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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

Time-lapse imaging of spines in prefrontal cortex. A, The same dendritic regions in the prefrontal cortex were imaged at 24 h intervals. The top panel shows an image acquired on day 0 (7 d after craniotomy), and the bottom panel shows an image acquired on day 1. Scale bar, 5 µm. B, The gained spines were identified by manual inspection of two images acquired at 24 h intervals. A filled rectangle indicates the position of an example of a gained spine. Scale bar, 1 µm. C, The same as B for lost spines. Filled triangles indicate the positions of lost spines. D, Box plots showing the spine turnover rate. The open circles in box plots indicate mean values. Black dots indicate values for each site. The whiskers extend to the largest and smallest values within 1.5 times the interquartile range. E, Cumulative distributions of spine length in persisting, gained, and lost populations.
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Figure 4: Time-lapse imaging of spines in prefrontal cortex. A, The same dendritic regions in the prefrontal cortex were imaged at 24 h intervals. The top panel shows an image acquired on day 0 (7 d after craniotomy), and the bottom panel shows an image acquired on day 1. Scale bar, 5 µm. B, The gained spines were identified by manual inspection of two images acquired at 24 h intervals. A filled rectangle indicates the position of an example of a gained spine. Scale bar, 1 µm. C, The same as B for lost spines. Filled triangles indicate the positions of lost spines. D, Box plots showing the spine turnover rate. The open circles in box plots indicate mean values. Black dots indicate values for each site. The whiskers extend to the largest and smallest values within 1.5 times the interquartile range. E, Cumulative distributions of spine length in persisting, gained, and lost populations.

Mentions: We then acquired images repeatedly from the same region over time. Figure 4 shows time-lapse images of the dorsolateral prefrontal cortex, presumably area 8B or 9. The images of the same region of the dendrites were taken over time at 24 h intervals. During these imaging sessions, the clarity of the imaging window was maintained. Because our samples contained a relatively small number of hrGFP-positive neurons per injected site, we were able to easily identify the same dendrite that we observed in the previous imaging session. The overall shapes of dendrites did not change over this imaging period (Fig. 4A, top and bottom). We marked each spine on the two images (day 0 and day 1) by comparing these images side by side, and identified the spines that were “gained” or “lost” during this time interval (Fig. 4B,C). In our experiments, we analyzed 779 spines (12 sites; 34 dendrites; 3 animals; total dendrite length, 2238 µm); of these spines, 51 were gained (mean across sites, 6.4%; SD, 4.2%) and 49 were lost (mean across sites, 5.6%; SD, 3.6%; Fig. 4D). The loss or gain rate at the 1 d interval observed in this study was similar to those in previous studies of layer 5 neurons of the somatosensory cortex of transgenic mice (∼12% in 3 d for both loss and gain; Kim and Nabekura, 2011) and layer 2/3 neurons of ferret V1 by the virus vector method (∼4% in 1 d for both loss and gain; Yu et al., 2011). We measured spine length by manual tracing using Neurolucida software, and examined the difference between the distribution of the spines that persisted and that of the spines that changed (gained and lost) over the period of imaging. We observed the tendency that the changed spines were shorter than those that persisted (Wilcoxon rank sum test, changed vs persisted, p = 0.0009a), which means that shorter spines tend to be gained or lost (Fig. 4E).


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)

Time-lapse imaging of spines in prefrontal cortex. A, The same dendritic regions in the prefrontal cortex were imaged at 24 h intervals. The top panel shows an image acquired on day 0 (7 d after craniotomy), and the bottom panel shows an image acquired on day 1. Scale bar, 5 µm. B, The gained spines were identified by manual inspection of two images acquired at 24 h intervals. A filled rectangle indicates the position of an example of a gained spine. Scale bar, 1 µm. C, The same as B for lost spines. Filled triangles indicate the positions of lost spines. D, Box plots showing the spine turnover rate. The open circles in box plots indicate mean values. Black dots indicate values for each site. The whiskers extend to the largest and smallest values within 1.5 times the interquartile range. E, Cumulative distributions of spine length in persisting, gained, and lost populations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Time-lapse imaging of spines in prefrontal cortex. A, The same dendritic regions in the prefrontal cortex were imaged at 24 h intervals. The top panel shows an image acquired on day 0 (7 d after craniotomy), and the bottom panel shows an image acquired on day 1. Scale bar, 5 µm. B, The gained spines were identified by manual inspection of two images acquired at 24 h intervals. A filled rectangle indicates the position of an example of a gained spine. Scale bar, 1 µm. C, The same as B for lost spines. Filled triangles indicate the positions of lost spines. D, Box plots showing the spine turnover rate. The open circles in box plots indicate mean values. Black dots indicate values for each site. The whiskers extend to the largest and smallest values within 1.5 times the interquartile range. E, Cumulative distributions of spine length in persisting, gained, and lost populations.
Mentions: We then acquired images repeatedly from the same region over time. Figure 4 shows time-lapse images of the dorsolateral prefrontal cortex, presumably area 8B or 9. The images of the same region of the dendrites were taken over time at 24 h intervals. During these imaging sessions, the clarity of the imaging window was maintained. Because our samples contained a relatively small number of hrGFP-positive neurons per injected site, we were able to easily identify the same dendrite that we observed in the previous imaging session. The overall shapes of dendrites did not change over this imaging period (Fig. 4A, top and bottom). We marked each spine on the two images (day 0 and day 1) by comparing these images side by side, and identified the spines that were “gained” or “lost” during this time interval (Fig. 4B,C). In our experiments, we analyzed 779 spines (12 sites; 34 dendrites; 3 animals; total dendrite length, 2238 µm); of these spines, 51 were gained (mean across sites, 6.4%; SD, 4.2%) and 49 were lost (mean across sites, 5.6%; SD, 3.6%; Fig. 4D). The loss or gain rate at the 1 d interval observed in this study was similar to those in previous studies of layer 5 neurons of the somatosensory cortex of transgenic mice (∼12% in 3 d for both loss and gain; Kim and Nabekura, 2011) and layer 2/3 neurons of ferret V1 by the virus vector method (∼4% in 1 d for both loss and gain; Yu et al., 2011). We measured spine length by manual tracing using Neurolucida software, and examined the difference between the distribution of the spines that persisted and that of the spines that changed (gained and lost) over the period of imaging. We observed the tendency that the changed spines were shorter than those that persisted (Wilcoxon rank sum test, changed vs persisted, p = 0.0009a), which means that shorter spines tend to be gained or lost (Fig. 4E).

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