無傷の3 - D脳の回路のウイルス標的のためのスケーラブルな流体インジェクタの配列

Two rectangular pieces of acrylic are laser cut and then assembled to form the stereotaxic clamp, which serves to hold the injector array tightly with respect to the stereo attacks. A syringe pump is loaded with syringes connected to polyethylene tubing for a desired brain circuit. A mill is used to drill holes through which fused silica tubing is inserted, thus forming injectors.

The injectors are reversibly mounted in the clamp and then trimmed to target the brain circuits of interest. Each injector is attached to a single polyethylene channel. Preparation of the system for use involves filling the system via the syringes with silicone oil and then loading fluidic reagents into the injectors.

The parallel injector is then inserted into the brain targets and reagents infused. Hi, I am Stephanie Chan from the Synthetic Neurobiology Group at MIT Up till now. Expressing genes in spatially complex brain circuits has required either the slow and expensive creation of transgenic animals or the painstaking process of viral injections, which must be done one side at a time.

Today we're going to show you how to build and make use of a parallel injector array that can deliver fluids to complex three-dimensional patterns in the brain. We use this array to rapidly make circuit specific transgenic mice delivering viruses in parallel through the set of injectors to hit all of the targets at once. Our injector can be used to deliver any fluidic reagent to a complex distribution of brain sites.

Our goal today is to deliver viruses that encode for light sensitive proteins so that we can turn on and off in a temporally precise fashion specific cell types in specific circuits. In our lab, we use this technology in conjunction with arrays of optical fibers that deliver light in a circuit specific way. So let's get started.

We first build a stereotaxic clamp that holds the injector array and can be mounted on the stereo tax. To make the clamp these items are needed. A two inch steel cannula with a 1.5 millimeter outer diameter or OD with one end flattened a small approximately 0.5 by 0.5 centimeter piece of PCB Protoboard and two identical rectangles.

One half by three eighths inch cut from one eighth inch acrylic sheeting in each of the two acrylic rectangles. Cut two circular holes at opposite corners. The first hole should have a diameter of 1.5 millimeters and be just large enough to hold the metal cannula tightly.

The second hole should have a diameter of one 16th of an inch. Insert the metal cannula into the 1.5 millimeter hole of one rectangle, then through the 1.5 millimeter hole of the second rectangle, align the bottom end of the cannula with the bottom face of the bottom rectangle to form the shape of a hockey stick, place the spacer between the two rectangles. Modeling clay is helpful to hold things together during this process while keeping the spacer tightly held between the two rectangles.

Cement this structure together by using hot glue around the cannula and 1.5 millimeter holes. Be careful not to glue the spacer to the rectangles after the glue has dried center. A 1 72 hex nut over the one 16th hole, and then screw in a 1 72 screw from the opposite side of the rectangles.

Drop small amounts of five minute epoxy around the edges of the nut to fix it to the rectangle. Be careful not to allow the epoxy to get into the threads of the nut or the screw. Once the epoxy has hardened, insert a 1 72 screw into the one 16th inch hole from the top side of the rectangles and screw it into the nut.

Remove the modeling clay and confirm that the spacer can be held firmly in place by tightening the screw. The injector array system can be customized to virtually any set of coordinates in the brain. First, locate the coordinates of the desired injection sites in a brain atlas of the species of interest and convert them to the appropriate coordinates used by the stereotypes.

In this demonstration, we'll use a mouse and there will be three injection sites, and thus there will be three tubes for injectors. The X, y, and Z coordinates will correspond to the media lateral, anterior, posterior, and or so ventral coordinates of a mouse brain atlas. Next place, three 10 microliter Hamilton syringes in an injection withdrawal syringe pump, such as this one from Harvard apparatus securely attach three foot long pieces of polyethylene tubing to the needle of each Hamilton syringe for each piece of polyethylene tubing.

Slide an F 2 52 tubing sleeve over the open end and attach it to a P 6 27 tubing adapter using the P 2 35 nut and P 200 fal. To construct the customized injector array, use a modela mini mill and a 0.011 inch diameter drill bit to drill three holes into a one 32nd inch thick PCB protoboard space. The three holes according to the relative X and Y coordinates of the injection sites before removing the board from the mill, drill a rectangle around the small holes using a drill bit of diameter, one 32nd of an inch.

Next, the individual injectors are inserted halfway into each hole so that they are held tightly and parallel to one another. Epoxy them to the board to form the of the injector array. To attach the injector array to the stereotaxic clamp.

Place one corner of the protoboard between the acrylic rectangles and tighten the screw of the stereotaxic clamp. After that, attach the metal cannula of the clamp to the stereo tax using the attachment mechanism of the stereotypes. These following steps are performed under a microscope for one of the outer injectors that can be approached from the side with a straight edge without bumping into any of the other injectors.

Cut the tip extending beyond the bottom of the protoboard. Flatten the tip with a dremel tool. Next, choose a stable reference point within the range of the stereotaxic arm and move the injector array so that the flattened tip of the outer injector is at this reference point.

Choose a second injector along with its corresponding coordinates. Move the injector array along the Z axis by the relative difference in height or Z direction between the first and second injector coordinates. Trim this second injector so that the tip is now at the height of the reference point and flatten it with a Dremel tool.

The injector array can be moved in the X and Y directions to facilitate comparison of the injector tip with the reference point. After repeating this process of trimming for the remaining injectors, the injector array is ready for assembly into the entire system. Assembling the entire system requires the previously constructed stereotactic clamp and customized injector array.

After sterilizing the injector array, insert the backend of each injector, the end that has not been trimmed into a blue F two 40 tubing sleeve. Using a P 2 35 nut and P 200 freal, attach the back end of the injector to the threaded adapter that is already connected to the Hamilton pump by polyethylene tubing. With a 27 gauge needle, inject sterile silicone oil into the back of the Hamilton syringes so that the entire system is filled from the Hamilton syringes to the injector tips.

Be very careful not to introduce any air bubbles, otherwise the volume of virus injected will be incorrect or the virus may not inject at all. As the Hamilton syringes are replaced in the Hamilton pump, the greatest possible volume of silicone oil is maintained in the syringes. If the experiment requires more than two syringes, spacers can be placed in the Hamilton pump as we have done here.

Alternatively, a multi rack upgrade kit can be purchased from Harvard apparatus to hold up to 10 syringes at once. To inject the virus into the mouse brain, begin by administering analgesics as appropriate for your animal protocol. Place the anesthetized mouse in the stereo attacks and confirm by a toe pinch that the animal is sufficiently anesthetized.

Then make a single incision down the midline of the skin from between the eyes to between the ears. Pull back the skin to expose the skull and clean off the fascia. Attach a pulled glass pipette to the stereo attacks.

Adjust the positions of the ear bars until bgma and lambda are aligned to the same height and the line connecting them is parallel to the Y axis of the stereo attacks. Zero the stereo attacks with the tip of the glass pipette at bgma, and then position the tip slightly above the skull at the x and Y coordinates of one of the injection sites. Using a dental drill, carefully drill down the skull below the tip until an extremely thin layer of bone remains with a 30 gauge needle, gently pick off a tiny piece of the layer at the correct x and y coordinates to expose the dura mater.

This hole should be just large enough to fit one of the injectors in this way. Small 0.25 millimeter wide holes are made in the skull at the x and y coordinates corresponding to each injection site. After completion of drilling, discard the glass alignment pipette and attach the customized injector array to the stereo tax using the stereotaxic clamp.

In order to correctly set the angle of the injector array, choose two outer injectors to be calibrated to a given reference point After one of the injectors is matched with the reference point, consider the relative X accent Y distances between the injection sites for this injector and one another. Move the injector array in the x and y directions by these relative distances. If the second injector is not aligned with the reference point, the metal cannula is loosened and rotated.

Repeat this process iteratively until the injector array is angled correctly. Set the refill rate of the Hamilton pump to one microliter per minute and refill the injectors with silicone oil until the syringes are at the two microliter point or greater. This provides a buffer amount of oil so that any air bubbles or clogging in the tip can be easily removed by pushing oil forward using the Hamilton pump without having to refill the entire system with silicone oil from the back of the syringes as described previously.

Place a clean piece of paraform on the skull and gently lower the injectors onto the paraform. If one microliter of virus is intended to be delivered to each site, pipette 1.5 microliters of virus onto the parfum for each site with the same refill rate of one microliter per minute. Refill 1.2 microliters of the virus into each injector.

Zero the tip of the longest injector at bgma, and then shift the injector array to the x and Y coordinates of that injector. Lower the injector array through the small holes made with the 30 gauge needles to correct Z depth. Inject one microliter of virus through each injector at 0.1 microliters per minute or slower.

Leave the injections alone for 30 minutes for the to diffuse away. After 30 minutes, slowly extract the injector array from the brain. Then set the Hamilton pump to infuse at the same rate of 0.1 microliters per minute in order to check for clogging in each injector.

After that, clean the injectors by refilling and infusing two microliters of ethanol at a rate of two microliters per minute. Finally, refill the injectors with silicone oil to maintain the two microliter buffer zone in each Hamilton syringe. This figure shows the result of using the injector array just shown containing three cannulas to deliver viral reagents to distributed parts of the mouse brain.

In this case, the genetic payload of the virus contains the fluorescent molecule GFP, resulting in three fluorescent volumes of labeled cells labeled in a single step. In principle, an arbitrary number of cannulas can be used to build up a virus injector array. Here, for example, is an injector array appropriate for bilaterally, targeting the ca one of the hippocampus with 16 injectors.

Using the protocol that we've shown you today, you'll be able to deliver a virus in a three-dimensional pattern to the brain, effectively creating circuit level transgenics. In addition, the parallel injector array can be used to deliver any fluidic reagent to the brain, perhaps allowing, for example, dopaminergic agonists to be delivered in a pattern fashion to specific neural circuits in a head fixed behaving animal. Thank you for watching and good luck with your experiments.