The influence of the mammalian retinal circadian clock on retinal physiology and function has been widely recognized, yet the cellular locations and neural regulation of retinal circadian pacemaking remains unclear. Therefore, the focus of this study is to localize the retinal circadian clocks within the mouse retina and to determine the key neural mechanisms regulating the mouse retinal clock. By combining several experimental strategies including single-cell real-time PCR, quantitative PCR, and real-time gene expression reporting, I have demonstrated that retinal neurons in the inner nuclear layer (INL) and ganglion cell layer express the complete molecular basis for rhythms generation and comprise an endogenous circadian clock that oscillates independent of the photoreceptors and of the brain. To study the neural regulation of retinal clock, I have developed a protocol for long-term culture (> 10 days) of intact retinas from adult mice, which allows retinal circadian rhythms to be monitored in real-time as luminescence rhythms from a clock gene reporter (PERIOD2::LUCIFERASE). With this assay, I have studied the characteristics and location within the retina of circadian PER2::LUC rhythms and the influence of major retinal neurotransmitters. Imaging of vertical retinal slices demonstrated that the rhythmic luminescence signals were concentrated in the INL. Interruption of cell communication via the major neurotransmitter systems of photoreceptors and ganglion cells (melatonin and glutamate), and the INL (dopamine, acetylcholine, GABA, glycine, glutamate), did not disrupt generation of retinal circadian PER2::LUC rhythms, nor did interruption of intercellular communication through sodium-dependent action potentials or connexin 36-containing gap junctions, indicating that PER2::LUC rhythms generation in the INL is likely cell-autonomous. However, dopamine, acting through D1 receptors, and GABA, acting through membrane hyperpolarization, set the phase and amplitude of retinal PER2::LUC rhythms, respectively. Collectively, my dissertation studies have shown that the inner retina is the primary location of mouse retinal circadian clock, and have indicated that dopamine and GABA act at the molecular level of PER proteins to play key roles in the organization of the retinal circadian clock, reinforcing the autonomous generation of retinal day and night states.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-07072008-232918 |
| Date | 23 July 2008 |
| Creators | Ruan, Guoxiang |
| Contributors | Dr. Laurence Zwiebel, Dr. Douglas McMahon, Dr. Carl Johnson, Dr. John Penn, Dr. Terry Page |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-07072008-232918/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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