Immunohistochemical analysis of neuron types in the mouse small intestine

Abstract

The definition of the nerve cell types of the myenteric plexus of the mouse small intestine has become important, as more researchers turn to the use of mice with genetic mutations to analyze roles of specific genes and their products in enteric nervous system function and to investigate animal models of disease. We have used a suite of antibodies to define neurons by their shapes, sizes, and neurochemistry in the myenteric plexus. Anti-Hu antibodies were used to reveal all nerve cells, and the major subpopulations were defined in relation to the Hu-positive neurons. Morphological Type II neurons, revealed by anti-neurofilament and anti-calcitonin gene-related peptide antibodies, represented 26% of neurons. The axons of the Type II neurons projected through the circular muscle and submucosa to the mucosa. The cell bodies were immunoreactive for choline acetyltransferase (ChAT), and their terminals were immunoreactive for vesicular acetylcholine transporter (VAChT). Nitric oxide synthase (NOS) occurred in 29% of nerve cells. Most were also immunoreactive for vasoactive intestinal peptide, but they were not tachykinin (TK)-immunoreactive, and only 10% were ChAT-immunoreactive. Numerous NOS terminals occurred in the circular muscle. We deduced that 90% of NOS neurons were inhibitory motor neurons to the muscle (26% of all neurons) and 10% (3% of all neurons) were interneurons. Calretinin immunoreactivity was found in a high proportion of neurons (52%). Many of these had TK immunoreactivity. Small calretinin neurons were identified as excitatory neurons to the longitudinal muscle (about 20% of neurons, with ChAT/calretinin/+/- TK chemical coding). Excitatory neurons to the circular muscle (about 10% of neurons) had the same coding. Calretinin immunoreactivity also occurred in a proportion of Type II neurons. Thus, over 90% of neurons in the myenteric plexus of the mouse small intestine can be currently identified by their neurochemistry and shape.

Involvement of TRPV2 Activation in Intestinal Movement through Nitric Oxide Production in Mice

“…It has been reported that 79.3% of nNOS-IR neurons are inhibitory circular muscle motor neurons, 10.3% are inhibitory longitudinal muscle motor neurons, and 10.3% are descending interneurons (Qu et al, 2008). Thus, it is likely that the majority of the nNOS-IR neurons observed in our study were inhibitory circular muscle motor neurons, whereas the TRPV2-negative nNOS-IR neurons may be descending interneurons.…”

“…Immunohistochemical methods were modified from previous reports (Ward et al, 2003;Furness et al, 2004b;Qu et al, 2008). All experiments were repeated on specimens from at least three mice.…”

“…LMMP preparations were incubated with primary antibodies at 4°C overnight and then washed (three times, 10 min each) in 0.3% PBS-T before incubation with secondary antibodies at room temperature for 1 h. Preparations were then washed (three times, 10 min each) in PBS and mounted on glass slides using a commercially available mounting medium (Diagnostic BioSystems). To visualize cellular CGRP as an IPAN marker, tissues were incubated in oxygenized DMEM containing colchicine before fixation as described previously (Qu et al, 2008).…”

“…To test whether TRPV2-IR, nNOS-negative neurons (Fig. 2 E) could be regarded as IPANs, we performed double staining for TRPV2 and IPAN markers CGRP or neurofilament 145 (NF145) in the mouse myenteric plexus (Qu et al, 2008) (Fig. 2 F, G).…”

“…2). To more effectively detect TRPV2-positive cells, we used ChAT-eGFP mice that express GFP mainly in IPANs or excitatory motor neurons (Tallini et al, 2006;Qu et al, 2008). When we compared 2-APB responses in myenteric neurons with and without GFP expression, the rates of 2-APB-sensitive cells were significantly larger in GFP-negative cells than in GFPpositive cells, which suggested that TRPV2 functions mainly in GFP-negative inhibitory motor neurons (Fig.…”