Rod Photoreceptors Avoid Saturation in Bright Light by the Movement of the G Protein Transducin

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Rod Photoreceptors Avoid Saturation in Bright Light by the Movement of the G Protein Transducin Research Articles: Cellular/Molecular Rod photoreceptors avoid saturation in bright light by the movement of the G protein transducin https://doi.org/10.1523/JNEUROSCI.2817-20.2021 Cite as: J. Neurosci 2021; 10.1523/JNEUROSCI.2817-20.2021 Received: 6 November 2020 Revised: 21 January 2021 Accepted: 26 January 2021 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2021 the authors 1 Rod photoreceptors avoid saturation in bright light by the 2 movement of the G protein transducin 3 4 5 Running title: Rods in bright light avoid saturation 6 7 Rikard Frederiksen1, Ala Morshedian1, Sonia A. Tripathy1, Tongzhou Xu1, Gabriel H. Travis1, 8 Gordon L. Fain1,2, and Alapakkam P. Sampath1 9 10 1Department of Ophthalmology and Jules Stein Eye Institute, University of California, Los 11 Angeles, CA 90095–7000, USA 12 2Department of Integrative Biology and Physiology, University of California, Los Angeles, CA 13 90095–7239, USA 14 15 Corresponding authors (*) Submitting author (†): 16 17 Gordon L. Fain* 18 Stein Eye Institute, 100 Stein Plaza 19 University of California, Los Angeles 20 Los Angeles, CA 90095-7000 21 Telephone: (310) 206-4281 22 Email: gfain@ucla.edu 23 ORCID ID: 0000-0002-9813-0342 24 25 Alapakkam P. Sampath*† 26 Stein Eye Institute, 100 Stein Plaza 27 University of California, Los Angeles 28 Los Angeles, CA 90095-7000 29 Telephone: (310) 825-2024 30 Email: asampath@jsei.ucla.edu 31 ORCID ID: 0000-0002-0785-9577 32 33 Key words: Rod, photoreceptor, vision, transducin, light adaptation, visual pigment, saturation, 34 transduction, G protein 35 36 7 Figures and 1 Table 37 38 Acknowledgements: We thank Ekaterina Bikovtseva for technical assistance, Marie Burns for 39 providing a Gnat2-/- mating pair, and Carter Cornwall and Jürgen Reingruber for comments on 40 the manuscript. Khris Griffis wrote the data analysis package Iris DVA. This work was 41 supported by NEI, NIH Grants EY29817 (A.P.S.), EY001844 (G.L.F.), EY024379 (G.H.T.), an 42 unrestricted grant from Research to Prevent Blindness USA to the UCLA Department of 43 Ophthalmology, and NEI Core Grant EY00311 to the Jules Stein Eye Institute. G.H.T. is the 44 Charles Kenneth Feldman Professor of Ophthalmology at UCLA. 45 46 The authors declare no competing financial interests. 47 ABSTRACT 48 49 Rod photoreceptors can be saturated by exposure to bright background light, so that no flash 50 superimposed upon the background can elicit a detectable response. This phenomenon, called 51 increment saturation, was first demonstrated psychophysically by Aguilar and Stiles and has 52 since been shown in many studies to occur in single rods. Recent experiments indicate, however, 53 that rods may be able to avoid saturation under some conditions of illumination. We now show 54 in ex vivo electroretinogram and single-cell recordings that in continuous and prolonged 55 exposure even to very bright light, the rods of mice from both sexes recover as much as 15% of 56 their dark current and that responses can persist for hours. In parallel to recovery of outer 57 segment current is an approximately 10-fold increase in the sensitivity of rod photoresponses. 58 This recovery is greatly decreased in transgenic mice with reduced light-dependent translocation 59 of the G protein transducin. The reduction in outer-segment transducin together with a novel 60 mechanism of visual-pigment regeneration within the rod itself enable rods to remain responsive 61 over the whole of the physiological range of vision. In this way, rods are able to avoid an 62 extended period of transduction channel closure, which is known to cause photoreceptor 63 degeneration. 64 65 66 67 68 69 70 2 71 72 SIGNIFICANCE 73 Rods are initially saturated in bright light so that no flash superimposed upon the background can 74 elicit a detectable response. Frederiksen and colleagues show in whole retina and single-cell 75 recordings that if the background light is prolonged, rods slowly recover and can continue to 76 produce significant responses over the entire physiological range of vision. Response recovery 77 occurs by translocation of the G protein transducin from the rod outer to the inner segment, 78 together with a novel mechanism of visual-pigment regeneration within the rod itself. Avoidance 79 of saturation in bright light may be one of the principal mechanisms the retina uses to keep rod 80 outer-segment channels from ever closing for too long a time, which is known to produce 81 photoreceptor degeneration. 82 83 3 84 INTRODUCTION 85 86 There are two kinds of photoreceptors in vertebrate retina: rods with quantum sensitivity 87 mediating vision in dim light; and less sensitive but kinetically more rapid cones, which permit 88 rapid estimation of light intensity enabling wavelength discrimination and sensitivity to motion. 89 Aguilar and Stiles (1954) first showed that rods saturate and no longer function when exposed to 90 bright background light. They measured light adaptation in human observers under conditions 91 that maximized the contribution of rods and minimized those of cones, and they found that rod 92 sensitivity decreased according to a Weber-Fechner relation in dim backgrounds; but as the light 93 was made brighter, sensitivity fell at a much more rapid rate. Eventually the observer could no 94 longer detect any flash superimposed on the background, no matter how bright the flash was 95 made. Subsequent results have confirmed these observations in human (see Makous, 2003) and 96 behaving mice (Naarendorp et al., 2010), and recordings from single rods in a variety of 97 vertebrate species show a similar effect (Fain, 1976; Tamura et al., 1991; Mendez et al., 2001; 98 Makino et al., 2004; Morshedian and Fain, 2017). Saturation of rods is one of the pillars of our 99 understanding of the duplex retina and is described in any elementary treatment of visual 100 behavior (see for example https://webvision.med.utah.edu/book/part-viii-psychophysics-of- 101 vision/light-and-dark-adaptation/). Rods set the visual threshold in dim light, but rod signals are 102 then thought to diminish as the light is made brighter to permit the kinetically faster cones to 103 mediate detection of more rapidly changing features of the visual scene. 104 This simple scheme has recently been challenged by work showing that rods continue to 105 respond in bright light provided the background illumination is maintained for a sufficiently long 106 duration (Tikidji-Hamburyan et al., 2017; Borghuis et al., 2018). To provide clarification of this 107 phenomenon, and to characterize its nature and mechanism, we have undertaken a detailed study 4 108 of mouse rod responses in bright background light. We show in ex vivo electroretinogram (ERG) 109 and single-cell recordings that rods indeed recover a significant fraction of their photocurrent 110 during long-duration light exposure and continue to respond for several hours under these 111 conditions, even in the absence of the retinal pigment epithelium (RPE) and when no visual 112 pigment is calculated to remain. Our experiments indicate that avoidance of saturation is a 113 consequence primarily of the light-dependent translocation of the G protein transducin from the 114 rod outer segment to the rod inner segment, which reduces the gain of phototransduction and 115 allows outer-segment channels to reopen (Sokolov et al., 2002). This process, together with the 116 recovery of sufficient visual pigment to enable continued excitation of the phototransduction 117 cascade, can make it possible for rods to maintain responsivity over the entire physiological 118 range of vision and to avoid a prolonged period of channel closure, which is known to produce 119 photoreceptor degeneration (Fain, 2006). 120 5 121 122 MATERIALS AND METHODS 123 Animals 124 This study was carried out in accordance with the recommendations of the Guide for the Care 125 and Use of Laboratory Animals of the National Institutes of Health, and the Association for 126 Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and 127 Vision Research. The animal-use protocol was approved by the University of California, Los 128 Angeles, Animal Research Committee (Protocol Number: 14-005). Euthanasia was performed by 129 cervical dislocation. Every effort was made to minimize pain and discomfort in mice used in this 130 study. 131 All mice were reared under 12-hour cyclic light. Gnat2-/- mice were generously provided 132 by Marie Burns. This strain and the details of its genotyping have been previously described 133 (Ronning et al., 2018). Gnat1-/-;A3C+ mice were provided by Nikolai Artemyev and bred with 134 Gnat2-/- mice to produce Gnat2-/-;Gnat1-/-;A3C+ mice used in this study. Details about the 135 Gnat1-/-;A3C+ strain and its genotyping can be found in a previous publication (Majumder et al., 136 2013) Genotyping of these strains was performed by Transnetyx (Gnat2-/-) and Laragen Inc. 137 (Gnat1-/-;A3C+). Wild-type (129/SV-E) mice were purchased from Charles River. All animals 138 used in this study were between 1 and 6 months old. Both sexes were used in approximately 139 equal numbers. 140 141 Dissections and tissue preparation 142 Eyes from mice were enucleated in darkness by means of infrared image converters (ITT 143 Industries, White Plains, NY). The anterior portion of the eye was cut, and the lens and cornea 144 were removed in darkness with a dissection microscope (Zeiss, Oberkochen, Germany) equipped 6 145 with infrared image converters (B.E.
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