Fig. 5.

The presence of presynaptic VGluT2 or VGAT and postsynaptic PSD-95 or Gephyrin (Geph) proteins in contacts between BDA-labeled varicosities and Orx+ neurons. A: Rendered 3D confocal image (16 serial 0.50-μm thick optical sections) of a BDA-labeled axon (in green, Cy2), whose varicosities (in yellow) are positive for VGluT2 (in red, Cy3). One of these varicosities is in apposition (arrowhead) to an Orx+ neuron (in pseudocolor blue, Cy5), as evident in the rendered zoom images on the right (6 serial 0.5-μm thick optical sections) depicting in detail the relation between the BDA+ varicosity (top) or the contained VGluT2 (middle) with the Orx+ neuron surface and showing the three elements together in the merged image (bottom). Note also on the left other VGluT2+ varicosities in contact with the Orx+ neuron. B: Rendered 3D confocal image (9 serial 0.33-μm thick optical sections) of a BDA+ varicosity (arrowhead, in green, Cy2) facing a PSD-95+ profile (opposite pointer, in pseudocolor red, Cy5) located between the varicosity and the surface of the Orx+ neuron (in pseudocolor blue, Cy3). As evident in the zoom rendered images on the right (13 serial 0.33-μm thick optical sections), the BDA+ varicosity is apposed to the Orx+ process (top image), opposite the PSD-95+ punctum, which is on the surface of the Orx+ process (pointer, middle image) and between the BDA+ varicosity and the Orx+ process (pointer in the merged image, bottom). Note the presence of other PSD-95+ puncta over the Orx+ neuron (upper pointer) at the left panel. C: Rendered 3D confocal image (28 serial 0.5-μm thick optical sections) of a BDA-labeled axon (in green, Cy2), from which most varicosities (in yellow) are positive for VGAT (in red, Cy3) and come into contact (large arrowhead) with the dendrites and soma of the Orx+ neuron. As seen in the rendered zoom images on the right (8 serial 0.5-μm thick optical sections), the BDA+ varicosity (top image) containing VGAT (middle image) appears to be in contact with the surface of the Orx+ neuron (merged image, bottom). Note in the left image other VGAT+ varicosities also appear to appose the Orx+ neuron. D: Rendered 3D confocal image (14 serial 0.33-μm thick optical sections) of BDA-labeled axonal varicosities (arrowhead, green, Cy2) facing Geph+ puncta (facing pointer, pseudocolor red, Cy5) over an Orx+ neuron (pseudocolor blue, Cy3). As evident in the zoom rendered images at the right (12 serial 0.33-+m thick optical sections), the apposition of the BDA+ varicosity with the Orx+ neuron (top image) is associated with a Geph+ punctum on the surface of the Orx+ neuron (pointer, middle image) and between the BDA+ varicosity and the Orx+ neuron (merged image, pointer, bottom). Note the presence of other Geph+ puncta over the Orx+ neuron (upper pointers) in the left panel. Deconvolution iterative restoration (see Materials and Methods) was applied to the Cy5 channel (in B and C, left panels, and in A–D, right panels), the Cy3 channel (in C, left panel, and in C,D, right panels) and the Cy2 channel (in B–D). Tonal range adjustments for each individual RGB channel were made for all pictures. Scale bars = 10 μm in left large panel in D (applies to large left panels in A–C); 1 μm in D, zoom bottom image (applies to zoom images in A–C).