Fahrenfort I , Steijaert M , Sjoerdsma T , Vickers E , Ripps H , van Asselt J , Endeman D , Klooster J , Numan R , ten Eikelder H , von Gersdorff H , Kamermans M .

2 T32 DK007680-16 NIDDK NIH HHS , EY-06516 NEI NIH HHS , EY14043 NEI NIH HHS , R01 EY014043 NEI NIH HHS , T32 DK007680 NIDDK NIH HHS , F32 EY006516 NEI NIH HHS , R01 EY006516 NEI NIH HHS

	Figure 1. Schemeatic representation of the two feedback hypothesis.A) the ephaptic hypothesis and B) the pH hypothesis. The cone terminal is depicted with the invaginating HC and BC dendrites. The synaptic ribbon with the synaptic vesicles is indicated by “R”. The voltage gated Ca-channels in the cone synaptic terminal are depicted in red. Hemichannels in the HC dendrites are indicated by the blue symbol. The white symbol indicate the extracellular resistance and the gray symbol the potassium channels in the HCs. The green symbol indicates an unidentified proton transport system.
	Figure 2. HEPES reduces feedback-mediated responses in the cone/horizontal cell network in the isolated retina of goldfish.A, Feedback responses elicited by a 500 ms full-field (3000 µm) stimulus (I = 0 log) delivered in the presence of a 20 µm spot of light (I = 0 log) centered on a cone voltage-clamped at −45 mV. Current responses are shown in control (left), in the presence of 20 mM HEPES (middle), and after washout (right). HEPES reduces the amplitude of the feedback-mediated responses. B, Responses of a monophasic horizontal cell to 550 nm light (I = 1 log) in control (left), and after application of 20 mM HEPES (right). HEPES hyperpolarizes horizontal cells and strongly reduces the light-induced hyperpolarization and the feedback-induced roll-back response (arrow). C, Responses of a biphasic horizontal cell to light of 550 nm (I = 1 log) and 650 nm (I = 1 log) in control (left), after application of 20 mM HEPES (middle), and after washout (right). HEPES reduces the feedback-induced depolarizing response due to deep red light stimulation (arrows).
	Figure 3. HEPES does affect the kainate-DNQX induced shift of the Ca2+-current in a dose-dependent manner in the isolated retina of goldfish.A, Ca2+-current of the cone in control bicarbonate-based Ringer's solution (filled circles), after depolarization of horizontal cells with 30 µM kainate (open circles), and after subsequent hyperpolarization of horizontal cells by the addition of 50 µM DNQX (filled triangles). B, Boltzmann fits through the activation functions of the Ca2+-current from the same cell shown in (A). 30 µM kainate shifts the Ca2+-current to more positive potentials, whereas a subsequent application of 50 µM DNQX shifts the Ca2+-current to more negative potentials. The difference of the half activation in kainate and in DNQX is 9.3±1.1 mV (n = 7) and is a measure for the feedback strength independent of light responses. C, Inhibition of maximal shift of activations of the Ca2+-current in control, and in the presence of various concentrations of HEPES (open symbols) or Tris (closed symbols). The solid line represents a fitted Hill equation though the HEPES data points. Compared to Tris, HEPES is a more potent inhibitor of feedback.
	Figure 4. HEPES inhibits Cx55.5 hemichannel-mediated currents in Xenopus oocytes.A, Mean currents of 9 oocytes are plotted as function of potential. The control current (filled squares) is inhibited by application 25 mM HEPES (open circles). No current is present in antisense injected oocytes (filled triangles). Error bars =  ±sem. B. The mean reduction in Cx55.5 hemichannel current at potentials between −55 and −35 mV. At a concentration of 4 mM HEPES, the hemichannel mediated current decreased to 37.9±0.07% (n = 7; p<0.001). All tested concentrations of HEPES induced a significant reduction of the Cx55.5 hemichannel current (40 µM HEPES: p = 0.0065, n = 5; 0.4 mM HEPES: p = 0.0118, n = 4; 4.0 mM HEPES: p = 0.00165, n = 5).
	Figure 5. HEPES and Tris lead to intracellular acidification in the isolated retina of goldfish.A, BCECF fluorescence of horizontal cells in the flat mounted retina in control conditions. Horizontal cells stain yellow. B and C, ΔF/F as function of time. Data points are the average of 7 cells (regions of interest) and error bars represent sem. Increase in fluorescence signal indicates acidification. Complete exchange of solution in recording chamber took about 2 minutes. Frames were sampled every 2 minutes. Application of 25 mM acetate, 20 mM HEPES and 20 mM TRIS all lead to a reduction of intracellular pH.
	Figure 6. Intracellular acidification reduces feedback-mediated responses without hyperpolarizing horizontal cells in the isolated retina of goldfish.A, Feedback-responses elicited by a 500 ms full-field stimulus (I = 0 log) delivered in the presence of a 20 µm spot of light (I = 0 log) centered on a cone clamped at −38 mV. Current responses are shown in control (left), after application of 25 mM acetate (middle), and after washout (right). Acetate reduces feedback-induced inward currents in cones. B, Responses of a monophasic horizontal cell to 550 nm light (I = 0 log), in control, after application of 25 mM acetate (middle), and after washout (right). Intracellular acidification blocks the feedback-induced roll-back response without affecting the resting membrane potential of the horizontal cells. C, Responses of a biphasic horizontal cell to light of 550 nm (I = 1 log) and 600 nm (I = 1 log) in control (left), and after application of acetate (right). Acetate reduces the feedback-induced depolarization. D, Boltzmann fits through the activation function of the Ca2+-current of a cone in 25 mM acetate. 30 µM kainate shifts the Ca2+-current to more positive potentials, whereas a subsequent application of 50 µM DNQX shifts the Ca2+-current to more negative potentials.
	Figure 7. Localization of extracellular carbonic anhydrase XIV in the goldfish retina.A, Distribution of the extracellular carbonic anhydrase in the goldfish retina. Immunostaining by an antibody raised against the mouse extracellular carbonic anhydrase XIV was found throughout the goldfish retina. Extensive labeling (red) was found throughout the OPL, suggesting a role for extracellular carbonic anhydrase in synaptic transmission between cones and horizontal cells. The blue label is due to the nuclear marker DAPI. ONL: outer nuclear layer, OPL: outer plexiform layer, INL: Inner nuclear layer, IPL: inner plexiform layer. B, No label remains after pre-adsorption of the primary antibody with the immunizing recombinant carbonic anhydrase XIV. Scale bars in A and B = 20 µm. C, Western blot of membrane fractions of goldfish retina shows a band at about 57 kDA and a band at 25 kDa. The 25 kDa band represent a proteolytically nicked form of carbonic anhydrase (A. Waheed and W. Sly, personal communication). D, Distribution of carbonic anhydrase (red) relative to GluR2 (green) in the cone synaptic terminal. GluR2 labels invaginating dendrites of horizontal cells in cone terminals. Yellow (arrows) indicates a close association between carbonic anhydrase and GluR2, showing synaptic localization of carbonic anhydrase. Scale bar = 2 µm. E, Distribution of carbonic anhydrase (red) and PKC-α (green) in the bipolar cell synaptic terminal. Yellow (arrows) indicates a close association between carbonic anhydrase and PKC, showing synaptic localization of carbonic anhydrase. Scale bar = 5 µm.
	Figure 8. Benzolamide enhances pH-dependent inhibition of L-type Ca2+ currents in isolated goldfish Mb bipolar cell terminals.A, Bath application of 100 µM benzolamide and 100 µM picrotoxin enhances the “pH-nose” inhibition of L-type Ca2+ currents elicited by 100 ms steps from −60 mV to −10 mV. This inhibition is mediated by the exocytosis of protons co-packaged with glutamate. Upper traces: Individual voltage-clamp traces from two different isolated terminals in the same slice. In the continuous presence of picrotoxin (100 µM; a: blue trace), a 100 ms step to −10 mV produces an inward Ca2+ current of nearly 400 pA that is inhibited by a rapidly decaying pH-nose (indicated by arrow) immediately following the onset of depolarization. Addition of benzolamide (100 µM; b: red trace) enhances the amplitude and area of the pH-nose. Voltage-clamp sine waves before and after the depolarizing step allow for the measurement of membrane capacitance change (ΔCm; exocytosis). Lower traces: Measurements of ΔCm corresponding to the traces shown above. The ΔCm in control is 158 fF (blue trace) and it is 132 fF (red trace) in the presence of 100 µM benzolamide. B, The pH-nose is absent from a pure Ca2+ current, elicited by a 100 ms step to −10 mV following nearly complete run-down of exocytosis. Upper traces: Depolarization of an isolated terminal to 10 mV for 100 ms produces an inward Ca2+ current of nearly 400 pA (c: red trace). Lower trace: The corresponding capacitance trace shows that exocytosis is almost completely absent. C, The exact charge transfer for each pH-dependent inhibition of ICa2+ in (A) was calculated by scaling the original traces (traces a,b) to match the amplitude of the Ca2+ current in (B), subtracting the pure Ca2+ current trace (trace c) from the Ca2+ current traces with pH noses (a,b), and integrating the area under the difference curve during the 100 ms depolarization to −10 mV. The pH-nose charge in the presence of 100 µM benzolamide (b–c) is 3.878 pC, and the pH-nose in control (a–c) is 2.854 pC. D, Bath application of 100 µM benzolamide (red triangles; n = 29, 6 cells; control is blue squares; n = 53, 8 cells) significantly increases the positive slope of the relationship between the size of the charge of pH-dependent inhibition of ICa2+ and ΔCm. The slope of the linear regression for 100 µM benzolamide (red line; 0.018+/−0.004) was significantly non-zero (p<0.0001, r2 = 0.45) and significantly larger than the slope for control (p = 0.017). The slope for control (blue line; 0.007+/−0.002) was also significantly non-zero (p = 0.004, r2 = 0.15).
	Figure 9. Benzolamide does not increase the feedback-induced inward currents in cones in the isolated goldfish retina.A, Feedback-responses elicited by a 500 ms full-field stimulus (I = 0 log) delivered in the presence of a 20 µm spot of light (I = 0 log) centered on a cone clamped at −43 mV. Current responses are shown in control (top), after application of 500 µM benzolemide (middle), and after washout (bottom). B. Feedback induced shift of the Ca-current of a cone in 100 µm benzolamide. 30 µM kainate shifts the Ca2+-current to more positive potentials, whereas a subsequent application of 50 µM DNQX shifts the Ca2+-current to more negative potentials. The difference of the half activation in kainate and in DNQX is 7.0±1.6 mV (n = 6).
	Figure 10. Effects of acetate on horizontal cells and cones in the isolated goldfish retina after chemical isolation.A, A horizontal cell was stimulated with full field 500 ms flashes of 500 nm light. Two time scales are given: one for the light flashes (top) and one for the whole trace (bottom). Removing synaptic input to a monophasic horizontal cell with 50 µM DNQX causes the cell to hyperpolarize. A subsequent application of acetate leads to a further hyperpolarization. B, Acetate exerts a direct effect on the cone Ca2+-current after pharmacologically isolating the cone from H-cell input with 100 µM Co2+. Boltzmann fits through activation function of the Ca2+-current in the presence of Co2+ (filled circles), after application of 25 mM acetate (open circles), and after washout (filled triangles). Boltzman fits were obtained as described in Fig. 3a. In the presence of Co2+, acetate shifts the Ca2+-current to more negative potentials. C, Responses of a monophasic horizontal cell to 550 nm light (I = 0 log) in control solution (left), after application of 100 µM Co2+ (middle), and after subsequent application of 25 mM acetate (right). The low concentration of Co2+ hyperpolarizes the horizontal cell and strongly reduces the amplitude of its light response. The addition of 25 mM acetate depolarized the cell, and increased the amplitude of the light response, indicative of an acetate-induced increase in neurotransmitter release.
	Figure 11. Glutamate-gated channels are able to mediate feedback in the presence of acetate in the isolated goldfish retina.Responses of a monophasic horizontal cell to 550 nm light (I = 1 log) during application of acetate and kainate. Expanded light responses at the time points indicated by the arrows, are given below. 1) light response in control with a feedback-induced roll-back response. 2) light-responses in the presence of acetate. The feedback-induced roll-back response has disappeared without significant changes in the light response amplitude. 3) light-responses after additional application of 10 µM kainate. Despite a reduction in the amplitude of the light response, the feedback-induced roll-back response reappears again after opening a fraction of the glutamate-gated channels with a non-saturating concentration of kainate. 4) Light responses after longer superfusion of kainate. As horizontal cells depolarize further, their light responses get smaller, but there is still a substantial feedback-induced roll-back response. 5) Light response after washout of kainate; note that the horizontal cell hyperpolarizes, the amplitude of the light response increases, and the roll-back response is again no longer present.
	Figure 12. Model for ep[haptic feedback from horizontal cells to cones.A, Network of components essential to model ephaptic feedback from horizontal cells to cones. The ephaptic influence of the horizontal cell onto the cones is through changes in the extracellular potential of the synaptic cleft. is the output of the model and affects the position of the Ca2+-current of the cone along the voltage axis and thereby modulates the amount of calcium flowing into the cone. The horizontal cell has two compartments, 1) the neuropil and soma, and 2) the dendritic tips. The neuropil and soma compartment has the following components: , reversal potential of combined potassium and leak channels; , the combined potassium and leak conductance. , glutamate conductance in the neuropil and soma. The dendritic components are: , the conductance of the extracellular space in the synaptic cleft between cones and horizontal cells; , the hemichannel conductance, and , the conductance of the glutamate-gated channels present in the tip of the horizontal cell dendrites. B, The relation between the fraction of glutamate-receptors in the tip of the horizontal cell dendrite (p) and the size of the voltage drop in the synaptic cleft when horizontal cells are polarized from their resting membrane potential (−34.7 mV) to −71.4 mV. The smaller the fraction of glutamate-gated channels on the tips of the horizontal cells, the larger the horizontal cell polarization-induced voltage drop in the synaptic cleft. C, Voltage drop elicited in the synaptic cleft at the different membrane potentials of horizontal cells when stimulated with a full-field (solid line) or with an annulus (dashed line). When horizontal cells are maximally polarized in the full-field condition, a 6 mV potential drop is elicited; an annular stimulus gives a 15 mV potential drop. D, The relation between the horizontal cell membrane potential and the change in potential deep in the synaptic cleft for annular stimulation (solid line) and in a condition when all hemichannels are closed () (dashed line) and when all glutamate gated channels are closed () (dotted line). These simulations show that about 40% of the feedback response remains when hemichannels are closed, illustrating that glutamate gated channels contribute about 40% to the ephaptic feedback mechanism.

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