Prolonged light exposure induces widespread phase shifting in the circadian clock and visual pigment gene expression of the Arvicanthis ansorgei retina.

PURPOSE: Prolonged periods of constant lighting are known to perturb circadian clock function at the molecular, physiological, and behavioral levels. However, the effects of ambient lighting regimes on clock gene expression and clock outputs in retinal photoreceptors--rods, cones and intrinsically photosensitive retinal ganglion cells--are only poorly understood. METHODS: Cone-rich diurnal rodents (Muridae: Arvicanthis ansorgei) were maintained under and entrained to a 12 h:12 h light-dark cycle (LD; light: ~300 lux). Three groups were then examined: control (continued maintenance on LD); animals exposed to a 36 h dark period before sampling over an additional 24 h period of darkness (DD); and animals exposed to a 36 h light period before sampling over an additional 24 h period of light (~300 lux, LL). Animals were killed every 3 or 4 h over 24 h, their retinas dissected, and RNA extracted. Oligonucleotide primers were designed for the Arvicanthis clock genes Per1, Per2, Cry1, Cry2, and Bmal1, and for transcripts specific for rods (rhodopsin), cones (short- and mid-wavelength sensitive cone opsin, cone arrestin, arylalkylamine N-acetyltransferase) and intrinsically photosensitive retinal ganglion cells (melanopsin). Gene expression was analyzed by real-time PCR. RESULTS: In LD, expression of all genes except cone arrestin was rhythmic and coordinated, with acrophases of most genes at or shortly following the time of lights on (defined as zeitgeber time 0). Arylalkylamine N-acetyltransferase showed maximal expression at zeitgeber time 20. In DD conditions the respective profiles showed similar phase profiles, but were mostly attenuated in amplitude, or in the case of melanopsin, did not retain rhythmic expression. In LL, however, the expression profiles of all clock genes and most putative output genes were greatly altered, with either abolition of daily variation (mid-wavelength cone opsin) or peak expression shifted by 4-10 h. CONCLUSIONS: These data are the first to provide detailed measures of retinal clock gene and putative clock output gene expression in a diurnal mammal, and show the highly disruptive effects of inappropriate (nocturnal) lighting on circadian and photoreceptor gene regulation.

Regulation of retinal photoreceptor phagocytosis in a diurnal mammal by circadian clocks and ambient lighting.

PURPOSE: To characterize light and circadian control of photoreceptor phagocytosis in a diurnal cone-rich rodent. METHODS: Diurnal Arvicanthis ansorgei were maintained under standard cyclic light (12 hours 300 lux white light [L]/12 hours dark [D]) and were divided into five groups: 1, maintained in LD; 2, transferred to constant darkness (DD); 3, transferred to constant light (LL, 300 lux); 4, subjected to 6-hour advance in light onset; and 5, subjected to 6-hour delay in light onset. Animals were killed every 3 hours during 24 hours, and their eyes were rapidly enucleated and fixed. Cryosections were stained using specific rod (rhodopsin) and cone (MW cone opsin) antibodies. Immunopositive inclusions within the retinal pigment epithelium layer were quantified for each time point. RESULTS: In LD, both rod and cone phagocytosis showed coincident synchronized profiles with sharp peaks 1 to 2 hours after light onset. In groups 2 and 3, phagocytic activity shifted partially to the new light schedules. In DD, the temporal phagocytic profile resembled largely that of LD. On the contrary, LL animals exhibited large alterations in both rod and cone phagocytosis. There was no longer any peak, with both showing relatively uniform profiles. In addition, the number of cone phagosomes was much higher ( approximately 250% increase) compared with LD or DD. CONCLUSIONS: These data are the first to measure photoreceptor phagocytosis in a diurnal mammal under different lighting conditions and to highlight the disruptive effects of constant light, especially on cone photoreceptor function.

Photoreceptor organisation and phenotypic characterization in retinas of two diurnal rodent species: potential use as experimental animal models for human vision research.

To characterize rod and cone distribution and composition in two diurnal mouse-like rodents, retinas from adult Arvicanthis ansorgei and Lemniscomys barbarus were processed for immunohistochemistry using multiple rod- and cone-specific antibodies. Antibodies tested included rhodopsin, cone opsins, pan-arrestin and cone arrestin, recoverin, and cGMP dependent ion channel. In both species, retinas were composed of approximately 33% cones, and most antibodies gave similar staining patterns. Data show these two diurnal rodents possess large numbers of cones, organised in a strict anatomical array. This suggests that diurnal rodents in general possess elevated cone numbers and could constitute valuable models for investigating cone pathophysiology.

Photoreceptor organization and rhythmic phagocytosis in the nile rat Arvicanthis ansorgei: a novel diurnal rodent model for the study of cone pathophysiology.

PURPOSE: To characterize rod and cone distribution, organization, and phagocytosis in the diurnal mouse-like rodent Arvicanthis ansorgei. METHODS: Retinas of adult A. ansorgei were processed for histology, electron microscopy and immunohistochemistry using rod- and mouse cone-specific antibodies. For phagocytosis studies, retinas were sampled every 3 hours under a 12-hour light-dark cycle and processed for double-label immunohistochemistry. The number of phagosomes in the retinal pigmented epithelium were quantified with a morphometric system. RESULTS: A. ansorgei retinas were composed of 33% cones and 67% rods, approximately 10 times more cones than mice and rats. Cones were arranged in two cell layers at the scleral surface, distributed uniformly across the entire retina. Cone arrestin was distributed throughout the dark-adapted cones, from outer segments to synapses, whereas short- and mid-wavelength cone opsins were restricted to outer segments. Short-wavelength cone density was mapped in wholemounted retinas, in a significantly higher number in the central region. Rhodopsin immunopositive (rod) phagosomes showed a small peak late in the dark phase, then a large burst 1 to 2 hours after light onset, after decreasing to low baseline levels by 12 AM. Mid-wavelength cone opsin immunopositive (cone) phagosomes were 10 times less numerous than rods, and demonstrated a broad peak 1 to 2 hours after light onset. CONCLUSIONS: The diurnal rodent A. ansorgei possesses a large number of cones, organized in a strict anatomic array. Rod and cone outer segment phagocytosis and shedding can be monitored simultaneously and show similar profiles but different amplitudes. This species may constitute a valuable novel animal model for investigating cone pathophysiology.