April 2012

Document Type


Degree Name



Oregon Health & Science University


The role of inhibitory signaling in CNS microcircuits depends on the specific connectivity and frequency-dependent plasticity of synapses from interneurons onto principle cells. Feedforward, feedback, and lateral inhibition in general are thought to subserve gating, dynamic range tuning, and spatial sharpening functions, respectively, in multiple brain areas including cortex, striatum, the olfactory bulb, and the retina. In the retina, inhibition is mediated either by horizontal cells (HCs) in the OPL, or by amacrine cells (ACs) in the IPL. Both classes of cells have been shown to mediate feedback and lateral inhibition, and ACs are known to mediate feedforward inhibition onto ganglion cells (GCs), the output neurons of the retina. ACs also mediate lateral feedback inhibition at bipolar cell (BC) presynaptic terminals, although the synaptic mechanisms, timing, and frequency dependence of these inhibitory inputs in response to light stimulation had not been previously well characterized, due to the relative inaccessibility of BC presynaptic terminals to direct electrophysiological recording. Here, we recorded directly from axotomized, large presynaptic terminals of ON-type, mixed rod/cone input (Mb) bipolar cells in a slice preparation of goldfish retina. By stimulating with light, we were able to electrophysiologically isolate lateral feedback inhibition in the form of lateral inhibitory post-synaptic currents (L-IPSCs). In order to investigate the role that these L-IPSCs may play in the regulation of vesicular glutamate release from Mb terminal ribbon synapses onto ACs and GCs, we first examined their timing, light sensitivity, and pharmacology in response to single, full-field light flashes. We hypothesized that L-IPSCs, mediated by a tri-synaptic circuit from photoreceptors to BCs to ACs to BC terminals, would depend on different combinations of rod and cone inputs under different background light conditions, would rely on diverse glutamatergic signaling at BC to AC synapses, and would be mediated primarily by GABAergic signaling at Mb terminals. Furthermore, we expected that L-IPSCs would be delivered by a set of AC synaptic inputs distinct from those that mediate reciprocal inhibition at the Mb terminal, and thus constitute an independently regulated pathway for control of Mb glutamate release in response to spatiotemporal modulation of surround light stimulation. First, we showed that L-IPSCs at the Mb terminal consist of an ON component, with an onset roughly 50 ms following the initiation of light flash, and an OFF component, occurring 100-150 ms following the offset of the light flash. We found that ON L-IPSCs were driven by a combination of rod and cone input, and that OFF L-IPSCs were driven primarily by cone inputs. Next, we showed that ON and OFF L-IPSCs are both mediated by a combination of signaling at GABA[subscript A]R and GABA[subscript C]R and rely on a combination of signaling at AMPARs and NMDARs at BC to AC synapses, although ON L-IPSCs were, unexpectedly, enhanced in the presence of the specific AMPAR antagonist, NBQX. Both ON and OFF L-IPSCs were disinhibited following application of SR-95531, a specific GABA[subscript A]R antagonist, consistent with the idea that serial inhibitory signaling between ACs plays a major role in regulating L-IPSC strength. Blockade of voltage-gated Na[superscript +] channels with TTX markedly reduced OFF L-IPSCs but had mixed effects on ON LIPSCs, suggesting that the AC class providing OFF inputs signals via regenerative Na[superscript +] action potentials, while ON ACs may propagate lateral inhibition via analog, subthreshold depolarization. Finally, we showed that step-depolarization-evoked reciprocal inhibitory feedback and light-evoked L-IPSCs did not cross-depress, suggesting that they are mediated by distinct classes of ACs. Next, we sought to examine the short-term plasticity (STP) of ON and OFF LIPSC size and timing. Such plasticity would indicate a modulation of lateral feedback inhibition by surround temporal contrast, and provide a novel mechanism for adaptation or sensitization of local, feed-forward light-responses by dynamic presynaptic inhibition at BC terminals. We hypothesized that STP of strength and timing for convergent ON and OFF L-IPSCs would provide a framework for precise regulation of both analog EPSPs and regenerative Ca[superscript 2+] action potentials at the Mb terminal. In order to address the mechanisms that would regulate this STP, we first examined the role of diverse GABAergic signaling and background light adaptation on L-IPSC amplitude, charge transfer, and onset latency. We found that block of signaling at GABA[subscript A]Rs enhanced amplitude and charge transfer and increased onset latencies. Under scotopic background conditions, ON L-IPSC exhibited an increase in amplitude and onset latency relative to mesopic background conditions, while OFF L-IPSCs showed an increase in amplitude and a decrease in onset latency. We used a paired light flash protocol to address STP, and found that ON L-IPSCs exhibited short-term depression (STD) of amplitude and charge transfer that recovered over 2 s, and showed delay (increase) of onset latency at short intervals (50 - 300 ms). OFF L-IPSCs also exhibited STD of amplitude and charge transfer, but this STD recovered over 1 s and transformed into short-term facilitation (STF) of amplitude for intervals from 1-2 s. In addition, OFF LIPSCs showed advance (decrease) of onset latency at short intervals (300 ms). Because L-IPSC timing is likely critical for proper regulation of glutamate release from Mb terminals, we examined the role that ACs play in determining L-IPSC latencies. We found that block of voltage-gated Na[superscript +] channels with TTX caused an increase in OFF LIPSC onset latencies, and, with paired recordings of Mb terminals and AC somata, we showed that long L-IPSC onset latencies, as well as their sustained and asynchronous nature, are likely driven by processes intrinsic to ACs. In order to pursue additional mechanisms that may regulate plasticity of GABAergic L-IPSCs, we examined the role of endogenous ascorbic acid (Asc) in the regulation of GABA[subscript A]R and GABA[subscript C]R mediated currents. We found that Asc acts as a stereospecific reducing agent at the two cysteines in the extracellular cys-loop, and at histidine 141, of heterologously expressed GABA[subscript C]Rs, to reversibly enhance GABA[subscript C]R mediated Cl[superscript -] current in a manner dependent on the concentration of both GABA and Asc. We showed that Asc applied to retinal slices reversibly enhanced standing GABA[subscript C]R mediated leak current, puff-evoked GABA[subscript C]R mediated currents, and GABA[subscript A] mIPSC amplitudes, and that intracellular Asc prevented run-down of puff-evoked GABA[subscript C] currents over a period of 15 minutes in axotomized Mb terminals. The presence of high endogenous concentrations of Asc in the retina (~0.2 mM), along with studies that show glutamate-evoked release of Asc and the retinal expression of SVCT2, a Na[superscript +] / Asc cotransporter, suggest that Asc may play a role in activity dependent regulation of STP of L-IPSCs at BC presynaptic terminals.




Neuroscience Graduate Program


School of Medicine



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