Presentations

Abstract

Introduction:

Neurexins are a complex family of synaptic proteins known to mediate synapse assembly and function in the brain. There are three Nrxn family members (Nrxn1, Nrxn2, Nrxn3), each with a longer alpha isoform and shorter beta isoform expressed from independent promoters, with different properties ascribed to each. Moreover, knockout mouse models have shown that they regulate synaptic function in the brain in a cell-type and synapse- specific manner. Recent studies have shown that Nrxns are expressed in the retina, but very little is known about their role. This experiment was intended to learn the importance of Nrxn1β and Nrxn3β in the synapse stability of the mouse retina.

Methods:

Eyes from male and female wild type (WT) and Nrxn1β and Nrxn3β knockout mice were harvested at 6-8 weeks of age, fixed in 2% paraformaldehyde, and processed for sectioning. Immunostaining was performed on 8um frozen retina sections using primary antibodies targeting synaptophysin (SYP) and PSD95 to detect synapses. Brn3a was used as a retinal ganglion cell marker. The following secondary antibodies were used: 488 donkey anti-rabbit, Cy5 donkey anti-mouse, and DAPI to stain the nuclei. Images were taken at four areas along each retinal section on a Zeiss 710 confocal microscope at 63X magnification using an oil objective. Synaptic puncta for SYP were quantified with the SynQuant Fiji plug-in and the PSD96 puncta were counted manually using Fiji. Separate sections were also stained with hematoxylin and eosin (H&E), and images were captured along the entire length of each section with a light microscope at 20X magnification. The images were stitched to create a composite and manually quantified using Fiji.

Results:

Sections from four WT, two Nrxn1β-/-, and five Nrxn3β-/- mice were evaluated. SYP-positive puncta were detected in both the inner plexiform layer (IPL) and outer plexiform layer (OPL). Average SYP-positive puncta were 1558, 581, and 850 for WT, Nrxn1β and Nrxn3β respectively. The p values for the Nrxn1β was 0.09 and 0.11 for Nrxn3β. Puncta of PSD95 were detected in the ganglion cell layer (GCL) and IPL with a strong signal in the OPL. Average PSD95-positive puncta were 58, 43, and 36 for WT, Nrxn1β and Nrxn3β respectively. The p values for the Nrxn1β was 0.43 and 0.14 for Nrxn3β. The average number of cells in the GCL of the H&E stains for WT, Nrxn1β and Nrxn3β were 719, 441, and 588 respectively. The p values for H&E staining were 0.008 and 0.02 for Nrxn1β and Nrxn3β respectively.

Conclusion:

In both knockout models, there were decreased cells in the GCL at 6 weeks of age and a trend towards a decreased number of puncta. While there were fewer SYP and PSD95 puncta in the knockout mice, the difference did not reach statistical significance.The lack of statistical significance between the number of synaptic puncta of Nrxn1β and Nrxn3β knockouts may be due to variability and a small sample size. The H&E staining showed a statistically significant decrease of nuclei in the GCL between the WT and the Nrxn1β and Nrxn3β mice. Further analysis with more mice per group is currently underway. This preliminary data suggests that Nrxn1β and Nrxn3β play a role in retinal synapses.