Stereotactic injection of AAVs
For investigations in cells in their intact brain environment the method of stereotactic injections of adeno associated viruses into the adult mouse brain were established in the group. The original protocol published by Cetin et al. was adjusted to follow the local regulations and fit the experimental setup (Cetin et al., 2006). The application for the required animal testing licence was granted by the Lower Saxony State Office for Consumer Protection and Food Safety (LAVES). All injected AAVs were diluted in phosphate buffered saline (PBS) and originated from self-cloned plasmid constructs.
For all AAVs the number of viral genomes (vg)/µl was calculated from the relative standard using the quantitative real-time PCR approach. An amount of 106-108 vg/µl according to the desired expression density was injected into the dorsal hippocampus using following coordinates relative to bregma: anterior/posterior -0.19, lateral ±0.15, ventral -0.16. The final procedure included the following steps:
Preparation of virus for injection: Rinse stereotactic injector needle once with ethanol and three times with NaCl. Load needed amount of AAV +50 nl buffer volume to syringe and avoid bubbles. Mount syringe to stereotactic frame.
Check the animals’ condition and determine the body weight. Inject anaesthetics (Ketamine 100 mg/kg, Medetomidine 0.25 mg/kg; 0.1 ml/10 g) intraperitoneally (i.p.; lower left quadrant). Bring animal back into the cage to fall asleep (5-10 min).
Place the animal on a warming plate covered with tissues. Check paw reflexes.
Apply additional analgesia (Carprofen, 4 mg/kg) i.p. (lower right quadrant).
Remove hair from the head (from front end of ears until beginning of the eyes) with big bent scissors. Use a tissue wetted with antiseptic spray to disinfect the head and remove small hair.
Put the animal to the stereotactic frame using bent tweezers to guide the teeth.
Gently pull the tail back. Fix the head with the nose bow from above and bring pressure with the ear bars from the sides until the top of the head moves slightly upwards and stays in a fixed position. Supervise the breathing and immediately remove ear bars when the animal is breathing hard.
Check and adjust the position of the head. It should be as straight as possible.
Apply protective cream to the eyes. Apply local anaesthetic spray to the shaved skin to prevent pain; wait at least 3 min for it to work. Check paw reflexes again.
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Hold up the head’s skin with tweezers and cut straight from the end of the ears to the beginning of the eyes, just as much as necessary (about 1 cm).
Pull the skin aside and hold it away with the bent needles. Take the scalpel and gently remove the skin above the skull.
Dry skull with cotton tips and if necessary with air spray. It is easier to locate the sutures when the skull is dry. Set up binoculars to work with from here.
Locate bregma and lambda. Lower the tip of the syringe to the skull and carefully push with the needle onto bregma. The plates will slightly move. Note down the ventral coordinates and then withdraw the needle slightly and go back to the position of lambda. Note down the ventral coordinates. If they are more than 0.3 mm apart readjust the skull’s angle by changing the pressure on the nose bar.
It is also possible to add a certain depth to the ventral coordinates later.
Go back to bregma. Note the anterior/posterior, ventral and lateral coordinates and calculate with the given coordinates the injection point.
Apply the coordinates to the stereotactic frame. Lower the needle to the point of injection on the skull, remember and pull back. Mark the spot on both lateral sides of the skull with a tissue marker, then retract the syringe.
Drill through the skull, carefully to prevent bleeding.
Lower the needle and check the position of the hole. Gently touch the top of the brain and check ventral coordinates, which should be 0.05 mm lower now.
Pull the needle back up and test if the syringe is unblocked by releasing 50 nl of virus. If there is a drop at the tip of the needle everything is fine. Simply wipe the drop with a tissue. If there is no liquid coming from the syringe, build up pressure trying to release more. If still nothing comes out, the syringe is seriously blocked and has to be washed. Collect the virus in a tube and rinse the needle excessively but carefully. Then try to load the virus again.
Slowly lower the needle into the brain to the calculated ventral coordinates.
Inject a viral amount of 100 nl/min into the brain. After finishing the injection wait for three minutes, and then slowly withdraw the needle.
Move to the other lateral side and repeat the injection. If injection of a different AAV is required, wash the syringe and prepare the other virus. Put the syringe back to the frame, calculate new coordinates and proceed with injection.
After finishing the injections, release the animal from the stereotactic frame.
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Apply a few µl of NaCl onto the skull and the surrounding skin to smoothen it.
Take the needle and stitching material to close the skin beginning from the anterior side. Take three loops around the holder for the first knot followed by three more knots with one loop. A total of about 4-5 stitches are required.
Inject 500 µl prewarmed NaCl i.p. to restore the bodies’ liquid content.
Apply antibiotics cream to the stitched wound.
Inject Atipamezol subcutaneously (s.c.; 1 mg/kg; 0.1 ml/10 g into the neck).
Keep the animal on a warming plate until it shows signs of wake-up. Apply additional oxygen for further support and supervise the wake-up.
Put the moving animal back to the cage on a warming plate and on a tissue. Offer soaked pellets in a petri dish on the ground.
Check animals every day and protocol with a score sheet.
Three weeks after stereotactic injections the animals were subjected to experiments and acute slices were prepared. Subsequent investigations included confocal microscopy, 2-photon excitation microscopy and electrophysiological recordings.
Slices were further fixed with 4% paraformaldehyde (PFA) and used for immunohistochemical stainings (Figure 4.1).
Figure 4.1: tdTomato expression in hippocampal astrocytes after stereotactic injection.
a) Coronal slices with 350 µm thickness were cut three weeks after stereotactic injection of AAV-hGFAP-tdTomato (red) to the CA1 region of the hippocampus. To confirm astrocyte specific expression, slices were fixed with 4% PFA and stained against GFAP (green) to label astrocytes, and neuronal marker NeuN (white).
Expression of AAV-hGFAP-tdTomato is restricted to the hippocampus and distributed around the injection site.
Scale bar 1 mm. b) Magnification of the white box in a). Visualization using confocal microscopy showed colocalization of tdTomato signal in GFAP positive cells in stratum oriens and stratum radiatum layers of the CA1 hippocampus. Scale bar 200 µm. c) Magnification of the white box in b) reveals the major astrocyte branches by GFAP staining and the more complex and fine structures by cytosolic expression of tdTomato. Scale bar 100 µm.
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