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In Vivo Two-Photon Imaging of Dendritic Spines in Marmoset Neocortex(1,2,3).

Sadakane O, Watakabe A, Ohtsuka M, Takaji M, Sasaki T, Kasai M, Isa T, Kato G, Nabekura J, Mizukami H, Ozawa K, Kawasaki H, Yamamori T - eNeuro (2015)

Bottom Line: Our results demonstrated that short spines in the marmoset cortex tend to change more frequently than long spines.The comparison of in vivo samples with fixed samples showed that we did not detect all existing spines by our method.Although we found glial cell proliferation, the damage of tissues caused by window construction was relatively small, judging from the comparison of spine length between samples with or without window construction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Molecular Analysis of Higher Brain Function, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan ; Division of Brain Biology, National Institute for Basic Biology , Aichi 444-8585, Japan.

ABSTRACT
Two-photon microscopy in combination with a technique involving the artificial expression of fluorescent protein has enabled the direct observation of dendritic spines in living brains. However, the application of this method to primate brains has been hindered by the lack of appropriate labeling techniques for visualizing dendritic spines. Here, we developed an adeno-associated virus vector-based fluorescent protein expression system for visualizing dendritic spines in vivo in the marmoset neocortex. For the clear visualization of each spine, the expression of reporter fluorescent protein should be both sparse and strong. To fulfill these requirements, we amplified fluorescent signals using the tetracycline transactivator (tTA)-tetracycline-responsive element system and by titrating down the amount of Thy1S promoter-driven tTA for sparse expression. By this method, we were able to visualize dendritic spines in the marmoset cortex by two-photon microscopy in vivo and analyze the turnover of spines in the prefrontal cortex. Our results demonstrated that short spines in the marmoset cortex tend to change more frequently than long spines. The comparison of in vivo samples with fixed samples showed that we did not detect all existing spines by our method. Although we found glial cell proliferation, the damage of tissues caused by window construction was relatively small, judging from the comparison of spine length between samples with or without window construction. Our new labeling technique for two-photon imaging to visualize in vivo dendritic spines of the marmoset neocortex can be applicable to examining circuit reorganization and synaptic plasticity in primates.

No MeSH data available.


Related in: MedlinePlus

Dendritic spines imaged by in vivo two-photon microscopy. A, Maximum intensity projection of the images acquired by in vivo two-photon imaging of marmoset cortex. B, Side view of three-dimensional reconstruction of the images of the same site shown in A. The depths of the areas shown in F and G are indicated by dashed lines. C, Image plane near pial surface. D, Magnified image of the boxed area in C. E, Magnified image of boxed area in D showing dendritic spines. F, Image plane at a depth of 220 µm showing soma and basal dendrites. G, Image plane at a depth of 330 µm. Scale bars: A, B, 100 µm; C, 50 μm; D, 5 μm; E, 2 μm; F, G, 50 µm.
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Figure 3: Dendritic spines imaged by in vivo two-photon microscopy. A, Maximum intensity projection of the images acquired by in vivo two-photon imaging of marmoset cortex. B, Side view of three-dimensional reconstruction of the images of the same site shown in A. The depths of the areas shown in F and G are indicated by dashed lines. C, Image plane near pial surface. D, Magnified image of the boxed area in C. E, Magnified image of boxed area in D showing dendritic spines. F, Image plane at a depth of 220 µm showing soma and basal dendrites. G, Image plane at a depth of 330 µm. Scale bars: A, B, 100 µm; C, 50 μm; D, 5 μm; E, 2 μm; F, G, 50 µm.

Mentions: To test the feasibility of our viral expression system in vivo, we injected the virus into the marmoset neocortex. After 2 weeks of an expression period, we acquired images from living animals under anesthesia induced by isoflurane. Figure 3 shows in vivo captured images. We were able to visualize each dendritic spine using our virus constructs (Fig. 3C–E). We observed the dendritic spines of apical dendrites located in layer 1. We were also able to observe the cell bodies located at a depth of ∼300 µm from the pia (Fig. 3F, at 220 µm; Fig. 3G, at 330 µm).


In Vivo Two-Photon Imaging of Dendritic Spines in Marmoset Neocortex(1,2,3).

Sadakane O, Watakabe A, Ohtsuka M, Takaji M, Sasaki T, Kasai M, Isa T, Kato G, Nabekura J, Mizukami H, Ozawa K, Kawasaki H, Yamamori T - eNeuro (2015)

Dendritic spines imaged by in vivo two-photon microscopy. A, Maximum intensity projection of the images acquired by in vivo two-photon imaging of marmoset cortex. B, Side view of three-dimensional reconstruction of the images of the same site shown in A. The depths of the areas shown in F and G are indicated by dashed lines. C, Image plane near pial surface. D, Magnified image of the boxed area in C. E, Magnified image of boxed area in D showing dendritic spines. F, Image plane at a depth of 220 µm showing soma and basal dendrites. G, Image plane at a depth of 330 µm. Scale bars: A, B, 100 µm; C, 50 μm; D, 5 μm; E, 2 μm; F, G, 50 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4596018&req=5

Figure 3: Dendritic spines imaged by in vivo two-photon microscopy. A, Maximum intensity projection of the images acquired by in vivo two-photon imaging of marmoset cortex. B, Side view of three-dimensional reconstruction of the images of the same site shown in A. The depths of the areas shown in F and G are indicated by dashed lines. C, Image plane near pial surface. D, Magnified image of the boxed area in C. E, Magnified image of boxed area in D showing dendritic spines. F, Image plane at a depth of 220 µm showing soma and basal dendrites. G, Image plane at a depth of 330 µm. Scale bars: A, B, 100 µm; C, 50 μm; D, 5 μm; E, 2 μm; F, G, 50 µm.
Mentions: To test the feasibility of our viral expression system in vivo, we injected the virus into the marmoset neocortex. After 2 weeks of an expression period, we acquired images from living animals under anesthesia induced by isoflurane. Figure 3 shows in vivo captured images. We were able to visualize each dendritic spine using our virus constructs (Fig. 3C–E). We observed the dendritic spines of apical dendrites located in layer 1. We were also able to observe the cell bodies located at a depth of ∼300 µm from the pia (Fig. 3F, at 220 µm; Fig. 3G, at 330 µm).

Bottom Line: Our results demonstrated that short spines in the marmoset cortex tend to change more frequently than long spines.The comparison of in vivo samples with fixed samples showed that we did not detect all existing spines by our method.Although we found glial cell proliferation, the damage of tissues caused by window construction was relatively small, judging from the comparison of spine length between samples with or without window construction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Molecular Analysis of Higher Brain Function, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan ; Division of Brain Biology, National Institute for Basic Biology , Aichi 444-8585, Japan.

ABSTRACT
Two-photon microscopy in combination with a technique involving the artificial expression of fluorescent protein has enabled the direct observation of dendritic spines in living brains. However, the application of this method to primate brains has been hindered by the lack of appropriate labeling techniques for visualizing dendritic spines. Here, we developed an adeno-associated virus vector-based fluorescent protein expression system for visualizing dendritic spines in vivo in the marmoset neocortex. For the clear visualization of each spine, the expression of reporter fluorescent protein should be both sparse and strong. To fulfill these requirements, we amplified fluorescent signals using the tetracycline transactivator (tTA)-tetracycline-responsive element system and by titrating down the amount of Thy1S promoter-driven tTA for sparse expression. By this method, we were able to visualize dendritic spines in the marmoset cortex by two-photon microscopy in vivo and analyze the turnover of spines in the prefrontal cortex. Our results demonstrated that short spines in the marmoset cortex tend to change more frequently than long spines. The comparison of in vivo samples with fixed samples showed that we did not detect all existing spines by our method. Although we found glial cell proliferation, the damage of tissues caused by window construction was relatively small, judging from the comparison of spine length between samples with or without window construction. Our new labeling technique for two-photon imaging to visualize in vivo dendritic spines of the marmoset neocortex can be applicable to examining circuit reorganization and synaptic plasticity in primates.

No MeSH data available.


Related in: MedlinePlus