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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

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

Published: April 24th, 2013

DOI:

10.3791/50339

1Department of Biomolecular Genetics, University of Rochester Medical Center

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.

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.

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....

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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 Ta.......

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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 .......

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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.......

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We thank Leo Pallanck for fly stocks, Christine Sommers for technical assistance and Gerasimos P. Sykiotis for helpful discussions. This work was funded by the NIH training grant T32CA009363 to M.C.B.

....

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Name Company Catalog Number Comments
Name Company Catalogue number Comments (optional)
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.

  1. Bayersdorfer, F., Voigt, A., Schneuwly, J., Botella, Dopamine-dependent neurodegeneration in Drosophila models of familial and sporadic Parkinson's disease. Neurobiol. Dis. 40, 113-119 (2010).
  2. Feany, M. B., Bender, W. W. A Drosophila model of Parkinson's disease. Nature. 404, 394-398 (2000).
  3. Friggi-Grelin, F., Coulom, H., Meller, M., Gomez, D., Hirsh, J., Birman, S. Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase. J. Neurobiol. 54, 618-627 (2003).
  4. Gargano, J. W., Martin, I., Bandari, P., Grotewiel, M. S. Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila. Exp. Gerontol. 40, 386-395 (2005).
  5. Hirth, F. Drosophila melanogaster in the study of human neurodegeneration. CNS Neurol. Disord. Drug Targets. 9, 504-523 (2010).
  6. Lundell, M., Hirsh, J. Temporal and spacial development of serotonin and dopamine neurons in the Drosophila CNS. Dev. Biol. 165, 385-396 (1994).
  7. Mao, Z., Davis, R. L. Eight different types of dopaminergic neurons innervate the Drosophila mushroom body neuropil: anatomical and physiological heterogeneity. Front Neural Circuits. 3, 5 (2009).
  8. Mizuno, H., Fujikake, N., Wada, K., Nagai, Y. Alpha-Synuclein Transgenic Drosophila As a Model of Parkinson's Disease and Related Synucleinopathies. Parkinsons Dis. 2011, 212706 (2011).
  9. Neckameyer, W. S., Woodrome, S., Holt, B., Mayer, A. Dopamine and senescence in Drosophila melanogaster. Neurobiol. Aging. 21, 145-152 (2000).
  10. Trinh, K., Moore, K., et al. Induction of the phase II detoxification pathway suppresses neuron loss in Drosophila models of Parkinson's disease. J. Neurosci. 28, 465-472 (2008).
  11. White, K. E., Humphrey, D. M., Hirth, F. The dopaminergic system in the aging brain of Drosophila. Front Neurosci. 4, 205 (2010).
  12. Whitworth, A. J., Wes, P. D., Pallanck, L. J. Drosophila models pioneer a new approach to drug discovery for Parkinson's disease. Drug Discov. Today. 11, 119-126 (2006).
  13. Wu, J. S., Luo, L. A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining. Nat. Protoc. 1, 2110-2115 (2006).
  14. Barone, M. C., Sykiotis, G. P., Bohmann, D. Genetic activation of Nrf2 signaling is sufficient to ameliorate neurodegenerative phenotypes in a Drosophila model of Parkinson's disease. Dis. Model Mech. 4 (5), 701-707 (2011).

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