Expression of genes encoding glutamate receptors and transportersin rod and cone bipolar cells of the primate retina determined bysingle-cell polymerase chain reaction

[摘要] Purpose: Light signals from rod and cone photoreceptors traversedistinct types of second-order, bipolar neurons that carry these signalsfrom the outer to inner retina. Anatomical and physiological studiessuggest that the specialization of rod and cone bipolar cells involvesthe differential expression of proteins involved in glutamatergicsignaling. In a previous study, we compared the expression of genes forthe AMPA- (GluR1-4) and kainate-sensitive (GluR5-7, KA1-2) ionotropicglutamate receptors, the metabotropic glutamate receptors (mGluR1-8),and five non-vesicular glutamate transporters (EAAT1-5) infull-complement cDNA constructed from fresh and aldehyde-fixed macaqueretina using a technique suitable for amplification of a variety ofdifferentially expressed transcripts. Here we apply the same protocol tocompare expression of these genes in cDNA constructed from single rodand cone bipolar cells previously-labeled for morphologicalidentification in fixed slices of macaque retina.Methods: We used immunocytochemical labeling and uniquemorphological features in lightly fixed slices of macaque retina totarget the rod bipolar or the DB3 cone OFF bipolar cell. Under visualcontrol, we used a micropipette to target and extract labeled cells, andwe isolated mRNA from each through enzymatic digestion. Full-length cDNAwas synthesized using 3'-end amplification (TPEA) PCR, in which thehighly diverse 3' regions were amplified indiscriminately to ensuredetection of both high and low abundance genes. We used gene-specificRT-PCR to probe the cDNA of each bipolar cell both for expression ofknown genes to confirm cell identification as well as expression ofgenes encoding glutamate receptors GluR1-7, KA1-2, and mGluR1-8 and fortransporters EAAT1-5.Results: Of 27 rod bipolar cells confirmed to express the genes forthe a subunit of protein kinase C, mGluR6, and its G proteinGαo, 26 expressed at least one AMPA GluR subunit gene, 16expressed at least two, and nine expressed three or more. Nearly everycell expressed the GluR4 gene (23/27), followed by GluR2 (16/27) andGluR1 (11/27). In addition to mGluR6, 20/27 cells also expressed themGluR3 gene. Nearly every rod bipolar cell also expressed the genes forthe EAAT2 (23/27) and EAAT4 (21/27) transporters. Of 26 DB3 cellsconfirmed by expression of calbindin D-28 and absence of GAD-65/67, eachexpressed the gene for the AMPA subunit GluR4, followed by GluR2(22/26), and GluR1 (15/26), the only kainate subunit gene expressed wasGluR6 (18/26). Nearly every DB3 cell also expressed the gene for theEAAT2 transporter (25/26), but no others.Conclusions: Rod bipolar cells in the Macaca monkey retinaexpressed not only the mGluR6 gene, a subunit necessary for transmissionof light-ON signals, but also nearly always GluR4 in combination withthe glutamate transporter EAAT4 (21/27 cells). The DB3 cell involved inprocessing light-OFF signals from cones expressed most highly thecombination of GluR4 and the transporter EAAT2 (25/26). These resultssuggest that glutamatergic signaling in rod and cone circuits in theprimate retina depends upon complex molecular interactions, involvingnot only multiple glutamate receptor subunits, but also glutamatetransporters. Our data demonstrate a consistent primary pattern for eachcell type with subtle variability involving other genes. Thus, likeneuronal cell types in other brain regions, morphological andphysiological homogeneity among retinal bipolar cell types does notexclude variations in expression that could serve to adjust thestimulus-response profile of each cell.

[学科分类] 生物化学/生物物理