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Dennis Tse, Inyoung Chung, Samuel Wu; Possible roles of glutamate transporter EAAT5 in mediating mouse cone on-bipolar cell light responses. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6150.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous study (ARVO 2012 #3160) found that intravitreally injected inhibitor of EAAT5 suppressed the ERG b-wave in living mice. We now sought to study its underlying mechanism and site.
Various solutions were injected unilaterally on dark-adapted mice (8-12wk of age) under the following conditions: (1) TBOA(EAAT5 inhibitor; Tocris#1223) in wild-type(WT) mice, (2)TBOA in bhlhb4 KO mice lacking rod bipolar cells(BCs), (3) a mixture of TBOA and GABA antagonists in WT mice, (4) saturating LAP4(Tocris#0103), TBOA and their mixture in WT mice. Scotopic flash ERGs were recorded 5m after the injection for all conditions, and repeated every 15m for 1hr for (1). Dual flash ERG was recorded for (4). Fellow eyes acted as controls. All handling was performed under infrared light. Immunohistochemistry(IHC) was performed using standard procedures on retinal sections of WT & bhlhb4 mice with antibodies for EAAT5, Goα(on-BC) and PSD95(rod spherules).
(1)TBOA inhibited the b-wave maximally at the 5m time point. Such inhibition dissipated with time. The b-wave was abolished at a stimulus of 1.8 log Rh*/rod with an IC50 of 23.5µM (calc. retinal conc.), and was reduced to 25% of the fellow eye at a saturating stimulus of 6.17 log Rh*/rod with an IC50 of 114 µM. (2)The b-wave was inhibited by TBOA in a similar manner in bhlhb4 mice, or (3)when GABA antagonists were co-injected. This suggested that the inhibitory mechanism was separated from the rod pathway and GABA feedback. (4)Mean mixed rod/cone b-wave decreased to 24% of control when saturating LAP4 was injected alone, and 13% when co-injected with TBOA. The cone b-wave (dual flash) was 45% with LAP4 alone and suppressed to 5% when co-injected with TBOA. This suggested that TBOA inhibition of b-wave is supplementary to that of LAP4, particularly on cone-driven on-BCs. EAAT5 immuno-labeling in the OPL was partly co-localized with PSD95 and partly with Goα, suggesting that EAAT5 may present on rod terminals and BC dendrites. Bhlhb4 retina showed a pattern similar to that of WT, suggesting that EAAT5 expressing dendrites comprise cone BCs.
Similar to previous results from fish retina, our result suggested the presence of a TBOA-sensitive postsynaptic mechanism underlying light responses of mouse cone-driven on-BCs. IHC indicates that such a mechanism is likely mediated by EAAT5 on dendrites of subpopulations of cone on-BCs.
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