Evolution of neuronal cell classes and types in the vertebrate retina.

The basic plan of the retina is conserved across vertebrates, yet species differ profoundly in their visual needs1. Retinal cell types may have evolved to accommodate these varied needs, but this has not been systematically studied. Here we generated and integrated single-cell transcriptomic atlases of the retina from 17 species: humans, two non-human primates, four rodents, three ungulates, opossum, ferret, tree shrew, a bird, a reptile, a teleost fish and a lamprey. We found high molecular conservation of the six retinal cell classes (photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (RGCs) and Müller glia), with transcriptomic variation across species related to evolutionary distance. Major subclasses were also conserved, whereas variation among cell types within classes or subclasses was more pronounced. However, an integrative analysis revealed that numerous cell types are shared across species, based on conserved gene expression programmes that are likely to trace back to an early ancestral vertebrate. The degree of variation among cell types increased from the outer retina (photoreceptors) to the inner retina (RGCs), suggesting that evolution acts preferentially to shape the retinal output. Finally, we identified rodent orthologues of midget RGCs, which comprise more than 80% of RGCs in the human retina, subserve high-acuity vision, and were previously believed to be restricted to primates2. By contrast, the mouse orthologues have large receptive fields and comprise around 2% of mouse RGCs. Projections of both primate and mouse orthologous types are overrepresented in the thalamus, which supplies the primary visual cortex. We suggest that midget RGCs are not primate innovations, but are descendants of evolutionarily ancient types that decreased in size and increased in number as primates evolved, thereby facilitating high visual acuity and increased cortical processing of visual information.

Electrophysiology and transcriptomics reveal two photoreceptor classes and complex visual integration in Hirudo verbana.

Among animals with visual processing mechanisms, the leech Hirudo verbana is a rare example in which all neurons can be identified. However, little is known about its visual system, which is composed of several pigmented head eyes and photosensitive non-pigmented sensilla that are distributed across its entire body. Although several interneurons are known to respond to visual stimuli, their response properties are poorly understood. Among these, the S-cell system is especially intriguing: it is multimodal, spans the entire body of the leech and is thought to be involved in sensory integration. To improve our understanding of the role of this system, we tested its spectral sensitivity, spatial integration and adaptation properties. The response of the S-cell system to visual stimuli was found to be strongly dependent on the size of the area stimulated, and adaptation was local. Furthermore, an adaptation experiment demonstrated that at least two color channels contributed to the response, and that their contribution was dependent on the adaptation to the background. The existence of at least two color channels was further supported by transcriptomic evidence, which indicated the existence of at least two distinct groups of putative opsins for leeches. Taken together, our results show that the S-cell system has response properties that could be involved in the processing of spatial and color information of visual stimuli. We propose the leech as a novel system to understand visual processing mechanisms with many practical advantages.

A Comparative Perspective on Extra-retinal Photoreception.

Ubiquitous in non-mammalian vertebrates, extra-retinal photoreceptors (ERPs) have been linked to an array of physiological, metabolic, behavioral, and morphological changes. However, the mechanisms and functional roles of ERPs remain one of the enduring questions of modern biology. In this review article, we use a comparative framework to identify conserved roles and distributions of ERPs, highlighting knowledge gaps. We conclude that ERP research can be divided into two largely unconnected categories: (i) identification and localization of photoreceptors and (ii) linkage of non-retinal light reception to behavioral and physiological processes, particularly endocrine systems. However, the emergence of novel gene editing and silencing techniques is enabling the unification of ERP research by allowing the bridging of this divide.

Biophysical Variation within the M1 Type of Ganglion Cell Photoreceptor.

Intrinsically photosensitive retinal ganglion cells of the M1 type encode environmental irradiance for functions that include circadian and pupillary regulation. Their distinct role, morphology, and molecular markers indicate that they are stereotyped circuit elements, but their physiological uniformity has not been investigated in a systematic fashion. We have profiled the biophysical parameters of mouse M1s and found that extreme variation is their hallmark. Most parameters span 1-3 log units, and the full range is evident in M1s that innervate brain regions serving divergent functions. Biophysical profiles differ among cells possessing similar morphology and between neighboring M1s recorded simultaneously. Variation in each parameter is largely independent of that in others, allowing for flexible individualization. Accordingly, a common stimulus drives heterogeneous spike outputs across cells. By contrast, a population of directionally selective retinal ganglion cells appeared physiologically uniform under similar conditions. Thus, M1s lack biophysical constancy and send diverse signals downstream.

Shaping of Signal Transmission at the Photoreceptor Synapse by EAAT2 Glutamate Transporters.

Photoreceptor ribbon synapses tonically release glutamate. To ensure efficient signal transmission and prevent glutamate toxicity, a highly efficient glutamate removal system provided by members of the SLC1 gene family is required. By using a combination of biophysical and in vivo studies, we elucidate the role of excitatory amino acid transporter 2 (EAAT2) proteins in synaptic glutamate homeostasis at the zebrafish photoreceptor synapse. The main glutamate sink is provided by the glial EAAT2a, reflected by reduced electroretinographic responses in EAAT2a-depleted larvae. EAAT2b is located on the tips of cone pedicles and contributes little to glutamate reuptake. However, this transporter displays both a large chloride conductance and leak current, being important in stabilizing the cone resting potential. This work demonstrates not only how proteins originating from the same gene family can complement each other's expression profiles and biophysical properties, but also how presynaptic and glial transporters are coordinated to ensure efficient synaptic transmission at glutamatergic synapses of the central nervous system.

Eye evolution and its functional basis.

Eye evolution is driven by the evolution of visually guided behavior. Accumulation of gradually more demanding behaviors have continuously increased the performance requirements on the photoreceptor organs. Starting with nondirectional photoreception, I argue for an evolutionary sequence continuing with directional photoreception, low-resolution vision, and finally, high-resolution vision. Calculations of the physical requirements for these four sensory tasks show that they correlate with major innovations in eye evolution and thus work as a relevant classification for a functional analysis of eye evolution. Together with existing molecular and morphological data, the functional analysis suggests that urbilateria had a simple set of rhabdomeric and ciliary receptors used for directional photoreception, and that organ duplications, positional shifts and functional shifts account for the diverse patterns of eyes and photoreceptors seen in extant animals. The analysis also suggests that directional photoreception evolved independently at least twice before the last common ancestor of bilateria and proceeded several times independently to true vision in different bilaterian and cnidarian groups. This scenario is compatible with Pax-gene expression in eye development in the different animal groups. The whole process from the first opsin to high-resolution vision took about 170 million years and was largely completed by the onset of the Cambrian, about 530 million years ago. Evolution from shadow detectors to multiple directional photoreceptors has further led to secondary cases of eye evolution in bivalves, fan worms, and chitons.

Intrinsically photosensitive retinal ganglion cells: classification, function and clinical implications.

PURPOSE OF REVIEW: The discovery of a new class of intrinsically photosensitive retinal ganglion cells (ipRGCs) revealed their superior role for various nonvisual biological functions, including the pupil light reflex, and circadian photoentrainment. RECENT FINDINGS: Recent works have identified and characterized several anatomically and functionally distinct ipRGC subtypes and have added strong new evidence for the accessory role of ipRGCs in the visual system in humans. SUMMARY: This review summarizes current concepts related to ipRGC morphology, central connections and behavioural functions and highlights recent studies having clinical relevance to ipRGCs. Clinical implications of the melanopsin system are widespread, particularly as related to chronobiology.

The retinal mosaics of opsin expression in invertebrates and vertebrates.

Color vision is found in many invertebrate and vertebrate species. It is the ability to discriminate objects based on the wavelength of emitted light independent of intensity. As it requires the comparison of at least two photoreceptor types with different spectral sensitivities, this process is often mediated by a mosaic made of several photoreceptor types. In this review, we summarize the current knowledge about the formation of retinal mosaics and the regulation of photopigment (opsin) expression in the fly, mouse, and human retina. Despite distinct evolutionary origins, as well as major differences in morphology and phototransduction machineries, there are significant similarities in the stepwise cell-fate decisions that lead from progenitor cells to terminally differentiated photoreceptors that express a particular opsin. Common themes include (i) the use of binary transcriptional switches that distinguish classes of photoreceptors, (ii) the use of gradients of signaling molecules for regional specializations, (iii) stochastic choices that pattern the retina, and (iv) the use of permissive factors with multiple roles in different photoreceptor types.

Receptive fields of retinal bipolar cells are mediated by heterogeneous synaptic circuitry.

Center-surround antagonistic receptive field (CSARF) organization is the basic synaptic circuit that serves as elementary building blocks for spatial information processing in the visual system. Cells with such receptive fields converge into higher-order visual neurons to form more complex receptive fields. Retinal bipolar cells (BCs) are the first neurons along the visual pathway that exhibit CSARF organization. BCs have been classified according to their response polarities and rod/cone inputs, and they project signals to target cells at different sublaminae of the inner plexiform layer. On the other hand, CSARFs of various types of BCs have been assumed be organized the same way. Here we examined center and surround responses of over 250 salamander BCs, and demonstrated that different types of BCs exhibit different patterns of dye coupling, receptive field center size, surround response strength, and conductance changes associated with center and surround responses. We show that BC receptive field center sizes varied with the degree of BC-BC coupling, and that surround responses of different BCs are mediated by different combinations of five lateral synaptic pathways mediated by the horizontal cells and amacrine cells. The finding of heterogeneous receptive field circuitry fundamentally challenges the common assumption that CSARFs of different subtypes of visual neurons are mediated by the same synaptic pathways. BCs carrying different visual signals use different synaptic circuits to process spatial information, allowing shape and contrast computation be differentially modulated by various lighting and adaptation conditions.

Four cone types characterized by anti-visual pigment antibodies in the pigeon retina.

Using three antibodies to visual pigments (monoclonal antibodies COS-1 and OS-2, and a polyclonal anti-opsin serum), four different types of cone cells could be distinguished in the red area (dorsoposterior part with the highest density of cones) of the pigeon retina. Both members of the double cone and the single cone with the red oil droplet were labelled with our monoclonal antibody COS-1 (type I cone). The single cone with the orange oil droplet was positive both with anti-opsin and monoclonal antibody OS-2 (type II cone). The single cone exhibiting a yellowish-green oil droplet, fluorescent in ultraviolet light, also reacted with anti-opsin but lacked the antigenic determinant recognized by OS-2 (type III cone). The thin cone with the small colorless oil droplet was negative with both COS-1 and anti-rhodopsin (type IV cone). We propose that the four immunologically distinguishable cone types correspond to cones expressing visual pigments with different (long-, middle-, short-wavelength and ultraviolet) color sensitivities.

Spectral sensitivities of seven morphological types of photoreceptors in the retina of the turtle, Geoclemys reevesii.

Spectral sensitivities of photoreceptors in the turtle (Geoclemys) retina were studied by intracellular recording, and each cell was filled with Lucifer yellow (LY). Photoreceptors were classified into seven morphological types: rod, four types of single cones, and two members of a double cone. Single cones contained one of four different oil droplets: red, pale-green, orange, and clear. Double cones consisted of two apposed cones; principal members contained yellow oil droplets, while accessory members contained no oil droplet. Spectral sensitivities recorded from these seven types of photoreceptors were classified into one type of rod and three chromatic types of cones. Rods (n = 19) showed peak sensitivity at 520 nm. Single cones containing either a red (n = 51) or a pale-green (n = 9) oil droplet were red-sensitive (lambda max at 620 nm). Single cones containing an orange oil droplet (n = 14) were green-sensitive (lambda max at 540 nm). Single cones containing a clear oil droplet (n = 3) were blue-sensitive (lambda max at 460 nm). Both members of the double cone, principal (n = 22) and accessory (n = 15), were red-sensitive (lambda max at 620 nm). No diffusion of LY was detected between the apposed members of double cones. Red-sensitive cones, therefore, consisted of four different morphological types of cones, and they occupy about 70% of the photoreceptor mosaic in the turtle retina.

Electrophysiological measurements of the spectral sensitivity of three types of cones in the carp retina.

The spectral sensitivities of red-, green-, and blue-sensitive cones were measured by intracellular recording in the carp retina. The responses from all cones were univariant, i.e., the waveform of a response to any wavelength and at any intensity could be superimposed on the response to other wavelengths if the intensity was properly selected. Red-sensitive cones showed a maximum sensitivity at about 620 nm, green-sensitive cones at about 520 nm, and blue-sensitive cones at about 460 nm. The peak wavelengths and general forms of the spectral sensitivity curves agreed with those of the spectral absorption curves measured by microspectrophotometry (MSP), but the sensitivity of red-sensitive cones at both ends of the spectrum was significantly lower than the spectral absorption determined by MSP.

Microspectrophotometric demonstration of four classes of photoreceptor in an old world primate, Macaca fascicularis.

1. Microspectrophotometric measurements reveal four classes of photoreceptor in the retina of the cynomolgus monkey, Macaca fascicularis, which is known to possess colour vision similar to that of a normal human trichromat. 2. Although the eyes were removed in bright illumination, the densities of pigment were comparable to those we have measured in dark-adapted rhesus retinae. 3. The mean wave-lengths of peak sensitivity (lambda max) for the four classes of photoreceptor were 415, 500, 535 and 567 nm. 4. The band widths of the absorbance spectra decreased linearly as the wave-number of peak sensitivity decreased. 5. If, by assuming a reasonable value for the axial density of the rod outer segment and correcting for lens absorption, a spectral sensitivity for human vision is reconstructed from the P500 pigment, it is found to be systematically broader than the CIE scotopic sensitivity function. 6. Given explicit assumptions, it is possible from the P535 and P567 pigments to reconstruct human psychophysical sensitivities that resemble the pi 4 and pi 5 mechanisms of W. S. Stiles. 7. Although the P415 pigment has a lambda max much shorter than that of the psychophysically measured blue mechanisms, the two spectral-sensitivity functions are brought into proximity when the microspectrophotometric data are corrected for absorption by the optic media.

Visual pigments of rods and cones in a human retina.

1. Microspectrophotometric measurements have been made of the photopigments of individual rods and cones from the retina of a man. The measuring beam was passed transversely through the isolated outer segments. 2. The mean absorbance spectrum for rods (n = 11) had a peak at 497.6 +/- 3.3 nm and the mean transverse absorbance was 0.035 +/- 0.007. 3. Three classes of cones were identified. The long-wave cones ('red' cones) had a lambda max of 562.8 +/- 4.7 nm (n = 19) with a mean transverse absorbance of 0.027 +/- 0.005. The middle-wave cones ('green' cones) had a lambda max of 533.8 +/- 3.7 nm (n = 11) with a mean transverse absorbance of 0.032 +/- 0.007. The short-wave cones ('blue' cones) had a lambda max of 420.3 +/- 4.7 nm (n = 3) with a mean transverse absorbance of 0.037 +/- 0.011. 4. If assumptions are made about the length of cones and about pre-receptoral absorption, it is possible to derive psychophysical sensitivities for the cones that closely resemble the appropriate pi mechanisms of W. S. Stiles. 5. If assumptions are made about the length of rods and about pre-receptoral absorption, however, the psychophysical sensitivity derived for the rods is considerably broader than the C.I.E. scotopic sensitivity function.