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.

1. Startle-induced Negative Geotaxis Assay
<ol>
 <li>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.</li>
 <li>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. 
 Note. The recovery time from anesthesia can be varied from few minutes to several hours (e.g. O/N) and should be optimized for the specific genotypes tested.</li>
 <li>Mark different heights (from 1 to 20 cm) on a piece of blotting paper and tape the paper on a vertical surface, perpendicular to the bench.</li>
 <li>Place the tubes in front of the paper: the tubes should be all aligned and as close as possible to the paper to allow accurate height measurement.</li>
 <li>Place the digital camera (see the "Special Equipment" Table) in front of the tubes, at a distance of 20-30 cm from the paper, on a stand (e.g. a pipette tip box), select the "movie" option and start recording; at this time you should also start a timer to keep track of the time between consecutive trials.</li>
 <li>Allow the flies to collect at the bottom of the tube by tapping the tube for few seconds (e.g. 10 sec). This step should be performed consistently (i.e. same duration, same strength, same operator) throughout the whole experiment.</li>
 <li>Record flies' movements with the digital camera for 10 sec.</li>
 <li>Repeat steps 1.5-1.7 fourteen times at one-min intervals.</li>
 <li>Analyze data:
 <ol type="a">
 <li>Count the number of flies that are above the 2 cm mark after 10 sec by visual inspection of the recorded movies. 
 Note: In our assay conditions the flies' negative geotaxis behavior didn't last beyond the first 10 sec after startling (i.e. flies started moving in all directions 10 sec after startling them). The minimum distance climbed by control flies (i.e. 1 week old flies expressing the THGal4 driver, see legend of Figure 1) in this time window was 2 cm. Thus, we used the 2 cm mark as a reference for analyzing the climbing activities of flies of different ages and genotypes 10 sec after initiating the behavior. These parameters may require adjusting to account for differences in behavior of the flies tested (due for example to different genetic backgrounds or laboratory conditions). Notice that in our assay conditions, all the genotypes tested performed worse or similarly to the flies of the control genotype.</li>
 <li>Take the average of the results from the fifteen trials.</li>
 <li>Express average as a percentage of the total number of flies in the tube (= % climbing activity); the number of repeats (N) is given by the number of independent cohorts tested for each genotype</li>
 <li>Assess statistical significance between different genotypes over time. This can be accomplished with a Student's t test (when comparing two genotypes) or a 2-way ANOVA analysis followed by Bonferroni post-hoc test (when performing multiple comparisons) using commercial softwares such as GraphPad (GraphPad Software, Inc.La Jolla, CA, USA).</li>
 </ol>
 </li>
</ol>
Note
We perform all our assays at the same time of the day, at RT and under the same light conditions. Keeping the flies in a 12 hr light/dark cycle environment is advisable to control for the effect of circadian rhythms on the flies' locomotor behavior.
2. Tyrosine Hydroxylase Immunofluorescence Assay of Whole Brain Mounts
Solutions and buffers
Fixative solution: 4% Paraformaldehyde (PFA) in phosphate buffered saline (PBS)
Washing buffer: 0.1% Triton X-100 in PBS
Blocking buffer: 0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.1% Triton X-100 and 0.5% BSA
Mounting media: Mowiol-Dabco (see protocol described in Harlow E, Lane D., Antibodies- A laboratory manual, Cold Spring Harbor Laboratories Press, Cold Spring Harbor, NY, USA, 10:418 (1988))
Procedure
<ol>
 <li>Put flies in a dissection dish filled with 70% EtOH for about 1 min to remove the cuticle wax.</li>
 <li>Transfer flies to another dissection dish filled with ice-cold PBS.</li>
 <li>Dissect the brain:
 <ol type="a">
 <li>Place the fly with the ventral side up (optional: remove wings and legs).</li>
 <li>Pull the head away from the body: use one set of forceps to hold on to the body and another one to hold on to the cuticle under the eye to prevent damaging the brain</li>
 <li>Remove the proboscis.</li>
 <li>Hold on to the head cuticle on opposite sides of the slit left open after removing the proboscis and pull in opposite directions until the brain is removed from the head capsule.</li>
 <li>Remove all cuticle from the brain.</li>
 <li>Remove all the air-filled tracheal tissue; this will prevent the brain from floating during the following incubation steps.</li>
 </ol>
 </li>
 <li>Cut a few millimeters off the tip of a P-200 pipette tip and equilibrate it with a 0.1% Triton X-100/PBS solution</li>
 <li>Using the prepared P-200 pipette tip, transfer the brains to a 0.5 ml Eppendorf tube filled with 0.5 ml of 4% PFA/PBS and keep on ice until the dissection is complete.</li>
 <li>Fix the brains at RT for 20 min: apply a gentle rotation by putting the Eppendorf tubes on a rack and placing the rack on a nutator.</li>
 <li>Remove the fixative using a P-1000 micro pipette, add 0.5 ml of 0.1% Triton X-100/PBS, mix by inversion and let incubate for 1 min; repeat this washing step once.</li>
 <li>Remove the buffer from the second wash, add 0.5 ml of 0.1% Triton X-100/PBS and let incubate at RT for 20 min; repeat this step twice.</li>
 <li>Remove the washing buffer and let the brains incubate in blocking buffer (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.1% Triton X-100 and 0.5% BSA) for 1 hr at RT.</li>
 <li>Incubate the brains with a 1:100 dilution of anti-TH antibody (see the "Table of specific reagents") in blocking solution, for two days, at 4 &deg;C, with gentle rotation (see step 2.6.).</li>
 <li>Repeat washing steps 2.7. and 2.8..</li>
 <li>Equilibrate the brains with a 1:200 dilution of secondary antibodyin blocking solution, for two days, at 4 &deg;C, with gentle rotation.</li>
 <li>Repeat washing steps 2.7. and 2.8..</li>
 <li>Prepare the mounting slides as illustrated in Figure 2A:
 <ol type="a">
 <li>Add 2 X 25 &mu;l drops of mounting media to a cover glass (24 x 60 mm, no. 1.5).</li>
 <li>Place two cover glasses (size 18 x 18 mm, no. 2) on the mounting media leaving a gap in the middle of the slide and let dry.</li>
 </ol>
 Note. Check the sample holder on the stage of your confocal microscope. You can make the cover glass the same size of a 25 x 75 mm slide by attaching a 22 x 22 mm 1.5 cover glass to one of the ends (use nail polish and tape).</li>
 <li>Remove the washing buffer, add 50 &mu;l of mounting media and allow the brains to equilibrate with it by pipetting up and down.</li>
 <li>Using a cut P-200 pipette tip (see step 2.4) transfer the brains to the slide in the space between the two coverslips and cover them with a coverslip no. 1.5 (see Figure 2A; to orient the brains on the slide see 7). 
Note: the samples are mounted between two cover glasses to allow visualization of both the anterior and the posterior side of the brain.</li>
 <li>Fill the whole space between the cover glasses with mounting media; pipette from one corner to remove all the air; let dry and seal with nail polish.</li>
 <li>To assess the number of dopaminergic neurons in a specific cluster acquire a series of optical sections along the z-axis with a 1.0 &mu;m step-size using a confocal microscope (for the anatomical identification of dopaminergic clusters in the Drosophila adult brain see 7).</li>
</ol>

<ol>
	<li>Bayersdorfer, F., Voigt, A., Schneuwly, J., Botella, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine-dependent+neurodegeneration+in+Drosophila+models+of+familial+and+sporadic+Parkinson's+disease.">Dopamine-dependent neurodegeneration in Drosophila models of familial and sporadic Parkinson's disease.</a> Neurobiol. Dis. 40, 113-119 (2010).</li><li>Feany, M. B., Bender, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+of+Parkinson's+disease.">A Drosophila model of Parkinson's disease.</a> Nature. 404, 394-398 (2000).</li><li>Friggi-Grelin, F., Coulom, H., Meller, M., Gomez, D., Hirsh, J., Birman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+in+Drosophila+dopaminergic+cells+using+regulatory+sequences+from+tyrosine+hydroxylase.">Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase.</a> J. Neurobiol. 54, 618-627 (2003).</li><li>Gargano, J. W., Martin, I., Bandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Exp. Gerontol. 40, 386-395 (2005).</li><li>Hirth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+in+the+study+of+human+neurodegeneration.">Drosophila melanogaster in the study of human neurodegeneration.</a> CNS Neurol. Disord. Drug Targets. 9, 504-523 (2010).</li><li>Lundell, M., Hirsh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spacial+development+of+serotonin+and+dopamine+neurons+in+the+Drosophila+CNS.">Temporal and spacial development of serotonin and dopamine neurons in the Drosophila CNS.</a> Dev. Biol. 165, 385-396 (1994).</li><li>Mao, Z., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eight+different+types+of+dopaminergic+neurons+innervate+the+Drosophila+mushroom+body+neuropil:+anatomical+and+physiological+heterogeneity.">Eight different types of dopaminergic neurons innervate the Drosophila mushroom body neuropil: anatomical and physiological heterogeneity.</a> Front Neural Circuits. 3, 5 (2009).</li><li>Mizuno, H., Fujikake, N., Wada, K., Nagai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-Synuclein+Transgenic+Drosophila+As+a+Model+of+Parkinson's+Disease+and+Related+Synucleinopathies.">Alpha-Synuclein Transgenic Drosophila As a Model of Parkinson's Disease and Related Synucleinopathies.</a> Parkinsons Dis. 2011, 212706 (2011).</li><li>Neckameyer, W. 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M., Hirth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dopaminergic+system+in+the+aging+brain+of+Drosophila.">The dopaminergic system in the aging brain of Drosophila.</a> Front Neurosci. 4, 205 (2010).</li><li>Whitworth, A. J., Wes, P. D., Pallanck, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+models+pioneer+a+new+approach+to+drug+discovery+for+Parkinson's+disease.">Drosophila models pioneer a new approach to drug discovery for Parkinson's disease.</a> Drug Discov. Today. 11, 119-126 (2006).</li><li>Wu, J. S., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+dissecting+Drosophila+melanogaster+brains+for+live+imaging+or+immunostaining.">A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining.</a> Nat. Protoc. 1, 2110-2115 (2006).</li><li>Barone, M. C., Sykiotis, G. P., Bohmann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+activation+of+Nrf2+signaling+is+sufficient+to+ameliorate+neurodegenerative+phenotypes+in+a+Drosophila+model+of+Parkinson's+disease.">Genetic activation of Nrf2 signaling is sufficient to ameliorate neurodegenerative phenotypes in a Drosophila model of Parkinson's disease.</a> Dis. Model Mech. 4 (5), 701-707 (2011).</li></ol>

We declare that we have no competing financial interests.

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...

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 transgenic expression not only in DA neurons, but also in serotoninergic neurons and subsets of glial cells 3.
Feany and Bender (2000) first observed that pan-neuronal expression of the PD-linked human &alpha;-synuclein gene accelerates the progressive loss of startle-induced negative geotaxis behavior in Drosophila. We have observed similar results using the DA-neuron specific THGal4 line to drive the expressionof an &alpha;-synuclein transgene 10 and used this functional read-out to study the neuroprotective role of the Nrf2 pathway in this Drosophila model of PD 14. The specific assay described here is adapted from 2 and 3.
The Drosophila brain contains more than 100 DA neurons, arranged in at least 5 different clusters (PPL1, PPL2, PAL, PPM1/2, PPM3) which can be visualized in whole brain mounts by confocal microscopy (see for example 11 and 7). It is still controversial whether or not the number of DA neurons in the Drosophila brain declines with age 9, 11; however, age-dependent neurodegeneration is observed in flies where PD-linked genes have been mutated/overexpressed to model human diseases 5. The expression of human &alpha;-synuclein in DA neurons has such an effect and, hence, has been used as a model for PD. Here we describe an assay for DA neurons count in whole brain mounts using TH as a cell marker. This assay has been adapted from 13 and 10.

<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Rabbit anti-tyrosine hydroxylase antibody</td>
			<td>Millipore</td>
			<td>AB152</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Donkey TRITC-labeled anti rabbit IgG antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>711-026-152</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mowiol</td>
			<td>Calbiochem</td>
			<td>475904</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DABCO</td>
			<td>Sigma</td>
			<td>D2522</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Polystyrene vials</td>
			<td>VWR</td>
			<td>89092-734</td>
			<td>28.5 x 95 mm</td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Canon</td>
			<td>PowerShot A3100IS (model)</td>
			<td>12.1 Megapixels 4X Optical Zoom</td>
		</tr>
		<tr>
			<td>Glass coverslips no. 1.5</td>
			<td>VWR</td>
			<td>48366 205 48393 252</td>
			<td>Sizes: 18 x 18 mm, 24 x 60 mm</td>
		</tr>
		<tr>
			<td>Glass coverslips no. 2</td>
			<td>VWR</td>
			<td>48368-040</td>
			<td>Size: 18 x 18 mm</td>
		</tr>
		<tr>
			<td>Dissection dishes</td>
			<td>Electron Microscopy Sciences</td>
			<td>70543-01</td>
			<td>Size: 30 mm O.D.</td>
		</tr>
		<tr>
			<td>Dumostar No. 5 tweezers</td>
			<td>Electron Microscopy Sciences</td>
			<td>72705-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi 2000 (model)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td>SP2 or SP5 (model)</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Materials Table.

assessing neurodegenerative phenotypes in drosophila dopaminergic neurons by climbing assays and whole brain immunostaining

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 &alpha;-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.

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 &alpha;-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 ...

Watch this Scientific Journal Video about Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining at JoVE.com

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

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.

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

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

assessing-neurodegenerative-phenotypes-drosophila-dopaminergic

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19.0K Views.  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.

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

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Olfactory Assays for Mouse Models of Neurodegenerative Disease

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson&#8217;s disease as well as in testing potential disease-modifying therapies.

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.