The physiology and behavior of virtually all eukaryotes, including humans, exhibits a pervasive daily rhythmicity. Many critical daily rhythms are endogenous, driven by an internal master pacemaker or biological clock, with a free-running period of about a day (i.e., circadian). The suprachiasmatic nuclei (SCN) of the hypothalamus are the apparent site of the master biological clock in mammals. Recent research has revealed that (i) the ability to generate neurophysiological circadian rhythms reside in single SCN neurons, and (ii) the fundamental mechanisms for rhythms resides in single SCN neurons, and (ii) the fundamental mechanisms for rhythms generation within these neurons involve transcriptional negative feedback loops among putative circadian clock genes. A critical for circadian neurobiology is to establish means to monitor real time circadian gene expression in single SCN neurons so that the precise roles of putative clock genes, and the linkage of the intracellular molecular clock with its neurophysiological output, can be investigated in detail. Toward these ends we have created three kinds three of transgenic mice incorporating the reporter constructs fosdGFP. Toward these ends we have created three kinds of transgenic mice incorporating the reporter constructs fosdGFP, (dGFP) appropriate for assaying dynamic gene expression. Our hypothesis is that SCN neurons from fosdGFP and perdGFP mice will display circadian gating of induction and circadian rhythms in dGFP fluorescence correlating with the previously documented endogenous rhythms in c-fos and mper1 RNAs. We propose three specific aims to test and expand our hypothesis.
Aim 1 -Intact Animal Induction Experiments-to validate the function of the transgene reporters.
Aim 2 In Vitro Induction Experiments-to test for acute induction of transgene reporters.
Aim 2 - In Vitro Induction Experiments-to test for acute induction of transgene reporters in vitro.
Aim 3 -In Vitro Circadian Experiments-to test for circadian rhythms in fos or per-driven gene expression. Successful completion of these aims will aims will establish promoter driven dGFP transgene reporters as an approach for assessing the molecular circadian rhythms in individual SCN neurons. This will provide a methodological foundation for expanded future research on the molecular basis of the mammalian SCN clock which should increase our understanding of the mechanism of seasonal affective disorder, age-related disruption of biological timing, and jet lag.
| Kuhlman, S J; Quintero, J E; McMahon, D G (2000) GFP fluorescence reports Period 1 circadian gene regulation in the mammalian biological clock. Neuroreport 11:1479-82 |
