Ganglion-Cell Photoreceptors and Non-Image-Forming Vision

Historical roots

For most contemporary retinal scientists, the recent discovery of photoreceptors in the inner retina may have come as a shock, but the roots of the idea are actually traceable to the very beginnings of retinal science (see reviews ). Beginning with Descartes and through the first half of the 19th century, it was assumed that the photoreceptive elements of the retina lined its inner surface, nearest the incoming light. For example, Treviranus, the first to conduct systematic microscopic studies of the retina, suggested that optic fibers passed through all retinal layers to end as photosensitive papillae at the vitreal surface. Bidder accurately described the outer retinal location of the rods, but hypothesized that they served a reflective function, like a tapetum, and continued to view optic fibers in the inner retina as the locus of phototransduction. The lack of photosensitivity of the fibers of the optic disk, as indicated by the blind spot, eventually undermined this view, and prompted Bowman and Helmholtz to propose ganglion cells as the true photoreceptors. It was not until the latter half of the 19th century that photoreception was convincingly localized to the outer retina. The key insights were made by Heinrich Müller, who inferred the retinal depth of the photoreceptors by a clever geometric analysis of the parallactic displacement of shadows of retinal vasculature (the “Purkinje tree”). He also detected “visual purple” or rhodopsin in the rods, which Franz Boll soon thereafter demonstrated was a photosensitive pigment.

Fifty years would pass before interest in the possibility of inner-retinal photoreception would re-emerge. The catalyst was the groundbreaking work of Clyde Keeler, who identified the first animal model of inherited retinal degeneration. This strain of mice – which Keeler termed “ rodless ” – is allelic with the rd1 strain (formerly rd ); both carry the Pde6b ^rd1 mutation and phenotypically resemble human autosomal recessive retinitis pigmentosa (OMIM 180072). ^, Keeler eventually achieved wide acclaim for this seminal contribution to the genetics of retinal disease, but a key observation he made in these mice received little notice at the time. Though his rodless mice were apparently blind when tested behaviorally or electroretinographically, they unexpectedly retained robust pupillary responses to light. This led him to suggest that “in mammals the iris may function independent of vision” either through intrinsic photosensitivity of the smooth muscle itself or by “direct stimulation of the internal nuclear or ganglionic cells” by light. ^,

Over the next 70 years, a series of studies in retinally degenerate mammals confirmed the persistence of the pupillary response and suggested that the functional blindness might not be as complete as Keeler had believed. Various photic effects on behavior or physiology were noted, despite the absence of an outer retina in histological material or a detectable electroretinogram (see reviews ^, ). These included avoidance of a visual cliff, photic suppression of activity in open field test, visually guided avoidance of shock in a shuttlebox, chromatic discrimination, entrainment of circadian rhythms, suppression of pineal melatonin synthesis, and suppression of spontaneous firing in the superior colliculus. A number of these studies demonstrated that eye removal abolished these residual photoresponses and overtly echoed Keeler's speculation about the possible existence of inner retinal photoreceptors, including ganglion cells. These suggestions gained only limited currency, probably because improved anatomical studies had begun to cast doubt on the completeness of the loss of outer retinal photoreceptors, especially in the peripheral retina. These studies showed that the mouse retina, once believed to possess only rod photoreceptors, actually had a modest population of cones as well, and that these degenerated far more slowly than rods in the rd1 model. Perhaps 20 percent survived beyond 80 days of age, albeit without intact outer segments, and a few lasted at least a year. This clouded the interpretation of the behavioral studies, virtually all of which had been conducted in mice young enough to have had many surviving cones. With the benefit of hindsight, however, it seems plausible that at least some of the residual light-driven effects reported in these early studies were indeed mediated by inner retinal photoreceptors. Even in rd1 animals as young as 6–10 weeks old, surviving rods or cones seem unable to support visual function: silencing ganglion-cell phototransduction in such animals (as described below) abolishes the photic influences on circadian rhythms, the pupil, locomotor activity, and melatonin synthesis that these mice would otherwise exhibit.

In the 1990s, Russell Foster and colleagues took up the work on retinally degenerate mice, applying more rigorous methods to provide compelling evidence for inner retinal photoreception. Their innovations included appropriate controls for genetic background and the use of very old rd1 animals and several genetically modified mouse strains in which they confirmed nearly complete loss of cones as well as rods. They also showed that there was no loss of sensitivity of circadian photoreception as rod and cone degeneration progressed and that the residual visually evoked behaviors extended beyond circadian regulation to other outputs of what they called the non-image-forming (NIF) visual centers of the brain. These included pupillary constriction, acute suppression of locomotor activity and acute suppression of melatonin release by light. The case for novel inner photoreceptors was bolstered by parallel observations in human patients with advanced outer retinal disease and by assessment of the spectral tuning of residual photoresponses in retinally degenerate animals. Yoshimura & Ebihara showed that the photic influence on circadian rhythms in retinally degenerate mice was most effective at 480 nm, clearly distinct from the optimal wavelength for the known rod and cone photopigments. ^, Remarkably similar spectral data were obtained for residual pupillary responses.

Discovery of melanopsin and ganglion-cell photoreceptors

The identities of the mysterious inner-retinal photoreceptors and their photopigment were established in a flurry of studies in the years 2000 to 2005. These developments have been thoroughly reviewed elsewhere, so we provide only an abbreviated summary here. A pivotal contribution was the discovery of melanopsin. ^, This novel opsin (coded by the opn4 gene) derives its name from the dermal melanophores of frogs, the photosensitive cells of the skin in which the gene was first identified. Ignacio Provencio and colleagues showed that in mice and primates, including humans, the protein was expressed solely in a small minority of cells in the inner retina, mainly in the ganglion cell layer. They speculated that melanopsin could be the photopigment of the postulated inner-retinal photoreceptors and that the neurons expressing it might be the cells of origin of the retinohypothalamic tract. These ideas were at odds with an alternative suggestion, introduced a few years earlier, that the relevant photopigment might not be an opsin but, rather, a cryptochrome, a blue-light-absorbing flavoprotein (see reviews ^, ). However, overwhelming evidence for melanopsin's central role soon emerged from studies in rodents and in heterologous expression systems.

Two key studies in this cohort focused on rat retinal ganglion cells shown by retrograde axonal tracing to innervate the suprachiasmatic nucleus (SCN) of the hypothalamus, the brain's circadian pacemaker. The first showed that these ganglion cells expressed melanopsin, while the second demonstrated that they generated robust electrical responses to light even when pharmacologically or mechanically isolated from other retinal neurons. ^, Because this capacity for autonomous phototransduction distinguishes these cells from all other RGCs, they are termed intrinsically photosensitive retinal ganglion cells (ipRGCs) or ganglion-cell photoreceptors. Both mice and primates were also soon shown to possess ipRGCs with structural and functional properties much like those in the rat (see Box 26.1 ).

The presence of melanopsin within physiologically identified ipRGCs was first demonstrated directly by Hattar et al . The opsin was found not only in the cell body, but also in their dendrites ^, which, like the soma, are directly photosensitive. Further support for the view that melanopsin was the sensory photopigment in these cells came from the opsin-like action spectrum of the light response in ipRGCs and from the observation that the intrinsic photosensitivity of these cells was abolished in melanopsin knock-out mice. ^, The capacity of melanopsin to function as a sensory photopigment was first demonstrated biochemically, and subsequently confirmed by electrophysiology and calcium imaging in heterologous expression systems. There is remarkably good concordance in the spectral sensitivity in this system, as assessed from absorbance of heterologously expressed or purified melanopsin, from the action spectrum of ipRGCs, and from behaviors mediated by inner retinal photoreceptors; all closely adhere to a retinaldehyde template function with a best wavelength near 480 nm ² (but see refs & ).

Distinctive functional properties of ipRGCs

Ganglion-cell photoreceptors have physiological properties that are optimized for their roles in NIF vision and contrast markedly with those of the rod and cone photoreceptors that feed the cortical circuits mediating the perception of form, color and motion.

Spectral tuning

As noted above, melanopsin has peak sensitivity in the blue region at around 480 nm, different from the spectral sensitivities of the rod and cone visual pigments ( Fig. 26.1 ). This wavelength roughly coincides with the peak of solar emission, perhaps the result of evolutionary pressures to optimize the sensitivity of this system to daylight. Though melanopsin is often described as a blue-light-sensitive pigment, it is important to recognize that, like other opsin-based photopigments, its spectral tuning is rather broad. Throughout the spectral range from 340 to 590 nm, sensitivity is within 2 log units of that seen with the optimal wavelength ( Fig. 26.1 ). Though there is evidence that ipRGCs project to the lateral geniculate nucleus (LGN) and could thus contribute to cortical function, ^, we are unaware of any evidence that the unique spectral tuning of melanopsin is exploited in the conscious appreciation of color, which is well accounted for by the three cone photopigments and trichromatic theory (see Box 26.3 ).

Box 26.3

Spectral Issues in the Design of Intraocular Lens Implants

Designers of intraocular lens implants must grapple with the fact that there are both harmful and salutary effects of short-wavelength visible light. The goal must be to strike the appropriate balance between photoprotection and photoreception. High-pass filters that block blue as well as ultraviolet wavelengths offer better retinal photoprotection than those that block only ultraviolet light, while leaving rod and cone photoresponses (which peak near 500 nm and 555 nm respectively) relatively unaffected. However, because melanopsin is most sensitive to blue light with peak sensitivity at 480 nm, lenses that block blue wavelengths can compromise NIF photoreception. On the other hand, reducing NIF photoreception in this manner might have the benefit of reducing the harmful effects of light exposure at night on the NIF visual system, as discussed in the main text below. These factors need to be considered in the design and selection of lens implants. A recent study showed that for the typical person, long-pass filtering with a sharp filter cutoff near 445 nm may provide the best compromise between photoprotection and light reception.

The spectral behavior of the ipRGC output signal is much more complex than predicted simply from the action spectrum of the intrinsic light response as measured in dark-adapted ipRGCs. This is in part because melanopsin appears to be a bistable photopigment (see the previous section). The absorption spectrum of the activated form of the pigment, and thus of photoreversal, is shifted to longer wavelengths with respect to that of melanopsin in the dark state (e.g. refs & ). How this shapes the ipRGC output signal under physiologically relevant lighting conditions is still in dispute, but it suggests the possibility of a form of spectral opponency under some conditions. A second source of complexity and potential spectral opponency is the functional input to the ipRGCs from classical rod and cone photoreceptors, discussed in detail below. Because the threshold for melanopsin activation is above that for rods and cones, the spectral tuning of ipRGCs can be expected to be intensity dependent. Furthermore, there is evidence in primates that the cone input to ipRGCs is itself spectrally opponent, with activation of short-wavelength cones driving OFF responses in ipRGCs, and activation of mid- and long-wavelength cones driving ON responses.

Invertebrate-like phototransduction cascade

In a photoreceptor cell, the light-absorbing pigment signals through an intracellular biochemical pathway to transduce light into electrical activity across the cell membrane. Interestingly, in ipRGCs this phototransduction cascade is more similar to those in invertebrate photoreceptors than to those in rods and cones. We have already noted that melanopsin has greater sequence homology with invertebrate photopigments than with the rod and cone opsins. ^, Upon stimulation by light, melanopsin activates a G-protein belonging to the G _q/11 family, which in turn signals to the effector enzyme phospholipase C (PLC). PLC then somehow causes the opening of a cation channel, which has been proposed to belong to the family of transient receptor potential (TRP) channels. While the signaling intermediates coupling PLC to the cation channel remain to be elucidated, the critical components are likely to be within or closely associated with the plasma membrane because patches of cell membrane isolated from ipRGCs continue to exhibit light-evoked electrical responses. Thus, the entire ipRGC phototransduction cascade is very similar to that in Drosophila photoreceptors but differs drastically from the rod/cone cascade (see also Chapter 18 ) ( Fig. 26.2 ). This invertebrate-like phototransduction cascade has prompted speculation that ipRGCs and invertebrate photoreceptors share a common evolutionary origin. It also confers upon ipRGCs properties well suited to NIF vision, as discussed below.

Depolarizing photoresponse with action potentials

The endpoint of the melanopsin phototransduction cascade is the opening of ion channels in the plasma membrane. These behave like cation-selective channels. ^, The interior of an ipRGC is negatively charged relative to the extracellular space so the opening of light-gated channels results in net influx of cations and membrane depolarization. In this ipRGCs once again resemble invertebrate rhabdomeric receptors, which likewise depolarize when illuminated, and differ from the vertebrate rods and cones, which are hyperpolarized by light. If the depolarizing ipRGC light response is large enough, it brings the membrane potential above the threshold for activating voltage-gated sodium channels, triggering action potentials ( Fig. 26.3 ). This represents yet another divergence from the rod/cone photoreceptors, which do not spike. Action potentials are necessary for ipRGCs, as for other RGCs, to propagate electrical information faithfully along the optic nerve and tract to relatively distant central visual centers. By contrast, rod and cone axons are very short, and passive spread of the electrical signals generated in the outer segment is sufficient for appropriate voltage modulation of the axon terminal.