Abstract
Purpose :
Although the patterns of connections between horizontal cells and photoreceptors are well-described, the developmental mechanism of how horizontal cells find their correct target remains unclear. To study this question, we developed an in vitro system coupled with live imaging to track and determine the cellular mechanisms responsible for neurite outgrowth and synaptic specificity of horizontal cells during development.
Methods :
Retinas were isolated from a horizontal cell-specific fluorescent reporter transgenic mouse line (Cx57icre;Ai14) and plated at a 50,000-cell density in a 384 well-plate at early developmental time points (P8-10) and at adult stages (P30). Horizontal cells were imaged for 2 days in vitro to capture cell-cell interactions and fixed after 4 days in culture to assess neurite outgrowth. Fluorescence-activated cell sorting (FACS) was used to isolate horizontal cells using Cx57icre;Ai14 transgenic mice. FACS-isolated horizontal cells (HCs) were then plated with the remaining cells of the retina at varying ratios to replicate biological conditions: 1:10, 1:250, 1000 HCs alone, 2500 HCs alone. The cells were fixed after 4 days in vitro, and horizontal cell neurite outgrowth was quantified using Imaris imaging software. One-way ANOVA was used for statistical analysis.
Results :
Horizontal cells from adult P30 mixed cell cultures exhibited significantly lower neurite outgrowth compared to P8 mixed cell cultures. Interestingly, FACS-isolated HCs exhibited little to no neurite outgrowth compared to HCs plated at a 1:10 ratio with other retinal cells. These data suggest that other retinal cells secrete signals to promote neurite outgrowth of horizontal cells at early developmental time points but not at adult stages. Horizontal cells were isolated via FACS and plated with conditioned media from young P8 retinas. Our findings showed that horizontal cells cultured with conditioned media showed significant neurite outgrowth compared to those cultured alone.
Conclusions :
These data demonstrates that cultured horizontal cells respond to secreted factors from other retinal cells to promote neurite outgrowth at early developmental stages but these signals are lost later in the adult. Future work will use this in vitro system coupled with live imaging to uncover the cellular and molecular mechanisms responsible for neurite outgrowth and synaptic specificity in the developing retina.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.