Limits...
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

Activation of glial cells. A, Confocal image of a sample immunohistochemically stained with anti-GFAP antibody. Scale bar, 500 µm. B, Magnified image of left boxed area in A, near the injection site. C, Magnified image of the right boxed area in A, distal from the injection site. Scale bar, 200 µm. D, Confocal image of a sample immunohistochemically stained with anti-Iba1 antibody. Scale bar, 500 µm. E, Magnified image of the left boxed area in C, near the injection site. F, Magnified image of the right boxed area in C, distal from the injection site. Scale bar, 200 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4596018&req=5

Figure 5: Activation of glial cells. A, Confocal image of a sample immunohistochemically stained with anti-GFAP antibody. Scale bar, 500 µm. B, Magnified image of left boxed area in A, near the injection site. C, Magnified image of the right boxed area in A, distal from the injection site. Scale bar, 200 µm. D, Confocal image of a sample immunohistochemically stained with anti-Iba1 antibody. Scale bar, 500 µm. E, Magnified image of the left boxed area in C, near the injection site. F, Magnified image of the right boxed area in C, distal from the injection site. Scale bar, 200 µm.

Mentions: One of the concerns raised in the two-photon imaging of dendritic spines in mice studies was the activation of glial cells under invasive procedures. Because our methods in the marmoset neocortex include invasive procedures of virus injection and dura opening, we checked the activation of glial cells in our sample of the prefrontal cortex that was used for the in vivo imaging study. We observed the activation of both astrocytes (GFAP; Fig. 5A–C) and microglias (Iba1; Fig. 5D–F) around the injection site. This observation indicated that experimenters should carefully choose the experimental paradigm when applying the method presented in this article (see Discussion).


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)

Activation of glial cells. A, Confocal image of a sample immunohistochemically stained with anti-GFAP antibody. Scale bar, 500 µm. B, Magnified image of left boxed area in A, near the injection site. C, Magnified image of the right boxed area in A, distal from the injection site. Scale bar, 200 µm. D, Confocal image of a sample immunohistochemically stained with anti-Iba1 antibody. Scale bar, 500 µm. E, Magnified image of the left boxed area in C, near the injection site. F, Magnified image of the right boxed area in C, distal from the injection site. Scale bar, 200 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Activation of glial cells. A, Confocal image of a sample immunohistochemically stained with anti-GFAP antibody. Scale bar, 500 µm. B, Magnified image of left boxed area in A, near the injection site. C, Magnified image of the right boxed area in A, distal from the injection site. Scale bar, 200 µm. D, Confocal image of a sample immunohistochemically stained with anti-Iba1 antibody. Scale bar, 500 µm. E, Magnified image of the left boxed area in C, near the injection site. F, Magnified image of the right boxed area in C, distal from the injection site. Scale bar, 200 µm.
Mentions: One of the concerns raised in the two-photon imaging of dendritic spines in mice studies was the activation of glial cells under invasive procedures. Because our methods in the marmoset neocortex include invasive procedures of virus injection and dura opening, we checked the activation of glial cells in our sample of the prefrontal cortex that was used for the in vivo imaging study. We observed the activation of both astrocytes (GFAP; Fig. 5A–C) and microglias (Iba1; Fig. 5D–F) around the injection site. This observation indicated that experimenters should carefully choose the experimental paradigm when applying the method presented in this article (see Discussion).

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