Neuroscience
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Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings
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Summary August 5th, 2021
This protocol describes a procedure for constructing carbon fiber microelectrode arrays for chronic and acute in vivo electrophysiological recordings in mouse (Mus musculus) and ferret (Mustela putorius furo) from multiple brain regions. Each step, following the purchase of raw carbon fibers to microelectrode array implantation, is described in detail, with emphasis on microelectrode array construction.
Transcript
The bench top construction described here is one of the few currently available electrode technologies that allow recordings from dense clusters of neurons and follow many of those neurons over time. Carbon fiber microelectrode arrays are inexpensive to build. They're small, biocompatible, and move with the brain, which allows healthy and stable recordings.
Carbon fibers are extremely thin and initially, they're very difficult to see. However, after a few days of working with them, they become easier to manipulate. To begin with, lay a piece of double-sided tape on two long sides of the cassette, aligning the edge of the tape with the inner edge of the cassette.
To prepare a carbon fiber manipulation tool, wrap a small piece of adhesive film around a 30 gauge needle to form a sharp but flexible point with the film. Next, separate individual fibers from the baked bundle and lay them parallel with the short side of the cassette keeping a two to three millimeter distance. After ensuring the fitting of 20 to 30 fibers on each cassette, seal the fibers in place by laying clear tape over the double stick tape, and place the filled cassettes in the cassette holder.
To assemble the carbon fiber microelectrode array, start feeding each grounding wire through the appropriate channel on the connector end of the jig and continue until two ends are equal in length. Then twist them together to secure them on the jig. Apply UV-cured dental cement to secure the wire while ensuring not to get any dental cement within the open channel from where the wire is fed through.
Use the UV curing wand to cure the dental cement for 20 seconds and align the jig and vice such that the connector end, the basin, and the funnel tip are visible. Next, orient the jig so the funnel points away from the user and the connector ends facing the user. Then slide the 25 gauge needle tip against the cassette at the potential fiber removal site and cut a single carbon fiber out of the cassette.
Hold the freshly cut free end of the fiber using a carbon fiber tool and using the needle, cut the other end of the fiber away from the cassette. Then again use the carbon fiber tool to pick up the carbon fiber such that one end is about one centimeter in length from the tool. Use the fiber-attached carbon fiber tool to feed the shorter end of the fiber through the funnel piece from the middle basin of the jig.
Continue to feed the fiber through the jig funnel until most of the length of the fiber is through. Feed the back portion of the fiber through an available channel using the carbon fiber tool. Continue to feed through the back until about five millimeters of fiber is sticking out or cut to size if necessary.
To remove the insulation at the connector end, use a standard spark wheel lighter to pass the flame over the exposed fibers, then feed the flamed fiber through the jig so that the flamed fiber portion is now within the channel while ensuring that no fibers are sticking out of the back of the jig. Apply UV-cured dental cement to the basin fibers of the jig to fill the entire basin, covering the openings of both channels and funnel. After removing the jig from the vice and orienting the jig so that the funnel points down, secure the jig with the connector end pointing up.
Fill the needle with silver print and carefully insert the needle into one channel until stopped by the dental cement. To fill the channel with paint, slowly depress the syringe while removing the needle from the channel, then dip a cotton tip applicator in the paint thinner and clean the base of the jig surface of any paint. Now insert the head stage connector in the proper orientation by aligning the pins with the channels.
Once the head stage connector sits straight, upright, and is as flushed to the jig as possible, secure the head stage connector to the jig by applying dental cement along the edge where the head stage connector meets the jig, then UV cure for 20 seconds. In order to cut the electrode tip to the desired length, lower the electrode into a beaker of deionized or distilled water until the funnel tip is fully submerged and held normal to the surface. Now bring the individual carbon fibers together by removing the electrode from the water.
Lay the electrode so that the fibers lay flush on the guide surface and cut the fibers to the desired length with a scalpel in a rolling motion, then attach the electrode to the multielectrode impedance tester using the appropriate adapter and test the impedance. Now lower the electrode tip about two millimeters into a microcentrifuge tube of 0.1 molar PBS, and insert grounding wire into the microcentrifuge tube. To reduce the impedance of the electrode tip, inject a positive current with the chosen amplitude and duration, and then rinse the fibers in deionized or distilled water to clean.
Next, to perform electroplating in the gold-plating solution, lower the electrode bundle tip about two millimeters into the microcentrifuge tube in the plating mixture and insert the grounding wire into the microcentrifuge tube. Then set appropriate parameters for electroplating. Rinse the fibers thoroughly with deionized or distilled water after electroplating.
The representative SEM image of prepared carbon fiber showed a gold-plating solution concentrated at the tip. The fiber electroplating in gold resulted in decreased impedances suitable for the recording. Impedance values from 300 channels after initial cutting, positive current injection, and electroplating showed decreased impedance values after each processing step.
Moderate gold electroplating durations produced the small and rounded deposits on the carbon fiber bundle tips. In addition, stable 64-channel electrophysiological activity in the retrosplenial cortex of a freely behaving adult male mouse was analyzed. The results demonstrated the consistency of recording quality, robust single unit detection, consistency over time as shown by non-normalized spike waveforms, and stable noise floor.
Also, raw voltage traced 11 months after implantation showed robust local field potential. A representative example of an acute 16-channel carbon fiber microelectrode array recording acquired from the primary visual cortex of an adult female ferret is also shown here. Carbon fiber microelectrode arrays allow the short-term or chronic recording of nearby neurons in the brain over long periods of time, which is essential for understanding neural circuits.
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