Histochemical localization of cholinesterases and monoamines in the central heart of Sepia officinalis L. (Cephalopoda)

The central heart of the coleoid cephalopod, Sepia officinalis, was studied using acetylcholinesterase and fluorescence histochemistry. Using histo- and cytochemical reactions, acetylcholinesterase was localized in the axolemma and axoplasm of specific cardiac nerve fibres, as well as in the sarcolemma and within the sarcotubular system of the muscle cells. Butyrylcholinesterase exhibited a different distribution, being found only in the luminal trabecular muscle layer. Glyoxylic-acid-induced fluorescence indicated the presence of catecholamines (emission maximum, 470 nm) in cardiac nerve axons. These histochemical findings support the hypothesis that noradrenaline and/or dopamine and acetylcholine act antagonistically as natural transmitters. Fluorophores indicating the presence of serotonin were not observed. The present results are discussed in the light of previous pharmacological findings.     

Localization of putative neurotransmitters in the mantle and siphuncle of the mollusc Nautilus pompilius L. (Cephalopoda)

The neurotransmitter supply in the nerve endings of the mantle and the siphuncle, i.e. in organs that are responsible for the shell formation in the ectocholeate Nautilus pompilius, were investigated with electron microscopical, fluorescence-, immuno- and enzyme histochemical methods as well as with high pressure liquid chromatography (HPLC). Using antibodies against serotonin and the tetrapeptide FMRF-amide, positive reactions were demonstrated immunohistochemically within the terminal nerve fibres of the mantle and the vessels of the siphuncle. Enzyme histochemical proof of the presence of specific acetylcholinesterase yielded positive results in the muscle fibres of the mantle and siphuncle. Additionally, in the mantle, glyoxylic acid-induced fluorescence was shown within the nerve endings indicating catecholamines as neurotransmitters, whereas in the siphuncle such fluorescence did not appear. However, the HPLC-analyses showed that in the mantle and also in the siphuncle the content of dopamine is higher than that of noradrenaline whereas only traces of adrenaline occur in both organs suggesting dopamine as a putative neurotransmitter. Transmission electron microscopical examination of the nerve endings of both organs revealed that different types of vesicles were distinguished that could be considered as cholinergic, aminergic and peptidergic structures.     

The autonomic innervation of rat jugular vein

Summary The autonomic innervation of rat jugular vein was studied using glyoxylic acid fluorescence and acetylcholinesterase histochemical methods. The rat jugular vein is provided with both adrenergic and cholinergic nerve fibers organized in plexuses located at the adventitial-medial border. The existence of these nerve plexuses does not seem to support biochemical findings that suggest a lack of innervation in the rat jugular vein and which propose this blood vessel as a model for the analysis of drug-smooth muscle cell interaction without the interference of neuronal uptake mechanisms.     

Evidence for exclusive adrenergic innervation of feather muscles (mm. pennati) in the chicken

Summary Feather follicles in the avian skin are interconnected by well-defined bundles of smooth muscle cells, which are responsible for the erection and depression of feathers and thus play an important role in thermoregulation. The depressing and erecting muscle bundles were found to receive a very dense supply of unmyelinated nerve fibres that displayed ultrastructural and histochemical characteristics of noradrenergic axons (formaldehyde- and glyoxylic acid-induced catecholamine fluorescence; uptake to 5-hydroxydopamine). No nerve fibres were encountered showing histochemical acetylcholinesterase activity. There was no indication of the presence of peptidergic or purinergic nerve endings.The neuromuscular space usually ranged from 40–60 nm in width and contained a basal lamina. Occasionally, this space was reduced to approximately 20 nm. At such close neuromuscular contacts a basal lamina was lacking, and focal densities beneath the pre- and postsynaptic plasma membrane were observed. Since no gap junctions between muscle cells were detected, the dense supply with noradrenergic nerve fibres indicates a high amount of directly innervated smooth muscle cells.An additional finding of the present study was the observation that high local concentrations of 5-hydroxydopamine led to degeneration of noradrenergic nerve endings.Supported by a grant from the Deutsche Forschungsgemeinschaft (Dr. 91)     

Intrinsic innervation of the ewe cervix and its variations during pregnancy

The course of the cholinergic and adrenergic nerve fibers in the cervix of the ewe was investigated in nonpregnant and pregnant animals using an acetylcholinesterase method and fluorescence histochemistry. Both technics in nonpregnant animals revealed a rich network of acetylcholinesterase and norepinephrine positive nerves around the blood vessels while the muscular innervation was moderately positive. Acetylcholinesterase fibers were also concentrated beneath the surface epithelium forming a plexus-like arrangement where isolated ganglion cells could be seen. At mid pregnancy cholinergic and adrenergic fibers decreased in density. The intensity of fluorescence was weaker and nerve fiber morphology was modified. We endeavoured to relate our findings to the problem of the neural control of contractions and the opening of the cervix of the ewe which is poorly supplied in nerve fibers, particularly at mid pregnancy.     

Intrinsic innervation of the ewe cervix and its variations during pregnancy

Summary The course of the cholinergic and adrenergic nerve fibers in the cervix of the ewe was investigated in nonpregnant and pregnant animals using an acetylcholinesterase method and fluorescence histochemistry. Both technics in nonpregnant animals revealed a rich network of acetylcholinesterase and norepinephrin positive nerves around the blood vessels while the muscular innervation was moderately positive. Acetylcholinesterase fibers were also concentrated beneath the surface epithelium forming a plexuslike arrangement where isolated ganglion cells could be seen. At mid pregnancy cholinergic and adrenergic fibers decreased in density. The intensity of fluorescence was weaker and nerve fiber morphology was modified. We endeavoured to relate our findings to the problem of the neural control of contractions and the opening of the cervix of the ewe which is poorly supplied in nerve fibers, particularly at mid pregnancy.     

Light and electron microscopic histochemical observations on cholinesterase-containing sympathetic nerve fibres in the pineal body of the rat

Synopsis Pineal glands of adult albino rats were examined histochemically using, first, formaldehyde-induced fluorescence to study monoamines and, second, copper thiocholine or copper ferrocyanide methods to study acetylcholinesterase and non-specific cholinesterase by light and electron microscopy. Cholinesterase was determined quantitatively by a constant pH titration assay.Fluorescent and acetylcholinesterase-positive nerve nets formed identical patterns. Nonspecific cholinesterase was observed only in nerve trunks outside the pineal. Bilateral removal of superior cervical ganglia resulted in complete disappearance of fluorescence and acetylcholinesterase from nerve fibres. Electron microscopically, acetylcholinesterase was found on sympathetic axons containing small granular vesicles. Quantitative cholinesterase determinations suggested that the pineal activity was mainly due to acetylcholinesterase. Comparison of the incubation times required for equal histochemical acetylcholinesterase reactions suggested that the activity of the sympathetic nerve fibres in the pineal is of the same order of magnitude as that in the nerve fibres of the iris.     

TRANSMITTER METABOLIZING ENZYMES AND FREE AMINO ACID LEVELS IN SENSORY AND MOTOR NERVES AND GANGLIA OF THE SHORE CRAB (CARCINUS MAENAS)

—The distribution of ChAT (choline acetyltransferase), GAD (glutamate decarboxylase) and acetylcholinesterase in some sensory and motor nerves of the shore crab, Carcinus maenas, has been investigated using micro-assay techniques. ChAT was concentrated in the afferent nerve fibres of the thoracic-coxal muscle receptor as well as in the coxo-basal chordotonal receptor nerve and other leg sensory fibres. GAD was found in leg motor nerves including the promotor and remotor muscle nerves, being undetectable in the sensory nerves. Acetylcholinesterase was found in similar levels in both sensory and motor nerves assayed. Amino acid analysis using a micro-dansylation technique showed that sensory nerves had low GABA levels, whereas the leg nerve including motor fibres had substantially higher GABA concentrations. GAD and GABA were also found in low amounts in the leg promoter mucle, which is consistent with GABA being a neuromuscular transmitter.     

Studies on the distribution of calcitonin gene-related peptide-like and substance P-like immunoreactivities in rat hind limb muscles

Summary The tibialis anterior, extensor digitorum longus and soleus muscles in the rat were examined with respect to the presence of calcitonin gene-related peptide-like as well as substance P-like immunoreactivity. In some of the motor endplates in these muscles, identified by staining for acetylcholinesterase activity, calcitonin gene-related peptide-like immunoreactivity was detected, but in others it was not. Calcitonin gene-related peptide-like immunoreactivity was found to coexist with substance-P-like immunoreactivity in nerve fibres located outside and inside the capsule of the muscle spindles, as well as in nerve fibres located in nerve fascicles. These fibres presumably represent sensory nerve fibres. Calcitonin gene-related peptide-like immunoreactivity, but not substance P-like immunoreactivity, was also detected, in cap-like structures located on the surface of the intrafusal muscle fibres in the polar regions of the spindles, structures which are likely to correspond to motor plate endings. The observations suggest that calcitonin gene-related peptide is heterogeneously present in the endplates of rat hind limb muscles, and gives for the first time immunohistochemical evidence for the presence of calcitonin gene-related peptide and substance P in the innervation of muscle spindles.     

In vivo regulation of acetylcholinesterase insertion at the neuromuscular junction

The efficiency of synaptic transmission between nerve and muscle depends on the number and density of acetylcholinesterase molecules (AChE) at the neuromuscular junction. However, little is known about the way this density is maintained and regulated in vivo. By using time lapse and quantitative fluorescence imaging assays in living mice, we demonstrated that insertion of new AChEs occurs within hours of saturating pre-existing AChEs with fasciculin2, a snake toxin that selectively labels AChE. In the absence of muscle postsynaptic activity or evoked nerve presynaptic neurotransmitter release, AChE insertion was decreased significantly, whereas direct stimulation of the muscle completely restored AChE insertion to control levels. This activity-dependent AChE insertion is mediated by intracellular calcium. In muscle stimulated in the presence of a Ca2+ channel blocker or calcium-permeable Ca2+ chelator, AChE insertion into synapses was significantly decreased, whereas ryanodine or ionophore A12387 treatment of blocked and unstimulated synapses significantly increased AChE insertion. These results demonstrated that synaptic activity is critical for AChE insertion and indicated that a rise in intracellular calcium either through voltage-gated calcium channels or from intracellular stores is critical for proper AChE insertion into the adult synapse.     

Cholinesterases and choline acetyltransferase in the longitudinal muscle of the guinea pig ileum

Summary In order to gain insight into the possible role of the ACh-system in the smooth muscle cell, the presence of choline acetyltransferase, acetylcholinesterase and butyrylcholinesterase was studied in the longitudinal muscle of the guinea-pig ileum after the mechanical removal of Auerbach's plexus. Such treatment completely removes all nerve elements as confirmed by histochemistry and electron-microscopic examination.It was found that in the longitudinal muscle devoid of all nervous elements a substantial percentage of the activity of all three enzymes still remained. Ultrastructural localization of acetylcholinesterase and butyrylcholinesterase was observed on the sarcolemma, sarcoplasmic reticulum, nuclear membrane and invaginations of the sarcolemma. The localization of cholinesterases coincides with sites which are presumably involved in calcium movements during contraction and relaxation. It is well known that the depolarized smooth muscle responds to exogenous ACh with a reversible, calcium dependent contraction and it was suggested that ACh may act by increasing the influx of calcium through the cell membrane or by liberating calcium from its bound form.The presence of choline acetyltransferase and cholinesterase activities in the muscle cell proper, as well as the localization of cholinesterases on structures connected with calcium movements, support the coexistence of an intrinsic cholinergic mechanism in the smooth muscle.Part of this work was presented at the 6^th International Meeting of the International Society for Neurochemistry, Copenhagen, 1977     

Acetylcholinesterase activity in nerve endings of tails of Rana japonica and R. catesbeiana during metamorphosis

Summary In anuran tadpole tails, the myelinated motor nerve fibers branch in the myoseptum to innervate both red and white muscle fibers at, or near, their ends. There are no significant ultrastructural differences between the nerve endings of the two types of muscle fibers.Intense acetylcholinesterase reaction product was observed in synaptic clefts and junctional folds, as well as in transverse tubules. As metamorphosis proceeded, the junctional folds of the nerve endings disappeared, however, acetylcholinesterase reaction product was still observed in the synaptic clefts. As muscle fibers began to degenerate, nerve endings began to separate from them. However, after nerve endings were completely separated from the surfaces, degenerated muscle fibers, synaptic and cored vesicles were still well preserved although no acetylcholinesterase reaction product was found. It seems clear that the mechanism of the muscle degeneration in the tadpole tail during metamorphosis is not the result of the degeneration of its nerve endings.     

Monoclonal Antibody Tor 23 Recognizes a Determinant of a Presynaptic Acetylcholinesterase

A significant proportion of the acetylcholinesterase that is present in the electric organ of Torpedo californica exists as a presynaptic membrane molecule. The monoclonal antibody Tor 23 binds the Torpedo presynaptic nerve membrane where it recognizes a polypeptide of 68,000 daltons. Our present studies indicate that Tor 23 identifies acetylcholinesterase. From the homogenates of Torpedo nerve terminals, Tor 23 immunoprecipitates measurable esterase activity. Esterase precipitation was not observed with no Tor 23 added; nor was it observed with any other test antibodies, including other Tor antibodies, in particular, Tor 70, which binds, as does Tor 23, to the presynaptic nerve membrane. The esterase activity was specific for acetylcholinesterase. Our studies indicate the molecule defined by Tor 23 has the solubility properties described for that of presynaptic acetylcholinesterase: it is soluble in detergent-treated electroplax homogenates and insoluble in high-salt extractions. In sections of Torpedo back muscle, both nerve and endplate acetylcholinesterase can be detected histochemically. Tor 23 localizes to the nerve and is not clustered at the endplate. The utility of the antibody Tor 23 thus includes biochemical and histological analyses of the multiple forms of acetylcholinesterase.     

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.     

Cholinesterases and choline acetyltransferase in the longitudinal muscle of the guinea pig ileum

In order to gain insight into the possible role of the ACh-system in the smooth muscle cell, the presence of choline acetyltransferase, acetylcholinesterase and butyrylcholinesterase was studied in the longitudinal muscle of the guinea-pig ileum after the mechanical removal of Auerbach's plexus. Such treatment completely removes all nerve elements as confirmed by histochemistry and electron-microscopic examination. It was found that in the longitudinal muscle devoid of all nervous elements a substantial percentage of the activity of all three enzymes still remained. Ultrastructural localization of acetylcholinesterase and butyrylcholinesterase was observed on the sarcolemma, sarcoplastic reticulum, nuclear membrane and invaginations of the sarcolemma. The localization of cholinesterases coincides with sites which are presumably involved in calcium movements during contraction and relaxation. It is well known that the depolarized smooth muscle responds to exogenous ACh with a reversible, calcium dependent contraction and it was suggested that ACh may act by increasing the influx of calcium through the cell membrane or by liberating calcium from its bound form. The presence of choline acetyltransferase and cholinesterase activities in the muscle cell proper, as well as the localization of cholinesterases on structures connected with calcium movements, support the coexistence of an intrinsic cholinergic mechanism in the smooth muscle.     

The ultrastructure and distribution of 5-HT-containing neurons in the intestine of a teleost fish,Aldrichetta forsteri

Summary The intestine of Aldrichetta forsteri was examined ultrastructurally for evidence of 5-HT-containing intrinsic neurons. A population of nerve cell bodies and processes characterized by the presence of amine-containing vesicles was found after the destruction of adrenergic processes with 6-hydroxydopamine. The distribution of the neurons matched that of the 5-HT-containing neurons previously seen with formaldehyde-induced fluorescence and 5-HT immunohistochemistry in A. forsteri and other teleosts. The axons made close contacts with the circular smooth muscle layer and the muscularis mucosae. The axons also lay exposed to the epithelial cells of the mucosa but did not make contact with neurons or with the longitudinal smooth muscle layer.     

Biochemical and Histochemical Evidence of 16S Acetylcholinesterase in Salivary Glands

Abstract: Three forms of acetylcholinesterase (AChE) have been reported: the 4S, 10S, and 16S forms. It was suggested previously that 16S AChE is characteristic of the end-plate region; subsequently its presence has also been demonstrated in sciatic nerve, vagus nerve, several nerve trunks, cardiac atria, and distal ileum of rat. The purpose of the present study was to investigate further the occurrence of 16S AChE. We found that not only is it present in motor axons but that it may also occur in the secretory postganglionic parasympathetic fibers in the synapse-free parotid and submandibular glands.     

Acceleratory nervous regulation of juvenile myogenic hearts in the isopod crustacean Ligia exotica

We examined regulation of the myogenic heart by two identified cardioacceleratory neurons (CA1, CA2) in early juveniles of the isopod Ligia exotica. Repetitive stimulation of either the CA1 or CA2 axon increased the frequency and plateau amplitude of the action potential and decreased the maximum hyperpolarization of the cardiac muscle. These effects were larger with increasing stimulus frequency. The rate of increase in the frequency caused by CA1 stimulation was significantly larger than that by CA2. No impulse activity of the cardiac ganglion was induced by acceleratory nerve stimulation. The frequency of the muscle activity was decreased by injection of a hyperpolarizing current into the muscle during stimulation of the acceleratory nerve. In a quiescent heart, acceleratory nerve stimulation caused an overall depolarization in the muscle membrane and the amplitude of the depolarization induced by CA1 stimulation was significantly larger than that by CA2. These results suggest that CA1 and CA2 neurons regulate the myogenic heart affecting directly the cardiac muscle; the CA1 neuron produces more potent effects than does the CA2 neuron.     

Scanning electron microscopy of nerve-muscle contacts in embryonic cell culture.

Scanning electron microscopy (SEM) of cell cultures of dissociated nerve and muscle from chick embryos has shown that developing muscle fibers can be contacted at many sites by one or more than one neuron, and that a single nerve can send branches to several myofibers. At these contact regions of nerve with muscle, the neurons send out terminal or lateral sprouts with fine tips which initially lack terminal swellings, but later acquire small “bouton”-like structures in contact with the sarcolemma, which resemble embryonic synapses. At these points, the sarcolemma does not appear to differ in ultrastructure from other surface regions of the myofiber. Transmission electron microscopy (TEM) has revealed the presence of both electron lucent and dense-cored vesicles at some nerve terminals. However, fluorescence histochemistry (Falck-Hillarp technique) failed to detect the presence of catecholamines in these cultures. The SEM pictures at substantially higher resolutions than the light microscope, and the enhanced three dimensional perspective of this technique, provide additional information about the developmental morphology of the nerve-muscle cell culture system. The results are correlated with previous findings by light microscopy, TEM and electrophysiology, and discussed in relationship to proposed innervation processes of skeletal muscle fibers in vivo.     

Neurotrophic control of 16S acetylcholinesterase from mammalian skeletal muscle in organ culture

The effects of rat obturator nerve extracts on total and 16S acetylcholinesterase (AChE) activity were studied in endplate regions of denervated anterior gracilis muscles maintained in organ culture for 48 hr. The decrease of total AChE activity in cultured muscles was similar to that observed in denervated muscles in vivo. This decrease in activity was partly prevented by addition of either 100 or 200 μl nerve extract (2.7 mg/ml protein) to the nutrient medium. Nerve extract treatment also decreased the release of AChE activity from the muscle into the bathing medium. Conversely, rat serum (20 μl; 90 mg/ml protein) had no effect on total AChE activity in muscle endplates, nor on release of the enzyme by the muscle. The 16S form of AChE was confined to motor endplate muscle regions and its activity was drastically decreased by denervation in both organ culture and in vivo preparations in a comparable manner. Nerve-extract supplemented cultures contained a significantly (p ? 0.001) larger amount of endplate 16S AChE activity (140–145%) than the corresponding controls (100-). Our results suggest that some nerve soluble substance, other than serum contaminants or 16S AChE itself, affects the maintenance of 16S AChE at the neuromuscular junction.