To begin, take a slide and add two thin strips of double-sided adhesive tape to it to prepare for imaging. Pipette approximately 10 to 15 microliters of mounting medium between the two strips of tape for brain mounting. transfer the label drosophila brains onto a microscope slide with a P20 pipette tip prerinsed with PBS and 0.2%Triton X-100.
Once the brains are transferred, use a fine paintbrush to align them, ensuring the antennal lobes face upwards. After orienting the brains, cover them with a glass coverslip. Then, fill in the sides of the coverslip with additional mounting medium.
Seal the edges of the cover slip with clear nail polish. After drying the slide thoroughly, store it in the refrigerator for subsequent imaging. Use a laser scanning confocal microscope equipped with a 63X oil immersion objective for imaging.
Employ an argon 488 and helium neon 543 laser for imaging the an antennal lobe's synaptic glomeruli and Or42a olfactory sensory neurons. Determine the optical gain and offset for both channels. Position the imaging to the center of the brain and focus on the VM7 glomeruli.
Locate approximately to the hole in the middle of the brain. Select the imaging resolution of 1024 by 1024. Capture an entire confocal Z stack projection through the antennal lobe to ensure the full Or42a neuron innervation of the VM7 glomeruli is captured.
Load the genotype or condition blinded image into Fiji. To split the laser line channels, go to image, click color, and select split channels. In the Or42a olfactory sensory channel, scroll through the Z stack to determine which slices contain the Or42a neuron innervation, identifying the beginning and end of fluorescence.
To create a sum slices projection that includes only slices with Or42a neuron innervation, click image and go to stacks. Then, click Z project, select sum slices, and enter the desired range. Use the lasso tool in Fiji to trace the outline of the Or42a neuron innervation in the VM7 glomerulus.
Multiply the circumference of the traced area by the number of Z stack slices and the thickness of each slice to calculate the VM7 synaptic glomerulus innervation volume. After 24 hour exposure to a control odorant vehicle, dense Or42a MCD:GFP innervation persists in VM7 glomeruli of both antennal lobes. In contrast, a 24 hour exposure to 25%EB odorant causes significant pruning and loss of synaptic glomerular volume.
Increasing EB odorant concentration causes progressively greater synaptic glomeruli pruning. Representative quantifications for this range of EB odorant concentrations showed similar results for fluorescence intensity and innervation volume.
