A protocol to detect neurodegeneration in Drosophila melanogaster whole-brain mounts using advanced microscopy

Abstract

Drosophila melanogaster is an excellent model organism to study neurodegeneration. Assessing evident neurodegeneration within the fly brain involves the laborious preparation of thin-sectioned H&E-stained heads to visualize brain vacuole degeneration. Here, we present an advanced microscopy-based protocol, without the need for sectioning, to detect vacuole degeneration within whole fly brains by applying commonly used stains to reveal the brain parenchyma. This approach preserves the whole-brain architecture and enables rapid, reproducible, and quantitative analyses of vacuole-like degeneration associated with specific brain regions. For complete details on the use and execution of this protocol, please refer to Behnke et al. (2021).

Keywords: Microscopy; Model Organisms; Neuroscience.

Graphical abstract

Figure 1

Organization of Drosophila whole-brain mounts (A) Brains are positioned in a uniform fashion at 45° to maximize the field of view for microscopy and minimize space on the microscope slide. Alternatively, for mispositioned brains, most microscope imaging software have a rotation function that can be used to fully capture the entire brain’s field of view. Scale bar = 1 mm. (B) Cartoon schematic of physiologically normal holes. For additional reference, please refer to Virtual Fly Brain Resource (Milyaev, Osumi-Sutherland et al. 2011):https://v2.virtualflybrain.org/org.geppetto.frontend/geppetto?id=FBbt_00045046&i=VFB_00101567

Figure 2

Detecting neurodegeneration in Drosophila whole-brain mounts (A) Representative max projection of whole-brain mount stained with DAPI (blue) and phalloidin (magenta) and imaged using two-photon microscopy. White scale bar =100 μm. (B) Representative montage of Z-stack showing range of acquired brain slices. White scale bar =100 μm. (C) Validation of vacuoles showing representative brain slice from young (3–5 day old) nSyb-GAL4, aged (5-week-old) nSyb-GAL4 and nSyb>hTau flies. Green outline corresponds to physiologically normal holes. Yellow outline corresponds to pathological degenerative vacuoles. Yellow scale bar =50 μm. (D) Shaded region from (C) showing representative vacuoles (yellow arrows) and physiologically normal hole (green arrow). Gray scale bar =24 μm. Vacuoles correspond to loss of neuropil. Co-labeling with neuropil-specific marker, discs large (magenta; DLG, a post-synaptic density 95 homolog) reveals region devoid of DAPI and phalloidin (gray) is found within region of neuropil. (E and F) Boxplot quantification of (E) vacuole number and (F) area from young and aged nSyb-GAL4 and young and aged nSyb>hTau flies. Mann–Whitney U test between young and aged groups and between aged nSyb-GAL4 and nSyb>hTau, with Bonferroni correction. Boxplots in (E and F) contain individually plotted values with whiskers corresponding to the maximum 1.5 interquartile range.

Figure 3

Comparison of vacuole imaging using confocal and two-photon microscopy (A–C) Anterior and (A′–C′) middle regions of the same representative brain imaged under (A, A′) 5% confocal laser power, (B, B′) 5%–10% z-corrected confocal laser power, and (C,C′) 3% two-photon laser power. Whole-brain mounts stained with DAPI to detect nuclei and phalloidin to detect actin (brain parenchyma). Yellow arrows indicate the presence of vacuoles. Scale bar = 100 μm.