Expression of genes encoding glutamate receptors and transporters in rod and cone bipolar cells of the primate retina determined by single-cell polymerase chain reaction

Abstract

Purpose: Light signals from rod and cone photoreceptors traverse distinct types of second-order, bipolar neurons that carry these signals from the outer to inner retina. Anatomic and physiologic studies suggest that the specialization of rod and cone bipolar cells involves the differential expression of proteins involved in glutamatergic signaling. In a previous study, we compared the expression of genes for the AMPA- (GluR1-4) and kainate-sensitive (GluR5-7, KA1-2) ionotropic glutamate receptors, the metabotropic glutamate receptors (mGluR1-8), and five non-vesicular glutamate transporters (EAAT1-5) in full-complement cDNA constructed from fresh and aldehyde-fixed macaque retina using a technique suitable for amplification of a variety of differentially expressed transcripts. Here we apply the same protocol to compare expression of these genes in cDNA constructed from single rod and cone bipolar cells previously-labeled for morphological identification in fixed slices of macaque retina.

Methods: We used immunocytochemical labeling and unique morphological features in lightly fixed slices of macaque retina to target the rod bipolar or the DB3 cone OFF bipolar cell. Under visual control, we used a micropipette to target and extract labeled cells, and we isolated mRNA from each through enzymatic digestion. Full-length cDNA was synthesized using 3'-end amplification (TPEA) PCR, in which the highly diverse 3' regions were amplified indiscriminately to ensure detection of both high and low abundance genes. We used gene-specific RT-PCR to probe the cDNA of each bipolar cell both for expression of known genes to confirm cell identification as well as expression of genes encoding glutamate receptors GluR1-7, KA1-2, and mGluR1-8 and for transporters EAAT1-5.

Results: Of 27 rod bipolar cells confirmed to express the genes for the a subunit of protein kinase C, mGluR6, and its G protein Galpha(o), 26 expressed at least one AMPA GluR subunit gene, 16 expressed at least two, and nine expressed three or more. Nearly every cell expressed the GluR4 gene (23/27), followed by GluR2 (16/27) and GluR1 (11/27). In addition to mGluR6, 20/27 cells also expressed the mGluR3 gene. Nearly every rod bipolar cell also expressed the genes for the EAAT2 (23/27) and EAAT4 (21/27) transporters. Of 26 DB3 cells confirmed by expression of calbindin D-28 and absence of GAD-65/67, each expressed the gene for the AMPA subunit GluR4, followed by GluR2 (22/26), and GluR1 (15/26), the only kainate subunit gene expressed was GluR6 (18/26). Nearly every DB3 cell also expressed the gene for the EAAT2 transporter (25/26), but no others.

Conclusions: Rod bipolar cells in the Macaca monkey retina expressed not only the mGluR6 gene, a subunit necessary for transmission of light-ON signals, but also nearly always GluR4 in combination with the glutamate transporter EAAT4 (21/27 cells). The DB3 cell involved in processing light-OFF signals from cones expressed most highly the combination of GluR4 and the transporter EAAT2 (25/26). These results suggest that glutamatergic signaling in rod and cone circuits in the primate retina depends upon complex molecular interactions, involving not only multiple glutamate receptor subunits, but also glutamate transporters. Our data demonstrate a consistent primary pattern for each cell type with subtle variability involving other genes. Thus, like neuronal cell types in other brain regions, morphological and physiologic homogeneity among retinal bipolar cell types does not exclude variations in expression that could serve to adjust the stimulus-response profile of each cell.