만성에 대한 마이크로 드라이브 배열 생체내에 녹화 : Tetrode 조립

In this protocol, we describe the fabrication and installment of Tetros bundles of four electrodes for chronic in vivo recording in the awake rodent to begin tetro fabrication. A strand of microelectrode wires folded twice upon itself to make a bundle of four parallel wires. These wires are then twisted together, infused with a hot air gun.

Each finished tero is placed in a storage box. While more teros are made, each tero is inserted down a dedicated pathway entering through an opening in a linear actuator and exiting through the base of the micro drive array. Each of the four wires of the tero are connected to separate pins on an output connector board affixed to the top of the micro drive array.

The impedance of each channel of the tero is lowered using a gold electroplating process. Once all the teros have been installed and electroplated, two ground wires are attached to the respective pins on the connector board, and a protective cone is affixed to the frame of the micro drive array for protection after the cone has been grounded, A Foley functioning Tero micro drive arrays ready for implantation. Hi, I'm Dave Nian from the laboratory of Nat Wilson in the Pickout Institute for Learning and Memory.

At MIT today, we will show you a procedure for fabricating teros, loading teros, and completing the final stages of the micro drive reconstruction. We use this procedure in our laboratory to study coordinated neural activity between large populations and neurons in an awake, behaving animal preparation. So let's get started.

To build the tero for our standard micro drive array, we begin with a 50 centimeter length of insulated 12 micron microme wire. Fold the wire in half, run your fingers along the pair to make them stick together. Make sure the pair of wires have good contact throughout their entire length.

Fold the pair again by holding the two ends together. Make sure that the loop formed on one end is not kinked. Cut the four wires on the non looped end so that the four tips are aligned.

For the next step, you'll need a modified alligator clip, a motorized turning device, and a horizontal bar above the turning device. The alligator clip is modified by gluing a plastic bar to the base of the clip. When the tips are exposed, clamp the four wires together with the modified alligator clip.

Hang the loop of wire over the horizontal bar. Place the alligator clip into the motorized stage. Apply 80 clockwise twists followed by 40 counterclockwise twists to the wire bundle over the course of approximately three minutes.

These parameters can be varied according to your needs. After tero twisting is completed, fuse the wires together by heating from three different angles with a heat gun using medium to low flow for each angle, begin one to two centimeters below where the wire bundle splits. Then run the heat gun down and up at a two centimeter distance from the wires for about five seconds.

Use caution as high temperatures will completely melt the insulation and lead to short circuits. Now that the wires have fused together, remove the tero from the twisting apparatus by gently lifting the alligator clip to relieve tension on the tero and cut the tero near the alligator clip At the other end, cut the loop such that there are four non-branded strands of wire of equal length. Next, separate the individual strands at the top by gently bending the wires with a soft tipped pair of tweezers.

The tero is now ready for loading into the micro drive array. Make 21 to 25 more Tet roads and store them in a dust-free box until it is time for the tero loading process to proceed. You will need a complete micro drive array if you have not yet built one.

Refer to the video micro electrode drive array for chronic in vivo recording micro drive array fabrication in our design plan. The tero will be attached at one end to connector hardware and will run through the poly mid carrier tube in the micro drive such that the electrode tips extend below the base of the micro drive array. We will now demonstrate this loading process for one tero.

Before beginning build the drive holder that can be attached to the connector board on one end and on the other end can be clamped by a pan of ice. In this example, the drive holder is a milax connector glued to one end of an X exacto knife handle holding the tero with soft tip tweezers and using a stereoscope, push the tip of the tero into the poly mid carrier tube of one of the micro drives. Push the Tet road into the tube until the individual electrode wires are close to the connector board at the top of the drive array bere, be careful not to ker, bend the wires as this will cause the tetro to enter the brain at an angle rather than perpendicular and will weaken the integrity of the tero.

Now gently feed the other end of the four wires into their respective holes in the electrode interface board using soft tip tweezers. Again, avoid kinking or bending the wires. With all four wires in place, push the gold pins into their holes with a pair of pliers that have a shortened lower jaw.

When the pins are pushed into the hole, they will strip the wire insulation and create an electrical connection for later reference. Keep track of the mapping between micro drives and pin position on the electrode interface board. Continue loading a total of 18 tetros.

The three remaining micro drives on the array will be used to house the reference electrodes. Each reference electrode is made of a tero that uses only one of the four unbonded yns. To attach the reference electrodes, follow the loading procedure, but attach only one of the four electrodes to the respective reference electrode pin on the connector board.

If you are planning multi-site recordings, it may be worthwhile to plan out the connections from the teros to the connector board ahead of time. For example, cluster tetros that are targeted to one anatomical region together on the electrode interface board. This should minimize the possibility of adjusting the wrong tetro during experiments.

Once tero loading is completed, make two ground wires, one for the animal and one for the protective cone. Cut one six inch long insulated steel wire and remove three millimeters of insulation using metal tweezers from each end of the wire. Then cut a four inch long insulated steel wire.

Remove three millimeters of insulation at one end and one centimeter at the other end. Route the six inch ground wire through the hole on the side of the micro drive array and up towards the interface board. Connect the exposed wire to the electrode interface board with a gold pin at the designated ground hole.

This wire will connect to the animal skull. Route the four inch ground wire parallel to the previously installed ground wire. Connect the three millimeter stripped end to the electrode interface board using a gold pin at another designated ground hole.

This wire will later be connected to a protective cone, which will act as a small faraday cage to reduce noise pickup. We are now finished connecting wires and can proceed with refining our teros. Our goal in this process is to cut all the teros so that they are slightly longer than the depth of the target brain structure and our example.

The micro drives are designed for a maximum travel of five to six millimeters sufficient to reach many neocortical areas and the dorsal hippocampus of an adult rat. First, lower all the micro drives so that the tetros are maximally exposed simultaneously. Cut all the tetros to lengths that are approximately five millimeters longer than the desired final length, using a pair of sharp fine scissors for the final cut.

Prepare a pair of serrated scissors in a pan of ice clamp one handle with a slight angle downwards and with serrations facing upwards while leaving the other handle. Dangling now completely withdraw all the Tet roads into their guide cannula by turning the micro drives up. Using a stereoscope fully extend one tero out from its cannula.

Using a ruler position the serrated scissors at the desired distance from the guide to cannula. Slowly and gently cut the tero with one smooth movement. After this tero has been cut, withdraw the micro drive up by three to four millimeters.

Repeat this process for each tero until all have been cut. At this point, you are ready for gold plating. Gold plating.

The ro teros is critical for long-term stable recordings. It prevents corrosion and improves biocompatibility. Set up the impedance meter in external mode in the current generator and DC check mode with audio output.

Touch the two leads of the current generator to the six possible pairs of channels within each tero and use the audio feedback to determine if shorts exist between pairs. A short between channel pairs sounds like this. If a short exists, recut the tero with serrated scissors and test again.

If the problem persists, discard the Tet road and replace it. Move all the micro drives down, extending all the Tet roads as far out as possible. Modify the equipment configuration for checking electrode impedance.

Now dip the tips of all the teros into a bath of standard gold plating solution. The gold bath is electrically connected to the negative lead of the impedance meter. Check the impedance of the electrode by touching the positive lead of the impedance meter to the corresponding pin on the connector board.

Measure and record the impedance of every wire on the micro drive array. Normal values range from one to three mega ohms on the current generator. Set the current between one to three microamps.

Next securely. Attach the positive lead of the impedance meter to the pin on the connector board. On the impedance meter.

Quickly switch from normal mode to bypass mode and back while in bypass mode. Current will pass through the Tet road and gold solution, and gold will be plated on the electrode tip. The impedance after each round of plating should be less than before.

Repeat the current pulse. If the impedance did not drop below one mega ohm. A conservative range of acceptable impedances are 250 kilo ohms to 350 kilo ohms.

If the impedance drops below 200 kilo ohms, this may indicate a short circuit. If after repeated pulses, the impedance does not drop sufficiently, the electrode tip may be obstructed. Recut the tero check for shorts and repeat the gold plating process.

If recutting doesn't help, replace the tero and start again. After plating all teros, dip them in ethanol and let them drive for three to five minutes. Test for short circuits within teros using the current generator with the tero tips exposed to air.

If a DC connection exists between two or more electrodes of a tero, recut the tero and plate with gold again. After gold plating of all the teros is complete, move all the teros into the drive. As a final step at the top of each micro drive, add a drop of medium thickness cyanoacrylate glue to the interface between each tero and its poly tubing.

This will secure the tero to its micro drive. The protective con's purpose is to shield micro drives and the exposed tero wire from the environment. It also provides support for handling during an experiment and reduces electrical noise.

We use a 3D printed plastic cone. However it is possible to make the cone from other materials like a bent piece of soda. Can take the plastic cone and fix some aluminum foil tape to the inner or outer surface.

First insert and partially turn 3 1 72 size screws, three sixteenths inches in length into the sides of the cone with the drive array suspended. Insert the cone over the drive. Make sure both ground wires extend from the bottom of the drive array.

Wrap the exposed part of the four inch ground wire around one of the 1 72 screws several times and fastened the screw Tightly secure the cone to the drive base by tightening the remaining two cone screws. Micro drive array production is now complete. Your micro drive array is loaded with 18 teros in three references.

It is encased in a protective cone and cap for durability. The finished drive usually weighs 20 to 25 grams. On average.

The finished drive array is implanted using standard surgical procedure. As you can see, the drive is attached to a pre amplifier chip and a bundle of wires that carry the signal to data acquisition hardware and software. We have just shown you how to make teros and complete the micro drive array construction procedure.

In this video, we showed you how to fabricate teros, load and condition the teros for in vivo recording. In addition, we showed you how to add a cone to the micro driver array to make it robust over time. There are many steps in the micro driver array construction, so be patient, be gentle with the electrodes and make sure all the intermediate steps are properly completed.

Thank you for watching and good luck with your experiments.