This study clearly shows a marked difference in the response of cells in the GCL to optic nerve transection and excitotoxic stimuli. We wanted to investigate potential causes of this phenomenon. Expressions of the key apoptotic mediators in the GCL of P6 and P60 were compared. A population of retinal ganglion cells was purified by sequential immunopanning,
20 and total RNA was extracted. This was then reverse transcribed to cDNA, and semiquantitative PCR was performed using sequence-specific primers and α-tubulin as a loading control. As shown in
Figure 6(a) , the levels of transcript of caspase-3 and Apaf-1 decreased between both ages, whereas the level of caspase-9 was reduced slightly. Measuring levels of inner nuclear layer (INL), outer nuclear layer (ONL), and GCL markers confirmed the purity of the immunopanned population. As shown in
Figure 6(a) , the INL (gfap, chx 10) and ONL (blue cone opsin, rhodopsin) markers were absent in the immunopanned population (IP) at P60 (IP P60) and at P6 (data not shown), whereas Thy1 was present, confirming a pure ganglion cell population. We wanted to confirm our results at the protein level. IHC staining of whole eye sections was carried out using antibodies specific for the proform of the caspase proteins and Apaf-1. As shown in
Figure 6(b) , staining was obvious in the GCL of all three proteins at P6 (
Figs. 6(b) a–c), but this staining was not detectable at P60 for caspase-3 and Apaf-1 (
Figs. 6(b) d, f). Staining for caspase-9 remained, consistent with the mRNA result (
Fig. 6(b) e). Absence of staining in secondary antibody-only controls showed that there was no nonspecific binding by the secondary antibodies (
Figs. 6(b) g–i). Both figures comprehensively showed that caspase-3, the main effector of apoptosis, and Apaf-1, a key mediator in caspase-3 activation, are downregulated during the development of the mouse retina to maturity.