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(a) Image of the fully labeled neuron using biocytin and streptavidin on the 300-µm-thick coronal slice taken with a confocal microscope. (b) In high-magnification image, dendritic spines in the layer of the neocortex are clearly observed. (c) A three-dimensional model was reconstructed from the data acquired by the IMARIS software using three-dimensional modeling software Blender. (d) A portion of the reconstructed three-dimensional model of a dendrite with four different spines is shown.330ders such as Down Syndrome and Tuberous Scle-rosis are reported to show distinct spine patterns that deviate from normal trends3). Patients with down syndrome are known to have spines with large heads4) and longer necks while having poorer synaptogenesis in childhood and stronger synapse atrophy in adulthood compared to normal subjects5). Tuberous Sclerosis is known to cause an increase in spine density because spine pruning is impaired6). Further analysis with animal experiments is neces-sary to clarify the link between spine morphology and pathology. There have been attempts to reveal the spine morphology of model mice with neurolog-ical disorders using electron microscopes. Although remarkable advances in electron microscopy have allowed for analysis of multiple synapses in three dimensions7), large-scale analysis of neuronal spines remain impractical. Development of new observa-tional technologies such as structured illumination microscopy combined with machine learning has been proposed to observe spines with electron microscopy grade resolution in high volume8), but these require new observational equipment which limit institutions that can do such research. To overcome these limitations, we investigated an efficient approach using confocal microscopy and computer software to make simultaneous anal-Figure 2　Images and Models of the Dendritesysis of spines throughout the dendrite possible. Although images acquired from confocal micros-copy have a significantly lower resolution than images acquired from electron microscopy, immu-nofluorescent markers allow spines to be clearly visible three-dimensional analysis under confocal microscopy as seen in figure 2a. We used 300 µm slices of B6 mouse including the somatosen-sory cortex and injected biocytin into a few pyra-midal neurons on the slice. After fixation of the slices with 4% paraformaldehyde, the slices were incubated with streptavidin conjugated with a fluo-rescent label (Alexa Flour® dye). Then, we observed the structures of the labeled neurons under a confocal laser scanning microscope. The full protocol is available on figure 3. Further, we employed the following clearing reagents to increase the quality of the confocal images for 3D reconstruction. We used CUBIC-L solution9) to clear the tissue before applying strepta-vidin. We applied SCALE S410) solution as the medium to observe the tissues under the micro-scope. Both solutions preserve tissue structure and prevent light scattering within the tissues which are necessary to create high-quality three-dimen-sional reconstruction of spines. The compiled confocal images such as one shown for