VAChT+, VGluT2+ or VGAT+, BDA-labeled varicosities in relation to Orx+ neurons viewed by epifluorescent microscopy. In each case, BDA is green (Cy2), VTP red (Cy3), and Orx blue (AMCA in A or Cy5 in B and C). A: BDA+ [VAChT+] terminals in the vicinity of an Orx+ neuron. BDA+ terminals (solid arrowheads in A′) located in the vicinity, yet not close to an Orx+ neuron (left), are positive for the VAChT protein (solid arrowheads in A″), as evident in the merged image (in yellow, solid arrowheads in A‴). A VAChT+ terminal that is not BDA+ (open arrowhead) is closer to the Orx+ cell body. Note also the sparse number of VAChT+ varicosities in the vicinity of Orx+ neurons. B: BDA+ [VGluT2+] terminals in close proximity to Orx+ neurons. Two small BDA+ terminals (solid arrowheads in B′) over an Orx+ cell body are positive for the VGluT2 protein (solid arrowheads in B″), as evident in the merged image (in yellow, solid arrowheads in B‴). Note also the relatively large number of VGluT2+ varicosities in close proximity to the Orx+ neurons. C: BDA+ [VGAT+] terminals over Orx+ neurons. Two BDA+ terminals (solid arrowheads in C′) in close proximity to an Orx+ neuron are positive for VGAT (solid arrowheads in C″), as evident in the merged image (in yellow, solid arrowheads in C‴). In all images, tonal range was adjusted for each RGB channel individually. Scale bar = 10 μm in C‴ (applies to A–C).