Expression of Period1-driven Green Fluorescent Protein in the Mammalian Retina

Abstract

Abstract: : Purpose:Although a circadian clock appears to be localized to the photoreceptor cell layer in amphibians, in mammals, the cells responsible for circadian pacemaker activity in the retina have not been identified with certainty. Here we identify inner retinal cells that may be involved in the retinal circadian clock using a line of transgenic mice harboring a transgene containing clock gene promoter Period1 driving a destabilized form of Green Fluorescent Protein (GFP). Methods:We used mice hemizygous for the Per1-driven GFP transgene that were 3 weeks and older and housed in a light dark cycle. Prior to removing the eyes, the animals were maintained in constant darkness for 30 min. to 2 hours. The isolated retina was prepared for imaging, at different times during the circadian cycle, either as a wholemount or, using a McIlwain-type tissue chopper, the retina was cut to 150-200 µm transverse sections and placed in a HEPES buffered extracellular solution. The wholemounted retina or retinal sections were imaged using either a conventional fluorescence microscope equipped with a narrowband EGFP filter set or a confocal microscope using the 488nm line of the argon laser to excite the GFP and a bandbass of 500nm-550nm to detect emission. Results: Per1-driven GFP expressing cells were detected in the retina at different times in the circadian cycle. In transverse sections, GFP containing cells were detectable in the amacrine region of the inner nuclear layer as well as in the ganglion cell layer. On the ganglion side of retinal wholemounts, we detected Per1-driven GFP fluorescent cells in a minority of ganglion and amacrine cells in all regions of the retina. Imaging of the photoreceptor side of the wholemount failed to reveal Per1-driven GFP fluorescence among rods or cones. Conclusion:We have identified Per1-driven GFP containing cells in the ganglion and inner nuclear layers of the retina. These cells may represent the cellular location of a circadian clock in the retina and/or cells involved in the photoentrainment pathway of the suprachiasmatic nucleus. The a priori identification of retinal cells involved in circadian pacemaking will aid in addressing future questions about the mechanisms involved in circadian rhythm generation in the mammalian retina.

Keywords: 349 circadian rhythms • 312 amacrine cells • 415 ganglion cells