Insights into the evolutionary origin of the pineal color discrimination mechanism from the river lamprey.

BACKGROUND: Pineal-related organs in cyclostomes, teleosts, amphibians, and reptiles exhibit color opponency, generating antagonistic neural responses to different wavelengths of light and thereby sensory information about its "color". Our previous studies suggested that in zebrafish and iguana pineal-related organs, a single photoreceptor cell expressing both UV-sensitive parapinopsin and green-sensitive parietopsin generates color opponency in a "one-cell system." However, it remains unknown to what degree these opsins and the single cell-based mechanism in the pineal color opponency are conserved throughout non-mammalian vertebrates. RESULTS: We found that in the lamprey pineal organ, the two opsins are conserved but that, in contrast to the situation in other vertebrate pineal-related organs, they are expressed in separate photoreceptor cells. Intracellular electrophysiological recordings demonstrated that the parietopsin-expressing photoreceptor cells with Go-type G protein evoke a depolarizing response to visible light. Additionally, spectroscopic analyses revealed that parietopsin with 11-cis 3-dehydroretinal has an absorption maximum at ~570 nm, which is in approximate agreement with the wavelength (~560 nm) that produces the maximum rate of neural firing in pineal ganglion cells exposed to visible light. The vesicular glutamate transporter is localized at both the parietopsin- and parapinopsin-expressing photoreceptor terminals, suggesting that both types of photoreceptor cells use glutamate as a transmitter. Retrograde tracing of the pineal ganglion cells revealed that the terminal of the parietopsin-expressing cells is located close enough to form a neural connection with the ganglion cells, which is similar to our previous observation for the parapinopsin-expressing photoreceptor cells and the ganglion cells. In sum, our observations point to a "two-cell system" in which parietopsin and parapinopsin, expressed separately in two different types of photoreceptor cells,  contribute to the generation of color opponency in the pineal ganglion cells. CONCLUSION: Our results indicate that the jawless vertebrate, lamprey, employs a system for color opponency that differes from that described previously in jawed vertebrates. From a physiological viewpoint, we propose an evolutionary insight, the emergence of pineal "one-cell system" from the ancestral "multiple (two)-cell system," showing the opposite evolutionary direction to that of the ocular color opponency.

Color opponency with a single kind of bistable opsin in the zebrafish pineal organ.

Lower vertebrate pineal organs discriminate UV and visible light. Such color discrimination is typically considered to arise from antagonism between two or more spectrally distinct opsins, as, e.g., human cone-based color vision relies on antagonistic relationships between signals produced by red-, green-, and blue-cone opsins. Photosensitive pineal organs contain a bistable opsin (parapinopsin) that forms a signaling-active photoproduct upon UV exposure that may itself be returned to the signaling-inactive "dark" state by longer-wavelength light. Here we show the spectrally distinct parapinopsin states (with antagonistic impacts on signaling) allow this opsin alone to provide the color sensitivity of this organ. By using calcium imaging, we show that single zebrafish pineal photoreceptors held under a background light show responses of opposite signs to UV and visible light. Both such responses are deficient in zebrafish lacking parapinopsin. Expressing a UV-sensitive cone opsin in place of parapinopsin recovers UV responses but not color opponency. Changes in the spectral composition of white light toward enhanced UV or visible wavelengths respectively increased vs. decreased calcium signal in parapinopsin-sufficient but not parapinopsin-deficient photoreceptors. These data reveal color opponency from a single kind of bistable opsin establishing an equilibrium-like mixture of the two states with different signaling abilities whose fractional concentrations are defined by the spectral composition of incident light. As vertebrate visual color opsins evolved from a bistable opsin, these findings suggest that color opponency involving a single kind of bistable opsin might have been a prototype of vertebrate color opponency.

Color vision in insects: insights from Drosophila.

Color vision is an important sensory capability that enhances the detection of contrast in retinal images. Monochromatic animals exclusively detect temporal and spatial changes in luminance, whereas two or more types of photoreceptors and neuronal circuitries for the comparison of their responses enable animals to differentiate spectral information independent of intensity. Much of what we know about the cellular and physiological mechanisms underlying color vision comes from research on vertebrates including primates. In insects, many important discoveries have been made, but direct insights into the physiology and circuit implementation of color vision are still limited. Recent advances in Drosophila systems neuroscience suggest that a complete insect color vision circuitry, from photoreceptors to behavior, including all elements and computations, can be revealed in future. Here, we review fundamental concepts in color vision alongside our current understanding of the neuronal basis of color vision in Drosophila, including side views to selected other insects.

Spectral response properties of higher visual neurons in Drosophila melanogaster.

The fruit fly Drosophila melanogaster can process chromatic information for true color vision and spectral preference. Spectral information is initially detected by a few distinct photoreceptor channels with different spectral sensitivities and is processed through the visual circuit. The neuroanatomical bases of the circuit are emerging. However, only little information is available in chromatic response properties of higher visual neurons from this important model organism. We used in vivo whole-cell patch-clamp recordings in response to monochromatic light stimuli ranging from 300 to 650 nm with 25-nm steps. We characterized the chromatic response of 33 higher visual neurons, including their general response type and their wavelength tuning. Color-opponent-type responses that had been typically observed in primates and bees were not identified. Instead, the majority of neurons showed excitatory responses to broadband wavelengths. The UV (300-375 nm) and middle wavelength (425-575 nm) ranges could be separated at the population level owing to neurons that preferentially responded to a specific wavelength range. Our results provide a first mapping of chromatic information processing in higher visual neurons of D. melanogaster that is a suitable model for exploring how color-opponent neural mechanisms are implemented in the visual circuits.

Behavioral color vision in a cichlid fish: Metriaclima benetos.

Color vision is the capacity to discriminate color regardless of brightness. It is essential for many fish species as they rely on color discrimination for numerous ecological tasks. The study of color vision is important because it can unveil the mechanisms that shape coloration patterns, visual system sensitivities and, hence, visual signals. In order to better understand the mechanisms underlying color vision, an integrative approach is necessary. This usually requires combining behavioral, physiological and genetic experiments with quantitative modeling, resulting in a distinctive characterization of the visual system. Here, we provide new data on the color vision of a rock-dwelling cichlid from Lake Malawi: Metriaclima benetos. For this study we used a behavioral approach to demonstrate color vision through classical conditioning, complemented with modeling of color vision to estimate color contrast. For our experiments we took into account opsin coexpression and considered whether cichlids exhibit a dichromatic or a trichromatic visual system. Behavioral experiments confirmed color vision in M. benetos; most fish were significantly more likely to choose the trained over the distracter stimuli, irrespective of brightness. Our results are supported by visual modeling that suggests that cichlids are trichromats and achieve color vision through color opponency mechanisms, which are a result of three different photoreceptor channels. Our analyses also suggest that opsin coexpression can negatively affect perceived color contrast. This study is particularly relevant for research on the cichlid lineage because cichlid visual capabilities and coloration patterns are implicated in their adaptive radiation.

Three distinct blue-green color pathways in a mammalian retina.

In mammalian retinae, the first steps in the process of discrimination of color are mediated by color-opponent neurons that respond with opposite polarity to signals from short (S, blue) and longer wavelength (M, green or L, red) cones. Primates also contain a second system that is different from M and L cones. Although pathways responding to the onset of S-cone stimulation (S-ON) are well known, the existence of bipolar cells and retinal ganglion cells that respond to the offset of S-cone stimulation (S-OFF) has been controversial. We have recorded from and stained three different types of S/M color-opponent ganglion cells in the rabbit retina that are distinguished by the polarity of their responses to S-cone stimulation, the stratification pattern of their dendrites, and the distinct mechanisms underlying their color-opponent responses. We describe an S-ON and an S-OFF pathway formed by amacrine cells inverting the S-ON signal. Most importantly, we also provide both anatomical and physiological evidence for a direct S-OFF pathway dependent on an S-OFF cone bipolar cell. The results indicate a greater diversity of pathways for processing of signals from S-cones than previously suspected.

Horizontal-cell like Dm9 neurons in Drosophila modulate photoreceptor output to supply multiple functions in early visual processing.

Dm9 neurons in Drosophila have been proposed as functional homologs of horizontal cells in the outer retina of vertebrates. Here we combine genetic dissection of neuronal circuit function, two-photon calcium imaging in Dm9 and inner photoreceptors, and immunohistochemical analysis to reveal novel insights into the functional role of Dm9 in early visual processing. Our experiments show that Dm9 receive input from all four types of inner photoreceptor R7p, R7y, R8p, and R8y. Histamine released from all types R7/R8 directly inhibits Dm9 via the histamine receptor Ort, and outweighs simultaneous histamine-independent excitation of Dm9 by UV-sensitive R7. Dm9 in turn provides inhibitory feedback to all R7/R8, which is sufficient for color-opponent processing in R7 but not R8. Color opponent processing in R8 requires additional synaptic inhibition by R7 of the same ommatidium via axo-axonal synapses and the second Drosophila histamine receptor HisCl1. Notably, optogenetic inhibition of Dm9 prohibits color opponent processing in all types of R7/R8 and decreases intracellular calcium in photoreceptor terminals. The latter likely results from reduced release of excitatory glutamate from Dm9 and shifts overall photoreceptor sensitivity toward higher light intensities. In summary, our results underscore a key role of Dm9 in color opponent processing in Drosophila and suggest a second role of Dm9 in regulating light adaptation in inner photoreceptors. These novel findings on Dm9 are indeed reminiscent of the versatile functions of horizontal cells in the vertebrate retina.

Circuit Mechanisms Underlying Chromatic Encoding in Drosophila Photoreceptors.

Spectral information is commonly processed in the brain through generation of antagonistic responses to different wavelengths. In many species, these color opponent signals arise as early as photoreceptor terminals. Here, we measure the spectral tuning of photoreceptors in Drosophila. In addition to a previously described pathway comparing wavelengths at each point in space, we find a horizontal-cell-mediated pathway similar to that found in mammals. This pathway enables additional spectral comparisons through lateral inhibition, expanding the range of chromatic encoding in the fly. Together, these two pathways enable efficient decorrelation and dimensionality reduction of photoreceptor signals while retaining maximal chromatic information. A biologically constrained model accounts for our findings and predicts a spatio-chromatic receptive field for fly photoreceptor outputs, with a color opponent center and broadband surround. This dual mechanism combines motifs of both an insect-specific visual circuit and an evolutionarily convergent circuit architecture, endowing flies with the ability to extract chromatic information at distinct spatial resolutions.

A Color Vision Circuit for Non-Image-Forming Vision in the Primate Retina.

Melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs) synchronize our biological clocks with the external light/dark cycle [1]. In addition to photoentrainment, they mediate the effects of light experience as a central modulator of mood, learning, and health [2]. This makes a complete account of the circuity responsible for ipRGCs' light responses essential to understanding their diverse roles in our well-being. Considerable progress has been made in understanding ipRGCs' melanopsin-mediated responses in rodents [3-5]. However, in primates, ipRGCs also have a rare blue-OFF response mediated by an unknown short-wavelength-sensitive (S)-cone circuit [6]. Identifying this S-cone circuit is particularly important because ipRGCs mediate many of the wide-ranging effects of short-wavelength light on human biology. These effects are often attributed to melanopsin, but there is evidence for an S-cone contribution as well [7, 8]. Here, we tested the hypothesis that the S-OFF response is mediated by the S-ON pathway through inhibitory input from an undiscovered S-cone amacrine cell. Using serial electron microscopy in the macaque retina, we reconstructed the neurons and synapses of the S-cone connectome, revealing a novel inhibitory interneuron, an amacrine cell, receiving excitatory glutamatergic input exclusively from S-ON bipolar cells. This S-cone amacrine cell makes highly selective inhibitory synapses onto ipRGCs, resulting in a blue-OFF response. Identification of the S-cone amacrine cell provides the missing component of an evolutionarily ancient circuit using spectral information for non-image forming visual functions.

Cones Support Alignment to an Inconsistent World by Suppressing Mouse Circadian Responses to the Blue Colors Associated with Twilight.

In humans, short-wavelength light evokes larger circadian responses than longer wavelengths [1-3]. This reflects the fact that melanopsin, a key contributor to circadian assessments of light intensity, most efficiently captures photons around 480 nm [4-8] and gives rise to the popular view that "blue" light exerts the strongest effects on the clock. However, in the natural world, there is often no direct correlation between perceived color (as reported by the cone-based visual system) and melanopsin excitation. Accordingly, although the mammalian clock does receive cone-based chromatic signals [9], the influence of color on circadian responses to light remains unclear. Here, we define the nature and functional significance of chromatic influences on the mouse circadian system. Using polychromatic lighting and mice with altered cone spectral sensitivity (Opn1mwR), we generate conditions that differ in color (i.e., ratio of L- to S-cone opsin activation) while providing identical melanopsin and rod activation. When biased toward S-opsin activation (appearing "blue"), these stimuli reliably produce weaker circadian behavioral responses than those favoring L-opsin ("yellow"). This influence of color (which is absent in animals lacking cone phototransduction; Cnga3-/-) aligns with natural changes in spectral composition over twilight, where decreasing solar angle is accompanied by a strong blue shift [9-11]. Accordingly, we find that naturalistic color changes support circadian alignment when environmental conditions render diurnal variations in light intensity weak/ambiguous sources of timing information. Our data thus establish how color contributes to circadian entrainment in mammals and provide important new insight to inform the design of lighting environments that benefit health.

Toward a Mechanistic Understanding of Color Vision in Insects.

Many visual animals exploit spectral information for seeking food and mates, for identifying preys and predators, and for navigation. Animals use chromatic information in two ways. "True color vision," the ability to discriminate visual stimuli on the basis of their spectral content independent of brightness, is thought to play an important role in object identification. In contrast, "wavelength-specific behavior," which is strongly dependent on brightness, often associates with foraging, navigation, and other species-specific needs. Among animals capable of chromatic vision, insects, with their diverse habitats, stereotyped behaviors, well-characterized anatomy and powerful genetic tools, are attractive systems for studying chromatic information processing. In this review, we first discuss insect photoreceptors and the relationship between their spectral sensitivity and animals' color vision and ecology. Second, we review recent studies that dissect chromatic circuits and explore neural mechanisms of chromatic information processing. Finally, we review insect behaviors involving "true color vision" and "wavelength-specific behaviors," especially in bees, butterflies, and flies. We include examples of high-order color vision, such as color contrast and constancy, which are shared by vertebrates. We focus on Drosophila studies that identified neuronal correlates of color vision and innate spectral preferences. We also discuss the electrophysiological studies in bees that reveal color encoding. Despite structural differences between insects' and vertebrates' visual systems, their chromatic vision appears to employ the same processing principles, such as color opponency, suggesting convergent solutions of neural computation to common problems.

The M5 Cell: A Color-Opponent Intrinsically Photosensitive Retinal Ganglion Cell.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) combine direct photosensitivity through melanopsin with synaptically mediated drive from classical photoreceptors through bipolar-cell input. Here, we sought to provide a fuller description of the least understood ipRGC type, the M5 cell, and discovered a distinctive functional characteristic-chromatic opponency (ultraviolet excitatory, green inhibitory). Serial electron microscopic reconstructions revealed that M5 cells receive selective UV-opsin drive from Type 9 cone bipolar cells but also mixed cone signals from bipolar Types 6, 7, and 8. Recordings suggest that both excitation and inhibition are driven by the ON channel and that chromatic opponency results from M-cone-driven surround inhibition mediated by wide-field spiking GABAergic amacrine cells. We show that M5 cells send axons to the dLGN and are thus positioned to provide chromatic signals to visual cortex. These findings underscore that melanopsin's influence extends beyond unconscious reflex functions to encompass cortical vision, perhaps including the perception of color.

Variations in normal color vision. VI. Factors underlying individual differences in hue scaling and their implications for models of color appearance.

Observers with normal color vision vary widely in their judgments of color appearance, such as the specific spectral stimuli they perceive as pure or unique hues. We examined the basis of these individual differences by using factor analysis to examine the variations in hue-scaling functions from both new and previously published data. Observers reported the perceived proportion of red, green, blue or yellow in chromatic stimuli sampling angles at fixed intervals within the LM and S cone-opponent plane. These proportions were converted to hue angles in a perceptual-opponent space defined by red vs. green and blue vs. yellow axes. Factors were then extracted from the correlation matrix using PCA and Varimax rotation. These analyses revealed that inter-observer differences depend on seven or more narrowly-tuned factors. Moreover, although the task required observers to decompose the stimuli into four primary colors, there was no evidence for factors corresponding to these four primaries, or for opponent relationships between primaries. Perceptions of "redness" in orange, red, and purple, for instance, involved separate factors rather than one shared process for red. This pattern was compared to factor analyses of Monte Carlo simulations of the individual differences in scaling predicted by variations in standard opponent mechanisms, such as their spectral tuning or relative sensitivity. The observed factor pattern is inconsistent with these models and thus with conventional accounts of color appearance based on the Hering primaries. Instead, our analysis points to a perceptual representation of color in terms of multiple mechanisms or decision rules that each influence the perception of only a relatively narrow range of hues, potentially consistent with a population code for color suggested by cortical physiology.

Chromatic clocks: Color opponency in non-image-forming visual function.

During dusk and dawn, the ambient illumination undergoes drastic changes in irradiance (or intensity) and spectrum (or color). While the former is a well-studied factor in synchronizing behavior and physiology to the earth's 24-h rotation, color sensitivity in the regulation of circadian rhythms has not been systematically studied. Drawing on the concept of color opponency, a well-known property of image-forming vision in many vertebrates (including humans), we consider how the spectral shifts during twilight are encoded by a color-opponent sensory system for non-image-forming (NIF) visual functions, including phase shifting and melatonin suppression. We review electrophysiological evidence for color sensitivity in the pineal/parietal organs of fish, amphibians and reptiles, color coding in neurons in the circadian pacemaker in mice as well as sporadic evidence for color sensitivity in NIF visual functions in birds and mammals. Together, these studies suggest that color opponency may be an important modulator of light-driven physiological and behavioral responses.