Analyzing Dendritic Morphology Across Drosophila Medulla Layers and Columns

Begin with a glycerol-cleared Drosophila brain in antifade mounting media.

The brain contains medulla neurons arranged in layers, which organize synaptic connections, and columns, which coordinate neural circuits.

A few of these neurons are labeled with green fluorescent markers, contrasting with the red fluorescent photoreceptor axons.

Position the brain ventral side up. Place a coverslip with small clay patches on the sides to prevent crushing the brain sample.

Under a microscope, obtain the horizontal view image stack, containing the green-labeled neuron.

Remove the coverslip and reposition the brain, anterior side up.

Capture an image stack of the frontal view of the same neuron.

Using the software, combine these images, remove the photoreceptor channel, and visualize the 3D image of the neuron.

Trace the axon and then the dendrites, following each branch to its termination, to map dendritic organization across medulla layers and columns.

To mount the brain in the horizontal orientation, transfer a glycerol-cleared brain into a 20-microliter drop of anti-fade mounting medium, deposited centrally on the microscope slide. Then, on a coverslip, attach small patches of clay at the four cor