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Double Virus Vector Infection to the Prefrontal Network of the Macaque Brain.

Oguchi M, Okajima M, Tanaka S, Koizumi M, Kikusui T, Ichihara N, Kato S, Kobayashi K, Sakagami M - PLoS ONE (2015)

Bottom Line: The retrograde vector incorporates the sequence which encodes Cre recombinase and the local vector incorporates the "Cre-On" FLEX double-floxed sequence in which a reporter protein (mCherry) was encoded. mCherry thus came to be expressed only in doubly infected projection neurons with these vectors.We applied this method to two macaque monkeys and targeted two different pathways in the prefrontal network: The pathway from the lateral prefrontal cortex to the caudate nucleus and the pathway from the lateral prefrontal cortex to the frontal eye field.As a result, mCherry-positive cells were observed in the lateral prefrontal cortex in all of the four injected hemispheres, indicating that the double virus vector transfection is workable in the prefrontal network of the macaque brain.

View Article: PubMed Central - PubMed

Affiliation: Brain Science Institute, Tamagawa University, Machida, Tokyo, Japan.

ABSTRACT
To precisely understand how higher cognitive functions are implemented in the prefrontal network of the brain, optogenetic and pharmacogenetic methods to manipulate the signal transmission of a specific neural pathway are required. The application of these methods, however, has been mostly restricted to animals other than the primate, which is the best animal model to investigate higher cognitive functions. In this study, we used a double viral vector infection method in the prefrontal network of the macaque brain. This enabled us to express specific constructs into specific neurons that constitute a target pathway without use of germline genetic manipulation. The double-infection technique utilizes two different virus vectors in two monosynaptically connected areas. One is a vector which can locally infect cell bodies of projection neurons (local vector) and the other can retrogradely infect from axon terminals of the same projection neurons (retrograde vector). The retrograde vector incorporates the sequence which encodes Cre recombinase and the local vector incorporates the "Cre-On" FLEX double-floxed sequence in which a reporter protein (mCherry) was encoded. mCherry thus came to be expressed only in doubly infected projection neurons with these vectors. We applied this method to two macaque monkeys and targeted two different pathways in the prefrontal network: The pathway from the lateral prefrontal cortex to the caudate nucleus and the pathway from the lateral prefrontal cortex to the frontal eye field. As a result, mCherry-positive cells were observed in the lateral prefrontal cortex in all of the four injected hemispheres, indicating that the double virus vector transfection is workable in the prefrontal network of the macaque brain.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0132825.g004: 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.

Mentions: Locally infected eGFP-positive cells were observed near the lateral ventricle in the left Cd of Monkey TA across 4.3mm along the AP direction (Fig 4A, 4B, 4D and 4E). These cells were infected from their cell bodies and/or from their axon terminals in the injection sites. eGFP-positive cells were highly frequent in the Cd head (over 100 cells in several corresponding slides) and gradually decreased with distance from the Cd head along the AP direction. We could not find eGFP-positive cells in the posterior part of Cd injection sites. In the left LPFC of the same monkey, we observed double-infected mCherry-positive cells in the inferior bank of the principal sulcus across 1.0 mm along the AP direction (in total, 81 cells: Fig 4C, 4F and 4G). We found relatively large number of mCherry-positive cells (50 cells) in the slide that is around the most posterior AAV5 injection site. We could not find mCherry-positive cells in the anterior part of LPFC injection sites. Theoretically, mCherry-positive cells also could be eGFP-positive, but all mCherry-positive cells we found were eGFP-negative, presumably because the amount of eGFP expression was insufficient for fluorescence observation in these cells.


Double Virus Vector Infection to the Prefrontal Network of the Macaque Brain.

Oguchi M, Okajima M, Tanaka S, Koizumi M, Kikusui T, Ichihara N, Kato S, Kobayashi K, Sakagami M - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4507872&req=5

pone.0132825.g004: 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.
Mentions: Locally infected eGFP-positive cells were observed near the lateral ventricle in the left Cd of Monkey TA across 4.3mm along the AP direction (Fig 4A, 4B, 4D and 4E). These cells were infected from their cell bodies and/or from their axon terminals in the injection sites. eGFP-positive cells were highly frequent in the Cd head (over 100 cells in several corresponding slides) and gradually decreased with distance from the Cd head along the AP direction. We could not find eGFP-positive cells in the posterior part of Cd injection sites. In the left LPFC of the same monkey, we observed double-infected mCherry-positive cells in the inferior bank of the principal sulcus across 1.0 mm along the AP direction (in total, 81 cells: Fig 4C, 4F and 4G). We found relatively large number of mCherry-positive cells (50 cells) in the slide that is around the most posterior AAV5 injection site. We could not find mCherry-positive cells in the anterior part of LPFC injection sites. Theoretically, mCherry-positive cells also could be eGFP-positive, but all mCherry-positive cells we found were eGFP-negative, presumably because the amount of eGFP expression was insufficient for fluorescence observation in these cells.

Bottom Line: The retrograde vector incorporates the sequence which encodes Cre recombinase and the local vector incorporates the "Cre-On" FLEX double-floxed sequence in which a reporter protein (mCherry) was encoded. mCherry thus came to be expressed only in doubly infected projection neurons with these vectors.We applied this method to two macaque monkeys and targeted two different pathways in the prefrontal network: The pathway from the lateral prefrontal cortex to the caudate nucleus and the pathway from the lateral prefrontal cortex to the frontal eye field.As a result, mCherry-positive cells were observed in the lateral prefrontal cortex in all of the four injected hemispheres, indicating that the double virus vector transfection is workable in the prefrontal network of the macaque brain.

View Article: PubMed Central - PubMed

Affiliation: Brain Science Institute, Tamagawa University, Machida, Tokyo, Japan.

ABSTRACT
To precisely understand how higher cognitive functions are implemented in the prefrontal network of the brain, optogenetic and pharmacogenetic methods to manipulate the signal transmission of a specific neural pathway are required. The application of these methods, however, has been mostly restricted to animals other than the primate, which is the best animal model to investigate higher cognitive functions. In this study, we used a double viral vector infection method in the prefrontal network of the macaque brain. This enabled us to express specific constructs into specific neurons that constitute a target pathway without use of germline genetic manipulation. The double-infection technique utilizes two different virus vectors in two monosynaptically connected areas. One is a vector which can locally infect cell bodies of projection neurons (local vector) and the other can retrogradely infect from axon terminals of the same projection neurons (retrograde vector). The retrograde vector incorporates the sequence which encodes Cre recombinase and the local vector incorporates the "Cre-On" FLEX double-floxed sequence in which a reporter protein (mCherry) was encoded. mCherry thus came to be expressed only in doubly infected projection neurons with these vectors. We applied this method to two macaque monkeys and targeted two different pathways in the prefrontal network: The pathway from the lateral prefrontal cortex to the caudate nucleus and the pathway from the lateral prefrontal cortex to the frontal eye field. As a result, mCherry-positive cells were observed in the lateral prefrontal cortex in all of the four injected hemispheres, indicating that the double virus vector transfection is workable in the prefrontal network of the macaque brain.

No MeSH data available.


Related in: MedlinePlus