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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

Construction of imaging window. A, Craniotomy and durotomy over the target region around the somatosensory cortex. The exposed target region of the marmoset cortex is shown. Scale bar, 500 µm. B, Illustration of the metal plate used in this study. C, Picture showing the metal plate for fixation, attached to the marmoset head. Scale bar, 10 mm. D, Experimental schedule.
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Figure 2: Construction of imaging window. A, Craniotomy and durotomy over the target region around the somatosensory cortex. The exposed target region of the marmoset cortex is shown. Scale bar, 500 µm. B, Illustration of the metal plate used in this study. C, Picture showing the metal plate for fixation, attached to the marmoset head. Scale bar, 10 mm. D, Experimental schedule.

Mentions: Before an imaging session, we constructed an imaging window on the head of the animal. The hole in the skull used for virus injection was expanded to a size of ∼2 × 3 mm2 using a dental drill, and part of the dura above the injection site was deflected and resected, yielding an incision ∼1 mm in diameter (Fig. 2A). A small coverglass ∼4 × 4 mm2 in size was fixed with dental cement on top of the skull, and the space between the coverglass and the cortex was filled with an agarose gel (1.5% in artificial CSF; type III-A; Sigma-Aldrich) to minimize vibration. A custom-made metal plate with a hole having an 11 mm inner diameter was glued to the skull (Figs. 2B,C). This plate was used to fix the head of the animal during the imaging sessions. The same antibiotic, analgesic, and anti-inflammatory agents as those used for the virus injections were administered immediately after window construction and on 2 subsequent days. We started the imaging sessions from 1 to 7 d after the imaging window construction (Fig. 2D).


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)

Construction of imaging window. A, Craniotomy and durotomy over the target region around the somatosensory cortex. The exposed target region of the marmoset cortex is shown. Scale bar, 500 µm. B, Illustration of the metal plate used in this study. C, Picture showing the metal plate for fixation, attached to the marmoset head. Scale bar, 10 mm. D, Experimental schedule.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Construction of imaging window. A, Craniotomy and durotomy over the target region around the somatosensory cortex. The exposed target region of the marmoset cortex is shown. Scale bar, 500 µm. B, Illustration of the metal plate used in this study. C, Picture showing the metal plate for fixation, attached to the marmoset head. Scale bar, 10 mm. D, Experimental schedule.
Mentions: Before an imaging session, we constructed an imaging window on the head of the animal. The hole in the skull used for virus injection was expanded to a size of ∼2 × 3 mm2 using a dental drill, and part of the dura above the injection site was deflected and resected, yielding an incision ∼1 mm in diameter (Fig. 2A). A small coverglass ∼4 × 4 mm2 in size was fixed with dental cement on top of the skull, and the space between the coverglass and the cortex was filled with an agarose gel (1.5% in artificial CSF; type III-A; Sigma-Aldrich) to minimize vibration. A custom-made metal plate with a hole having an 11 mm inner diameter was glued to the skull (Figs. 2B,C). This plate was used to fix the head of the animal during the imaging sessions. The same antibiotic, analgesic, and anti-inflammatory agents as those used for the virus injections were administered immediately after window construction and on 2 subsequent days. We started the imaging sessions from 1 to 7 d after the imaging window construction (Fig. 2D).

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