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P. D. Lukasiewicz, T. Ichinose; Hepes Blocks Outer, but Not Inner Retinal Surround Inhibition in Mouse. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5794. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The two inhibitory pathways in the retina are located in the outer (OPL) and the inner plexiform layers (IPL). Both pathways contribute to the receptive field surround of ganglion cells (GCs); however, their relative roles remain unknown. Previously, we showed that light-evoked excitatory postsynaptic currents (L-EPSCs) in GCs were enhanced with HEPES. This could occur by either blocking OPL inhibition or reducing proton-mediated feedback to bipolar cell terminals in IPL. Here, we determined the effects of HEPES in each plexiform layer.
L-EPSCs, evoked by full-field light, were recorded from GCs and cone bipolar cells (BCs). We also directly activated ON BCs by briefly switching from the mGluR6 agonist AP-4 to the antagonist, CPPG (0.5-3 sec), and measured EPSCs in GCs and IPSCs in BCs and GCs. HEPES (20 mM) was added to bicarbonate buffer Ringer solution to examine whether it blocked lateral inhibition in the OPL and/or IPL. Lateral inhibition in the IPL was blocked by bicuculline and TPMPA. Strychnine was always present to block glycine receptors. Cells were morphologically characterized by including sulforhodamine in the pipette.
HEPES enhanced L-EPSCs in ON cone BCs. CNQX, which blocks transmission between cones and horizontal cells, similarly enhanced the L-EPSCs. These findings suggest that HEPES blocks OPL surround inhibition. To examine whether HEPES affected IPL inhibition, we activated ON cone BCs directly with CPPG to avoid any contribution of OPL inhibition. CPPG-evoked IPSCs in cone BCs and in GCs were not affected by HEPES, but were eliminated with GABA receptor blockers, bicuculline and TPMPA, indicating that HEPES does not affect IPL inhibition. We also examined whether HEPES enhanced L-EPSCs in GCs by reducing IPL inhibition to BCs. HEPES did not affect the CPPG-evoked EPSCs, which were eliminated by glutamate receptor blockers, suggesting that HEPES acts in the OPL, but not in the IPL, to block inhibition.
We found HEPES exclusively and reversibly blocks OPL surround inhibition in the mouse retina. Although HEPES transiently enhances electrically-evoked release from BCs in goldfish (Palmer MJ. et al., 2003), it does not affect light-evoked release in mouse BCs. Our findings suggest that HEPES is a useful pharmacological tool to dissociate OPL and IPL surround inhibition.
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