There is increasing evidence in man and mice that the altered ERG observed in muscular dystrophy patients and in corresponding mouse models is due to a disturbed synaptic communication within the retinal OPL.
19 21 40 It is, therefore, of considerable interest to determine the precise distribution of the DGC within this retinal layer. We present three different lines of evidence that within the OPL dystroglycan is exclusively found presynaptically in photoreceptor terminals and not associated with dendrites of postsynaptic horizontal or bipolar cells and that within the photoreceptor terminals, the immunoreactivity is not concentrated directly at ribbon synapses but instead is found perisynaptically. First, at the light-microscopic level, dystroglycan immunoreactivity was adjacent to, but did not overlap with Bassoon immunoreactivity, which marks the anchoring area of the ribbon to the presynaptic photoreceptor plasma membrane,
35 39 suggesting that the DGC is not directly associated with the ribbon synapse. Second, at the ultrastructural level, no immunoreactivity was associated with the invaginating bipolar or horizontal cell dendrites, demonstrating that β-dystroglycan in the OPL was exclusively concentrated in photoreceptor terminals. Third, three-dimensional reconstruction of several photoreceptor terminals confirmed that the active zone and the area directly adjacent to the ribbon synapses were devoid of label throughout their three-dimensional extension. Instead, β-dystroglycan was concentrated in processes, extending from the photoreceptor terminals into the OPL. Because the distribution of β-dystroglycan immunoreactivity in single sections of chick, mouse, and rat retinas was very similar,
23 25 it appears likely that the results from this study apply to all three species. The expression of β-dystroglycan mRNA by photoreceptors,
32 the appearance of dystrophin and β-dystroglycan immunoreactivity concomitant with the formation of presynaptic specializations within the photoreceptors,
31 the colocalization of dystroglycan with dystrophin and β-dystrobrevin,
29 31 together with the presynaptic concentration of dystrophin and the nonoverlapping staining pattern of dystroglycan and synaptophysin
29 are consistent with our results. However, this distribution is in contrast to other studies that report a concentration of dystroglycan in bipolar and horizontal cell processes, as well as a direct association of β-dystroglycan immunoreactivity with the photoreceptor ribbon synapse.
25 26 29 Because most of the studies used the 43DAG/8D5 monoclonal antibody and because this antibody is specific for β-dystroglycan in all species tested by using Western blotting and immunohistochemistry, and because the staining pattern was similar, using antibodies directed against other proteins of the dystrophin-associated protein complex, different results are unlikely to be caused by cross-reactivity of the antibody to unrelated proteins. Moreover, we used the sensitive method of silver intensification combined with gold toning to detect β-dystroglycan immunoreactivity, and we analyzed the immunoreactivity within a major part of the OPL covering four cone terminals, each of which contained several ribbon synapses. Thus, it is equally unlikely that the differences in the distribution and subcellular concentration of β-dystroglycan are due to the sensitivity of the staining method or due to the analysis of only single individual sections. Instead, we consider two possibilities to explain the different results regarding the precise localization of β-dystroglycan in the retinal OPL. One possibility is that dystroglycan has a different distribution in different species. Whereas in mouse, rat, and chick retinas, β-dystroglycan immunoreactivity is concentrated perisynaptically in photoreceptor processes, the immunoreactivity in rabbit, bovine, and human retinas could be concentrated in the processes of bipolar and horizontal cells. The second possibility is that the distribution is variable even within one species and may depend, for example, on the day/night cycle. In this respect, it is interesting to note that the processes extending from the photoreceptor terminals, which contain the vast majority of β-dystroglycan labeling, have been detected in a number of different species and have been shown to be highly dynamic and plastic structures, forming and disappearing reversibly during light/dark cycles.
41 42 Moreover, the localization of dystroglycan has been shown to be variable, even in retinas of a single species, i.e., the mouse. While β-dystroglycan had a perisynaptic localization in normal wild-type mice, the
mdx 3Cv mouse strain, which carries a mutation in the dystrophin gene and has an altered ERG, showed a strongly reduced immunoreactivity with antibodies against β-dystroglycan and dystrophin, as well as a redistribution of the immunoreactivity and a concentration at the active zone,
23 suggesting that the localization of the DGC is flexible and is dependent on structures within the photoreceptor terminal. On the other hand, there is no apparent correlation between dystroglycan distribution and preponderance of a certain photoreceptor type, i.e., a similar distribution of the dystroglycan labeling was observed in the diurnal chick retinas, which are cone dominated, and in nocturnal rat and mouse, which have a rod-dominated retina.
25