Intrinsically photosensitive melanopsin retinal ganglion cell contributions to the pupillary light reflex and circadian rhythm

Recently discovered intrinsically photosensitive melanopsin retinal ganglion cells contribute to the maintenance of pupil diameter, recovery and post-illumination components of the pupillary light reflex and provide the primary environmental light input to the suprachiasmatic nucleus for photoentrainment of the circadian rhythm. This review summarises recent progress in understanding intrinsically photosensitive ganglion cell histology and physiological properties in the context of their contribution to the pupillary and circadian functions and introduces a clinical framework for using the pupillary light reflex to evaluate inner retinal (intrinsically photosensitive melanopsin ganglion cell) and outer retinal (rod and cone photoreceptor) function in the detection of retinal eye disease.

Intrinsically photosensitive melanopsin retinal ganglion cell contributions to the post-illumination pupil response and circadian rhythm

Intrinsically photosensitive retinal ganglion cells (ipRGCs) in the eye transmit the environmental light level, projecting to the suprachiasmatic nucleus (SCN) (Berson, Dunn & Takao, 2002; Hattar, Liao, Takao, Berson & Yau, 2002), the location of the circadian biological clock, and the olivary pretectal nucleus (OPN) of the pretectum, the start of the pupil reflex pathway (Hattar, Liao, Takao, Berson & Yau, 2002; Dacey, Liao, Peterson, Robinson, Smith, Pokorny, Yau & Gamlin, 2005). The SCN synchronizes the circadian rhythm, a cycle of biological processes coordinated to the solar day, and drives the sleep/wake cycle by controlling the release of melatonin from the pineal gland (Claustrat, Brun & Chazot, 2005). Encoded photic input from ipRGCs to the OPN also contributes to the pupil light reflex (PLR), the constriction and recovery of the pupil in response to light. IpRGCs control the post-illumination component of the PLR, the partial pupil constriction maintained for > 30 sec after a stimulus offset (Gamlin, McDougal, Pokorny, Smith, Yau & Dacey, 2007; Kankipati, Girkin & Gamlin, 2010; Markwell, Feigl & Zele, 2010). It is unknown if intrinsic ipRGC and cone-mediated inputs to ipRGCs show circadian variation in their photon-counting activity under constant illumination. If ipRGCs demonstrate circadian variation of the pupil response under constant illumination in vivo, when in vitro ipRGC activity does not (Weng, Wong & Berson, 2009), this would support central control of the ipRGC circadian activity. A preliminary experiment was conducted to determine the spectral sensitivity of the ipRGC post-illumination pupil response under the experimental conditions, confirming the successful isolation of the ipRGC response (Gamlin, et al., 2007) for the circadian experiment. In this main experiment, we demonstrate that ipRGC photon-counting activity has a circadian rhythm under constant experimental conditions, while direct rod and cone contributions to the PLR do not. Intrinsic ipRGC contributions to the post-illumination pupil response decreased 2:46 h prior to melatonin onset for our group model, with the peak ipRGC attenuation occurring 1:25 h after melatonin onset. Our results suggest a centrally controlled evening decrease in ipRGC activity, independent of environmental light, which is temporally synchronized (demonstrates a temporal phase-advanced relationship) to the SCN mediated release of melatonin. In the future the ipRGC post-illumination pupil response could be developed as a fast, non-invasive measure of circadian rhythm. This study establishes a basis for future investigation of cortical feedback mechanisms that modulate ipRGC activity.

The post-illumination pupil response of melanopsin expressing intrinsically photosensitive retinal ganglion cells in diabetes

Purpose: This study investigates the clinical utility of the melanopsin expressing intrinsically photosensitive retinal ganglion cell (ipRGC) controlled post-illumination pupil response (PIPR) as a novel technique for documenting inner retinal function in patients with Type II diabetes without diabetic retinopathy. Methods: The post-illumination pupil response (PIPR) was measured in seven patients with Type II diabetes, normal retinal nerve fiber thickness and no diabetic retinopathy. A 488 nm and 610 nm, 7.15º diameter stimulus was presented in Maxwellian view to the right eye and the left consensual pupil light reflex was recorded. Results: The group data for the blue PIPR (488 nm) identified a trend of reduced ipRGC function in patients with diabetes with no retinopathy. The transient pupil constriction was lower on average in the diabetic group. The relationship between duration of diabetes and the blue PIPR amplitude was linear, suggesting that ipRGC function decreases with increasing diabetes duration. Conclusion: This is the first report to show that the ipRGC controlled post-illumination pupil response may have clinical applications as a non-invasive technique for determining progression of inner neuroretinal changes in patients with diabetes before they are ophthalmoscopically or anatomically evident. The lower transient pupil constriction amplitude indicates that outer retinal photoreceptor inputs to the pupil light reflex may also be affected in diabetes.

Melanopsin expressing intrinsically photosensitive Retinal Ganglion cells in retinal disease

Melanopsin containing intrinsically photosensitive Retinal Ganglion Cells (ipRGCs) are a class of photoreceptors with established roles in non-image forming processes. Their contributions to image forming vision may include the estimation of brightness. Animal models have been central for understanding the physiological mechanisms of ipRGC function and there is evidence of conservation of function across species. ipRGCs can be divided into 5 ganglion cell subtypes that show morphological and functional diversity. Research in humans has established that ipRGCs signal environmental irradiance to entrain the central body clock to the solar day for regulating circadian processes and sleep. In addition, ipRGCs mediate the pupil light reflex (PLR), making the PLR a readily accessible behavioural marker of ipRGC activity. Less is known about ipRGC function in retinal and optic nerve disease, with emerging research providing insight into their function in diabetes, retinitis pigmentosa, glaucoma and hereditary optic neuropathy. We briefly review the anatomical distributions, projections and basic physiological mechanisms of ipRGCs, their proposed and known functions in animals and humans with and without eye disease. We introduce a paradigm for differentiating inner and outer retinal inputs to the pupillary control pathway in retinal disease and apply this paradigm to patients with age-related macular degeneration (AMD). In these cases of patients with AMD, we provide the initial evidence that ipRGC function is altered, and that the dysfunction is more pronounced in advanced disease. Our perspective is that with refined pupillometry paradigms, the pupil light reflex can be extended to AMD assessment as a tool for the measurement of inner and outer retinal dysfunction.

Assessing rod, cone and melanopsin contributions to human pupil flicker responses

Purpose: To determine the relative contributions of rods, cones and melanopsin to pupil responses in humans using temporal sinusoidal stimulation for light levels spanning the low mesopic to photopic range. Methods: A four-primary Ganzfeld photostimulator controlled flicker stimulations at seven light levels (-2.7 to 2 log cd/m2) and five frequencies (0.5 to 8Hz). Pupil diameter was measured using a high-resolution eyetracker. Three kinds of sinusoidal photoreceptor modulations were generated using silent substitution: 1) rod modulation, 2) cone modulation, and 3) combined rod and cone modulation in phase (Experiment 1) or phase shifted (Experiment 2) from a fixed rod phase. The melanopsin excitation was computed for each condition. A vector sum model was used to estimate the relative contribution of rods, cones and melanopsin to the pupil response. Results: From Experiment 1, the pupil frequency response peaked at 1Hz at two mesopic light levels for the three modulation conditions. Analyzing the rod-cone phase difference for the combined modulations (Experiment 2) identified a V-shaped response amplitude with a minimum between 135° and 180°. The pupil response phases increased as cone modulation phase increased. The pupil amplitude increased with increasing light level for cone and combined in-phase rod and cone modulation, but not for the rod modulation. Conclusions: These results demonstrate that cone- and rod-pathway contributions are more predominant than melanopsin contribution to the phasic pupil response. The combined rod, cone and melanopsin inputs to the phasic state of the pupil light reflex follow linear summation.

Temporal characteristics of melanopsin inputs to the human pupil light reflex

Rods, cones and melanopsin containing intrinsically photosensitive retinal ganglion cells (ipRGCs) operate in concert to regulate pupil diameter. The temporal properties of intrinsic ipRGC signalling are distinct to those of rods and cones, including longer latencies and sustained signalling after light offset. We examined whether the melanopsin mediated post-illumination pupil response (PIPR) and pupil constriction were dependent upon the inter-stimulus interval (ISI) between successive light pulses and the temporal frequency of sinusoidal light stimuli. Melanopsin excitation was altered by variation of stimulus wavelength (464 nm and 638 nm lights) and irradiance (11.4 and 15.2 log photons cm(-2) s(-1)). We found that 6s PIPR amplitude was independent of ISI and temporal frequency for all melanopsin excitation levels, indicating complete summation. In contrast to the PIPR, the maximum pupil constriction increased with increasing ISI with high and low melanopsin excitation, but time to minimum diameter was slower with high melanopsin excitation only. This melanopsin response to briefly presented pulses (16 and 100 ms) slows the temporal response of the maximum pupil constriction. We also demonstrate that high melanopsin excitation attenuates the phasic peak-trough pupil amplitude compared to conditions with low melanopsin excitation, indicating an interaction between inner and outer retinal inputs to the pupil light reflex. We infer that outer retina summation is important for rapidly controlling pupil diameter in response to short timescale fluctuations in illumination and may occur at two potential sites, one that is presynaptic to extrinsic photoreceptor input to ipRGCs, or another within the pupil control pathway if ipRGCs have differential temporal tuning to extrinsic and intrinsic signalling.

Melanopsin mediated post-illumination pupil response in early age-related macular degeneration

Purpose To determine whether melanopsin expressing intrinsically photosensitive Retinal Ganglion Cell (ipRGC) inputs to the pupil light reflex (PLR) are affected in early age-related macular degeneration (AMD). Methods The PLR was measured in 40 participants (20 early AMD and 20 age-matched controls) using a custom-built Maxwellian-view pupillometer. Sinusoidal stimuli (0.5 Hz, 11.9 s duration, 35.6° diameter) were presented to the study eye and the consensual pupil response was measured for stimuli with high melanopsin excitation (464nm; blue) and with low melanopsin excitation (638 nm; red) that biased activation to the outer retina. Two melanopsin PLR metrics were quantified: the Phase Amplitude Percentage (PAP) during the sinusoidal stimulus presentation and the Post-Illumination Pupil Response (PIPR). The PLR during stimulus presentation was analyzed using latency to constriction, transient pupil response and maximum pupil constriction metrics. Diagnostic accuracy was evaluated using receiver operating characteristic (ROC) curves. Results The blue PIPR was significantly less sustained in the early AMD group (p<0.001). The red PIPR was not significantly different between groups (p>0.05). The PAP and blue stimulus constriction amplitude were significantly lower in the early AMD group (p < 0.05). There was no significant difference between groups in the latency or transient amplitude for both stimuli (p>0.05). ROC analysis showed excellent diagnostic accuracy for the blue PIPR metrics (AUC>0.9). Conclusions This is the initial report that the melanopsin controlled PIPR is dysfunctional in early AMD. The non-invasive, objective measurement of the ipRGC controlled PIPR has excellent diagnostic accuracy for early AMD.

Effect of age and refractive error on the melanopsin mediated Post-Illumination Pupil Response (PIPR)

Melanopsin containing intrinsically photosensitive Retinal Ganglion cells (ipRGCs) mediate the pupil light reflex (PLR) during light onset and at light offset (the post-illumination pupil response, PIPR). Recent evidence shows that the PLR and PIPR can provide non-invasive, objective markers of age-related retinal and optic nerve disease, however there is no consensus on the effects of healthy ageing or refractive error on the ipRGC mediated pupil function. Here we isolated melanopsin contributions to the pupil control pathway in 59 human participants with no ocular pathology across a range of ages and refractive errors. We show that there is no effect of age or refractive error on ipRGC inputs to the human pupil control pathway. The stability of the ipRGC mediated pupil response across the human lifespan provides a functional correlate of their robustness observed during ageing in rodent models.

The post-illumination pupil response of melanopsin expressing retinal ganglion cells

Purpose The post-illumination pupil response (PIPR) has been quantified in the literature by four metrics. The spectral sensitivity of only one metric is known and this study quantifies the other three. To optimize the measurement of the PIPR in humans, we also determine the stimulus protocol producing the largest PIPR, the duration of the PIPR, and the metric(s) with the lowest coefficient of variation. Methods The consensual pupil light reflex (PLR) was measured with a Maxwellian view pupillometer (35.6° diameter stimulus). - Experiment 1: Spectral sensitivity of four PIPR metrics [plateau, 6 s, area under curve (AUC) early and late recovery] was determined from a criterion PIPR (n = 2 participants) to a 1 s pulse at five wavelengths (409-592nm) and fitted with Vitamin A nomogram (ƛmax = 482 nm). - Experiment 2: The PLR was measured in five healthy participants [29 to 42 years (mean = 32.6 years)] as a function of three stimulus durations (1 s, 10 s, 30 s), five irradiances spanning low to high melanopsin excitation levels (retinal irradiance: 9.8 to 14.8 log quanta.cm-2.s-1), and two wavelengths, one with high (465 nm) and one with low (637 nm) melanopsin excitation. Intra and inter-individual coefficients of variation (CV) were calculated. Results The melanopsin (opn4) photopigment nomogram adequately described the spectral sensitivity derived from all four PIPR metrics. The largest PIPR amplitude was observed with 1 s short wavelength pulses (retinal irradiance ≥ 12.8 log quanta.cm-2.s-1). Of the 4 PIPR metrics, the plateau and 6 s PIPR showed the least intra and inter-individual CV (≤ 0.2). The maximum duration of the sustained PIPR was 83.4 ± 48.0 s (mean ± SD) for 1 s pulses and 180.1 ± 106.2 s for 30 s pulses (465 nm; 14.8 log quanta.cm-2.s-1). Conclusions All current PIPR metrics provide a direct measure of intrinsic melanopsin retinal ganglion cell function. To measure progressive changes in melanopsin function in disease, we recommend that the intrinsic melanopsin response should be measured using a 1 s pulse with high melanopsin excitation and the PIPR should be analyzed with the plateau and/or 6 s metrics. That the PIPR can have a sustained constriction for as long as 3 minutes, our PIPR duration data provide a baseline for the selection of inter-stimulus intervals between consecutive pupil testing sequences.

Melanopsin ganglion cell function in early age-related macular degeneration

Purpose Melanopsin-expressing retinal ganglion cells (mRGCs) have non-image forming functions including mediation of the pupil light reflex (PLR). There is limited knowledge about mRGC function in retinal disease. Initial retinal changes in age-related macular degeneration (AMD) occur in the paracentral region where mRGCs have their highest density, making them vulnerable during disease onset. In this cross-sectional clinical study, we measured the PLR to determine if mRGC function is altered in early stages of macular degeneration. Methods Pupil responses were measured in 8 early AMD patients (AREDS 2001 classification; mean age 72.6 ± 7.2 years, 5M, and 3F) and 12 healthy control participants (mean age 66.6 ± 6.1 years, 8M and 4F) using a custom-built Maxwellian-view pupillometer. Stimuli were 0.5 Hz sinewaves (10 s duration, 35.6° diameter) of short wavelength light (464nm, blue; retinal irradiance = 14.5 log quanta.cm-2.s-1) to produce high melanopsin excitation and of long wavelength light (638nm, red; retinal irradiance = 14.9 log quanta.cm-2.s-1), to bias activation to outer retina and provide a control. Baseline pupil diameter was determined during a 10 s pre-stimulus period. The post illumination pupil response (PIPR) was recorded for 40 s. The 6 s PIPR and maximum pupil constriction were expressed as percentage baseline (M ± SD). Results The blue PIPR was significantly less sustained (p<0.01) in the early AMD group (75.49 ± 7.88%) than the control group (58.28 ± 9.05%). The red PIPR was not significantly different (p>0.05) between the early AMD (84.79 ± 4.03%) and control groups (82.01 ± 5.86%). Maximum constriction amplitude in the early AMD group for blue (43.67 ± 6.35%) and red (48.64 ± 6.49%) stimuli were not significantly different to the control group for blue (39.94 ± 3.66%) and red (44.98 ± 3.15%) stimuli (p>0.05). Conclusions These results are suggestive of inner retinal mRGC deficits in early AMD. This non-invasive, objective measure of pupil responses may provide a new method for quantifying mRGC function and monitoring AMD progression.

Melanopsin and Clock Genes: Regulation by Light and Endothelin in the Zebrafish ZEM-2S Cell Line

It is well known that clocks are present in brain regions other than the suprachiasmatic nucleus and in many peripheral tissues. In the teleost, Danio rerio, peripheral oscillators can be directly synchronized by light. Danio rerio ZEM-2S embryonic cells respond to light with differential growth: cells kept in constant light exhibited a strong inhibition of proliferation, whereas in cells kept in light:dark (LD) cycles (14L:10D and 10L:14D) or in constant darkness (DD), the doubling times were not statistically different. We demonstrated by RT-PCR followed by PCR that ZEM-2S cells express two melanopsins, Opn4x and Opn4m, and the six Cry genes. The presence of the protein OPN4x was demonstrated by immunocytochemistry. The pattern of temporal expression of the genes Opn4x, Per1, Cry1b, and Clock was studied in ZEM-2S cells kept for five days in 12L:12D or DD. In 12L:12D, the clock genes Per 1 and Cry1b exhibited robust circadian expression, while Opn4x and Clock expression seemed to vary in an ultradian pattern. Both Per1 and Cry1b genes had higher expression during the L phase; Clock gene had an increase in expression coincident with the D phase, and during the subjective night. In DD, the temporal variation of Per1 and Cry1b genes was greatly attenuated but not extinguished, and the higher expressions were shifted to the transition times between subjective day and night, demonstrating that Per and Cry1b were synchronized by the LD cycle. Clock and Opn4x kept the ultradian oscillation, but the rhythm was not statistically significant. As endothelins (ET) have been reported to be a potent stimulator of Per genes in rodents, we investigated the effect of endothelin on ZEM-2S cells, which express ETA receptors. Cells were kept in 12D:12L for five days, and then treated with 10-11 to 10-8M ET-1 for 24h. ET-1 exhibited a biphasic effect on Opn4x expression. At 10-11M, the hormone exerted a highly significant stimulation of Opn4x expression during the L phase and introduced a circadian oscillatory pattern. At 10-10M, a significant increase was seen at ZT21 and ZT0 (i.e., at the end of the D phase and beginning of the L phase), whereas 10-9 and 10-8M ET-1 inhibited the expression of Opn4x at most ZTs. Clock expression was unaffected by 10-8M ET-1; however, in the presence of lower concentrations, the expression was enhanced at some ZTs, strengthening the ultradian oscillation. ET-1 at 10-11 and 10-10M had no effect on Per1 circadian expression; however, 10-9 and 10-8M ET-1 reduced the amplitude of Per1 expression in the beginning of the L phase. ET-1 effects were less evident on Cry 1b. For both genes, the reduction in expression was not sufficient to abolish the circadian oscillatory pattern. Based on these results and data in the literature, a link between ET-1 stimulation of ETA receptors may be established by E4BP4 binding to the promoters and consequent inhibition of gene expression.

Toward a Clinical Protocol for Assessing Rod, Cone, and Melanopsin Contributions to the Human Pupil Response

PURPOSE. To better understand the relative contributions of rod, cone, and melanopsin to the human pupillary light reflex (PLR) and to determine the optimal conditions for assessing the health of the rod, cone, and melanopsin pathways with a relatively brief clinical protocol. METHODS. PLR was measured with an eye tracker, and stimuli were controlled with a Ganzfeld system. In experiment 1, 2.5 log cd/m(2) red (640 +/- 10 nm) and blue (467 +/- 17 nm) stimuli of various durations were presented after dark adaptation. In experiments 2 and 3, 1-second red and blue stimuli were presented at different intensity levels in the dark (experiment 2) or on a 0.78 log cd/m(2) blue background (experiment 3). Based on the results of experiments 1 to 3, a clinical protocol was designed and tested on healthy control subjects and patients with retinitis pigmentosa and Leber`s congenital amaurosis. RESULTS. The duration for producing the optimal melanopsin-driven sustained pupil response after termination of an intense blue stimulus was 1 second. PLR rod-and melanopsin-driven components are best studied with low-and high-intensity flashes, respectively, presented in the dark (experiment 2). A blue background suppressed rod and melanopsin responses, making it easy to assess the cone contribution with a red flash (experiment 3). With the clinical protocol, robust melanopsin responses could be seen in patients with few or no contributions from the rods and cones. CONCLUSIONS. It is possible to assess the rod, cone, and melanopsin contributions to the PLR with blue flashes at two or three intensity levels in the dark and one red flash on a blue background. (Invest Ophthalmol Vis Sci. 2011; 52: 6624-6635) DOI: 10.1167/iovs.11-7586

The expression of melanopsin and clock genes in Xenopus laevis melanophores and their modulation by melatonin

Vertebrates have a central clock and also several peripheral clocks. Light responses might result from the integration of light signals by these clocks. The dermal melanophores of Xenopus laevis have a photoreceptor molecule denominated melanopsin (OPN4x). The mechanisms of the circadian clock involve positive and negative feedback. We hypothesize that these dermal melanophores also present peripheral clock characteristics. Using quantitative PCR, we analyzed the pattern of temporal expression of Opn4x and the clock genes Per1, Per2, Bmal1, and Clock in these cells, subjected to a 14-h light:10-h dark (14L:10D) regime or constant darkness (DD). Also, in view of the physiological role of melatonin in the dermal melanophores of X. laevis, we determined whether melatonin modulates the expression of these clock genes. These genes show a time-dependent expression pattern when these cells are exposed to 14L:10D, which differs from the pattern observed under DD. Cells kept in DD for 5 days exhibited overall increased mRNA expression for Opn4x and Clock, and a lower expression for Per1, Per2, and Bmal1. When the cells were kept in DD for 5 days and treated with melatonin for 1 h, 24 h before extraction, the mRNA levels tended to decrease for Opn4x and Clock, did not change for Bmal1, and increased for Per1 and Per2 at different Zeitgeber times (ZT). Although these data are limited to one-day data collection, and therefore preliminary, we suggest that the dermal melanophores of X. laevis might have some characteristics of a peripheral clock, and that melatonin modulates, to a certain extent, melanopsin and clock gene expression.

Melanopsin Retinal Ganglion Cells: relevance to circadian rhythms and sleep in neurodegeneration

In this PhD thesis 3 projects were addressed focusing on the melanopsin retinal ganglion cells (mRGCs) system and its relevance for circadian rhythms and sleep in neurodegeneration. The first project was aimed at completing the characterization of mRGCs system in hereditary optic neuropathies (LHON and DOA). We confirmed that mRGCs are relatively spared also in post-mortem retinal specimens of a DOA case and pupillometric evaluation of LHON patients showed preservation of the pupillary light reflex, with attenuated responses compared to controls. Cell studies failed to indicate a protective role exerted by melanopsin itself. The second project was aimed at characterizing the possible occurrence of optic neuropathy and rest-activity circadian rhythm dysfunction in Alzheimer (AD) and Parkinson disease (PD), as well as, at histological level, the possible involvement of mRGCs in AD. OCT studies demonstrated a subclinical optic neuropathy in both AD and PD patients, with a different pattern involving the superior and nasal quadrants in AD and the temporal quadrant in PD. Actigraphic studies demonstrated a tendency towards an increased intradaily variability (IV) and reduced relative amplitude (RA) of rest-activity circadian rhythm in AD and a significant increased IV a reduced RA in PD. Immunohistochemical analysis of post-mortem retinal specimens and optic nerve cross-sections of neuropathologically confirmed AD cases demonstrated a significant loss of mRGCs and a nearly significant loss of axons in AD compared to controls. The mRGCs were affected in AD independently from age and magnitude of axonal loss. Overall these results suggest a role of the mRGCs system in the pathogenesis of circadian dysfunction in AD. The third project was aimed at evaluating the possible association between a single nucleotide polymorphism of the OPN4 gene and chronotype or SAD, failing to find any significant association with chronotype, but showing a non-significant increment of TT genotype in SAD.