Circadian Photoreception in Mammals MCB 186: Circadian Biology Joshua Gooley MCB 186: Circadian Biology October 26, 2005

The retinohypothalamic tract

Laminar Organization of the Retina

Rods and Cones are not required for light entrainment Freedman et al., 1999

Light entrainment and the pupillary light reflex are mediated by a short wavelength-sensitive, vitamin A1-based photopigment Phase Shifting Pupillary light reflex rdta/cl Hattar et al., 2003 Lucas et al., 2001

Cryptochromes are expressed in the inner retina Miyamoto and Sancar, 1998

Melanopsin is expressed in retinal ganglion cells

RGCs that project to the SCN contain melanopsin SCN injection PACAP mRNA Opn4 mRNA OPN4 immuno Retro label Gooley et al., 2001 Hannibal et al., 2002

Gene-targeted tracing in OPN4-tauLacZ mice reveals an intense projection to the SCN Hattar et al., 2002

Retinal ganglion cells that project to the SCN are intrinsically photosensitive Berson et al., 2002

Intrinsically photosensitive RGCs contain melanopsin Lucifer yellow merged

Some intrinsically photosensitive RGCs show a rapid and transient response to light Effects of synaptic blockade on the intrinsic response We have shown that rod/cone circuits do contact at least some photosensitive ganglion cells, that is, they provide a second mechanism, independent of the intrinsic phototransduction, by which light can excite these cells. But we have also seen evidence of a possible suppressive influence. David Berson

ipRGCs are most sensitive to short-wavelength light Berson et al., 2002

Melanopsin cells project to the IGL and olivary pretectal nucleus (involved in the pupillary light reflex) IGL Hattar et al., 2002 Gooley et al., 2003

OPN4 cells send collateralized projections to the SCN and olivary pretectal nucleus Tracer injection in SCN Tracer injection in OPT Retros from SCN Retros from OPT OPN4 mRNA Gooley et al., 2003

A broad role for OPN4 in non-visual photoreception Gooley et al., 2003

Melanopsin is necessary for intrinsic photosensitivity Hattar et al., 2003

Phase shifting is attenuated, but not absent in OPN4 knockout mice Panda et al., 2002

Pupillary light reflex is attenuated, but not absent in OPN4 knockout mice Lucas et al., 2003

OPN4 cells likely receive input from the classical visual photoreceptors

Light entrainment abolished in mice without rods, cones, and melanopsin Panda et al., 2003

Pupillary light reflex abolished in mice without rods, cones, and melanopsin Hattar et al., 2003

Negative masking and light-induced suppression of NAT absent in mice without rods, cones, and melanopsin Masking absent NAT suppression absent Panda et al., 2003

Mammalian Photoreceptors

The vertebrate phototransduction cascade Whether or not melanopsin is the pigment, want to understand what is downstream in the transduction cascade, what second messengers couple the photoactivation step to the voltage change across the cell’s membrane. STEPHANIE There are two attractive models for the transduction process, those in the vertebrate and invertebrate photoreceptors. Here’s the vertebrate. Attractive starting point because apparently used in all known vertebrate photoreceptors. Light activation of opsin leads to activation of the Gprotein transducin and activation of PDE.. This hydrolyzes cGMP which is high in darkness, reducing its concentration and closing plasma membrane ion channels that are gated by cGMP, so call CNG channels. All vertebrate photoreceptors use a cyclic nucleotide cascade, although at least one inverts the sign of the response by inhibiting PDE, so that the cell depolarizes.

The invertebrate phototransduction cascade The other plausible model is that used by invertebrate photoreceptors. Photoactivation of pigment activates a different Gprotein, this time in the Gq/11 family. This leads to activation of phospholipase C to cleave PIP2, releasing IP3 and DAG. In some invertebrates (such as Limulus), the major signalling pathway involves the soluble factor IP3, which acts at IP3 receptors to release Ca from stores and through unknown mechanisms opening of channels of the TRP family. In others, e.g., in Drosophila, the DAG branch appears central, perhaps through signalling through polyunsaturated fatty acids

Human melanopsin confers photosensitivity to light-insensitive neuro-2a cells GFP OPN4 Light-evoked current Melyan et al., 2005

OPN4 may function as a bireactive opsin Xenopus oocytes Panda et al., 2005

HEK293 cells that co-express OPN4 and TRPC3 channels are photosensitive Qui et al., 2005

OPN4 retinal ganglion cells in primate What is the photopigment that gives rise to this curve? Again we are following in the footsteps of the work done in rat retina - this time led by King W. Yau. In rat we know that these ganglion cells contain the opsin like protein - Melanopsin. Recently, Yau made an antibody against human melanopsin and we can now show conclusively that the phototransducing giant ganglion cells that are retrogradely labelled from LGN tracer contain melanopsin. In macaque monkey as well as human the melanopsin stains a population of ganglion cells with large sparsely branching dendritic trees as you see here in a wholemount view. The MOP antibody labels both the cell body and the complete dendritic tree. This second panel shows a photostained luminance coding cell after retrograde label from tracer injections made in the LGN. In the next panel you can see that superimposing the pictures that the MOP containing cell are as we knew would be the case just from the morphological identity - are the corresponding cells. Dacey et al., 2005

Distribution of primate OPN4-containing ganglion cells Dacey distribution ~3000 cells (0.3% of all GCs) There are about only 3000 MOP containing cells in the entire retina - here in this wholemount each one is indicated by a dot. You can see that the density peaks around the fovea - it is important to note however that there is only about 1 MOP cell for every 2000 ganglion cells in the central retina (and only 1/500 overall. Despite this extremely low density there is in fact a photoreceptive plexus dramatically wraps round the foveal margins creating distinct meswork. In the periphery also the dendritic network provide a uniform coverage over the retinal surface despite the extremely low density of cells. Dacey et al., 2005