Optogenetic micro-electrocorticography for modulating and localizing cerebral cortex activity

Abstract

Objective: Spatial localization of neural activity from within the brain with electrocorticography (ECoG) and electroencephalography remains a challenge in clinical and research settings, and while microfabricated ECoG (micro-ECoG) array technology continues to improve, complementary methods to simultaneously modulate cortical activity while recording are needed.

Approach: We developed a neural interface utilizing optogenetics, cranial windowing, and micro-ECoG arrays fabricated on a transparent polymer. This approach enabled us to directly modulate neural activity at known locations around micro-ECoG arrays in mice expressing Channelrhodopsin-2. We applied photostimuli varying in time, space and frequency to the cortical surface, and we targeted multiple depths within the cortex using an optical fiber while recording micro-ECoG signals.

Main results: Negative potentials of up to 1.5 mV were evoked by photostimuli applied to the entire cortical window, while focally applied photostimuli evoked spatially localized micro-ECoG potentials. Two simultaneously applied focal stimuli could be separated, depending on the distance between them. Photostimuli applied within the cortex with an optical fiber evoked more complex micro-ECoG potentials with multiple positive and negative peaks whose relative amplitudes depended on the depth of the fiber.

Significance: Optogenetic ECoG has potential applications in the study of epilepsy, cortical dynamics, and neuroprostheses.

Figure 1

Micro-ECoG array fabricated on a transparent substrate and chronically implanted under a cranial window. (a) The photolithography process involved a lift-off method to pattern metal layers onto a Parylene C coated wafer, deposition of an additional layer of Parylene, and then exposure of the electrode sites with plasma etching. (b) A micro-ECoG electrode array with platinum electrode sites patterned on Parylene C. Electrode sites 150 μm in diameter are arranged in a 4x4 grid with 500 μm between sites. (c) The electrode array was assembled with a custom printed circuit board to route traces from the FPC connector to a zero insertion force connector. The array was implanted under a cranial window in mice as illustrated in panel (d). (e) Blood vessels, labeled with rhodamine-B dextran, were visible through the cranial window and electrode for several weeks following implantation.

Figure 2

Optogenetically evoked micro-ECoG potentials. (a) An unfiltered micro-ECoG signal shows large negative potentials associated with every photostimulus (465 nm, 3 ms, 0.75 mW/mm², 5.3 mW applied to entire window). (b) Wild type mice did not show any response to stimulation with blue light photostimulus (465 nm, 0.75 mW/mm²), while ChR2 positive mice were responsive to blue (465 nm, 0.75 mW/mm²) but not red (625 nm, 0.81 mW/mm²) photostimuli. A single channel is shown with 50 trials averaged for each condition. Control figures with all 16 channels can be found in supplementary figure 2. Trial-averaged evoked potentials are mapped according to channel location on the array (1 or 5 ms, 465 nm, 0.75 mW/mm², 5.3 mW total across the window). Longer stimuli evoked larger negative potentials. These potentials were spatially uniform with the entire window illuminated. 10 trials were averaged for each condition. (d) The amplitude of optogenetically evoked potential depended on the duration and irradiance (i.e. brightness) of the photostimulus. The stimulus duration and irradiance parameter space was systematically explored to generate a 2D interpolated contour plot.

Figure 3

Optogenetically evoked micro-ECoG potentials in response to pairs of light pulses. Averaged potentials for a single channel are shown in response to pairs of photostimuli applied at 0.75 mW/mm². The duration of each pulse and the inter-stimulus interval was varied. Pairs of brief stimuli (1 ms) caused negative potentials that superimposed linearly, while longer photostimuli (4 ms) did not evoke a linear response as the second peak was not twice as large as the first. Photostimuli spaced by more than 2 ms could be readily distinguished, but photostimuli seperated by less than 1 ms appeared as a single event based on the recorded signals. Between 13 and 28 trials were averaged for each condition.

Figure 4

Spatially mapped micro-ECoG potentials in response to focally applied optogenetic stimuli. One or two focal photostimuli were applied repeatedly to the cortical surface using an objective lens. The photostimuli were 200 μm in diameter, for 5 ms in duration - and 1 mW in power (32 mW/mm²). (a) The amplitude of the evoked micro-ECoG potentials was greatest nearest the stimulus location. Surrounding electrode sites had similar but smaller amplitude waveforms with minimal phase delay suggesting electrostatic volume conduction. (b,c) Stimultaneously applied focal photostimuli evoke spatially separable potentials at distances greater than twice the electrode spacing (c), but at closer electrode spacings the evoked potentials were less separable. 90 trials were averaged for each.

Figure 5

Spatiotemporal photostimulation and localization. (a) Photostimuli were applied at four locations in sequence. Four LEDs were focused on the cortex and illuminated in a clockwise sequence at 2 cycles per second. (b) The corresponding cycle averaged micro-ECoG potentials (100 cycles averaged) are plotted in white for each position. The peak potential for each stimulus location is marked , and these values were pseudocolored using linear interpolation. Spatially localized potentials were observed for each stimulus location. Potentials were bandpass filtered from 5 to 500 Hz.

Figure 6

The spatio-spectral cortical response. A frequency-ramped photostimulus was applied to an area 500 μm in diameter between the electrode sites . Spectrograms of the signals at each micro-ECoG electrode site show that the cortical response decreases as distance and frequency increase. 5 trials were averaged.

Figure 7

Optogenetically evoked micro-ECoG potentials in response to photostimuli applied intracortically with a fiber-coupled LASER or at depths below the cortical surface with a LASER coupled fiber. (a) A cross section diagram of a micro-ECoG electrode array on the dura and a fiber inserted into the cortex. Expression of ChR2/H134R-YFP under the Thy1 promoter in found in a subset of cortical layers. (b) Epifluorescence of an entire coronal slice 1.5 mm posterior to bregma shows expression in the cortex, hipocampus, and thalamus. Greater cortical expression is found medially, but the implanted region (0.5 to 3.5 mm lateral) has more uniform expression. (c) A two-photon image of the outlined region in (b) shows expression in cortical layers 2/3 and 5. Layer 1 also has expression but no cell bodies, suggesting expression in apical dendrites. (d,e) Averaged potentials recorded from the micro-ECoG array in response to photostimuli - (3 ms, 473 nm) applied next to the array at multiple depths within the cortex. Photostimulus power was 2.5 mW (78 mW/mm² ) in (d) and 0.8 mW (25.5 mW/mm²) in (e). Stronger photostimuli (d) caused potentials with multiple negative and positive peaks, while weaker photostimuli (e) evoked only an initial negative peak. These subsequent peaks followed several miliseconds after the photostimulus ceased. Similar to figure 4, the amplitude of the micro-ECoG potentials (d,e) were spatially related to the stimulus location. 50 trials were averaged for each condition in (d) and (e).