Stereotaxic Adeno-associated Virus Injection and Cannula Implantation in the Dorsal Raphe Nucleus of Mice

Optogenetic methods are now widespread in neuroscience research. Here we present a detailed surgical procedure to inject adeno-associated viruses and implant optic fiber cannulas in the dorsal raphe nucleus (DRN) of living mice. Combined with transgenic mouse lines, this protocol allows specific targeting of serotonin-producing neurons in the brain. It includes fixing a mouse in a stereotaxic frame, performing a craniotomy, virus injection and fiber implantation. Animals can be later used in behavioral experiments, combined with optogenetic manipulations (Dugué et al., 2014; Correia et al., 2017) or monitoring of neuronal activity (Matias et al., 2017). The described procedure is a fundamental step in both optogenetic and fiber photometry experiments of deep brain areas. It is optimized for serotonin neurons in the DRN, but it can be applied to any other cell type and brain region. When using transgenic mouse lines that express functionally relevant levels of optogenetic tools or reporter lines, the virus injection step can be skipped and the protocol is reduced to the cannula implantation procedure.


Background
With the advent of optogenetic methods, the use of optical fibers and genetically encoded probes to manipulate or monitor brain activity has rapidly expanded. Optogenetic tools are particularly useful to study neuromodulatory systems, as they are usually characterized by clusters of neurons located in deep brain regions, with long and wide projections to a multitude of brain areas. Virus injections and fiber cannula implantations have been previously described for diverse areas in the brain (e.g., ventral tegmental area [Tsai et al., 2009], locus coeruleus [Carter et al., 2010]).
Targeting the dorsal raphe nucleus (DRN, the main source of serotonin projections to the forebrain) can be complex, given its deep anatomical location below the aqueduct and the This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). superior sagittal sinus. Using standard surgical procedures might cause extensive bleeding and low success rate, resulting in small sample sizes (Ranade and Mainen 2009;Cohen et al., 2015). Optogenetic studies targeting the DRN have been previously described (

Procedure
The surgery setup consists of a stereotaxic frame connected to a gas anesthesia system, situated on top of a surgery table. A heating pad covered by a surgical drape is placed on the stereotaxic frame, below the mouse mask (where the animal will be placed). The picospritzer for the virus injection is located on a shelf, close to the surgery table. The microscope is attached to the wall, allowing movements in different angles. The aseptic surgical field is the disinfected skin and exposed surgical wound. All materials necessary for

1.
Using one needle mounted on a stereotaxic holder in the right arm (32° angled), mark bregma and calculate target coordinates (DRN is -4.7 AP, -2.9 DV from bregma), using the following equation.
Note: This equation is used to calculate the corrected target coordinates after having a defined angle for the implantation. When no angle is used in the stereotaxic arm, it is enough to sum the coordinates of the target to the coordinates read in the stereotaxic frame while touching bregma. However, when using an angle, the target coordinates need to be Note: Wait 5 min between removing the pipette from each anteriorposterior location.

7.
When injections are finished, pull the arm up. If fiber cannula implantation is to be performed, do not remove the right arm from the stereotaxic frame. Replace the stereotaxic holder with the cannula fiber holder.

8.
Cover craniotomy with a wet sponge in saline.

E.
Optical fiber implantation

1.
Mount the fiber on the stereotaxic holder in the right arm.

2.
Position the fiber tip on bregma and calculate target coordinates.

3.
Remove wet sponge from the craniotomy and keep it wet with saline ( Figure 3A).

4.
In case of additional check for exact fiber location (see Data analysis section), apply a fluorescent dye (e.g., DiI) to the fiber sides before implantation, using a syringe with a 30 G needle (be careful and do not cover the tip of the fiber).

5.
Move the right arm to the anterior-posterior target position ( Figure 3B).

6.
Insert the fiber slowly into the dorsal-ventral position ( Figure 3C). For optogenetic experiments (not for fiber photometry) a 180 μm retraction along the dorsal-ventral axis is recommended for the final target.
Note: Before reaching the final target, apply eye ointment in the craniotomy (just enough to cover the space between the fiber and the skull). Alternatively, agarose gel (1%, 10 mg in 1 ml water, Sigma-Aldrich) can be used to cover the craniotomy.

F.
Finalization and post-operative care

1.
Apply dental acrylic, in small quantities each time, until fiber implant is firmly fixed to skull.
Note: After the acrylic is dry and hard, remove any sharp edges (with the drill if necessary).

2.
Suture wound, with approximately two sutures in the back and two in the front.

3.
Remove the fiber cannula holder and place the cap on the optical fiber.

4.
Prepare and apply a mixture of wound powder and 0.3% gentamicin above the sutures and head skin.

6.
Remove mouse from the stereotaxic frame and let it recover on the heating pad.

7.
Once the mouse is locomoting, transfer it to its home cage.

8.
Monitor the mouse daily for the first four postsurgical days.

9.
After surgery, animals are single housed. Photostimulation or recordings of neural activity with fiber photometry can start 2-3 weeks post-surgery, but if necessary, behavioral training can start 5 days after surgery.

Data analysis
Viral expression and fiber location can be analyzed using standard histological analysis (please refer to Correia

1.
If the virus pipette is clogged before injection, put a saline drop around it and generate some pulses with the picospritzer to unclog it. If this does not work, it might be necessary to perform a fine cut on the tip. In the former option, do not forget to re-mark bregma and adjust the target coordinates.

2.
For head-fixed experiments (Matias et al., 2017), a head-bar needs to be fixed to the skull. In this case, we recommend applying Super Bond only after the fiber cannula implantation step. Once the fiber cannula is held at the target location, apply Super Bond above the skull and place the head-bar above bregma. Then cover it with more Super Bond and finally, once it is dry, apply dental acrylic above all implants and Super Bond.

Super bond C&B
Use the super bond kit to prepare one small spoon of polymer L-type clear, four drops of monomer and one drop of catalyst. Stir gently and apply immediately (< 2 min) using the brush. It is very important to apply the super bond immediately after preparation. Keep the dispensing dish at 4 °C before using (recommended temperature range of the dish is 10-16 °C) and clean it immediately after usage.