Peter Bałuk

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.

Glial cells in the guinea pig myenteric plexus are dye coupled.

Glial cells in the myenteric plexus of the guinea pig small intestine were stained intracellularly with Lucifer yellow and horseradish peroxidase. The cells were identified by both their electrophysiological characteristics and by their morphology. Injection of Lucifer yellow, which is known to cross gap junctions, resulted in the staining of many (up to about 100) glial cells. The staining pattern was comparable to the immunostaining of glia with an antiserum for S-100 protein. In contrast to Lucifer yellow, horseradish peroxidase (which does not cross these junctions), was confined to the injected cell. It is concluded that enteric glia are coupled, presumably by gap junctions. This finding indicates that in addition to structural and biochemical similarities, enteric glia may share certain physiological characteristics with central nervous system astrocytes.

Afferent nerve endings in the tracheal muscle of guinea-pigs and rats.

SummaryThe trachea of guinea-pigs was stained as a whole-mount preparation with the zinc iodide-osmium technique. A distinct class of nerve endings was observed associated with the tracheal muscle. The endings, issued from myelinated fibres of the vagus nerve via the recurrent laryngeal nerve, are distributed on either side of the midline and ventral to the tips of cartilages. They are interpreted as afferent nerve endings that may correspond to slow adapting stretch receptors identified by physiological studies. Each nerve contributes predominantly, but not exclusively, to the receptors of the ipsilateral side. There are 120–180 receptors along the full length of the guinea-pig trachea, their density being higher at the cranial end. The receptors are variable in size and structural complexity, and, to some extent, also in spatial orientation, but distinct subtypes are not recognizable. Receptors of similar morphology and distribution are found also in the rat trachea. The receptors can also be visualized with a cytochrome oxidase method for nerve endings, but they do not stain with immunohistochemistry for the neuropeptides substance P, calcitonin gene-related peptide, vasointestinal polypeptide and neurotensin.

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.

An ultrastructural study of nerve profiles in the myenteric plexus of the rabbit colon.

The ultrastructure of the myenteric plexus from the rabbit colon was examined in both conventionally fixed tissue and also material fixed with the chromaffin method. Montages of the ganglia were analysed semi-quantitatively. Six main types of axon profile are described and classified on a morphological consideration of the vesicle population. Most axon types formed synapses with myenteric neurons. Two kinds of chromaffin-positive nerve fibre were seen, one containing a predominance of small granular vesicles, the other containing many flattened vesicles. The difficulties in relating axon profile types to putative transmitters are discussed.

Myenteric neurons express electrophysiological and morphological diversity in tissue culture

Intracellular recordings were taken from neurons of the myenteric plexus of the guinea-pig caecum grown in tissue culture. Subsequently horseradish peroxidase (HRP) was injected into the same neurons for examination by light and electron microscopy. Neurons showed a variety of electrophysiological responses, including S- and AH-cell type behavior, similar to those reported for myenteric neurons in situ, and depolarization to iontophoresis of acetylcholine. Neurons resembling Dogiel type I, type II and intermediate forms, and nerve varicosities which contained small agranular, large granular or large opaque vesicles were observed.

Some parasympathetic neurons in the guinea-pig heart express aspects of the catecholaminergic phenotype in vivo

SummaryIn a histochemical study of intrinsic cardiac ganglia of the guinea-pig in whole-mount preparations, it was found that some 70–80% of the neurons express aspects of the catecholaminergic phenotype. These neurons have an uptake mechanism for L-DOPA, and contain the enzymes for converting L-DOPA, (but not D-DOPA) to dopamine and noradrenaline, i.e. aromatic L-aminoacid decarboxylase and dopamine β-hydroxylase. Monoamine oxidase is also present within some of the neurons. In these respects, the neurons resemble noradrenergic neurons of sympathetic ganglia, so we refer to them as intrinsic cardiac amine-handling neurons. However, these neurons do not contain tyrosine hydroxylase and show little or no histochemically detectable uptake of α-methyldopa, dopamine or noradrenaline, even after depletion of endogenous stores of amines by pre-treatment with reserpine. Noradrenergic fibres from the sympathetic chain form pericellular baskets around nerve cell bodies. The uptake of L-DOPA into nerve cell bodies is not prevented by treatment with 6-hydroxydopamine sufficient to cause transmitter-depletion or degeneration of the extrinsic noradrenergic fibres. Such degeneration experiments suggest that axons of the amine-handling neurons project to cardiac muscle, blood vessels and other intrinsic neurons. The cardiac neurons do not show any immunohistochemically detectable serotonergic characteristics; there is no evidence for uptake of the precursors L-tryptophan and 5-hydroxytryptophan or 5-HT itself, whereas the extrinsic noradrer ergic nerve fibres within the ganglia can take up 5-HT when it is applied in high concentrations.

Scanning electron microscopy of the muscle coat of the guinea-pig small intestine

SummaryWe have studied the layers of the muscular coat of the guinea-pig small intestine after enzymatic and chemical removal of extracellular connective tissue. The cells of the longitudinal muscle layer are wider, have rougher surfaces, more finger-like processes and more complex terminations, but fewer intercellular junctions than cells in the circular muscle layer. A special layer of wide, flat cells with a dense innervation exists at the inner margin of the circular muscle layer, facing the submucosa. The ganglia of the myenteric and submucosal plexuses are covered by a smooth basal lamina, a delicate feltwork of collagen fibrils, and innumerable connective tissue cells. The neuronal and glial cell processes at the surface of ganglia form an interlocking mosaic, which is loosely packed in newborn and young animals, but becomes tightly packed in adults. The arrangement of glial cells becomes progressively looser along finer nerve bundles. Single varicose nerve fibres are rarely exposed, but multiaxonal bundles are common. Fibroblast-like cells of characteristic shape and orientation are found in the serosa; around nerve ganglia; in the intermuscular connective tissue layer and in the circular muscle, where they bridge nerve bundles and muscle cells; at the submucosal face of the special, flattened inner circular muscle layer; and in the submucosa. Some of these fibroblast like cells correspond to interstitial cells of Cajal. Other structures readily visualized by scanning electron microscopy are blood and lymphatic vessels and their periendothelial cells. The relationship of cellular elements to connective tissue was studied with three different preparative procedures: (1) freeze-cracked specimens of intact, undigested intestine; (2) ‘stretch preparations’ of longitudinal muscle with adhering myenteric plexus; (3) sheets of submucosal collagen bundles from which all cellular elements had been removed by prolonged detergent extraction.

Tracheal parasympathetic neurons of rat, mouse and guinea pig: Partial expression of noradrenergic phenotype and lack of innervation from noradrenergic nerve fibres

Noradrenergic nerves were studied in whole-mount preparations of the rat, mouse and guinea pig trachea by means of glyoxylic acid-induced catecholamine fluorescence and dopamine beta-hydroxylase immunoreactivity. In an effort to raise tissue levels of catecholamines, some specimens were also treated with the monoamine oxidase inhibitor pargyline, and with L-DOPA, a precursor of noradrenaline. Noradrenergic nerve fibres were detected around blood vessels, within the tracheal smooth muscle and in the mucosa, but never around or in the proximity of neurons of the tracheal ganglia, even after amine precursor loading. These parasympathetic ganglion cells did not show catecholamine fluorescence under control conditions. In the rat and mouse, but not in the guinea pig, some tracheal neurones were dopamine beta-hydroxylase immunoreactive and showed uptake and metabolism of amine precursors, thus expressing aspects of the catecholaminergic phenotype.

Scanning electron microscopic studies of bullfrog sympathetic neurons exposed by enzymatic removal of connective tissue elements and satellite cells

SummaryThe ninth and tenth abdominal sympathetic ganglia of bullfrogs were studied by light microscopy and transmission and scanning electron microscopy after the removal of the connective tissue elements overlying the neurons. Digestion of tissues with trypsin and subsequent acid hydrolysis exposed the unipolar neurons, which remained covered by their satellite cells. The preganglionic innervation was visible on the proximal segment and axon hillock region of the postganglionic neurite. Clusters of small cells seen at the periphery of ganglia probably corresponded to groups of cells with abundant catecholamine-containing granules (SIF cells). Digestion with collagenase and protease removed some or all of the satellite cells in addition to the connective tissue. The true neuronal surfaces had short finger-like processes, whereas the external surfaces of satellite cells were smooth. Preganglionic nerve varicosities were clearly visible on the proximal segment of the postganglionic neurite, on the axon hillock and on the cell body of neurons. A few axonal varicosities were fractured to reveal the synaptic vesicles within. The possible effects of the distribution and glial ensheathment of nerve varicosities on their function are discussed.