Reference #: FUK-1017-211060
Submit Date: 03/26/2002 23:59:14-0500

Pineal photoreception and opsins.

ABSTRACT:
The chicken pineal oscillator regulates a daily rhythm of melatonin production, and the rhythm synchronizes to the environmental light-dark cycle via endogenous photic input pathway. The presence of an opsin-type photoreceptor, pinopsin, in the chicken pinealocytes (clock cells) suggests a G-protein-mediated photic input pathway to the oscillator. We cloned cDNAs encoding the alpha-subunits of Gt1 and G11 from the chick pineal cDNA library, and these two alpha-subunits were immunohistochemically colocalized with pinopsin in the luminal membranes of all the pineal follicles. These G-proteins interacted with opsins in a light-dependent manner in vitro, raising a possibility that they contribute to the pineal phototransduction. Upon ectopic expression of a Gq/G11-coupled m1-type muscarinic acetylcholine receptor (m1 mAChR) in the cultured pineal cells, carbamylcholine stimulation of m1 mAChR and the consequent activation of endogenous G11 induced phase-dependent phase shifts of the melatonin rhythm in a manner very similar to the effect of light. This contrasts well with no effect of carbamylcholine on the cells expressing Gi/Go/Gt-coupled m2 mAChR. These results together demonstrate the G11-mediated opsin signaling pathway contributing to the photic entrainment of the circadian clock. We also investigated the light-signal transduction process to the nucleus where the clock gene expression is regulated, and we found that mitogen-activated protein kinase (MAPK) is rapidly dephosphorylated upon light illumination during the nighttime. In constant darkness, MAPK exhibited a circadian rhythm in tyrosine phosphorylation and in enzymatic activity with a peak during subjective night. Importantly MAPK kinase inhibitor PD98059 treatment during the subjective night shifted the peak-time of MAPK phosphorylation and induced a phase-delay of the oscillator regulating the melatonin production. In cultured 293EBNA cells, BMAL1:CLOCK-induced transcription was negatively regulated by MAPK-mediated phosphorylation of cBMAL1 at Thr-534, and these observations support an important role of MAPK not only for the photic input pathway but also for circadian time keeping mechanism in the pineal cells.

Keywords: pineal gland, pinopsin, G11, mitogen-activated protein kinase (MAPK)

Reference #: FOS-1017-173809
Submit Date: 03/26/2002 08:37:31-0500

Irradiance detection and the role of non-rod, non-cone vertebrate opsins.

ABSTRACT:
Varied aspects of vertebrate physiology and behaviour are regulated by gross changes in environmental light. For example, over the past 10 years evidence has accumulated in both man and rodents that some of these irradiance changes are detected by novel ocular photoreceptors. The photoreceptive mechanisms underlying these, non-rod, non-cone photoreceptors remain largely uncharacterised. Yet their importance may be profound. It is now clear that light exposure can influence alertness and sleep propensity. Furthermore, light regulates the phase of circadian clocks, and thus the timing of rhythmic functions such as digestion, sleep and performance. The first part of this presentation will consider: (i) the extent to which non-rod, non-cone ocular photoreceptors might regulate mammalian physiology and behaviour, (ii) our progress to-date in identifying these novel photoreceptor mechanisms, and (iii) the extent to which information from both novel and classical photoreceptors is integrated. In contrast to mammals, isolated organs and cell lines from zebrafish exhibit circadian oscillations in clock gene expression that can be entrained to a 24h-light/dark cycle. In this case we know even less about the mechanisms underlying zebrafish cellular photosensitivity. We have recently identified a novel opsin family, tmt-opsin, that has a genomic structure characteristic of vertebrate photopigments, an amino acid identity equivalent to the known photopigment opsins, and the essential residues required for photopigment function. Significantly tmt-opsin is expressed in a wide variety of neural and non-neural tissues, including a zebrafish embryonic cell line that exhibits a light entrainable clock. The second part of this presentation will assess tmt-opsins candidacy as a photopigment with reference to other vertebrate opsins, and address the extent to which functionally related opsin molecules share a close phylogenetic relationship based upon both amino acid identity and a conserved genomic structure.

Keywords: Circadian, Opsin, Irradiance, Vertebrate

Reference #: 034534
Submit Date:

Break

ABSTRACT:

Keywords:

Reference #: DAV-1017-696686
Submit Date: 04/01/2002 15:18:50-0500

Mechanisms for the inhibition of locomotor activity

ABSTRACT:
It has long been thought that the wheel-running behavior of nocturnal rodents is inhibited by a circadian signal. The best evidence for this comes from the "temporal chimera" experiments of Vogelbaum and Menaker (1992). Other experiments, especially those of Silver and colleagues (1991,1996), indicate that the circadian regulation of locomotor activity is mediated by a diffusible signal. Light entrains the circadian pacemaker so that activity is inhibited at some times of day and expressed at others. In addition to entrainment, light also has an acute inhibitory effect on activity called "masking". Little is know about the mechanisms of either circadian or acute inhibition of locomotor activity, or about the extent to which the mechanisms are similar. Recently, in collaboration with Charles Weitz's laboratory, we identified a factor, transforming growth factor -alpha (TGFalpha) with properties of an inhibitory signal. TGFalpha is strongly expressed in the suprachiasmatic nucleus (SCN) and infusion of TGFalpha into the third ventricle of hamsters reversibly inhibits locomotor activity (Kramer et al., 2001). Although TGFalpha is a good candidate for a circadian signal, genetic evidence indicated that its receptor, the EGF receptor (EGFR) is also involved in masking. Thus EGF signaling could be involved in both the circadian and acute inhibition of locomotor activity. Integration of these mechanisms is also suggested by the persistence of both entrainment and masking in mice lacking rods (Freedman et al., 1999; Mrsovsky et al., 1999). New evidence concerning the role of TGFalpha in circadian timing and masking will be presented. Supported by NIH Grant HD18686.

Keywords: circadian, suprachiasmatic, growth factor

Reference #: HAT-1017-172472
Submit Date: 03/26/2002 13:45:49-0500

Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity.

ABSTRACT:
ganglionThe 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 cells (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, beta-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 clock and initiate other non-image-forming visual functions.

Keywords: Melanopsin, Photoentrainment, circadian, SCN

Reference #: GOO-1017-644473
Submit Date: 04/01/2002 00:29:00-0500

A broad role for melanopsin in non-visual photoreception based on neuroanatomical evidence in rats

ABSTRACT:
The rod and cone photoreceptors that mediate visual phototransduction in mammals are not required for light-induced circadian entrainment, suppression of pineal melatonin, negative masking, or the pupillary light reflex. The novel photoreceptor(s) that mediates these light-driven responses, however, remains unknown. The photopigment melanopsin is expressed in retinal ganglion cells (RGCs) that project to the suprachiasmatic nucleus (SCN), intergeniculate leaflet (IGL) and olivary pretectal nucleus (OPN). Furthermore, these RGCs are directly photosensitive and depolarize in response to light. However, it is not known whether melanopsin is expressed in RGCs that project to other retinorecipient areas of the brain. We injected the SCN, ventral subparaventricular zone (vSPZ), ventrolateral preoptic nucleus (VLPO), pretectal area, lateral geniculate nucleus (LGN), and superior colliculus (SC) with FluoroGold or cholera toxin (beta subunit) (CTB), and examined retrogradely labeled RGCs for melanopsin mRNA. Melanopsin was expressed in the majority of RGCs that project to the SCN, vSPZ, and VLPO, and in a subpopulation of RGCs that innervate the pretectal area. In animals injected with FluoroGold and CTB in the SCN and pretectal area, respectively, a subpopulation of retrogradely RGCs was positive for both tracers and melanopsin mRNA, demonstrating that retinohypothalamic RGCs bifurcate and project to the pretectal area. FluoroGold injections in the LGN that included the IGL also labeled RGCs that express melanopsin, suggesting that melanopsin may modulate SCN activity directly, via the monosynaptic retinohypothalamic pathway, and indirectly via the IGL and the geniculohypothalamic tract. Melanopsin mRNA was nearly absent in RGCs projecting to the dorsal lateral geniculate (DLG) and the SC. Our results suggest that melanopsin may have a broad role in the regulation of non-visual photoreception, and could mediate circadian entrainment, negative masking, the regulation of sleep-wake states, and the pupillary light reflex.

Keywords: melanopsin, ganglion cells, circadian, suprachiasmatic nucleus

Reference #: 034743
Submit Date:

Introductory Remarks

ABSTRACT:

Keywords: