Neuroscience
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Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice
Chapters
Summary August 10th, 2019
This protocol describes the construction of a hybrid microdrive array that allows implantation of nine independently adjustable tetrodes and one adjustable opto-silicon probe in two brain regions in freely moving mice. Also demonstrated is a method for safely recovering and reusing the opto-silicon probe for multiple purposes.
Transcript
Our hybrid microdrive design allows experimenters to not only record from multiple brain regions, but also to choose different types of electrodes to optimize yield and stability. We combined nine tetrodes, a silicon linear probe, and fiber optics that are adjustable and reusable in a microdrive array for in vivo experiments with freely moving mice. This technique can easily be adapted to other larger animals such as rats or marmosets.
Silicon probes are extremely fragile. We need to be careful when dealing with the probes, for example, when adapting the optical fiber and when rotating the shuttle assembly into the microdrive. The microdrive array consists of five parts.
To prepare the microdrive screw for the silicon probe first 3D print a plastic pattern of the microdrive and place tape around the pattern to make a temporary wall around the pattern. Pour liquid silicon gel into the pattern and remove air bubbles with gentle shaking. When the silicon has cured, carefully remove the silicon gel mold from the pattern.
Next use a rotary grinder to cut 18 and 9.5mm lengths of 23 gauge stainless steel wire tube and use the grinder to roughen the top 2 to 3mm of the tubes. Apply a small volume of silicon oil to a custom screw and set the tubes and screw into the mold. Pour dental acrylic into the mold and use a syringe to eliminate any air bubbles around the tubes and the screw.
When the dental acrylic has fully cured remove the microdrive screw from the mold and use pliers to bend 6mm from one end of the 18mm wire tip to a 60 degree angle. Using a turning tool rotate the microdrive screw to check the quality then install the microdrive screw into the microdrive array body and turn the screw to check whether it is able to move up and down smoothly. To prepare the shuttle for the silicon probe use sharp scissors to cut two 5mm lengths of PEEK and align the tubes at both sides of the shuttle.
Glue the tubes to the shuttle with epoxy and apply a small volume of silicon oil to the guide posts. When the epoxy has dried, place the shuttle over the guide posts of the microdrive array body to check the quality. The shuttle should move smoothly without excessive friction.
To prepare the optorode use a Rubi cutter to cleave a piece of optical fiber to 21mm in length and grind the fiber tip until it is flat and shiny. When the tip is ready, gently place the optical fiber on the front side of the silicon probe 200 to 300 micrometers above the top of the electrode sites and temporarily secure the fiber with transparent tape then glue the optical fiber to the base of the silicon probe with a small volume of epoxy and allow the epoxy to cure for five hours. To attach the shuttle to the silicon probe apply a small volume of epoxy at the back of the silicon probe base and gently hold the bottom part of the shuttle against the silicon probe base for two to three minutes to avoid the formation of a gap between the shuttle and the silicon probe base.
When the epoxy has completely cured hold the groove of the shuttle with fine tweezers and use a microscope to carefully place the shuttle tubes on to the guide posts of the main body. Turn the screw to insert the microdrive screw into the screw hole and insert the tip of the L shaped tube into the groove of the shuttle head to engage the silicon probe and microdrive screw. Next cut two number 0 screws to a 3.5mm thread length and grind the tips to remove burrs.
Place the probe connector holder on the array body and insert the silicon probe electrical connector into the holder. Insert the number 0 screws to hold the probe connector holder. Use epoxy to secure the silicon probe connector in the holder taking care not to glue the probe to the microdrive array body.
To attach the ferrule holder to the opto-silicon probe and the microdrive array body, cut two number 0 screws to a 6mm thread length and grind the tips. Grind the outside of two number 0 machine screw nuts to make small hex nuts with 2.5 to 3mm outer diameters and insert the number 0 screw into component A of the holder. Glue the screw heads with epoxy.
Apply a small volume of silicon grease to components A and B to reduce the friction with the body. Use inverse tweezers to temporarily insert component A into the body and place component B on to the screws of component A.Thread the customized nuts on to the screws and use pliers to tighten the nuts to secure the ferrule holder on to the body. Insert the fiber ferrule into the groove of the fiber ferrule holder taking care that the fiber ferrule is sticking 4 to 5mm out from the holder.
Apply a small amount of epoxy between the ferrule and the holder groove. When the epoxy is cured, loosen the nuts and turn the microdrive screw to check the shuttle and ferrule holder for smooth motion then confirm that the probe tip completely retracts into the body when the ferrule holder is at the top position and the shuttle tubes are still associated with the guide posts. To attach the shield cone, fasten two 3.5mm number 0 screws from the outside of the cone to hold the microdrive in place.
The microdrive array can be set up within five days if the construction is completed according to the timeline outlined in the table. After the tetrode adjustment behavioral performance can be tested on a linear track and in an open field. In both types of experiments the mouse is allowed to explore freely for approximately 30 minutes while the electrophysiological signals are recorded without severe motion related noise throughout the recording session.
Light stimulation can then be performed at the medial entorhinal cortex to stimulate the medial entorhinal cortex layer 3 neurons that project to the CA1 subfield of the hippocampus. Spontaneous spiking activities and local field potentials are then recorded from the tetrodes and the silicon probe when the mouse is sleeping. In this representative experiment the left field potentials recorded in the tetrodes demonstrated large ripple activities suggesting that all of the tetrodes were positioned in the vicinity of the CA1 pyramidal cell layer.
Light induced responsive activities in this assay were first observed in the medial entorhinal cortex followed by activities within CA1 with 13 to 18 milliseconds of latency. Take care to insert the opto-silicon probe into the microdrive body in a electrostatic free environment. The shielding Cone can be replaced with materials such as a sheet of paper and tape.
This will decrease the rate of the microdrive by up to 20%Our microdrive design provides flexible choices for effective combination of recording electrodes to measure and manipulate multiple brain regions and to investigate the dynamics and interactions of different brain structures.
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