1): MBN, midpoint between the
bregma and nasion; AB, 1 cm anterior to the
bregma; B,
bregma; LB, 1 cm to the left of the
bregma; RB, 1 cm to the right of the
bregma; PB, 1 cm posterior to the
bregma; MBI, midpoint between the
bregma and inion; I, inion; LTL, left temporal line; and RTL, right temporal line (the cranial thickness at the temporal line was measured at the site of intersection between the frontal plane and temporal line).
KA was infused with a 5 [micro]L Hamilton syringe over a 2-min period according to the following coordinates: SNc (n=27): -4.3 mm posterior to
bregma and 2.2 mm from the midline, with infusions to a depth of 7.4 mm from the skull surface (0.25 [micro]L per site) (19).
Six sections per animal were selected with 150 [micro]m interval according to anatomical landmarks corresponding to −1.46 and −2.46 mm posterior to the
bregma with a reference to the mouse brain atlas.[24] As previously described,[25] in short, the sections were treated with 10% normal donkey serum (in 0.05 mol/L PBS) for 30 min and incubated with rat anti-BrdU (1:200, BioSource International, Camarillo, CA, USA) and goat anti-DCX (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 12 h at 4[degrees]C.
Grafts were found at AP coordinates between 0.7 and -0.4 relative to the
bregma. Due to this considerable size, at least three sections were used from each animal.
To overexpress [alpha]-syn, recombinant adeno-associated viral (rAAV) vector containing human [alpha]-syn gene, SNCA, (10 [micro]g/10 [micro]L/rat) was injected into the left lateral cerebral ventricle using the following coordinates adapted from the rat brain atlas: AP: -0.8 mm, ML: -1.5 mm, and DV: -3.6 mm from the
bregma, midline, and skull surface, respectively.
Briefly, the optimal stimulation site was 3.0-5.0 mm rostral to
bregma and 2.0-4.0 mm lateral from the midline (shown as the supplementary Figure 1).
A stainless steel needle (0.4 mm diameter) was inserted through a small burr hole on the right side of the skull, and the needle tip was placed in the right medial forebrain bundle (4.5 mm posterior to the
bregma, 1.2 mm lateral to the sagittal suture, and 8.5 mm ventral to the periosteum surface) according to the atlas of Paxinos and Watson [13].
A burr hole was made, and a 30-gauge needle was inserted through the burr hole into the striatum (coordinates: 0.2 mm posterior, 5.0 mm ventral, and 3.0 mm lateral to the
bregma).
All sections along the entire dorsal/ventral axis of the hippocampus that contained the DG subregion (i.e., from 1.34 mm posterior to the
bregma to 3.52 mm posterior to the
bregma [62]) were used for the analysis, resulting in 10-12 DG-containing sections per brain.
A small hole was drilled in the skull over the CnF according to the stereotaxic coordinates of the Rat Brain in Stereotaxic Coordinates by Paxinos and Watson (7.6-8.5 mm caudal to the
bregma, 1.7-2.2 mm lateral to the midline suture, and 5.5-6.2 mm ventral from the
bregma).