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.