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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 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.
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pone.0132825.g005: 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.

Mentions: Locally infected eGFP-positive cells were observed along several track traces in the right FEF of Moneky TA across 4.8 mm along the AP direction (Fig 5A, 5B, 5E and 5F). The frequency of eGFP expression in the area was higher (over 1,000 cells in several corresponding slides) than that in the left Cd of the same monkey. In the ipsilateral LPFC, we observed a low frequency of double-infected mCherry-positive cells in the superior bank of the principal sulcus and the VLPFC across 3.4 mm along the AP direction (in total, 24 cells: Fig 5C, 5D, 5G and 5H). All mCherry-positive cells we found were eGFP-negative.


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 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.
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Related In: Results  -  Collection

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pone.0132825.g005: 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.
Mentions: Locally infected eGFP-positive cells were observed along several track traces in the right FEF of Moneky TA across 4.8 mm along the AP direction (Fig 5A, 5B, 5E and 5F). The frequency of eGFP expression in the area was higher (over 1,000 cells in several corresponding slides) than that in the left Cd of the same monkey. In the ipsilateral LPFC, we observed a low frequency of double-infected mCherry-positive cells in the superior bank of the principal sulcus and the VLPFC across 3.4 mm along the AP direction (in total, 24 cells: Fig 5C, 5D, 5G and 5H). All mCherry-positive cells we found were eGFP-negative.

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