Mass Histology to Quantify Neurodegeneration in Drosophila

Summary

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

Abstract

Progressive neurodegenerative diseases like Alzheimer's disease (AD) or Parkinson's disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the 'collar' method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Introduction

With the increase in life expectancy, neurodegenerative diseases like Alzheimer's or Parkinson's have become an increasing health threat for the general population. According to the National Institutes of Health, 115 million people worldwide are predicted to be affected by dementia in 2050. Although significant progress has been made in identifying genes and risk factors involved in at least some of these diseases, for many of them, the underlying molecular mechanisms are still unknown or not well understood.

Simple invertebrate model organisms like Caenorhabditis elegans and Drosophila melanogaster offer a variety....

Protocol

1. Fixing the Head on Collars and Embedding in Paraffin

Note: All of the steps in the fixation process should be done in a fume hood. Methylbenzoate, while not posing a health risk, has a highly distinct odor, which can be overwhelming if not handled in a fume hood.

1. Before anesthetizing the flies, make up 50 mL of Carnoy solution by adding 15 mL of chloroform and 5 mL of glacial acetic acid to 30 mL of 99% ethanol (do not mix the chloroform and acetic acid). Pour it in a glass container with a flat bottom, such as a crystalizing dish, to ensure that the collars can lay flat and are completely covered by the solution.
2. Anesthetize the flies ....

Results

Using the described method results in serial sections stained by the eye pigment³³ that encompass the entire fly head. A part of this is shown in Figure 1B, where the sections from an individual head are shown from top to bottom. The sections from different flies are seen left to right in this example. To facilitate orientation and identification of the flies, an eyeless fly (sine oculis) is inserted as a marker at position 3 (arrow, Figure 1B<.......

Discussion

The described method provides a means to quantify neurodegeneration in the brain of Drosophila. While other methods, like counting a specific cell type, can be used to identify neurodegeneration, the advantage of this method is that it can be applied more generally. Counting cells requires that these cells can be reliably identified using either a specific antibody or the expression of a cell-specific marker, which is not always available. Furthermore, it has been shown that dramatically different results can be.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Name of the Reagent/Equipment	Company	Catalog Number
Collar	Genesee Scientific	TS 48-100	We are using custom made collars that are made from one piece of metal instead of layers as the ones by Genesee. A description on how to make collars can be found at http://flybrain.neurobio.arizona.edu/Flybrain/html/atlas/fluorescent/index.html
Rubber ice cube tray for embedding	Household store		The size can be made-to-fit by glueing in additional walls
Crystallizing dish	Fisher Scientific company	08-762-3
Ether	Fisher Scientific Company	E138-1
Ethanol	Decon Laboratories Inc.	2701
Choloroform	Fisher Scientific Company	C298-500
Glacial Acetic Acid	Fisher Scientific Company	A38-212
Methylbenzoate	Fisher Scientific Company	M205-500	Distinct Odor - Use in fume hood!
Low Melting Point Paraffin Wax	Fisher Scientific Company	T565	Make sure to keep extra melted in a 65°C waterbath
Microtome	Leica Biosystems	Reichert Jung 2040 Autocut
Microscope Slide	Fisher Scientific Company	12-550D
Microscope Cover Glass	Fisher Scientific Company	12-545-M
SafeClear	Fisher Scientific Company	314-629	Three different containers for washes
Vertical Staining Jar with Cover	Ted Pella Inc.	432-1
Permount	Fisher Scientific Company	SP15-500
Poly-L-Lysine Solution (PLL)	Sigma Life Science	P8290-500