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M. J. Rowan, W. Shen; Glycine Feedback Enhances Glutamate Synapses in Cones through Regulation of Excitatory Amino Acid Transporter II (EAAT2). Invest. Ophthalmol. Vis. Sci. 2009;50(13):5174.
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A specific EAAT2 antibody was used to localize the glutamate transporter in tiger salamander retina. Whole cell patch clamp was performed on cones and bipolar cells in dark- and light-adapted retinal slices. Transporter currents were elicited with 1mM glutamate in the bath solution. GABA receptor antagonists SR95531, CGP55845, and TPMPA blocked feedback in the distal retina while testing transporter currents in cones. 100uM Glycine was applied in the bath and mimicked glycine feedback to the distal retina. DHKA and TBOA were used to block EAAT2 currents and overall EAAT currents, respectively.
Immunoantibody labeling indicated that both cones and Off-bipolar cells were EAAT2-positive. Application of glutamate in cones elicited large currents that were partially blocked by DHKA and fully blocked by TBOA, a broad spectrum EAATs inhibitor, indicating that the current produced was exclusively from transporters. Glycine reduced glutamate transporter currents approximately 30%; however, in presence of DHKA, glycine had no inhibitory effect on the remaining glutamate current. This indicates that glycine directly inhibits the activation of EAAT2 in cones. In Off-bipolar cells, blockage of EAAT2 produced an inward current in dark and enhanced the light offset evoked overshoot response. We also found that EAAT2 activation was suppressed in bright light-adapted retinas and enhanced in fully dark adapted retina.
We determined that the EAAT2 subtype participates in glutamate transmission from cones to bipolar cells, and is involved in the fast uptake of glutamate in the OPL. Cone EAAT2 inhibition by network glycine feedback leads to an increase in local glutamate levels in the OPL. This finding supports our previous hypothesis that glycine feedback serves as a positive control in glutamate transmission. A possible mechanism is through internal Cl- regulation in cones, as glycine alters local Cl- levels, and because EAAT2 is coupled to a Cl- current when activated. Considering that the activation of EAAT2 was suppressed by bright light, this transporter may be more susceptible to glyinergic regulation in dark or dim light conditions.
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