A neurochemical characterisation of the golden hamster myenteric plexus

The neurochemical composition of nerve fibres and cell bodies in the myenteric plexus of the proventriculus, stomach and small and large intestines of the golden hamster was investigated by using immunohistochemical and histochemical techniques. In addition, the procedures for localising nitric-oxide-utilising neurones by histochemical (NADPH-diaphorase) and immunohistochemical (nitric oxide synthase) methods were compared. The co-localisation of vasoactive intestinal polypeptide and nitric oxide synthase in the myenteric plexus of all regions of the gut was also assessed. The results demonstrated the presence of nerve fibres and nerve cell bodies immunoreactive to protein gene product, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, tyrosine hydroxylase, 5-hydroxytryptamine and nitric oxide synthase in all regions of the gastrointestinal tract examined. The pattern of distribution of immunoreactive nerve fibres and nerve cell bodies containing the above markers was found to vary in different regions of the gut. Myenteric neurones and nerve fibres containing immunoreactivity to nitric oxide synthase and NADPH-diaphorase reactivity, however, were shown to have an identical distribution throughout the gut. In contrast to some studies on the guinea-pig and rat, the co-existence of vasoactive intestinal polypeptide and nitric oxide synthase was seen in only a small population of myenteric neurones.     

Histochemical characterization of myenteric plexus in domestic fowl small intestine

The myenteric plexus of the domestic fowl (Gallus domesticus) small intestine was studied by means of silver staining, glyoxylic acid-induced fluorescence, the modified Koelle-Friedenwald method for the detection of acetylcholinesterase, NADH-diaphorase techniques and the unlabelled antibody method involving the use of an antiserum raised against GABA conjugated by glutaraldehyde to bovine serum albumin. The majority of the perikarya were in the ganglia, with an average density of 3370 +/- 942 nerve cells/cm2. Cholinesterase-positive and a few GABA-immunoreactive nerve cell bodies were seen in the myenteric ganglia, while fluorescent ganglion cells were not observed. In addition to AChE and GABA-positive nerve fibres, a rich fluorescent network of varicose and nonvaricose nerve fibres was detected, pointing to the presence of an extrinsic aminergic system in the domestic fowl myenteric plexus. Electron microscopic observations on nerve cells, axon profiles and varicosites with various vesicle populations were in good agreement with the histochemical findings.     

Vascularization of the myenteric plexus in the small intestine of pigeons]

By means of light and electron microscopy vascularization of the myenteric plexus has been studied in the pigeon small intestine. Ganglia of the plexus, their cell composition, ultrastructure of neurons have been described. Links of the microcirculatory bed of the intramural ganglia are characterized, interrelations of capillaries with neurons are described, quantitative estimation of microhemovessels, surrounding the microcirculatory bed of the myenteric plexus in the intestinal wall in birds and mammalia.     

Chemical coding of neurons in the myenteric plexus and external muscle of the small and large intestine of the mouse

Immunohistochemical techniques were used to examine the presence and co-localisation of a range of putative neurotransmitters and other neuronal markers in the myenteric plexus of the small and large intestine of the mouse. Distinct sub-populations of myenteric neurons were identified, based on the combinations of substances they contained and the distribution of their fibres. In the small intestine, there were two major classes of circular muscle motor neurons; one class was characterised by the presence of nitric oxide synthase, vasoactive intestinal peptide plus neuropeptide Y (NOS/VIP/NPY), and the second class contained calretinin plus substance P (CalR/SP). There were seven classes of neurons that innervated myenteric ganglia; these contained NOS, VIP, NOS/VIP, NPY, CalR/calbindin (CalB), SP or 5-HT. In the large intestine, there were five major classes of motor neurons that contained NOS, NOS/VIP, GABA, SP, or CalR/SP, and seven major classes of neurons that innervated myenteric ganglia and contained NOS, VIP, CalR/CalB, CalR, SP, GABA or 5-HT. Although some aspects of the patterns of co-localisation are similar to those in other species, this study re-inforces recent analyses that indicate significant species differences in neurochemical patterns in the enteric neurons of different species. Received: 28 August 1995 / Accepted: 30 November 1995     

The action of substance P on neurons of the myenteric plexus of the guinea-pig small intestine.

Extracellular and intracellular recordings were made in vitro from single neurons of the myenteric plexus of the guinea-pig small intestine. Synthetic substance P was applied to the neurons by means of the perfusing solution or by electrophoresis from micropipettes. Extracellular recording showed that substance P (100 pm-30 nm), applied by perfusion, increased the firing rate of myenteric neurons. Intracellular recording indicated that perfusion with substance P caused a dose-dependent membrane depolarization which was unaffected by hexamethonium, hyoscine, naloxone or baclofen. The depolarization was also evoked by electrophoretic application of substance P. It was associated with an increase in membrane resistance, augmented by membrane depolarization and reduced by membrane hyperpolarization. The relation between the substance P reversal potential and the logarithm of the extracellular potassium concentration was linear with a slope of 54 mV/log10[K+], which indicates that substance P inactivates the resting potassium conductance of the myenteric neurons. This effect on ion conductance is the same as that of an unknown substance that mediates slow synaptic excitations with the myenteric plexus.     

Structure of the tertiary component of the myenteric plexus in the guinea-pig small intestine

The tertiary component of the myenteric plexus consists of interlacing fine nerve fibre bundles that run between its principal ganglia and connecting nerve strands. It was revealed by zinc iodide-osmium impregnation and substance P immunohistochemistry at the light-microscope level. The plexus was situated against the inner face of the longitudinal muscle and was present along the length of the small intestine at a density that did not vary markedly from proximal to distal. Nerve bundles did not appear to be present in the longitudinal muscle as judged by light microscopy, although numberous fibre bundles were encountered within the circular muscle layer. At the ultrastructural level, nerve fibre bundles of the tertiary plexus were found in grooves formed by the innermost layer of longitudinal smooth muscle cells. In the distal parts of the small intestine, some of these nerve fibre bundles occasionally penetrated the longitudinal muscle coat. Vesiculated profiles in nerve fibre bundles of the tertiary plexus contained variable proportions of small clear and large granular vesicles; they often approached to within 50–200 nm of the longitudinal smooth muscle cells. Fibroblast-like cells lay between strands of the tertiary plexus and the circular muscle but were never intercalated between nerve fibre varicosities and the longitudinal muscle. These anatomical relationships are consistent with the tertiary plexus being the major site of neurotransmission to the longitudinal muscle of the guinea-pig small intestine.     

The projections of 5-hydroxytryptamine-accumulating neurones in the myenteric plexus of the small intestine of the guinea-pig

Retrograde tracing, combined with immunohistochemistry, was used to study the projections of 5-hydroxytryptamine (5-HT)-accumulating neurones within the ileum of the guinea-pig, with confocal microscopy being used to characterise further their morphology. Two classes of neurones in the myenteric plexus, capable of taking up 5-HT or analogues, were distinguished. One class had Dogiel type I morphology with lamellar dendrites, was located on the edge or in the middle of ganglia and lacked immunoreactivity for somatostatin (SOM). The other class had smooth ovoid cell bodies with multiple filamentous dendrites and a single axon and represented a subset of the SOM-immunoreactive interneurones in the myenteric plexus. Varicosities immunoreactive for 5-HT alone, 5-HT/SOM or SOM alone were present in the myenteric ganglia. Both classes of 5-HT-accumulating neurones had long aboral projections within the myenteric plexus (up to 100 mm long) and to the submucous plexus and probably function as descending interneurones.     

Quantitative estimation of putative primary afferent neurons in the myenteric plexus of human small intestine

This study aimed at estimating the proportion of human myenteric Dogiel type II neurons, putative intrinsic primary afferent neurons (IPANs), in relation to the entire myenteric neuron population. Since, at present, there is no known single marker, which specifically labels these neurons, we tried to identify the most appropriate marker combination based on the results of an earlier study. For this purpose, 10 wholemounts derived from human small intestinal segments were immunohistochemically triple-stained for calretinin (CALR), somatostatin (SOM) and neurofilaments (NF) and 9 were stained for substance P (SP), SOM and NF. In each wholemount, 15 ganglia selected randomly were evaluated. On the basis of their NF-reactivity, neurons reactive for one or co-reative for both of the other two markers, respectively, were morphologically classified as type II or non-type II neurons. We found that the majorities of neurons co-reactive for CALR/SOM and SP/SOM, respectively, were type II neurons whereas this was not the case for neurons, which were reactive for only one of the two markers. One of the statistical parameters estimated was the positive predictive value, the probability that a neuron displaying CALR/SOM- or SP/SOM-co-reactivity, respectively, is a type II neuron. This value was 97% in case of CALR/SOM- and 95% in case of SP/SOM-co-staining. Although the difference of the statistical parameters between the two stainings was not significant, CALR and SOM were used to estimate indirectly the proportion of type II neurons, in wholemounts co-stained with the pan-neuronal marker neuronal protein HuC/HuD (HU). In these wholemounts, altogether 9.1% of neurons were coreactive for CALR/SOM. We suggest that the proportion of myenteric type II neurons in the human small intestine is related to the proportion of CALR/SOM-co-reactive neurons and may be near to one tenth of the total myenteric neuronal population.     

Ultrastructural studies of the myenteric plexus and smooth muscle in organotypic cultures of the guinea-pig small intestine

External muscle and myenteric plexus from the small intestine of adult guinea-pigs were maintained in vitro for 3 or 6 days. Myenteric neurons and smooth muscle cells from such organotypic cultures were examined at the electron-microscopic level. An intact basal lamina was found around the myenteric ganglia and internodal strands. Neuronal membranes, nuclei and subcellular organelles appeared to be well preserved in cultured tissues and ribosomes were abundant. Dogiel type-II neurons were distinguishable by their elongated electron-dense mitochondria, numerous lysosomes and high densities of ribosomes. Vesiculated nerve profiles contained combinations of differently shaped vesicles. Synaptic membrane specializations were found between vesiculated nerve profiles and nerve processes and cell bodies. The majority of nerve fibres were well preserved in the myenteric ganglia, in internodal strands and in bundles running between circular muscle cells. No detectable changes were found in the ultrastructure of the somata and processes of glial cells. Longitudinal and circular muscle cells from cultured tissue had clearly defined membranes with some close associations with neighbouring muscle cells. Caveolae occurred in rows that ran parallel to the long axis of the muscle cells. These results indicate that the ultrastructural features of enteric neurons and smooth muscle of the guinea-pig small intestine are well preserved in organotypic culture.     

The effects of cold-stress,hibernation, and prolonged inactivity on bone dynamics in the golden hamster,Mesocricetus auratus

The effects of cold-stress and hibernation on bone dynamics in the femurs of hamsters were investigated using histometric analyses. Control animals were maintained at 27° C for 90 days; experimental animals were kept at 5° C and hibernated for 7, 15, 21, 50, or 90 days. Histometric analyses of cross sections indicated that bone diameter and cortical thickness at the femoral midshaft increased after 83 days of extreme cold and 7 days of hibernation but decreased significantly after 69 days of cold stress and 21 days of hibernation. Osteoporosis was evident although the number of osteons per unit area of bone increased during hibernation. An initial decrease in the number of non-Haversian longitudinal vessels per unit area of bone was seen in experimental animals which was apparently related to a corresponding reduction in cortical thickness. Lacunar area increased in these animals, suggesting that osteocytic osteolysis may be a significant mechanism for calcium regulation during hibernation.     

Image analysis of the sympathetic innervation of the myenteric plexus in the small intestine of mammalian species

1. The arrangement of the sympathetic innervation of the myenteric plexus varies between mammalian species. 2. In larger mammals the density of sympathetic innervation of the myenteric plexus is significantly less than in small (less then 1 kg) species. 3. The number of varicosities on the terminal parts of sympathetic neurons innervating the gut is significantly less in larger mammals.     

Developmental pattern of three vesicular glutamate transporters in the myenteric plexus of the human fetal small intestine

Three vesicular glutamate transporters (VGLUT1-3) have previously been identified in the central nervous system, where they define the glutamatergic phenotype, and their expression is tightly regulated during brain development. In the present study we applied immunocytochemistry to examine the distribution of the immunoreactivity of all three VGLUTs during prenatal development of the myenteric plexus in the human small intestine. We also investigated changes in their localization in the different segments of the small intestine and in the different compartments of the developing myenteric ganglia. Immunoreactivity against all three VGLUTs was found predominantly in the ganglionic neuropil, interganglionic varicose fibers and perisomatic puncta, but cytoplasmic labeling with different intensities also occurred. Each transporter displayed a characteristic spatiotemporal expression pattern, with the transient increase or decrease of immunoreactive cell bodies, varicosities or perisomatic puncta, depending on the fetal age, the gut segment or the ganglionic compartment. Throughout gestational weeks 14-23, VGLUT1 immunoreactivity always predominated over VGLUT2 immunoreactivity, though both peaked around week 20. VGLUT3 immunoreactivity was less abundant in the developing myenteric plexus than those of VGLUT1 and VGLUT2 immunoreactivity. It was mainly expressed in the ganglionic neuropil and in the perisomatic puncta throughout the examined gestational period. Neuronal perikarya immunoreactive for VGLUT3 were restricted to between weeks 18 and 20 of gestation and exclusively to the oral part of the small intestine.     

Quantitative analysis of inputs to somatostatin-immunoreactive descending interneurons in the myenteric plexus of the guinea-pig small intestine

Somatostatin immunoreactivity occurs in a specific subgroup of cholinergic descending interneurons in the myenteric plexus of the guinea-pig small intestine. In the present work, we made light- and electron-microscopic investigations of chemically defined inputs to these neurons, in order that the origins of the connections of other neurons with them could be deduced. Somatostatin-immunoreactive synapses and close contacts were found on the cell bodies and filamentous processes of somatostatin neurons; these were 84% of all inputs. It is thus confirmed that this class of interneuron forms chains that project anally. Descending interneurons with immunoreactivity for nitric oxide synthase provided 14% of inputs to somatostatin-immunoreactive descending interneurons. An antiserum against a calcium-binding protein, calbindin, was used as marker for the majority of intrinsic primary afferent neurons, AH/Dogiel type II neurons; this class of neurons provided only 2.5% of the inputs to somatostatin-immunoreactive descending interneurons. We conclude that somatostatin-immunoreactive descending interneurons are involved in the conduction of impulses distally along the full length of the small intestine, but receive only a minor input from calbindin-immunoreactive primary afferent neurons.     

Mechanosensitive enteric neurons in the myenteric plexus of the mouse intestine

Background

Within the gut the autonomous enteric nervous system (ENS) is able to sense mechanical stimuli and to trigger gut reflex behaviour. We previously proposed a novel sensory circuit in the ENS which consists of multifunctional rapidly adapting mechanosensitive enteric neurons (RAMEN) in the guinea pig. The aim of this study was to validate this concept by studying its applicability to other species or gut regions.

Methodology/Principal Findings

We deformed myenteric ganglia in the mouse small and large intestine and recorded spike discharge using voltage sensitive dye imaging. We also analysed expression of markers hitherto proposed to label mouse sensory myenteric neurons in the ileum (NF145kD) or colon (calretinin). RAMEN constituted 22% and 15% of myenteric neurons per ganglion in the ileum and colon, respectively. They encoded dynamic rather than sustained deformation. In the colon, 7% of mechanosensitive neurons fired throughout the sustained deformation, a behaviour typical for slowly adapting echanosensitive neurons (SAMEN). RAMEN and SAMEN responded directly to mechanical deformation as their response remained unchanged after synaptic blockade in low Ca⁺⁺/high Mg⁺⁺. Activity levels of RAMEN increased with the degree of ganglion deformation. Recruitment of more RAMEN with stronger stimuli may suggest low and high threshold RAMEN. The majority of RAMEN were cholinergic but most lacked expression of NF145kD or calretinin.

Conclusions/Significance

We showed for the first time that fundamental properties of mechanosensitive enteric neurons, such as firing pattern, encoding of dynamic deformation, cholinergic phenotype and their proportion, are conserved across species and regions. We conclude that RAMEN are important for mechanotransduction in the ENS. They directly encode dynamic changes in force as their firing frequency is proportional to the degree of deformation of the ganglion they reside in. The additional existence of SAMEN in the colon is likely an adaptation to colonic motor patterns which consist of phasic and tonic contractions.