Protocol

Imaging the Deep Brain of Freely Moving Transgenic Mice

Ver 1.0

Imaging the Deep Brain of Freely Moving Transgenic Mice

Materials and Methods Mouse Model Fluorophore/Marker Promoter Adult fluorescent mice (B6 background) YFP Thy-1

Note: There are a number of commercially available transgenic animals expressing fluorescent proteins under the direction of neural- or glial-specific promoters. For further information, please contact applications support at VisualSonics (apps@visualsonics.com), or visit The Jackson Laboratory website: http://jaxmice.jax.org/research/fluorescent_proteins_or_lacZ.html Equipment

Cellvizio LAB 488 ImageCellTM software QuantiKitTM 488 calibration kit NeuroPak

Laboratory material Anesthesia and appropriate delivery method (i.e. vaporizer or syringe) Hair clippers Gauze PBS or 0.9% NaCl Anesthetic (gas or solution) Dissecting microscope Ice 70% ethanol solution Betadine Topical analgesic Topical antibiotic Scalpel Surgical scissors Forceps Stereotaxic brain atlas Stereotactic frame (for mouse) with electrode and implant holders Ear bars Needle Drilling tool (with small drill bits: 0.25mm 0.5mm) Dental cement/acrylic Lead pencil Bone wax

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Protocol Step 1: Implantation of guide-cannula (Day 1) NOTE: See also Appendix I for visual instructions 1. Anesthetize the mouse according to the method of the lab. Pinch the hind foot - the mouse is ready when it is not responsive. 2. Clean all surgical instruments with 70% ethanol solution to disinfect. 3. Shave the head to expose skin. 4. Fix the mouse head into the stereotaxic frame with ear bars and bite plate. Ensure that the animal is breathing normally. 5. Make a longitudinal incision in the middle of the head with scalpel or scissors to expose Lambda, Bregma, and the approximate X, Y position of the target structure. Clean the skull with PBS or 0.9% NaCl. Wipe with betadine and 70% alcohol. Depending on your facility, add topical analgesic and/or topical antibiotic. 6. Under a dissecting microscope, use forceps to apply pressure and ensure that the skull remains immobile. Identify anatomical landmarks Lambda (caudal aspect) and Bregma (frontal aspect). Mark them with the lead pencil. Refer to Appendix I for visual instructions. 7. Use the drilling tool to make 3 holes for the anchorage microscrews contralateral to the image target. The diameter of the holes should be adapted to the size of the screws. To ensure stability of the guide-cannula, one microscrew should be fixed in the frontal bone as far away as possible from Bregma, one in the parietal bone, and one on the occipital bone. Allow the top of the microscrews to remain above the skull: they will better serve as anchorage points if dental cement can be put all around them. Refer to Appendix I for visual instructions. Tip: Gently blow bone dust away rather than wiping it away with a damp piece of gauze.

Positioning of the skull 1. Fix a needle onto the electrode holder of the stereotactic frame. Move the tip of the needle using the micromanipulators of the stereotaxic frame to ensure that Bregma and Lambda are aligned along the X, Y axis. If they are not aligned, reposition the skull using the ear bar micromanipulators until these points are perfectly aligned. Similarly, note the Z positions of Lambda and Bregma and reposition the skull using the ear bar and bite plate micromanipulators until the Z coordinates are the same. 2. Move 1mm away from Lambda on each side. Note the Z for these two points. Adjust skull position to get these points at the same level. This procedure aims to ensure that the skull is perfectly placed, a need to perform a good probe placement.

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3. Reposition the needle tip at Bregma and note its position by placing the tip of the needle exactly on this point. Note all coordinates for this point (X, Y, Z). This is the reference point (0, 0, 0). 4. Use the brain atlas to determine the appropriate coordinates of the structure that you want to reach, according to Bregma. Use the coordinates to calculate the target position (X, Y). Using the micromanipulators of the stereotaxic frame, move the tip of the needle to reach this point. Mark the skull with the pencil on this precise location. 5. Use the drilling tool to make a hole in the marked location. A hole at least 1,250m in diameter is required to allow insertion of the guide-cannula. Tip: If a blood vessel is damaged and blood begins to leak, use dry gauze to absorb the blood and rinse with ice-cold PBS or 0.9% NaCl.

6. Use a standard 25 G needle or pointed forceps to punch through and remove the meninges. This will facilitate insertion of the dedicated implant. 7. Fix a guide-cannula implant onto the dedicated implant holder provided in the NeuroPak kit. Exchange the stereotaxic electrode holder with the dedicated implant holder and rotate such that the implant is 45 degrees from the midline as described on the following diagrams.

Schematic representation of skull and implant for insertion of guide cannula within the left hemisphere

Schematic representation of skull and implant for insertion of guide cannula within the right hemisphere

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Lateral view of the implant turned 45 degrees away from the midline (implantation on the left side). Note that the bevel allows visualization of the guide-cannula as it enters the predrilled hole during implantation. The guide-cannula of the implant is represented in blue, while the red dot represents the anchor column to which the CerboFlex will be secured.

8. Using the micromanipulators on the stereotaxic frame, position the center of the guide-cannula above Bregma. Note the X, Y and Z0 positions. This is the new reference coordinate (0,0,0). Using the micromanipulators on the stereotaxic frame, place the guide-cannula in the X and Y positions according to the stereotaxic atlas, above the image target (should be centered in the hole previously done). 9. Place the guide-cannula directly onto the brain. Note the Z1 position. 10. Insert the guide-cannula into the brain. For subcortical areas, insert the tube 300 micrometers below the brain surface. For deeper brain areas, insert the tube deeper below the brain surface (600 micrometers) Note the final position Z2. Knowing Z0 for Bregma and final Z2 position allows precise calculation of the depth of implantation within the brain from Bregma (= distance of the exit part of the cannula from Bregma). 11. This point will be used later to reach the target with precision. 12. Apply a small amount of bone wax on the underside of the implant around the base of the guide-cannula to avoid bleeding and prevent dental cement from entering the guide-cannula. Note: This step is not required but it should also help to prevent brain infection by closing the hole around the cannula. Prepare the dental cement (as described in the preparation guide, follow the safety instructions). 1. Begin by adding small amounts of dental cement. Begin by the screws then, as the cement begins to polymerize, finish by the immediate periphery of the cannula and include the implant. Be very careful to avoid blockage of the guide-cannula with cement (It should not be of concern if the end of the guide-cannula is inserted correctly into the brain). Allow the cement dry for 10 minutes. NOTE: Avoid contact between the skin/fur and the dental cement.

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2. Once the cement has hardened, disconnect the guide-cannula implant from the implant holder. 3. Insert the anti-contamination plug into the guide-cannula to prevent infection. Suture the skin around the base of the implanted guide-cannula. 4. Remove the animal from the stereotaxic frame and allow the animal to recover from the surgery for at least one week prior to imaging. Follow guidelines from your institution for post operative animal care (use of analgesics, antibiotics, etc.). Step 2: In vivo imaging (Day 7) 1. Turn on the Cellvizio LAB LSU488 imaging system and the connected computer. Insert and lock the proximal end of the CerboFlex into the connector on the LSU. For detailed instructions regarding the Cellvizio LAB hardware, consult the hardware user manual. 2. Launch the ImageCell program. For detailed instructions regarding the ImageCell software, consult the software user manual. Ensure that the CerboFlex microprobe is detected by turning on the laser. If the probe is not detected, remove it from the LSU, insert the CerboFlex installation CD (included in the NeuroPak kit) into the computer and follow the onscreen instructions. Launch ImageCell anew and start the laser to ensure that the microprobe is detected. Turn on the laser and leave it on for at least 20 minutes immediately prior to the imaging session. 3. Prior to mounting the CerboFlex into the electrode holder of the stereotaxic frame, calibrate the system using the QuantiKit 488 calibration kit. Set up the desired storage folder and prefix for the images to be acquired. Rinse the tip in 70% EtOH and then rinse with distilled water. 4. Fix the CerboFlex into the electrode holder of the stereotaxic frame. Remove the anti-contamination plug. Carefully align the tip of the CerboFlex with the center of the guide-cannula implant. Ensure that the orientation of both the fiberoptic microprobe and the guide-cannula are such that both parts will fit together perfectly. Be very careful to avoid any contact between the tip and the walls of the cannula (risk of damage). If the parts are not aligned, refer to the positioning of the skull section above. Ensure that the Z-lock screw on the CerboFlex is sufficiently loose to allow the CerboFlex to fit around the stabilization column of the guide-cannula implant. 5. Using dissecting microscope and the micromanipulators on the stereotaxic frame, center the tip of the CerboFlex above the entrance to the guide-cannula. Be very careful to avoid any contact between the tip and the walls of the cannula (risk of damage). Lower the CerboFlex until the tip of the microprobe reaches the entrance of the tube. Note the Z3 position.

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6. Calculate the target position distance from this point (Z3) according to cannula length and the depth of the cannula base in the brain. Knowing that the guidecannula is 7.5 mm long and that the end of the tube is inserted 300 or 600 micrometers into the brain, calculate how far away from the entrance of the cannula the CerboFlex should be lowered to reach the targeted cells (Calculation: 7.5 mm + distance of target from base of the cannula). 7. Slowly and incrementally insert the CerboFlex through the cannula and into the brain tissue. If there is any resistance while inserting the CerboFlex into the cannula, Zlock screw on the CerboFlex is sufficiently loose to allow the CerboFlex to fit around the stabilization column of the guide-cannula implant and ensure that there is nothing blocking the cannula using a needle under stereotaxic guidance to clean the port. Turn on the laser to guide the CerboFlex to the image target under image guidance. Tip: The image target may move slightly once reached. Once cells are identified, turn the laser off and wait 5 minutes to let the tissue to adjust to the probe. If movement is detected in the field of view, very fine adjustment of the CerboFlex in the Z direction toward brain surface may be helpful.

8. Carefully secure the CerboFlex onto the stabilization column by tightening the Z-lock screw. Tip: The field of view may have moved slightly when tightening the Z-lock screw. Turn the laser off and wait 5 minutes. If the field of view changed, adjust the position of the CerboFlex. Repeat this process until the field of view remains stable.

9. Remove the animal from the stereotactic frame and allow it to recover from the anesthetic. Put it in a resting place to recover from anaesthesia or directly within your experimetal set up. Ensure that the CerboFlex does not become twisted by monitoring the animal and manually manipulating the animal if necessary. 10. From this point, images can be captured continuously or intermittently using time lapse acquisition according to your needs. Step 4: Removal of the CerboFlex 1. At the end of the imaging session, turn off the laser beam. 2. Anaesthetize and position the animal into the stereotactic frame to remove the CerboFlex. Use caution and slowly remove the CerboFlex using the micromanipulators of the stereotaxic frame to prevent damage to the probe tip. It is equally important to use caution when removing the probe as when inserting the probe. 3. Clean the CerboFlex immediately thoroughly using the QuantiKit according to instructions outlined in the user guide.

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