A, schematic diagram showing the experimental arrangement. Membrane-permeable rhod-2 AM was pressure-ejected into the stratum lucidum, resulting in selective loading of the presynaptic terminals through the mossy fibre pathway. Fluorescence from the labelled region, which had a diameter of about 100 μm and was about 500 μm away from the ejection site, was measured with a single photodiode. B, transmission (upper) and fluorescence (lower) images of the slice in which the mossy fibres were labelled with the Ca2+ indicator rhod-2 AM. Images were obtained with a × 10 objective. A bright fluorescent band extended bidirectionally away from the ejection site (arrow), reflecting the antero- as well as retrograde labelling of the mossy fibre pathway. C, transmission (upper), fluorescence (middle) and superimposed (bottom) images (× 40 objective) of the area indicated by the box in B. These panels show that fluorescence is localized only in the stratum lucidum (Luc). Pyr, stratum pyramidale. Rad, stratum radiatum. D, fluorescence image obtained with a × 100 objective. Relatively large mossy fibre terminals of approximately 3-5 μm in diameter are seen at the single bouton level. Scale bars represent 200 μm (B), 50 μm (C) and 10 μm (D), respectively.