```markdown
# Goal/Experiment:
The goal of this experiment is to inject Adeno-Associated Virus (AAV) into the nodose ganglia in mice to study the role of specific cell populations in the bidirectional communication between the brain and body via the vagus nerve. This protocol aims to describe a practical surgical approach to access the vagal trunk and the jugular-nodose ganglion (JNG) complex in mice.

# AAV Injection in the Nodose Ganglia in Mouse
### Santiago Unda¹, Michael G. Kaplitt¹
¹Weill Cornell Medicine

#### ASAP Collaborative Research Network

#### Kaplitt Protocols

#### Eileen Ruth Torres
#### Weill Cornell Medicine

#### DOI:
[dx.doi.org/10.17504/protocols.io.5qpvobjmdl4o/v1](https://dx.doi.org/10.17504/protocols.io.5qpvobjmdl4o/v1)

#### Protocol Status:
Working - We use this protocol and it is working

### Abstract
The gut-brain axis links the visceral organs to the medulla oblongata via the vagus nerve. Accessing the afferent vagal pathway is crucial to dissect the role of cell populations in the communication between the brain and body. The jugular-nodose ganglion (JNG) complex has varied neural subpopulations responsible for sensing physiological conditions of the thoracic and abdominal organs. Studying these ganglia is challenging in small animals due to their size and location. Herein, we describe a practical surgical approach to the vagal trunk and the JNG complex in mice.

## Materials

| Equipment | Type | Brand | SKU | Link |
| --------- | ---- | ----- | --- | ---- |
| 10μl, Neuros Syringe, Model 1701 RN, 33 gauge | Syringe | Hamilton | 65460-06 | |
| Sub-Microliter Injection System | Injection System | World Precision Instruments | N/A  | [wpiinc.com](https://www.wpiinc.com/var-3167-sub-microliter-injection-system) |
| NanoFil Application Kits | Application Kit | World Precision Instruments | IO-KIT | [wpiinc.com](https://www.wpiinc.com/var-3327-nanofil-application-kits) |
| 36-gauge Beveled NanoFil needle | Needle | World Precision Instruments | NF36BV-2 | |
| Micromanipulator | Manipulator | Miller Design | P#10 | |

## Before Start Instructions

1. Disinfect the surgical work surface with 70% ethanol and prepare sterile instruments (e.g., fine scissors, forceps, retractor).
2. Use gauzes, staples, and swabs sterilized by autoclaving.
3. For multiple surgeries, clean and re-sterilize instruments with 70% ethanol or a dry bead sterilizer between mice.
4. A surgical mask, clean lab coat, hair bonnet, and sterile gloves should be worn.
5. These ganglia are approximately 1mm wide and located deep in the cervical carotid triangle. A surgical microscope will be needed for the entire procedure.

## Preparation of the Surgical Setup

1. **Turn on the heating pad to 37°C.**
2. **Position the surgical microscope.**
3. **Prepare the AAV aliquot:**
   - Thaw the aliquot, mix well, and keep on ice.
   - For intraneural injections (vagus trunk), use the mosaic AAVrg/rh10 for better efficiency and mainly transduce afferent neurons with minimal impact on the efferent vagal pathway. Titer: 1-3×10¹²vg/ml; Volume: 4-6μl.
   - For intraganglionic injections, use AAV9. Titer: 1-3×10¹²vg/ml; Volume: 2-3μl.
4. **Withdraw the AAV with a 10μl 33G syringe.**
5. **Remove locking cap and gasket from the 10μl syringe.**
6. **Connect the NanoFil sub-microliter injection system.**
7. **Attach the SilFlex tubing to the 10μl syringe and the other end to the Neuros Syringe.**
8. **Secure the 36G beveled NanoFil needle to the injection holder.**
9. **De-gas the NanoFil system by slowly pushing the plunger.**

## Surgery

1. **Anesthetize mice** using a mixture of ketamine (110mg/kg) and xylazine (8mg/kg) or isoflurane.
2. **Shave** the whole anterior cervical area with an electric razor or shaving cream.
3. **Lay the mouse flat** on supine position on a heating pad.
4. **Apply ophthalmic ointment** to the mouse’s eyes.
5. **Sterilize the surgical area** with 70% alcohol, complex iodine, and 70% alcohol again. Place a surgical gauze in the sterile area.
6. **Make a small incision** in the skin with straight thin scissors or a scalpel.
7. **Retract the submandibular glands** laterally to expose the cervical musculature.
8. **Dissect the sternocleidomastoid muscle** laterally and the omohyoid muscle medially.
9. **Identify the carotid bifurcation** and gently dissect the connective tissue surrounding the area.
10. **Remove the connective tissue** surrounding the vagus trunk above the carotid bifurcation (Proceed if intra neural injections are required).
11. **Identify the temporal bone** underneath the posterior belly of the digastric muscle.
12. **Dissect the muscle fibers**, next to the mastoid notch of the temporal bone to reveal the JNG swelling.

## Stepwise Procedure

1. **Make a small incision** in the middle of the neck.
2. **Retract the submandibular glands** laterally.
3. **Dissect the sternocleidomastoid muscle** laterally and the omohyoid muscle medially.
4. **Identify the carotid bifurcation** and gently dissect the connective tissue surrounding the vagus trunk above it.
5. **Remove the connective tissue** around the vagus trunk above the carotid bifurcation. (Intra neural injections proceed).
6. **Identify the temporal bone** and dissect the muscle fibers to reveal JNG swelling.
7. **Visualize JNG**, proceed with intraganglionic injection.
8. **Open the muscle fibers** behind the posterior belly of the digastric muscle for clear view if needed.
9. **Injection volume and method**: Use a micromanipulator or free-handed; inject using 10μl syringe at 2nl/s infusion rate with a total volume of 500nl.

## Post-Surgery Procedures

1. **Close the skin** with sterile suture, apply povidone-iodine.
2. **Apply antibiotic ointment** and inject 5mg/kg Carprofen for analgesia.
3. **Maintain the mouse under a heat lamp** until fully awake; return to the cage when fully recovered.

## Visual Guide
![Visual Guide](surgical_steps_image.jpg)
1. Midline incision
2. Submandibular glands
3. Muscle dissection
4. Carotid bifurcation
5. Left vagus trunk
6. Intra neural injection site

End of Protocol.

---
End of Output.
```
endofoutput