Volume 89, Issue 5, Pages 927-939 (March 2016) A Large-Scale Interface for Optogenetic Stimulation and Recording in Nonhuman Primates  Azadeh Yazdan-Shahmorad, Camilo Diaz-Botia, Timothy L. Hanson, Viktor Kharazia, Peter Ledochowitsch, Michel M. Maharbiz, Philip N. Sabes  Neuron  Volume 89, Issue 5, Pages 927-939 (March 2016) DOI: 10.1016/j.neuron.2016.01.013 Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 CED Infusion of Viral Vector (A and B) Top (A) and side (B) views of MRI-compatible infusion cylinder with fixed grid position, used for monkey G. See Figures S1D and S1E for modified infusion cylinder used for monkey J. (C) MR image of the infusion chamber and the saline-filled cannula grid. (D) Photo of the reflux-resistant injection cannula tip. (E and F) MRI surface rendering of the cortical surface below the cylinder for monkeys G (E) and J (F). The S1 infusion locations are shown in blue, M1 locations in red. (G) Spread of the viral vector in coronal sections of monkey G for one injection site in S1 (shown with an arrow in E). Neuron 2016 89, 927-939DOI: (10.1016/j.neuron.2016.01.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 MR-Based Estimates of Virus Distribution Volume (A) MR volume reconstruction of the spread of viral vector during CED infusion. Brain is shown in light gray; S1 and M1 Vd are shown in blue and red, respectively. (B) Plots of MR-based estimate of Vd versus Vi over time for each infusion site. Neuron 2016 89, 927-939DOI: (10.1016/j.neuron.2016.01.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Cylinder and Artificial Dura (A) Design of the titanium cylinder was based on coronal and sagittal curvatures of the skull. Curvature measurements of monkey G are shown here. (B) Photograph of the artificial dura. (C) Schematic of the implanted artificial dura with flange placed between the dura mater and the arachnoid. (D) Photograph of the implanted titanium cylinder used with monkey G. (E) Photograph of cylinder with artificial dura. The artificial dura does not look transparent because it is viewed at an oblique angle. Neuron 2016 89, 927-939DOI: (10.1016/j.neuron.2016.01.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Surface Imaging (A) Images of the cortical surface taken over a time period of 3 months for both monkeys. The images on the left were taken shortly after the infusion, before initial placement of the artificial dura. The other images were taken with the artificial dura in place. For monkey G, a neomembrane removal was performed between months 1 and 2 (see Figure S4C). The surface of the cortex remains healthy and clear following infusion and chronic artificial dura placement. (B) Left panels: epifluorescence images were obtained approximately 3 months postinfusion (see Experimental Procedures). Right panels: epifluorescence images were thresholded (21% of full saturation; value selected by eye) in order to estimate surface area of expression (see text) and to facilitate comparison with the MR-based estimates of the spread of viral vector (dashed lines; cf. Figure 2). White dots indicate injection sites. Neuron 2016 89, 927-939DOI: (10.1016/j.neuron.2016.01.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Histological Analysis (A) Baseline coronal MR image. (B) Spread of contrast agent after the infusion for the same MR coronal slice as in (A). (C) A coronal tissue section from approximately the same site as in (A) and (B); peroxidase staining reflects expression of the EYFP reporter (see Experimental Procedures). (D) Example higher-magnification image showing EYFP expression in individual cells. (E) A second example, as in (D). (F) Good alignment is observed between the area of EYFP expression measured with surface epifluorescence (dark green areas) or with histological staining (light green lines). These include the region of vector spread estimated from MR images (white line); white dots indicate injection sites. Neuron 2016 89, 927-939DOI: (10.1016/j.neuron.2016.01.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Electrophysiological Recording of Light-Evoked Activity (A) Microphotographs of the μECoG array. The array has 96 electrodes (40 μm diameter), as well as perforations for fiber optic and penetrating electrode insertion (shown in the right panel; not used in this study). (B) Placement of a μECoG array in the cylinder of monkey G. (C) Placement of two μECoG arrays in the cylinder of monkey J. (D and E) μECoG recordings from monkey G (D) and monkey J (E) during pulsed optical stimulation. Recording traces were from the closest electrode to the site of stimulation for examples of M1 (red) and S1 (green) stimulations shown in the upper left panels. Shaded areas around the traces represent SE across 25 trials. The blue squares on the traces show the timing of stimulation. The pseudocolor maps on the left show the spatial distribution of the high-gamma energy of evoked responses across the array(s) for the corresponding M1 stimulation. (F) The full set of stimulus-triggered waveforms, superimposed on the mean waveform, for the four sample pairs of stimulation and recording sites shown in (D) and (E). Neuron 2016 89, 927-939DOI: (10.1016/j.neuron.2016.01.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 Spatiotemporal Analysis of Responses to Different Stimulation Parameters in M1 (A) Filtered evoked activity (60–200 Hz) during 1 s train of pulsed light stimulation. Black line shows the average across 50 trials. Stimulation pulses are shown in blue (filled). (B and C) Filtered evoked activity (60–200 Hz) during 1 pulse of stimulation for 50 trials for the corresponding stimulation parameters shown in (A). Black line shows the average across trials: 50 trials for constant stimuli, 150 trials for sinusoidal stimuli. Stimulation light pulses are shown in blue (filled). (B) shows temporal responses for all pulses, and (C) shows only responses to the first pulse in a 1 s train. (D) Distribution of high-gamma and gamma activity across the μECoG array averaged across 50 trials. The pseudocolor axis represents the power of the response in the high gamma (60–200 Hz) and gamma (30–60 Hz) bands, divided by the power in the same band during the baseline period (500 ms) before the stimulation train. The stimulation site is indicated as a black arrow, and the recording site shown in (A) is indicated as a black dot in upper left panel. Neuron 2016 89, 927-939DOI: (10.1016/j.neuron.2016.01.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 8 Network Connectivity Observed in the Response to Optical Stimulation (A) Light-evoked responses, filtered in the high-gamma band (60–200 Hz) for two sites in S1, one near the site of stimulation (red) and located 3 mm away (green), as well as one site located in M1. The responses are averaged across 50 trials, and shaded areas represent SE. The stimulation pulse shape is shown in blue. (B) Pseudocolor maps show the spatiotemporal distribution of high-gamma power following stimulation averaged across 50 trials. The stimulation and recordings sites (same colors as in A) are shown in the first panel. Black line marks the central sulcus. Neuron 2016 89, 927-939DOI: (10.1016/j.neuron.2016.01.013) Copyright © 2016 Elsevier Inc. Terms and Conditions