Recently, melanopsin has emerged as the leading candidate for the elusive photopigment of the mammalian circadian system. This novel opsin-like protein is expressed in retinal ganglion cells that form the retinohypothalamic tract, a neuronal connection between the retina and the suprachiasmatic nucleus. These hypothalamic structures contain the circadian pacemaker, which generates daily rhythms in physiology and behavior. In mammals, proper synchronization of these rhythms to the environmental light-dark cycle requires retinal input. Surprisingly, rod and cone photoreceptors are not required. Instead, the melanopsin-containing ganglion cells are intrinsically sensitive to light, perhaps responding via a melanopsin-based signaling pathway. To test this hypothesis, we have characterized melanopsin following heterologous expression in COS cells. We found that melanopsin absorbed maximally at 424 nm after reconstitution with 11-cis-retinal. Furthermore, melanopsin activated the photoreceptor G-protein, transducin, in a light-dependent manner. In agreement with the measured absorbance spectrum, melanopsin was most efficiently excited by blue light (420-440 nm). In contrast, published action spectra suggest that the photopigment underlying the intrinsic light sensitivity of SCN-projecting RGCs has an absorption maximum near 484 nm. In summary, our experiments constitute the first direct demonstration that melanopsin forms a photopigment capable of activating a G-protein, but its spectral properties are not consistent with the action spectrum for circadian entrainment.

In mammals, light entrainment of the circadian clock, located in the suprachiasmatic nuclei (SCN), requires retinal input. Traditional rod and cone photoreceptors, however, are not required. Instead, the SCN-projecting retinal ganglion cells (RGCs) function as autonomous photoreceptors and exhibit light responses independent of rod-and cone-driven input. Using whole-cell patch-clamp recording techniques, we have investigated the morphological and electrophysiological properties of this unique class of RGCs. Although SCN-projecting RGCs resemble Type III cells in form, they display strikingly different physiological properties from these neurons. First, in response to the injection of a sustained depolarizing current, SCN-projecting cells fired in a transient fashion, in contrast to most RGCs which fired robust trains of action potentials. Second, in response to light, SCN-projecting RGCs exhibited an intensity-dependent transient depolarization in the absence of rod and cone input. This depolarization reached a peak within 5 s and generated increased spiking activity before decaying to a plateau. Voltage-clamp recordings were used to characterize the light-activated conductance which generated this depolarization. In response to varying light intensities, SCNprojecting RGCs exhibited a graded transient inward current which peaked within 5 s and decayed to a plateau. The voltage dependence of the light-activated current was obtained by subtracting currents elicited by a voltage ramp before and during illumination. The light-activated current displayed both inward and outward rectification and was largely unaffected by substitution of extracellular Na + with choline. In both respects, the intrinsic light-activated current observed in SCNprojecting RGCs resembles currents carried by ion channels of the transient receptor potential (trp) family, which are known to mediate the light response of invertebrate photoreceptors.

Melanopsin-containing retinal ganglion cells (RGCs) project to the suprachiasmatic nuclei (SCN) and mediate photoentrainment of the circadian system. Melanopsin is a novel retinal-based photopigment that renders these cells intrinsically photosensitive (ip). Although genetic ablation of melanopsin abolishes the intrinsic light response, it has a surprisingly minor effect on circadian photoentrainment. This and other non-visual responses to light are lost only when the melanopsin deficiency is coupled with mutations that disable classical rod and cone photoreceptors, suggesting that melanopsin-containing RGCs also receive rod-and cone-driven synaptic inputs. Using wholecell patch-clamp recording, we demonstrate that light triggers synaptic currents in ipRGCs via activation of ionotropic glutamate and γ-aminobutyric acid (GABA) receptors. Miniature postsynaptic currents (mPSCs) were clearly observed in ipRGCs, although they were less robust and were seen less frequently than those seen in non-ip cells. Pharmacological treatments revealed that the majority of ipRGCs receive excitatory glutamatergic inputs that were blocked by DNQX and/or kynurenic acid, as well as inhibitory GABAergic inputs that were blocked by bicuculline. Other ipRGCs received either glutamatergic or GABAergic inputs nearly exclusively. Although strychnine (Strych)-sensitive mPSCs were evident on many non-ipRGCs, indicating the presence of glycinergic inputs, we saw no evidence of Strych-sensitive events in ipRGCs. Based on these results, it is clear that SCN-projecting RGCs can respond to light both via an intrinsic melanopsin-based signaling cascade and via a synaptic pathway driven by classical rod and/or cone photoreceptors. It remains to be determined how the ipRGCs integrate these temporally distinct inputs to generate the signals that mediate circadian photoentrainment and other non-visual responses to light.

The time course of G-protein-coupled responses is largely determined by the kinetics of GTP hydrolysis by the G protein alpha subunit, which is accelerated by interaction with regulator of G-protein signaling (RGS) proteins. Light responses of ON-bipolar cells of the vertebrate retina require rapid inactivation of the G protein Galphao, which is activated in the dark by metabotropic glutamate receptor, mGluR6, in their dendritic tips. It is not yet known, however, which RGS protein(s) might be responsible for rapid inactivation kinetics. By immunofluorescence and co-immunoprecipitation, we have identified complexes of the Galphao-selective RGS proteins RGS7 and RGS11, with their obligate binding partner, Gbeta5, that are localized to the dendritic tips of murine rod and cone ON-bipolar cells, along with mGluR6. Experiments using pre- and post-synaptic markers, and a dissociated bipolar cell preparation, clearly identified the location of these complexes as the ON-bipolar cell dendritic tips and not the adjacent photoreceptor terminals or horizontal cell dendrites. In mice lacking mGluR6, the distribution of RGS11, RGS7 and Gbeta5 shifts away from the dendritic tips, implying a functional relationship with mGluR6. The precise co-localization of Gbeta5-RGS7 and Gbeta5-RGS11 with mGluR6, and the dependence of localization on the presence of mGluR6, suggests that Gbeta5-RGS7 and Gbeta5-RGS11 function specifically in the mGluR6 signal transduction pathway, where they may stimulate the GTPase activity of Galphao, thus accelerating the ON-bipolar cell light response, in a manner analogous to the acceleration of photoreceptor light responses by the Gbeta5-RGS9-1 complex.