Heavy staining of the cone OS (COS) region is seen, corresponding to concentration of S-cone pigment in this cellular compartment

Heavy staining of the cone OS (COS) region is seen, corresponding to concentration of S-cone pigment in this cellular compartment. and knockout mice using a specific MerTK antibody. == Results == In LD, cone phagocytosis showed a statistically significant peak of activity 1 h after light onset, 23 fold higher than at other times. Rabbit polyclonal to Caspase 10 In constant darkness, the temporal phagocytic profile resembled that of LD (significant peak at 1 h of subjective day), but the number of phagosomes was decreased at all time points. Immunostaining of MerTK in wild-type andNrl/mice showed expression at the apical surface of the RPE. == Conclusions == Cone-like outer segment phagocytosis inNrl/mice shows a similar profile to that of rods in normal mice and other species. These data are the first to quantify blue cone-like photoreceptor phagocytosis under different lighting conditions in mice, and suggest this model may constitute a valuable system for investigating circadian regulation of cone function. == Introduction == Retinal photoreceptors (RP) are composed of two different populations, rods, and cones. ML349 Rods are used for nocturnal vision because of their high light sensitivity [1], whereas cones require a relatively high light level to activate them, and are used for diurnal vision [2]. The latter are responsible for color discrimination and high-acuity vision, and are of utmost importance in human vision. In humans and Old World primates, there are three types of cones, made up of blue, green, and red light-sensitive pigments (or short wavelength sensitive (S) cones [3], and middle/long wavelength sensitive cones, respectively). In the great majority of mammals, there are only two cone types: a short wavelength-sensitive and a mid/long wavelength-sensitive populace [4]. Both rods and cones are known to undergo continuous cyclic turnover, involving the addition of new membranes at the base of the outer segment (OS) and the removal of aged membranes at the distal pole [5]. This removal is usually achieved by the opposing retinal pigmented epithelium (RPE), which phagocytose and digest the shed OS membranes [6]. In recent years, much progress has been made in identifying molecular components of the phagocytic pathway, including membrane-bound receptors such as MerTK and V5 integrin, and ligands [7-9]. Mutations in the Mertk receptor lead to inherited retinal degeneration in animals [7,10] and humans [11]. The great majority of data have been obtained for rods, in part because conventional laboratory rodent (mouse and rat) retinas are ML349 composed of 97% rods and only ~3% cones [12,13]. A genetically altered mouse line has become available for the study of rod differentiation, the neural retina leucine ML349 zipper gene (Nrl) knockout mouse (Nrl/) [14]. Deletion of the transcription factorNrlresults in the complete absence of rods, as revealed by histology, immunocytochemistry, electrophysiology, and gene expression analysis [14,15]. Morphological, molecular, and electrophysiological features of theNrl/photoreceptors seem to be identical to blue or short wavelength light-sensitive S cones [14]. Hence, this retina provides a potential means for the investigation of blue cone function and cone-specific genes [16]. In the present study, we have used homozygousNrl/mice to inquire whether cone phagocytosis shows daily rhythms, if any such rhythms resemble or differ from those known for rods, and whether these rhythms are maintained under constant darkness. We show that rhythmic phagocytosis does indeed occur, in both cyclic light and constant darkness, and that it resembles rod behavior. == Methods == == Animals and handling == This study was conducted withNrl/mice, raised in our animal facilities. The strain was originally obtained from Dr. C. Grimm at the Laboratory of Retinal Cell biology, University Hospital, Switzerland, with permission from Dr. A. Swaroop (NIH, Bethesda, MD). The colony was maintained on a 12 h:12 h light-dark (LD) cycle in an ambient temperature of 22 C for four weeks. Small adult wild-type C57Bl/6 mice (n=3), the strain on which the knockout line was created, were used as a positive control for antibody binding and specificity. All experiments were conducted according to ethical guidelines in operation at the institute, and adhered to theARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Two experimental series were made (shown schematically inFigure 1): in.