The protocol studies cerebellar granule neurons across different stages of development in order to visualize key morphological growth. The advantages of the technique include cell specific targeting, fast expression of transfected constructs, and sparse labeling of cells which allows for the study of cell autonomous effects. Start with cutting an 11.2 millimeter segment from a loading pipette tip and place the cut part over the tip of the Hamilton syringe as a spacer to limit the injection depth to 1.5 millimeters.
Secure the spacer on the syringe tip with adhesive or parafilm. To study the morphology of single electroporated CGNs from sagittal brain sections of the experimental pup, take Z-stack images at 0.5 micrometers per stack on a confocal microscope. Image one cell per image window to allow for easy image analysis and 3D reconstruction.
Analyze neurite length and dendritic claw formation in a blinded manner using simple neurite tracer. Upload single channel Z-stack images of electroporated CGNs into Fiji and click on Plugins"Segmentation"and Simple Neurite Tracer"Select Create New 3D Viewer"from the dropdown menu. Scroll to the base of a dendrite connecting to cell soma and start a path by clicking on the junction.
Manually trace the path by clicking through the sections where the cell fill signal is brightest and pressing Y to keep the trace. Trace until the end of the dendrite and confirm the path by pressing F.Alternatively, trace until the base of the claw. Next, trace the claw from the base of the structure until the end of the longest neurite.
Trace secondary and tertiary branches by holding down Control on windows or ALT on a macOS and clicking the path. Confirm the path by pressing F.Observe that measurements for the traces are visible on a separate window. Add up all the sizes of the claw branches to obtain the total length for each claw.
In the representative analysis, projection images of electroporated CGNs from 3 to 14 days post-injection showed a progressive decrease in number of dendrites. CGNs underwent a phase of dendritic growth followed by refinement from 3 DPI to 7 DPI that resulted in the pruning of more than 50%of excess dendrites. This event coincides with the gradual lengthening of the remaining arbors in the formation of claw-like structures at the end of each dendrite indicating that these developmental processes are happening concurrently.
By 7 DPI, claws were found on roughly 75%of dendrites. Each labeled CGN was reconstructed in NMRS to quantify the total somato-dendritic surface area and volume. No significant difference in CGN size was observed across development.
Though at 7 DPI, CGNs exhibited a significant 20%decrease in volume compared to 3, 5, and 10 DPI. The most important thing in the procedure is to accurately locate the cerebellum before the injection. The method can be adapted to genetically manipulate genes in vivo to study their role in granule neuron development by transfection of either shRNAs, siRNAs, or Cre Recombinase.
