Simon Jonathan Brookes

Neurochemical classification of myenteric neurons in the guinea-pig ileum

A strategy has been developed to identify and quantify the different neurochemical populations of myenteric neurons in the guinea-pig ileum using double-labelling fluorescence immunohistochemistry of whole-mount preparations. First, six histochemical markers were used to identify exclusive, non-overlapping populations of nerve cell bodies. They included immunoreactivity for the calcium binding proteins calbindin and calretinin, the neuropeptides vasoactive intestinal polypeptide, substance P and somatostatin, and the amine, 5-hydroxytryptamine. The sizes of these populations of neurons were established directly or indirectly in double-labelling experiments using a marker for all nerve cell bodies. Each of these exclusive populations was further subdivided into classes by other markers, including immunoreactivity for enkephalins and neurofilament protein triplet. The size of each class was then established directly or by calculation. These distinct, neurochemically-identified classes were related to other published work on the histochemistry, electrophysiology and retrograde labelling of enteric neurons and to the simple Dogiel morphological classification. A classification scheme, consistent with previous studies, is proposed. It includes 14 distinct classes of myenteric neurons and accounts for nearly all neurons in the myenteric plexus of the guinea-pig ileum.

Classes of enteric nerve cells in the guinea-pig small intestine

The guinea‐pig small intestine has been very widely used to study the physiology, pharmacology and morphology of the enteric nervous system. It also provides an ideal, simple mammalian preparation for studying how nerve cells are organised into functional circuits underlying simple behaviours. Many different types of nerve cells are present in the enteric nervous system and they show characteristic combinations of morphological features, projections, biophysical properties, neurochemicals, and receptors. To identify the different functional classes is an important prerequisite for systematic analysis of how the enteric nervous system controls normal gut behaviour. Based on combinations of multiple‐labelling immunohistochemistry and retrograde tracing, it has been possible to account quantitatively for all of the neurones in the guinea‐pig small intestine. This article summarises that account and updates it in the light of recent data. A total of 18 classes of neurones are currently distinguishable, including primary afferent neurones, motor neurones, interneurones, secretomotor and vasomotor neurones. It is now possible to take an individual nerve cell and use a few carefully chosen criteria to assign it to a functional class. This provides a firm anatomical foundation for the systematic analysis of how the enteric nervous system normally functions and how it goes wrong in various clinically important disorders. Anat Rec 262:58–70, 2001.

Intraganglionic laminar endings are mechano-transduction sites of vagal tension receptors in the guinea-pig stomach.

1 Distension‐sensitive vagal afferent fibres from the cardiac region of the guinea‐pig stomach were recorded extracellularly, then filled with biotinamide, using an anterograde tracing technique. 2 Most of the stretch‐sensitive units of the guinea‐pig stomach (41 out of 47; number of animals N= 26) had low thresholds (less than 1 mm) to circumferential stretch and showed slow adaptation. Twenty of these units fired spontaneously under resting conditions (mean: 1.9 ± 0.3 Hz, n = 20, N = 14). 3 Adaptation of firing during slow or maintained stretch correlated closely with accommodation of intramural tension, but tension‐independent adaptation was also present. 4 Nicardipine (3 μm) with hyoscine (3 μm) reduced stretch‐evoked firing of gastric vagal afferents, by inhibiting smooth muscle contraction. Gadolinium (1 mm) blocked distension‐evoked firing. 5 Focal stimulation of the stomach muscle wall with a von Frey hair (0.4 mN) identified one to six punctate receptive fields in each low threshold vagal distension‐sensitive afferent. These were marked on the serosal surface of the stomach wall. 6 Anterograde filling of recorded nerve trunks revealed intraganglionic laminar endings (IGLEs) within 142 ± 34 μm (n = 38; N = 10) of marked receptive fields. The mean distance from randomly generated sites to the nearest IGLE was significantly greater (1500 ± 48 μm, n = 380, N = 10, P < 0.0001). Viscerofugal nerve cell bodies, intramuscular arrays and varicose axons were not associated with receptive fields. The results indicate that IGLEs are the mechanotransduction sites of low threshold, slowly adapting vagal tension receptors in the guinea‐pig upper stomach.

Sensory transmission in the gastrointestinal tract

Abstract  The gastrointestinal (GI) tract must balance ostensibly opposite functions. On the one hand, it must undertake the process of digestion and absorption of nutrients. At the same time, the GI tract must protect itself from potential harmful antigenic and pathogenic material. Central to these processes is the ability to ‘sense’ the mechanical and chemical environment in the gut wall and lumen in order to orchestrate the appropriate response that facilitates nutrient assimilation or the rapid expulsion through diarrhoea and/or vomiting. In this respect, the GI tract is richly endowed with sensory elements that monitor the gut environment. Enteric neurones provide one source of such sensory innervation and are responsible for the ability of the decentralized gut to perform complex reflex functions. Extrinsic afferents not only contribute to this reflex control, but also contribute to homeostatic mechanisms and can give rise to sensations, under certain circumstances. The enteric and extrinsic sensory mechanisms share a number of common features but also some remarkably different properties. The purpose of this review is to summarize current views on sensory processing within both the enteric and extrinsic innervation and to specifically address the pharmacology of nociceptive extrinsic sensory pathways.

Identification and immunohistochemistry of cholinergic and non-cholinergic circular muscle motor neurons in the guinea-pig small intestine

Motor neurons which innervate the circular muscle layer of the guinea-pig small intestine were retrogradely labelled, in vitro, with the carbocyanine dye, DiI, applied to the deep muscular plexus. By combining retrograde tracing and immunohistochemistry, the chemical coding of motor neurons was investigated. Five classes of neuron could be distinguished on the basis of the co-localization of immunoreactivity for the different antigens; the five classes were also characterized by different lengths and polarities of their axonal projections and by their cell body shapes. Two classes with local or orally directed axons were immunoreactive for choline acetyltransferase and substance P and are likely to be cholinergic excitatory motor neurons. Two other classes had anally directed axons; they were immunoreactive for vasoactive intestinal polypeptide and are likely to be inhibitory motor neurons. A small proportion of neurons with short projections to the circular muscle were immunoreactive for neither substance P nor for vasoactive intestinal polypeptide, but are likely to be cholinergic. The morphological and histochemical identification of excitatory and inhibitory motor neurons provides a neuroanatomical basis for the final motor pathways involved in the polarized reflex motor activity of the gut.

Neuroanatomy of extrinsic afferents supplying the gastrointestinal tract

Here we discuss the neuroanatomy of extrinsic gastrointestinal (GI) afferent neurones, the relationship between structure and function and the role of afferents in disease. Three pathways connect the gut to the central nervous system: vagal afferents signal mainly from upper GI regions, pelvic afferents mainly from the colorectal region and splanchnic afferents from throughout. Vagal afferents mediate reflex regulation of gut function and behaviour, operating mainly at physiological levels. There are two major functional classes − tension receptors, responding to muscular contraction and distension, and mucosal receptors. The function of vagal endings correlates well with their anatomy: tracing studies show intramuscular arrays (IMAs) and intraganglionic laminar endings (IGLEs); IGLEs are now known to respond to tension. Functional mucosal receptors correlate with endings traced to the lamina propria. Pelvic afferents serve similar functions to vagal afferents, and additionally mediate both innocuous and noxious sensations. Splanchnic afferents comprise mucosal and stretch‐sensitive afferents with low thresholds in addition to high‐threshold serosal/mesenteric afferents suggesting diverse roles. IGLEs, probably of pelvic origin, have been identified recently in the rectum and respond similarly to gastric vagal IGLEs. Gastrointestinal afferents may be sensitized or inhibited by chemical mediators released from several cell types. Whether functional changes have anatomical correlates is not known, but it is likely that they underlie diseases involving visceral hypersensitivity.

Anatomy and physiology of the enteric nervous system

The enteric nervous system (ENS) is a quasi autonomous part of the nervous system and includes a number of neural circuits that control motor functions, local blood flow, mucosal transport and secretions, and modulates immune and endocrine functions. Although these functions operate in concert and are functionally interlinked, it is useful to consider the neural circuits involved in each separately.1 This short summary will concentrate mainly on the neural circuits involved in motor control.2 The enteric neural circuits are composed of enteric neurones arranged in networks of enteric ganglia connected by interganglionic strands. Most enteric neurones involved in motor functions are located in the myenteric plexus with some primary afferent neurones located in the submucous plexus. As in all nervous systems involved in sensory-motor control, the ENS comprises primary afferent neurones, sensitive to chemical and mechanical stimuli, interneurones and motorneurones that act on the different effector cells including smooth muscle, pacemaker cells, blood vessels, mucosal glands, and epithelia, and the distributed system of intestinal cells involved in immune responses and endocrine and paracrine functions. The digestive tract is unique among internal organs because it is exposed to a large variety of physicochemical stimuli from the external world in the form of ingested food. As a consequence, the intestine has developed a rich repertoire of coordinated movements of its muscular apparatus to ensure the appropriate mixing and propulsion of contents during digestion, absorption, and excretion. The oro-aboral transit of the intestinal contents can be regarded as a form of adaptive locomotion that occurs over a wide range of spatial and temporal domains.3 The movements of the intestine are the result of interaction of the neural apparatus and the muscular apparatus.4 The muscular apparatus is organised in muscle layers made up of large collections of smooth muscle cells …

Calretinin immunoreactivity in cholinergic motor neurones, interneurones and vasomotor neurones in the guinea-pig small intestine

SummaryImmunoreactivity for calretinin, a calcium-binding protein, was studied in neurones in the guinea-pig small intestine. 26±1% of myenteric neurones and 12±3% of submucous neurones were immunoreactive for calretinin. All calretinin-immunoreactive neurones were also immunoreactive for choline acetyltransferase and hence are likely to be cholinergic. In the myenteric plexus, two subtypes of Dogiel type-I calretinin-immunoreactive neurones could be distinguished from their projections and neurochemical coding. Some calretinin-immunoreactive myenteric neurones had short projections to the tertiary plexus, and hence are likely to be cholinergic motor neurones to the longitudinal muscle. Some of these cells were also immunoreactive for substance P. The remaining myenteric neurones, immunoreactive for calretinin, enkephalin, neurofilament protein triplet and substance P, are likely to be orad-projecting, cholinergic interneurones. Calretinin immunoreactivity was also found in cholinergic neurones in the submucosa, which project to the submucosal vasculature and mucosal glands, and which are likely to mediate vasodilation. Thus, calretinin immunoreactivity in the guinea-pig small intestine is confined to three functional classes of cholinergic neurones. It is possible, for the first time, to distinguish these classes of cells from other enteric neurones.

Immunohistochemical identification of cholinergic neurons in the myenteric plexus of guinea-pig small intestine

It is well established that acetylcholine is a neurotransmitter at several distinct sites in the mammalian enteric nervous system. However, identification of the cholinergic neurons has not been possible due to an inability to selectively label enteric cholinergic neurons. In the present study an immunohistochemical method has been developed to localize choline acetyltransferase, the synthetic enzyme for acetylcholine, in order that cholinergic neurons can be visualized. The morphology, neurochemical coding and projections of cholinergic neurons in the guinea-pig small intestine were determined using double-labelling immunohistochemistry. These experiments have revealed that many myenteric neurons are cholinergic and that they can be distinguished by their specific combinations of immunoreactivity for neurochemicals such as calretinin, neurofilament protein triplet, substance P, enkephalin, somatostatin, 5-hydroxytryptamine, vasoactive intestinal peptide and calbindin. On the basis of their previously described projections, functional roles could be attributed to each of these populations. The identified cholinergic neurons are: motorneurons to the longitudinal muscle (choline acetyltransferase/calretinin); motorneurons to the circular muscle (choline acetyltransferase/neurofilament triplet protein/substance P, choline acetyltransferase/substance P and choline acetyltransferase alone); orally directed interneurons in the myenteric plexus (choline acetyltransferase/calretinin/enkephalin); anally directed interneurons in the myenteric plexus (choline acetyltransferase/somatostatin, choline acetyltransferase/5-hydroxytryptamine, choline acetyltransferase/vasoactive intestinal peptide); secretomotor neurons to the mucosa (choline acetyltransferase/somatostatin); and sensory neurons mediating myenteric reflexes (choline acetyltransferase/calbindin). This information provides a unique opportunity to identify functionally distinct populations of cholinergic neurons and will be of value in the interpretation of physiological and pharmacological studies of enteric neuronal circuitry.

Identification of myenteric neurons which project to the mucosa of the guinea-pig small intestine

Myenteric neurons which innervate the mucosa of the guinea-pig ileum were characterized by combining retrograde transport of DiI in vitro with immunohistochemistry. Of DiI-labelled myenteric neurons, 43% were immunoreactive for calbindin and substance P, 25% were immunoreactive for calbindin alone, and 18% were immunoreactive for substance P alone. These 3 classes of neurons had Dogiel Type II morphology and are probably sensory neurons. Two classes of probable secretomotor neurons were characterized by immunoreactivity for neuropeptide Y (4%) and vasoactive intestinal peptide (2%). These 5 classes of myenteric neurons represent over 90% of the retrogradely labelled myenteric neurons that project to the mucosa.