Differential expression of Brn3 transcription factors in intrinsically photosensitive retinal ganglion cells in mouse.

Several subtypes of melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs) have been reported. The M1 type of ipRGCs exhibit distinct properties compared with the remaining (non-M1) cells. They differ not only in their soma size and dendritic arbor, but also in their physiological properties, projection patterns, and functions. However, it is not known how these differences arise. We tested the hypothesis that M1 and non-M1 cells express Brn3 transcription factors differentially. The Brn3 family of class IV POU-domain transcription factors (Brn3a, Brn3b, and Brn3c) is involved in the regulation of differentiation, dendritic stratification, and axonal projection of retinal ganglion cells during development. By using double immunofluorescence for Brn3 transcription factors and melanopsin, and with elaborate morphometric analyses, we show in mouse retina that neither Brn3a nor Brn3c are expressed in ipRGCs. However, Brn3b is expressed in a subset of ipRGCs, particularly those with larger somas and lower melanopsin levels, suggesting that Brn3b is expressed preferentially in the non-M1 cells. By using dendritic stratification to distinguish M1 from non-M1 cells, we found that whereas nearly all non-M1 cells expressed Brn3b, a vast majority of the M1 cells were negative for Brn3b. Interestingly, in the small proportion of the M1 cells that did express Brn3b, the expression level of Brn3b was significantly lower than in the non-M1 cells. These results provide insights about how expression of specific molecules in a ganglion cell could be linked to its role in visual function.

Expression of osteopontin in the rat retina: effects of excitotoxic and ischemic injuries.

PURPOSE: The cytokine osteopontin (OPN) has been localized to the retinal ganglion cell layer in the normal rodent retina, prompting the suggestion that it could serve as a useful marker for identifying and quantifying such neurons in models of retinal and optic nerve neurodegeneration. In the present study, we characterized the time course and cellular localization of OPN expression in the rat retina after excitotoxic and ischemic injuries. METHODS: Excitotoxicity and ischemia-reperfusion experiments were performed by using standard techniques. Rats were killed at various time points, and the retinas were removed either for mRNA analysis or to be processed for immunohistochemistry. RESULTS: In the normal retina, double-labeling immunofluorescence indicated that OPN is expressed by the majority of, if not all, RGCs, since OPN was associated with more cells than Brn-3, but was colocalized with Thy1.1. NMDA, kainic acid, and ischemia-reperfusion all caused decreases in the total retinal levels of Thy1 and Brn-3 mRNAs, reflecting injury to RGCs, but a dramatic, short-lived upregulation in OPN mRNA. The source of the increased OPN signal after excitotoxic-ischemic insults is unlikely to be injured RGCs, as no alteration in the intensity of OPN immunostaining in RGCs was apparent. Instead, additional cells, mostly contained within the IPL, were identified as positive for OPN. Double-labeling immunofluorescence showed that ED1 always colocalized with OPN in these cells, indicating their status as activated microglia. CONCLUSIONS: OPN is exclusively expressed by RGCs in the physiological retina, but in response to retinal neurodegeneration is synthesized de novo by endogenous, activated microglia.