View Project Greene-5P50NS038399-050001

Project Summary
Status:	Public
Publications:	1 Published
Project Detail		Data Detail
Platform:	Affymetrix	MIAME Areas	Compliance
Species:	Rat	Array Design Detail	true
Organ/Tissue Type:	brain	Experiment Detail	false
Organ Region:	SNc and VTA	Sample Detail	false
Cell Type:	mixed neural cells	Hybridization Detail	true
Study Type:	subclassification	Measurement Detail	true
Disease/Condition:	Parkinson's Disease
Replicates:	8
Expected Samples:

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Investigator Contact Detail
Name	James G Greene
Street Address:	5002 Rollins Research Building
	1510 Clifton Road
City, State/Province:	Atlanta , GA
Zip/Postal Code:	30322
Country:	United States
Work Phone:	404-727-5635
Fax:
E-mail:	jggreen@emory.edu

Proposal Detail

Grant:

5P50NS038399-050001

Status:

Public

Service Type:

Start to Finish Profiling

IACUC:

222-2001Y

IACUC date:

2003-11-19

Study Relevance:

The cardinal clinical features of Parkinson's disease (PD) (rigidity, rest tremor, bradykinesia, and postural instability) result from selective loss of midbrain dopaminergic neurons. More specifically, dopaminergic neurons in the substantia nigra pars compacta (SNc) are much more susceptible to damage than the adjacent dopaminergic neurons in the ventral tegmental area (VTA). This dichotomy is not only seen in human Parkinsons disease, but also in many animal models of PD, including administration of the mitochondrial toxin rotenone to rats, which replicates many of the behavioral and neuropathological features of PD. The factors underlying this selective vulnerability are unknown, but could be related to differences in neuronal circuitry, differences in glial support, or intrinsic differences between the neuronal populations of the two regions. Elucidation of these factors may lead to a greater understanding of the pathogenesis and treatment of Parkinson's disease.

Hypothesis:

There are intrinsic differences in gene expression between dopaminergic neurons in the rat SNc and VTA that result in greater susceptibility of SNc neurons to degeneration in experimental parkinsonism. These differences may be related to dopamine metabolism, oxidative metabolism and stress, protein aggregation, or other unforseen pathways.

Specific Aim:

We will determine gene expression profiles of untreated rat SNc and VTA dopaminergic neurons using laser capture microscopy to obtain region-specific neuronal mRNA.

Experimental Procedure and Design:

We will compare gene expression profiles between SNc and VTA dopaminergic neurons in normal rats. No treatment or time points will be studied in this experiment. Animals will be anesthetized, sacrificed by decapitation, and brains frozen on dry ice. Frozen sections will be collected onto glass microscope slides and rapidly immunostained for tyrosine hydroxylase to identify dopaminergic neurons. SNc and VTA neurons (approx. 200 per sample) will be isolated using laser capture microscopy. Total RNA will be extracted and poly-A RNA will be amplified using a modified Eberwine protocol. aRNA will be sent to the centers for labeling and hybridization to Affymetrix rat U34A arrays. We have confirmed with the center that our aRNA protocol is compatible with the centers amplification protocols; in fact, it is essentially identical. We will be providing a two-round amplification product to the center for labeling and hybridization. We recognize that using RNA after three rounds of amplification may decrease sensitivity for low copy number transcripts, but favor this approach versus pooling our samples (which are inherently paired) at this point. We have discussed this point in detail with the center. SNc and VTA samples from eight animals (16 samples total) will be provided to mitigate differences specific to individual animals. With the assisatnce of the center, paired t-tests will be used to determine differential expression between the two regions. Permutational t-test analysis and/or Benjamini and Hochberg analysis of expression ratios will be used to protect against multiple comparisons. Selected differentially expressed genes will be validated on separate tissue samples using quantitative RT-PCR or in situ hybridization.

Experimental Factors:

Conditions that are tested in the experiment. At least one is required. Experimental factors are the independent variables in the experiment.


Experimental Factors is empty.

Project Samples

Samples associated with this project.

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Name	Description	Bio-Source	Extracts
SNc1	Control rat 1 - substantia ...	BioSource15	1
VTA1	Control rat 1 ventral tegm...	BioSource16	1
SNc2	Control rat 2 substantia n...	BioSource17	1
VTA2	Control rat 2 ventral tegm...	BioSource18	1
SNc3	Control rat 3 substantia n...	BioSource19	1
VTA3	Control rat 3 ventral tegm...	BioSource20	1
SNc4	Control rat 4 substantia n...	BioSource21	1
VTA4	Control rat 4 ventral tegm...	BioSource22	1
SNc5	Control rat 5 substantia n...	BioSource23	1
VTA5	Control rat 5 ventral tegm...	BioSource24	1
SNc6	Control rat 6 substantia n...	BioSource25	1
VTA6	Control rat 6 ventral tegm...	BioSource26	1
SNc7	Control rat 7 substantia n...	BioSource27	1
SNc8	Control rat 8 - substantia ...	BioSource28	1
VTA8	Control rat 8 ventral tegm...	BioSource29	1

Project Hybridizations

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