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

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

1. Perform Drosophila open book preparations of third instar wandering larvae (L3), as previously described.
2. Co-immunolabel Drosophila NMJ terminals using a combination of two markers: Dlg-1 or Hrp together with Brp for analysis with "Drosophila NMJ Morphometrics", and Syt or Csp together with Brp for analysis with "Dr....

Results

Figure 1: Examples of Inappropriate Macro Segmentation Results. Result images after running "Drosophila NMJ Morphometrics" or "Drosophila NMJ Bouton Morphometrics". Parts o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
<strong>Immunostaining</strong>			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
<strong>Equipment</strong>
Confocal microscope or fluorescence microscope	Leica SP5
Zeiss Axio imager
Computer	Mac or Pc
<strong>Software</strong>
FIJI