Automated Quantification of Synaptic Fluorescence in C. elegans

Summary

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

Abstract

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.

Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.

The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Protocol

1. Preparation of Worms for Imaging

This segment of the protocol is based on published C. elegans culture techniques^1,2, and is outlined in Figure 1.

1. Grow worms on high peptone NGM agar plates (10 cm) seeded with NA22 E. coli bacteria until nearly starved. If immunostaining, one plate will reliably produce enough worms for 1 individual stain, taking into account losses along the way, and the strict criteria used to select worms for imaging (see Part 2).
2. Harvest worms by pouring a few ml of ddH₂0 onto plate, swirling briefly, and pouring liquid into a 15 ml conica....

Discussion

The method presented here is designed to extract quantitative multi-parameter data for large populations of synapses in C. elegans, while maximizing consistency within treatment groups. Three features contribute to these objectives. First, immunostaining is performed on synchronous worm populations to ensure that all animals are the same age. This step is critical because developmental regulation of expression levels may obscure effects of the experimental treatment (e.g. UNC-49 GABA receptor immunofluorescence .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Volocity v4.0 or higher	PerkinElmer/Improvision		Check your local imaging core facility for access to this software. Demo software is available at the PerkinElmer website. This method requires only the Quantitation module of Volocity.
Table 2. Specific reagents and equipment.
Egg buffer NaCl KCl CaCl2 MgCl2 HEPES (Adjust pH to 7.3) 118 mM 48 mM 2 mM 2 mM 24 mM Alkaline hypochlorite (for 5 ml) Fresh household bleach (discard bottle 30 days after opening) 10 N NaOH ddH2O 1.0 ml 0.25 ml 3.75 ml
Table 1. Solutions.