Protocol

Imaging the Deep Brain of Freely Moving Mice

Ver 1.0

Imaging the Deep Brain of Freely Moving Mice

Materials and Methods Mouse Model Fluorophore/Marker Equipment Adult wildtype mouse Oregon green BAPTA 1 AM

Cellvizio LAB 488 ImageCellTM software QuantiKitTM 488 calibration kit NeuroPakTM

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 Oregon Green BAPTA 1 solution Precision pump 36 G tube (5 cm) Hamilton syringe (5 or 10 microliters) Tubing 0.011 x 0.24 x 0.0065 Krazy glue Lead pencil Bone wax Dental cement/acrylic 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)

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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 NeuroPakTM 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: Dye injection (Day 7) Note: This protocol describes the use of Oregon Green BAPTA 1 AM, but other 488nm excitable dyes can be used with minimal modification to the protocol. Note: This protocol is also appropriate for 660nm excitable dyes however a Cellvizio LAB 660 system is required. Dye preparation: Reference: Stosiek C et al. (2003) Use one vial of OGB 488 BAPTA-1AM (Oregon Green 488 BAPTA AM-1, MW 1258.07 g, available from Invitrogen #O-6807) a. b. c. d. e. f. g. h. i. Add 4l of 20% pluronic acid (Invitrogen #P-3000MP) in DMSO Vortex for 3 mins After this step, the color of the solution should be slightly yellow Add 36L of Ca2+-free ACSF Add 1L of SR101 (2.5mM or 2mM mixed in ACSF) Vortex for about 3min Sonicate on ice for 5min If dye sits longer than 30min, sonicate again for 5min Pipette dye into centrifuge filter (Ultrafree MC, available from Fisher Scientific; UFC30GV25) j. Centrifuge for 30 sec k. Dilute 1: 10 in a solution containing (in mM): 150 NaCl, 2.5 KCl, 10 Hepes, pH 7.4. l. The final solution concentration is 1mM m. Fill pipette with approximately 8l

Practically add 3.97 microliters of DMSO pluronic corresponding to a 10 mM solution) in a vial of OGB1AM and dilute the solution in 35.7 microliters of Hepes solution to obtain a 1mM solution

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Injection needle preparation 1. Cut a 20 cm long piece of tubing and glue it onto the 36 Gauge needle. Fix the other extremity of the tubing onto the needle of the Hamilton syringe. 2. Anesthetize the animal using inhaled or injected anesthetic. Inhaled anesthetic is preferred as it provides continuous anesthesia, however if this is not possible, adjust the dose so that it provides approximately 90min of immobilization. 3. Remove the anti-contamination plug from the guide-cannula. 4. Position and secure the animal within the stereotaxic frame (see positioning of the skull, above). Ensure that the skull is flat and that the guide cannula is parallel to the descending stereotaxic arm. 5. Clean the tip of the needle with 70% EtOH. 6. Secure the injection needle and tubing onto the descending arm of the stereotaxic frame perpendicular to the lateral arm of the stereotaxic frame using the electrode holder. Using the micromanipulators of the stereotaxic frame, position the tip of the needle on the rim of the guide-cannula opening. Repeat this step for a point on the opposite side of the rim. Verify that the Z position is the same for both points; adjust if necessary. This step ensures that the guide-cannula is truly parallel with the length of the needle. 7. Use the brain atlas to determine the appropriate Z coordinates of the target structure according to Bregma. 8. Center the injection needle within the guide-cannula. Slowly lower the needle until it reaches the level of the entrance of the tube. Note the Z3 position. 9. Knowing that the guide-cannula 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 injection needle should be lowered to reach the targeted cells (Calculation: 7.5 mm + distance of target from base of the cannula) 10. Fill the injection needle and the tubing with the dye. Lower the needle to the appropriate location. Set up the perfusion pump according to manufacturers directions to deliver one microliter over the course of 10 minutes (0.1 l/minute) and inject the dye. To allow appropriate diffusion of the dye, do not remove the needle for 10 minutes following dye delivery. Retraction should be incremental over a 5 minute period. Use caution when removing the needle from the guide-cannula of the implant. Following retraction, wait at least one hour prior to imaging to ensure that appropriate cell loading is achieved. 11. Replace the needle and tubing with the fiberoptic CerboFlex imaging microprobe.

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Step 3: In vivo imaging While waiting for cellular uptake of the injected dye: 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 Z4 position. 6. Calculate the target position distance from this point (Z4) according to cannula length and the depth of the cannula base in the brain. The calculation is similar to the one calculated previously for the dye delivery (7.5mm + the distance of the target from the 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, Z-lock 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.

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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 Zlock 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. 4. As Oregon Green Bapta 1 AM is neurotoxic, the animal should be sacrificed at the end of the imaging session. References Stosiek C, Garaschuk O, Holthoff K, Konnerth A (2003). Proc Natl Acad Sci USA; (100):7319-732

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