Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

Understanding the regulation of retinal synapses is a key step in elucidating the processing of information in the retina. Our lab has previously shown that nitric oxide (NO) can alter the synaptic response properties of amacrine cells by releasing Cl- from internal acidic compartments. This alteration in the Cl- gradient brings about a positive shift in the reversal potential of the GABA-gated current, which can convert inhibitory synapses into excitatory synapses. Recently, we have shown that the cystic fibrosis transmembrane regulator (CFTR) Cl- channel is involved in the Cl- release. Here, we test the hypothesis that (acidic) synaptic vesicles are a source of NO-releasable Cl- in chick retinal amacrine cells. If SVs are a source of Cl-, then depleting synaptic vesicles should decrease the NO-dependent shift in the reversal potential of the GABA-gated current. The efficacy of four inhibitors of dynamin (a GTPase required for some forms of endocytosis) were evaluated. In order to deplete synaptic vesicles, voltage-steps were used to activate voltage-gated Ca2+ channels and stimulate the synaptic vesicle cycle either under control conditions or after treatment with the dynamin inhibitors. Voltage-ramps were used to measure the NO-dependent shift in the reversal potential of the GABA-gated currents under both conditions. Our results reveal that activating the synaptic vesicle cycle in the presence of dynasore and dyngo 4a blocked the NO-dependent shift in Erev-GABA. However, we also discovered that some dynamin inhibitors reduced Ca2+ signaling and L-type Ca2+ currents. Conversely, dynasore also increased neurotransmitter release at autaptic sites. To try to resolve the mechanism underlying the inhibition of the NO-dependent shift in the reversal potential for the GABA-gated currents, and to verify the effects of inhibiting endocytosis on the NO-dependent release of Cl-, we also tested the effects of the clathrin assembly inhibitor Pitstop 2 and found that this compound also inhibited the shift. These data provide evidence that dynamin inhibitors have multiple effects on amacrine cell synaptic transmission. These data also suggest that inhibition of endocytosis disrupts the ability of NO to elicit Cl- release from internal stores, which may in part be due to depletion of synaptic vesicles.

Date

6-29-2018

Committee Chair

Gleason, Evanna

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