Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Summary

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

Abstract

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's ⁵.

Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human α-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function ². Accordingly, this model has been successfully used to identify potential therapeutic targets in PD ⁸.

Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line ^{3, 10}.

Introduction

The specificity of the assays described here relies on the use of a Gal4 fly line which exploits the regulatory sequences of the tyrosine hydroxylase gene to achieve specific expression in dopaminergic neurons ³. Tyrosine hydroxylase catalyses the first and rate-limiting step in dopamine synthesis. TH immunoreactivity in the adult fly brain overlaps with that of dopamine, making TH a good candidate to identify DA neurons in vivo (see for example ⁶). Moreover, the expression pattern of THGal4 is more specific than that of other Gal4 lines such as DdcGal4, which contains regulatory sequences from the dopa decarboxylase gene and drives tran....

Protocol

1. Startle-induced Negative Geotaxis Assay

1. Select flies of the desired genotype, separate them by gender, randomly group them in cohorts of 20-30 animals and flip them into new food vials every 2-3 days; test each set of flies (i.e. age-matched cohorts, one cohort/genotype) at least once a week.
2. Thirty minutes before the negative geotaxis test, anesthetize the flies with CO₂ and place them in pre-labeled clear plastic tubes made with two empty food vials (see Materials Table) joined at their openings with clear tape (chamber dimensions: 19 cm x 2.85 cm). In our assays we typically use 25 flies/tube.
   

Results

We have used the assays described here to study the role of the stress protective Nrf2 pathway in a Drosophila model of PD ¹⁴. This model relies on the expression of the human α-synuclein gene in DA neurons using a TH Gal4 driver ^{2, 10}.

Figure 1 shows a representative result of a startle-induced negative geotaxis (climbing) assay in male flies expressing different transgenes under the control of the TH Gal4 driver. All the genotypes tested exhibi.......

Discussion

The assays described here provide a useful approach to study the role of specific genes, signaling pathways or small compounds in the maintenance of DA neurons in aging as well as in different disease-linked genetic backgrounds (reviewed in ⁵).

The startle-induced negative geotaxis behavior of Drosophila has been extensively used as a functional read-out for the functionality of DA neurons in different genetic backgrounds, particularly in the presence of PD-linked mutation.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Rabbit anti-tyrosine hydroxylase antibody	Millipore	AB152
Donkey TRITC-labeled anti rabbit IgG antibody	Jackson ImmunoResearch	711-026-152
Mowiol	Calbiochem	475904
DABCO	Sigma	D2522
Equipment
Polystyrene vials	VWR	89092-734	28.5 x 95 mm
Digital camera	Canon	PowerShot A3100IS (model)	12.1 Megapixels 4X Optical Zoom
Glass coverslips no. 1.5	VWR	48366 205 48393 252	Sizes: 18 x 18 mm, 24 x 60 mm
Glass coverslips no. 2	VWR	48368-040	Size: 18 x 18 mm
Dissection dishes	Electron Microscopy Sciences	70543-01	Size: 30 mm O.D.
Dumostar No. 5 tweezers	Electron Microscopy Sciences	72705-01
Stereomicroscope	Carl Zeiss	Stemi 2000 (model)
Confocal microscope	Leica	SP2 or SP5 (model)
Materials Table.