View Project Fukuhara-2U54NS034194-09

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:	pineal gland	Experiment Detail	false
Organ Region:	adrenal medulla	Sample Detail	false
Cell Type:	pineal gland cultured cells	Hybridization Detail	true
Study Type:	time_series_design	Measurement Detail	false
Disease/Condition:	Anesthetics
Replicates:	0
Expected Samples:

Available Actions

Investigator Contact Detail
Name	Chiaki Fukuhara
Street Address:	Neurosci Inst, Morehouse School of Medicine
	720 Westview Dr SW
City, State/Province:	Atlanta , GA
Zip/Postal Code:	30310-1495
Country:	United States
Work Phone:	404-756-6697
Fax:	404-752-1041
E-mail:	fukuhac@msm.edu

Proposal Detail

Grant:

2U54NS034194-09

Status:

Public

Service Type:

Start to Finish Profiling

IACUC:

02-25

IACUC date:

2002-09-19

Study Relevance:

There is growing appreciation that the feeling of well-being, alterations in mood and susceptibility to a variety of medical disorders depend on the proper expression of the master circadian clock and the synchrony among the other oscillators found in many peripheral tissues. To improve our understanding of the role of peripheral oscillators, an improved understanding of the molecular machinery sub-serving the circadian variation in gene expression is essential. Our long-term goal is to understand the molecular mechanisms that initiate circadian gene expression following external stimulation in the mammalian pineal gland.

Hypothesis:

A failure of NE to initiate circadian rhythms is due to failure of activation of certain "circadian clock" genes. We found that circadian rhythms are initiated by stimulation of cAMP or cGMP analogue in the rat pineal gland, while norepinephrine (NE) stimulation only moderately induce Period1 mRNA (one of "circadian clock" genes) 24 h after start of stimulation. From these results we hypothesize that external stimulation activates some "circadian clock" genes simultaneously, and that failure of circadian rhythm initiation following NE stimulation is due to insufficient "circadian clock" genes activations.

Specific Aim:

Are all or just some of the "circadian clock" genes induced by NE, cAMP, or cGMP? In this project we compare a series of gene expression patterns using DNA microarray in response to cAMP, cGMP and NE stimulation to understand why NE does not initiate circadian rhythms in the rat pineal gland. As a preliminary experiment we will compare gene expression 0, 1, and 4 h after start of chemical stimulation.

Experimental Procedure and Design:

Male rats of wistar strain are kept in 12h-12h light dark cycles at least for one week before start of experiments. Pineal glands are removed from animals and placed in the culture dish. Rat pineal glands are cultured for 3 days before chemical stimulation. On the day of experiment, the pineal cultures are stimulated using one of NE, cAMP and cGMP analogues for 1 or 4 h before harvest. The samples are immediately frozen and total RNA are extracted from each group which are from a pool of 8 pineal glands using Trizol reagent (Invitrogen). Concentrations of total RNA are determined using spectrophotometer, and six microgram of total RNA is aliquoted in each sample tube for further analysis. Since we already have Affymetrix Rat Genome U34A array GeneChips, we would like to send Chips as well as our total RNA samples.

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	Extracts
Ctrl Pt 1 (none 0h)	rat	1
Exp pt 1 (EtOH 1h)	rat	1
Exp pt 2 (Melatonin 1h)	rat	1
Exp pt 3 (Melatonin 4h)	rat	1

Project Hybridizations

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Name	Array	Labeled Extract	Hybridization Protocol
Hybridization3	Human Genome U133 Plus 2.0 Array_1	sample #2_e1_le1	Affymetrix
Hybridization4	Human Genome U133 Plus 2.0 Array_2	sample #3_e1_le1	Affymetrix
Hybridization5	Human Genome U133 Plus 2.0 Array_3	sample #1_e1_le1	Affymetrix

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