Abstract
Abstract: :
Purpose: Extracellular matrix (ECM) elements regulate many aspects of development in the CNS including cell fate, migration, guidance, and synapse formation. We have shown previously that laminin deletion produces profound disruptions in the synaptic connectivity in the outer retina (Libby et al., '99; Manglapus et al., '03). We are interested in determining if inner retinal changes accompany this disruption. Methods: We used standard immunohistochemical techniques to determine if amacrine cell markers are changed in mutant lines: ß2, γ3, netrin–4 and ß2γ3 double nulls. We used computer–assisted methods to determine the density of cells and used nearest–neighbor analysis to examine the distribution of cells. Results:Our survey of a wide variety of markers showed no difference; however, one amacrine marker, tyrosine hydroxylase (TH), was altered profoundly. In radial sections, the frequency and number of TH+ cells were dramatically increased in both the ß2 and ß2γ3 double nulls compared to wild–type controls. We used flat–mounted whole retina to quantify our results. In the wild–type retina, one class of TH+ cells was present and 448 ± 83 cells were seen (N=3). In the ß2 null retina, the number of TH+ cells increased to 840 ± 30; interestingly two classes of TH+ cells were evident; large bodied, well–labeled cells (Type I) and smaller bodied, more faintly labeled cells (Type II). In contrast, there appears to be no change in TH expression in either γ3 or netrin–4 null retina. We see similar alterations in TH expression in other CNS regions. Conclusions: These data demonstrate that laminin depletion by targeted mutagenesis results in disruptions of the inner retina as well as the outer retina. Other studies have demonstrated that TH expression is regulated by activity. Thus, we theorize that altered TH expression is a consequence of alterations in the retinal network.
Keywords: retinal development • extracellular matrix • amacrine cells