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A small subset of retinal ganglion cells projecting to the suprachiasmatic nucleus and other brain areas, is implicated in non-image forming visual responses to environmental light such as the pupillary light reflex, seasonal adaptations in physiology, photic inhibition of nocturnal melatonin release, and modulation of sleep, alertness and activity. These cells are intrinsically photosensitive (ipRGCs) and express an opsin-like photopigment called melanopsin. Two recent studies utilizing selective genetic ablation of ipRGCs demonstrate the key role of these inner retinal cells in conveying luminance signals to the brain for non-image forming visual processing. These findings advance our understanding of functional organization of a novel photosensory system in the mammalian retina, demonstrating well-defined roles for ipRGCs in circadian timing and other homeostatic functions related to ambient illumination.
The retina is the sensory organ that mediates both image and non-image forming vision (NIFV). The high spatial and temporal resolution of image-forming vision allows discrimination of object shapes, colors and motion within the visual field. NIFV, on the other hand, relays the overall luminance level of the external environment to allow photoentrainment of the endogenous biological clock, pupillary light reflex and regulation of pineal melatonin secretion. The neuronal signal for NIFV is relayed from a subset of retinal ganglion cells that send projections to brain areas such as suprachiasmatic nucleus (SCN, involved in circadian entrainment) and olivary pretectal nucleus (OPN, involved in the pupillary light reflex; Pickard, 1980; Moore et al., 1995; Young & Lund, 1998).
Melanopsin (Opn4) was first discovered in the light sensitive cells of frog skin (Provencio et al., 1998), and its expression was later discovered to be restricted to a few cells within the ganglion cell layer of the mammalian retina (Provencio et al., 2000). This inner retinal distribution of melanopsin within a small population of retinal ganglion cells shared a remarkable resemblance to the cohort of retinal ganglion cells known to project to the SCN and entrain the circadian rhythm. These findings implied a role for the photopigment melanopsin in NIFV. Indeed, Hattar et al. (Hattar et al., 2002) later demonstrated that melanopsin-expressing ganglion cells do indeed project axons to the SCN and OPN. Concurrently, physiological experiments by Berson et al. (Berson et al., 2002) showed that SCN-projecting retinal ganglion cells are intrinsically photosensitive, and thus named these cells intrinsically photosensitive retinal ganglion cells (ipRGCs; Figure 1). Unlike other photoreceptors, ipRGCs depolarize in response to a light stimulus and, unlike other RGCs, these cells are capable of responding to light even in the absence of input from rods and cones (Berson et al., 2002).
Gene ablation studies in mice where the melanopsin protein is knocked out (Opn4−/−) have confirmed a role for melanopsin in both circadian entrainment and the pupillary light reflex (Hattar et al., 2002; Panda et al., 2002). However, deficits in both of these behaviors in Opn4−/− mice was seen only at high luminance levels, implying a role for rods and cones in both of these systems that could compensate to some degree for the loss of melanopsin. Together, rods, cones, and melanopsin account for all light detection by the retina because when Opn4−/− mice are crossed with mice lacking rods and cones, all retinal responses to light are lost (Hattar et al., 2003). Additionally, ipRGCs themselves receive synaptic inputs, and combine these extrinsic light signals from the outer retina with those of their intrinsic system (Dacey et al., 2005; Wong et al., 2007; Schmidt et al., 2008).
Therefore it is clear that ipRGCs are key elements in photoentraiment and the pupillary light reflex, but are they the sole pathway through which NIFV occurs? Conceivably, there could be some redundancy in the system, whereby if ipRGCs themselves and not just melanopsin are ablated, rods and cones could still mediate various NIFV functions via other RGCs (Figure 2). In support of this idea, a number of studies have indicated that regular RGCs send afferents to the SCN and other areas of the brain involved in processing NIFV responses (Gooley et al., 2003; Morin et al., 2003; Sollars et al., 2003). This key question has now been addressed by two groups of investigators using elegant transgenic methods in the mouse (Guler et al., 2008; Hatori et al., 2008). These groups independently investigated the role of ipRGCs in NIFV by genetically ablating these cells via selective expression of an attenuated form of diphtheria toxin (aDTA) within ipRGCs or via selective expression of the receptor for diphteria toxin (DT) and subsequent intraperitoneal injection of the toxin itself. Both of these methods result in a drastic reduction in the number of ipRGCs in the retina.
In the first of these studies, Guler et al. (2008) selectively expressed DT in ipRGGs. The DT gene product is highly cytotoxic and even relatively low DT expression can ablate the target cell with high efficiency (Yamaizumi et al., 1978). The genetic ablation of ipRGCs in the mouse line Opn4aDTA presumably did not impair the development or function of regular ganglion cells and other neurons in the retina as these animals displayed normal performance on visual tasks testing image-forming vision and their electroretinograms were indistinguishable from wild type mice. However, in tests for NIFV functions such as pupillary light reflexes and circadian entrainment, the Opn4aDTA animals performed far poorer than their wild-type counterparts. Opn4aDTA homozygous mice constricted their pupils only to ~ 40% following a light stimulus that caused more than 90% of constriction in wild type mice. Monitoring of wheel running activity showed that most Opn4aDTA mice fail to entrain the endogenous circadian clock following pulses of light. Finally, under an ultradian cycle which alternates 7 hours of light with 7 hours of dark under which wild type mice confine activity mainly to the dark periods, Opn4aDTA mice were active apparently independent of the current light conditions. Collectively, these results strongly support the notion that ipRGCs are the principal type of ganglion cells that relay the sensory information for NIFV functions.
Though generally conclusive, there are lingering questions from the Guler et al. study. One interesting issue relates to the fact that at high luminance levels, the Opn4aDTA animals still responded with significant pupillary constriction. In addition, some Opn4aDTA were weakly light responsive on wheel running activity assays. One straightforward explanation for this would be the existence of alternative pathways whereby rods and cones signal via other types of RGCs for NIFV functions which can compensate to some degree for the loss of ipRGCs. A second, possibility, favored by the authors, is that the survival of few ipRGCs in the mutant mice may have supported these residual responses, and alternative supported by the results obtained by Hatori et al.
In this second study, Hatori et al. (2008) employed a conditional approach for the genetic ablation of ipRGCs. Mice were engineered to express the DT receptor, and ablation of ipRGCs was achieved via intraperitoneal injection of diphtheria toxin, which crosses the blood brain/retina barrier and can therefore reach the target ipRGCs in the retina. This approach allowed a more complete and uniform level of ipRGC cell ablation than that obtained by Guler et al. and avoided any problems associated with homeostatic compensations during development. Overall, there was remarkable agreement between the Guler et al. and Hatori et al. studies, with Hatori et al. finding impairment in pupillary light reflex, photoentraiment, and light-suppression of locomotor activity in the mice which were injected with DT that expressed the DT receptor in ipRGCs. Indeed, these animals displayed almost complete impairment of all the tested non-imaging forming functions (including pupillary light reflex, for which Guler et al. saw only partial impairment) reinforcing the necessary role of ipRGCs in relaying the irradiance information to the brain areas involved in NIFV behaviors.
The requirement of ipRGCs for the light signaling of non-image forming visual functions highlights the importance and unique role of this small subpopulation of RGCs, and should spur new efforts to define their function. Of particular interest is the recent observation of a considerable degree of morphological and physiological heterogeneity within the ipRGC population. At least two major morphological populations of ipRGCs with differential dendritic stratification in the inner plexiform layer of the retina have been described (Viney et al., 2007; Schmidt et al., 2008)and demonstrated to project differentially to the SCN and OPN (Baver et al., 2008). In addition, calcium imaging and multi-electrode array studies have described functional diversity in ipRGC light responses (Sekaran et al., 2003; Tu et al., 2005). Do distinct morphological subtypes of ipRGCs subserve distinct functional roles? Are the signal transduction pathways that couple light simulation with electrical activity similar in these cell types? These and many other intriguing questions and mechanistic details will likely be addressed in the future not only by extending the elegant experimental studies described in these papers, but also by the application of novel single cell electrical and optical technologies.