Fig 2: GluA2 is retained intracellularly and is expressed less at post-synaptic sites in the granule cell layer of Tg(PG14) mice.(A) HeLa cells were co-transfected with plasmids encoding WT or PG14 PrP-EGFP fusion protein, and the AMPAR subunit GluA2. After 48h cells were fixed, permeabilized, stained with anti-GluA2 (red), and reacted with Hoechst 33258 (blue) to stain the nuclei. (B) The fluorescent density of GluA2 on the cell surface was measured and corrected for background (Corrected IntDen). Each bar indicates the mean ± SEM of 12 WT and 14 PG14 PrP-transfected cells from three independent experiments; *p<0.01 by Student’s t-test. (C) HeLa cells were co-transfected with WT or PG14 PrP and EGFP-tagged GluA4. After 48 h cells were fixed, permeabilized, stained with anti-PrP monoclonal antibody 98A3 (green), and reacted with Hoechst 33258 (blue) to stain the nuclei. (D) The fluorescent density of GluA4 on the cell surface was measured and background corrected. Each bar indicates the mean ± SEM of 6 WT and 6 PG14 PrP-transfected cells. (E) CGNs from WT and PG14 mice were fixed, permeabilized and immunostained with mouse monoclonal anti-GluA2 (green) and chicken polyclonal anti-MAP2 (red). Cells were reacted with Hoechst 33258 (blue) to stain the nuclei, and analyzed by confocal microscopy using sequential Z-stack acquisition mode and 3D reconstruction. The GluA2 signal on dendrites (F) and cell bodies (G) was analyzed by NIH ImageJ software. Data are the mean ± SEM of 18 WT cells (28 dendrites) and 18 PG14 cells (27 dendrites) from three independent experiments. (H-K) Brain sections of WT and PG14 mice were stained with anti-GluA2 (H) or GluA4 (J) (red) and anti-VGLUT1 (green) antibodies. The GluA2 (I) and GluA4 (K) in each cerebellar glomerulus were measured and expressed as the ratio between the area of the AMPA receptor subunit and VGLUT1 signals. The dotted line identifies a single glomerulus used for quantification. Signal outside the ROI has been cleared with ImageJ for clarity. Bars indicate the mean ± SEM of three mice per group (1098–1392 glomeruli). *p < 0.05 by Student’s t-test. Scale bars 20 µm in A, C and E; and 125 µm in H and J.

Fig 3: Transcripts encoding ionotropic glutamate receptors are expressed in mouse blastocysts and oocytes. Subunits of ionotropic glutamate receptors are listed in the table on the left. The most frequently used common names of subunits are given in parentheses. Mouse gene symbols are used (human gene symbols are the same but written with all letters capitalized, e.g., GRIA1). In transcripts which were consistently expressed in both blastocysts and oocytes, fold regulation values (“+" means upregulation and “-" means downregulation in blastocysts compared to oocytes) and corresponding P-values are shown. Transcripts were detected by reverse transcription-polymerase chain reaction (RT-PCR) and representative agarose gels with separated PCR products are shown in the panels on the right. Lanes: Gria1, Glutamate receptor, ionotropic, AMPA1; Gria2, Glutamate receptor, ionotropic, AMPA2; Gria3, Glutamate receptor, ionotropic, AMPA3; Gria4, Glutamate receptor, ionotropic, AMPA4; Grik1, Glutamate receptor, ionotropic, kainate 1; Grik2, Glutamate receptor, ionotropic, kainate 2; Grik3, Glutamate receptor, ionotropic, kainate 3; Grik4, Glutamate receptor, ionotropic, kainate 4; Grik5, Glutamate receptor, ionotropic, kainate 5; Grin1, Glutamate receptor, ionotropic, NMDA1; Grin3a, Glutamate receptor, ionotropic, NMDA3A; Grin3b, Glutamate receptor, ionotropic, NMDA3B; Grin2a, Glutamate receptor, ionotropic, NMDA2A; Grin2b, Glutamate receptor, ionotropic, NMDA2B; Grin2c, Glutamate receptor, ionotropic, NMDA2C; Grin2d, Glutamate receptor, ionotropic, NMDA2D; MW, molecular weight markers; (A) positive control tissue; (B) ovulated oocytes; (C) blastocysts. The MWs in base pairs (bp) are indicated to the right of the panels. *Primers for Grik3, Grin2d, Grin3a, and Grin3b subunits were not included in the Mouse GABA & Glutamate RT2 profiler PCR array, and commercial primer sets from Qiagen were used (see Materials and methods). Delta receptors (formed from delta 1 and delta 2 subunits), classified by sequence homology as ionotropic glutamate receptors, do not bind glutamate, and were not investigated in this study.