E. Borroni

Putative cholinergic-specific gangliosides in guinea pig forebrain.

The nature of the cholinergic‐specific antigen Chol‐1 recognized by an antiserum raised against Torpedo cholinergic electromotor synaptosomal plasma membranes was investigated in guinea pig forebrain to establish whether it has a gangliosidic nature in guinea pig as in Torpedo. Gangliosides extracted from guinea pig forebrain and extensively purified to eliminate peptide contaminants were effective in inhibiting the selective lysis of the cholinergic subpopulation of cortical synaptosomes induced by the antiserum. Neuraminidase, protease, alkali, and heat treatment did not impair the inhibitory activity of gangliosides. Whereas the antiserum recognized many gangliosides from Torpedo electric organ, the immunostaining of guinea pig forebrain gangliosides separated on TLC showed only two immunopositive bands migrating close to GT1b and GQ. After affinity purification on Torpedo electric organ gangliosides the activity of the antiserum in inducing complement‐mediated lysis was increased and it still recognized the two ganglioside bands on TLC. These results strongly suggest the existence of two polysialogangliosides bearing antigenic determinants specific for the cholinergic neurons.

THE DEVELOPMENTAL EXPRESSION OF THE CHOLINERGIC-SPECIFIC ANTIGEN CHOL-1 IN THE CENTRAL AND PERIPHERAL NERVOUS-SYSTEM OF THE RAT

Antisera raised by the injection into sheep of presynaptic plasma membranes isolated from the purely cholinergic electromotor nerve terminals of Torpedo marmorata recognize a cholinergic-specific epitope, designated Chol-1 which has been shown to be gangliosidic in nature both in Torpedo (Richardson et al., 1982) and guinea-pig brain (Ferretti and Borroni, 1986). In rat brain the serum recognizes a group of antigenically-related minor gangliosides (Chol-1 alpha, beta and gamma) which migrate just below the standard gangliosides GQ, GT1B and between GD1b and GD1a, respectively. We have studied the developmental expression of these gangliosides in rat brain and hippocampus and in the neuromuscular junction of rat intercostal muscle in an attempt to correlate their expression with specific events in the development of the cholinergic neuron. The period in which Chol-1 is first detected suggests that it is expressed relatively late during the maturation process of the cholinergic synapse. This is supported by the finding: (a) that it is not detected in the growth cones (immature nerve terminals) in 5-day-old rat brain but is in the whole brain implying that only the more mature nerve terminals present at this stage express Chol-1; and (b) that Chol-1 is first expressed in the neuromuscular junction at a time in which functional synapses are already present. These results argue against a role for the Chol-1 antigens as recognition molecules in the formation of cholinergic synapses. The expression of Chol-1 in both the hippocampus and the neuromuscular junction correlates well with the establishment of the adult pattern of innervation; thus the Chol-1 antigens may be seen as markers for mature cholinergic terminals.

Cholinergic Surface Antigen Chol-1 Is Present in a Subclass of VIP-Containing Rat Cortical Synaptosomes

Abstract: The colocalization of vasoactive intestinal polypeptide (VIP) with the cholinergic specific surface antigen Chol‐1 was investigated in synaptosomes derived from the rat cerebral cortex. Immunoaffinity purification of cortical synaptosomes using antisera to Chol‐1 resulted in the copurification of VIP and cholinergic nerve terminals. VIP was purified with a yield of 75% of that of choline acetyltransferase (ChAT). These results suggest that approximately 53% of the cortical cholinergic terminals contain VIP, whereas 75% of the cortical VIP content is present in these cholinergic terminals. Both hypotonic lysis and depolarization of the nerve terminals resulted in the differential release of VIP and acetylcholine (ACh), indicating the different compartmentalization in the same nerve terminal. Complement‐mediated lysis of cholinergic nerve terminals, using antisera to Chol‐1, resulted in the release of 64% of the ChAT, 71% of ACh, and 27% of the VIP. The application of our method enables quantifying and mapping, with a fast, efficient, and specific technique, the coexisting peptides in cholinergic neurons of distinct brain areas.

Effect of denervation on a cholinergic-specific ganglioside antigen (Chol-1) present in Torpedo electromotor presynaptic plasma membranes.

Abstract: The presence of Chol‐1, an antigen identified in the plasma membrane of cholinergic electromotor nerve terminals of Torpedo marmorata, was investigated in Torpedo electric organ after 3, 6, and 9 weeks’denervation. Denervation was monitored by the cessation of stimulus‐evoked discharge potentials, by the reduction in nerve terminals seen morphologically, and by the decrease in ACh and ChAT contents. The content of ganglioside‐bound sialic acid did not show any appreciable change with time. Some modification of ganglioside pattern on TLC was observed after 9 weeks’denervation. The presence of Chol‐1 after denervation was assayed by its activity in inhibiting the selective complement‐induced lysis of the cholinergic subpopulation of guinea pig cortical synaptosome which is mediated by the anti‐Chol‐1 antiserum. Denervation did not affect Chol‐1 immunoreactivity although it did alter the distribution of the immunoreactivity among gangliosides. The possible significance of the results is discussed.

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 compounds 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 compounds 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.

An immunohistochemical study of synaptogenesis in the electric organ of Torpedo marmorata by use of antisera to vesicular and presynaptic plasma membrane components.

SummarySynaptogenesis has been studied in the electric organ of embryonic Torpedo marmorata by use of two antisera directed against components of synaptic vesicles (anti-SV) and presynaptic plasma membranes (ap-anti-TSM), respectively. The anti-SV serum was previously shown to recognize a proteoglycan specific for synaptic vesicles. The ap-anti-TSM serum was raised to plasma membranes of synaptosomes derived from the electromotor nerve terminals and affinity-purified on electric-organ gangliosides. The vesicular antigen was first detectable at the 81-mm stage of development, which is 1–2 weeks earlier than the formation of morphologically mature presynaptic terminals, but is coincident with a rise in choline acetyltransferase levels and the ability of the electric organ to generate discharges. The gangliosidic antigen recognized by the ap-anti-TSM was first detectable on the ventral electrocyte surface at the 93-mm stage of development. This indicates that specific carbohydrate epitopes, not present on the growth cones, are expressed during maturation of the nerve terminal. The nerve terminal components recognized by these sera arose pari passu with neurite coverage of the ventral surface of the electrocyte, reaching a maximum in the adult. In contrast, postsynaptic aggregates of acetylcholine receptor, rendered visible with rhodamine-labeled α-bungarotoxin, arose previous to the presynaptic antigens, reaching a maximum surface density at 110 mm and then declining in the adult.

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.

Role of Creatine Phosphate in the Discharge of the Electric Organ of Torpedo marmorata

Abstract: The role of creatine phosphate and adenosine triphosphate, as high energy phosphate sources, has been investigated during the discharge and recovery of the electric organ of Torpedo. ATP serves as the immediate source of energy for the biochemical process supporting the electrical activity of the electric organ. Under repetitive stimulation, when the energy demands exceed production, ATP levels are maintained constant at the expense of creatine phosphate. Only when the reservoir of creatine phosphate is depleted do the levels of ATP decrease, and this point corresponds to the state of maximal fatigue of the electric organ. Recovery studies show that the electric organ rapidly recovers the capacity to respond to single pulse stimuli. This recovery is statistically related to the recovery of the levels of ATP and acetylcholine. However, in this phase, the fatiguability of the electric organ is very high since its energy reservoir is still depleted. The complete recovery of the electric organ requires several hours and is closely related to the restoration of the levels of creatine phosphate.

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.

Characterization of a monoclonal antibody directed against a sulphoglycolipid that is evolutionarily conserved and developmentally regulated in rat brain.

SummaryMonoclonal antibodies (MABs) have been raised against acidic glycolipids extracted from the electric organ of Torpedo marmorata. One of these, designated L9, appears to recognize acidic glycolipids in adult T. marmorata electric organ, electromotor nerves and brain, adult rat sciatic nerve, and in embryonic and neonatal rat brain, starting at embryonic day (ED) 15 and disappearing by the 20th day of post-natal life. The epitope is present in growth cones isolated from 4-day-old rats; its proportion relative to total gangliosides is, however, no higher than that found in whole neonatal brain membranes. Desialidation of the acidic glycolipid fraction modifies neither the immunoreactivity nor the RF value following thin-layer chromatography (TLC) of the antigen; it is concluded that the antigen is not a ganglioside. The MAB, HNK-1, recognizes the L9 antigen. Both HNK-1 and L9 recognize a sulphoglycolipid of the same RF in TLC. The function of the L9 antigen is not known but its evolutionary conservation, presence in growth cones and its developmental regulation in the mammalian central nervous system indicate that it plays an important role in nervous system maturation.