Experimental setups for behavioral analyses.

<p>A) Morphology of Degu hands. Degus have thumb-like structure (top) that allow them to manipulate tools in a dexterous fashion (bottom). B) The homecage of the degus. The degus lived in an “enriched environment” that contained a running wheel, a den, and a tunnel. C) Different levels of tool-use training processes. At Level 1, the degus simply pulled the tools (arrow) toward themselves. At Level 2, the degus had to make lateral movements (arrow*) before pulling the tool toward themselves (arrow**). At Level 2b, the degus had to place the tool beyond the reward before pulling. The degu has grabbed the tool and is about to make a lateral movement and pull the food (down). D) The experimental platform (middle), the TFT monitor for event display (left), and the numeric pad for recording (right). The scale bar is inserted as a rough reference because the photograph is tilted. The degus were placed behind the transparent fence and the experimenter sat facing them from outside of the enclosure. E) A schematic of the radial maze apparatus and the spatial cues. F) A photograph of the radial maze set-up. The experimental platform (middle), the PC for event display (right), and the spatial cues (yellow star, red square and green apple) are visible.</p

The photograph of tool use and the learning curves.

<p>A) A photograph of a degu using tool during training. The degu has grabbed the tool and is about to pull it to retrieve the food reward. An upward arrow with an asterisk indicates a food reward (a half of sunflower seed). The scale bar indicates 1 cm. B) Left: The learning curve (median ± limits of variation) observed during the tool use training phase. The success rates (ordinate) were plotted for every three sessions (abscissa) of Level 2. Sessions were numbered independently for Level 2 and were aligned at day 0 (abscissa). Each data point represents the average of four animals. Right: The success rate (median ± limits of variation) of the tool use task during the BrdU injection phase C) Left: The learning curve (median ± limits of variation) observed during the radial maze training phase. The average success rates (ordinate) were plotted for every three sessions (abscissa). Each data point represents the average of three animals. Right: The success rate (median ± limits of variation) of the radial maze task during the BrdU injection phase. For both B Left and C left, the criterion for success was an 80% or higher success rate in consecutive sessions. Day 0 is the date the criterion was satisfied. Sessions are numbered based on their alignment at the origin of the abscissa. For both B right and C right, BrdU injections started at Day 0.</p

Tool use learning, but not radial maze learning, promoted maturation of newly generated neurons.

<p>A) Confocal images showing double-labeling for Synapsin-I (green) and PSA-NCAM (red) in the CA3 subfield of the hippocampus. Low magnification images are shown with high magnification insets. Representative PSA-NCAM⁺/Synapsin-I⁺ puncta are indicated by arrowheads in the inset images. B) Effects of tool use or radial maze tasks on formation of clusters of presynaptic vesicles in the CA3 subfield. The numbers of PSA-NCAM⁺/Synapsin-I⁺ puncta in the CA3 subfield were plotted along the y-axis. A schematic of the hippocampus is shown in B (<i>top</i>). Target areas (square) were randomly selected and the images were acquired in the <i>stratum lucidum</i> of the CA3 subfield. Asterisks refer to statistical significance: *p<0.01. Scale bars indicate 50 µm in the low magnification images and 5 µm in the high magnification images.</p

Schematic of the whole slide imaging (WSI) system with darkfield internal reflection illumination (DIRI).

<p>DIRI was incorporated into the WSI system’s motorized stage. Three color light-emitting diodes (LEDs) illuminate the slide glass from the side, and the specimen scatters this light. The scattered light is then incident on the objective lens above the stage. The dichromatic mirror on the motorized turret of the microscope can be removed from the light path when acquiring darkfield images. A tube lens above the dichromatic mirror focuses the sample image onto the imaging device. A charge-coupled device camera then captures the image. A sharp cut-off filter is placed between the field diaphragm and the mirror.</p

DIRI with three-color-LED array.

<p>(a) The three-color-LED array and controller, (b) a sample illuminated with green-LED array, (c) a schematic diagram of the three-color LEDs and a slide glass sample, and (d) a sample illuminated with red (left), green (middle), and blue (right) LEDs in the <i>E gracilis</i> experiment.</p

Comparison of Hausmann’s darkfield, white-LED DIRI, and three-color LED DIRI.

<p>Comparison of Hausmann’s darkfield, white-LED DIRI, and three-color LED DIRI.</p

Diaminobenzidine (DAB)-stained brain slices.

<p>(a) Brightfield image. (b) Color DIRI image with red 150, green 225, and blue 100. Images were taken with PlanApon 2× (NA 0.08) (a), and UPlanSapo20× (NA 0.75) (b). The exposure times are (a) 4 ms, and (b) 150 ms. Both are using the VC50 color camera. Scale bars indicate 0.5 mm.</p

Cheek cell image using brightfield and darkfield microscopy.

<p>(a) Schematic diagrams of a darkfield condenser, (b) brightfield microscopy image, and (c) darkfield microscopy image of a cheek cell. Scale bars in the Figs represent 5 μm. Images were taken with UplanSapo 60× oil (NA 1.35). All images (b, c) were taken using the color camera.</p

Maximum intensity illumination of color DIRI and color balance modification.

<p>(a1) Image taken with the maximum intensity of color DIRI with red 255, green 255, and blue 255. (a2) Photoshop color chart of a1 showing the location in the color balance. (b1) Image taken with the modified color DIRI with red 150, green 255, and blue 100. (b2) Photoshop color chart of b1 showing the location in the color balance. All images (a1, b1) were taken with UplanSapo 40× oil (NA 0.95) using the VC50 color camera. The exposure time is 400 ms. The sample was skin tissue. Scale bars in the Figs represent 200 μm.</p

Brightfield image and red, green, and blue darkfield internal reflection illumination (DIRI) images of tissue microarray (TMA) sample.

<p>(a) Illuminated by brightfield, (b) red, (c) green, and (d) blue. All images were taken with UplanSapo 40× oil (NA 0.95) using the VC50 color camera. The exposure times are; (a) 8.7 ms, (b-d) 700 ms. The sample was skin tissue. Scale bars in the Figs represent 200 μm.</p