Visualizing the Synaptogenesis of Cerebellar Granule Neurons at Different Developmental Stages

Begin with pre-fixed brain tissue slices collected from early and late postnatal stages of mice. 

The slice contains cerebellar granule neurons, or CGNs, expressing a fluorescent protein.

Using a confocal microscope, focus on a single fluorescent CGN.

Capture z-stack images at varying depths to create a three-dimensional view of the neuron.

Using imaging software, trace the neurons' neurites or long projections, including dendrites and axons.

Measure dendrite length from the cell body to its tip.

Analyze dendritic claw formation, specialized structures at dendrite tips where signals from other neurons are received.

Also, assess the neuron's surface area and volume.

In the early postnatal stage, neurons show an increased number of dendrites, with no claw formation. 

Meanwhile, in the late postnatal stage, there are fewer dendrites, but their lengths and the number of claws increase.

Thus, with age, CGNs undergo synaptogenesis and form connections with other cells for signal transmission.

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 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 bath 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 Mac OS 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.