
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Lee, J., Ozden, I., Song, YK. et al. Transparent intracortical microprobe array for simultaneous spatiotemporal optical stimulation and multichannel electrical recording. Nat Methods 12, 1157–1162 (2015). https://doi.org/10.1038/nmeth.3620
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DOI: https://doi.org/10.1038/nmeth.3620
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