Representative images of autofluorescence and a range of markers for retinal cell types at P30 and P240 in WT and
Cln6nclf mice are shown in
Figure 6. Cone photoreceptors were labeled using fluorescein-bound peanut agglutinin (PNA; green) and sections imaged for autofluorescence (Auto; 633-nm excitation; pseudo-colored red) and cell nuclei (DAPI, blue;
Figs. 6A–D). WT mice at P30 and P240 (
Figs. 6A,
6C) showed normal cone photoreceptor labeling and no autofluorescence (
Figs. 6A,
6C, respectively). At P30,
Cln6nclf mice exhibited similar PNA labeling to WT retina, and despite some rare autofluorescent deposits, there was no apparent colocalization with cone photoreceptors (
Fig. 6B). However, by P240 autofluorescent debris was apparent in all retinal layers of
Cln6nclf mice, including the RPE (
Fig. 6D). Furthermore, despite the loss of many photoreceptor nuclei in the ONL (see
Fig. 4K), there was a relative preservation of cone numbers; however, their morphology was altered (
Fig. 6D). Specifically, there was a decrease in cone inner and outer segment length (IS/OS) and occasional colocalization of autofluoresence with PNA-labeled cone photoreceptors (
Fig. 6D, arrow). Amacrine and ganglion cells in WT mice at P30 and P240 showed normal calretinin labeling (CalR; green) of subsets of amacrine and ganglion cells and no autofluorescence (red;
Fig. 6E,
6G, respectively). While colocalization of calretinin and lipofuscin was absent in P30
Cln6nclf mice (
Fig. 6F), autofluorescent deposits within intact amacrine and ganglion cells were apparent at P240 (
Fig. 6H, arrow in magnified inset Hi). Retinal Müller cells were labeled using an antibody against glutamine synthetase (GS; green;
Figs. 6I–J). At P240, Müller glia displayed normal morphology in WT retina (
Fig. 6I); however, in
Cln6nclf mice, isolated autofluorescent deposits were apparent within Müller cells adjacent to cell nuclei (
Fig. 6J, arrow in magnified inset Ji). Despite this accumulation of autofluorescent debris, there was no effect on cell survival, as Müller cell number did not change between WT and
Cln6nclf mice at this age (
Fig. 6K). In summary, at P240, there was a distinct thinning of the photoreceptor layers in
Cln6nclf mice; however, lipofuscin accumulation was apparent throughout the retina and had little adverse effect on retinal structure and cellular survival of the inner retinal cell types investigated, including amacrine cells, ganglion cells, and Müller glia.