To begin, acquire the confocal images of mouse hippocampal slices. After opening the image file in Neurolucida 360 software, left-click the move/pivot point tool located in the top toolbar of the 3D environment window. Then, left-click on the desired dendrite to establish the new pivot point.
After that, left-click the reset orientation icon to restore the original view. Then, click the move/pivot point tool again to continue tracing the dendrite. Left-click the tree tab within the 3D environment window.
Select User Guided"for the tracing mode and directional kernels, as the method under User Guided"tracing options. For initiating dendrite tracing, left-click on the dendrite as soon as a circular kernel appears. Then, move the cursor along the dendritic segment.
To ensure continuous kernel population, gently move the cursor back and forth over the dendrite. Then, left-click to secure any kernels that have been detected. If the kernels seize populating, left-click where a kernel appears further along the dendrite to place it manually, and right-click to stop tracing.
Left-click and drag within the 3D environment window to check the precision of the dendritic tracing in all three dimensions, pitch, yaw, and roll. Locate any points where the dendrite tracing deviates from the intended path. For correcting any inaccuracies in the dendrite tracing, navigate to the edit tab located within the tree menu.
Left-click on the dendrite of interest and then click on points to make necessary adjustments. Then, move improperly placed points back onto the dendrite segment, via click and drag. Left-click on the extraneous point to remove it and then click the delete button.
If the points do not adequately cover the dendrite segment, adjust their size by left-clicking on them and modifying the thickness using the slider. After that, navigate to the spine tab within the 3D environment window. Set the detection settings for outer range, minimum height, and minimum voxel count.
Adjust the detector sensitivity to 70%and then left-click Detect All"to identify spines on all dendrites according to the set sensitivity level. For a dendrite specific selection, take the option Click Image"to detect all on nearest branch, and manually left-click on each dendrite to apply different detector sensitivities. After detecting spines, click and drag the dendrite in all three directions to exam the coverage.
If the detected spines are underfilled, increment the detector sensitivity by 5%to 10%and click Detect All"again to replace them at a higher sensitivity level. If overfilled spines are detected, reduce the detector sensitivity by 5%to 10%and select Detect All"once more to update the spine detection with a lower sensitivity. Then, left-click Keep Existing Spines"in the spine tab of the 3D environment.
If the option, Click image to detect all on nearest branch"is active, make sure to deselect it. To focus on a specific dendrite for further spine analysis, left-click Move Pivot Point"and then click on the desired dendrite to set the pivot point. Once the pivot point is set, deselect Move Pivot Point"Identify a dendritic spine that appears unfilled.
Increase the detector sensitivity by 10%to 20%beyond the previous setting, and left-click directly on the spine. If the spine does not populate, the message, Unable to detect a spine at the selected location"will appear. Increase the detector sensitivity incrementally, potentially exceeding 100%until a spine is detected and adequately filled.
To correct an underfilled spine, go to the edit tab and left-click on it. Choose Remove"then deselect the edit tab. Increase the sensitivity by 5%to 10%and left-click on the spine again.
For an overfilled spine, navigate to the edit tab and select it. Click Remove"then deselect the edit tab. Reduce the sensitivity by 5%to 10%and left-click on the spine.
Inspect the dendrite for any spines that may belong to neighboring dendrites, false positives, or dendritic segments incorrectly identified as spines. Utilize Remove"to delete these inaccuracies. Then, access the spines identified on the dendrite.
In cases where multiple spines might appear merged into a single conglomerate, access the edit tab, select the conglomerate spine, and click Hide Selection"to evaluate. If confirmed, within the edit tab, choose Show Selection"and then split to separate the spines. With all spines visually identified and correctly depicted, move to the spine tab and select Classify All"to categorize them into thin, mushroom, stubby, and filopodia.
After completing all spine detections and classifications, in the top toolbar of the 3D environment window, select Save and View"in Neurolucida Explorer to save the data gathered from the tracings. Within the Explorer window, navigate to the view tab and left-click Select all"to highlight all dendrites and spines. Then, proceed to the analyze tab located in the upper toolbar.
Left-click the structure dropdown menu and select Branch Structure Analysis"to initiate a detailed structural analysis of the highlighted elements. For spine density and average spine volume analysis, select each tree and each dendrite. Then, click Spines"followed by Spine Details"Click Okay"to execute the analysis and the results will be displayed in two distinct windows.
