This document provides a protocol for imaging the deep brain of freely moving mice. It describes: 1) Implanting a guide cannula into the mouse skull to access the brain region of interest. This involves drilling holes, inserting screws, and cementing the cannula in place. 2) Precisely positioning the cannula using stereotactic coordinates to target a specific brain structure. 3) Inserting the cannula 300-600 micrometers into the brain depending on the target depth.

1. Protocol
Imaging the Deep Brain of
Freely Moving Mice
Ver 1.0

2. Imaging the Deep Brain of Freely Moving Mice
Materials and Methods
Mouse Model Adult wildtype mouse
Fluorophore/Marker Oregon green BAPTA 1 AM
Equipment
 Cellvizio® LAB 488
 ImageCellTM software
 QuantiKitTM 488 calibration kit
 NeuroPakTM
Laboratory material
Anesthesia and appropriate delivery Betadine
method (i.e. vaporizer or syringe) Topical analgesic
Topical antibiotic
Hair clippers Scalpel
Gauze Surgical scissors
PBS or 0.9% NaCl Forceps
Anesthetic (gas or solution) Stereotaxic brain atlas
Dissecting microscope Stereotactic frame (for mouse) with electrode and
implant holders
Ice Ear bars
70% ethanol solution Needle
Oregon Green BAPTA 1 solution Drilling tool (with small drill bits: 0.25mm – 0.5mm)
Precision pump
36 G tube (5 cm) Dental cement/acrylic
Hamilton syringe (5 or 10
microliters)
Tubing 0.011” x 0.24” x 0.0065” Lead pencil
Krazy glue Bone wax
Protocol: Imaging the Deep Brain of Freely Moving Mice Page 1

3. 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.
Protocol: Imaging the Deep Brain of Freely Moving Mice Page 2

4. 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 1,250µm
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
Protocol: Imaging the Deep Brain of Freely Moving Mice Page 3

5. 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.
Protocol: Imaging the Deep Brain of Freely Moving Mice Page 4

6. 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. Add 4μl of 20% pluronic acid (Invitrogen #P-3000MP) in DMSO
b. Vortex for 3 mins
c. After this step, the color of the solution should be slightly yellow
d. Add 36μL of Ca2+-free ACSF
e. Add 1μL of SR101 (2.5mM or 2mM mixed in ACSF)
f. Vortex for about 3min
g. Sonicate on ice for 5min
h. If dye sits longer than 30min, sonicate again for 5min
i. 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 8μl
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
Protocol: Imaging the Deep Brain of Freely Moving Mice Page 5

7. 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 manufacturer’s
directions to deliver one microliter over the course of 10 minutes (0.1 μl/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.
Protocol: Imaging the Deep Brain of Freely Moving Mice Page 6

8. 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.
Protocol: Imaging the Deep Brain of Freely Moving Mice Page 7

9. 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.
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
Protocol: Imaging the Deep Brain of Freely Moving Mice Page 8