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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

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.
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pone.0132825.g001: 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.

Mentions: The double-infection technique utilizes two different virus vectors in monosynaptically connected areas that constitute some particular projection pathway. One is a vector which can locally infect cell bodies of projection neurons (a “local vector”) and the other is a vector which can retrogradely infect from axon terminals of projection neurons to their cell nuclei (a “retrograde vector”). In this study, the two retrograde vectors that we used (AAV9/HiRet) incorporated the sequence which encodes Cre-recombinase and eGFP, while the local vector that we used (AAV5) incorporated the “Cre-On” FLEX double-floxed sequence in which mCherry and hM4Di were included. The FLEX sequence reverses under Cre-existence condition and the constructs in the FLEX sequence become readable. mCherry and hM4Di thus come to be expressed only in neurons which were doubly infected by both local and retrograde vectors (Fig 1).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4507872&req=5

pone.0132825.g001: 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.
Mentions: The double-infection technique utilizes two different virus vectors in monosynaptically connected areas that constitute some particular projection pathway. One is a vector which can locally infect cell bodies of projection neurons (a “local vector”) and the other is a vector which can retrogradely infect from axon terminals of projection neurons to their cell nuclei (a “retrograde vector”). In this study, the two retrograde vectors that we used (AAV9/HiRet) incorporated the sequence which encodes Cre-recombinase and eGFP, while the local vector that we used (AAV5) incorporated the “Cre-On” FLEX double-floxed sequence in which mCherry and hM4Di were included. The FLEX sequence reverses under Cre-existence condition and the constructs in the FLEX sequence become readable. mCherry and hM4Di thus come to be expressed only in neurons which were doubly infected by both local and retrograde vectors (Fig 1).

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