Fig 1.
Schematic diagram of the pathway specific gene transfer using the double virus vector infection.
The retrograde vector was injected into the destination area: where the axon terminal of the target cell was. The retrograde vector was then transported to the cell body of the target cell through axonal transport. The target cell expressed Cre and eGFP if retrogradely infected by the retrograde vector. The local vector was injected into the departure area: where the soma of the target cell was. Only doubly infected target cell expressed mCherry and possibly hM4Di because of the “Cre-on” system. In our study local vector and retrograde vector were alternately injected over a period of weeks.
Fig 2.
Target pathway and injected local and retrograde virus vectors.
AAV5 was injected into the bilateral LPFC of both monkeys. AAV9 was injected into the left Cd and the right FEF of Monkey TA. HiRet was injected into the right Cd and the left FEF of Monkey TO.
Fig 3.
Anatomical location of insertion sites for the two monkeys.
Distribution of insertion sites in the LPFC-Cd pathway (A), and in the LPFC-FEF pathway (B). The yellow circles represent insertion sites of local vector (AAV5 for the both monkeys). The blue circle represents insertion sites of retrograde vectors (AAV9 for Monkey TA; HiRet for Monkey TO). Three different depths along each injection track in the Cd were indicated by the blue circles on the T1 weighted anatomical images (A).
Fig 4.
mCherry and eGFP expressions in the departure and the destination areas of the frontostriatal pathways.
(A-C). Distribution of mCherry- and eGFP-positive cells in the left Cd and the ipsilateral LPFC of Monkey TA. Red dots represent mCherry-positive cells and green dots represent eGFP-positive cells. mCherry- and eGFP-positive cells were aggregated from several slides across ~2 mm along the AP direction and then superimposed on a corresponding wide-area photomicrograph. (D). eGFP-expressing cells in the boxed area of figure B as observed with a NIBA filter cube. (E). The micrograph of the same area as (D) as observed with a WIG filter cube. (F). mCherry-expressing cells in the boxed area of figure C as observed with a WIG filter cube. (G). The micrograph of the same area as (F) as observed with a NIBA filter cube. (H-O). Distribution of mCherry- and eGFP-positive cells in the right Cd and the ipsilateral LPFC of Monkey TO. Black arrows indicate where mCherry-expressing cells are. (P). eGFP-expressing cells in the boxed area of figure I as observed with a NIBA filter cube. (Q). The micrograph of the same area as (P) as observed with a WIG filter cube. (R). A mCherry-expressing cell in the boxed area of figure N as observed with a WIG filter cube. (S) The micrograph of the same area as (R) as observed with a NIBA filter cube. Cd: The caudate Nucleus, Pt: The putamen, lv: The lateral ventricle, PS: The principal sulcus.
Fig 5.
mCherry and eGFP expressions in the departure and the destination areas of the frontofrontal pathways.
(A-D). Distribution of mCherry- and eGFP-positive cells in the right FEF and the ipsilateral LPFC of Monkey TA. (E). eGFP-expressing cells in the boxed area of figure B as observed with a NIBA filter cube. (F) The micrograph of the same area as (E) as observed with a WIG filter cube. (G). A mCherry-expressing cell in the boxed area of figure C as observed with a WIG filter cube. (H) The micrograph of the same area as (G) as observed with a NIBA filter cube. (I-N). Distribution of mCherry- and eGFP-positive cells in the left FEF and the ipsilateral LPFC of Monkey TO. (O). eGFP-expressing cells in the boxed area of figure K as observed with a NIBA filter cube. (P) The micrograph of the same area as (O) as observed with a WIG filter cube. (Q). mCherry-expressing cells in the boxed area of figure M as observed with a NIBA filter cube. (R) The micrograph of the same area as (Q) as observed with a NIBA filter cube. ASs: The superior ramus of the arcuate sulcus, ASi: The inferior ramus of the arcuate sulcus, PS: The principal sulcus.