RdgB2 is required for dim-light input into intrinsically photosensitive retinal ganglion cells

A subset of retinal ganglion cells is intrinsically photosensitive (ipRGCs) and contributes directly to the pupillary light reflex and circadian photoentrainment under bright-light conditions. ipRGCs are also indirectly activated by light through cellular circuits initiated in rods and cones. A mammalian homologue (RdgB2) of a phosphoinositide transfer/exchange protein that functions in Drosophila phototransduction is expressed in the retinal ganglion cell layer. This raised the possibility that RdgB2 might function in the intrinsic light response in ipRGCs, which depends on a cascade reminiscent of Drosophila phototransduction. Here we found that under high light intensities, RdgB2^−/− mutant mice showed normal pupillary light responses and circadian photoentrainment. Consistent with this behavioral phenotype, the intrinsic light responses of ipRGCs in RdgB2^−/− were indistinguishable from wild-type. In contrast, under low-light conditions, RdgB2^−/− mutants displayed defects in both circadian photoentrainment and the pupillary light response. The RdgB2 protein was not expressed in ipRGCs but was in GABAergic amacrine cells, which provided inhibitory feedback onto bipolar cells. We propose that RdgB2 is required in a cellular circuit that transduces light input from rods to bipolar cells that are coupled to GABAergic amacrine cells and ultimately to ipRGCs, thereby enabling ipRGCs to respond to dim light.     

The Visual Cycle in the Inner Retina of Chicken and the Involvement of Retinal G-Protein-Coupled Receptor (RGR)

The vertebrate retina contains typical photoreceptor (PR) cones and rods responsible for day/night vision, respectively, and intrinsically photosensitive retinal ganglion cells (ipRGCs) involved in the regulation of non-image-forming tasks. Rhodopsin/cone opsin photopigments in visual PRs or melanopsin (Opn4) in ipRGCs utilizes retinaldehyde as a chromophore. The retinoid regeneration process denominated as “visual cycle” involves the retinal pigment epithelium (RPE) or Müller glial cells. Opn4, on the contrary, has been characterized as a bi/tristable photopigment, in which a photon of one wavelength isomerizes 11-cis to all-trans retinal (Ral), with a second photon re-isomerizing it back. However, it is unknown how the chromophore is further metabolized in the inner retina. Nor is it yet clear whether an alternative secondary cycle occurs involving players such as the retinal G-protein-coupled receptor (RGR), a putative photoisomerase of unidentified inner retinal activity. Here, we investigated the role of RGR in retinoid photoisomerization in Opn4x (Xenopus ortholog) (+) RGC primary cultures free of RPE and other cells from chicken embryonic retinas. Opn4x (+) RGCs display significant photic responses by calcium fluorescent imaging and photoisomerize exogenous all-trans to 11-cis Ral and other retinoids. RGR was found to be expressed in developing retina and in primary cultures; when its expression was knocked down, the levels of 11-cis, all-trans Ral, and all-trans retinol in cultures exposed to light were significantly higher and those in all-trans retinyl esters lower than in dark controls. The results support a novel role for RGR in ipRGCs to modulate retinaldehyde levels in light, keeping the balance of inner retinal retinoid pools.     

Intrinsic Photosensitive Retinal Ganglion Cells in the Diurnal Rodent,Arvicanthis ansorgei

Intrinsically photosensitive retinal ganglion cells (ipRGCs) represent a new class of photoreceptors which support a variety of non-image forming physiological functions, such as circadian photoentrainment, pupillary light reflex and masking responses to light. In view of the recently proposed role of retinal inputs for the regulation of diurnal and nocturnal behavior, we performed the first deep analysis of the ipRGC system in a diurnal rodent model, Arvicanthis ansorgei , and compared the anatomical and physiological properties of ipRGCs with those of nocturnal mice. Based on somata location, stratification pattern and melanopsin expression, we identified two main ipRGC types in the retina of Arvicanthis : M1, constituting 74% of all ipRGCs and non-M1 (consisting mainly of the M2 type) constituting the following 25%. The displaced ipRGCs were rarely encountered. Phenotypical staining patterns of ganglion cell markers showed a preferential expression of Brn3 and neurofilaments in non-M1 ipRGCs. In general, the anatomical properties and molecular phenotyping of ipRGCs in Arvicanthis resemble ipRGCs of the mouse retina, however the percentage of M1 cells is considerably higher in the diurnal animal. Multi-electrode array recordings (MEA) identified in newborn retinas of Arvicanthis three response types of ipRGCs (type I, II and III) which are distinguished by their light sensitivity, response strength, latency and duration. Type I ipRGCs exhibited a high sensitivity to short light flashes and showed, contrary to mouse type I ipRGCs, robust light responses to 10 ms flashes. The morphological, molecular and physiological analysis reveals very few differences between mouse and Arvicanthis ipRGCs. These data imply that the influence of retinal inputs in defining the temporal niche could be related to a stronger cone input into ipRGCs in the cone-rich Arvicanthis retina, and to the higher sensitivity of type I ipRGCs and elevated proportion of M1 cells.     

Melanopsin as a Sleep Modulator: Circadian Gating of the Direct Effects of Light on Sleep and Altered Sleep Homeostasis in Opn4−/− Mice

Light influences sleep and alertness either indirectly through a well-characterized circadian pathway or directly through yet poorly understood mechanisms. Melanopsin (Opn4) is a retinal photopigment crucial for conveying nonvisual light information to the brain. Through extensive characterization of sleep and the electrocorticogram (ECoG) in melanopsin-deficient (Opn4^−/−) mice under various light–dark (LD) schedules, we assessed the role of melanopsin in mediating the effects of light on sleep and ECoG activity. In control mice, a light pulse given during the habitual dark period readily induced sleep, whereas a dark pulse given during the habitual light period induced waking with pronounced theta (7–10 Hz) and gamma (40–70 Hz) activity, the ECoG correlates of alertness. In contrast, light failed to induce sleep in Opn4^−/− mice, and the dark-pulse-induced increase in theta and gamma activity was delayed. A 24-h recording under a LD 1-h1-h schedule revealed that the failure to respond to light in Opn4^−/− mice was restricted to the subjective dark period. Light induced c-Fos immunoreactivity in the suprachiasmatic nuclei (SCN) and in sleep-active ventrolateral preoptic (VLPO) neurons was importantly reduced in Opn4^−/− mice, implicating both sleep-regulatory structures in the melanopsin-mediated effects of light. In addition to these acute light effects, Opn4^−/− mice slept 1 h less during the 12-h light period of a LD 1212 schedule owing to a lengthening of waking bouts. Despite this reduction in sleep time, ECoG delta power, a marker of sleep need, was decreased in Opn4^−/− mice for most of the (subjective) dark period. Delta power reached after a 6-h sleep deprivation was similarly reduced in Opn4^−/− mice. In mice, melanopsin's contribution to the direct effects of light on sleep is limited to the dark or active period, suggesting that at this circadian phase, melanopsin compensates for circadian variations in the photo sensitivity of other light-encoding pathways such as rod and cones. Our study, furthermore, demonstrates that lack of melanopsin alters sleep homeostasis. These findings call for a reevaluation of the role of light on mammalian physiology and behavior.     

Melanopsin as a Sleep Modulator: Circadian Gating of the Direct Effects of Light on Sleep and Altered Sleep Homeostasis in Opn4?/? Mice

Light influences sleep and alertness either indirectly through a well-characterized circadian pathway or directly through yet poorly understood mechanisms. Melanopsin (Opn4) is a retinal photopigment crucial for conveying nonvisual light information to the brain. Through extensive characterization of sleep and the electrocorticogram (ECoG) in melanopsin-deficient (Opn4^−/−) mice under various light–dark (LD) schedules, we assessed the role of melanopsin in mediating the effects of light on sleep and ECoG activity. In control mice, a light pulse given during the habitual dark period readily induced sleep, whereas a dark pulse given during the habitual light period induced waking with pronounced theta (7–10 Hz) and gamma (40–70 Hz) activity, the ECoG correlates of alertness. In contrast, light failed to induce sleep in Opn4^−/− mice, and the dark-pulse-induced increase in theta and gamma activity was delayed. A 24-h recording under a LD 1-h∶1-h schedule revealed that the failure to respond to light in Opn4^−/− mice was restricted to the subjective dark period. Light induced c-Fos immunoreactivity in the suprachiasmatic nuclei (SCN) and in sleep-active ventrolateral preoptic (VLPO) neurons was importantly reduced in Opn4^−/− mice, implicating both sleep-regulatory structures in the melanopsin-mediated effects of light. In addition to these acute light effects, Opn4^−/− mice slept 1 h less during the 12-h light period of a LD 12∶12 schedule owing to a lengthening of waking bouts. Despite this reduction in sleep time, ECoG delta power, a marker of sleep need, was decreased in Opn4^−/− mice for most of the (subjective) dark period. Delta power reached after a 6-h sleep deprivation was similarly reduced in Opn4^−/− mice. In mice, melanopsin''s contribution to the direct effects of light on sleep is limited to the dark or active period, suggesting that at this circadian phase, melanopsin compensates for circadian variations in the photo sensitivity of other light-encoding pathways such as rod and cones. Our study, furthermore, demonstrates that lack of melanopsin alters sleep homeostasis. These findings call for a reevaluation of the role of light on mammalian physiology and behavior.     

Melanopsin regulates visual processing in the mouse retina

The discovery of melanopsin-dependent inner retinal photoreceptors in mammals has precipitated a fundamental reassessment of such non-image forming (NIF) light responses as circadian photoentrainment and the pupil light reflex. By contrast, it remains unclear whether these new photoreceptors also play a role in classical image-forming vision. The retinal ganglion cells that subserve inner retinal photoreception (ipRGCs) project overwhelmingly to brain areas involved in NIF responses, indicating that, in terms of central signaling, their predominant function is non-image forming. However, ipRGCs also exhibit intraretinal communication via gap junction coupling, which could allow them to modulate classical visual pathways within this tissue. Here, we explore this second possibility by using melanopsin knockout (Opn4-/-) mice to examine the role of inner retinal photoreceptors in diurnal regulation of retinal function. By using electroretinography in wild-type mice, we describe diurnal rhythms in both the amplitude and speed of the retinal cone pathway that are a function of both prior light exposure and circadian phase. Unexpectedly, loss of the melanopsin gene abolishes circadian control of these parameters, causing significant attenuation of the diurnal variation in cone vision. Our results demonstrate for the first time a melanopsin-dependent regulation of visual processing within the retina, revealing an important function for inner retinal photoreceptors in optimizing classical visual pathways according to time of day.     

Deletion of Metabotropic Glutamate Receptors 2 and 3 (mGlu2 & mGlu3) in Mice Disrupts Sleep and Wheel-Running Activity,and Increases the Sensitivity of the Circadian System to Light

Sleep and/or circadian rhythm disruption (SCRD) is seen in up to 80% of schizophrenia patients. The co-morbidity of schizophrenia and SCRD may in part stem from dysfunction in common brain mechanisms, which include the glutamate system, and in particular, the group II metabotropic glutamate receptors mGlu2 and mGlu3 (encoded by the genes Grm2 and Grm3). These receptors are relevant to the pathophysiology and potential treatment of schizophrenia, and have also been implicated in sleep and circadian function. In the present study, we characterised the sleep and circadian rhythms of Grm2/3 double knockout (Grm2/3^-/-) mice, to provide further evidence for the involvement of group II metabotropic glutamate receptors in the regulation of sleep and circadian rhythms. We report several novel findings. Firstly, Grm2/3^-/- mice demonstrated a decrease in immobility-determined sleep time and an increase in immobility-determined sleep fragmentation. Secondly, Grm2/3^-/- mice showed heightened sensitivity to the circadian effects of light, manifested as increased period lengthening in constant light, and greater phase delays in response to nocturnal light pulses. Greater light-induced phase delays were also exhibited by wildtype C57Bl/6J mice following administration of the mGlu2/3 negative allosteric modulator RO4432717. These results confirm the involvement of group II metabotropic glutamate receptors in photic entrainment and sleep regulation pathways. Finally, the diurnal wheel-running rhythms of Grm2/3^-/- mice were perturbed under a standard light/dark cycle, but their diurnal rest-activity rhythms were unaltered in cages lacking running wheels, as determined with passive infrared motion detectors. Hence, when assessing the diurnal rest-activity rhythms of mice, the choice of assay can have a major bearing on the results obtained.     

Physiologic diversity and development of intrinsically photosensitive retinal ganglion cells

Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate numerous nonvisual phenomena, including entrainment of the circadian clock to light-dark cycles, pupillary light responsiveness, and light-regulated hormone release. We have applied multielectrode array recording to characterize murine ipRGCs. We find that all ipRGC photosensitivity is melanopsin dependent. At least three populations of ipRGCs are present in the postnatal day 8 (P8) murine retina: slow onset, sensitive, fast off (type I); slow onset, insensitive, slow off (type II); and rapid onset, sensitive, very slow off (type III). Recordings from adult rd/rd retinas reveal cells comparable to postnatal types II and III. Recordings from early postnatal retinas demonstrate intrinsic light responses from P0. Early light responses are transient and insensitive but by P6 show increased photosensitivity and persistence. These results demonstrate that ipRGCs are the first light-sensitive cells in the retina and suggest previously unappreciated diversity in this cell population.     

视网膜中的自主感光神经节细胞

视网膜中少数神经节细胞能够合成感光蛋白--黑视素(melanopsin),因此具备了自主感光的能力,被称为自主感光神经节细胞(intrinsically photosensitive retinal ganglion cells,ipRGCs).ipRGCs可根据树突形态和分层位置的差异分为五个不同的亚型,其轴突主要投...     

Clock Genes and Behavioral Responses to Light Are Altered in a Mouse Model of Diabetic Retinopathy

There is increasing evidence that melanopsin-expressing ganglion cells (ipRGCs) are altered in retinal pathologies. Using a streptozotocin-induced (STZ) model of diabetes, we investigated the impact of diabetic retinopathy on non-visual functions by analyzing ipRGCs morphology and light-induced c-Fos and Period 1–2 clock genes in the central clock (SCN). The ability of STZ-diabetic mice to entrain to light was challenged by exposure animals to 1) successive light/dark (LD) cycle of decreasing or increasing light intensities during the light phase and 2) 6-h advance of the LD cycle. Our results show that diabetes induces morphological changes of ipRGCs, including soma swelling and dendritic varicosities, with no reduction in their total number, associated with decreased c-Fos and clock genes induction by light in the SCN at 12 weeks post-onset of diabetes. In addition, STZ-diabetic mice exhibited a reduction of overall locomotor activity, a decrease of circadian sensitivity to light at low intensities, and a delay in the time to re-entrain after a phase advance of the LD cycle. These novel findings demonstrate that diabetes alters clock genes and behavioral responses of the circadian timing system to light and suggest that diabetic patients may show an increased propensity for circadian disturbances, in particular when they are exposed to chronobiological challenges.     

Localization of the ultraviolet-sensor Opn5m and its effect on myopia-related gene expression in the late-embryonic chick eye

Recent studies show that exposure to ultraviolet (UV) light suppresses ocular elongation, which causes myopia development. However, the specific mechanisms of this process have not been elucidated. A UV-sensor, Opsin 5 (Opn5) mRNA was shown to be present in extraretinal tissues. To test the possibility that UV-signals mediated by Opn5 would have a direct effect on the outer connective tissues of the eye, we first examined the expression patterns of a mammalian type Opn5 (Opn5m) in the late-embryonic chicken eye. Quantitative PCR showed Opn5m mRNA expression in the cornea and sclera. The anti-Opn5m antibody stained a small subset of cells in the corneal stroma and fibrous sclera. We next assessed the effect of UV-A (375 nm) irradiation on the chicken fibroblast cell line DF-1 overexpressing chicken Opn5m. UV-A irradiation for 30 min significantly increased the expression of Early growth response 1 (Egr1), known as an immediate early responsive gene, and of Matrix metalloproteinase 2 (Mmp2) in the presence of retinal chromophore 11-cis-retinal. In contrast, expression of Transforming growth factor beta 2 and Tissue inhibitor of metalloproteinase 2 was not significantly altered. These results indicate that UV-A absorption by Opn5m can upregulate the expression levels of Egr1 and Mmp2 in non-neuronal, fibroblasts. Taken together with the presence of Opn5m in the cornea and sclera, it is suggested that UV-A signaling mediated by Opn5 in the extraretinal ocular tissues could influence directly the outer connective tissues of the chicken late-embryonic eye.     

Critical Endothelial Regulation by LRP5 during Retinal Vascular Development

Vascular abnormalities in the eye are the leading cause of many forms of inherited and acquired human blindness. Loss-of-function mutations in the Wnt-binding co-receptor LRP5 leads to aberrant ocular vascularization and loss of vision in genetic disorders such as osteoporosis-pseudoglioma syndrome. The canonical Wnt-β-catenin pathway is known to regulate retinal vascular development. However, it is unclear what precise role LPR5 plays in this process. Here, we show that loss of LRP5 function in mice causes retinal hypovascularization during development as well as retinal neovascularization in adulthood with disorganized and leaky vessels. Using a highly specific Flk1-Cre^Breier line for vascular endothelial cells, together with several genetic models, we demonstrate that loss of endothelium-derived LRP5 recapitulates the retinal vascular defects in Lrp5^-/- mice. In addition, restoring LRP5 function only in endothelial cells in Lrp5^-/- mice rescues their retinal vascular abnormalities. Furthermore, we show that retinal vascularization is regulated by LRP5 in a dosage dependent manner and does not depend on LRP6. Our study provides the first direct evidence that endothelium-derived LRP5 is both necessary and sufficient to mediate its critical role in the development and maintenance of retinal vasculature.     

Photoreceptor Proteins Initiate Microglial Activation via Toll-like Receptor 4 in Retinal Degeneration Mediated by All-trans-retinal

Although several genetic and biochemical factors are associated with the pathogenesis of retinal degeneration, it has yet to be determined how these different impairments can cause similar degenerative phenotypes. Here, we report microglial/macrophage activation in both a Stargardt disease and age-related macular degeneration mouse model caused by delayed clearance of all-trans-retinal from the retina, and in a retinitis pigmentosa mouse model with impaired retinal pigment epithelium (RPE) phagocytosis. Mouse microglia displayed RPE cytotoxicity and increased production of inflammatory chemokines/cytokines, Ccl2, Il1b, and Tnf, after coincubation with ligands that activate innate immunity. Notably, phagocytosis of photoreceptor proteins increased the activation of microglia/macrophages and RPE cells isolated from model mice as well as wild-type mice. The mRNA levels of Tlr2 and Tlr4, which can recognize proteins as their ligands, were elevated in mice with retinal degeneration. Bone marrow-derived macrophages from Tlr4-deficient mice did not increase Ccl2 after coincubation with photoreceptor proteins. Tlr4^−/−Abca4^−/−Rdh8^−/− mice displayed milder retinal degenerative phenotypes than Abca4^−/−Rdh8^−/− mice. Additionally, inactivation of microglia/macrophages by pharmacological approaches attenuated mouse retinal degeneration. This study demonstrates an important contribution of TLR4-mediated microglial activation by endogenous photoreceptor proteins in retinal inflammation that aggravates retinal cell death. This pathway is likely to represent an underlying common pathology in degenerative retinal disorders.     

Postnatal Constant Light Compensates Cryptochrome1 and 2 Double Deficiency for Disruption of Circadian Behavioral Rhythms in Mice under Constant Dark

Clock genes Cryptochrome (Cry1) and Cry2 are essential for expression of circadian rhythms in mice under constant darkness (DD). However, circadian rhythms in clock gene Per1 expression or clock protein PER2 are detected in the cultured suprachiasmatic nucleus (SCN) of neonatal Cry1 and Cry2 double deficient (Cry1 ^-/-/Cry2 ^-/-) mice. A lack of circadian rhythms in adult Cry1 ^-/-/Cry2 ^-/- mice is most likely due to developmentally disorganized cellular coupling of oscillating neurons in the SCN. On the other hand, neonatal rats exposed to constant light (LL) developed a tenable circadian system under prolonged LL which was known to fragment circadian behavioral rhythms. In the present study, Cry1 ^-/-/Cry2 ^-/- mice were raised under LL from postnatal day 1 for 7 weeks and subsequently exposed to DD for 3 weeks. Spontaneous movement was monitored continuously after weaning and PER2::LUC was measured in the cultured SCN obtained from mice under prolonged DD. Surprisingly, Chi square periodogram analysis revealed significant circadian rhythms of spontaneous movement in the LL-raised Cry1 ^-/-/Cry2 ^-/- mice, but failed to detect the rhythms in Cry1 ^-/-/Cry2 ^-/- mice raised under light-dark cycles (LD). By contrast, prolonged LL in adulthood did not rescue the circadian behavioral rhythms in the LD raised Cry1 ^-/-/Cry2 ^-/- mice. Visual inspection disclosed two distinct activity components with different periods in behavioral rhythms of the LL-raised Cry1^-/-/Cry2^-/- mice under DD: one was shorter and the other was longer than 24 hours. The two components repeatedly merged and separated. The patterns resembled the split behavioral rhythms of wild type mice under prolonged LL. In addition, circadian rhythms in PER2::LUC were detected in some of the LL-raised Cry1^-/-/Cry2^-/- mice under DD. These results indicate that neonatal exposure to LL compensates the CRY double deficiency for the disruption of circadian behavioral rhythms under DD in adulthood.     

Effects of obesity on circadian photic entrainment of locomotor activity in wild mice Neotomodon alstoni

Obesity is increasing in industrialized countries at an alarming rate. Recent studies have linked this condition with changes in the circadian regulation, and circadian clock dysfunctions have also been linked to metabolic disorders. When in captivity and fed a regular rodent chow diet ad libitum some volcano mice, Neotomodon alstoni (endemic species of Mexico) become overweight and display symptoms equivalent to metabolic syndrome. The aim of this work was to observe whether there are significant changes in the functional properties of the circadian system, namely in the entraining circadian locomotor activity rhythm, between mice that became obese and normal adult mice. Freely moving circadian rhythms of locomotor activity were tested under constant conditions as well as under conditions of discrete and continuous entrainment. Our results show that volcano lean mice present a phase response curve with larger delays than advances, indicating that re-entrainment is more easily achieved by delays. Volcano obese mice are less efficient at re-entraining because they show smaller phase shifts in delays than control mice. These results indicate that obesity in N. alstoni has a negative effect on the circadian mechanisms that integrate the photic entrainment.     

Altered Phase-Relationship between Peripheral Oscillators and Environmental Time in Cry1 or Cry2 Deficient Mouse Models for Early and Late Chronotypes

The mammalian circadian system is composed of a light-entrainable central clock in the suprachiasmatic nuclei (SCN) of the brain and peripheral clocks in virtually any other tissue. It allows the organism to optimally adjust metabolic, physiological and behavioral functions to the physiological needs it will have at specific time of the day. According to the resonance theory, such rhythms are only advantageous to an organism when in tune with the environment, which is illustrated by the adverse health effects originating from chronic circadian disruption by jetlag and shift work. Using short-period Cry1 and long-period Cry2 deficient mice as models for morningness and eveningness, respectively, we explored the effect of chronotype on the phase relationship between the central SCN clock and peripheral clocks in other organs. Whereas the behavioral activity patterns and circadian gene expression in the SCN of light-entrained Cry1^-/- and Cry2^-/- mice largely overlapped with that of wild type mice, expression of clock and clock controlled genes in liver, kidney, small intestine, and skin was shown to be markedly phase-advanced or phase-delayed, respectively. Likewise, circadian rhythms in urinary corticosterone were shown to display a significantly altered phase relationship similar to that of gene expression in peripheral tissues. We show that the daily dissonance between peripheral clocks and the environment did not affect the lifespan of Cry1^-/- or Cry2^-/- mice. Nonetheless, the phase-shifted peripheral clocks in light-entrained mice with morningness and eveningness-like phenotypes may have implications for personalized preventive and therapeutic (i.e. chronomodulation-based) health care for people with early and late chronotypes.     

Vesicular glutamate transporter 2 (VGLUT2) is co-stored with PACAP in projections from the rat melanopsin-containing retinal ganglion cells

The retinal ganglion cell layer of the eye comprises a subtype of cells characterized by their intrinsic photosensitivity and expression of melanopsin (ipRGCs). These cells regulate a variety of non-image-forming (NIF) functions such as light entrainment of circadian rhythms, acute suppression of locomotor activity (masking), and pupillary light reflex. Two neurotransmitters have been identified in ipRGCs, glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP). To date, little is known about their release and interplay. Here, we describe the presence and co-localization of vesicular glutamate transporter 2 (VGLUT2; a marker of glutamate signaling) and PACAP in ipRGCs and their projections in the brain. Nine adult male Wistar rats were assigned to one of three groups; anterograde tracing (n = 3), eye enucleation (n = 3), and untreated (n = 3). Under anaesthesia, rats were transcardially perfusion-fixated, after which the brains and eyes were removed for double immunohistochemical staining using a polyclonal anti-VGLUT2 antibody and a mouse monoclonal anti-PACAP antibody. Results revealed that VGLUT2- and PACAP-immunoreactivity (-ir) were present in ipRGCs and co-localized in their projections in the suprachiasmatic nucleus, the intergeniculate leaflet, and the olivary pretectal nucleus. We conclude that there is evidence to support the use of glutamate and PACAP as neurotransmitters in NIF photoperception by rat ipRGCs, and that these neurotransmitters are co-stored and probably released from the same nerve terminals. Furthermore, we conclude that VGLUT2 is the preferred subtype of vesicular transporter used by these cells.