Visualizing Synaptic Degeneration in Adult <em>Drosophila</em> in Association with Neurodegeneration

Abstract

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused on neuromuscular junctions (NMJs) in Drosophila larvae, assessing synaptic integrity in adult Drosophila has received much less attention. Here we provide a straightforward method for dissection of the dorsal longitudinal muscles (DLMs), which are required for flight ability. In addition to flight as a behavioral readout, this dissection allows for the both DLM synapses and muscle tissue to be amenable to structural analysis using fluorescently labeled antibodies for synaptic markers or proteins of interest. This protocol allows for the evaluation of the structural integrity of synapses in adult Drosophila during aging to model the progressive, age-dependent nature of most neurodegenerative diseases.

Genome-wide analysis reveals novel regulators of synaptic maintenance in Drosophila

“…To identify genes required to maintain synaptic integrity, we first screened for a progressive loss of flight ability with age as an initial readout. Our previous work demonstrated a strong link between impaired flight ability and the loss of DLM synaptic integrity ( Sidisky and Babcock 2020 ; Sidisky et al 2021 ), suggesting that this strategy would likely uncover genes associated with synaptic maintenance. We assessed flight ability in 198 DGRP fly stocks at both Day 3 and Day 21.…”

“…We chose Day 3 for our initial measurements to allow the DLM innervation pattern to stabilize following pruning that occurs during metamorphosis ( Hebbar and Fernandes 2005 ). We used Day 21 since our previous findings revealed synaptic defects in a model of amyotrophic lateral sclerosis (ALS) at this time ( Sidisky and Babcock 2020 ). The average landing height for each genotype was scored at both timepoints ( Fig.…”

“…DLMs were dissected as previously described ( Sidisky and Babcock 2020 ). Briefly, preps were fixed in 4% paraformaldehyde for 30 min and washed with 1 × phosphate-buffered saline (PBS) four times.…”

Mayday sustains trans-synaptic BMP signaling required for synaptic maintenance with age

“…Thoraces were then flash frozen with liquid nitrogen and bisected down the midline in ice cold PBS. Tissues were then incubated in blocking buffer (PBS with 0.1% normal goat serum and 0.2% Triton X-100) for at least 1 hr at 4°C ( Sidisky and Babcock, 2020 ). The following primary antibodies were used to treat samples overnight at 4°C unless otherwise specified; rabbit polyclonal anti-DsRed 1:500 (Takara Bio USA Inc), chicken polyclonal anti-GFP 1:500 (ThermoFisher), rabbit monoclonal anti-pMad 1:500 (Abcam ab52903) and rat anti-elav 1:20 (Developmental Studies Hybridoma Bank Elav-9F8A9).…”

Cellular And Small Animal Models Of Neurodegenerative Diseases

“…Sidiski and Babcock present a method to quantify synaptic integrity in an age-dependent manner in the neuromuscular junctions of the dorsal longitudinal muscle (DLM) tissue in Drosophila melanogaster 10 . Importantly, their dissection protocol, which involves the flash freezing of tissue, preserves both the neuronal and muscle tissue and, due to the ease of genetic manipulation of the fly, enables the study of…”