Arcachon and cholinergic transmission.

The cholinergic nature of transmission at the electromotor synapse of Torpedo marmorata was established at Arcachon in 1939 by Feldberg, Fessard and Nachmansohn (J. Physiol. (Lond.) 97 (1939/1940) 3P-4P) soon after transmission at the neuromuscular junction had been shown to be cholinergic. In 1964, after a quarter of a century of neglect, workers in Cambridge, then in Paris, Göttingen and elsewhere, began to use this system, 500-1000 times richer in cholinergic synapses than muscle, for intensive studies of cholinergic transmission at the cellular and molecular level.

Interferon-gamma alters nerve-induced redistribution of acetylcholine receptors in cultured rat skeletal muscle cells.

The influence of recombinant interferon-gamma (rIFN-gamma) on the development of acetylcholine receptor (AChR) aggregates in cocultures of rat embryonic muscle cells and spinal cord neurons was studied by counting the number of AChR aggregates in relation to cholinergic nerve fibers coming to the muscle fibers. rIFN-gamma caused no decrease in the number of cholinergic nerve fibers, but inhibited the increase in the number of AChR aggregates that occurs early during cocultivation and is an early sign in the development of neuromuscular junctions. rIFN-gamma stimulated release of nitric oxide, but no effects on aggregation of AChRs occurred after exposure to a nitric oxide synthase inhibitor, L-NG-monomethylarginine, or by the addition of nitroprusside, a generator of nitric oxide. No effect was seen on the number of AChR aggregates when the cultures were exposed to rIFN-gamma at later time points of cocultivation, when the increase in number of AChRs had already occurred. These studies indicate that the key immunoregulatory cytokine IFN-gamma can cause alterations in the early process of synapse formation and that these effects are independent of the nitric oxide release caused by the cytokine.

Cholinergic-specific glycoconjugates.

Cholinergic nerve terminals utilize glycoconjugates in several ways, as surface markers and as structural components of the synaptic vesicles present within them. The surface markers have been discovered immunochemically: antibodies raised against them are able specifically to sensitize the cholinergic subpopulation of mammalian brain synaptosomes to complement-mediated lysis. One such group of antigens (Chol-1) have been identified as a novel series of minor gangliosides having in common a sialylated N-acetylgalactosamine residue. These gangliosides may constitute the major gangliosides at cholinergic terminals. A second surface antigen (Chol-2) is thought to be a protein with an epitope in common with a Torpedo electric organ ganglioside. Cholinergic synaptic vesicles are rich in a proteoglycan which appears to assist in the sequestration of acetylcholine within the vesicle and to stabilize the vesicle membrane during cycles of exocytosis and recovery. It may be the cholinergic equivalent of the chromogranins.

Thirty years of synaptosome research.

Detached synapses (synaptosomes), first isolated by the author in 1958 and identified as such in 1960, are sealed presynaptic nerve terminals often with a portion of the target cell--sometimes amounting to a complete dendritic spine--adhering to their external surface. They can be prepared in high yield from brain tissue and also in decreasing yield from spinal cord, retina, sympathetic ganglia, myenteric plexus and electric organs. They are sealed structures which, under metabolizing conditions, respire, take up oxygen and glucose, extrude Na+, accumulate K+, maintain a normal membrane potential and, on depolarization, release transmitter in a Ca(2+)-dependent manner. They thus provide an excellent preparation with which to investigate synaptic function without the complications encountered with synapses in situ. They also serve as the parent fraction for preparations of synaptic vesicles and other synaptic components.

A novel cholinergic-specific antigen (Chol-2) in mammalian brain.

Three new antisera have been raised in sheep against cholinergic electromotor presynaptic plasma membranes prepared from the electric organs of the electric ray, Torpedo marmorata. They all recognized one or more cholinergic-specific antigens in the mammalian nervous system by the following criteria: they sensitized the cholinergic subpopulation of rat-brain synaptosomes--and only this subpopulation--to lysis by the complement system and, in an immunocytochemical study, selectively stained choline acetyltransferase-positive cholinergic neurons in the rat spinal cord. However, two of the three antisera failed to recognize Chol-1 alpha and -beta, two closely related minor gangliosides already identified as the cholinergic-specific antigens recognized by previous anti-Torpedo presynaptic plasma membrane antisera or indeed any other ganglioside and the third recognized only Chol-1 alpha. A further investigation of the antigen(s) recognized by the most antigenic of the new antisera indicated that it is proteinaceous in nature, but has epitopes in common with electric organ gangliosides.

Vesamicol blocks the recovery, by recycling cholinergic electromotor synaptic vesicles, of the biophysical characteristics of the reserve population.

The effect of vesamicol on the ability of recycling cholinergic synaptic vesicles to recover, during a period of post-stimulation rest, the biophysical properties of the reserve pool was studied in prestimulated perfused blocks of the electric organ of the electric ray, Torpedo marmorata, a tissue rich in cholinergic synapses. The effect of the drug was analysed by high-resolution centrifugal density-gradient fractionation in a zonal rotor of the extracted vesicles. The two vesicle fractions were identified by their ATP and acetylcholine content and the recycled vesicles by their acquisition of [3H]acetylcholine derived from [3H]acetate in the perfusate. Vesamicol (10 microM) blocked the uptake of tritiated acetylcholine by recycled vesicles and also prevented them from rejoining the reserve pool. This is consistent with a previously formulated model of the recovery process, whereby the increase in the acetylcholine and ATP content of the recycled vesicles which takes place during a post-stimulus period of rest increases their osmotic load and thus their content of free water. Vesamicol, by blocking acetylcholine uptake, also blocks rehydration of the recycled vesicles and thus the accompanying decrease in their density to the value characteristic of fully charged vesicles.

The cholinergic marker Chol-1 is selectively localized in nerve terminals.

The subcellular distribution of the cholinergic-specific gangliosidic Chol-1 antigens in rat brain has been compared with that of total ganglioside as a marker for neural plasma membranes, particle-bound choline acetyltransferase (ChAT) as a synaptosomal marker, and supernatant ChAT as a marker for neuronal perikaryal cytoplasm. Over 50% of brain Chol-1 was recovered in a synaptosome fraction prepared by a standard density-gradient method. The specific concentration of Chol-1 relative to protein or major gangliosides was also highest in the synaptosome fraction and low in a supernatant fraction which contained most of the neuronal plasma membrane fragments. ChAT was likewise mainly recovered in the synaptosome fraction but its relative concentration there was significantly less than that of Chol-1 (p < 0.01).

Distribution of the cholinergic-specific antigen Chol-1 in mouse spinal cord neurons developing in culture.

The distribution of the cholinergic-specific ganglioside antigen Chol-1 has been studied by indirect fluorescence immunohistochemistry in mouse spinal cord neurons developing in vitro. Chol-1 is first detected after 9 days of culture where it can be seen in the cell bodies of neurons and in the proximal part of their processes. In cultures of the same age, staining with antisynaptophysin antibody revealed that synapse formation has already taken place and the level of choline acetyltransferase activity was found to have reached a plateau. After 12 days in culture Chol-1 can still be seen in the cytoplasm of certain neurons; however, the anti-Chol-1 staining is now more intense in the region of nerve terminals. Anti-neurofilament staining of cultures at this stage reveals that the neurons are highly differentiated and possess an extensive network of processes. These results show that Chol-1 is expressed late in the development of cholinergic neurons after they have formed synapses; it then appears to be transported to the nerve terminal where it accumulates.

Structural characterization of a novel cholinergic neuron-specific ganglioside in bovine brain.

A new ganglioside antigen, termed Chol-1 alpha-b, recognized by cholinergic neuron-specific antibody, Chol-1 alpha, was isolated from bovine brain ganglioside mixture using Q-Sepharose. The yield was approximately 1.3 mg from 5 g of the total ganglioside. The chemical structure was characterized as a novel ganglioside by means of gas-liquid chromatography, a permethylation study, mild acid hydrolysis, thin layer chromatography-enzyme immunostaining, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. The ganglioside has the following unique structure. [formula: see text] When examined by thin layer chromatography immunostaining and enzyme-linked immunosorbant assays, this ganglioside has the most intense immunoreactivity with Chol-1 alpha antibody among bovine brain gangliosides. As combined with our previous results (Hirabayashi, Y., Hyogo, A., Nakao, T., Tsuchiya, K., Suzuki, Y., Matsumoto, M., Kon, K., and Ando, S. (1990) J. Biol. Chem. 265, 8144-8151; Ando, S., Hirabayashi, Y., Kon, K., Inagaki, F., Tate, S., and Whittaker, X. (1992) J. Biochem. (Tokyo), 111, 287-290), the present study indicates the occurrence of a new series of gangliosides containing N-acetylneuraminic acid residue attaching to N-acetylgalactosamine as cholinergic specific antigens.

Chol-1 is a cholinergic marker in the human central nervous system.

The cholinergic-specific gangliosides Chol-1 alpha and beta were detected in human brain and spinal cord by immune staining of thin-layer chromatography (TLC) plates on which ganglioside extracts had been separated. The colocalization of choline acetyltransferase (ChAT) and the Chol-1 antigens in ventral horn motoneurons was demonstrated immunocytochemically. In analytical studies, Chol-1 was found to be more concentrated in dorsal cord than ventral but the reverse was true of ChAT. This difference was explained by differences in the subcellular location of the two markers. Within each region of the thoracic cord the levels of ChAT and Chol-1 in different cords showed covariance. The expected fall of ChAT and Chol-1 in amyotrophic lateral sclerosis (ALS) cords was not seen and possible reasons for this are discussed.

A trisialoganglioside containing a sialyl alpha 2-6 N-acetylgalactosamine residue is a cholinergic-specific antigen, Chol-1 alpha.

Cholinergic-specific antigens termed the Chol-1 family have been suggested to be of a ganglioside nature by Richardson et al. (J. Neurochem. 38, 1605-1614, 1982). Two molecular species of polysialogangliosides among bovine brain gangliosides were found to react with anti-Chol-1 alpha antiserum. One of them, Chol-1 alpha-a, was isolated and characterized as a trisialoganglioside containing the gangliotetraose backbone in which 1 mol of sialic acid was attached to each of the reducing end galactose, N-acetylgalactosamine and internal galactose residues, respectively. The chemical structure of Chol-1 alpha-a was determined for the first time, being as follows: IV3NeuAc III6NeuAc II3NeuAc-GgOse4 Cer.

Nerve growth factor enhances the expression of the cholinergic-specific ganglioside Chol-1 in cocultures of rat septum and hippocampus.

Cocultures of septal and hippocampal tissues taken from 6- to 7-day-old rats were maintained in culture for up to 30 days in the presence and absence of nerve growth factor (NGF), and their Chol-1 contents determined at varying time intervals by a modified enzyme-linked immunosorption assay (ELISA). The major brain gangliosides were determined densitometrically after spraying chromatograms with resorcinol reagent. There was little change in the contribution of the major gangliosides to the total ganglioside content of the explants either with time or the presence or absence of NGF, the only exception being an NGF-insensitive fall in the contribution of GM1 to about 60% of its initial value at 20 and 30 days. By contrast, the concentration of Chol-1 expressed either per unit weight of ganglioside sialic acid or protein increased considerably in culture and this increase was enhanced by NGF. The effect of NGF resembles that on other cholinergic markers, choline acetyltransferase and acetylcholinesterase, and may be attributed to an NGF-stimulated hippocampal ingrowth of cholinergic fibres and enhanced survival of cholinergic septal neurons. The Chol-1 concentration finally attained in the presence of NGF and the time course of its increase parallel those previously found in vivo and indicate the potential usefulness of septal-hippocampal cocultures for investigating the function of Chol-1.

Further studies on the gangliosidic nature of the cholinergic-specific antigen, Chol-1.

The antigen designated as Chol-1 beta, detected by an antiserum specific for cholinergic neurons, has been purified to homogeneity from ganglioside mixtures extracted from Torpedo electric organ and pig brain. The final products from the two sources behaved identically in a wide range of tests and gave coincident immunopositive and Ehrlich-positive spots after thin layer chromatography in seven different solvent systems; they were thus considered to be identical and to constitute a single, pure chemical species. Gas-chromatographic analysis revealed the presence of long-chain bases, glucose, galactose, N-acetylgalactosamine, and sialic acid in integral molar ratios of 1:1:2:1:3; the compound's reactivity to cholera toxin after Vibrio cholerae sialidase treatment on thin layer chromatography and the recovery of GM1 as sole product of exhaustive sialidase treatment identified it as a member of the gangliotetrahexosyl series. From the products of partial enzymatic desialylation and treatment with beta-galactosidase and a comparison of the compound's immunoreactivity to anti-Chol-1 antisera with that of other trisialogangliosides of defined molecular structure, we were able to assign a disialosyl residue alpha-Neu5Ac-(2----8)-alpha-Neu5Ac-(2----3)- to the inner galactose, and we suggest GalNAc as a possible site of linkage of the third sialic acid.

Cholinergic synaptic vesicles are metabolically and biophysically heterogeneous even in resting terminals.

The metabolic heterogeneity of synaptic vesicles in the cholinergic nerve terminals of the electromotor neurons of Torpedo marmorata has been studied in resting tissue by evaluating the molecular acetylcholine content (MAC) of synaptic vesicles after extraction from frozen and crushed tissue and high-resolution centrifugal density gradient separation in a zonal rotor. Although vesicular acetylcholine was distributed in the gradient as a single, more or less symmetrical peak, 3 subpopulations of synaptic vesicles could be identified: a small, relatively light subpopulation of low MAC on the ascending limb of the acetylcholine peak, designated V0, a main population of fully charged vesicles designated V1, and a small, denser subpopulation also of low MAC on the descending limb of the acetylcholine peak, designated V2. The mean proportions and MACs of the 3 pools were: V0, 13%, 58,000; V1, 53%, 246,000; V2, 34%, 79,000. When tritiated acetate was perfused through excised blocks of electric organ for 1-2 h before vesicle isolation, the specific radioactivity of the acetylcholine in the V0 and V2 pools was 10-30 times higher than in the V1 pool. This suggests that both the V0 and V2 pools are not generated by the isolation procedure but are present in the intact endings and are functionally active. On the basis of their density and uptake of newly synthesized acetylcholine, the V0 and V2 pools were identified with the previously described VP0 pool of axonal vesicles and the VP2 pool of recycling vesicles in stimulated nerve terminals respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of gastrin-releasing peptide immunoreactivity in distinct storage particles in guinea pig myenteric and Torpedo electromotor neurones.

Using high resolution centrifugal density-gradient separation of cytoplasmic extracts of guinea pig myenteric plexus and Torpedo electric tissue, we have succeeded in isolating fractions of storage particles rich in gastrin-releasing peptide (GRP). In extracts of myenteric plexus and gradients derived therefrom, the 10-amino acid GRP peptide (GRP-10) was the sole form present; this was bimodally distributed in the gradients, one peak copurifying with Golgi membranes and apparently consisting of immature storage particles, the other with other synaptophysin-rich neuropeptide-containing particles. In extracts of electric organ, a tissue rich in cholinergic electromotor nerve terminals, and gradients derived therefrom, GRP-like immunoreactivity behaved in gel permeation and reversed phase high performance liquid chromatography like the 27-amino acid peptide (GRP-27). About half of the immunoreactivity sedimented in the centrifugal gradient to a region rich in particles containing vasoactive intestinal polypeptide-like immunoreactivity; the remainder was recovered in a very dense region of the gradient containing larger membrane fragments, including synaptosomes. The electromotor nerves and cell bodies also contained GRP-27-like immunoreactivity in relatively high concentration as did the Torpedo gut. It is concluded that this GRP-like peptide is packaged in dense storage particles in the electromotor neurones.