TITLE: Virus injections and lens placement (Mendonça et al 2024)
AUTHORS: Marcelo D Mendonça

[DESCRIPTION]
Protocol for viral injections and GRIN lens placement used in Mendonça et al 2024. 

[BEFORE_START]
Mice were kept in deep anesthesia using 1-3% isoflurane and oxygen (at 1L/min flow rate). 

[GUIDELINES]
All procedures were done in aseptic conditions. 

Experiments were done on male mice-C57BL/6 or B6.SJL-Slc6a3tm1.1(Cre)Bkmn/J (Jax labs stock #006660). 

[STEPS]
SECTION: Stereotaxic Surgery
1. Place the anesthetized mouse in the stereotaxic apparatus then stabilize the head. 
SECTION: Stereotaxic Surgery
2. Make a skin incision to expose the skull.
SECTION: Stereotaxic Surgery
3. Carefully remove any connective and muscle tissue from the exposed area. 
SECTION: Stereotaxic Surgery
4. Level the skull surface to be at less than 0.05mm by comparing the height of bregma and lambda, and also in medial-lateral directions. 
SECTION: AAV Virual Injections
5. Inject GCaMP6f stock viral solution (AAV2/5.SYN.FlexGCaMP6fWPRE.SV40 - University of Pennsylvania). Note-The virus solution is kept at -80 ºC and thawed at room temperature just before the injection. 
SECTION: AAV Virual Injections
4.1. Find the injection coordinates and mark area for drilling: Substantia nigra compacta was injected at the following coordinates: -3.16 mm anteroposterior, 1.40mm lateral from bregma and 4.20 deep from the brain surface.
SECTION: AAV Virual Injections
4.2. Drill Burr-holes in marked areas carefully.
SECTION: AAV Virual Injections
4.3. Load glass pipette into the pipette holder (Nanojet II or Nanojet III (Drummond Scientific)) and affix to the stereotaxic frame. 
SECTION: AAV Virual Injections
4.4. Set rate of injection to 4.6 nL per 5 seconds. 
SECTION: AAV Virual Injections
6. After the injection finishes, leave the pipette in place for 10-15 minutes.
SECTION: GRIN Lens Placement (if desired)
7.  Please initially follow stereotaxic surgery section. 
SECTION: GRIN Lens Placement (if desired)
10. The lens is then fixed in place using cyanoacrilate, quick adhesive cement (C&B Metabond) and black dental cement (Ortho-Jet).
SECTION: GRIN Lens Placement (if desired)
8. A blunt 28 G needle was lowered to 3 mm deep from the brain surface to facilitate the lowering of the GRIN lens.
SECTION: AAV Virual Injections
6. After the injection finishes, leave the pipette in place for 10-15 minutes.
SECTION: GRIN Lens Placement (if desired)
9. The GRIN lens was then lowered at the same coordinates as the AAV injection coordinates (-3.16 mm anteroposterior, 1.40mm lateral from bregma and 4.20 deep from the brain surface.)
SECTION: GRIN Lens Placement (if desired)
9.1. GRIN Lens is A 500-um diameter, 8.2-mm long gradient index (GRIN) lens (GLP-0584, Inscopix).
SECTION: GRIN Lens Placement (if desired)
11. Mouse recovers for 3 weeks post-GRIN placement, then a baseplate was positioned. 
SECTION: GRIN Lens Placement (if desired)
11.1. Mouse is anesthetized with isoflurane and fixed with head bars. 
SECTION: GRIN Lens Placement (if desired)
11.2. A baseplante (BPC-2, Inscopix) attached to a mini epifluorescence microscope (nVista HD, Inscopix) is then positioned above the GRIN lens.
SECTION: GRIN Lens Placement (if desired)
11.3. To correctly position the baseplate, brain tissue was imaged through the lens to find the appropriate focal plane using 40% LED power, a frame rate of 10 Hz and a digital gain of 4. 
SECTION: GRIN Lens Placement (if desired)
11.4. Once the focal plane was set, the baseplate was cemented to the rest of the cap using the same dental cement. 
SECTION: GRIN Lens Placement (if desired)
11.5. Imaging with the GRIN lense started 2–3 days after this final step. 
SECTION: GRIN Lens Placement (if desired)
7.  Please initially follow stereotaxic surgery section. 
SECTION: GRIN Lens Placement (if desired)
8. A blunt 28 G needle was lowered to 3 mm deep from the brain surface to facilitate the lowering of the GRIN lens.
SECTION: GRIN Lens Placement (if desired)
9. The GRIN lens was then lowered at the same coordinates as the AAV injection coordinates (-3.16 mm anteroposterior, 1.40mm lateral from bregma and 4.20 deep from the brain surface.)
SECTION: GRIN Lens Placement (if desired)
9.1. GRIN Lens is A 500-um diameter, 8.2-mm long gradient index (GRIN) lens (GLP-0584, Inscopix).
SECTION: GRIN Lens Placement (if desired)
10. The lens is then fixed in place using cyanoacrilate, quick adhesive cement (C&B Metabond) and black dental cement (Ortho-Jet).
SECTION: GRIN Lens Placement (if desired)
11. Mouse recovers for 3 weeks post-GRIN placement, then a baseplate was positioned. 
SECTION: GRIN Lens Placement (if desired)
11.1. Mouse is anesthetized with isoflurane and fixed with head bars. 
SECTION: GRIN Lens Placement (if desired)
11.2. A baseplante (BPC-2, Inscopix) attached to a mini epifluorescence microscope (nVista HD, Inscopix) is then positioned above the GRIN lens.
SECTION: GRIN Lens Placement (if desired)
11.3. To correctly position the baseplate, brain tissue was imaged through the lens to find the appropriate focal plane using 40% LED power, a frame rate of 10 Hz and a digital gain of 4. 
SECTION: GRIN Lens Placement (if desired)
11.4. Once the focal plane was set, the baseplate was cemented to the rest of the cap using the same dental cement. 
SECTION: GRIN Lens Placement (if desired)
11.5. Imaging with the GRIN lense started 2–3 days after this final step.