Drosophila Neuromuscular Junction (NMJ) Quantification: A Method to Assess Synaptic Morphology and Function

Published: April 30, 2023

Abstract

Source: Castells-Nobau, A., et al. Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology. J. Vis. Exp. (2017).

The Drosophila neuromuscular junction (NMJ) is used as a model to study the morphology and function of synapses. This video describes important features that researchers quantify to characterize the NMJ. The example protocol demonstrates a software able to perform the quantification automatically.

Protocol

This protocol is an excerpt from Castells-Nobau et al., Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology, J. Vis. Exp. (2017). 1. Requirements Prior to Image Processing Perform Drosophila open book preparations of third instar wandering larvae (L3), as previously described. Co-immunolabel Drosophila NMJ terminals using a com…

Representative Results

Figure 1: Examples of Inappropriate Macro Segmentation Results. Result images after running "Drosophila NMJ Morphometrics" or "Drosophila NMJ Bouton Morphometrics". Parts of the synaptic terminal are not included in the yellow outline (A). Parts of the background are included in the synaptic terminal by the yellow outline (B). Blue skeleton line exte…

Materials

Immunostaining Dilution
Mouse anti-discs large 1 Developmental Studies Hybridoma Bank AFFN-DLG1-4D6 1/25 (conjungated using the Zenon Alexa Fluor 528 Labeling Kit)
Rabbit anti-horseradish peroxidase Jackson IR 323-005-021 1/500
Rabbit anti-Synaptotagmin Gift from Hugo Bellen Jan-00
Mouse anti-Cysteine string protein Developmental Studies Hybridoma Bank DCSP-1(ab49) 1/10 (conjungated using the Zenon Alexa Fluor 528 Labeling Kit)
Mouse anti-Bruchpilot Developmental Studies Hybridoma Bank nc82 Jan-50
Goat anti-mouse Alexa Fluor 488 Life technologies A11029 1/200
Goat anti-rabbit Alexa Fluor 568 Life technologies A11011 1/500
Zenon Alexa Fluor 568 Mouse IgG1 Labeling Kit ThermoFisher Z25006
ProLong Gold Antifade Mountant ThermoFisher P36930
Equipment
Confocal microscope or fluorescence microscope Leica SP5
Zeiss Axio imager
Computer Mac or Pc
Software
FIJI

Play Video

Diesen Artikel zitieren
Drosophila Neuromuscular Junction (NMJ) Quantification: A Method to Assess Synaptic Morphology and Function. J. Vis. Exp. (Pending Publication), e20107, doi: (2023).

View Video