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 of experimental advantages to study the mechanisms of neurodegenerative diseases, including a short life cycle, large number of progeny, and the availability of well-established and sometimes unique genetic and molecular methods^1-12. Furthermore, these organisms are amenable to unbiased interaction screens that can identify factors contributing to these diseases by their aggravating or ameliorating effects on neurodegenerative phenotypes.

Analyzing such genetic interactions and assessing aging effects requires quantitative protocols to detect neurodegeneration and to measure its severity. This assessment can be done relatively easily when measuring behavioral aspects in Drosophila, such as olfactory learning, negative geotaxis, or fast phototaxis, which provide a numeric performance value^13-21. It is also possible to determine the effects on neuronal survival by counting neurons. However, this is only possible when focusing on a specific population that is clearly identifiable, like the dopaminergic neurons that are affected in PD, and even then, the results have been controversial^22-24.

The protocol described here uses the collar method to perform paraffin serial sections, a method that was originally developed by Heisenberg and Böhl, who used it to isolate anatomical brain mutants in Drosophila²⁵. The use of the collar method has subsequently been adapted, including in cryosections, vibratome sections, and plastic sections^26-28. Here, this method is employed to obtain serial sections of the entire fly head, which can then be used to measure the vacuoles that develop in flies with neurodegenerative phenotypes^16,21,29-32. These measurements can be done in specific brain areas or can cover the entire brain; the latter approach allows one to identify even weak degenerative phenotypes, as observed during aging. Finally, when using the collars, up to 20 flies can be processed as one preparation, which is not only less time-consuming, but also allows for the analysis of control and experimental flies in the same preparation, minimizing artifacts due to slight changes in the preparation.

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 with CO₂ or ether.
3. Thread flies (up to 20 with most collars) by their necks into the collars using forceps. Remember to align all of the heads in the same orientation, as seen in Figure 1A, and be gentle to ensure that no damage occurs to the head or eyes.
4. Include sine oculis flies (arrows, Figure 1A) at random positions so that the order of the flies can be easily identified in the sections. In addition, if the experimental flies have a light or white eye color, thread some red-eyed flies, such as wild type, between them to ensure that sufficient pigment is present to stain the slide. Record the order of the flies on a protocol sheet together with the collar number if using more than one collar.
5. Once a collar has been finished, place it in the prepared Carnoy solution for 3.5 - 4 h.
6. Dump out the Carnoy solution into the appropriate disposal canister and begin the ethanol washes. Make sure to pour slowly so as not to disturb the placement of the collars in the container.
7. Wash the collars for 30 min in 99% ethanol twice.
8. Wash the collars in 100% ethanol for 1 h. Be sure to change the washes on time to prevent overdehydration.
9. Put the collars in methylbenzoate O/N at RT. Seal the container with parafilm to prevent the evaporation of the methylbenzoate.
10. Pour the methylbenozate into the proper disposable container in the fume hood. Add a previously-prepared mixture of 1:1 low melting point (56 - 57 °C) paraffin wax and methylbenzoate for 1h. From this point on, the collars need to be kept in an incubator at 65 °C to make sure that the paraffin does not harden.
11. Pour out the methylbenozate and paraffin mixture into the proper disposable container, and pour molten pure paraffin wax, kept at 65 °C, onto the collars.
12. Change the paraffin after 30 min and repeat this at least 5 times. At least 6 - 8 washes should be performed.
13. Once the washes are complete, place the collars into a rubber ice cube tray with slots approximately the size of the collars. Pour molten paraffin over them until completely covered and allow it to harden O/N (try to avoid air bubbles).
14. Remove the paraffin blocks containing the collars from the ice cube tray. Separate the paraffin block from the collar using a razor blade, gently breaking off the collar. The heads will be in the paraffin block while the bodies will stay in the collar. The blocks can be kept at room temperature.
15. To clean the collars, soak them in a deparafinization agent at 65 °C to remove the paraffin, clean with light scrubbing, and wash in ethanol before reusing.

2. Sectioning and Mounting

1. Warm a heating plate to 50 °C. Place the object holders (or metal mounting blocks) and razor blades on the plate and let them warm up.
2. Depending on the desired orientation for sectioning, attach the paraffin block either with the heads towards the side (for horizontal sections) or facing upwards (for frontal sections) to a heated mounting block (briefly melting the block at the contact side). Remove the block from the heating plate and allow it to cool for at least 10 min to ensure that the paraffin is hardened enough for a proper seal onto the mounting block. Keep the row of heads aligned in parallel with the surface of the block as much as possible to prevent uneven sections.
3. Take a razor blade and trim the excess paraffin away from the fly heads so that only a small row with the embedded heads remains (the razor blade can be warmed up for easier trimming). Make sure not to trim too much so that the paraffin does not break during sectioning (more trimming can be done during sectioning).
4. Place the mounting block into the object holder of the microtome and ensure that the alignment of the row of heads is as parallel as possible to the edge of the blade.
5. Prepare microscope slides by covering them with a thin layer of poly-L-Lysine (PLL) solution and let them dry for 5 min. Cover them with water shortly before use.
6. Cut 7 µm sections and transfer the ribbon of sections to the slide floating on the water.
   NOTE: To obtain the entire brain for horizontal sections, we collect the ribbon from when starting to cut into the eye until the head has been completely cut (cutting from the proboscis into the brain). More than one slide may be needed for the entire head.
7. Place the slide on a heat plate at 37 °C and allow the ribbon to expand for about 1 min.
8. Remove excess water (by pouring it off or using a tissue) and dry the slides O/N.
9. Remove the paraffin wax from the slide by placing the slides in a tall, vertical slide-staining jar filled with a deparafinization agent (completely covering the sections). Perform 3 washes of 30 min - 60 min each.
10. Remove the slide from the final wash. Place 2 drops of embedding media onto the slide and cover it with a large coverslip.

3. Photographing and Analyzing the Sections

1. Allow the prepared slides to dry for 1 - 2 d. Then, examine them on a fluorescence microscope under blue light.
2. Use a lower magnification to determine the orientation of the flies and to find the region of interest if focusing on a specific region.
   NOTE: For the sws flies (see Figure 2), we find the section that contains the great commissure and take an image (usually at 40X magnification). When analyzing the entire brain (as in Figure 3), we scroll through all the sections from a head and either photograph the section with the most severe phenotype or all sections that show vacuoles.
3. For a double-blind analysis, take and number images without knowing the genotype and record the collar number and position of the head in the row to identify them later.
4. Once the images have been taken, analyze them using an imaging software.
5. Count the number of vacuoles per section or per head. To measure the vacuole size, open the images in a software program and select the vacuoles with a selection tool. Determine the amount of pixels in the selected vacuoles.
6. For conversion to µm², take a photo of a stage micrometer calibration slide at the magnification used for acquiring photos. Determine the amount of pixels in 100 µm² to calculate a conversion factor.
7. Convert the total pixel number into µm² by dividing the number of pixels by the conversion factor calculated in the above step.

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