NRG3 mRNA expression in distinct hippocampal neuron subtypes in the adult brain and in dissociated culture. Expression of NRG3 transcripts in distinct neuronal subtypes was analyzed in adult mouse hippocampal coronal sections and cultured rat hippocampal neurons using multiplex fluorescent in situ hybridization (RNAscope). (A) Low magnification micrograph showing merged signals for probes corresponding to NRG3 (white), VGluT1 (red; glutamatergic neurons) and GAD (green; GABAergic interneurons). (B) Abundant NRG3 expression is detected in granule neurons of the dentate gyrus (DG) and in pyramidal cell layer neurons (CA1–CA3). (C–F) Higher magnification micrograph of CA1 neurons showing merged (C) and separate signals for NRG3 (D), VGluT1 (E), and GAD (F); examples of glutamatergic neurons co-expressing NRG3 and VGluT (filled arrowheads) or GABAergic co-expressing NRG3 and GAD (open arrowheads) are indicated. (G–J) Micrographs of cultured dissociated hippocampal neurons co-hybridized with probes for NRG3 and VGluT1 (G and H) or NRG3 and GAD (I and J) illustrate NRG3 expression in both cultured excitatory and inhibitory neuron types. Sections and cultures were additionally stained with DAPI to label nuclei. Scale bars: A and B, 200 µm; C–J, 20 µm.