Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice

In the mammalian retina, besides the conventional rod-cone system, a melanopsin-associated photoreceptive system exists that conveys photic information for accessory visual functions such as pupillary light reflex and circadian photo-entrainment. On ablation of the melanopsin gene, retinal ganglion cells that normally express melanopsin are no longer intrinsically photosensitive. Furthermore, pupil reflex, light-induced phase delays of the circadian clock and period lengthening of the circadian rhythm in constant light are all partially impaired. Here, we investigated whether additional photoreceptive systems participate in these responses. Using mice lacking rods and cones, we measured the action spectrum for phase-shifting the circadian rhythm of locomotor behaviour. This spectrum matches that for the pupillary light reflex in mice of the same genotype, and that for the intrinsic photosensitivity of the melanopsin-expressing retinal ganglion cells. We have also generated mice lacking melanopsin coupled with disabled rod and cone phototransduction mechanisms. These animals have an intact retina but fail to show any significant pupil reflex, to entrain to light/dark cycles, and to show any masking response to light. Thus, the rod-cone and melanopsin systems together seem to provide all of the photic input for these accessory visual functions.     

Melanopsin-expressing ganglion cells in primate retina signal colour and irradiance and project to the LGN

Human vision starts with the activation of rod photoreceptors in dim light and short (S)-, medium (M)-, and long (L)- wavelength-sensitive cone photoreceptors in daylight. Recently a parallel, non-rod, non-cone photoreceptive pathway, arising from a population of retinal ganglion cells, was discovered in nocturnal rodents. These ganglion cells express the putative photopigment melanopsin and by signalling gross changes in light intensity serve the subconscious, 'non-image-forming' functions of circadian photoentrainment and pupil constriction. Here we show an anatomically distinct population of 'giant', melanopsin-expressing ganglion cells in the primate retina that, in addition to being intrinsically photosensitive, are strongly activated by rods and cones, and display a rare, S-Off, (L + M)-On type of colour-opponent receptive field. The intrinsic, rod and (L + M) cone-derived light responses combine in these giant cells to signal irradiance over the full dynamic range of human vision. In accordance with cone-based colour opponency, the giant cells project to the lateral geniculate nucleus, the thalamic relay to primary visual cortex. Thus, in the diurnal trichromatic primate, 'non-image-forming' and conventional 'image-forming' retinal pathways are merged, and the melanopsin-based signal might contribute to conscious visual perception.     

Induction of photosensitivity by heterologous expression of melanopsin

Melanopsin has been proposed to be the photopigment of the intrinsically photosensitive retinal ganglion cells (ipRGCs); these photoreceptors of the mammalian eye drive circadian and pupillary adjustments through direct projections to the brain. Their action spectrum (lambda(max) approximately 480 nm) implicates an opsin and melanopsin is the only opsin known to exist in these cells. Melanopsin is required for ipRGC photosensitivity and for behavioural photoresponses that survive disrupted rod and cone function. Heterologously expressed melanopsin apparently binds retinaldehyde and mediates photic activation of G proteins. However, its amino-acid sequence differs from vertebrate photosensory opsins and some have suggested that melanopsin may be a photoisomerase, providing retinoid chromophore to an unidentified opsin. To determine whether melanopsin is a functional sensory photopigment, here we transiently expressed it in HEK293 cells that stably expressed TRPC3 channels. Light triggered a membrane depolarization in these cells and increased intracellular calcium. The light response resembled that of ipRGCs, with almost identical spectral sensitivity (lambda(max) approximately 479 nm). The phototransduction pathway included Gq or a related G protein, phospholipase C and TRPC3 channels. We conclude that mammalian melanopsin is a functional sensory photopigment, that it is the photopigment of ganglion-cell photoreceptors, and that these photoreceptors may use an invertebrate-like phototransduction cascade.     

Melanopsin signalling in mammalian iris and retina

Non-mammalian vertebrates have an intrinsically photosensitive iris and thus a local pupillary light reflex (PLR). In contrast, it is thought that the PLR in mammals generally requires neuronal circuitry connecting the eye and the brain. Here we report that an intrinsic component of the PLR is in fact widespread in nocturnal and crepuscular mammals. In mouse, this intrinsic PLR requires the visual pigment melanopsin; it also requires PLCβ4, a vertebrate homologue of the Drosophila NorpA phospholipase C which mediates rhabdomeric phototransduction. The Plcb4(-/-) genotype, in addition to removing the intrinsic PLR, also essentially eliminates the intrinsic light response of the M1 subtype of melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (M1-ipRGCs), which are by far the most photosensitive ipRGC subtype and also have the largest response to light. Ablating in mouse the expression of both TRPC6 and TRPC7, members of the TRP channel superfamily, also essentially eliminated the M1-ipRGC light response but the intrinsic PLR was not affected. Thus, melanopsin signalling exists in both iris and retina, involving a PLCβ4-mediated pathway that nonetheless diverges in the two locations.     

Addition of human melanopsin renders mammalian cells photoresponsive

A small number of mammalian retinal ganglion cells act as photoreceptors for regulating certain non-image forming photoresponses. These intrinsically photosensitive retinal ganglion cells express the putative photopigment melanopsin. Ablation of the melanopsin gene renders these cells insensitive to light; however, the precise role of melanopsin in supporting cellular photosensitivity is unconfirmed. Here we show that heterologous expression of human melanopsin in a mouse paraneuronal cell line (Neuro-2a) is sufficient to render these cells photoreceptive. Under such conditions, melanopsin acts as a sensory photopigment, coupled to a native ion channel via a G-protein signalling cascade, to drive physiological light detection. The melanopsin photoresponse relies on the presence of cis-isoforms of retinaldehyde and is selectively sensitive to short-wavelength light. We also present evidence to show that melanopsin functions as a bistable pigment in this system, having an intrinsic photoisomerase regeneration function that is chromatically shifted to longer wavelengths.     

A cyclic GMP-activated channel in dissociated cells of the chick pineal gland.

Phototransduction in the vertebrate retina is dependent in part on a cyclic GMP-activated ionic channel in the plasma membrane of rods and cones. But other vertebrate cells are also photosensitive. Cells of the chick pineal gland have a photosensitive circadian rhythm in melatonin secretion that persists in dissociated cell culture. Exposure to light causes inhibition of melatonin secretion, and entrainment of the intrinsic circadian oscillator. Chick pinealocytes express several 'retinal' proteins, including arrestin, transducin and a protein similar to the visual pigment rhodopsin. Pinealocytes of lower vertebrates display hyperpolarizing responses to brief pulses of light. Thus it is possible that some of the mechanisms of phototransduction are similar in retinal and pineal photoreceptors. We report here the first recordings of cyclic GMP-activated channels in an extraretinal photoreceptor. Application of GMP, but not cyclic AMP, to excised inside-out patches caused activation of a 15-25 pS cationic channel. These channels may be essential for phototransduction in the chick pineal gland.     

Light adaptation in cone vision involves switching between receptor and post-receptor sites

We see over an enormous range of mean light levels, greater than the range of output signals retinal neurons can produce. Even highlights and shadows within a single visual scene can differ approximately 10,000-fold in intensity-exceeding the range of distinct neural signals by a factor of approximately 100. The effectiveness of daylight vision under these conditions relies on at least two retinal mechanisms that adjust sensitivity in the approximately 200 ms intervals between saccades. One mechanism is in the cone photoreceptors (receptor adaptation) and the other is at a previously unknown location within the retinal circuitry that benefits from convergence of signals from multiple cones (post-receptor adaptation). Here we find that post-receptor adaptation occurs as signals are relayed from cone bipolar cells to ganglion cells. Furthermore, we find that the two adaptive mechanisms are essentially mutually exclusive: as light levels increase the main site of adaptation switches from the circuitry to the cones. These findings help explain how human cone vision encodes everyday scenes, and, more generally, how sensory systems handle the challenges posed by a diverse physical environment.     

Photoreceptive net in the mammalian retina. This mesh of cells may explain how some blind mice can still tell day from night

We have discovered an expansive photoreceptive 'net' in the mouse inner retina, visualized by using an antiserum against melanopsin, a likely photopigment. This immunoreactivity is evident in a subset of retinal ganglion cells that morphologically resemble those that project to the suprachiasmatic nucleus (SCN), the site of the primary circadian pacemaker. Our results indicate that this bilayered photoreceptive net is anatomically distinct from the rod and cone photoreceptors of the outer retina, and suggest that it may mediate non-visual photoreceptive tasks such as the regulation of circadian rhythms.     

视神经损伤引起斑马鱼视网膜神经细胞凋亡的研究

用石蜡连续切片苏木精染色法,通过定量分析研究夹伤和切断视神经后,斑马鱼视网膜神经节细胞、视杆和视锥细胞密度的变化。结果发现,在损伤视神经7～21d后,上述3种细胞的细胞核密度均呈减少趋势,节细胞减少的比率大于感光细胞,而感光细胞中视锥细胞所受影响比视杆细胞更为明显;在夹伤和切断视神经两种情况中,后者引起视网膜神经节细胞核密度的减少更为显著。上述结果表明,损伤视神经不但影响与其相连的神经节细胞,而且可逆向跨神经元地影响感光细胞的变化。由上述结果推测,由于损伤视神经使视网膜神经节细胞失去靶组织而引起的各种神经细胞密度减少是视网膜中神经细胞凋亡的表现。     

Transmembrane semaphorin signalling controls laminar stratification in the mammalian retina

In the vertebrate retina, establishment of precise synaptic connections among distinct retinal neuron cell types is critical for processing visual information and for accurate visual perception. Retinal ganglion cells (RGCs), amacrine cells and bipolar cells establish stereotypic neurite arborization patterns to form functional neural circuits in the inner plexiform layer (IPL), a laminar region that is conventionally divided into five major parallel sublaminae. However, the molecular mechanisms governing distinct retinal subtype targeting to specific sublaminae within the IPL remain to be elucidated. Here we show that the transmembrane semaphorin Sema6A signals through its receptor PlexinA4 (PlexA4) to control lamina-specific neuronal stratification in the mouse retina. Expression analyses demonstrate that Sema6A and PlexA4 proteins are expressed in a complementary fashion in the developing retina: Sema6A in most ON sublaminae and PlexA4 in OFF sublaminae of the IPL. Mice with null mutations in PlexA4 or Sema6A exhibit severe defects in stereotypic lamina-specific neurite arborization of tyrosine hydroxylase (TH)-expressing dopaminergic amacrine cells, intrinsically photosensitive RGCs (ipRGCs) and calbindin-positive cells in the IPL. Sema6A and PlexA4 genetically interact in vivo for the regulation of dopaminergic amacrine cell laminar targeting. Therefore, neuronal targeting to subdivisions of the IPL in the mammalian retina is directed by repulsive transmembrane guidance cues present on neuronal processes.     

Release of endogenous excitatory amino acids from turtle photoreceptors

Responses to light are transmitted from photoreceptors to second-order retinal neurons by chemical synapses that may use an excitatory amino acid (EAA) as the neurotransmitter. This hypothesis is based primarily on the pharmacological actions of EAA agonists and antagonists on the membrane potentials and light responses of second-order neurons. But the release of endogenous EAAs, which is a critical criterion for the identification of EAAs as transmitters, has not been demonstrated. Here we report the use of outside-out membrane patches excised from rat hippocampal neurons to detect the release of EAAs from synaptic terminals of isolated turtle photoreceptors. Electrical stimulation of or application of lanthanum chloride to photoreceptors induced an increase in the frequency of opening of 50-pS channels in the patches. These channels were identified as the class of glutamate-activated channels that are also gated by aspartate and NMDA (N-methyl-D-aspartate). In several photoreceptor-patch pairs, spontaneous channel activity was observed near the synaptic terminals. These results provide strong evidence to support the hypothesis that both rods and cones of the turtle use an EAA as their neurotransmitter.     

Segregation of object and background motion in the retina

An important task in vision is to detect objects moving within a stationary scene. During normal viewing this is complicated by the presence of eye movements that continually scan the image across the retina, even during fixation. To detect moving objects, the brain must distinguish local motion within the scene from the global retinal image drift due to fixational eye movements. We have found that this process begins in the retina: a subset of retinal ganglion cells responds to motion in the receptive field centre, but only if the wider surround moves with a different trajectory. This selectivity for differential motion is independent of direction, and can be explained by a model of retinal circuitry that invokes pooling over nonlinear interneurons. The suppression by global image motion is probably mediated by polyaxonal, wide-field amacrine cells with transient responses. We show how a population of ganglion cells selective for differential motion can rapidly flag moving objects, and even segregate multiple moving objects.     

Molecular identification of a retinal cell type that responds to upward motion

The retina contains complex circuits of neurons that extract salient information from visual inputs. Signals from photoreceptors are processed by retinal interneurons, integrated by retinal ganglion cells (RGCs) and sent to the brain by RGC axons. Distinct types of RGC respond to different visual features, such as increases or decreases in light intensity (ON and OFF cells, respectively), colour or moving objects. Thus, RGCs comprise a set of parallel pathways from the eye to the brain. The identification of molecular markers for RGC subsets will facilitate attempts to correlate their structure with their function, assess their synaptic inputs and targets, and study their diversification. Here we show, by means of a transgenic marking method, that junctional adhesion molecule B (JAM-B) marks a previously unrecognized class of OFF RGCs in mice. These cells have asymmetric dendritic arbors aligned in a dorsal-to-ventral direction across the retina. Their receptive fields are also asymmetric and respond selectively to stimuli moving in a soma-to-dendrite direction; because the lens reverses the image of the world on the retina, these cells detect upward motion in the visual field. Thus, JAM-B identifies a unique population of RGCs in which structure corresponds remarkably to function.     

4种变温动物松果腺复合体的形态结构比较

本文采用光镜和透射电镜，对日本七鳃鳗，黑斑蛙和丽斑麻蜥４种变动物的松果腺复合体，进行了观察和比较，并对其结构与功能的关系作了初步探讨，变温动物的松果腺复合体，由副松果体和松果体构成，在系统发育中，副松果体始终是光感受器，松果体则从光感觉器向着内分泌腺发展，从这４种动物松果腺复合体的变化揭示；最早脊椎动物的松果眼是２个，以后在动物从低等向高等进化中，其中一个保留在变温动物，始终是光感觉器，另一个变成     

Photoreceptor excitation and adaptation by inositol 1,4,5-trisphosphate

A central question concerning vision is the identity of the biochemical pathway that underlies phototransduction. The large size of the ventral photoreceptors of Limulus polyphemus renders them a favourite preparation for investigating this problem. The fact that a single photon opens approximately 1,000 ionic channels in these photoreceptors suggests the need for an internal transmitter. We have investigated whether inositol 1,4,5-trisphosphate (InsP3) functions as such an internal transmitter, given that InsP3 may act as an intracellular messenger in other cellular processes. Here we report that in Limulus, intracellular pressure injection of InsP3 both excites and adapts ventral photoreceptors in a manner similar to light.     

Spatial structure of cone inputs to receptive fields in primate lateral geniculate nucleus.

Human colour vision depends on three classes of cone photoreceptors, those sensitive to short (S), medium (M) or long (L) wavelengths, and on how signals from these cones are combined by neurons in the retina and brain. Macaque monkey colour vision is similar to human, and the receptive fields of macaque visual neurons have been used as an animal model of human colour processing. P retinal ganglion cells and parvocellular neurons are colour-selective neurons in macaque retina and lateral geniculate nucleus. Interactions between cone signals feeding into these neurons are still unclear. On the basis of experimental results with chromatic adaptation, excitatory and inhibitory inputs from L and M cones onto P cells (and parvocellular neurons) were thought to be quite specific (Fig. 1a). But these experiments with spatially diffuse adaptation did not rule out the 'mixed-surround' hypothesis: that there might be one cone-specific mechanism, the receptive field centre, and a surround mechanism connected to all cone types indiscriminately (Fig. 1e). Recent work has tended to support the mixed-surround hypothesis. We report here the development of new stimuli to measure spatial maps of the linear L-, M- and S-cone inputs to test the hypothesis definitively. Our measurements contradict the mixed-surround hypothesis and imply cone specificity in both centre and surround.     

Functions of the ON and OFF channels of the visual system

In the mammalian eye, the ON-centre and OFF-centre retinal ganglion cells form two major pathways projecting to central visual structures from the retina. These two pathways originate at the bipolar cell level: one class of bipolar cells becomes hyperpolarized in response to light, as do all photoreceptor cells, and the other class becomes depolarized on exposure to light, thereby inverting the receptor signal. It has recently become possible to examine the functional role of the ON-pathway in vision by selectively blocking it at the bipolar cell level using the glutamate neurotransmitter analogue 2-amino-4-phosphonobutyrate (APB)1. APB application to monkey, cat and rabbit retinas abolishes ON responses in retinal ganglion cells, the lateral geniculate nucleus and the visual cortex but has no effect on the centre-surround antagonism of OFF cells or the orientation and direction selectivities in the cortex2-5. These and related findings6-11 suggest that the ON and OFF pathways remain largely separate through the lateral geniculate nucleus and that in the cortex, contrary to some hypotheses, they are not directly involved in mechanisms giving rise to orientation and direction selectivities. We have examined the roles of the ON and OFF channels in vision in rhesus monkeys trained to do visual detection and discrimination tasks. We report here that the ON channel is reversibly blocked by injection of APB into the vitreous. Detection of light increment but not of light decrement is severely impaired, and there is a pronounced loss in contrast sensitivity. The perception of shape, colour, flicker, movement and stereo images is only mildly impaired, but longer times are required for their discrimination. Our results suggest that two reasons that the mammalian visual system has both ON and OFF channels is to yield equal sensitivity and rapid information transfer for both incremental and decremental light stimuli and to facilitate high contrast sensitivity.     

Functional interaction of phytochrome B and cryptochrome 2

Light is a crucial environmental signal that controls many photomorphogenic and circadian responses in plants. Perception and transduction of light is achieved by at least two principal groups of photoreceptors, phytochromes and cryptochromes. Phytochromes are red/far-red light-absorbing receptors encoded by a gene family of five members (phyA to phyE) in Arabidopsis. Cryptochrome 1 (cry1), cryptochrome 2 (cry2) and phototropin are the blue/ultraviolet-A light receptors that have been characterized in Arabidopsis. Previous studies showed that modulation of many physiological responses in plants is achieved by genetic interactions between different photoreceptors; however, little is known about the nature of these interactions and their roles in the signal transduction pathway. Here we show the genetic interaction that occurs between the Arabidopsis photoreceptors phyB and cry2 in the control of flowering time, hypocotyl elongation and circadian period by the clock. PhyB interacts directly with cry2 as observed in co-immunoprecipitation experiments with transgenic Arabidopsis plants overexpressing cry2. Using fluorescent resonance energy transfer microscopy, we show that phyB and cry2 interact in nuclear speckles that are formed in a light-dependent fashion.     

Spectral sensitivity of a novel photoreceptive system mediating entrainment of mammalian circadian rhythms

Environmental light cycles are the dominant synchronizers of circadian rhythms in the field, and artificial light cycles and pulses are the major tools used in the laboratory to analyse properties of circadian systems. It is therefore surprising that few studies have analysed the physical parameters of light stimuli that affect circadian rhythms. There have previously been no spectral sensitivity measurements for phase shifting the circadian rhythms of mammals and only two preliminary reports on the wavelength dependence of this response exist. Using the magnitude of phase shift caused by a single 15-min pulse of monochromatic light given 6 h after activity onset, we have now characterized the spectral sensitivity of the photoreceptors responsible for phase shifting the locomotor rhythm of the hamster (Mesocricetus auratus). The sensitivity curve for this response has a maximum near 500 nm and is similar to the absorption spectrum for rhodopsin. Although the spectral sensitivity is consistent with a rhodopsin-based photopigment, two features of the photoreceptive system that mediates entrainment are unusual: the threshold of the response is high, especially for a predominantly rod retina like that of the hamster, and the reciprocal relationship between intensity and duration holds for extremely long durations (up to 45 min). These results suggest that the photoreceptive system mediating entrainment is markedly different from that involved in visual image formation.