Different subpopulations of cholinergic and nitrergic myenteric neurones project to mucosa and circular muscle of the guinea-pig gastric fundus

Since the stomach lacks a well-developed ganglionated submucous plexus, the somata of enteric neurones innervating the muscle or the mucosa have to be localised within the myenteric plexus. The aim of this study was to determine the projection pathways and the neurochemical coding of myenteric neurones innervating these different targets in the gastric fundus. Myenteric cell bodies projecting to the mucosa or the circular muscle were retrogradely labelled by mucosa or muscle application of the fluorescent tracer DiI and subsequently characterised by their immunoreactivity for choline acetyltransferase (ChAT), nitric oxide synthase (NOS), substance P (SP) and/or neuropeptide Y (NPY). On average 143±91 and 89±49 myenteric neurones were labelled from the mucosa and the circular muscle, respectively. DiI-labelled neurones were either ChAT- or NOS-positive. DiI-labelled ChAT-positive neurones were mainly ascending and outnumbered NOS-positive neurones, which were mainly descending (79.3±6.2% vs 20.7±6.2% for mucosa neurones; 69.3±11.1% vs 30.7±11.1% for muscle neurones). Three ChAT-positive subpopulations (ChAT/–, ChAT/SP, ChAT/NPY) and two NOS-positive subpopulations (NOS/–, NOS/NPY) were found. ChAT/SP neurones projected mainly to the circular muscle (36.1±11.9% of the cholinergic muscle neurones; mucosa projection: 8.0±2.1%), whereas ChAT/NPY neurones projected mainly to the mucosa (38.1±9.2% of the cholinergic mucosa neurones; muscle projection: 5.7±2.4%). NOS/– cells projected predominantly to the muscle. This study demonstrates polarised pathways in the myenteric plexus consisting of ascending ChAT and descending NOS cells that innervate the circular muscle and the mucosa of the gastric fundus. The ChAT/SP neurones might function as circular muscle motor neurones, whereas ChAT/NPY neurones might represent secretomotor neurones.     

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.     

Aspects of opiate dependence in the myenteric plexus of the guinea-pig.

Myenteric plexus-longitudinal muscle strips prepared from tolerant/dependent guinea-pigs and continuously exposed to normorphine, display a contracture upon naloxone challenge. This phenomenon represents a sign of abstinence. Removal of the opiate by extensive washing resulted in the failure of naloxone to induce the abstinence sign, while the plexus still displayed considerable, although reduced, tolerance to morphine. Reexposure of withdrawn preparations to normorphine reinduced the ability to display the abstinence sign. Highly tolerant preparations exhibited a 30 fold increase in sensitivity to serotonin and prostaglandin E₁ when tested a few minutes after naloxone-precipitated withdrawal. Supersensitivity rapidly declined when normorphine was washed off the preparation, while reincubation of withdrawn tissues with the opiate resulted in reinduction of supersensitivity. The data confirms a close relationship between a state of tolerance and dependence (including display of the abstinence sign) and supersensitivity to putative neurotransmitters or neuromodulators, becoming evident following administration of naloxone.     

Evidence for a non-opioid sigma binding site in the guinea-pig myenteric plexus

The presence of a binding site to (+)-(3H)SKF 10,047 was demonstrated in a guinea-pig myenteric plexus (MYP) membrane preparation. Specific binding to this receptor was saturable, reversible, linear with protein concentration and consisted of two components, a high affinity site (KD = 46 +/- 5 nM; Bmax = 3.4 +/- 0.5 pmole/g wet weight) and a low affinity site (KD= = 342 +/- 72 nM; Bmax = 22 +/- 3 pmole/g wet weight). Morphine and naloxone 10(-4) M were unable to displace (+)-(3H)SKF 10,047 binding. Haloperidol, imipramine, ethylketocyclazocine and propranolol were among the most potent compounds to inhibit this specific binding. These results suggest the presence of a non-opioid haloperidol sensitive sigma receptor in the MYP of the guinea-pig.     

Multiple mechanisms contribute to myenteric plexus ablation induced by benzalkonium chloride in the guinea-pig ileum

Ablation of rat myenteric plexus with benzalkonium chloride has provided a model of intestinal aganglionosis, but the degenerative responses are not well understood. We examined the effects of this detergent on neurons and glia, including expression of c-Myc, c-Jun, JunB, and c-Fos, and on immunocytes in the guinea-pig ileum. Benzalkonium chloride (0.1%) or saline was applied to the serosal surface of distal ileum. Tissues were analyzed 2, 3, or 7 days later and compared with cyclosporine-treated and untreated animals. More than 90% of myenteric neurons were destroyed in ileal segments 3–7 days after benzalkonium-chloride treatment. Glia withdrew processes from around neurons after 2 days and were mostly gone after 3 days. Neuronal c-Myc began to disappear while c-Fos, c-Jun, and JunB were evident in some neuronal nuclei after 2 or 3 days. After 3 days, widespread apoptosis was evident in the myenteric plexus. Populations of T cells, B cells, and macrophage-like cells in untreated and saline-treated myenteric plexuses were substantially increased 3 and 7 days after benzalkonium-chloride treatment. Cyclosporine delayed significant neuronal loss. We conclude that a variety of degenerative mechanisms may be active in this model, including an immune response which may actively contribute to tissue destruction. Received: 13 September 1996 / Accepted: 20 January 1997     

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.     

Storage and release of labelled acetylcholine in the myenteric plexus of the guinea-pig ileum.

Guinea-pig ileum myenteric plexus-longitudinal muscle preparation was superfused with [3H]choline for 15 min either without being stimulated or during field stimulation at 0.1 or 16 Hz; the preparation was then either removed immediately or after 75- or 135-min superfusion with hemicholinium-3 (HC-3) and the total acetylcholine (ACh) and [3H]ACh contents were determined. For measuring the release of [3H]ACh the preparation was stimulated for 60 min the second time at 0.1 or 16 HZ in the presence of hemicholinium. Exposure to [3H]choline without stimulation resulted in the formation of [3H]ACh stores which were maintained in the first 75 min but decreased therafter. Labelling during stimulation at 16 Hz produced the largest and best maintained [3H]ACh content. Following labelling during 0.1-Hz stimulation, more label could be released than following labelling in the absence of stimulation. Labelling during 16-Hz stimulation did not increase any further in fool of [3H]ACh accessible to release by 0.1-Hz stimulation, but caused a 2.5 times increase in the pool from which Hz stimulation released [3H]ACh. These results suggest that two populations of cholinergic neurons exist in the myenteric plexus, one activated only by high frequency stimulation, the other by both high and low frequency stimulation.     

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.     

Tetraethylammonium facilitates the stimulation-evoked loss of the enkephalins from the myenteric plexus of guinea-pig ileum

The effects of electrical field stimulation on the contents of [Met]enkephalin and [Leu]enkephalin were determined in myenteric plexus-longitudinal muscle preparations of the guinea-pig small intestine. Cycloheximide (0.1 mM) was present in all experiments to prevent de nouveau biosynthesis. The two enkephalins were separated by high performance liquid chromatography and assayed on the mouse vas deferens. Stimulation with submaximal pulses (50 mA, 0.5 ms) at a frequency of 10 Hz caused maximal losses of about 35% of [Met]enkephalin and [Leu]enkephalin after 3 h (108000 pulses). The plot of log (enkephalin content) against number of pulses was steeper during the first 30 min than during the later periods. Tetraethylammonium bromide (TEA, 10 mM) increased the [Met]enkephalin and [Leu]enkephalin contents of the non-stimulated preparations by about 50%. When the preparations were stimulated in the presence of TEA at 50 mA and 1 Hz, the plots of loss of enkephalins against number of pulses were linear until the maximum of about 50% was reached. Compared with stimulation in the absence of TEA, the rate constant was 8 times greater for [Leu]enkephalin and 20 times greater for [Met]enkephalin. The absolute losses per pulse were about 13 times greater for [Leu]enkephalin and 27 times greater for [Met]enkephalin than in the absence of TEA. In the presence of bacitracin and a mixture of dipeptides, the enzymatic degradation of the enkephalins was sufficiently suppressed to cause an overflow of 30-60% of the enkephalins lost from their stores into the perifusing Krebs solution. Until it is possible to determine the preformed precursors, which are present in large quantities, the kinetics relationship between these precursors and the enkephalins cannot be investigated. A similar dilemma exists for the relationship between "released' enkephalins and the losses from their stores.     

Neuropeptide Y is an inhibitor of neural function in the myenteric plexus of the guinea-pig ileum

Neuropeptide Y (NPY) reduced the resting tension of the myenteric plexus-longitudinal muscle preparation (MP-LM) of the guinea-pig ileum (GPI). NPY in a dose-dependent manner also reduced the neurally-mediated excitatory effect of cholecystokinin octapeptide (CCK8) sulfated form on this preparation. However, NPY, at the concentration used in the study, did not modify the effect of exogenous acetylcholine (ACh). All these features were also shared by other inhibitory peptides, like somatostatin (SOM) and the enkephalin derivative FK 33-824. The preparation developed a degree of tachyphylaxis to the inhibitory effect of NPY more rapidly than it did to SOM. Moreover, the inhibitory effect of neuropeptide Y was of longer duration than the one seen for somatostatin. A faster metabolic rate might account for the lower development of tachyphylaxis to somatostatin. The presence of the opioid antagonist naloxone did not alter the inhibitory features of NPY or SOM. Therefore, the involvement of any endogenous opioid in the action of these two inhibitory peptides can be disregarded.     

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.     

Evidence that myenteric neurons of the gastric corpus project to both the mucosa and the external muscle: myectomy operations on the canine stomach

Summary The distribution of nerve cell bodies and fibres in the canine stomach was investigated using antibodies to the general neuronal marker, neuron-specific enolase. Prominent ganglia containing many reactive nerve cells were found in the myenteric plexus of the gastric corpus and antrum. Nerve cells were absent from the submucosa of the corpus and were extremely rare in the antrum. Renoval of areas of longitudinal muscle and myenteric plexus from the corpus (myectomy), with 7 days allowed for axon degeneration, resulted in the loss of fibres reactive for galanin, gastrin-releasing peptide, substance P and vasoactive intestinal peptide from both the circular muscle and mucosa in the area covered by the lesion. Combined vagotomy and sympathetic denervation did not significantly affect these fibres, but did cause fibres reactive for calcitonin gene-related peptide to degenerate. It is concluded that the myenteric plexus of the gastric corpus, like the myenteric plexus of the small intestine and colon, is the source of nerve fibres innervating the circular muscle, but, in contrast to other regions of the gastrointestinal tract, myenteric ganglia, not submucous ganglia, are the major, or sole, source of the intrinsic innervation of the mucosa.     

Morphology of horseradish peroxidase (HRP)-injected glial cells in the myenteric plexus of the guinea-pig

Glial cells of the myenteric plexus from guinea pig small intestine were intracellulary filled with horseradish peroxidase (HRP), and histochemically stained. Camera lucida-like drawings of twenty cells were morphologically and morphometrically analyzed. The cells have very small ellipsoid, somata (85±0.7 m equivalent diameter, i.e., about 330 m³ volume), and send up to 20 thin and short processes (less than 26 to about 110 m in length). The morphology of the cells appears to depend on their location within the plexus. Glial cells located within the ganglia are similar to CNS protoplasmic astrocytes; they are star-shaped, and their very short processes are irregularly, branched. In contrast, glial cells within the interganglionic fiber tracts resemble CNS fibrous astrocytes. They extend longer processes that are parallel to the fiber tracts, and show less tendency to branch. We propose that the morphology of enteric glia is determined by the structure of the microenvironment. Both cell types form several flat endfeet at a basal lamina either surrounding blood vessels or at the ganglionic border. Furthermore, the occurrence of holes in the glial cell processes suggests that particular neuronal cell processes may be enwrapped in a specific manner. Fractal analysis of camera lucida-like drawings of the cells showed that the cells have a highly complex surface structure, comparable to that of protoplasmic astrocytes in the brain. These tiny cells may possess a membrane surface area of 2000 m², almost 90% of which are contributed by the cell processes. This geometry may enable an intense exchange of metabolites and ions between neurons, glial cells, and the capillaries and/or environment of enteric ganglia.     

Phosphatase activity in the myenteric plexus

Intense and very intense reactions were obtained for acid phosphatase, calcium activated ATP-ase (pH 9.4), magnesium activated ATP-ase (pH 7.2) and glucose-6-phosphatase in the cytoplasms of the myenteric plexus nerve cells of the small intestine of Macacus rhesus and rabbit. Nucleotidase activity was moderate or slight and unspecific alkaline phosphatase activity absent. Both ATP-ases presented an intense activity in the myenteric plexus nerve cells of human fetuses 30, 33, and 34 weeks old; 5-nucleotidase activity, slight in the 30-week-old fetuses became more intense in the 33- and 34-week-old fetuses. The satellite neuroglial cells, nerve fibers and blood capillaries presented negative alkaline phosphatase reactions and intense or very intense activities of the other phosphatases.     

Comparative studies of quinacrine-positive neurones in the myenteric plexus of stomach and intestine of guinea-pig,rabbit and rat

Summary The number of quinacrine-fluorescent nerve cell bodies and the percentage of the ganglion area occupied by this fluorescence within stretch preparations of the myenteric plexus of the stomach and ileum of the guineapig, rabbit and rat were assessed. The number of quinacrine-positive cell bodies per cm² of plexus varied between 1045 in the rabbit ileum to 2633 in the rat stomach, whilst the percentage of the ganglionic area occupied by fluorescence was approximately 10 %. The distribution of quinacrine-fluorescent nerve fibres and cell bodies in the myenteric plexus was compared to the distribution of nerves revealed by catecholamine fluorescence and by staining for acetylcholinesterase in the stomach and ileum of all three species. Quinacrine fluorescence appears to be selective for non-adrenergic, non-cholinergic nerves; the possibility that it binds to high levels of ATP is discussed.     

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.     

Action of morphine on guinea-pig myenteric plexus and mouse vas deferens studied by intracellular recording.

Morphine reduces the output of transmitter from the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum and from the mouse vas deferens. Intracellular recordings were made from ganglion cells of the myenteric plexus and smooth muscle cells of the vas deferens. Synaptic transmission within the myenteric plexus was blocked by hexamethonium. Morphine did not change the properties of the ganglion cells, nor did it affect synaptic potentials. 5-Hydroxytryptamine inhibited acetylcholine release at intraganglionic synapses by an action which was unaffected by morphine. In the vas deferens, excitatory junction potentials were elicited by stimulation of postganglionic adrenergic nerve fibres. The junction potentials were depressed by morphine and levorphanol but not by dextrorphan. This depression was reversed by naloxone. The results indicate that morphine acts directly to reduce transmitter release at the neuro-effector junctions in the myenteric plexus-longitudinal muscle preparation and in the vas deferens in these species.