Yes, it is advisable to routinely verify DV (Dorsal-Ventral) coordinates, even when targeting lateral brain regions like ML (Medial-Lateral) 3.5-4.0 mm, during bilateral stereotaxic injections in mice. While the ML coordinates define the side-to-side (left/right) position, the DV coordinate determines the depth of the injection. Variations in skull thickness, head position, or even subtle differences in the stereotaxic apparatus can lead to asymmetry in DV measurements. Therefore, verifying the DV coordinate for each hemisphere ensures precise targeting of the desired brain region on both sides.

DV coordinate variability is normal in bilateral stereotaxic injections. This variability arises from anatomical differences between individual animals, even within the same race and age group. Although it is highly recommended to use rats of the same breed, age, sex, and weight, a pilot study is still necessary. Brains vary slightly in size and shape, due to individual differences even within a strain. Subtle variations in brain structure and the position of key landmarks like bregma and lambda can influence targeting accuracy. The impact of DV variability is greater when targeting smaller brain areas or when studying animals with significant size differences (e.g., due to age or strain). For greater accuracy, especially in smaller brain regions, you can use the cryosectioning technique. It's crucial to adjust DV coordinates based on individual animal anatomy and potentially perform test injections to optimize the targeting depth. Please pay close attention to skull landmarks like bregma and lambda, ensuring they are properly aligned on the stereotaxic apparatus.

Abolfazl Akbari, thank you for your reply! I appreciate the information you provided.

To my knowledge, the recommended DV difference between lambda and bregma should be less than 0.1 mm, while the AP distance should be approximately 4.2 mm. Although I understand how to address some technical challenges (e.g., locating bregma/lambda and maintaining the exact AP distance between them), I’m still uncertain how to deal with specific issues when performing lateral injections.

For example:

1) What constitutes an acceptable difference between left and right DV coordinates?

2) How should I manage the lateral temporalis muscle when it’s positioned very close to the injection site?

I would greatly appreciate it if you (or someone else) could clarify these points.

Kind regards,

Ilia

Which nucleus or area is your bilateral injection being administered to? Can you provide the coordinates?

Which nucleus or area is your bilateral injection being administered to? Can you provide the coordinates?