Multiscale Investigations of Cortical Processing by Integrating Laminar Polytrodes and Optogenetics with Micro Electrocorticography in Rodents

How labs investigate how brain activity gives rise to functions, like perception and cognition, using ECOG and other tools to link local neuronal activity with cortical signals at a broader scale.

Extracellular spike recordings in two-photon imaging are used to measure population activity, but ECOG is one of the few techniques available in both humans and for basic research in animals.

Brain activity spans many spatial and temporal scales. No signal method captures them all at once, but ECOG has sufficient temporal and spatial resolution for many critical questions.

Our protocol bridges a key gap by combining ECOG with local recordings and optogenetics, effectively providing microscale information from the mesoscale cortical surface signals.

Our findings will expand the use of ECOG as part of multimodal experimental paradigms to study fundamental principles of neocortical organization and reveal biomarkers of specific cortical functions in humans.

[Instructor] To begin, place an anesthetized mouse on a surgical bed and use tweezers to lift a point on the skin over the skull. Then, using surgical scissors,, resect approximately a one centimeter diameter portion of the skin. With a scraper, clear away connective tissue and periosteum from the top of the skull. Flush saline onto the skull. Next, use a surgical drill set at low speed to drill a burr hole in the frontal part of the hemisphere ipsilateral to the recorded area. Drill a shallow trench on the perimeter to define the contour of the craniotomy. When the skull has been thinned to the point where extremely light pressure causes the entire window to visibly wiggle, remove the thinned portion. Apply saline solution regularly and use a hemostatic sponge to keep the brain moist. Now, insert the silver reference wire approximately one millimeter into the burr hole so that it contacts the brain surface without causing bleeding. After implantation of the head bar, place the animal in the recording setup. Now attach the micro-electrocorticography grid to the headstage using the zero insertion force clip connectors. Hold the headstage electronic board in place using a mechanical bar fixed to a micromanipulator. Lower the micro-electrocorticography grid horizontally to align flat over the craniotomy along the anteroposterior axis. Once the grid is close to the brain without touching it, attach the grid's reference wire to the implanted silver wire gold pin. Next, lower the grid further to make contact with the brain. Move the grid laterally to glide over the moist dura surface and continue adjusting until it is centered along the mediolateral axis. Use aspiration or a surgical sponge around the edges of the craniotomy to remove excess saline solution. Once the preparation is slightly drier, verify that the grid adheres more firmly to the dura and does not slide. Apply a lateral to medial movement to the grid to ensure contact with the most lateral electrodes while the grid's cable bends to match the brain contour. Observe the electrophysiological activity using the recording software. Under light anesthesia, monitor for variable brain signal patterns. Ensure the grid, reference, and ground wires are properly connected to yield high signal-to-noise ratio. Use Trodes software with band pass filtering between 300 and 6,000 hertz to monitor noise in the high frequency range and ensure it remains within tens of microvolts. Assess sensory responsiveness by generating noise stimuli, such as clapping or snapping fingers, and observe the corresponding cortical surface electrical potentials. Turn on the optogenetic light at low intensity to guide the light source and help position the fiber, then use the articulated arm to roughly position the optogenetic light toward the target area. Focus and fine-tune the fiber's position using a micromanipulator or fine adjustment screws before recording the signals. To clean the grid, if the brain is dry, apply a small drop of saline onto the brain surface using a syringe and allow it to sit for 30 seconds to one minute before lifting the grid. Working under the microscope, gently lift the grid from the brain surface using micromanipulators. A localized electrocorticographic response was observed approximately 10 milliseconds after single whisker stimulation, with peak deflection amplitudes around one millivolt. The strongest electrode response was evoked by stimulation of its corresponding whisker, while weaker or no response was observed from more distant whiskers. Optogenetic inhibition led to a suppression of whisker-evoked cortical response only in animals expressing inhibitory opsins. Optoelectric artifacts appeared only at the onset and offset of the five second light pulse stimulation using the large diameter fiber. The one millimeter optical fiber produced a large light artifact on the micro-electrocorticography grid, whereas the 200 micrometer fiber minimized this artifact significantly. Micro-electrocorticography recordings in rats showed strong auditory-evoked responses, with peak response around 25 to 30 milliseconds post-stimulus. High gamma, ultra high gamma, and multi-unit activity peaks were observed in the frequency spectrum of the evoked response, with high gamma as the dominant component. Micro-electrocorticography was recorded together with laminar probe recordings. Clear spike waveforms were detected in polytrode recordings, showing distinct waveform across multiple channels. Frequency response amplitude plots from surface micro-electrocorticography electrodes closely match those from laminar polytrode electrodes across depth, demonstrating consistent auditory tuning. A high-resolution tonotopic map generated from high gamma signals revealed the functional organization of auditory cortical fields, including primary, posterior, and ventral areas.