Selective EGFP expression in raphe 5 HTergic neurons in organotypic slice cultures. A. Confocal images from slice cultures of the dorsal raphe after transduction with the dual AVV system (AVV-G4BS-3.6TPH-EGFP and AVV-3.6TPH-GAL4p65) and immunostained for TPH-2 (centre panels). EGFP expression (left panels) was 5 HT-selective (overlay of green and red channel – right panels) and sufficiently potent for single-cell experiments on live 5 HTergic neurones. Middle row of images in a higher power image of the upper one. Lower row of images is from a different slice, closer to the ventral edge (white dotted line). Yellow dashed line in upper raw shows approximate midline. B. Distribution of EGFP-expressing 5 HTergic neurones in a midbrain slice culture. Left panel – EGFP fluorescence, right panel – overlay of fluorescence channel on the DIC image. Yellow arrow points at the edge of the aqueduct. Cells are largely located along midline (B7/B8 groups), some more lateral cells probably come from B9 group. C. Characteristic varicosities of EGFP-expressing 5 HTergic axons. Note that varicosities are predominantly small but few of a larger type can also be observed (red arrow). D. Morphometric analysis of living 5 HTergic varicosities (n = 106) in slice cultures. The histogram shows the distribution of varicosity lengths measured along the axis of the axon. While the majority of varicosities were small (1.5–2 μm) there was a distinct sub-population of larger varicosities (median ~4 μm, red arrow). Large varicosities are rare, but could represent a specific functional phenotype.