Enteric nerves harbour a wide array of neuropeptides playing a key role in the regulation of gastrointestinal tract functions. In this study, the distribution patterns of cocaine- and amphetamine-regulated transcript-immunoreactive (CART-IR) nerve fibres as well as the percentages of CART-positive enteric neurons were immunohistochemically assessed in the rumen, reticulum, omasum and abomasum of the sheep. Double staining were applied, to elucidate whether neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), substance P (SP), somatostatin or serotonin co-exist in CART-IR gastric structures. In the rumen, reticulum, omasum and abomasum, a majority of myenteric neurons identified by immunoreactivity to Hu C/D were CART-positive (47.1 +/- 3.6%, 45.1 +/- 3.0%, 41.6 +/- 2.6% and 40.9 +/- 2.9% respectively). The smooth musculature of the forestomachs as well as abomasum was innervated with numerous CART-IR nerve fibres. Blood vessels-associated CART-positive nerve terminals were identified in the submucosa of the reticulum only. Lamina muscularis mucosae of the omasum and abomasum was moderately innervated with CART-IR nerve terminals. In the abomasum sparse CART-IR nerve fibres were seen between mucosal glands. A small population of endocrine cells of the abomasum also exhibited the presence of CART. All neuronal elements as well as endocrine cells IR to CART were negative to somatostatin and/or serotonin. No immunoreactivity to VIP, NPY and/or SP was found in myenteric ganglia-projecting CART-positive nerve fibres. The co-localization of CART with VIP, NPY and/or SP was regularly observed in myenteric neurons as well as the smooth muscle layer- and lamina muscularis mucosae-projecting nerve fibres. CART-IR nerve terminals located between mucosal glands of the abomasum frequently co-stored VIP, NPY and/or SP. Although the exact function of CART in the ovine forestomachs/stomach has to be elucidated, several potential functions (like enteric nerves protection) have been suggested.

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ABSTRACT:Enteric neurons are able to alter their neurotransmitter content during adaptation to new artificial conditions. The aim of the present study was to investigate changes in vasoactive intestinal peptide (VIP), substance P (SP) and galanin expression during culture of myenteric neurons from the ovine abomasum. In order to accurately reflect the in vivo situation cryostat sections from the ovine abomasum were used. Cultured and non-cultured myenteric neurons were immunohistochemically stained with a mixture of antibodies raised against Hu C/D (neuronal marker) and VIP, SP or galanin. Double labeling revealed that Hu C/D-IR/VIP-IR myenteric neurons were very rare in cryostat sections (1.4 ± 0.2%) but significantly increased to 21.3 ± 1.7% when cultured for three days. A significant increase in Hu C/D-positive/VIP-positive myenteric neurons were also found in 6-and 9-days cultures (23.9 ± 1.9 and 24.5 ± 2.0%, respectively). In vivo, the expression of SP was found in 9.7 ± 1.0% of myenteric perikarya. After 3, 6 and 9 days of incubation the proportion of Hu C/D-IR/SP-IR myenteric perikarya significantly increased to 19.3 ± 1.3%, 22.3 ± 1.2% and 24.1 ± 1.7% (respectively). When compared to the in vivo situation the proportion of galanin-expressing myenteric neurons was unchanged after 3, 6 and 9 days of culturing. In conclusion, alterations in VIP and SP (but not galanin) expression occur during neuronal culturing. Our results supports the idea that both VIP and SP may act as factors which increase neuronal survival.