In this proposal we examine the effects of aging on mRNA expression in single neurons and glia isolated from the rat hippocampus. A profile of gene expression in these cells will be generated by using amplified antisense RNA (aRNA) made from single cells to probe Southern blots containing the cloned cDNAs for a number of molecules thought to be important in neuronal and glial cell development. Characterization of this profile for 50 individual neurons and glia will permit the categorization of neurons and glia into groups dependent upon their relative levels of expression of the selected mRNAs. Generation of aRNA probe from a single cell will be accomplished by injection of reverse transcriptase and the oligo-dT-T7 amplification oligonucleotide directly into dispersed calls using a patch-pipette. Simultaneous with this injection recordings will be taken from the cells to insure that they are viable. The cDNA made in this manner is of high complexity and is converted into aRNA by making it double-stranded and. adding T7 RNA polymerase to the template. Several million-fold amplification of mRNA levels have. been achieved using this technology. aRNA will be made from several dispersed cells that are isolated from rats ranging in age from 2 days to 33 months. These aRNAs will be used in the Southern blot profiling screen and will also be cloned so that cDNA libraries from single neurons and glia of these differing ages can be used to facilitate isolation of previously uncharacterized mRNAs whose abundance is age-related. The glucocorticoid model of aging will be examined using this same technology so that this model can be directly compared with aging in the single cell. Differences in age-related gene expression between hippocampal tissue and single cells will be examined by comparing the aRNA results with results generated using in situ transcription (1ST) of hippocampal brain section. Age-related and glucocorticoid regulated mRNAs will be isolated by 1ST-based aRNA differential hybridization and differential amplification. Clones will be characterized by DNA sequencing and in situ hybridization across the age range used to create the single cell gene expression profile.
| Buckley, Peter T; Khaladkar, Mugdha; Kim, Junhyong et al. (2014) Cytoplasmic intron retention, function, splicing, and the sentinel RNA hypothesis. Wiley Interdiscip Rev RNA 5:223-30 |
| Bartfai, Tamas; Buckley, Peter T; Eberwine, James (2012) Drug targets: single-cell transcriptomics hastens unbiased discovery. Trends Pharmacol Sci 33:9-16 |
| Buckley, Peter T; Lee, Miler T; Sul, Jai-Yoon et al. (2011) Cytoplasmic intron sequence-retaining transcripts can be dendritically targeted via ID element retrotransposons. Neuron 69:877-84 |
| Ross, John R; Porter, Brenda E; Buckley, Peter T et al. (2011) mRNA for the EAAC1 subtype of glutamate transporter is present in neuronal dendrites in vitro and dramatically increases in vivo after a seizure. Neurochem Int 58:366-75 |
| Kim, Junhyong; Eberwine, James (2010) RNA: state memory and mediator of cellular phenotype. Trends Cell Biol 20:311-8 |
| Sanchez-Alavez, Manuel; Tabarean, Iustin V; Osborn, Olivia et al. (2010) Insulin causes hyperthermia by direct inhibition of warm-sensitive neurons. Diabetes 59:43-50 |
| Bell, Thomas J; Miyashiro, Kevin Y; Sul, Jai-Yoon et al. (2010) Intron retention facilitates splice variant diversity in calcium-activated big potassium channel populations. Proc Natl Acad Sci U S A 107:21152-7 |
| Bell, Thomas J; Miyashiro, Kevin Y; Sul, Jai-Yoon et al. (2008) Cytoplasmic BK(Ca) channel intron-containing mRNAs contribute to the intrinsic excitability of hippocampal neurons. Proc Natl Acad Sci U S A 105:1901-6 |
| Wu, Chia-wen K; Zeng, Fanyi; Eberwine, James (2007) mRNA transport to and translation in neuronal dendrites. Anal Bioanal Chem 387:59-62 |
| Zielinski, Jennifer; Kilk, Kalle; Peritz, Tiina et al. (2006) In vivo identification of ribonucleoprotein-RNA interactions. Proc Natl Acad Sci U S A 103:1557-62 |
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