Transmission Electron Microscopy as the Visualization Technique for Analysis of Circadian Synaptic Plasticity in the Mouse Barrel Cortex

We are interested in neural plasticity, particularly circadian changes in plasticity of synapses, neurons, and glial cells. We are trying to answer the question how synapses are transformed during the day and night and under pathological conditions. The most recent developments in our field of studies is to use various genetic tools to manipulate with gene expression, also using methods to visualize changes in the brain, as well as there is a growing number of animal models to study neurodegenerative diseases. We have found, using electron transmission microscope, that the number of synapses in the barrel cortex of mice changes during the day and night. And this rhythm is also circadian, at least in case of inhibitory synapses. Because inhibitory synapses, they increase in the number during the night, while excitatory synapses increase during the day. Similar changes we have found in the brain of the fruit fly. In this species, also the number of synapses changes during the day and night. And in addition, we have found structural changes in shape of neurons and glial cells.

[Narrator] To begin, take the mouse's cerebellum and divide the brain into two hemispheres. Choose one hemisphere and orient it so that sections can be cut tangentially to the barrel cortex. Then, cut the sections at 60-micrometer thickness, and transfer them into a solution containing 0.1-molar phosphate buffer and fixative in a one-to-five ratio. Examine the sections under a light microscope, and collect only those sections that clearly show the barrel field cortex. Using a paintbrush, gently transfer the sections into a small glass Petri dish. Rinse the brain sections in 0.1-molar cacodylate. Then, fix the sections in 1% osmium tetroxide in 0.1-molar cacodylate buffer with 1.5% potassium ferricyanide. Once the sections are rinsed in distilled water, use a syringe with a 25-millimeter filter to slowly dispense 70% ethanol containing 1% uranyl acetate into each Petri dish with tissues. After overnight incubation in 70% ethanol at four degrees Celsius, wash tissues in 80%, 90%, and 100% ethanol. Then, wash the tissues twice in propylene oxide for 10 minutes each. Next, replace propylene oxide with two milliliters of a one-to-one mixture of Poly/Bed resin and propylene oxide. Cover with a lid and incubate for 40 minutes. Embed the brain sections in two milliliters of a three-to-one mixture of Poly/Bed resin and propylene oxide. Cover and leave for 1.5 hours. Then, cut the ACLAR film into pieces matching the size of a standard glass slide. Using a plastic pipette, apply a small volume of resin onto each ACLAR film piece. With a paintbrush, transfer the brain sections from the Petri dish to the resin on the ACLAR film. Embed each section between two ACLAR films, and incubate to polymerize. Using a light microscope with a two times objective, photograph the embedded brain sections. To open the images, click File and Open, select the images while holding Shift, and click Open. Begin stacking images from the cortex. Right-click on Layer 1 in each image, select Duplicate Layer, and enter the image number as the name in the dialogue box. Choose one image as the destination file and click OK. Next, use the Move tool to align the images manually, and adjust orientation with Edit, Transform, Rotate, or Flip as needed. For finer adjustments, use Edit, Transform, Distort, and move the corners individually to match anatomical landmarks. Set the blending mode of the top layer to overlay in the Layers panel to facilitate alignment. In each section that displays the barrel cortex, use a stereo microscope to identify the selected barrel, and cut it out along with the adjacent one using a razor blade. After embedding the section in resin block, collect the ultra-thin sections onto Formvar-coated single-slot nickel grids. After aligning the TEM images stack as demonstrated earlier, define the analysis area. Click Layer followed by New and Layer, then click OK. Select the Rectangle tool, go to Layer, Layer Style, Stroke. Set the stroke color, thickness, and position to inside, then click OK. Now, draw a rectangle to define the analysis boundary, and select Fill 0%. To add a new layer for annotations, select Layer, New, Layer, then click OK. Use the Brush tool to mark synapses on this layer. Use the Zoom tool and Hand tool to ensure precision while annotating. Now, open the image stack intended for 3D reconstruction. Select the Crop tool, mark the region containing the dendritic spine across the stack, and press Enter to crop that region in all visible layers. To make only one layer visible at a time, toggle the eye icon in the Layers panel. Click File, Save As, name the file, select JPG format, and click OK to save each cropped image separately. Open the 3D reconstruction software. Before uploading, click Z on the navigation gizmo or press numpad 7 to align the viewport perspective. Next, to upload the images, click Add, Image, Reference, then select the first image and double-click to confirm. To position the final TEM image along the z-axis, click on the image. In the Object Properties panel on the right sidebar, enter the appropriate value in the Location Z field. To distribute all images evenly along the z-axis, select all image objects. Press End to open the End panel. Navigate to the Edit tab and click Distribute Z to automatically space them based on section thickness. To manually outline the dendritic spine shape on each TEM image using a single curve per image, click Add, Curve, and Circle. To move the circle, hold g, drag the mouse, and then click to confirm the placement. Hold s to adjust the size, then click to confirm. Next, scale the object to align with the shape of the dendritic spine. Now, modify the curved shape to fit the spine boundary. Select a control point, press g, move the point, and click to confirm. Use the g or s keys as needed to adjust the handle points, refining the curve's direction and intensity. To add more details, hold Shift and select two neighboring control points. Then right-click and choose Subdivide to insert a new point between them. Once the curve accurately outlines the spine, enter the Layout tab, right-click the Bezier circle object, and choose Duplicate Object without moving the mouse. To connect the edges of the joined object and form a closed mesh, enter the Modeling tab. Press 2 to switch to Edge Select Mode. Click Select, All, right-click, and choose Bridge Edge Loops to link the edge rings together. To fill the gap at the top of the object, deselect all by clicking next to it. Hold Alt and click on one edge of the top ledge to select the entire loop. Right-click and choose Extrude Edges, drag upward along the z-axis. Now, press s, move the mouse inward to shrink the new loop, and click to confirm. With the loop still selected, right-click and choose New Face from Edges. To smooth the final structure, go to the Modifiers tab on the right sidebar. Click Add Modifier, navigate to Generate, and select Subdivision Surface. Set the viewport and render levels to one for consistent smoothing. To assign color to individual structures, select the desired object. Open the Material tab on the right sidebar, click New, and set the base color. Select the light object to adjust scene lighting. Press and hold g, move the mouse to reposition the light, and click to confirm its new location. To position the camera for rendering, orient the viewport to face the dendritic spine as it should appear in the final output. Press Control + Alt + numpad 0 to snap the camera to the current view. Adjust the camera frame by selecting it, holding g, and moving it to refine the framing. The mouse somatosensory cortex was identifiable even in unstained brain sections, and the entire barrel field could be reconstructed using digital tools despite the distribution of individual barrels across multiple sections. Consecutive ultra-thin TEM images enabled the reliable classification of synapses into excitatory or inhibitory types based on serial visual evidence. Three-dimensional reconstruction of dendritic spines from serial sections allowed for visualization of spine morphology, revealing different shapes, such as thin, mushroom, stubby spines, and intermediate. Spine shapes were quantified based on measurements, including spine length, neck length, and diameters of the spine, head, and neck.