Hey all,
Recently i am doing stereotaxic surgeries in mice tageting BLA with the following coordinates. AP= 1.4, ML= 3.1(left and right), DV= 3.9.
But still i am loosing quite a few animals(5-6 per experiment) due to misplacements at Dorso ventral.
Medial-lateral was good in all my surgeries, so i would not suspect the error during ear bar fixation.
Can someone please provide some inputs/your experience how could i improve further with dorso ventral region?
Thank you
I did Basolateral amygdala stereotaxic injection in mice with the coordinates: AP= 1.6, ML= 3.3, DV= 4.2 . For stereotaxic injection, the key points are 1) ear bar fixtion, 2) bregma identification 3) holes drilled through the skulls. Please take the considerations of these points during injection. By the way, my paper was attached.
Dear Dongliang,
Thank you very much for tips and informative article. I will take care of those tips next time.
You can find what the problem is primarily, and fix it by having the brains of those rats with supposedly misplaced cannulae dissected, and then looking at the cannulae tract(s) under a microscope or just capture a photo of them for future analysis.
In order to gain enhanced contrast, just microinject some 0.2-0.5 ul of cresyl violet, or if not possible, the same amount of a blue ink about 1 hour before you sacrifice the animals for brain removal and dissection.
I have done this and found the problem even for a small and unclear region like the VTA.
After you got your coronal dissection images ready, you can compare your placements and coordination with corresponding coordination of a brain atlas. You can easily see how much you miss in aiming for a correct placement of your cannulae as well as having a method in hand to substantiate your procedure for future histological verification if needed.
As Dongliang Ma also mentioned, correct ear bar placement is a must! and even well-experienced researchers doing stereotaxic surgeries on rodents for a long time may find a bit tricky as each animal may have diminutive individual or anatomical differences.
The eye reflex (eyelid flicker) can be a semi-reliable response for correct aural bar fixation. Just restating, though it seems that you do not have any problems with this part.
Bregma and lambda estimation and their correct subtraction is also crucial for your the whole procedure, bur hole drilling and cannulae insertion.
Your mid-procedure calculations should also be accurately done for all axes (DV is very important too). Recheck your calculations for BLA please. It is possible that you are repeating a miscalculation for any of the axes (AP, and in particular LP and DV).
My final suggestion is about when you pour the dentistry cement on the skull surface after the cannulae placement is done to fix them. Be very careful in that when you pour the material or during the time you wait for it to be hardened, check and watch always to avoid any unwanted movements of cannuale. I experienced a few instances that because the cement is very sticky and viscose, its viscosity or flow during the hardening or solidification process may make minute and invisible movements of cannulae either pushing them laterally or deeper in the tissue.
Dear Bahman, Thank you very much for your helpful suggestion. as you mentioned probably i will have recheck bregma lamda estimation and also cement hardness. I will follow up your suggestion.