TITLE: Multi-Site Optic Fiber Implants
AUTHORS: Avalon Amaya, Kenta M. Hagihara, Benjamin Ouellette, Conor Grasso

[DESCRIPTION]
This protocol describes the surgical procedure, instrumentation, and reagents necessary for implanting optic fiber probes involving injection(s) and headpost into an adult mouse brain for in-vivo fiber photometry and/or optogenetic manipulation 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: Expose and Prepare the Skull Surface for Fiber Implants
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: Align the Skull
5. 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: Expose and Prepare the Skull Surface for Fiber Implants
2. Using Vanna scissors, cut a teardrop shape of skin away, ensuring enough skull is exposed for implants.

 

SECTION: Expose and Prepare the Skull Surface for Fiber Implants
4. 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 for Fiber Implants
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: Align the Skull
6. 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: Align the Skull
5.1. 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: Perform Stereotaxic Injections
8. Reference protocol Stereotaxic Injection by Nanoject Protocol V.6 for performing stereotaxic injections via nanoject:
dx.doi.org/10.17504/protocols.io.bp2l6nr7kgqe/v6

Begin at step 8.2.6 "Mark the Injection Site"
Stop at step 8.5 "Suturing"

 

 

SECTION: Fiber Implantation
12. Place fiber in the stereotaxic cannula holder.

 

 

SECTION: Fiber Implantation
16. Slowly lower the tip of the optic fiber implant to the designated coordinate.
SECTION: Fiber Implantation
17. With the optic fiber implant still in the stereotaxic holder, apply several drops of Loctite 4305 light curing glue to the base of the fiber ferrule.
SECTION: Fiber Implantation
18. Flood area with blue light using the LED curing light until optical glue is hardened. This will take roughly 1 min minutes.

 

SECTION: Fiber Implantation
19. Carefully release the ferrule from the stereotaxic fiber holder.

 

SECTION: Fiber Implantation
20. Repeat steps   through   for all desired fiber optic implants.

SECTION: Fiber Implantation
11. If not using the same burr hole as the one drilled for virus injection, drill a burr hole for optic fiber implant.
SECTION: Fiber Implantation
14. Position fiber above the pre-drilled burr hole at the desired coordinates and angle for implantation
SECTION: Fiber Implantation
14.1. If burr hole is not wide enough to allow optic fiber implant to pass through, peel away excess bone using a microprobe, 1 mL insulin syringe, or fine forceps.
SECTION: Fiber Implantation
15. Lower the tip of the optic fiber implant to the brain surface and zero the Z coordinate.
SECTION: Fiber Implantation
15.1. Ensure that the skull thickness will allow the optic fiber implant to be lowered to desired depth. To do this, measure distance from the dorsal surface of skull to the pia surface, then subtract the measurement from optic fiber length.
SECTION: Headpost Placement
22. Attach Dovetail Clamp to stereotaxic arm and attach stylus. Center stylus over headframe fiducial point and then raise stylus slightly.

 

SECTION: Headpost Placement
23. Replace stylus with the appropriate custom headframe and lower the headframe until there is contact with the skull.

 


SECTION: Headpost Placement
24. Secure the headframe with metabond. Cover all exposed skull with metabond and place enough metabond around the metal of the headframe and base of fiber ferrules to secure them in place. Confirm that metabond does not cover the top   of the ferrule. Let dry completely (around 5 min minutes).

 

SECTION: Headpost Placement
25. Cover metabond with black cement.
SECTION: Headpost Placement
25.1. Add equal parts Jet Fast Curing Acrylic Resin Liquid and black cement powder to an empty well in the 24 well plate.
SECTION: Headpost Placement
25.2. Use the broken end of a swab to layer the black cement. Confirm that black cement does not cover the top   of the ferrule. Let dry completely (around 5 min minutes).

 


 

SECTION: Recover Mouse and Takedown
28. Obtain the mouse’s postoperative weight.
SECTION: Recover Mouse and Takedown
29. Place the mouse back in a recovery cage and put the cage on the 37 °C heat plate.
SECTION: Recover Mouse and Takedown
30. Reference General Setup and Takedown Procedures for Rodent Neurosurgery protocol for takedown procedures.

SECTION: Perform Stereotaxic Injections
7. Skip to section "Fiber implantation" if not performing virus injections.
SECTION: Fiber Implantation
11.1. Fibers will have a   diameter. In order for the fiber to pass through the burr hole, ensure that the skull is thin enough to be picked away at this width.


 

SECTION: Fiber Implantation
17.1. Glue should not cover more than roughly 1/3 of the ferrule. Most ferrules will have a notch   from the base. Use this notch as a reference for glue application.
SECTION: Headpost Placement
21. Skip headpost placement if not including headpost.
SECTION: Fiber Implantation
13. If target coordinates require an angled fiber, rotate mouse using stereotaxic controls.
SECTION: Perform Stereotaxic Injections
9. Reference protocol Stereotaxic Surgery for Delivery of Tracers by Iontophoresis V.6 for performing stereotaxic 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: Headpost Placement
26. Ensure that the metabond and black cement do not cover the top   of the ferrule, as it interferes with experimentation. Use the broken end of a dry swab to push down/remove any excess metabond or black cement.

 


 
 
 
 
 

 

SECTION: Headpost Placement
27. Remove mouse from stereotax once metabond and black cement are fully dry. 
SECTION: Headpost Placement
27.1. Detach Dovetail Clamp from headframe carefully.
SECTION: Headpost Placement
27.2. Remove earbars.
SECTION: Headpost Placement
27.3. Remove mouse teeth from bitebar by lightly scruffing mouse in a way that raises the teeth out of the bite bar.
SECTION: Fiber Implantation
10. Use forceps to disinfect optic fiber implant by dipping it in a 70% ethanol well. Rinse twice by dipping it into two wells of ACSF.