Feedback from horizontal cells to rod photoreceptors in vertebrate retina

Abstract

Retinal horizontal cells (HCs) provide negative feedback to cones, but, largely because annular illumination fails to evoke a depolarizing response in rods, it is widely believed that there is no feedback from HCs to rods. However, feedback from HCs to cones involves small changes in the calcium current (I(Ca)) that do not always generate detectable depolarizing responses. We therefore recorded I(Ca) directly from rods to test whether they were modulated by feedback from HCs. To circumvent problems presented by overlapping receptive fields of HCs and rods, we manipulated the membrane potential of voltage-clamped HCs while simultaneously recording from rods in a salamander retinal slice preparation. Like HC feedback in cones, hyperpolarizing HCs from -14 to -54, -84, and -104 mV increased the amplitude of I(Ca) recorded from synaptically connected rods and caused hyperpolarizing shifts in I(Ca) voltage dependence. These effects were blocked by supplementing the bicarbonate-buffered saline solution with HEPES. In rods lacking light-responsive outer segments, hyperpolarizing neighboring HCs with light caused a negative activation shift and increased the amplitude of I(Ca). These changes in I(Ca) were blocked by HEPES and by inhibiting HC light responses with a glutamate antagonist, indicating that they were caused by HC feedback. These results show that rods, like cones, receive negative feedback from HCs that regulates the amplitude and voltage dependence of I(Ca). HC-to-rod feedback counters light-evoked decreases in synaptic output and thus shapes the transmission of rod responses to downstream visual neurons.

Figure 1.

Feedback from HCs to rods studied using simultaneous whole-cell recordings from HCs and rods. Hyperpolarizing the HC membrane potential from −14 to −54, −84, and −104 mV caused a progressive hyperpolarizing activation shift and increased the amplitude of rod I _Ca. A, Confocal stacks of a simultaneously recorded rod (yellow) and HC (magenta) stained with Lucifer yellow (2 mg/ml) and sulforhodamine B (0.5 mg/ml), respectively. Images were obtained using a spinning disk confocal microscope system (UltraView; Perkin-Elmer) with a black and white camera (Orca ER; Hamamatsu). Colors were added using Adobe Photoshop. Confocal stacks of the two stained cells were superimposed on a bright-field image of the retinal slice. Scale bar, 10 μm. B, I _Ca recorded from a rod using a ramp voltage protocol. The steady HC holding potential was varied among −14 (purple trace), −54 (black trace), −84 (green), and −104 mV (blue). C, The average shift in V ₅₀ for I _Ca produced by changes in HC potential relative to V ₅₀ determined when the HC was held at −54 mV. The shift in V ₅₀ was fit by linear regression (slope = 0.0328 ± 0.00243; r ² = 0.73; n = 17). Addition of HEPES (10 mm) to the superfusate blocked the shift in V ₅₀ (open circles; n = 9). D, The change in I _Ca amplitude as a function of HC holding potential fit by linear regression (slope = −0.00244 ± 0.000344; r ² = 0.43; n = 17). Addition of HEPES blocked the increase in I _Ca amplitude as a function of HC hyperpolarization (open circles; n = 9).

Figure 2.

Hyperpolarizing HCs by light caused a negative shift in voltage dependence and increased the amplitude of rod I _Ca. A, Light response of a horizontal cell evoked by a 3 s white light flash. B, I _Ca recorded using a ramp voltage protocol from a rod lacking its outer segment in darkness (black trace) and white light (gray trace). C, The leftward shift in the V ₅₀ of I _Ca induced by light in control conditions was blocked by application of HEPES (10 mm; n = 11; p < 0.0001) or kynurenic acid (KynA; 1 mm; n = 9; p < 0.0001). D, The light-induced increase in I _Ca peak amplitude was also blocked by application of HEPES (10 mm; n = 11; paired t test, p = 0.04) or kynurenic acid (1 mm; n = 9; paired t test, p = 0.026). *p < 0.05.