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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.sup.-/-] 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., The discovery of ipRGCs has overturned the century-old belief that rods and cones are the only mammalian retinal photoreceptors (1-6). These ganglion-cell photoreceptors serve primarily non-image visual functions, one of [...]

Author(s): S. Hattar [1]; R. J. Lucas [2]; N. Mrosovsky [3]; S. Thompson [2]; R. H. Douglas [4]; M. W. Hankins [2]; J. Lem [5]; M. Biel [6]; F. Hofmann [...]

The primary circadian pacemaker, in the suprachiasmatic nucleus (SCN) of the mammalian brain, is photoentrained by light signals from the eyes through the retinohypothalamic tract. Retinal rod and cone cells are not required for photoentrainment. Recent evidence suggests that the entraining photoreceptors are retinal ganglion ceils (RGCs) that project to the SCN. The visual pigment for this photoreceptor may be melanopsin, an opsin-like protein whose coding messenger RNA is found in a subset of mammalian RGCs. By cloning rat melanopsin and generating specific antibodies, we show that melanopsin is present in cell bodies, dendrites, and proximal axonal segments of a subset of rat RGCs. In mice heterozygous for tau-lacZ targeted to the melanopsin gene locus, β-galactosidase--positive RGC axons projected to the SCN and other brain nuclei involved in circadian photoentrainment or the pupillary light reflex. Rat RGCs that exhibited intrinsic photosensitivity invariably expressed melanopsin. Hence, melanopsin is most likely the visual pigment of phototransducing RGCs that set the circadian dock and initiate other non--image-forming visual functions., Retinal rods and cones, with their light-sensitive, opsin-based pigments, are the primary photoreceptors for vertebrate vision. Visual signals are transmitted to the brain through RGCs, the output neurons whose axons [...]

The physiological role of protein kinase C (PKC) in phototransduction was investigated through monitoring the effects of agents that either activate or suppress PKC in single and mechanically dissociated rods of salamander. Application of PKC inhibitor, GF109203X did not affect rod sensitivity under the different light conditions. It was therefore suggested that PKC had no significant influence in the photosensitivity of rods particularly in light adaptation.

Phototransduction in retinal rods involves a G protein-coupled signaling cascade that leads to cGMP hydrolysis and the closure of cGMP-gated cation channels that are open in darkness, producing a membrane hyperpolarization as the light response. For many years there have also been reports of the presence of a phosphoinositide pathway in the rod outer segment, though its functions and the molecular identities of its components are still unclear. Using immunocytochemistry with antibodies against various phosphoinositide-specific phospholipase C (PLC) isozymes ([Beta]1-4, [Gamma]1-2, and [Delta]1-2), we have found PLC[Beta]4-like immunoreactivity in rod outer segments. Similar experiments with antibodies against the [Alpha]-subunits of the [G.sub.q] family of G proteins, which are known to activate PLC[Beta]4, have also demonstrated [G.sub.[Alpha]11]-like immunoreactivity in this location. Immunoblots of total proteins from whole retina or partially purified rod outer segments with anti-PLC[Beta]4 and anti-[G.sub.[Alpha]11] antibodies gave, respectively, a single protein band of the expected molecular mass, suggesting specific labelings. The retinal locations of the two proteins were also supported by in situ hybridization experiments on mouse retina with probes specific for the corresponding mouse genes. These two proteins, or immunologically identical isoforms, therefore likely mediate the phosphoinositide signaling pathway in the rod outer segment. At present, [G.sub.[Alpha]11] or a [G.sub.[Alpha]11]-like protein represents the only G protein besides transducin (which mediates phototransduction) identified so far in the rod outer segment. Although absent in the outer segment layer, other PLC isoforms as well as [G.sub.[Alpha]q] (another [G.sub.q] family member), are present elsewhere in the retina.

Exposure to light causes the rods and cones of the two lateral eyes of vertebrates to hyperpolarize. This can be attributed to a cyclic GMP cascade that results in cGMP hydrolysis and the consequent closure of cGMP-gated cation channels that open in the dark. In contrast, the parietal or third eye of lizards and other lower vertebrates depolarize when illuminated. Despite these opposite reactions, evidence is found indicating that both lateral eyes and parietal eyes use a cGMP signalling cascade for phototransduction without the involvement of inositol-1,4,5-trisphosphate.

The activity of guanylate cyclase in single retinal rod segments decreases in the presence of calcium ions. The phototransduction reaction in vertebrates uses the enzyme. Guanosine 3':5' cyclic monophosphate (cGMP) gates cationic channels on the plasma membrane of the photoreceptor outer segments. Guanylate cyclase produces the second messenger which is hydrolyzed by a light- and cGMP-dependent phosphodiesterase. A quantitative characterization of the role of guanylate cyclase is reported, along with exploration of the role of Ca2+, free Mg2+ and intracellular K+.

The regulation of the enzymes guanylate cyclase and phosphodiesterase by calcium ions affects the step response and sensitivity of intact retinal rods. A negative feedback by calcium ions regulates the phototransduction mechanism that results in adaptation to light. At low light intensities, calcium ions regulate the activity of guanylate cyclase, while regulation of the activity of phosphodiesterase becomes more important at higher intensities. The modulation by calcium ions of the guanosine 3':5'-cyclic monophosphate (cGMP)-gated channel has only a minor effect on rod sensitivity.

Analysis of cGMP-gated channels derived from the expressed cloned subunits in human embryonic kidney (HEK) reveals that the 240-kDa protein in the retinal rod channel comprises the subunit 2b of the channel. This subunit 2b is involved in the Ca2+-calmodulin regulation of the channel. The N-terminal of the protein 2b is implicated in the regulation. The subunit might also be a medium for calmodulin binding.

An investigation of the structure of the cyclic guanylic acid-gated cation channel in retinal rods is reported. Cloning procedures succeeded in cloning a protein from human retina that had 30% overall identity to the rod channeel subunit. The eprotein localizes to rod outer segments, but of itself cannot form a functional channel. Co-expression of the protein with human rod channel protein in functional cation channel. These results indicate that the protein is a key subunit of the cyclic guanylic acid-gated cation channel in retinal rods.