Hi, I always injected bilaterally, it seems your coordinates are ok based on the dye, but just use your x and -x coordinates next time

Dear Sir Al-Hamamy,

I see that you have posed several matters in the same topic. They must be spliced to explain better.

1 - The dye do not spread to the contralateral ventricle

ANSWER: This not probable to occur, unless you inject very large amounts of dye. This is not recommended why this would injury the animal. Even more, the normal flow of the cerebrospinal fluid tends to carry the dye rather and firstly to the 4th ventricle (that is a median cleft in the diencephalon). If you aim both ventricles, you must perform two separate injection in two separate coordinates.

2 - You target the medulla? Which structure specifically?

ANSWER: You must be very careful why the medulla contains the pre-Botzinger complex, that is the primary respiratory drive. This is complicated by the very small volume of the medulla, which is not as complacent as the telencephalon structures. So, injections of very small amounts, even less than one microliter, in this region can cause terminal apnoea. Also, to target specific nuclei in the medulla, you probable will need a stereotaxic micromanipulator with a precision of 1/100 micrometer or greater instead of the more widely available micromanipulators which have a precision of 1/10 micrometer.

3 - Please, do not begin the surgery before you are absolutely sure that the head is properly fixed and centered in the holder. You must begin putting the animal's incisor teeth in the incisor bar. After that, you must palpate the ear meatus and plug it with the ear bar. It is a little difficult for beginners. The gold standard is the blinking of the same side eye due to the compression of the facial nerve towards the border of the ear meatus. Once both meatuses are plugged with the ear bars, you must center the animal's head using the ear bar verniers.

4 - You must be skilled in vernier reading. You can train with a calliper.

5 - I strongly recommend you to read:

- FERRY, GERVASONI, VOGT 2014 - Stereotaxic Neurosurgery in Laboratory Rodent

- The preface of the Paxino's atlas, where the authors teach how to deal with craniometric points and coordinates.

I hope I have helped you.

Sincerely

Dear Dr. Ítalo Rosal Lustosa

Thanks a lot for your detailed reply. The spread of the dye through the whole ventricular system was recommended by my seniors to make sure I could find the correct coordinates and get used to the stereotaxic process, I found as you said that it only works after injecting a big volume of dye, previously I was only injecting 2 ul, after increasing the volume in the last few days it became visible in the whole ventricular system as I was trying.

We will be targeting the RTN in the ventral medulla, we have a new stereotaxic frame that will only be used for this, we haven't set up yet, I'm not sure it has the same accuracy as you recommended or not though.

I had a lot of trouble with fixating the head in the beginning, but now I'm better at it, and your point about checking the blink reflex of the same side eye is great, I will try using it as a gold standard from now on.

Vernier scale reading was completely new to me, it took me a while till I got used to it, and the new one we have is a digital one so I guess it will make life easier as well.

I was really looking for a resource like that! Thanks a lot for the recommendation!

Your reply definitely helped and cleared a lot of things to me, absolutely grateful!

Sincerely,