The overall goal of this methodology is to demonstrate a low-cost alternative protocol for constructing and recording from a chronic high-density transcranial electrode array. This method can help answer key questions in the neurosciences, such as large-scale organization of neuronal networks, and spatial and temporal features of sensory evoked responses. The main advantages of this technique are its low cost, reproducibility, and ease for experiment-specific customization.

Though this method can provide insight on the complex analyses of electroencephalographic data, its application is ideal for studies in anaesthesiology, epilepsy, and sleep neurobiology. To make the headpiece, begin by insulating the pins of a two-by-fifty pin brick with a very light coat of nail polish. Then, with a pair of tweezers, remove every eighth row of pins by pushing their receptacle end through the brick.

Once the polish has completely dried, remove the coating from the tips of the pins with a small cloth and acetone. Then, using a razor blade or wire cutters, trim the blocks to make two-by-seven pin bricks. Two of these bricks will function as the transcranial EEG electrodes.

Next, from each end of a two-by-fifty brick, prepare two one-by-two pin bricks to record the EMG signal using the same process. Each one-by-two brick should retain one of the outer smooth sides of the two-by-fifty brick. To complete the two halves of the full array, align the one-by-two pin holes and pins of one brick with the posterior-most row of pins of a two-by-seven brick.

Then, use a two-part epoxy to attach their smooth surfaces and reinforce the connection with more epoxy if necessary. Let the two halves cure overnight. To make two thoracic EMG wires, begin with a 3cm insulated silver wire.

Remove 1cm of insulation from one end and add two loops on the bare wire by wrapping it around a pair of tweezers. Then remove 25mm of insulation from the other end. Now, make the two similar cervical EMG wires, starting with a 1.5cm wire.

From each headpiece half, remove the lateral pin in the furthest anterior row, because this pin will not contact relevant tissues. To attach the wires, cut the pins of the one-by-two bricks 3mm from their tips. Then solder a cervical EMG wire to the anterior pin, and a thoracic EMG wire to the posterior pin.

Before proceeding, use a multimeter in continuity mode to ensure that each pin is electrically isolated. Connect the leads to different pin combinations and if a pair is electrically coupled, the meter will produce an audible beep. Once dry, bend the EMG wires until they are parallel to the AP axis with minimal lateral displacement.

Then cover the soldered joints with nail polish. Now, adjust the pin lengths using wire cutters and the aid of a brain atlas. Find the pin whose ventral distance from the bregma will be the largest.

Do no trim this pin, but trim all the other pins with respect to this pin. To complete the headpieces, cover all of the pin tips with a silver solution using an applicator pen. To prepare an adapter, begin with a 36 position dual row male nano connector.

First, razor down the connector wires to a uniform length of 2cm. And then strip 2.5mm of wire from the end of each wire. Finally, tin together the strands of fine wire, and trim off the stripped insulation from the tinned wire.

Next, create a matching male-male connector to the headpiece from a con-strip header two-by-fifty pin block. As before, prepare two two-by-sevens and two two-by-ones. Now, solder one of the ground wires from the nano-connector to the pin that is 0.6mm anterior to, and 1mm lateral of the bregma.

This pin functions as the reference, and as the ground when using an RHD 2132 amplifier chip. Next, solder the tinned nano-connector wires to the same side as the ground pin connection. Because each wire maps to a specific channel, consult a channel map diagram of the amplifier head stage and map the channels.

Once all the wires are soldered to their respective pin, cut off any unused wires. Then, use a voltmeter to insure that each pin is electrically isolated from the other pins. Once electrical isolation is confirmed, apply a thin coat of nail polish around each soldering joint to further insulate each pin.

Then, using a two-part epoxy, reinforce the matching nano-adapter to the bilateral pin arrays on the male-male bricks. There cannot be any excess epoxy on the medial portions of the adapters, as this will prevent the headpieces from being plugged in simultaneously. For increased durability, epoxy the base of the nano-connector.

Be sure to cover all soldering joints with epoxy. Once the epoxy has dried, confirm the channel mapping using impedance measurements. After the mouse is set up securely, and bregma is defined as the origin of the stereotaxic coordinate system, level the skull in the medial lateral axis and in the anterior posterior axis.

Then, using a 0.5mm diameter micro drill bit, drill burr holes 1.3mm apart, and 1mm from the midline, beginning 3.3mm anterior of the bregma and finishing 4.5mm posterior of the bregma. Make two rows bilaterally. Next, drill burr holes on both sides of the midline that are 1.3mm apart, beginning 2mm anterior of the bregma, and ending 4.5mm posterior of the bregma.

Now, implant the headpieces. With straight forceps, prepare EMG wire tunnels for the thoracic EMG wires. From the back of the mouse, tunnel 2.5cm below the skin, and place the EMG wires in the cavity.

Next, maneuver the EEG brick with curved forceps such that the pins align with the burr holes. Apply slight pressure and wiggle the pins into the skull. The EEG will be stable once properly inserted.

Next, adjust the EMG wires to their final positions. Once set in place, prepare a one-to-one mixture of methyl methacrylate with its cross-linking compound. Then, cover the exposed skull, the nail-polished parts of the pin electrodes, and the proximal portion of the EMG wires with cement.

Do not cover the female receptacles of the headpiece or get any cement into the animal's fur. Once the cement dries, the mounting procedure is complete. Remove the mouse from the stereotaxic frame and let it recover while monitoring vital signs.

Two weeks after surgery, a fully recovered mouse can be tethered to a recording cable through the adapter for habituation. After mice are habituated, they can be used in experiments. After connecting a mouse to the acquisition board through an adapter, amplifier and standard interface cable, open the Open E-Fizz recording software.

Therein, create a signal chain of rhythm FPGA and LFP viewer by dragging the modules into the signal chain. Next, obtain electrode impedance values of 30.0 kilo-samples per second. Then select the desired sampling rate for recording.

Filters can also be dragged into the signal chain if desired. Continue by selecting a save path for the data and the desired sampling rate. Then click on the LFP viewer window and press the play button.

Now, using the draw method to visualize the EEG, adjust the view by changing the signal amplitude, sweep speed, and the settings of any filters that are in use. Once all the settings have been adjusted, begin making recordings. Data was collected from a freely moving mouse implanted with a high-density EEG headpiece.

The thoracic EMG recording contains embedded electrical activity, originating from the heart. The cervical EMG contains information about muscle tone. The individual EEG waveforms correspond to the channel mapping scheme.

With this recording, it is possible to calculate the mouse's heart rate by measuring the time between electrocardiographic QRS spikes. Similarly, it is possible to measure the mouse's respiratory rate by calculating phasic variability of the QRS spike as the thoracic cavity expands and contracts with each breath. Hence, this setup permits for acquisition of murine polysomnography.

This setup enables cortical mapping of visual evoked potentials. When a ten millisecond pulse of light is delivered only to the left eye, classic responses are recorded in the contralateral primary visual cortex, followed by a delayed response in the contralateral secondary visual cortex. This is easily seen in the time varying electrical potentials across the entire cortical surface in contralateral V1 and V2.After watching this video, you should have a good understanding of how to construct high-density microelectrode arrays for a rodent model, and record from the implanted array.

Once mastered, the construction and implantation portions of this technique can be done in two hours if performed properly. While attempting this procedure, it's important to remember to create proper epidural electrode contact without penetrating the dura. Pin trimming, and attentiveness during implanting the headpieces, will create the best chances for suitable electrode contact.

Good luck!

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