Figure 4.

A finer characterization of the LN₂. (a) Lateral neurons LN₁ and LN₂ (overlay of 10 confocal stacks from a vibratome section, distance of z-stacks 1.0 µm). All LN₂ are co-stained with both anti-PER and anti-PDF. White arrows point to selected LN₂. Scale bar, 30 µm. (b) Magnification of the PDF-positive LN₂ (single confocal stack from a wholemount brain; 10× objective; numerical aperture: 0.30). The brain was scanned in z steps of 2.5 µm, and the present image is at a depth of 85 µm from the anterior surface. PER is confined to the nuclei of the LN₂. Scale bar, 10 µm. (c) Single confocal stack of a vibratome section (the same as in figure 3b) showing the LN₁ and LN₂ stained by anti-PER, anti-HRP and anti-PDF (same microscope settings as in (b)). HRP and PDF are shown in magenta and indicate the size of the neurons. Although HRP does not stain the cytoplasm membrane uniformly, it shows that the LN₁ are much smaller than the LN₂. All photos were taken from foragers' brains. Arrows point to single LN₁ neurons the cytoplasm of which is clearly labelled by the neuronal marker HRP. Arrowheads point to single LN₂ neurons the cytoplasm of which is HRP positive and additionally labelled by PDF. Scale bar, 10 µm.