Type of Document Dissertation Author Tekmen, Merve URN etd-06282011-112408 Title Calcium-Dependent Mechanisms in the Chicken Retina Degree Doctor of Philosophy (Ph.D.) Department Biological Sciences Advisory Committee
Advisor Name Title Gleason, Evanna Committee Chair Galvez, Fernando Committee Member Siebenaller, Joseph F. Committee Member Svoboda, Kurt R Committee Member Cheng, Henrique Dean's Representative Keywords
- L-type Channels
- chicken retina
Date of Defense 2011-06-16 Availability restricted AbstractMultiple classes of retinal amacrine cells use L-type Ca2+ channels to mediate synaptic transmission. I have used whole cell voltage clamp recordings from primary cultures of amacrine cells to investigate the regulation of these channels. In this study, I show that inhibiting mitochondrial calcium uptake (MCU) caused a reversible reduction in the Ca2+ current amplitude. Replacing external Ca2+ with Ba2+ minimized the effects of blocking MCU indicating that the Ca2+ influx is the primary source of the inhibition. With 1,2-bis-(o-aminophenonxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA) in the recording pipette, MCU inhibition caused an increase in the current amplitude indicating that the fast buffering capability of BAPTA minimizing the Ca2+-dependent inactivation of the channels, revealing a Ca2+-dependent enhancement, possibly through protein kinase A (PKA) activity. The effect of a PKA inhibitor was consistent with this possibility. Inhibiting the calcium-induced calcium release (CICR) also decreased the Ca2+ current amplitude. These results indicate that MCU, PKA and CICR are critical to maintain the availability of L-type Ca2+ channels for depolarization-dependent signaling in amacrine cells.
Production of nitric oxide (NO) by neuronal and endothelial nitric oxide synthases (nNOS; eNOS) is another Ca2+-dependent mechanism in the retina. In the chicken retina, I demonstrate the cell-autonomous nature of the NO signal by comparing the pattern of NO production to the expression of NOS. The NO indicator fluorescence dye, DAF was used to detect the pattern of NO production. A NOS inhibitor L-NAME suppressed the DAF signal suggesting that the source of DAF-signal was due to NOS activity. I also demonstrate the presence of NOS-immunoreactivity in the chicken retina. Neuronal NOS and eNOS antibodies labeled photoreceptors, amacrine cells and cells in the ganglion cell layer (GCL). Anti-e NOS also labeled horizontal cells, a small subset of bipolar cells and Müller cells. Different subsets of amacrine cells were labeled in dorsal and ventral retina with anti-nNOS. Endothelial NOS labeling did not show difference in dorsal and ventral retina but expression was more wide spread than nNOS. These results suggest that the potential for NO production is wide spread in the avian retina.
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