In the highly heterogeneous central nervous system, the genes of greatest interest are those involved in the unique functions of each neuronal phenotype, and with their dysfunctions related to disease, aging, and injury. The objective of this project is to implement a generalizable stategy to identify these genes.
The first aim i s to develop methods to isolate phenotypically similar neurons and specifically identify cDNAs from mRNAs that are critical to phenotype and function. Transgenic mice expressing degradable green fluorescent protein (dGFP) from the per1 promoter, a clock-element gene, or from the fos promoter, a clock-driven gene, allow automated isolation of suprachiasmatic nucleus neurons expressing a circadian rhythm. Developing methods to use antisense RNA amplification, a linear mechanism of cDNA amplification from single neurons, and representational difference analysis, a high fidelity method of differential cDNA subtraction, on single phenotypically sorted neurons will allow both broad characterization of the mRNAs they express and specific identification of mRNAs whose expression changes during the circadian rhythm. Rather than assuming that changes in mRNA expression necessarily lead to protein changes, the second aim will reduce to practice a novel method for improved sensitivity in Western blots and use it to test rhythms in protein expression.
The third aim seeks to develop a method to simultaneously measure multiple proteins from single neurons, using a high-throughput optical method that is predicted to detect antigens at levels sufficient for analysis of proteins in single neurons.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
1R01AG018229-01
Application #
6051790
Study Section
Special Emphasis Panel (ZNS1-SRB-P (01))
Project Start
1999-09-30
Project End
2002-06-30
Budget Start
1999-09-30
Budget End
2000-06-30
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Kentucky
Department
Physiology
Type
Schools of Medicine
DUNS #
832127323
City
Lexington
State
KY
Country
United States
Zip Code
40506
Sammeta, Neeraja; Yu, Tun-Tzu; Bose, Soma C et al. (2007) Mouse olfactory sensory neurons express 10,000 genes. J Comp Neurol 502:1138-56
Yu, Tun-Tzu; McIntyre, Jeremy C; Bose, Soma C et al. (2005) Differentially expressed transcripts from phenotypically identified olfactory sensory neurons. J Comp Neurol 483:251-62