Hippocampal neurons 3 jigsaw puzzle

Distribution of hippocampal neurons expressing EGFP from the Nr4a1/Nur77 promoter (Tg(Nr4a1-EGFP)GY139Gsat, www.gensat.org) in hippocampal CA1 colabelled for the vesicular glutamate transporter (VGluT1, red, Invitrogen polyclonal, 1:1000) and stained with DAPI (blue) to show cell layers. EGFP expression was amplified with chicken anti-GFP (Abcam, 1:2000). VGluT1 labels excitatory terminals synapsing in the molecular layer of the dentate gyrus, mossy fibre terminals in CA3 and dendritic fields of the pyramidal cells in stratum orien and stratum radiatum. Nr4a1-EGFP is heavily expressed by CA1 pyramidal cells and to a lesser extent in CA3 pyramidal cells with sparse cells in the dentate gyrus (see associated images). Section (40 um, sagittal) was mounted with Fluoromount G and imaged with a 5X, 0.15 NA objective on a Zeiss Axiovert microscope. AF568 was excited at 562/40 nm and emission collected at 624/40 nm with Semrock filters and a single band dichroic (530-585 nm reflected, 601-800 nm transmitted). AF488 was excited at 482/35 nm and emission collected at 536/40 nm with Semrock filters and a single band dichroic (446-499 nm reflected, 513-735 nm transmitted). DAPI was excited at 350/50 nm and emission collected at 460/50 nm with a CLP400 dichroic (Chroma). Single images were captured for each channel using a Zeiss Axiocam MR CCD and pseudo-coloured using Axiovision. Linear background subtraction was used on the presented image. See Davis and Puhl (2011), Pubmed ID 21305052, for a detailed staining protocol and images of EGFP expression in the striatum and amygdala in this strain. This image is part of a series characterising EGFP expression from the Nr4a1 promoter throughout the brain.