Morphology and Neurochemistry of Descending and Ascending Myenteric Plexus Neurons of Sheep Ileum

Abstract

The specific patterns of gastrointestinal motility in large herbivores may relate to differences in the organization of enteric nerve circuits, compared with other mammals. To investigate this possibility, we characterized the morphologies, chemical phenotypes, and projections of myenteric plexus (MP) neurons of the sheep ileum. Morphologies and projections were investigated after application of the carbocyanine dye (1,1 0 , di-octadecyl-3,3,3 0 ,3 0 ,-tetramethylindo-carbocyanine perchlorate, DiI) to fixed tissues. To study chemical phenotypes, the fluorescent tracer Fast Blue (FB) was injected into the wall of the ileum, in vivo, 12-14 cm oral to the ileo-caecal junction. Over 80% of the descending and ascending DiI-labeled neurons had typical Dogiel type I morphology, whereas only a few Dogiel type II neurons were observed. Nevertheless, there were long projections (up to 10 cm) of Dogiel type II neurons in both directions. Both type II and type I neurons were neurofilament immunoreactive (IR). We observed long projections of descending (up to 18 cm) and ascending (up to 12-14 cm) FB-labeled MP neurons. Nitric oxide synthase (NOS)-IR, peripheral choline acetyltransferase (pChAT)-IR, and substance P (SP)-IR occurred in both descending and ascending myenteric neurons. NOS-IR was in approximately 60% of FB-labeled descending and ascending neurons, whereas those expressing pChAT-IR were 67 6 15% and 60 6 14%, respectively. Descending neurons expressing SP-IR were 48 6 15% and ascending were 56 6 12%. NOS-IR and pChAT-IR, and SP-IR and pChAT-IR were commonly colocalized in both ascending and descending pathways. In descending pathways, almost all SP-IR neurons were also pChAT-IR (98 6 3%) and NOS-IR (99 6 2 NOS 1 /SP 1 /pChAT 2 ). Many FB-labeled descending neurons showed both NOS-and pChAT-IR. Descending neurons may represent inhibitory motor neurons (NOS 1 /SP 1 /pChAT 2 ) and two classes of interneurons (pChAT 1 /NOS 2 , and pChAT 1 /NOS

Intrinsic neuronal control of the pyloric sphincter of the lamb

“…This last population could represent a subpopulation of interneurons, expressing both phenotypes, similar to those neurons which we recently observed in the sheep ileum (Mazzuoli et al, 2007). The large percentage of nitrergic neurons innervating the sheep PS reinforces the hypothesis that NOS-IR FB-labelled neurons are responsible for inhibition of the PS musculature.…”

“…Almost all nitrergic neurons were also SP-IR (98%); among these cells, about 64% innervated the PS; these could be descending inhibitory motoneurons or interneurons because we have recently observed large subpopulations of neurons expressing both NOSand SP-IR in the sheep ileum (Mazzuoli et al, 2007). Of the neurons innervating the PS, it is possible to identify at least three distinct categories of neurons: NOS/SP-IR (64%), NOS/ChAT-IR (21%), and ChAT/SP-IR (2%); we should also consider dopaminergic neurons which probably expressed only TH-IR.…”

High- and medium-molecular-weight neurofilament proteins define specific neuron types in the guinea-pig enteric nervous system

“…The present observations show that NF immunoreactivity occurs in type II neurons in the guinea-pig, as it does in the small intestine of other species, including human (Ganns et al 2006;Brehmer 2007), pig (Barbiers et al 1995;Brehmer et al 2002), cattle (Balemba et al 1999), sheep (Mazzuoli et al 2007) and mouse (Qu et al 2008). Indeed, in each of these species, anti-NF antibodies are useful for identifying type II neurons.…”

“…Type II neurons, which are readily distinguished by their large round or oval cell bodies and multiple axons (Brehmer et al 1999;Furness 2006) are characteristically revealed by anti-NF-H antibodies in the human and pig small intestine, where this is one of the best ways to demonstrate type II neurons (Brehmer et al 2002;Ganns et al 2006;Brehmer 2007;Wolf et al 2007). Anti-NF antibodies are also ideal for the localisation of type II neurons in cattle (Balemba et al 1999), sheep (Mazzuoli et al 2007) and mice (Qu et al 2008). In marked contrast, two previous studies found that type II neurons in the guineapig enteric nervous system were not NF-immunoreactive (Costa et al 1996;Hu et al 2002), although other neurons with NF immunoreactivity were reported in both studies.…”

Adenosine A₁ and A₃ Receptor Agonists Inhibit Nonadrenergic, Noncholinergic Relaxations in the Guinea Pig Isolated Trachea

“…Specimens were embedded in OCT compound (Tissue-Tek Sakura, Torrance, Calif., USA), frozen in liquid nitrogen and cut into 12- to 14-µm-thick sections using a cryostat. Sections were then processed for double-labeling indirect immunofluorescence as described previously [39, 40]. Briefly, sections were washed in PB, incubated in 10% normal goat serum for 1 h at room temperature and then incubated overnight at 4°C with the following mixtures containing: rabbit polyclonal anti-A 1 adenosine receptor (1:100; Sigma-Aldrich Chemie, Steinheim, Germany) and either mouse monoclonal anti-HuC/D (1:200; Molecular Probes Inc., Eugene, Oreg., USA) or anti-nNOS (1:200, Santa Cruz Biotechnology, Santa Cruz, Calif., USA), a general neuronal marker and a major inhibitory neurotransmitter, respectively.…”