Two peptide transmitters co-packaged in a single neurosecretory vesicle

Abstract

Numerous neurosecretory cells are known to secrete more than one peptide, in both vertebrates and invertebrates. These co-expressed neuropeptides often originate from differential cleavage of a single large precursor, and are then usually sorted in the regulated pathway into different secretory vesicle classes to allow separable release dynamics. Here, we use immuno-gold electron microscopy to show that two very different neuropeptides (the nonapeptide crustacean cardioactive peptide (CCAP) and the 30 kDa heterodimeric bursicon) are co-packaged within the same dense core vesicles in neurosecretory neurons in the abdominal ganglia of Periplaneta americana. We suggest that this co-packaging serves a physiological function in which CCAP accelerates the distribution of bursicon to the epidermis after ecdysis to regulate sclerotization of the newly formed cuticle.

Figure 1

A–F: Electron micrographs of CCAP and PBURS immunoreactivity in Cell 27 of P. americana. A: Low magnification electron micrograph showing whole neuron processed by conventional electron microscopy, arrows point to clusters of electron dense vesicles; perineurium (P), neurilemma (N). The inset shows the same cell at the light microscope level, stained with toluidine blue. B: High magnification of Cell 27 cytoplasm. Arrows point to both single small and large electron dense vesicles. This image also shows multiple large vacuoles (V) and Golgi (G). C: Immunogold co-labeling of CCAP (large particles, 25 nm gold) and PBURS (small particles, 15nm gold). Arrows point to electron dense vesicles that are clearly co-labeled; mitochondria(M). D: High magnification view of co-labeled vesicle. Thick arrows point to 25nm gold conjugated to CCAP, and thin arrow points to 15nm gold conjugated to PBURS. E: Immunogold labeling of 6 nm gold granules conjugated to CCAP. Inset shows a magnification of the vesicle in the lower right. . F: High magnification of a co-labeled vesicle with 15 nm gold conjugated to PBURS (thick arrow) and 6 nm gold conjugated to CCAP (thin arrow). Scale bars: A: 50 microns; B, C: 250nm; D, E and F: 50nm.

Figure 2

Immuno-gold labeling of dense cored vesicles in a single Cell 27 in the abdominal ganglia of P. americana. A total of 953 vesicles were evaluated at 25,000X magnification for 15 nm gold particles detecting PBURS (beige), 25 nm gold particles detecting CCAP (green), co-labeling with both gold particles (red) or no gold granules (blue).