Pupil Constriction During Prolonged Exposure to Flickering Stimuli
Total Page:16
File Type:pdf, Size:1020Kb
Pupil Constriction During Prolonged Exposure to Flickering Stimuli: Evidence for Cholinergic ipRGC Stimulation THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Elizabeth Galko Graduate Program in Vision Science The Ohio State University 2019 Master's Examination Committee: Andrew Hartwick, OD, PhD, Advisor Donald Mutti, OD, PhD Michael Earley, OD, PhD Copyright by Elizabeth Galko 2019 Abstract Intrinsically photosensitive retinal ganglion cells (ipRGCs) express melanopsin, a Gq-coupled photopigment, and these neurons exhibit sustained action potential firing in response to light. ipRGCs have primarily non-visual functions, including an influence on the pupillary light reflex (PLR) to which they contribute to sustained pupil constriction. Acetylcholine is released in the retina by starburst amacrine cells in response to retinal image motion, and can be stimulated by flickering light and moving gratings. Light with a flicker frequency range of 3-10 Hz, peaking near 6 Hz, is especially effective at evoking retinal acetylcholine release. It has been previously demonstrated that rat ipRGCs fire sustained action potentials in response to cholinergic agonists. Sustained ipRGC spiking was also evoked by 6 Hz flickering light, presented at an irradiance below melanopsin’s activation threshold, mediated by a muscarinic acetylcholine receptor-mediated pathway. The purpose of this study is to evaluate human pupil responses during and after exposure to light of differing frequencies, hypothesizing that a 6 Hz flickering light will cause more sustained pupil constriction than other frequencies. Seven healthy subjects were exposed to blue (480 nm) and red (620 nm) light at different irradiances (1012 and 1010 photons/s/cm2) and frequencies (0, 0.1, 6 and 30 Hz) for five minutes. Light was presented to the dilated left pupil; the consensual response of ii the right pupil was recorded. Pupil constriction was normalized and compared amongst the different flicker frequencies within each wavelength and irradiance level. Pupil constriction during light exposure and pupil dilation after light offset were analyzed. For blue light at 1012 photons/s/cm2, the 6 Hz and 0.1 Hz light stimuli produced greater overall pupil constriction compared to the 30 Hz stimulus. For red light at 1012 photons/s/cm2, the 6 Hz and 0.1 Hz stimuli elicited greater overall pupil constriction relative to the 0 and 30 Hz stimuli. For blue light at 1010 photons/s/cm2, 0.1 Hz stimulated greater overall pupil constriction as compared to the 0 and 30 Hz stimuli. The 6 Hz stimulus evoked greater constriction than the 30 Hz stimulus during the second minute of light exposure. For red light at 1010 photons/s/cm2, there was no significant difference in pupil constriction amongst the stimuli of different flicker frequencies. When assessing the rate of pupil dilation after the first pulse of 0.1 Hz, there was no difference in the re- dilation rate between any of the stimuli, regardless of color or irradiance. My results show that flickering light at 0.1 and 6 Hz stimulate greater pupil constriction as compared to 0 and 30 Hz stimuli of similar irradiance and color. My results support the hypothesis that flickering light near peak sensitivity for retinal acetylcholine release causes a greater pupil constriction, supporting the presence of this pathway in the human retina. The potential presence of this cholinergic pathway could expand the range of light levels for which ipRGCs are active. iii Dedication This work is dedicated to my parents, Danny and Monica Galko. You have always valued my education and made sacrifices out of love to provide opportunities for success. All you have done has led to the completion of this document, and I can never thank you enough for your love and support. iv Acknowledgments I would like to thank my advisor, Dr. Andy Hartwick, for his guidance and support during this entire journey. Thank you to Dr. Phil Yuhas and Jaime Etterling for support in learning research methods, data collection, and data analysis. Thank you to my thesis committee members, Dr. Don Mutti and Dr. Michael Earley, for your mentorship and commitment to this work. Thank you to my family and friends for the support and encouragement throughout this process. There are too many people to personally name, which in itself is a blessing to be surrounded by an abundant, wonderful community. Finally, thank you to my colleagues and mentors at The Ohio State University College of Optometry. It is an honor to work alongside you and better the future of optometry and vision science. v Vita 2015……………………………………….B.A. Biochemistry, The Ohio State University Fields of Study Major Field: Vision Science vi Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments ............................................................................................................... v Vita ..................................................................................................................................... vi Fields of Study ................................................................................................................... vi Table of Contents .............................................................................................................. vii List of Figures ..................................................................................................................... x Introduction ......................................................................................................................... 1 Acetylcholine in the Central Nervous System ................................................................. 1 Acetylcholine in the Retina .............................................................................................. 4 Starburst Amacrine Cells ................................................................................................ 7 Intrinsically-Photosensitive Retinal Ganglion Cells ..................................................... 12 Functions of ipRGCs ..................................................................................................... 17 Neuromodulation of ipRGCs ......................................................................................... 19 Cholinergic Stimulation of ipRGCs .............................................................................. 21 Purpose of the Study ...................................................................................................... 23 vii Methods............................................................................................................................. 24 IRB Approval and Patient Recruitment ......................................................................... 24 Study Design and Equipment ........................................................................................ 25 Session Structure and Stimuli ........................................................................................ 26 Data and Statistical Analysis ........................................................................................ 27 Results ............................................................................................................................... 30 Pupil Constriction in “Bright” Light ............................................................................ 30 Pupil Constriction in “Dim” Light ............................................................................... 36 Pupil Dilation After Light Exposure ............................................................................. 41 Discussion ......................................................................................................................... 43 Flickering Light Caused Increased Pupil Constriction ................................................ 43 Melanopsin Activity Below Conventional Threshold .................................................... 45 Melanopsin Acts as Photon Counter ............................................................................. 47 Confounding Factors Regarding Pupillary Light Reflex .............................................. 48 Pathways of ipRGC Stimulation .................................................................................... 49 Expanded Range of ipRGC Activity .............................................................................. 51 Circadian Control of Cholinergic ipRGC Stimulation ................................................. 53 Retinal Acetylcholine Production in Real-World Conditions ....................................... 56 Impact of Expanded Range of ipRGC Activity on Daily Life ........................................ 57 viii Future Work .................................................................................................................. 58 Conclusion ..................................................................................................................... 59 References ......................................................................................................................... 60 ix List of Figures Figure 1. Resting and light-evoked release of acetylcholine in the rabbit retina. Incubation