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. As previously described, 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.
For intraventricular injection, the coordinates of the injections were as follows: anteroposterior (AP) -0.7 mm; mediolateral (ML) -1.7 mm from the bregma
; and depth, 5 mm from the skull surface .
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 .
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
) were used for the analysis, resulting in 10-12 DG-containing sections per brain.
Craniotomy was carried out on parietal bones, by drilling round holes 2 mm in diameter, with coordinates: 2-2,5 mm posterior to the bregma
and 2 mm lateral to the sagittal suture, and 10,5 mm posterior to the bregma
and 1-1,5 mm lateral to the sagittal suture .
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