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Imaging an optogenetic pH sensor reveals that protons mediate lateral inhibition in the retina

Nature Neuroscience volume 17, pages 262–268 (2014)Cite this article

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Abstract

The reciprocal synapse between photoreceptors and horizontal cells underlies lateral inhibition and establishes the antagonistic center-surround receptive fields of retinal neurons to enhance visual contrast. Despite decades of study, the signal mediating the negative feedback from horizontal cells to cones has remained under debate because the small, invaginated synaptic cleft has precluded measurement. Using zebrafish retinas, we show that light elicits a change in synaptic proton concentration with the correct magnitude, kinetics and spatial dependence to account for lateral inhibition. Light, which hyperpolarizes horizontal cells, causes synaptic alkalinization, whereas activating an exogenously expressed ligand-gated Na+ channel, which depolarizes horizontal cells, causes synaptic acidification. Whereas acidification was prevented by blocking a proton pump, re-alkalinization was prevented by blocking proton-permeant ion channels, suggesting that distinct mechanisms underlie proton efflux and influx. These findings reveal that protons mediate lateral inhibition in the retina, raising the possibility that protons are unrecognized retrograde messengers elsewhere in the nervous system.

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Figure 1: Design and expression of CalipHluorin, a genetically engineered fluorescent pH sensor.
Figure 2: Light induces alkalinization of the cone synaptic cleft.
Figure 3: Quantification of the light-induced change in pH.
Figure 4: The temporal properties of the pH change resemble those of the horizontal cell light response.
Figure 5: The spatial and pharmacological features of the pH change match those of the horizontal cell light response.
Figure 6: Activation of an exogenously expressed ligand-gated Na+ channel in horizontal cells acidifies the cone synaptic cleft.

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Acknowledgements

We thank R. Montpetit for help with molecular biology, J. LeDue for help with optical instrumentation, W. Thoreson for helpful discussions, C. Davenport for critical reading of the manuscript and S. Brockerhoff (University of Washington), R. Huganir (Johns Hopkins University School of Medicine), M. Kamermans (Netherlands Institute for Neuroscience) and A.R. McQuiston (Virginia Commonwealth University School of Medicine) for providing plasmids. This work was supported by grants from the US National Institutes of Health to R.H.K. (R01EY015514 and P30EY003176) and T.-M.W. (F32EY020095).

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Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA

    Tzu-Ming Wang, Lars C Holzhausen & Richard H Kramer

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  1. Tzu-Ming Wang
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Contributions

T.-M.W. and R.H.K. conceived the project. T.-M.W. performed most of the experiments, analyzed the data and developed plugins for the image acquisition software. L.C.H. made the FaNaC and the double transgenic fish lines, collected and analyzed the electrophysiological data and prepared Figure 6a and Supplementary Figure 4. T.-M.W. and R.H.K. prepared the manuscript, and all authors edited the manuscript.

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Correspondence to Richard H Kramer.

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Integrated supplementary information

Supplementary Figure 1 CalipHluorin on the cell surface is sensitive to pH.

(a) Fluorescence image of a dissociated cultured hippocampal neuron, transfected with CalipHluorin. The average pixel intensity within the ROI (red line) was plotted in (b). (b) The fluorescence intensity dropped upon applying acidic buffer solution (i.e. from pH 7.4 to 6) and recovered after washout. Treatment with thrombin (10 U/ml) cuts the CalipHluorin at its extracellular proteolytic cleavage site, removing the pHluorin and eliminating cell surface fluorescence. The decrease in fluorescence coincides with elimination of the response to application of acidic buffer.

Supplementary Figure 2 The CalipHluorin response was reliable across many retinal fields of view.

For each panel, the retinas were stimulated with a 1 mm spot light for 0.5 s. (a) We imaged 23 total retinal fields (i.e. n = 23) from 8 retinas and each individual retinal field (gray) and the average are shown. CalipHluorin fluorescence increased 5% after illumination. The CalipHluorin signal was never observed in the somata of cones (n = 20, b), and was blocked by pH buffer Tris (20mM, n = 21, c) and AMPA receptor antagonist DNQX (150 μM, n = 21, d).

Supplementary Figure 3 Line-scan analysis of the light-evoked transient acidification in retinas treated with DNQX to eliminate the HC contribution.

(a) Delayed transient decrease in fluorescence following termination of a 508 ms flash. The black trace shows measurements from successive scanned lines (2 ms/line), with each 30 μm line traversing several cone terminals. Data was averaged over 21 trials from regions of interest imaged from 6 retinas. The red trace shows the rolling average of the black trace (40 ms time window). The dotted line represents the average value of the first 40 ms of acquired data after the flash. (b) Comparison of mean line scan values (from the black trace) from the initial 40 ms after the flash (the period under the blue bar in a) and a later 40 ms (the period under the red bar in a), showing a significant decrease in fluorescence, reflecting transient acidification (**, p = 0.009). (c, d) The same experiment and analysis carried out after adding 20 mM HEPES (21 trials from 5 retinas) showed no transient acidification (p = 0.882). Error bars represent SEM.

Supplementary Figure 4 Effects of bafilomycin A1 (BFA1), carbenoxolone (CBX), and meclofenamic acid (MFA) on the Na+ conductance of FaNaC expressed in HEK cells.

BFA1 (a, b) and MFA (e, f) have no direct effect on the Na+ conductance of FaNaC. The peak current amplitude of FaNaC evoked by FMRFamide (FMRFa) is not significantly affected by the presence of BFA1 (n = 9 cells, p = 0.55) or MFA (n = 11 cells, p = 0.95). In contrast, CBX inhibited the current amplitude evoked by FMRFamide (by ∼64%, n = 8 cells, p = 0.02). (a, c, e) show representative traces and (b, d, f) show the average (red) and single responses (gray). Error bars represent SEM.

Supplementary Figure 5 Experimental and image analysis paradigm.

(a) ROI (red) that included cone terminals was created and pixels within it were analyzed. (b) Images were taken at a fixed rate while 3 episodes of light flashes were given in a single trial (black/yellow bar). The average pixel intensity of the ROI (gray line) was compensated (black line) for photobleaching according to the fitted exponential curve (red). (c) Light responses from the 3 flash episodes were averaged. The mean response is shown as the black line. (d) The mean traces from trials from each of 16 ROIs from 4 different retinas were averaged to generate the overall average response to light.

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Wang, TM., Holzhausen, L. & Kramer, R. Imaging an optogenetic pH sensor reveals that protons mediate lateral inhibition in the retina. Nat Neurosci 17, 262–268 (2014). https://doi.org/10.1038/nn.3627

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