Neurochemical characterization of zinc transporter 3-like immunoreactive (ZnT3(+)) neurons in the intramural ganglia of the porcine duodenum

Abstract

The SLC30 family of divalent cation transporters is thought to be involved in the transport of zinc in a variety of cellular pathways. Zinc transporter 3 (ZnT3) is involved in the transport of zinc into synaptic vesicles or intracellular organelles. As the presence of ZnT3 immunoreactive neurons has recently been reported in both the central and peripheral nervous systems of the rat, the present study was aimed at disclosing the presence of a zinc-enriched neuron enteric population in the porcine duodenum to establish a preliminary insight into their neurochemical coding. Double- and triple-immunofluorescence labeling of the porcine duodenum for ZnT3 with the pan-neuronal marker (PGP 9.5), substance P, somatostatin, vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS), leu-enkephalin, vesicular acetylcholine transporter (VAChT), neuropeptide Y, galanin (GAL), and calcitonin gene-related peptide were performed. Immunohistochemistry revealed that approximately 35, 43, and 48 % of all PGP9.5-postive neurons in the myenteric (MP), outer submucous (OSP), and inner submucous (ISP) plexuses, respectively, of the porcine duodenum were simultaneously ZnT3(+). In the present study, ZnT3(+) neurons coexpressed a broad spectrum of active substances, but co-localization patterns unique to the plexus were studied. In the ISP, all ZnT3(+) neurons were VAChT positive, and the largest populations among these cells formed ZnT3(+)/VAChT(+)/GAL(+) and ZnT3(+)/VAChT(+)/VIP(+) cells. In the OSP and MP, the numbers of ZnT3(+)/VAChT(+) neurons were two times smaller, and substantial subpopulations of ZnT3(+) neurons in both these plexuses formed ZnT3(+)/NOS(+) cells. The large population of ZnT3(+) neurons in the porcine duodenum and a broad spectrum of active substances which co-localize with this peptide suggest that ZnT3 takes part in the regulation of various processes in the gut both in normal physiology and during pathological processes.

Fig. 1

Control staining demonstrating duodenal neurons in the MP (a, a′), OSP (b), and ISP (c) stained with zinc selenide AMG and TSQ methods (d). Some neurons show intensity of AMG staining in the cytoplasm; High-magnification image of two ZnT3⁺ neurons (e); neurons in the MP, OSP, and ISP of the porcine duodenum immunostained for PGP 9.5 (f, g, h) and ZnT3 (f′, g′, h′). Scale bars = 100 μm (a), 15 μm (a′,b, c, d), 10 μm (e), and 25 μm (f–h′)

Fig. 2

Representative images of ZnT3⁺ neurons located in the duodenal MP of the pig. Merged images (d, h, l) are composites of images taken separately from red (a, c, i), green (b, f, j), and blue (c, g, k) fluorescent channels. Scale bar = 25 μm. a–d ZnT3⁺/VAChT⁻/NOS⁺—long arrows, ZnT3⁺/VAChT⁺/NOS⁻—small arrow; e–h ZnT3⁺/VAChT⁻/VIP⁺—long arrow, ZnT3⁺/VACHT⁺/VIP⁻—small arrow; i–l ZnT3⁺/SOM⁻/LENK⁺—long arrow, ZnT3⁺/SOM⁺/LENK⁻—small arrow

Fig. 3

Representative images of ZnT3⁺ neurons located in the duodenal OSP (a–h) and ISP (i–p) of the pig. Merged images (d, h, l, p) are composites of images taken separately from red (a, c, i, m), green (b, f, j, n), and blue (c, g, k, o) fluorescent channels. Scale bar = 25 μm. a–d ZnT3⁺/VAChT⁻/NOS⁺—long arrows, ZnT3⁺/VAChT⁺/NOS⁻—small arrows; e–h ZnT3⁺/SP⁻/VIP⁺—long arrow, ZnT3⁺/SP⁺/VIP⁻—small arrows; i–l ZnT3^+/VAChT⁺/SP⁺—long arrows, ZnT3⁺/VAChT⁺/SP⁻—small arrows; m–p ZnT3⁺/GAL⁺/VIP⁻—long arrows, ZnT3⁺/GAL⁻/VIP⁺—small arrows