1. Prepare virus
   
   • Combine 10 uL of virus stock with 30 uL of dH₂O and 10 uL of 0.4% trypan blue dye in a small (0.6 mL) centrifuge tube
     
     • This would dilute the virus 1:5, so that a ~5x10¹² vg/mL stock titer would become ~1x10¹² vg/mL
       
     • Trypan blue dye would become 0.08%
       
   • Vortex, centrifuge down and store on ice.
     

2. Prepare injection needle

   • Pull bee stinger-style glass needles from glass capillary tubes.

     • Use 10 uL Drummond Microdispenser capillaries (Cat# 3-000-210-G)

     • Use program #04 (named "adair beestinger") on the puller with settings:

       Heat	Pull	Vel	Delay	Pressure	Ramp
       631	100	70	90	200	631

     • Place the capillary tube asymmetrically in the puller centered at around 1 mm from farthest thin orange line (just make sure all orange lines are preserved):

       

   • Trim needle tip to about 5 mm 😏 (at least 3 mm)​.

     • You can either use scissors or stretch a kimwipe over a beaker and poke the needle tip through the kimwipe.
       
     • The tip has to be wide enough to front-fill with the virus but long enough to go into the ventricle.
       
     • If the tip is too fine, it will just break a little when you try to penetrate the skull of the animal. A little glass in the scalp of the rat doesn't seem to cause major issues and the part of the needle remaining will likely then be strong enough to penetrate the skull.
       
       

   • Mark needle with a fine-point marker at every 0.25 inch (corresponds to 2.5 uL).

     • The two thin orange lines are 1 inch (10 uL) apart. It can be evenly divided into 4 parts, then an extra mark can be made between the thin and thick orange line.
       

3. Set up your injection area.

   • Connect a needle holder (example) to the picospritzer. Usually, a short stretch of Tygon tubing suffice.
     
   • Tape the needle holder to an injection arm of the stereotax.
     
     • Alternatively, Peter used to use a Narishige manipulator and used some sort of metal plate to affix the magnetic base of the manipulator.
       
   • Set up the picospritzer:
     
     • Make sure it is at the gated mode (you want to be able to deliver continuous pressure, versus the quick pulses we use for normal virus injections).
       
     • Make sure it is on the lowest possible pressure: Under the Eject panel, turn the Regulator knob all the way off, then turn it back up 2 full turns.
       
   • Turn on the batteries for the hand pedal that can be used to activate the pressure by sending a 5V signal.
     
   • Store the underside illumination apparatus in the fridge.
     

4. Fill needles with virus.

   • Apply negative pressure to the back of the needle while the tip is submerged in the virus.
     
   • Load up to ~12.5 uL (5 markings) at a time.
     

5. Cryoanesthetize the rats.

   • Use rat pups aged P0-2.
     
   • Place a pup in a small plastic beaker and place it in the freezer for ~5-10 minutes.
     
     • Cool the pups until they are pale and stop moving.
       
     • You can intersperse each pup 4 minutes apart to maximize efficiency.
       
     • Don't worry if a pup stops breathing. 100% of Peter's rats fully recovered at the end of the procedure! However, try not to overdo it. Tissue in direct contact with the freezer may freeze solid and then may turn black and stop growing. Peter had a few rats end up with stunted tails when he was first figuring things out.
       

6. Inject virus.

   • Take out the underside illumination apparatus from the fridge and set it up on the stereotax platform.

   • Hold the rat pup on top of the light with one hand and visualize the skull features. This JOVE video explains that the injection site is about 0.25 cm lateral to the confluence of the sinuses:

   

   • With your other hand, lower the needle with the manipulator into the skull ~3-5 mm.

   • Press on the hand pedal (or the start button if you can reach) and see if virus starts entering the head.

   • If nothing dispenses, try raising or lowering the needle slightly. It should take less pressure to dispense virus if you are in the ventricle vs. in brain tissue.

   • If necessary, raise the needle out completely and repeat the above two steps.

   • Dispense ~2.5 uL of virus per hemisphere.

   • Wait 15-20 seconds before removing the needle to prevent backflow.

   • Repeat on the other hemisphere, if desired.

   • Label the injected rats. Peter used a marker to write a big number on the side of each animal. Peter found that red > blue > black in terms of how long the ink remains visible. You'll need to remark every 1-2 days because the dam will groom off the ink pretty quickly. Around P10 the ears will be big enough to hole punch if you want a more permanent ID.

7. Return the rats to their home cage.

   • You could let them recover on a heat pad or under a light first, but Peter found that the dams do a damn good job at rewarming the pups.
     

8. Wait for virus expression.

   • The earliest Peter checked was about 7 days after injection and he could already see decent GCaMP expression, although that will probably be virus dependent.
     
     
     

Excerpt of Peter's dissertation

Intracerebroventricular injections. In some instances, P0-2 rats received an intracerebroventricular (ICV) injection of an AAV9.Syn.GCaMP6s.WPRE.SV40 viral vector (Penn Vector Core, Philadelphia, PA, AV-1-PV2824; supplied by the GENIE Project, Janelia Research Campus, HHMI) (Glascock et al., 2011). Sterile microliter calibrated glass pipettes were filled with virus diluted to ~1x1013 GC/ml in 0.1% trypan blue dye (Bio-Rad, Raleigh, NC). Rats were cryoanesthetized and the pipette was lowered through the skull, into the lateral ventricle. A picospritzer (Picospritzer III, Parker Hannifin, Hollis, NH) was used to deliver 3 μl of virus solution into each lateral ventricle. Animals were then returned to the dam to allow time for GCaMP6s expression.