In my experience that distance is quite variable, but does not always reflect the brain underneath, merely the alignment. See attached for guide on how to define lambda and alignment. Also note that C57's, like some other inbred strains, have a ~15 degree slant to their skull relative to the brain, which can distort the coordinates and must be taken into account. Also see attached. That would explain why you are consistently too posterior.

See section 2.4.1 (pg. 11-12) and figure 8.4B (pg. 27) of attached paper.

Article Turning ON Caspases with Genetics and Small Molecules

In my experience that distance is quite variable, but does not always reflect the brain underneath, merely the alignment. See attached for guide on how to define lambda and alignment. Also note that C57's, like some other inbred strains, have a ~15 degree slant to their skull relative to the brain, which can distort the coordinates and must be taken into account. Also see attached. That would explain why you are consistently too posterior.

See section 2.4.1 (pg. 11-12) and figure 8.4B (pg. 27) of attached paper.

Article Turning ON Caspases with Genetics and Small Molecules

Hi Roland. I inject much deeper regions (>5mm) so I find that the 15 degree rotation has a larger effect, and I find an AP offset of ~1mm posterior. I typically have to adjust my AP-coordinate by ~1mm relative to the brain atlas (depending on the exact depth). However, I do not adjust from mouse to mouse based on body weight or BL distance. I am working with a normal mouse line without growth defects, and I find the body weight does not impact the size of the brain, and likewise BL distance is not necessarily correlated with body weight. Whenever I want to establish new coordinates I do several dye injections until I get the AP-coordinate where I want it, regardless of what the brain atlas says.

I also found that at one point we had maintenance done on our stereotax, after which all of my AP-coordinates changed by ~0.3mm, so every apparatus is different, and coordinates will need to be established on each one, and after each calibration.

As for your question about the lambda definition, I always use the same angles for my superimposed lines and call that intersection lambda.The actual sutures may wiggle around and not intersect there, but the using the dashed lines is a good proxy for the position of the brain underneath, and has given me consistent results. In my experience it is safe to define that intersection as lambda for all cases.

I treat bregma the same way I treat lambda, by superimposing those lines as best as possible and defining bregma as that intersection. I try to match the dashed lines to an average of the sutures a little further out from the intersection as the sutures do tend to wiggle a lot, and as you said, can make a deep V or a shallow V depending on the mouse.

Hmmm. I definitely agree with the top 2, the angle is perfect and the point is at the best compromise position. For the bottom 2, the angle is a bit shallower than I normally do. The left one is fine, although I might have picked a point slightly lower (not the bottom of the V, but maybe one dot-length lower), but I would do a sharper angle for the bottom right. I try to keep the angle basically the same from one mouse to another. The bottom right is the opposite of the example in the bottom left, where instead of a deep V, you get an inverted or extra shallow V, but they should be treated the same way. If the deep V gets defined slightly above the V, then the shallow V should get defined slightly below the V. I hope that makes sense.

Hi Roland,

Sorry for the delay. I forgot to check if you had written back.

I have marked what I would call bregma and lambda on all the skulls. For the most part it's an approximation, which is always going to be true anyway. I also use the blood vessel that runs down the midline to help define them if the midline suture is particularly crooked (as in the last one).