This protocol includes fabricating on optrode system for simultaneous optogenetic stimulation and electrophysiological recording. The proposed LED-based system enhances the light coupling efficiency through a microlens array. An LED light source has a simple lighting set up than a laser light source so that the system is readily applicable to the wireless system.
Optogenetics is a path technique for understanding the pathology of neurological disease and treatment mechanisms. Optogenetics can be applied to treat spinal cord injury, epilepsy, Parkinson's disease and Alzheimer disease. Our LED-based device can be implemented further as a wireless system with several advantages, such as low system complexity, cost effectiveness, and low power consumption.
First, connect the 1.27 millimeter pitch female connector to the head stage preamplifier. Place the LED in the 3D printed housing. Measure the light intensity at the end of the optical fiber tip using a photodiode.
Immerse the tungsten electrodes and optical fibers in alcohol for 15 minutes and air dry them. Shave the head skin and place the anesthetized mouse in the stereotactic frame. Position the head within the stereotactic frame and insert ear bars into the meatus.
Turn on the thermal heating pad embedded in the stereotactic frame and maintain the body temperature at 37 degrees Celsius throughout the surgical procedures. Adjust the incision bar for setting the height of the incision bar and tighten the nose clamp against the snout. Cover the eyes with petroleum jelly to prevent dryness.
Lift the skin in the head with forceps, secure an injection space and inject 1%lidocaine under the scalp. Perform a sagittal incision using a scalpel and fine scissors. Hold the incised skin with a microclamp to broaden the visibility of the surgical area.
Remove the periosteum using cotton swabs. If there is bleeding, cauterized the skull using a bovie to seal the blood vessels. Clean the skull with saline and mark the craniotomy sites using a manipulator arm.
Drill a hole above the cerebellum and insert a screw for the ground. Using a precision screw, put the ground screw 0.5 millimeters deep until it reaches the top of the cerebellum. Drill the marked area and remove a piece of the skull with forceps.
Bend a 26-gauge needle tip clockwise at an angle of 120 degrees and expose the brain area by inserting the bevel side of the needle facing upward. Fix the optrode array in the manipulator arm and move close to the exposed area. Slowly insert the optrode array and connect the ground screw to the silver wire attached to the system.
Insert gel foam between the exposed brain and the device to protect chemicals from direct contact with the brain. Carefully apply dental cement to the skull to fix the device and cover the gel foam. Before the dental cement is completely hardened, separate the scalp if attached to the dental cement.
Pull the incised skin with forceps to cover the hardened dental cement and suture the scalp. Place the anesthetized mouse in the stereotactic frame. Set the light pulse recipe to 4%duty cycle in 10 Hertz frequency.
Set up the light stimulation for two seconds during neural recording. Take off the plastic cover and attach the light delivery systems upper part with the reusable LED in circuits. Connect the head stage preamplifier to the implanted five-pin connector.
Open the software and set up the software filters. Next, click and set amplifier sampling rate. Then click change bandwidth.
Set amplifier lower bandwidth and amplifier upper bandwidth. Check software DAC High-Pass Filter. Open Spike Scope and check neural signals using voltage threshold.
Click Stop and record. Load the acquired data using MATLAB and check raw data. Then run spike sorting code which contains wave clust algorithm.
Check sorted spikes in raster plot. Now plot counting spikes and check evoked spikes during light stimulation. The figure shows whole recorded wave forms with exact conditions, including two seconds light on period in the middle.
The number of evoked individual neural spikes following each light stimulation pulse significantly increased compared with the baseline with different time bins of two seconds and 0.2 seconds. The figure shows a spike histogram for each channel before, during, and after light stimulation. The inset figure indicates the location of the implanted optrode array.
Further, the zoomed version shows a spike histogram with a 100 millisecond time bin. The blue bar represents the time period the LED light was on to record the response. To minimize the artifact noise from LED driving current process, the system should be designed to ensure enough distance between the electro signal pathway and the LED circuit.
The proposed LED-based optrode system can investigate the various neuro signaling related to animals'behaviors because it can deliver optogenetic stimulation and record the evoked neuro responses. An LED optrode array is compatible with the wireless optogenetics interface, which can be used in freely moving animal research.
