TITLE: Stereotactic Injections with Headframe Implant
AUTHORS: Avalon Amaya, Katrina Nguyen

[DESCRIPTION]
This protocol describes the surgical procedure, instrumentation, and reagents necessary for performing stereotactic injections and securing a headframe to an adult mouse brain for in-vivo recording procedures.

[BEFORE_START]
Reference protocol below for all general setup and takedown procedures for rodent neurosurgery:
 

Reference protocol(s) below if performing injections:

Stereotaxic Surgery for Delivery of Tracers by Iontophoresis V.6: dx.doi.org/10.17504/protocols.io.14egn8ewzg5d/v6

Stereotaxic Injection by Nanoject Protocol V.6: dx.doi.org/10.17504/protocols.io.bp2l6nr7kgqe/v6

[GUIDELINES]
Only perform this procedure in accordance with IACUC and veterinary requirements.

[STEPS]
SECTION: Perform Stereotactic Injections
13. Fill burr holes with bone wax or Kwick-Cast.

 

SECTION: Headframe Placement
16. Attach Dovetail Clamp to stereotaxic arm and attach stylus. Center stylus over headframe fiducial point and then raise stylus slightly.

 

SECTION: Headframe Placement
17. Replace the stylus with the headframe and lower until there is contact with the skull.

 

SECTION: Headframe Placement
18. Apply white Metabond around the outside of the headframe. Metabond should wick under and into the headframe. If it becomes too viscous, remake it. Place Metabond so the majority of the skull within the well remains exposed. Let dry completely (around 5 min).

SECTION: Headframe Placement
19. Use clear Metabond to seal the rest of the skull within the inside well. Allow all metabond to completely dry (around 5 min ).

 

SECTION: Recover mouse and takedown
21. Obtain the mouse’s postoperative weight.
SECTION: Recover mouse and takedown
22. Place the mouse back in a recovery cage and put the cage on the 37 °C heat plate.
SECTION: Recover mouse and takedown
23. Reference General Setup and Takedown Procedures for Rodent Neurosurgery protocol for takedown procedure.
SECTION: Expose and Prepare the Skull Surface
1. After hair removal and disinfection, create a midline incision with a scalpel blade from approximately behind the eyes to the front of the ears.


 

SECTION: Expose and Prepare the Skull Surface
2. Using Vanna scissors, cut a teardrop shape of skin away, ensuring enough skull is exposed for headframe placement.

 

SECTION: Expose and Prepare the Skull Surface
3. Remove exposed periosteum by rubbing it apart with cotton swabs and bunching it near the edges of the skin. Then cut away with vanna scissors at the skin edge. Use ACSF to rehydrate if necessary (i.e. periosteum dries out).

 

SECTION: Expose and Prepare the Skull Surface
6. Seal all along incision site with Vetbond. Use a Sugi Absorption Spear to absorb any excess fluid either prior or during. Extend vetbond seal 1-2mm past incision site along undamaged skin. 

 

SECTION: Expose and Prepare the Skull Surface
2.1.  On the anterior side expose the rostral rhinal vein between the eyes.
SECTION: Expose and Prepare the Skull Surface
2.2. On the left and right lateral sides stop at the interface between the skull and cheek muscle.

SECTION: Expose and Prepare the Skull Surface
2.3. Posteriorly expose about   -    of neck muscle. 
SECTION: Align The Skull
7. Locate Bregma and Lambda landmarks with Dovetail Clamp and Bregma Stylus, and use them to level the skull in the anterior-posterior axis within 0.1mm.
 
 

SECTION: Align The Skull
7.1. If Lambda-Bregma offset is greater than 0.1mm in X, use the yaw adjustment on the stereotaxic alignment system to adjust the yaw within 0.1mm.
SECTION: Align The Skull
8. At midline, approximately midway between Bregma and Lambda, measure 2mm laterally on both the left and right hemisphere, ensuring that the skull is level in the medial-lateral axis within 0.15mm.
SECTION: Perform Stereotactic Injections
9. Skip to section "Headframe Placement" if not performing virus injections.
SECTION: Perform Stereotactic Injections
10. Reference protocol Stereotaxic Injection by Nanoject Protocol V.6 for performing stereotactic injections via nanoject:
dx.doi.org/10.17504/protocols.io.bp2l6nr7kgqe/v6

Begin at step 8.2.5"Mark the Injection Site"
Stop at step 8.5 "Suturing"
SECTION: Perform Stereotactic Injections
11. Reference protocol Stereotaxic Surgery for Delivery of Tracers by Iontophoresis V.6 for performing stereotactic injections via Iontophoresis:
dx.doi.org/10.17504/protocols.io.14egn8ewzg5d/v6

Begin at step 8.2.6 "Mark the Injection Site"
Stop at step 8.5 "Suturing"
SECTION: Perform Stereotactic Injections
12. If target coordinates require an angled injection, rotate mouse before proceeding with the injection. Return to level once injection has been completed and pipette has been removed.
SECTION: Headframe Placement
15. If desired, mark coordinates on skull with Sharpie.

 

SECTION: Expose and Prepare the Skull Surface
5. Score entire skull with scalpel blade to provide greater surface area for bonding agents. Do not obstruct any landmarks.
SECTION: Expose and Prepare the Skull Surface
4. Detach visible neck muscles using dumont forceps.
SECTION: Headframe Placement
14. Seal entire skull with Vetbond.
SECTION: Headframe Placement
20. Remove mouse from stereotaxic instrument base once metabond is fully dry. 
SECTION: Headframe Placement
20.1. Detach Dovetail Clamp from headframe carefully.
SECTION: Headframe Placement
20.2. Remove earbars.
SECTION: Headframe Placement
20.3. Remove mouse teeth from bitebar by lightly scruffing mouse in a way that raises the teeth out of the bite bar.