Numerous separate commissural tracts of varying diameter cross the midline within the neuropil, being located at different levels along the antero-posterior as well as the dorso-ventral axes (Fig. 2a,b).
To minimize the bias from the 3D orientation of the calvaria, a spheric volume of interest (VOI of 2 mm diameter) with the midline suture of the skull in its center was defined, as previously described [ 6].
After a midline incision, a bipolar 75-µm-diameter twisted stainless steel wire (A&M Systems) was positioned in each dorsal hippocampus (2 mm posterior to Bregma, 2 mm lateral to the midline, 1.5 mm deep).
This example likely overestimates the variability by assuming a cylindrical vessel but highlights the need for further research into what the actual change in diameter is with drift off midline.
Following a midline scalp incision, a burr hole of diameter 1 mm was made in the skull 3.5 mm to the right of the bregma.
injections, a cannula was inserted in the lateral cerebral ventricle with the following specifications (coordinates: 0.5 mm posterior to bregma, 1.6 mm lateral to the midline; size: 5.6 mm length, 0.8 mm diameter, 4.5 mm depth; Fig. 1 D).
Probe movement off-midline results in measurement of a false diameter that is smaller than the true IVC diameter.
If we assume a cylindrical vessel of 27 mm in diameter, a 3.9-mm drift off midline would result in a measured new diameter of 12.9 mm, erroneously indicating a greater than 50% respiratory variation in diameter.
A midline incision was made, and a 1 mm diameter right parietal burr hole, centered 2 mm posterior to the coronal suture and 2 mm lateral to the sagittal suture, was drilled.
Finally, any narrowing of the spinal canal was recorded, using the midline sagittal images evaluating the inner tapering AP diameter.
