Doctor of Philosophy (PhD)
Amacrine cells are a class of retinal interneurons that process the visual signal in the inner retina. Several subtypes of amacrine cells express nitric oxide synthase and produce nitric oxide (NO), making NO a possible regulator of amacrine cell function. My dissertation research tests the hypothesis that NO alters amacrine cell GABAergic synaptic output. To investigate this, I made whole-cell voltage clamp recordings of cultured chick amacrine cells receiving synaptic input from other amacrine cells and Ca2+ imaging of amacrine cell dendrites, which can be presynaptic. I find that NO-dependent increases in GABAergic spontaneous postsynaptic current (sPSC) frequency are independent of soluble guanylate cyclase and action potentials. Removal of extracellular Ca2+ and buffering of cytosolic Ca2+ both inhibit the response to NO. In Ca2+ imaging experiments, I confirm that NO increases dendritic Ca2+ by activating a Ca2+ influx pathway. Neither NO-dependent dendritic Ca2+ elevation nor increase in sPSC frequency are dependent upon Ca2+ release from stores. NO also enhances evoked GABAergic responses, and because voltage-gated Ca2+ channel function is not altered by NO, the enhanced evoked release is likely due to the combination of voltage-dependent Ca2+ influx and the voltage-independent, NO-dependent Ca2+ influx. Insight into the identity of the Ca2+ channel involved in the NO response was provided by characteristics unique to the transient receptor potential canonical (TRPC) channel subunits 4 and 5: the NO-dependent increase in sPSC frequency was dependent on downstream activity of PLC, blocked by 2 mM La3+ and enhanced by 10 µM La3+. The TRPC inhibitor ML204, which preferentially blocks TRPC4, had no effect on the NO response at 10 µM, but 20 µM ML204 blocked the NO response. The TRPC inhibitor clemizole, which preferentially blocks TRPC5, blocked NO-dependent dendritic Ca2+ elevations and the increase in sPSC frequency. Genetic knockdown of TRPC5 in cultured amacrine cells using the CRISPR/Cas9 system confirms that TRPC5 mediates NO-dependent dendritic Ca2+ elevations and the increase in sPSC frequency. These results suggest that NO-dependent activation of TRPC5 at amacrine cell presynaptic sites will enhance vesicular GABA release and increase inhibition onto postsynaptic cells.
Document Availability at the Time of Submission
Release the entire work immediately for access worldwide.
Maddox, John Wesley, "A Presynaptic Role for Nitric Oxide at a GABAergic Synapse" (2017). LSU Doctoral Dissertations. 4450.