TITLE: Optimized Stereotactic Injection Protocol for Targeting the Locus Coeruleus with Minimal Neurotoxicity
AUTHORS: Csilla Novák, Rukhshona Kayomova, Matthias Prigge

[DESCRIPTION]
The Locus Coeruleus is a critical brain region known for its vulnerability to mechanical perturbations, neuroinflammation, and axonal disruption. This protocol presents a refined stereotactic injection procedure designed to target the LC while minimizing neurotoxicity. By positioning the injection site 0.5 mm lateral to the LC, we effectively deliver viral vectors without directly damaging the region. The protocol outlines the administration of anesthetics, including ketamine and xylazine, ensuring stable conditions during surgery. Key steps include precise head fixation, identification of anatomical landmarks, and careful drilling of the skull to facilitate accurate injections. The use of a beveled needle allows for controlled viral infusion, with a recommended volume of 500 nl per hemisphere at a rate of 50-100 nl/min. Post-operative care, including analgesic administration and monitoring for distress, is emphasized to ensure animal welfare. This optimized approach not only enhances the safety of stereotactic injections in the LC but also supports the integrity of experimental outcomes in neurobiological research.

[STEPS]
SECTION: Animal Preparation
2. Weigh the mouse to calculate the precise dosage of anesthetic required. Administer ketamine at a dosage of 10 mg/kg and xylazine at a dosage of 20 mg/kg. Alternatively, use isoflurane as approved by your institutional animal care and use protocols. Ensure adherence to all relevant guidelines and protocols for the safe and effective administration of anesthesia.
SECTION: Animal Preparation
3. Anesthetize the mouse using the ketamine-xylazine mixture about 10 minutes prior to the
surgery via intraperitoneal injection for stable  (reduced pain) surgery
SECTION: Animal Preparation
4. Confirm the depth of anesthesia by checking the pedal withdrawal reflex
SECTION: Pre-surgical procedure
5. Shave the area over the skull


 

SECTION: Pre-surgical procedure
6. Clean the skin with 70% ethanol and disinfect with Betadine
SECTION: Pre-surgical procedure
7. Cover the eyes of animals with moisturising balm
SECTION: Pre-surgical procedure
8. Put 2% Lidocaine gel in the clean skin of the head
SECTION: Head fixation and Exposure of the Skull
9. Place the mouse in the stereotactic frame


 

SECTION: Head fixation and Exposure of the Skull
10. position the nose in the nose clamp ensuring that the head is straight and stable
SECTION: Head fixation and Exposure of the Skull
11. secure the head by adjusting the ear bars into the external auditory canals
SECTION: Head fixation and Exposure of the Skull
12. check the pedal withdrawal reflex
SECTION: Head fixation and Exposure of the Skull
13. using sterile scalpel, make a midline incision (ca 1 cm) to exposed the skull
SECTION: Head fixation and Exposure of the Skull
15. clean the skull with sterile saline and 3% hydrogen peroxide
SECTION: Head fixation and Exposure of the Skull
14. retract the skin to exposed the bregma and lambda on the skull
SECTION: Measuring coordinates
16. Identify the lambda point and measure its depth (dorsoventral level) using a needle or injection syringe. To ensure accurate measurement, gently touch the skull with the needle or syringe, and confirm contact through the stereoscope.
SECTION: Measuring coordinates
18. zero the stereotactic manipulator on bregma and move to desired coordinates. For locus
coeruleus injections use 

AP -5.4, mm
ML +/- 1.5 mm
DV: - 3.75 mm   coordinates relative to bregma

Important: We are using a bevel needle, such as the WPI-NF34BV from World Precision Instruments (WPI) or equivalent needles from other companies.

Important: To prevent overexpression in the LC and subsequent degeneration, it is crucial to test the titer and efficiency of viral constructs by using different AAV serotypes and titers before conducting an experiment. The injection strategy described here can accommodate higher viral loads, as the injection site is 0.5 mm away from the LC. This distance reduces the amount of virus that reaches the LC, allowing for more controlled dosing.
 
Picture taken from WPI website (https://www.wpiinc.com/var-3184-nanofil-needles.html?srsltid=AfmBOooP8yjCs3-6cLYDJprVGTamPO5Ml3nUrzVFZ2qrAELROV6AXlF2)) to illustrate nature of. bevel neele
SECTION: Measuring coordinates
17. Move from lambda to bregma while maintaining the same mediolateral position of the arm. Measure the depth at bregma, ensuring that both lambda and bregma are aligned on the same midline. Adjust the head position in the left-right direction if needed to align them. Ensure that the dorsoventral (DV) levels of bregma and lambda are similar, allowing for a difference of up to 50 micrometers.
At the lambda position, verify the plane along the mediolateral axis. To do this, move 1.5 mm to the left and right, and check if the DV depths at these points are similar. A difference of up to 50 micrometers is acceptable between the left and right points.
SECTION: Craniotomy and Injections
19. Using a microdrill, carefully drill a small hole in the skull at the identified coordinates. Ensure
that the holes are clean and stop any bleedings, by treating the holes with sterile saline and
clean tissues.
SECTION: Craniotomy and Injections
20. Fill the Hamilton syringe or microinjector with the prepared injection material. Ensure that the opening of the bevel needle (e.g., WPI-NF34BV) is facing the midline, as the viral injection will diffuse in that direction. Confirm the needle's orientation using a stereoscope. Before starting the injection, expel a small amount of the viral solution outside the brain to check that the needle is not clogged and to confirm the bevel's orientation.
SECTION: Craniotomy and Injections
22. slowly inject the substance (For LC: 500 nl per hemisphere) at a rate of 50-100 nl / min
SECTION: Craniotomy and Injections
21. slowly lower the syringe needle into the brain to the desired depth  and allow the needle to rest for 2 minutes
SECTION: Craniotomy and Injections
23. after injection, allow the needle to rest for 5 minutes to allow for viral diffusion and prevent
reflux of the injected material
SECTION: Craniotomy and Injections
24. slowly withdraw the needle
SECTION: Post-surgical procedure
25. Use sterile saline to moisture the skull and clean the area around the craniotomy. Make sure no
lose hair are left on the skull or muscule tissue
SECTION: Post-surgical procedure
28. place the mouse back to the home cage placed on the heating pad. Monitor the animal until it
regains consciosness.
SECTION: Post-surgical procedure
27. Inject analgesic under the skin around the neck area (e.g. Carprofen 0.2 ml of 1mg/ml) and
inject saline (0.5 ml s.c.) to protect from dehydration.
SECTION: Post-surgical procedure
26. close the incision using sutures or surgical glue
SECTION: Post-surgical procedure
29. provide analgesic (e.g. Carprofen chewing tablets) for 3 consecutive days post surgery.
Monitor the animal for signs of distress and infection for a few days.
SECTION: Optimized Stereotactic Injection Protocol for Targeting the Locus Coeruleus with Minimal Neurotoxicity
1. One of the key challenges in studying the Locus Coeruleus (LC) is its high vulnerability to perturbations, including mechanical forces, neuroinflammation, and the disruption of axonal tracts. To address this, we have developed a refined stereotactic injection strategy that targets areas further away from the LC, allowing us to infuse viruses from a lateral position. This approach minimizes the risk of damaging the LC while still effectively delivering the viral vector.