Fig. 4. Hemolymph lacunae are coincident with areas of immunolabel avoidance in the anterior commissural organ (ACO). In the commissural ganglion, the substance P-immunopositive plexus is fenestrated by what appears to be a network of branched tubes. To determine if these tubular structures are hemolymph lacunae, several India ink-filled ganglia were immunoprocessed with the substance P antibody, and the ink and immunolabel were simultaneously imaged via confocal microscopy. As can be seen in this set of micrographs, ink-filling (A) was evident in numerous lacunae in the portion of the ganglion containing the substance P-immunopositive plexus (B). When micrographs of the individual labels were merged (C), it became apparent that the plexus is fenestrated by hemolymph vessels. As the merged micrograph shows, the substance P-immunopositive nerve terminals that form the plexus envelop the hemolymph lacunae. This organization, with nerve terminals in direct apposition to the hemolymph space, is considered the defining characteristic of a crustacean neuroendocrine site. We hypothesize that this plexus is a neuroendocrine organ and we have named it the anterior commissural organ based on its location. Each micrograph is a brightest pixel projection of nine optical sections taken at 0.5 µm intervals. A and B are shown at the same scale. Scale bars, 20 µm in B and C.