K.R. Jessen

Immunohistochemical studies of the enteric nervous system in tissue culture and in situ: Localization of vascoactive intestinal polypeptide (VIP), substance-P and enkephalin immunoreactive nerves in the guinea-pig gut

Abstract Immunofluorescence methods have been used to determine the detailed distribution of vasoactive intestinal polypeptide (VIP), substance-P and enkephalin nerve fibres in fixed cryostat sections from guinea-pig duodenum, jejunum, ileum, caecum at the site of the taenia coli, and proximal and distal colon. A novel method is used involving immunostaining of tissue culture preparations of both myenteric and submucous plexuses. These preparations allow each plexus to be studied in isolation from all axonal input for the first time, since they provide unequivocal extrinsic denervation together with severance of any intrinsic connections between the plexuses. In tissue sections the most prominent sites of VIP and substance-P immunoreactive fibres are the ganglia of the myenteric and submucous plexuses, the circular muscle layer and the longitudinal muscle of the taenia coli. In addition, VIP is prominent in the lamina propria of the submucosa except in the caecum. Enkephalin-immunopositive fibres are restricted to the ganglia of the myenteric plexus, the circular muscle layer and the longitudinal layer of the taenia coli. The culture preparations reveal that intrinsic ‘VIP neurons’ are common in the submucous plexus of the caecum and colon. They are also present, but in much lower numbers, in the myenteric plexus of the small intestine and colon but are not found in the myenteric plexus of the caecum. Intrinsic ‘substance-P neurons’ are present in the myenteric plexus from the small intestine, caecum and colon as well as in the submucous plexus of the colon; intrinsic ‘substance-P neurons’ are not found in the submucous plexus of the caecum. ‘Enkephalin neurons’ are numerous in the myenteric plexus of the small intestine, caecum and colon but are absent from the submucous plexus. Immunoreactivity is compared in the normal and denervated caecum by both the histochemical method and by radioimmunoassay of tissue extracts. In conjunction with the studies on tissue cultures, the results provide evidence for intrinsic reciprocal connections between the myenteric and submucous plexus of the caecum by neurons containing VIP and substance-P. An extensive comparison of these results with data from functional studies shows that the distribution of VIP, substance-P and enkephalin fibres in the gut is broadly in agreement with present knowledge of the action of these peptides on gut tissue, if it is assumed that they function as neurotransmitters or neuromodulators. In some instances, however, peptide-containing fibres and pathways are found which do not correlate with present knowledge obtained from functional studies. These observations provide new clues to the role of peptide neurons in gut function.

The enteric nervous system in tissue culture. I. Cell types and their interactions in explants of the myenteric and submucous plexuses from guinea pig, rabbit and rat

This paper describes methods for removing the ganglionated myenteric and submucous plexuses from the mammalian gut and maintaining them as explants in tissue culture. A detailed account is given of cell types, their interactions and the development of these cultures during 5 weeks in vitro. Three major cell types were identified in the cultures: neurons, glial cells and fibroblasts. The development of the plexuses in culture was studied in detail for the myenteric plexus from the guinea pig taenia coli. It followed a characteristic pattern, in which the merging of individual ganglia into a continuous monolayer of flattened neurons was accompanied and followed by the formation of an extensive outgrowth zone of flat glial cells covered by a dense mesh of outgrowing neurites. In older cultures, neuronal migration resulted in the reformation of discrete and compact aggregates, which consisted of neurons and glial cells, and were interconnected by thick neurite bundles. This arrangement resembles in many ways the original organization of enteric nervous tissue in vivo. This is the first time the enteric ganglia have been freed from the gut wall and grown in culture as explants of nervous tissue. These preparations open many new directions for investigations of the largest and most complex division of the peripheral nervous system, including studies of the molecular nature of neuronal and glial cell surfaces, analysis of cell-cell interactions, trophic factors and developmental signals.

GABA as an autonomic neurotransmitter: studies on intrinsic GABAergic neurons in the myenteric plexus of the gut

Abstract Outside the CNS, no part of the nervous system approaches the structural and functional complexity of the enteric ganglia of the gastrointestinal tract. Recently, it has become evident that there is a population of GABAergic neurons within the myenteric ganglia in the stomach, duodenum, ileum, colon and caecum. The GABAergic neurons of the gut nervous system project not only to other neurons within the myenteric ganglia, but also out into the circular muscle coat of the gut wall. Their presence has important implications for the intrinsic neuronal control of gastrointestinal function.

γ-Aminobutyrate as an autonomic neurotransmitter: release and uptake of [3H]γ-aminobutyrate in guinea pig large intestine and cultured enteric neurons using physiological methods and electron microscopic autoradiography

Abstract To provide further evidence that some enteric neurons use γ-aminobutyrate (GABA) as a neurotransmitter, we have demonstrated a depolarization-induced release of [3H]GABA from isolated myenteric ganglia in culture, and from segments of large intestine containing the myenteric plexus. In addition, light and electron microscopic autoradiography has been employed to visualize the putative GABAergic neurons and their projections, both in cultured ganglia and in sections from the gut wall. Explant cultures of the guinea-pig myenteric plexus, containing only neurons and glia intrinsic to the gut, were incubated with 0.14μ M [3H]GABA, washed and then repeatedly depolarized by 62 mM K+. The depolarizations always evoked release of [3H]GABA. The evoked release was reversibly blocked by 5mM Co2+, suggesting a dependence on Ca2+ influx, a characteristic of neuronal transmitter release. Strips of the guinea-pig taenia coli, containing the myenteric plexus, were incubated with 0.14 or 0.7μM [3H]GABA, washed and subjected to electrical field stimulation. This caused release of [3H]GABA, which could be evoked successively on repeated stimulation. The release was of neuronal origin and Ca2+ dependent, since it was abolished by 3μ M tetrodotoxin and reversibly blocked by 10 mM Co2+. By combined electrophoresis and chromatography the released tritium was identified as being attached to GABA. Autoradiography, following incubation with low concentrations of [3H]GABA, was used to identify specifically putative GABAergic neurons. Light microscopic autoradiography of cultured ganglia, and electron microscopic autoradiography of sections from the taenia coli including the myenteric plexus, were in good agreement, showing a selective and heavy labelling over a sub-population of neurons, and light labelling over glial cells. The majority of neurons and the non-neural cells were unlabelled. The electron microscopic autoradiographs also showed heavy labelling over some, but not all, axons in the fine axon bundles that innervate the longitudinal muscle of the taenia. These results strongly support our previous suggestion that a population of myenteric neurons are GABAergic, although more work is needed before this is fully established. Our data also suggest that some of the putative GABAergic neurons project out to the gut musculature, where their axons, together with axons from other neurons, run in bundles to form the intramuscular nerves. We therefore propose that one of the roles of these neurons in the gut is the presynaptic modulation of transmitter released from neighbouring axons in these nerves.

Transplantation of postnatal rat enteric ganglia into denervated adult rat hippocampus

These experiments explore the possible value of the myenteric plexus as a source of donor cells for autografting into the central nervous system. Neurons and glia from 10-12-day postnatal rat myenteric plexus survive for at least one month after transplantation into cholinergically denervated syngeneic adult rat hippocampus. A population of donor cholinergic neurons has acetylcholinesterase-positive processes, but these appear not to innervate host tissue. Host gliosis in response to these implants seems to be less than that seen with other peripheral ganglia, and unlike Schwann cells, the enteric glia form end-feet on brain capillaries.

The enteric nervous system in tissue culture. III. Studies on neuronal survival and the retention of biochemical and morphological differentiation

The maintenance of differentiated properties and survival rates of enteric neurons, grown in explant cultures for periods of up to 3 weeks, was studied. Using catecholamine fluorescence, immunohistochemistry and autoradiography, it was found that adrenergic neurons, VIP-containing neurons and putative GABAergic neurons, which constitute small subpopulations of guinea pig myenteric neurons in vivo, were all represented in plexus explants after maintenance in culture for 2-3 weeks. The pattern of expression of the transmitter-related enzymes, acetylcholinesterase and monoamine oxidase, paralleled that found in in situ studies. Investigation of neuronal structure by intracellular injection of horseradish peroxidase revealed that the cultured neurons continue to express the wide diversity in gross morphology which characterizes these cells in vivo. Employing autoradiography following uptake of [3H]GABA to label putative GABAergic neurons, their survival rate from days 1 to 15 of culturing was determined. No neuronal death was detected between days 1 and 5, while the number of neurons decreased between days 5 and 15. These observations suggest that enteric neurons maintained in explant cultures survive well and maintain to a high degree their histochemical and morphological properties.

Macrophage-like cells in muscularis externa of mouse small intestine: immunohistochemical localization of F4/80, M1/70, and Ia-antigen.

SummaryMacrophage-like cells (MLC) in mouse small intestine are situated in the muscularis externa in the subserosal layer at the level of Auerbachs plexus, and at the level of the deep muscular plexus. By combined labelling with FITC-dextran and immunohistochemical techniques, the MLC were shown to express the macrophage markers F4/ 80 and M1/70.15. The MLC appeared to be constitutively IE-antigen-positive, but did not contain lysozyme. It is suggested that MLC, like Langerhans cells, belong to a specialized class of cells in the mononuclear phagocyte system.

Distribution of neurons with high-affinity uptake sites for GABA in the myenteric plexus of the guinea-pig, rat and chicken

SummaryThe occurrence of neurons possessing high-affinity uptake sites for GABA was studied in the myenteric plexus of the guinea-pig ileum, caecum, and proximal and distal colon, the rat proximal colon, and the chicken gizzard with the use of 3H-GABA and autoradiography. Experiments were carried out on plexuses that had been freshly isolated from the gut wall or on isolated plexuses that had been maintained as expiant cultures for 7 to 14 days. Scattered neurons selectively labelled with 3H-GABA were found in the myenteric plexuses from all the areas examined. The results suggest that GABAergic neurons are widely distributed in the enteric nervous system.

The enteric nervous system in tissue culture. II. Ultrastructural studies of cell types and their relationships

Tissue culture preparations of the myenteric plexus from the guinea pig taenia coli have been studied by electron microscopy. Three main cell types can be identified: neurons, enteric glial cells and fibroblasts. The ultrastructure of these cells resembles that of the same cells in situ. Neuronal processes form close associations with other neurons and glial cells, but not with fibroblasts. After extended periods in culture, neurons and glial cells form aggregates of cells which resemble ganglia of the myenteric plexus in situ, having a compact neuropil and synapses between neuronal elements. Aggregates are connected to each other by thick bundles of neurites. Vesicle-containing nerve profiles are common; the majority contain a predominance of small agranular vesicles, but some contain many large granular or large opaque vesicles; profiles may also contain variable mixtures of these kinds of vesicles.

Intracellular calcium changes associated with cholinergic nicotinic receptor activation in cultured myenteric plexus neurones

Intracellular free calcium concentration ([Ca2+]i) was measured in cultured explants of myenteric plexus neurones by using the fluorescent calcium indicator Indol in combination with patch-clamp techniques. The basal [Ca2+]i was 94 nM and spontaneous oscillations in the internal free calcium concentration were recorded. These oscillations were associated with bursts of action potentials triggered by spontaneous nicotinic excitatory synaptic potentials. Under voltage clamp conditions, application of the selective nicotinic agonist m-hydroxyphenylpropyl-trimethylammonium iodide (10 microM) induced an inward current and increased the intracellular free calcium concentration. We conclude that cholinergic synaptic excitatory activity provide a regular calcium entry in myenteric neurone and suggest that the nicotinic channel might be significantly permeable to calcium.