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.
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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.
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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
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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
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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
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Future Work ...... 58
Conclusion ...... 59
References ...... 60
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List of Figures
Figure 1. Resting and light-evoked release of acetylcholine in the rabbit retina. Incubation in a medium with low calcium (0.2 mM Ca2+ and 20 mM Mg2+) completely inhibited the light-evoked acetylcholine release and decreased basal release in darkness. Acetylcholine concentration is expressed as 103 disintegrations/min/mL (Masland & Cassidy, 1987). ... 5
Figure 2. A starburst amacrine cell that has been electrode-filled with dye. Scale 50 um
(from Vaney, 1984)...... 8
Figure 3. The starburst a cell, residing in the inner nuclear layer, with its dendrites extending in the nearest sublamina of the inner plexiform layer. The dendrites arborize in the same two-dimensional plane of the IPL (from MacNeil & Masland, 1998)...... 8
Figure 4. The effect of flickering light on the release of (A) acetylcholine and (B) GABA from the rabbit retina in vivo. Each bar indicates a five-minute sampling period. The hatched bars represent periods of flickering light at the noted frequencies (from Neal et al., 1992)...... 10
Figure 5. Left) The effect of flicker frequency on acetylcholine release, expressed as evoked/resting release. Right) The effect of flicker frequency on acetylcholine release per flash during a five-minute period. Each point is both charts is the mean ± SEM (from
Neal et al., 1992)...... 11
Figure 6. Left) The spectral sensitivity of human melanopsin. The data was fit to a predicted absorbance spectrum of a vitamin A-based opsin with a �max = 479 nm (from
Bailies & Lucas, 2013)...... 13
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Figure 7. A) Normalized pupil constriction (0% = greatest pupil size and 100% = smallest pupil size) vs. time for “bright” blue light (480 nm, 1012 photons/s/cm2) at 0 Hz
(Continuous), 6 Hz, and 30 Hz. B) Normalized pupil constriction for bright blue light at
0.1 Hz, which was graphed separately to facilitate comparison between the 0, 6 and 30
Hz traces...... 31
Figure 8. Average normalized pupil constriction of each stimulus for bright blue (480 nm,
1012 photons/s/cm2). ** indicates p<0.01 ...... 32
Figure 9. Normalized pupil constriction for bright blue light (480 nm, 1012 photons/s/cm2) within A) first minute of light exposure, B) second minute of light exposure, C) third minute of light exposure, D) fourth minute of light exposure, E) fifth minute of light exposure. * indicates p<0.05, ** indicates p<0.01 ...... 32
Figure 10. A) Normalized pupil constriction (0% = greatest pupil size and 100% = smallest pupil size) vs. time for “bright” red light (620 nm, 1012 photons/s/cm2) at 0 Hz
(Continuous), 6 Hz, and 30 Hz. B) Normalized pupil constriction for bright red light at
0.1 Hz...... 34
Figure 11. Average normalized pupil constriction of each stimulus for bright red (620 nm, 1012 photons/s/cm2). * indicates p<0.05, ** indicates p<0.01 ...... 35
Figure 12. Normalized pupil constriction for bright red light (620 nm, 1012 photons/s/cm2) within A) first minute of light exposure, B) second minute of light exposure, C) third minute of light exposure, D) fourth minute of light exposure, E) fifth minute of light exposure. * indicates p<0.05, ** indicates p<0.01 ...... 35
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Figure 13. A) Normalized pupil constriction (0% = greatest pupil size and 100% = smallest pupil size) vs. time for “dim” blue light (480 nm, 1010 photons/s/cm2) at 0 Hz
(Continuous), 6 Hz, and 30 Hz. B) Normalized pupil constriction for dim blue light at 0.1
Hz...... 37
Figure 14. Average normalized pupil constriction of each stimulus for dim blue (480 nm,
1010 photons/s/cm2). ** indicates p<0.01 ...... 38
Figure 15. Normalized pupil constriction for dim blue light (480 nm, 1010 photons/s/cm2) within A) first minute of light exposure, B) second minute of light exposure, C) third minute of light exposure, D) fourth minute of light exposure, E) fifth minute of light exposure. * indicates p<0.05 ...... 38
Figure 16. A) Normalized pupil constriction (0% = greatest pupil size and 100% = smallest pupil size) vs. time for “dim” red light (620 nm, 1010 photons/s/cm2) at 0 Hz
(Continuous), 6 Hz, and 30 Hz. B) Normalized pupil constriction for dim red light at 0.1
Hz...... 39
Figure 17. Average normalized pupil constriction of each stimulus for dim red light (620 nm, 1010 photons/s/cm2)...... 40
Figure 18. Normalized pupil constriction for dim red light (620 nm, 1010 photons/s/cm2) within A) first minute of light exposure, B) second minute of light exposure, C) third minute of light exposure, D) fourth minute of light exposure, E) fifth minute of light exposure...... 40
Figure 19. A) Pupil re-dilation after the first pulse of the flashing 0.1 Hz stimulus for bright blue, bright red, dim blue, and dim red. Data was re-normalized to the first data
xii point after light offset. B) The decay constant for each stimulus, which shows no significant difference...... 42
Figure 20. A) Pupil re-dilation after light offset for the bright red 0, 6, and 30 Hz. Data was re-normalized to the first data point after light offset. B) The decay constants for each stimulus, which shows no significant difference...... 42
Figure 21. As the number of photons increased within a 10-s light stimulus, the total spikes elicited from ipRGCs increased in a linear fashion (from Dacey et al., 2005)...... 47
Figure 22. The known and proposed pathways of ipRGC stimulation. Light stimulates the melanopsin photopigment as well as rod and cone photoreceptors. Rods and cones stimulate ipRGCs via bipolar cells and starburst amacrine cells, which elicit prolonged signaling...... 50
Figure 23. The proposed shared pathway of both types of ipRGC stimulation. Both melanopsin and muscarinic acetylcholine receptors activate phospholipase C which generates second messengers that open transmembrane TRP channels. The shared pathway and utilization of the TRP channel is competitive in nature, and so the effects of light and cholinergic stimulation are not additive, as shown by Sodhi and Hartwick
(2016)...... 51
Figure 24. The proposed expanded range of sustained firing of ipRGCs. The cholinergic signaling pathway allows ipRGCs to fire prolonged action potentials at lower light levels.
This expanded range also shows greater overlap with the cone-driven, transient responses of ipRGCs...... 52
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Figure 25. A summarization of the relationships between light, the retinal circadian clock, dopamine, and melatonin (from Besharse & McMahon, 2016)...... 54
Figure 26. The effect of melatonin on retinal acetylcholine release, graphed as percent of acetylcholine released, per volume of choline incubation, over time. The black bar indicates the exposure to 3 Hz flickering light (from Mitchell & Redburn, 1991)...... 55
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Introduction
Acetylcholine in the Central Nervous System
Acetylcholine has long been known to be an integral neurotransmitter in both the central and peripheral nervous systems (Lahiri & Sen, 1968). In the central nervous system, cholinergic neurons are located in the basal forebrain, hippocampus, amygdala, brainstem, diencephalon, and striatum (Wevers, 2011). Cholinergic neurons are linked to spatial learning and memory, and the loss of cholinergic neurons in the basal forebrain has been linked to the development of Alzheimer’s disease (Fibiger, 1991).
Acetylcholine activates two different types of receptors, muscarinic and nicotinic, and the different receptor subtypes within these two broad classes have been linked to a variety of functions within the central nervous system. For example, M1 receptors have been linked to spatial learning and memory, and are the primary muscarinic receptors found in the cerebral cortex and hippocampus (Fisher, 2008). M2 and M4 receptors are the primary muscarinic receptors of the hippocampus; they regulate acetylcholine release in response to novel environments (Tzavara et al., 2003). There are many subtypes of nicotinic receptors, which consist of pentamers of different combinations of subunits
(labeled �2-�10 and �2-�4). These receptors have been found throughout the brain, including the cerebral cortex, thalamus, basal ganglia, brainstem, and cerebellum; and influence processes such as arousal, sleep, fatigue, anxiety, processing of pain, attention, learning, and memory (reviewed by Wevers, 2011). Nicotinic cholinergic receptors in the brain can be grouped into subsystems. The most significant system is the magnocellular basal complex, which provides input to the cortical and hippocampal regions; followed
1 by the pedunculopontine-laterodorsal tegmental complex, where cholinergic neurons from the pedunculopontine tegmental nucleus project to thalamic nuclei and midbrain dopaminergic neurons. These and other subsystems have been reviewed by Gotti &
Clementi (2004).
Nicotinic receptors are ligand-gated ion channels, where stimulation of the receptor causes the opening of the ion channel and cation (sodium and calcium) influx into the cell (Gotti & Clementi, 2004). The influx of cations leads to a direct depolarization of the cell membrane. This event cascade is different from that associated with muscarinic receptors, which are G-protein coupled receptors (GPCRs). Stimulation of these receptors causes an intracellular signaling cascade to be initiated that culminates in altered activity of enzymes such as adenylate cyclase and phospholipase C (Wevers,
2011). The intracellular signaling pathway utilized by each GPCR is dependent upon the subtype of the coupled G-protein: Gs, Gi or Gq.
There are 5 subtypes of muscarinic receptors expressed in the human brain, labeled M1 through M5 (Raiteri, 1994). The M1 through M4 subtypes were classified by
Peralta et al. (1987), who found that all subtypes are transmembrane proteins that are similar to rhodopsin and �-adrenergic receptors. The fifth muscarinic receptor was characterized by Bonner, Young, Brann, & Buckley (1988), though it appears to be expressed in a much smaller volume compared to the other muscarinic subtypes. M1, M3, and M5 receptors are coupled to Gq proteins that activate intracellular phospholipase C, which then activates inositol 1,4,5-triphosphate (IP3), a second messenger that increases intracellular calcium through release from the endoplasmic reticulum, as well as opening
2 of IP3-gated membrane calcium channels (Felder, 1995). In contrast, M2 and M4 receptors are coupled to Gi proteins, and their activation leads to an inhibition of adenylate cyclase.
This results in decreased levels of cyclic AMP (cAMP; Felder, 1995).
There are two theories regarding acetylcholine transmission in the central nervous system: traditional synaptic transmission via synaptic clefts, often called “wired” transmission; and passive diffusion of acetylcholine outside a synaptic cleft, referred to as
“volume transmission.” There are multiple studies supporting both modes of transmission
(Sarter, Parikh, & Howe, 2009). The presence of cholinergic terminals and acetylcholinesterase in synaptic clefts support wired transmission of acetylcholine; however, the presence of nicotinic and muscarinic receptors outside synaptic clefts and small detectable extracellular levels of acetylcholine support volume transmission (Sarter,
Parikh, & Howe, 2009). There is some question about whether the evidence for volume transmission is an artifact of experimental preparations, as the methods of extracellular recordings disrupt normal tissue structure. The process of microdialysis involves surgical implantation of a probe, which temporarily disrupts extracellular neurotransmitter levels.
These levels appear to stabilize near 24 hours after surgery, but it has been hypothesized that scar tissue develops around the probe. Since neurotransmitter sampling occurs within a small radius of the probe, the neurotransmitter levels of scarred tissue may not accurately reflect normal states (Westerink, 1995). A reasonable working theory, based on current available evidence, is that both modes of transmission exist but volume transmission likely only occurs over short distances.
3
Outside of the central nervous system, stimulation of muscarinic receptors by acetylcholine is an integral component of the pupil light reflex. Acetylcholine stimulates the iris sphincter muscle, causing it to constrict in response to light (Laties & Jacobowitz,
1966). This reflex is first initiated in response to light falling on the retina, resulting in the stimulation of retinal ganglion cells that project to the pretectal region of the brainstem.
Premotor neurons then project from the olivary pretectal nucleus to the oculomotor nucleus of cranial nerve III, specifically the Edinger-Westphal nucleus (Sun & May,
2014). Preganglionic parasympathetic neurons travel from the Edinger-Westphal nucleus and synapse within the ciliary ganglion (Akert, Glicksman, Lang, Grob, & Huber, 1980;
Burde & Loewy, 1980). Postganglionic neurons then project from the ciliary ganglion, through the short ciliary nerves, to the iris sphincter and ciliary muscles. Stimulation of the iris sphincter muscle through the parasympathetic nervous system causes constriction of the muscle and miosis. Acetylcholine release by the parasympathetic pathway also causes relaxation of the iris dilator muscle, furthering the pupil constriction in response to light (Suzuki, Oso, & Kobayashi, 1983). Blocking of muscarinic receptors on the iris through cholinergic antagonists, such as tropicamide and atropine, prevents contraction of the iris sphincter muscle. This temporary effect leads to an overall greater pupil size and the inability of the iris sphincter to constrict in response to light.
Acetylcholine in the Retina
Retinal acetylcholine is released at a basal level under dark conditions, and application of a flickering light causes an increase in acetylcholine release (Masland &
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Cassidy, 1987; Masland & Livingstone, 1976). Acetylcholine release, especially in response to light, is inhibited by low extracellular calcium levels, indicating synaptic involvement in light-evoked but not in resting release of acetylcholine (see Figure 1).
Increased release of acetylcholine begins soon after the beginning of a light stimulus and ends right after the end of the train of flickering light. The limit of resolution is approximately five seconds, which is slower than conventional synaptic transmission.
The effect began 1-5 seconds after application of the stimulant and then steadily increased in magnitude of effect (Straschill & Perwein, 1973).
Figure 1. Resting and light-evoked release of acetylcholine in the rabbit retina. Incubation in a medium with low calcium (0.2 mM Ca2+ and 20 mM Mg2+) completely inhibited the light-evoked acetylcholine release and decreased basal release in darkness. Acetylcholine concentration is expressed as 103 disintegrations/min/mL (Masland & Cassidy, 1987).
Acetylcholine leads to increased firing of ON and directionally-selective ganglion cells and decreases firing of OFF ganglion cells (Strang, Renna, Amthor, & Keyser,
2010). Atropine, a cholinergic antagonist, significantly decreases both excitation and
5 suppression of RGC firing, but it is much more effective at decreasing suppression.
Light-evoked release of acetylcholine is inhibited by GABA signaling (Massey & Neal,
1979).
All subtypes of muscarinic receptors have been located in the retina and labeled to subsets of bipolar, amacrine, and retinal ganglion cells (RGCs; Strang et al., 2010). The
M3 subtype was localized to cell bodies of horizontal and bipolar cells in the inner nuclear layer (INL), processes in the outer plexiform layer (OPL), and in both ON and
OFF sublamina of the inner plexiform layer (IPL). M2 and M5 subtypes were also localized to bipolar cells, though their immunoreactivity was much less than M3. The presence of muscarinic receptors in the OPL indicates a potential effect of acetylcholine on retinal processing in the outer retina.
Collectively, subsets of amacrine cells and RGCs expressed all muscarinic subtypes. M1 receptors were distributed in two broad bands in the ON and OFF sublamina of the IPL, indicating both ON and OFF RGCs express M1 receptors. Both M1 and M2 receptors are expressed by subsets of amacrine cells and RGCs, but there is little overlap between expression of these two receptors. Strang et al. (2010) also found that
Gq-coupled (M1, M3, M5) muscarinic receptors tended to be co-localized, as did Gi- coupled (M2, M4) muscarinic receptors.
Nicotinic receptors have been located on bipolar cells and ganglion cells, and nicotine has been found to increase intracellular calcium levels of retinal ganglion cells
(Baldridge, 1996). This effect is dependent upon extracellular calcium levels, indicating that the calcium mediating the signal was due to influx of the cation across the cell
6 membrane. The effects of nicotine were not inhibited by glutamatergic antagonists, indicating that the responses were due to direct effects of nicotine on the inner retinal neurons and not due to the release of pre-synaptic glutamate following stimulation of outer retinal neurons (Baldridge, 1996). Baldridge also found that a small subset of ganglion cells that responded to nicotine also responded to muscarine, increasing intracellular calcium levels, and the effect was stopped by a M1-receptor antagonist.
Acetylcholine-synthesizing neurons can be stained with antibodies raised against the enzyme choline acetyltransferase (ChAT), and this reveals cells with expansive dendritic fields that lay within the inner plexiform layer. These amacrine cells are located in both the inner nuclear layer, with dendritic branches extending into the outer third of the IPL, and in the ganglion cell layer, with dendritic branches extending into the inner third of the IPL with mirror-symmetry (Tauchi & Masland, 1984). Labeling of acetylcholine through autoradiography similarly reveals two discrete bands in the IPL, one in sublamina a (outer) and the other in sublamina b (inner) (Masland, Mills, &
Hayden, 1984). Vaney (1984) determined that the cholinergic amacrine cells are what had been morphologically-identified as “starburst” amacrine cells.
Starburst Amacrine Cells
Starburst amacrine cells (SBACs) make up 3% of the amacrine cell population in the retina (MacNeil & Masland, 1998). They are named for their wide array of dendrites and expanse of retinal area covered by each amacrine cell (see Figure 2).
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Figure 2. A starburst amacrine cell that has been electrode-filled with dye. Scale 50 um (from Vaney, 1984).
Starburst a cells reside in the inner nuclear layer and stratify in sublamina a (outer) of the inner plexiform layer (IPL), where they receive synaptic input from ON-type bipolar cells. Starburst b cells reside in the ganglion cell layer and stratify in sublamina b of the
IPL, where they receive synaptic input from OFF-type bipolars. Both sets of cells stratify in a narrow band of the IPL (Famiglietti, 1991; see Figure 3).
Figure 3. The starburst a cell, residing in the inner nuclear layer, with its dendrites extending in the nearest sublamina of the inner plexiform layer. The dendrites arborize in the same two-dimensional plane of the IPL (from MacNeil & Masland, 1998). 8
SBACs contain acetylcholine and gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter. Acetylcholine is released in response to flickering light, as discussed previously. A small subset of starburst amacrine cells express M1 receptors, supporting the hypothesis that acetylcholine release produces a feedback loop to decrease further release of acetylcholine (Strang et al., 2010). GABA signaling is essential to the activity of direction-selective ganglion cells (DSGCs), and the close proximity of the dendrites of DSGCs and SBACs indicates that SBACs are the source of GABA for this process. Furthermore, selective ablation of SBACs was found to inhibit the activity of
DSGCs (Taylor & Smith, 2012).
Neal and colleagues (1992) quantified the resting release of acetylcholine and
GABA from starburst amacrine cells. They then determined that while light evokes a greater release of acetylcholine, there was no change in GABA release. There was a significant change in acetylcholine release in response to 0.1 Hz light, but the greatest release was achieved by using light flickering at rates of 6-10 Hz. The amount of acetylcholine released then decreased as light flicker frequency increased, until at 30 Hz there was no difference from the basal acetylcholine release in the dark (see Figure 4).
They also found that onset or offset of a continuous light did not significantly change levels of acetylcholine or GABA release (Neal, Cunningham, Hutson, & Semark, 1992).
Neal then determined the quantity of acetylcholine release per flash at each tested flicker frequency, and determined the greatest release of acetylcholine per flash was at 0.1 Hz
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(see Figure 5). The average amount released per flash at 6 and 10 Hz was only near 10% of the release per flash of 0.1 Hz.
Figure 4. The effect of flickering light on the release of (A) acetylcholine and (B) GABA from the rabbit retina in vivo. Each bar indicates a five-minute sampling period. The hatched bars represent periods of flickering light at the noted frequencies (from Neal et al., 1992).
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Figure 5. Left) The effect of flicker frequency on acetylcholine release, expressed as evoked/resting release. Right) The effect of flicker frequency on acetylcholine release per flash during a five-minute period. Each point is both charts is the mean ± SEM (from Neal et al., 1992).
Studies have shown that starburst amacrine cells are most responsive to light flickering at a rate of 3-10 Hz (O’Malley & Masland, 1993). The cone system has a maximum flicker sensitivity near 10 Hz, whereas a rod system that is light adapted has a maximum flicker sensitivity near 7 Hz (Y. V. Wang, Weick, & Demb, 2011). At this range of temporal frequencies, both rod and cone photoreceptors respond to the flickering stimulus and excite starburst amacrine cells. Flickering light, because of the light onset and offset, stimulates both ON and OFF signaling pathways and thus is effective at stimulating both a and b types of SBACs. The decreased stimulation of SBACs at higher temporal frequencies of flickering light might be because of the loss of rod-driven input to the pathway.
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Intrinsically-Photosensitive Retinal Ganglion Cells
Intrinsically-photosensitive retinal ganglion cells, or ipRGCs, are a small subset of ganglion cells, making up 0.2% of ganglion cells in the primate retina (M T H Do &
Yau, 2010). ipRGCs express the photopigment melanopsin, a transmembrane protein that is similar in structure to other opsins in the retina (Graham et al., 2008). It is a Gq-coupled receptor (Panda et al., 2005; Qiu et al., 2005), similar to the invertebrate rhodopsin, that uses the messenger protein phospholipase Cß4 (Graham et al., 2008) to open a transient receptor potential (TRP) channel, likely the TRPC6 and TRPC7 subtypes in ipRGCs
(Hartwick et al., 2007; Xue et al., 2011). Phospholipase C cleaves phosphatidylinositol
4,5-bisphosphate (PIP2) to form inositol triphosphate (IP3) and diacylglycerol (DAG). In some invertebrate photoreceptors, IP3 acts as a second messenger to raise intracellular calcium levels, whereas other photoreceptors use PIP2, DAG, and/or other molecules to activate TRP channels. It is still unclear how phospholipase C connects and transmits the signal to the TRP channel in ipRGCs (M T H Do & Yau, 2010). The opening of the ipRGC TRP channels results in an influx of ions and depolarization of the membrane potential and the subsequent firing of action potentials. This differs from rod and cone opsins that are coupled to the transducin G-protein that, when activated, triggers a signaling cascade that culminates in to closing of a cyclic nucleotide-gated channel
(Schmidt, Chen, & Hattar, 2011). The mediator enzyme in this cascade is phosphodiesterase (PDE), which hydrolyzes cGMP. With the cation channels closing due to the decreased intracellular levels of cGMP, rod and cone photoreceptors hyperpolarize in response to light.
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Melanopsin has a unique spectral sensitivity with its peak near 480 nm, which is between the peak sensitivity of the short-wavelength cone opsin (437 nm) and rhodopsin
(498 nm; Berson, Dunn, & Takao, 2002; Dacey et al., 2005; Lucas, Douglas, & Foster,
2001). Light with a wavelength of 480 nm is in the blue spectrum, and melanopsin is
1000x more sensitive to blue light near its peak wavelength sensitivity compared to red light at the tail of its action spectrum (Bailies & Lucas, 2013). However, like other opsins, the action spectrum for melanopsin is relatively broad (see Figure 6) and red light can also stimulate ipRGCs if it has a sufficiently high irradiance.
Figure 6. Left) The spectral sensitivity of human melanopsin. The data was fit to a predicted absorbance spectrum of a vitamin A-based opsin with a �max = 479 nm (from Bailies & Lucas, 2013).
The threshold irradiance for melanopsin is higher than other photoreceptors and is near 1013 photons/s/cm2 for wavelengths of light around the peak sensitivity. At the peak wavelength of each photoreceptor’s action spectrum, ipRGCs are less sensitive than rods by a factor of six log units (1,000,000x) and less sensitive than cones by four log units
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(10,000x; Do & Yau, 2010). Part of this lower sensitivity is due to a lower density of melanopsin photopigment in the dendrites and soma of ipRGCs compared to the opsin proteins in rods and cones (reduced by a factor of 10,000; Do et al., 2009). ipRGCs also have a much slower response to a photon of light compared to rods and cones. When examining the single-photon response of ipRGCs by dividing the area under the response by the response peak, a mouse ipRGC has an integration time of eight seconds (Do et al.,
2009). In comparison, this is twenty times slower than rods and one hundred times slower than cones (Chen et al., 1999; Kraft, 1988). ipRGCs respond to steps of light differently based on the luminance. Dimmer steps of light produce a response that is the equivalent of a string of single-photon responses. When the step of light is brighter, these responses accumulate to form a sustained firing of action potentials. ipRGCs exhibit light adaptation similar to other photoreceptors in that the initial response to a bright light is the peak, which then relaxes back into a sustained response of lower voltage (Wong,
Dunn, & Berson, 2005). ipRGCs also adapt in having a quicker response to a single bright light or to a bright light with a steady background light. The purpose of light adaptation in ipRGCs is still unclear.
Continued work has found that there are five subtypes of ipRGCs that all express melanopsin to varying degrees and differ in their anatomy, signaling, and interaction with other retinal cells (Schmidt et al., 2011). The M1 subtype, the first to be studied and classified, has expansive dendrites that stratify in the outer sublamina of the inner plexiform layer (IPL). The M2 subtype, which has a larger soma and more expansive branching of its dendrites, stratifies in the inner sublamina of the IPL (Berson, Castrucci,
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& Provencio, 2010; Schmidt & Kofuji, 2009; Viney et al., 2007). The M3 subtype, similar in size and expanse of dendritic branching to the M2 subtype, is bistratified with dendrites extending into both laminae of the IPL. The specific branching of M3 dendrites can have more variability compared to other ipRGC subtypes (Schmidt & Kofuji, 2011;
Viney et al., 2007). The M4 and M5 subtypes of ipRGCs, though they cannot be easily stained with a melanopsin antibody, exhibit an intrinsic light response. Both subtypes stratify in the inner sublamina of the IPL. The M4 subtype has the largest soma and most expansive dendritic branching of all ipRGCs, whereas the M5 subtype has small and highly-branched dendrites that stay close to the cell’s soma (Ecker et al., 2010).
The ipRGC subtypes are as different in function as they are structure. The M1 subtype has a greater and more-sensitive light response compared to the M2 subtype, which is less sensitive to light by one log unit (Schmidt & Kofuji, 2009). The M1 subtype has a greater input resistance and more-depolarized resting membrane potential than M2, but M2 spikes at higher frequencies than the M1 subtype. The M3 cells, even with the variability in their dendritic structure, have a constant intrinsic light response (Schmidt &
Kofuji, 2011). The M4 and M5 subtypes, which physiologically express fewer melanopsin proteins, unsurprisingly have the smallest and least sensitive response to light
(Ecker et al., 2010). Research has also shown variability in how each subtype responds to input from the outer retina. Signaling by the M2 and M3 subtypes are more dependent upon input from rod and cone photoreceptors, whereas the M1 subtype has a greater intrinsic photosensitivity (Schmidt & Kofuji, 2010; Schmidt & Kofuji, 2011).
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As the dendrites of ipRGCs stratify in the inner plexiform layer of the retina, they are similar to other retinal ganglion cells in that they receive synaptic input from bipolar and amacrine cells. The outer sublamina of the IPL (closer to the inner nuclear layer) is named the OFF-sublamina due to its input from OFF-bipolar cells that respond to the removal of a light stimulus. The inner layer (closer to the ganglion cell layer), named the
ON-sublamina, receives input from ON-bipolar cells that respond to the introduction of a light stimulus. Due to the above description of each subtype’s stratification (M1 in OFF,
M2/M4/M5 in ON, and M3 in ON/OFF) we would expect the M1 to receive input from
OFF-bipolar cells, M2/M4/M5 cells to receive input from ON-bipolar cells, and M3 to receive input from both OFF- and ON-bipolar cells; however, it has been discovered that
M1, M2, and M3 cells all receive primarily ON input, with a very small OFF input to M1 cells (Berson et al., 2002; Wong, Dunn, Graham, & Berson, 2007). The interaction of
ON-bipolar cells with M1 ipRGCs is possible through en passant synapses, which are synapses that do not involve the axon terminal. This indicates that ipRGCs are much more responsive to light onset than to light offset.
The subtypes of ipRGCs also differ in where their axons synapse in the central nervous system. M1 cells project to the suprachiasmatic nucleus (SCN) for circadian photoentrainment and the olivary pretectal nucleus (OPN) to regulate the pupillary light reflex (PLR; Hattar, Liao, Takao, Berson, & Yau, 2002). They also project to other areas of the brain that are involved in circadian rhythms, including the intergeniculate leaflet
(IGL), ventral lateral geniculate nucleus (vLGN), the supraoptic nucleus, ventral subparaventricular zone, medial amygdala, and lateral habenula (Schmidt et al., 2011).
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Non-M1 ipRGCs have been shown to also travel to the OPN (for unknown reasons) as well as the dorsal lateral geniculate nucleus (dLGN), the processing center for information on its way to the visual cortex, and the superior colliculus. Dacey et al.
(2005) showed an ON-response of ipRGCs to long wavelength (L+M) cones and an OFF- response by short wavelength (S) cones, though the OFF response was overshadowed by the ON-signaling through melanopsin. This dichotomy regarding response to different wavelengths, and the axonal projections to the dLGN, indicate a potential involvement of ipRGCs to image-forming vision.
Functions of ipRGCs
The pupillary light reflex (PLR) occurs when the iris sphincter muscle constricts in response to light, and brighter light causes a greater pupil constriction. This phenomenon limits saturation of rods and cones, increasing depth of field, and improving visual resolution (Do & Yau, 2010). Lucas et al. (2001) showed that melanopsin- containing cells contribute to the pupillary light reflex in rodents without rod or cone photoreceptors. Their work, which contributed to the discovery of intrinsically- photosensitive retinal ganglion cells, showed that in rodents lacking rods and cones, the iris sphincter muscle was still constricting in response to light. The presence of the PLR in the absence of rods and cones indicated another photoreceptor was present and capable of inducing this reflex. In melanopsin-deficient mice, the minimum light-evoked pupil size was larger than in wild-type mice (Lucas et al., 2003), and PLR was transient and did
17 not maintain a sustained pupil constriction after tens of seconds of light exposure (Zhu et al., 2007).
The PLR in rodless/coneless mice (with intact melanopsin) was diminished at lower illumination levels, which is supported by the knowledge that melanopsin has a greater luminance threshold compared to rod and cone opsins. The pupil responses of the rodless/coneless mice also showed greater and more sustained constriction compared to rodents with rods and cones that lacked melanopsin (Lucas et al., 2003). The sustained firing of action potentials leads to sustained pupil constriction in the presence of light and is an in vivo indicator of ipRGC activity. Strong evidence, based its action spectrum and the persistence of the effect following intravitreal injections of synaptic blockers in non- human primates, indicates that the sustained pupil constriction that occurs with prolonged light exposure is primarily mediated by ipRGCs (Gamlin et al., 2007; McDougal &
Gamlin, 2010), and this is a key outcome measure in this work. As mentioned above, ipRGCs take longer to respond to a single photon of light compared to rod- and cone- driven responses. ipRGCs also exhibit a delay in the cessation of their action potential firing after a light stimulus is turned off. It is well established that there is a slower recovery in pupil re-dilation after exposure to a bright blue light as compared to a similarly bright red light (Gamlin et al., 2007; Kankipati, Girkin, & Gamlin, 2010; Park et al., 2011). This persistent post-illumination pupillary response reflects the contribution of the sluggishly responding ipRGCs (Gamlin et al., 2007).
The circadian pacemaker is the control center that regulates daily cycles of physiology and behavior, including sleep and wakefulness (Pittendrigh & Daan, 1976a).
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Under constant light and temperature conditions, an organism’s circadian pacemaker enters a “freerunning” cycle that is typically slightly less or longer than 24 hours
(Aschoff, 1979). The circadian pacemaker is regulated by light through a process called photoentrainment, when the shift from dark to light or light to dark changes the timing of the organism’s internal rhythms to match its external environment (Aschoff, 1979). In mice, which are nocturnal animals, the introduction of a light stimulus will cause the animal to be inactive or to begin its sleep cycle, whereas the removal of light will typically be associated with an increase in activity. Under free-running conditions, the introduction of light near either the beginning or end of a sleep cycle can cause a “phase shift” that results in the animal beginning or ending their next sleep cycle at an earlier or later time, respectively (Decoursey, 1986; Pittendrigh & Daan, 1976b). In humans, the sleep/wake cycle is influenced by the hormone melatonin, which is produced by the pineal gland and reaches a peak concentration at night. Melatonin production is reduced by light (Zawilska, Skene, & Arendt, 2009). Though melanopsin is not required for sleep regulation, it does influence the circadian pacemaker, as genetically modified mice without melanopsin exhibit altered phase shifts following exposure to light pulses (Panda et al., 2002). Mice lacking melanopsin show a phase shift of 40% lesser magnitude compared to wild-type mice (Ruby et al., 2002). Thus, melanopsin increases the effectiveness of an animal’s ability to photoentrain to its external environment.
Neuromodulation of ipRGCs
Previous studies have shown that ipRGCs are modulated in their response by
19 other retinal compounds. Retinal dopamine is produced by dopaminergic amacrine cells in response to light (Witkovsky, 2004). Dopamine levels are higher during the day due to light stimulation and circadian control (Doyle, McIvor, & Menaker, 2002; Pozdeyev &
Lavrikova, 2000). Dopamine is a neuromodulator that is involved in light adaptation, causing the visual processing pathway to be less sensitive to light during the day and more sensitive at night (Witkovsky, 2004). Within the inner plexiform layer, the dendrites of dopaminergic amacrine cells and ipRGCs are in close proximity (Vugler et al., 2007), and it has been proposed that there is a direct synapse between these two cells in the OFF-sublamina of the IPL (Viney et al., 2007). Dopamine depolarizes the resting membrane potential of ipRGCs through its action on D1-receptors expressed by these photoreceptors (Van Hook, Wong, & Berson, 2012). There is also evidence that melanopsin-based ipRGC phototransduction drives sustained signaling of dopaminergic amacrine cells (Zhang et al., 2008).
Adenosine is produced in the retina in response to darkness or at night by the circadian clock (Ribelayga & Mangel, 2005). It has been found that stimulation of A1- receptors, naturally responsive to adenosine, causes intracellular decreases of intracellular cAMP levels and protein kinase A (PKA) activity in retinal ganglion cells. In ipRGCs, this decrease of cAMP decreases the duration and frequency of light-evoked spiking in these cells, resulting in diminished responses to light (Sodhi & Hartwick, 2014). It has been previously found that ipRGCs do not alter their inherent light sensitivity throughout the day (Weng, Wong, & Berson, 2009), although there is in vivo data that suggests that the contribution of ipRGCs to the PLR exhibits a circadian rhythm, with a diminished
20 contribution at night (Zele, Feigl, Smith, & Markwell, 2011). The modulation of ipRGC signaling by adenosine during the night, resulting in less robust light-evoked ipRGC spiking responses, could be a mechanism to explain the reduction in the post-illumination pupil response at night.
Cholinergic Stimulation of ipRGCs
Previous work has shown that ipRGCs in rat retinas fire sustained action potentials in the presence of 100 µm carbachol, a cholinergic agonist (Sodhi & Hartwick,
2016). This effect was negated in the presence of atropine, a metabotropic muscarinic antagonist, indicating the presence of muscarinic receptors on ipRGC membranes. 4-
DAMP (M1/M3 antagonist) and AF-DX 384 (M2/M4 antagonist) were individually added to the retinas, and the response to carbachol was significantly reduced by 4-DAMP. This indicates the muscarinic receptors are specifically M1 or M3. These Gq-coupled receptors have a similar pathway to the melanopsin photopigment (D. A. Brown, 2010).
Melanopsin-driven stimulation of ipRGCs by light in the presence of carbachol were not additive, but occluded one another, indicating that these two mechanisms may share a common effector pathway. This theory of a common pathway is supported by Q. Wang et al. (2017), who studied melanopsin present in rodent irides and the local PLR. Their findings, which involved an effect of melanopsin signaling on the iris sphincter muscle, also showed a common pathway between melanopsin and acetylcholine signaling, involving a Gq-coupled receptor and phospholipase C to produce IP3 and increased intracellular calcium levels.
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The source of endogenous acetylcholine in the retina is from starburst amacrine cells, mentioned previously, that respond especially well to moving gratings or flickering light. Evidence of cholinergic stimulation of ipRGCs via endogenous acetylcholine was achieved through application of physostigmine, a cholinesterase inhibitor. In the presence of physostigmine with all other experiment parameters, ipRGC firing increased. This effect was not seen when the rat retinas were pre-treated with atropine, a cholinergic antagonist.
Since starburst amacrine cells best respond to flickering light at a rate of 3-10 Hz, flickering yellow light (6 Hz, 2 x 1012 photons/s, 585 nm) was applied to the retinas for five minutes. This irradiance was below melanopsin threshold and meant to stimulate a solely cone-driven response to light. There was prolonged ipRGC spiking in response to the light stimulus, similar in appearance to a melanopsin-driven response and distinctly different than the glutamate-driven response. To prove this was through cholinergic stimulation, atropine-treated retinas were also exposed to the flickering light, and the ipRGC response was significantly reduced. The study suggested that the ipRGC spiking responses to the relatively dim flickering yellow light was mediated through an acetylcholine-mediated pathway. The latency of response seen in this experiment could be explained by volume transmission of acetylcholine, as discussed before, compared to wired transmission. However, wired transmission can also be supported through the proximity of ipRGC and SBAC dendrites (Chang, Strang, & Gamlin, 2014, unpublished).
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Purpose of the Study
The purpose of my study was to investigate human pupil responses to flickering red and blue light stimuli of different frequencies and intensities and determine whether the responses support the existence of a cholinergic pathway for ipRGC stimulation in the human retina. As 6 Hz flickering light is known to be an effective stimulus for evoking retinal acetylcholine release, I hypothesize that a 6 Hz flickering light stimulus, presented at an irradiance below that required for melanopsin activation, will stimulate robust and sustained pupil constriction that is more commonly associated with ipRGC stimulation.
These responses will be compared to those evoked by higher (30 Hz) and lower (0 and
0.1 Hz) frequencies of light.
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Methods
IRB Approval and Patient Recruitment
This cross-sectional study from The Ohio State University College of Optometry was approved by the Institutional Review Board (IRB) in Biomedical Sciences and follows the tenants of the Declaration of Helsinki. Seven subjects completed the study, ranging in ages 23-43, and 71% were female. Each subject completed all sessions of the study. Subjects were recruited by word of mouth amongst faculty and students of the
Ohio State University College of Optometry. Subjects were educated on the purpose of the study and signed both informed consent and HIPAA authorization prior to continuing with the study. They were asked for verbal consent to continue the study at each subsequent visit, and could choose to leave the study at any time.
Requirements for inclusion in the study were ages ranging from 18-45 with good ocular health, open anterior segment angles indicating safety with pupil dilation, and a refractive error not exceeding a spherical equivalent of ±6 diopters with a best-corrected visual acuity of 20/25 or better on the days of each visit. Grounds for exclusion from the study included a diagnosis of glaucoma or other optic nerve disease, history of retinopathy or retinal detachment, history of traumatic brain injury requiring hospitalization, presence of significant cataract, taking medications that may affect pupil function, history of unreactive pupils, any history of retinal disease, and a history of head injury resulting in loss of consciousness.
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Study Design and Equipment
Light stimuli were presented using a custom-made optical system that is similar to one used by Kankipati et al. (2010). It involves an extended Maxwellian-view system.
The traditional Maxwellian optical system consists of placing a single lens between a light source and the viewer’s eye to focus the rays at the center of the pupil, maximizing illumination of the retina (Westheimer, 1966). The extended Maxwellian system consists of two equal-powered lenses separated by a distance equal to double their focal lengths
(Beer, MacLeod, & Miller, 2005). This study used two 3” x 3” Fresnel lenses separated by 6”. The end of the fiber optic light guide (Edmund Optics, Barrington, NJ) was placed at the focal plane of the first Fresnel lens. The subject was placed in a chin and forehead rest and situated so that the corneal plane of the left eye was in the focal plane of the second Fresnel lens. Traditional Maxwellian systems require precise placement of the subject through use of a bite bar, and the light stimulus coming from a traditional system is very bright. In the extended system, a 5° holographic diffuser (Edmund Optics) was placed between the second Fresnel lens and the subject’s eye to diffuse uniform light along the corneal plane with a visual angle of approximately 53 degrees. The extended
Maxwellian-view design allowed for some variability in the exact position of each subject’s eye and increased subject comfort during data collection, in comparison to the use of a bite bar.
Red light (peak �= 635 nm, dominant �= 625nm, full width at half maximum =
17 nm) and blue light (peak �= 465 nm, dominant �= 470 nm, full width at half maximum = 22 nm) were presented by a light-emitting diode (LED) microscope
25 illuminator (DiCon LED, Richmond, CA). Computer control (LightControl software,
DiCon LED) and neutral density filters were used to create irradiance values of 1 x 1012 photons/s/cm2 for blue light and 7 x 1012 photons/s/cm2 for red light, as these values have been previously shown in the laboratory to cause an equal initial pupil constriction. When the target irradiance was 1010 photons/s/cm2, the simple modification included the use of an additional neutral density filter to decrease the luminance of the light. Photon density at the corneal plane was determined using an optical power meter (Newport, Irvine, CA).
In each session, the subject’s left eye was dilated with 1% tropicamide to minimize fluctuations in retinal illumination in response to the flickering light and variations in pupil size. The consensual pupil constriction of the subject’s right eye was captured under infrared light conditions using a Sony HDR-XR500V high definition digital video camera (Tokyo, Japan). The camera captured images at 30 frames per second.
Session Structure and Stimuli
Subjects were recruited for four sessions, each including exposure to four test stimuli. All sessions began between 12:00 and 15:00 to negate circadian effects on ipRGC activity and pupil size. The sessions differed in color and photon density values and were as follows: blue light (480 nm) at 1012 photons/s/cm2 (hereafter referred to as
“bright” light), “bright” red light (620 nm), blue light at 1010 photons/s/cm2 (hereafter referred to as “dim” light), and “dim” red light. Each test stimulus within a session had the same wavelength and photon density but the four test stimuli used in each session
26 differed in its flicker frequency as follows: 0 Hz (continuous light), 0.1 Hz, 6 Hz, and 30
Hz. The order of the test stimuli was randomized for each subject but kept the same throughout the four sessions to better compare results. At the start of each session with
“dim” light, the subjects were exposed to a short “bright” test stimulus lasting 5 seconds to be used for data analysis.
At the start of each session, the subject’s left eye was dilated with 1% tropicamide; the first test stimulus was presented 30 minutes after dilation. Each test stimulus had a 5-minute duration and was preceded by 15 minutes of dark adaptation. In the “dim” sessions, the first 15-minute dark adaptation began immediately after instillation of tropicamide, and the “bright” test stimulus was presented after the first dark adaptation. This 5 second-long “bright” test stimulus was used to normalize the pupil constriction values (100% constriction reflected the smallest pupil size evoked after stimulation by the “bright” stimulus) in the “dim” light sessions, and to facilitate comparison of the data between the different sessions. The first test stimulus was presented 30 minutes after dilation in the “dim” session, as occurred in the “bright” session.
Data and Statistical Analysis
The recorded videos of each subject’s right eye were analyzed with MATLAB to determine the area of the pupil in pixels. These number were then entered into a
Microsoft Excel document and filtered to remove errors and eyelid blinks.
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For each session, the maximum pupil size and minimum pupil size over the first
10 seconds of each of the four stimuli were normalized to be 100% and 0% constriction, respectively, with the following equation: