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

2. The retinohypothalamic tract

3. Laminar Organization of the Retina

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

5. 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

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

7. Melanopsin is expressed in retinal ganglion cells

8. 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

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

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

11. Intrinsically photosensitive RGCs contain melanopsin
Lucifer yellow
merged

12. 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

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

14. 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

15. 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

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

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

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

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

20. OPN4 cells likely receive input from the classical visual photoreceptors

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

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

23. 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

24. Mammalian Photoreceptors

25. 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.

26. 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

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

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

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

30. 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

31. 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