Circadian Rhythm of Period1 Clock Gene Expression in NOS Amacrine Cells of the Mouse Retina

Abstract

Abstract: : Purpose: The vertebrate retina contains self–sustained circadian clocks that broadly influence retinal physiology. In the present study we have examined the relationship of nitric oxide, GABA–ergic and glycinergic inner retinal neurons with expression of a reporter for the circadian clock gene Period1 (Per1). Methods: Adult male mice used in this study were hemizygous for the Per1::GFP transgene. Immunocytochemistry was performed on both retinal vertical slices and wholemount retinas. The primary antibodies used were: 1:5000 rabbit polyclonal anti–GFP, 1:500 mouse monoclonal anti–bNOS, 1:5000 goat polyclonal anti–glycine transporter 1, 1:5 mouse monoclonal GAD65. The secondary antibodies (1:500) include Alexa Fluor 594 donkey anti–goat IgG (H+L), Alexa Fluor 488 donkey anti–rabbit IgG, Alexa Fluor 594 donkey anti–mouse IgG. Specimens were visualized using confocal microscopy at the excitation wavelength of 488 nm for Alexa 488 and 543nm for Alexa 594. Results: Using Per1::GFP transgenic mice, we found that 72% of brain nitric oxide synthase (bNOS) expressing amacrine cells (NOS amacrine cells) sampled during daytime were also immunoreactive for GFP. The number of bright GFP⁺ NOS⁺ cells was greater at zeitgeber time (ZT) 10 than at 22, and this pattern persisted in retinas from animals which were placed in constant darkness, [circadian time (CT) 10 vs 22]. Intensities of GFP–IR for individual NOS amacrine cells were analyzed at ZT4, 10, 16 and 22, with the peak value occurring at ZT10. Similar results were obtained from retinas sampled at CT4, 10, 16 and 22 in constant darkness, indicating that an endogenous circadian clock drives oscillations of Per1 clock gene transcription within NOS amacrine cells. The predominance of GFP⁺ amacrine cells, (82%), were also immunoreactive to glutamate decarboxylase 65, but no GFP⁺ amacrine cells colabeled with a glycine transporter 1 antibody. Conclusions: The results demonstrate circadian rhythms in Per1 promoter activation in nitric oxide (NO) and GABA secreting amacrine cells, and suggest that the synthesis, release and physiological function of NO and GABA could be controlled by circadian clock mechanisms in the mammalian retina.

Keywords: circadian rhythms • amacrine cells • gene/expression