만성에 대한 마이크로 드라이브 배열 생체내에 녹화 : 드라이브 제작

This video will cover the assembly of a lightweight micro drive array with 21 independently adjustable screw driven micro drives. Each micro drive controls the recording position of a single electrode. The micro drives are mounted in a computer designed plastic base that stabilizes them relative to the skull of the moving animal and collects the electrodes into a densely packed pattern that can pass through a small craniotomy into the brain.

The base also supports an electrode interface board for the connection to the recording instruments, which allows for the animal to be conveniently plugged in and unplugged at the beginning and end of an experiment. A plastic cone attached to the base protects the micro drives and electrodes from the elements in the rat cage. Hi, I'm Faia Postman from the laboratory of Matt Wilson in the PIKA Institute for Learning and Memory at the Massachusetts Institute of Technology.

Today we'll show you a protocol for how to fabricate one of those micro dive arrays. In our laboratory, we use those micro dive arrays to record from a large population of neurons in the brain of rats, for example, from the cortex or hippocampus. First, we will show you how we designed our micro dive array.

Then we will head over to the workshop where Steve and Stewart will show us how to process the raw materials and how to assemble them into a fully functional device. So let's get started. The plastic base is designed using computer rated design software and is manufactured using an inexpensive 3D printing process.

We also model all the other components of the micro drive array. This allows us to carefully control the size and weight of the finished drive. At the top of the plastic base are two rings of holes that will accept the individual micro drives.

The 3D model is sent to a service bureau that builds the design from a laser curable resin using stereo lithography. That should only take a few days and the resulting part is lightweight and reusable. Once the part arrives from the service bureau, we need to prepare it for use.

So let's head over to the workshop. The 3D printing process cannot reliably print small holes, so we specify smaller diameter holes in our models. Then drill and tap the holes to the required size in the workshop.

Three different size drill bits are used to prepare the plastic base, a number 61 bit, a number 65 bit, and a 1.55 millimeter bit in the outer ring of the base. Use a hand drill with a number 61 bit at very low speeds to prepare the pilot holes where later the micro drive screws will be inserted. Next, prepare the holes for the micro drive support tubes in the entering in the base by drilling out these holes with a number 65 bit.

Using the larger 1.55 millimeter bit, drill out the holes that will attach the base to the electrode interface board and protective cone. To ensure smooth movement in the micro drive, insert a stainless steel sleeve into the inner ring of holes. Each sleeve should be press fit with a pair of pliers until it is flushed with the top of the base.

The screw pilot holes now need to be threaded. Use a pin vice to hold the tap and apply a dry lubricant before tapping. Align the tap and it's all by making sure it is parallel to a 23 gauge tube you temporarily insert into the neighboring sleeve.

The holes that hold the interface board and the protective cone need to be threaded. Hand tap these holes the same way. The plastic based design features a tap starter for each screw pilot hole to make sure the initial threads are not damaged during the threading process.

After all pilot holes are threaded. These tap starters can be easily snapped off using a razor blade. The electrodes are guided toward a single site at the bottom of the drive by a number of electro guide tubes.

First, we need to make the collector cannula that will hold all the electro guide tubes. The collector cannula is made from a 1.5 centimeter long piece of stainless steel tube. The collector cannula will be held in with dental cement.

So to enhance adhesion carefully roughen the tube using a Dremel grinding wheel, insert this cannula into the bottom of the plastic base. Position it so that five to seven millimeters of the cannula remains outside of the micro drive array. To facilitate surgical implantation, attach the collector cannula to the bottom of the plastic base.

With dental acrylic, allow it to cure for at least 15 minutes. Now, prepare the guide tubes by cutting 21 pieces of polyamide with a razor blade. Begin this step by feeding all 21 guide tubes into the collector cannula at the bottom of the drive base.

This will make sure that the tubes are all parallel to one another. If tubes in the bundle are too loose or tight, you can start with fewer tubes or add extra tubes as needed. Now, remove each guide tube one at a time and feed it into one of the sleeves at the top of the drive.

Pass the tube down through the gap at left behind in the bundle of tubes. Inspect guide tubes for kinks or tight bends and replace as needed. Adjust each guide tubes position so it extends one to two millimeters from the top of the sleeve.

Now add a small drop of thin sano acro lake glue to the bottom of the collector cannula. Be very careful not to let the glue flow into the guide tubes. Next, use a fresh razor blade to cut off the bundle of any excess tubing at the bottom of the collector cannula.

Verify under a stereoscope that the tubes are neatly cut and flush with the cannula. Each electrode is advanced by its own micro drive, so we need to make 21 micro drives and then insert them into the plastic base. Each micro drive consists of a custom screw and a hollow support tube, which are attached to each other.

With dental acrylic, the electrode will pass through the support tube. The screw features a non threaded part with a small lip that is embedded in the dental acrylic and allows the screw to turn freely in this way. As the screw is turned into the plastic base, it advances or retracts the electrode.

The head of the screw is shaped like a half cylinder. Before we can make the micro drives, we need to make a screwdriver to turn our custom screws. Our makeshift screwdriver is constructed by taking a custom screw and completely grinding down the lip that surrounds the half cylinder head.

Insert the screw head out into a five centimeter long 15 gauge tube until it is fully encased. Now, crush the tube around the threads of the screw, securing it in the tube and preventing it from turning. Attach a mini pin vice to act as a handle in voila a screwdriver.

The micro drive is made by molding dental acrylic around the screw and the support tube. The molds we use are 3D printed plastic just like the plastic base and have holes in the bottom that keep the screw and the support post aligned as we pour the acrylic into the mold. First, we need to prepare the mold clear out the support tube hole using a 23 gauge needle, clear out the screw hole and create threads using a tap.

This is the same procedure we just used on the plastic base. Now the mold is ready to use to make micro drives. Line the mold with a Teflon based lubricant to prevent the dental acrylic from sticking to the mold.

Scuff the top two to three millimeters of a 14 millimeter long 23 gauge stainless steel support post with a grinding wheel so that it will be firmly secured in the depth of acrylic. Insert the support post into the smaller hole of the mold so that the top of the post is above the top of the.Well. Apply a thin film of Teflon based lubricant to a screw and insert it into the threaded hole until the threads are just below the bottom of the well.

Now mix up some dental acrylic and poured into the mold. Make sure it gets into all the nooks and crannies. A needle can help dislodge air bubbles once the acrylic is hardened.

You can remove the micro drive by backing out the screw. Do a strict quality check. There should be no cracks or bubbles in the acrylic.

The post and screws should be parallel and unable to move sideways. The screws should turn smoothly, but with no play any bad micro drives should be broken down in the parts reused. Repeat this process until you have 21 good micro drives.

If your mold has multiple wells, then you can make the micro drives and batches to save time. Carefully align each micro drive with a pair of holes in the plastic base. Slip the micro drive support tube over the poly me guide tube and into the metal sleeve while using the screwdriver to advance the micro drive screw.

This can be tricky. Keep your eye on the center of the base to make sure that the guide tube does not get caught in buckle as the micro drive is lowered. Once all the micro drive are in place and fully lowered, a small amount of acrylic is dropped into the center of the drive to lock the bouquet of guide tubes in place.

Allow the acrylic to cure for 15 minutes. Once the acrylic is dry, to retract each micro drive about three millimeters, create a top-down view schematic of the micro drives and give each micro drive a unique name or number. Now looking in the stereoscope, flip the drive upside down and take a picture of the bottom of the collector cannula and guide tubes.

Make a printout of the picture that can be used as a reference in the following steps. Next with the drive upside down, insert a narrow polyamide carrier tube into each guide tube until it comes out the top of a micro drive. Note on the photograph which guide tube corresponds to which micro drive.

The carrier tube will later protect and support the delicate electrode wires. It passes through the micro drive. Extend each carrier tube until it is one centimeter above the top of the micro drive support tube, and then glue the carrier tube to the support tube with five minute epoxy or thick sano acro lake glue.

Be very careful with this step. If glue drips down the inside of the support tube, it can bind the support tube to the stationary guide tube ruining that micro drive. This is the reason that we raise the micro drives before inserting the carrier tubes.

Once all the carrier tubes are glued in place, fully lower the micro drives again and cut all the carrier tubes off flush at the bottom of the drive using a fresh razor blade. Next, cut the carrier tubes one to two millimeters above the top of the micro drive using sharp scissors. Finally place the electrode interface board on top of the drive base and mount it with two screws.

At this point, the drive array is ready to be loaded with electrodes and our experiments. We typically use four channel microwire electrodes called tetros, but any type of electrode can be used. Fabrication of teros, how to load them into the micro drives and how to prepare the drive array for surgical implantation are covered in other Joe videos.

This protocol can be followed directly or modified to meet your needs. For example, some researchers will require longer screws to record from deeper brain structures. Others may wish to design a drive that targets more than one brain location.

The design can also be miniaturized for use in smaller animals like mice with slightly more modification, motorized micro drives may be used, which allow remote adjustment of the electrode positions. We just showed you how to design and fabricate a micro drive array that can be implanted onto the head of a rat and allows us to record from large populations of neurons while the animal is really moving. When doing this procedure, it's important to remember to carefully thread the holes in the plastic base so that the micro drive screws do not slip.

Perform a rigorous quality check of the fabricated micro drives. Make sure the screw and the support tube are parallel and that the screw turns smoothly without play. Be careful when using glue or dental acrylic to not accidentally clog any of the tubing.

Finally, remember that to complete the MyDrive array and before doing the surgery, you will have to fabricate tetros and low dose into the drive. This is covered in another job video article. So that's it.

Thank you for watching and good luck with your experiments.