Fig 1: Expression of the excitatory amino acid transporter 3 (EAAT3) in mouse brain and primary hippocampal neurons. (A–C) Expression pattern of EAAT3 in the adult mouse brain. Coronar cryosections of adult mouse brains were incubated with a polyclonal antibody directed against EAAT3. Protein expression was detected in neuronal perikarya and the neuropil of various brain areas, including caudate putamen, cortex and the hippocampus formation. Insets show enlargements of depicted areas. CP, Caudate putamen; Ctx, Cortex; Hpf, Hippocampus formation; Hy, Hypothalamus; Sp, Stratum pyramidale; Th, Thalamus. (D) Mouse hippocampal neurons were prepared at embryonic day 16, cells were grown on glass cover slips for 14 days, fixed and stained for EAAT3 and GFAP as marker for astrocytes present to some extent in culture. EAAT3 expression was restricted to neurons. (E,F) Mouse hippocampal neurons were prepared and cultured as described, fixed and stained for EAAT3, Map2 as dendritic (E) and NFP as axonal marker (F) by immunofluorescence methods and confocal imaging. EAAT3 staining was observed in a punctuate fashion throughout the cell body as well as dendritic and axonal processes [lower panels, see also insets (Ea,Fb)]. (G) Homogenates of whole mouse brains and cultured hippocampal neurons were subjected to Western blot procedure and probed for the expression of neuronal EAAT3. Actin was used as loading control. The antiserum detected a major band between the 55 and 70 kDa markers in both samples. Overall, the signals corresponded well with the expected molecular weight. (H) Murine astrocytes were cultured close to confluency, fixed and stained for the main glial glutamate transporter EAAT1 (GLAST) and EAAT2 (GLT-1) by immunofluorescence and confocal imaging. Astrocytes predominantly expressed EAAT1 at the plasma membrane. EAAT2 expression was observed at comparably low levels.