# The influence of melanopsin stimulation on sensitivity to cone isolating stimulation **Research team (alphabetical order):** Geoffrey K. Aguirre, David H. Brainard, Joris Vincent **Research project:** MeLMSens ## Purpose and Approach The broad purpose of this project is to study the interactions between signals originating with stimulation of melanopsin and signals originating with stimulation of the cones. The specific experiment preregistered here asks whether changes in the melanopsin stimulation level of a steady background affect thresholds for detecting light flicker seen only by the cones. The design of this experiment incorporates knowledge gained from a pilot version of the experiment run on author JV. ## Participants Three participants will be used in this experiment: the three authors JV, DHB, GKA. Author JV will also function as the experimenter and administer each session. The nature of task requires continued attention and head position, and consistency in perceptual judgement. This makes the use of trained psychophysical observers preferred over untrained naive observers. During any data collection, both the experimenter and the participant will not know the order of the adaptation conditions in that session, the flicker contrast on any trial, and which interval contains the target stimulus. We recognize that familiarity with the perceptual quality of the different conditions might still allow the participant to identify a particular condition. ## Stimulus design ### Stimuli The stimuli for this experiment will consist of cone-directed flicker on four backgrounds; all stimulus spectra are designed through silent substitution that will target selected photoreceptors while attempting to silence other photoreceptors. Visual stimuli will be tailored to the age of each individual participant, accounting for their predicted lens density in the prereceptoral filtering (see Spitschan 2014, 2015, 2016 for details). The four backgrounds come in two pairs: one pair of backgrounds (nominally) differs only in how much they excite the melanopsin photoreceptor (Mel-high vs. Mel-low), while keeping excitation of the cones constant; the other pair of backgrounds (nominally) differs only in how much they excite the long- (L), medium- (M) and short- (S) wavelength sensitive cones (LMS-high vs. LMS-low), while keeping excitation of melanopsin constant. For each pair, the change from the low to high excitation background constitutes a 350% contrast step upon the targeted photoreceptor mechanism(s). The cone-directed flicker on each background is designed to deliver a maximum 5% contrast both in the positive direction (increase in excitation compared to background) and the negative direction (decrease in excitation compared to background) directed at the LMS cone opsins (while keeping melanopsin excitation constant). This flicker contrast is adjusted between trials, (nominally) varying between 0% and 5% contrast in 0.1% contrast steps. ### Stimulus delivery All of the stimulus modulations will be generated with a digital light synthesis engine (OneLight Spectra) under computer control, admixed with steady light from a video projector. The stimulus component from the OneLight will be imaged onto a custom diffuser to produce a circular, uniform field of 27.5° diameter. The central 5° diameter of the field will be obscured to block the effects of the foveal macular pigment which can cause variation in photoreceptor spectral sensitivity. Light from the diffuser and will then pass through a beam splitter. Scattered light from the stimulus onto the black central blocker could confound the measurement. To counteract this, steady light from a video projector is imaged onto a custom diffuser, and admixed to the optical signal after the central blocker. The steady light consists of a central spot overlapping with and slightly exceeding the central blocker, and an annulus at and extending outward from the outer edge of the stimulus field. The steady light is dim (approx. 11 cdm-2), but bright enough that the largest LMS flicker contrast (5%) was undetectable in the center region during pilot testing. Light from the projector-aligned diffuser will then pass through the other side of the aforementioned beam splitter. The combined light from the beam splitter will be imaged to the eye through a 35 mm focal length lens. The participant will view the stimulus through a 6 mm diameter artificial pupil placed close to the eye. The participant’s right eye will be pharmacologically dilated with a 1% tropicamide ophthalmic solution following administration of 0.5% proparacaine as a local anesthetic. A rubber eyepiece and chin rest are used to stabilize eye position. ### Stimulus validation At the start of each session, we will take spectroradiometric measurements (PhotoResearch PR-670) of the projector spot, backgrounds, and flicker stimuli, and validate that these stimuli are within acceptable tolerances. Validation measurements will be made at the start (before any acquisitions) and at the end (after all acquisitions have been completed). We will report the results of these validation measurements. Scatter from the admixed steady light from the projector onto the stimulus field will be accounted for when generating the stimuli for a session. Spectroradiometric measurements of the stimulus field will be taken in four locations outside the central blocker and admixed projector light. These measurements are made with the projector light present and absent, and with the OneLight full-power output present or absent. The median spectrum added by the projector light, across locations and conditions, is taken to represent the scatter of the projector light onto the stimulus field. The experimental stimuli are then generated, taking into account this projector light. For each background-pair, contrast between the high and low background is calculated. Target contrast between high and low backgrounds is 350% contrast on the targeted photoreceptor(s). For each flicker stimulus measurements are taken only at nominally 5% contrast on the L, M, and S cone photoreceptors (and 0% contrast on melanopsin) – for practical considerations, we assume that the actual contrast of steps between 0% and this validated 5% contrast are well approximated by this single validation. ## Experiment design ### Trial design Sensitivity will be measured using a 2-interval forced-choice paradigm; on each trial participants are presented with two intervals, and after both intervals indicate which interval contained the target stimulus. One interval (the “target” interval) contains the stimulus, consisting of sinusoidal flicker (5 Hz) produced by the OneLight and directed at modulating L-, M- and S-cone activity while keeping melanopsin stimulation constant. The other (“reference”) interval contains no such modulation, just the steady background. Each interval is 500 ms in duration, with a 500 ms inter stimulus interval. Both the target and reference stimuli are ‘redrawn’ at the same nominal frame rate of 200 Hz, to avoid possible frame rate artifacts confounding measurement. After both intervals have been presented, the participant indicates which interval contains the target via a keypad button press. The response interval is untimed, and participants can take a break or blink before responding - the next trial starts immediately after response. Participants do not receive feedback. ### Acquisition design Flicker sensitivity under different conditions will be tested in separate acquisitions. Each acquisition starts with 5 minutes of adaptation, followed by 120 trials. The contrast of the LMS flicker stimulus is varied from trial to trial through an adaptive staircase method; correct responses by the participant lead to a decrease in nominal flicker contrast, while incorrect responses by the participant lead to an increase in flicker contrast. From the start of adaptation, until the end of the acquisition, the participant keeps their eye in the eyepiece to maintain retinal adaptation state. At the end of the acquisition, initial threshold estimates for each background will be obtained as the mean contrast level across staircase reversals. Spectroradiometric measurements will be taken of the stimuli at these estimated threshold contrasts at the end of the session; both the initial nominal threshold value and the measured spectral information will be reported. Final threshold estimates for each background will be obtained offline via a maximum likelihood fit of a Weibull psychometric function to the nominal contrast data from all 120 trials, using a 70.71% correct criterion to match the performance rate targeted by the staircase procedure. ### Session design Each session will consist of all four acquisitions: high and low melanopsin-directed backgrounds, and high and low cone-directed backgrounds. Each session starts with creation of stimuli tailored to the participant, followed by validation of these spectra. Then, acquisitions are tested in pseudo-random order, under computer control; both participant and experiment are kept blind to this order. The participant adapts to the background for an acquisition for five minutes, and then proceeds to complete 120 trials. Each participant will complete 4 full sessions. ## Planned analyses The primary test is whether cone-directed flicker sensitivity differs between high melanopic and low melanopic adaptation conditions. The difference in flicker sensitivity between high and low cone-direction adaptation conditions functions as a positive control. The primary measure is the nominal bipolar flicker contrast on the L, M and S cone photoreceptors at detection threshold. Each session yields this measure for all four conditions: low cone-directed adaptation, high cone-directed adaptation, low melanopic adaptation, high melanopic adaptation. The four sessions per participant yield four repetitions of each measurement. To account for unknown individual differences, data will be analyzed separately for each participant. We consider the measurements in the high and low conditions paired, or dependent, per adaptation axis. Per participant, per adaptation axis, this yields four paired samples (1 per session). Considering the low sample size per participant, we cannot check for or assume normality in the samples; nonparametric statistics are desired. Per participant, the median (across sessions) flicker sensitivity per condition will be calculated and reported, as well as the standard error of the median (SEM). To test for differences in flicker sensitivity between the high and low adaptation conditions, we will use the nonparametric Wilcoxon signed-rank test. Per participant, two Wilcoxon signed-rank tests will be run: - One will test the null hypothesis that nominal contrast at cone-directed flicker threshold will not be significantly different between the low and high melanopic adaptation condition. We make no prediction whether this null hypothesis will be rejected. - The other will test the null hypothesis that nominal contrast at cone-directed flicker threshold will be significantly different between the low and high cone-directed adaptation condition. We predict that this null hypothesis will not be rejected; we predict there will be no significant difference in nominal contrast at threshold, consistent with the prediction from Weber’s Law.