John B. Furness

Co-localization of calcitonin gene-related peptide-like immunoreactivity with substance P in cutaneous, vascular and visceral sensory neurons of guinea pigs.

Calcitonin gene-related peptide (CGRP) has been immunohistochemically co-localized with substance P (SP) in capsaicin-sensitive, varicose axons supplying the skin, viscera and cardiovascular system of the guinea pig. After treatment with colchicine in vitro, 82% of SP neurons in the dorsal root ganglia contained CGRP-like immunoreactivity while 96% of CGRP neurons were immunoreactive for SP. Both CGRP- and SP-like immunoreactive material are transported peripherally and centrally from dorsal root ganglia. Thus, in tissues such as the gut where there are intrinsic nerves containing SP but lacking CGRP, CGRP-like immunoreactivity is a useful means of specifically labelling axons of most sensory neurons containing SP.

Types of nerves in the enteric nervous system.

The enteric nervous system is one of the three divisions of the autonomic nervous system, the others being the sympathetic and parasympathetic. In contrast to the other divisions, it can perform many functions independently of the central nervous system. It consists of ganglionated plexuses, their connections with each other, and nerve fibres which arise from the plexuses and supply the muscle, blood vessels and mucosa of the gastrointestinal tract. The enteric nervous system contains a large number of neurons, approximately 107 to 108. About ten or more distinct types of enteric neurons have been distinguished on electrical, pharmacological, histochemical, biochemical and ultrastructural grounds as well as on the basis of their modes of action. Both excitatory and inhibitory nerves supply the muscle and there are inhibitory and excitatory interneurons within the enteric plexuses. There are also enteric nerves which supply intestinal glands and blood vessels, but these receive less emphasis in this commentary. Correlations between groups of neurons defined on different criteria are poor and in many cases the physiological roles of the nerves are not known. The functions of noradrenergic nerves which are of extrinsic origin are reasonably well understood, but cholinergic nerves in the intestine are the only intrinsic nerves for which both the transmitter and to some extent the functions are known. In the case of non-cholinergic, non-noradrenergic enteric inhibitory nerves, the functions are understood but the transmitter is yet to be determined, both adenosine 5′-triphosphate and vasoactive intestinal polypeptide having been proposed. Other nerves have been defined pharmacologically (non-cholinergic excitatory nerves to neurons and muscle, intrinsic inhibitory inputs to neurons, and enteric, non-cholinergic vasodilator nerves) and histochemically (intrinsic amine-handling neurons and separate neurons containing peptides: substance P, somatostatin, enkephalins, vasoactive intestinal polypeptide, gastrin cholecystokinin tetrapeptide, bombesin, neurotensin and probably other peptides). Little is known of the functions of these nerves, although a number of proposals which have been made are discussed.

The enteric nervous system and neurogastroenterology

Neurogastroenterology is defined as neurology of the gastrointestinal tract, liver, gallbladder and pancreas and encompasses control of digestion through the enteric nervous system (ENS), the central nervous system (CNS) and integrative centers in sympathetic ganglia. This Review provides a broad overview of the field of neurogastroenterology, with a focus on the roles of the ENS in the control of the musculature of the gastrointestinal tract and transmucosal fluid movement. Digestion is controlled through the integration of multiple signals from the ENS and CNS; neural signals also pass between distinct gut regions to coordinate digestive activity. Moreover, neural and endocrine control of digestion is closely coordinated. Interestingly, the extent to which the ENS or CNS controls digestion differs considerably along the digestive tract. The importance of the ENS is emphasized by the life-threatening effects of certain ENS neuropathies, including Hirschsprung disease and Chagas disease. Other ENS disorders, such as esophageal achalasia and gastroparesis, cause varying degrees of dysfunction. The neurons in enteric reflex pathways use a wide range of chemical messengers that signal through an even wider range of receptors. These receptors provide many actual and potential targets for modifying digestive function.

INTRINSIC PRIMARY AFFERENT NEURONS OF THE INTESTINE

After a long period of inconclusive observations, the intrinsic primary afferent neurons of the intestine have been identified. The intestine is thus equipped with two groups of afferent neurons, those with cell bodies in cranial and dorsal root ganglia, and these recently identified afferent neurons with cell bodies in the wall of the intestine. The first, tentative, identification of intrinsic primary afferent neurons was by their morphology, which is type II in the terminology of Dogiel. These are multipolar neurons, with some axons that project to other nerve cells in the intestine and other axons that project to the mucosa. Definitive identification came only recently when action potentials were recorded intracellularly from Dogiel type II neurons in response to chemicals applied to the lumenal surface of the intestine and in response to tension in the muscle. These action potentials persisted after all synaptic transmission was blocked, proving the Dogiel type II neurons to be primary afferent neurons. Less direct evidence indicates that intrinsic primary afferent neurons that respond to mechanical stimulation of the mucosal lining are also Dogiel type II neurons. Electrophysiologically, the Dogiel type II neurons are referred to as AH neurons. They exhibit broad action potentials that are followed by early and late afterhyperpolarizing potentials. The intrinsic primary afferent neurons connect with each other at synapses where they transmit via slow excitatory postsynaptic potentials, that last for tens of seconds. Thus the intrinsic primary afferent neurons form self-reinforcing networks. The slow excitatory postsynaptic potentials counteract the late afterhyperpolarizing potentials, thereby increasing the period during which the cells can fire action potentials at high rates. Intrinsic primary afferent neurons transmit to second order neurons (interneurons and motor neurons) via both slow and fast excitatory postsynaptic potentials. Excitation of the intrinsic primary afferent neurons by lumenal chemicals or mechanical stimulation of the mucosa appears to be indirect, via the release of active compounds from endocrine cells in the epithelium. Stretch-induced activation of the intrinsic primary afferent neurons is at least partly dependent on tension generation in smooth muscle, that is itself sensitive to stretch. The intrinsic primary afferent neurons of the intestine are the only vertebrate primary afferent neurons so far identified with cell bodies in a peripheral organ. They are multipolar and receive synapses on their cell bodies, unlike cranial and spinal primary afferent neurons. They communicate with each other via slow excitatory synaptic potentials in self reinforcing networks and with interneurons and motor neurons via both fast and slow EPSPs.

Pathway-specific patterns of the co-existence of substance P, calcitonin gene-related peptide, cholecystokinin and dynorphin in neurons of the dorsal root ganglia of the guinea-pig.

SummaryThe co-existence of immunoreactivities to substance P (SP), calcitonin gene-related peptide (CGRP), cholecystokinin (CCK) and dynorphin (DYN) in neurons of the dorsal root ganglion (DRG) of guinea-pigs has been investigated with a double-labelling immunofluorescence procedure. Four main populations of neurons could be identified that contained different combinations of these peptides and had distinctive peripheral projections: (1) Neurons that contained immunoreactivity to SP, CGRP, CCK and DYN were distributed mainly to the skin. (2) Neurons with immunoreactivity to SP, CGPR and CCK, but not DYN, were distributed mainly to the small blood vessels of skeletal muscles. (3) Neurons with immunoreactivity to SP, CGRP and DYN, but not CCK, were distributed mainly to pelvic viscera and airways. (4) Neurons containing immunoreactivity to SP and CGRP, but not CCK and DYN, were distributed mainly to the heart, systemic blood vessels, blood vessels of the abdominal viscera, airways and sympathetic ganglia. Other small populations of DRG neurons containing SP, CGRP or CCK alone also were detected. Perikarya containing these combinations of neuropeptides were not found in autonomic ganglia. The peripheral axons of neurons containing immunoreactivity to at least SP and CGRP were damaged by chronic treatment with capsaicin. However, some sensory neurons containing CCK alone were not affected morphologically by capsaicin.These results clearly show that individual DRG neurons can contain many different neuropeptides. Furthermore, the combination of neuropeptides found in any particular neuron is related to its peripheral projection.

Immunohistochemical localization of polypeptides in peripheral autonomic nerves using whole mount preparations.

SummaryA method is described for the immunohistochemical localization of peptides in whole-mount preparations. Tissue is fixed as laminae with a picric acid/formaldehyde mixture and then dehydrated, cleared and rehydrated before exposure to antibodies. This procedure ensures adequate penetration of the antibody molecules without the need to freeze and thaw the tissue or to use detergents, preserves antigenicity and lowers non-specific background staining. The laminae are incubated with the primary antisera for 16 h at room temperature and, after washing, with a second, fluorescent tagged, antiserum. This can be followed by a peroxidase-anti-peroxidase localization of the second antiserum, which acts as a bridge. The method gives a precise and reproducible localization of immunoreactive peptides, with good penetration and low background even in thick preparations. Large areas can be scanned and neuroeffector relationships studied more easily than in sections.

Substance P-like immunoreactivity in nerves associated with the vascular system of guinea-pigs

Substance P-like immunoreactivity was localized by an indirect immunohistochemical technique in whole mounts and sections of blood vessels from the guinea-pig. There was a widespread association of nerve fibres that had substance P-like immunoreactivity with blood vessels, extending into all vascular beds. The relative densities of supply of different vessels were assessed visually and a rating scale used to compare them. Large elastic arteries close to the heart had dense networks of immunoreactive nerves associated with them. The density decreased as more peripheral beds were approached, except that there was a particularly dense network of nerves with arteries of the splanchnic beds. Arteries to myocardial, central nervous system, renal, reproductive and skeletal muscle beds all had substance P-immunoreactive nerves associated with them to varying extents. The venae cavae near the heart were densely supplied, but there were few fibres with their more peripheral extensions. Some large veins (e.g. pulmonary, hepatic portal and superior mesenteric) had a few fibres with them, but veins of peripheral vascular beds had very few or no immunoreactive nerve fibres. Substance P-like immunoreactivity in vascular nerves was markedly reduced in guinea-pigs that were injected with capsaicin but was unaffected by the injection of 6-hydroxydopamine. It is concluded that the vascular substance P-immunoreactive nerves are likely to be of sensory origin.

Short-chain fatty acid receptor, GPR43, is expressed by enteroendocrine cells and mucosal mast cells in rat intestine

Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are the major anions in the large intestinal lumen. They are produced from dietary fiber by bacterial fermentation and are known to have a variety of physiological and pathophysiological effects on the intestine. In the present study, we investigated the expression of the SCFA receptor, GPR43, in the rat distal ileum and colon. Expression of GPR43 was detected by reverse transcriptase/polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry. mRNA for GPR43 was detected, by RT-PCR, in extracts of the whole wall and separated mucosa from the ileum and colon and from muscle plus submucosa from the ileum, but not from muscle plus submucosa preparations from the colon. We raised a rabbit antiserum against a synthesized fragment of rat GPR43; this was specific for rat GPR43. GPR43 protein was detected by Western blot analysis in extracts of whole wall and separated mucosa, but not in muscle plus submucosa extracts. By immunohistochemistry, GPR43 immunoreactivity was localized to enteroendocrine cells expressing peptide YY (PYY), whereas 5-hydroxytryptamine (5-HT)-immunoreactive (IR) enteroendocrine cells were not immunoreactive for GPR43. Mast cells of the lamina propria expressing 5-HT were also GPR43-IR. The results of the present study suggest that the PYY-containing enteroendocrine cells and 5-HT-containing mucosal mast cells sense SCFAs via the GPR43 receptor. This is consistent with physiological data showing that SCFAs stimulate the release of PYY and 5-HT from the ileum and colon.

Projections and chemical coding of neurons with immunoreactivity for nitric oxide synthase in the guinea-pig small intestine

The distribution of nitric oxide synthase (NOS) immunoreactivity was investigated in the guinea-pig small intestine. There were many immunoreactive nerve cell bodies in the myenteric plexus but very few in submucous ganglia. NOS immunoreactivity was not found in non-neuronal cells except for rare mucosal endocrine cells. Abundant immunoreactive nerve fibres in both myenteric and submucous ganglia, and in the circular muscle, arose from myenteric nerve cells whose axons projected anally along the intestine. NOS immunoreactivity coexisted with VIP-immunoreactivity, but not with substance P immunoreactivity. We conclude that nitric oxide synthase is located in a sub-population of enteric neurons, amongst which are inhibitory motor neurons that supply the circular muscle layer.

Co-localization of nitric oxide synthase immunoreactivity and NADPH diaphorase staining in neurons of the guinea-pig intestine

SummaryNeuronal nitric oxide synthase (NOS), an enzyme capable of synthesizing nitric oxide, appears to be identical to neuronal NADPH diaphorase. The correlation was examined between NOS immunoreactivity and NADPH diaphorase staining in neurons of the ileum and colon of the guinea-pig. There was a one-to-one correlation between NOS immunoreactivity and NADPH diaphorase staining in all neurons examined; even the relative staining intensities obtained were similar with each technique. To determine whether pharmacological methods could be employed to demonstrate that NADPH diaphorase staining was due to the presence of NOS, tissue was pre-treated with NG-nitro-l-arginine, a NOS inhibitor, or l-arginine, a natural substrate of NOS. In these experiments on unfixed tissue, it was necessary to use dimethyl thiazolyl tetrazolium instead of nitroblue tetrazolium as the substrate for the NADPH diaphorase histochemical reaction. Neither treatment caused a significant decrease in the level of NADPH diaphorase staining, implying that arginine and NADPH interact at different sites on the enzyme.