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S. H. DeVries, D. G. Ryan; Expression of AMPA and Kainate Receptors in Off Bipolar Cells of the Ground Squirrel Retina. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4798.
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© ARVO (1962-2015); The Authors (2016-present)
Parallel processing begins when a cone releases glutamate onto 8-10 anatomical subtypes of bipolar cells that fall into two broad functional classes: On and Off. In the ground squirrel, the subtypes of Off bipolar cells can be distinguished based on the properties of their postsynaptic glutamate receptors, with b2 cells using AMPA-type receptors and b3 and b7 cells using different kainate-type receptors. To better understand the function of the different glutamate receptors in parallel processing, we are characterizing their subunit composition.
We searched the database of ground squirrel genomic sequences with human and rat mRNAs of each of the AMPA (GluR1-4) and kainate (GluR5-7, KA1,2) receptor subunits. Exonic sequences were extracted and spliced together to produce longer contiguous transcripts. PCR primers were designed to discriminate between the 9 ground squirrel subunits. RT-PCR was performed on RNA prepared from the ground squirrel retina to test for the presence of each subunit. PCR products were used to generate subunit specific RNA probes. In situ hybridizations were performed on fixed frozen sections. In some cases, hybridized sections were labeled with antibodies to bipolar cell specific markers.
PCR reactions with retinal cDNA generated the expected products for all but the KA1 subunit. Translations of the cloned sequences revealed >98% homology to their rat and human counterparts. An anti-sense probe for the GluR5 subunit strongly labeled a band of somas in the center of the IPL which was further divided into upper (recoverin negative) and lower (recoverin positive) layers. Recoverin is a marker for subset of b3 cell. Double immunolabeling with a pan-bipolar cell marker, CHX10, and recoverin suggested that an additional 1-2 layers of Off bipolar cell somas were sandwiched between the GluR5 positive band and amacrine cells somas. A probe for the GluR6 subunit did not label bipolar cells. Additionally, antibodies to GluR6 only labeled processes in the IPL.
Recoverin positive b3 cells have synaptic kainate receptors based on physiological and pharmacological criteria. Molecular evidence suggests that their receptors contain the GluR5 subunit.
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