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Blake Hugo Fortes, Manuel Tapia, Luis Aranguren, Ines Agosto, Gabriela Smith, Luis E Vazquez; Biochemical characterization of the post-synaptic density isolated from whole mouse retina. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6025.
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© ARVO (1962-2015); The Authors (2016-present)
The post-synaptic density (PSD) is a protein complex anchored in the post-synaptic membrane of neurons that couples membrane receptors and effector proteins. Characterization of the PSD allows identification of signaling cascades regulating synaptic strength of neuronal connections. We describe a method for isolating PSDs from mouse retina lysates and describe major components therein.
The protocol to isolate PSDs from mouse retina was adapted from Siekevitz et al (PMID: 7410481). Modifications to the protocol include scaling down buffer volume and centrifuge tube size to fit mouse retina. Retinas and hippocampi from 12 C57/B6 mice are dissected and homogenized (whole retinal lysate) in a single experiment. Samples are collected after precipitating cell nuclei (S1), precipitating cell membranes (S2), and separating synaptic membranes by sucrose gradient centrifugation (SPM). Purified PSDs are prepared by washing the SPM fraction in Triton X-100 and high-speed ultracentrifugation as previously described. 30 micrograms of protein are loaded per lane for SDS-PAGE to visualize bands or Western Blots (WB). Primary antibodies against PSD95, NR1, NR2A, NR2B, synapsin, and rhodopsin, and secondary antibodies coupled with HRP were used for WB; a BioRad ChemiDoc was used for semi-quantitation of chemiluminescence.
SDS-PAGE resolved the retinal PSD into a pattern of bands that resembled those from hippocampi and other brain areas (published literature). There is an enrichment of PSD95 and all NMDA-type glutamate receptor subunits and a depletion of rhodopsin in retinal SPM and PSDs. PSD95 and NMDA receptor subunits were similarly abundant in the inner plexiform layer (IPL) and outer plexiform layer (OPL) of the retina by immunohistochemistry, suggesting retinal PSDs may represent a mixed population of synapses.
Established protocols to isolate PSDs from brain tissue can be adapted for effectively isolating PSDs from mouse retina. PSDs seem to represent post-synaptic terminals from both IPL and OPL. Further work including electron microscopy and mass spectrometry is needed to further characterize retinal PSDs.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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