The localization and rate of disappearance of a synaptic vesicle antigen following denervation

Summary A proteoglycan-specific antiserum has been used to monitor the effects of denervation in the electric organ of Torpedo marmorata. The antiserum was produced by injecting a highly purified synaptic vesicle fraction prepared from the electric organs of Torpedo marmorata. Following absorption the serum appears to be specific towards synaptic vesicles. The ultrastructural localization of the antigen determined by immuno-electron microscopy confirmed the specificity of the antiserum and showed that it did not crossreact with the proteoglycans of the basal lamina. The rate of disappearance of the vesicle proteoglycans following denervation was evaluated by means of the antiserum and was compared to the rate of disappearance of other vesicular and nerve terminal-associated markers. The results suggest that degeneration affects the vesicular constituents at varying rates resulting in a progressive disappearance of the entire functional capacity of the synaptic vesicles.     

Synaptic vesicles in electromotoneurones. II. Heterogeneity of populations is expressed in uptake properties; exocytosis and insertion of a core proteoglycan into the extracellular matrix.

The three populations of synaptic vesicles in electromotor nerve terminals were analysed quantitatively. Empty vesicles (VP0), fully charged vesicles (VP1) and charged but smaller VP2-type vesicles are present in approximately equal amounts in the nerve terminal. The populations show differences in the kinetics of in vitro uptake of acetylcholine, ATP and Ca2+. VP0 and VP2 accumulate acetylcholine and ATP but no Ca2+, whereas VP1 shows negligible acetylcholine and ATP but high Ca2+ uptake. Thus the expression of uptake properties of this secretory organelle depend on the stage it has reached in its life cycle and might constitute a signal for processing. VP2 was found to contain much less core proteoglycan than VP0 and VP1 indicating that part of it has been lost by exocytosis. In synaptic extracellular matrix containing fractions an antigen is detectable that cross-reacts with an antiserum against the vesicle proteoglycan. This material elutes upon gel filtration in a position similar to a smaller form of proteoglycan found in vesicles. We conclude that the electromotor nerve terminal releases a proteoglycan by the regulated secretory pathway that is deposited in the extracellular matrix. It might have a function in keeping pre- and postsynaptic structures in alignment constituting a transsynaptic signal. Based on the findings described, a model of the vesicles' life-cycle is discussed, whereby the VP2 population is the major source of quantal release of acetylcholine.     

A highly antigenic proteoglycan-like component of cholinergic synaptic vesicles

A monoclonal antibody, tor70, recognizes an antigenic determinant on the inside surface of synaptic vesicles, purified from the electric organ of Narcine brasiliensis. The antigenic determinant appears to be unique to vesicles since it co-purifies with vesicle content and is blocked by an antiserum specific for synaptic vesicle antigens. Immunoblotting of vesicle proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the antigen has a low heterogeneous electrophoretic mobility and corresponds to a major protein component of pure synaptic vesicles. Synaptic vesicles contain a proteoglycan-like material since proteolytic digestion yields a ruthenium red-binding material that migrates during electrophoresis with a mammalian heparin standard. The only major vesicle component with which the proteoglycan-like material co-elutes during chromatography on Sepharose 6B is the material recognized by tor70. The antigen adsorbs specifically to beads coated with the lectin wheat germ agglutinin. Isolation of the tor70 antigen by velocity sedimentation in sodium dodecyl sulfate-sucrose gradients shows it to contain glucosamine (0.75 nmol/microgram of protein) and uronic acid but no galactosamine. Earlier work has shown that specific antiserum to pure synaptic vesicles could be used to identify nerve terminals, quantitate vesicle components, purify membranes, and monitor exocytosis. We now know that one of the components recognized by the antiserum is a molecule with properties of a proteoglycan, attached to the inside surface of vesicle membranes.     

Membrane proteins of synaptic vesicles and cytoskeletal specializations at the node of Ranvier in electric ray and rat

Summary Binding sites for antibodies against membrane proteins of synaptic vesicles have been shown to be enhanced at nodes of Ranvier in electromotor axons of the electric ray Torpedo marmorata and sciatic nerve axons of the rat, using indirect immunofluorescence and monoclonal antibodies against the synaptic vesicle transmembrane proteins SV2 and synaptophysin (rat) or SV2 (Torpedo). In the electric lobe of Torpedo, vesicle-membrane constituents occurred at higher density in the proximal axon segments covered by oligodendroglia cells than in the distal axon segments where myelin is formed by Schwann cells. Antibody binding sites were enhanced at nodes forming the borderline of the central and peripheral nervous systems. Filamentous actin was present in the Schwann-cell processes covering both the nodal and the paranodal axon segments as suggested by the pattern of phalloidin labelling. Furthermore, in rat sciatic nerve, Schmidt-Lanterman incisures were intensely labelled by phalloidin. A similar nodal distribution was found for binding sites of antibodies against actin and myosin. Binding of antibodies to tubulin was enhanced at nodes in Torpedo electromotor axons. The apparent nodal accumulation of constituents of synaptic vesicle membranes and the presence of filamentous actin and of myosin are discussed in relation to the substantial constriction of the axoplasm at nodes of Ranvier.     

Characterization of a membrane protein from cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata

Rabbits were immunized with cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata. The resultant antiserum had one major antibody activity against an antigen called the Torpedo vesicle antigen. This antigen could not be demonstrated in muscle, liver or blood and is therefore, suggested to be nervous-tissue specific. The vesicle antigen was quantified in various parts of the nervous system and in subcellular fractions of the electric organ of Torpedo marmorata and was found to be highly enriched in synaptic vesicle membranes. The antigen bound to concanavalin A, thereby demonstrating the presence of a carbohydrate moiety. By means of charge-shift electrophoresis, amphiphilicity was demonstrated, indicating that the Torpedo vesicle antigen is an intrinsic membrane protein. The antigen was immunochemically unrelated to other brain specific proteins such as 14-3-2, S-100, the glial fibrillary acidic protein and synaptin. Furthermore, it was unrelated to two other membrane proteins, the nicotinic acetylcholine receptor and acetylcholinesterase, present in Torpedo electric organ. The antiserum against Torpedo synaptic vesicles did not react with preparations of rat brain synaptic vesicles or ox adrenal medullary chromaffin granules.     

VAT-1: an abundant membrane protein from Torpedo cholinergic synaptic vesicles

Expression screening was used to isolate cDNA clones encoding a synaptic vesicle membrane protein, VAT-1, which is specifically expressed in the electric lobe of marine rays. The predicted protein has a molecular weight of 41,572 daltons and contains several hydrophobic regions. An antibody raised against a fusion protein synthesized in E. coli recognizes an abundant 42 kd protein that copurifies largely with synaptic vesicles. Trypsin digestion of intact and lysed vesicles as well as membrane extractions suggests that VAT-1 is an integral membrane protein. The VAT-1 RNA is localized to the electromotor nucleus, and the fusion protein antibody stains the electric organ, demonstrating that the protein is transported to nerve terminals. These studies define a novel synaptic vesicle protein that is likely to play a central role in the functions mediated by specific classes of synaptic vesicles.     

Presynaptic plasma membranes and synaptic vesicles of cholinergic nerve endings demonstrated by means of specific antisera

Summary Antisera were raised to cholinergic presynaptic plasma membranes and synaptic vesicles isolated from the electric organ of Torpedo marmorata and tested by immunochemical and immunohistochemical methods. The antisera responded to many antigens not specific to nerve endings, but it was possible to eliminate these antibodies by means of simple absorption procedures with fractions containing the unwanted antigens. After absorption, staining of thin sections of electric organ by immunofluorescence was limited to the region of nerve endings in the tissue.The remaining antibodies responded in the case of the plasma membrane antisera predominantly to a 33,000 molecular-weight polypeptide and a chloroform/methanol-soluble antigen. In cross reactivity studies it was found that this antiserum not only stains cholinergic nerve endings in Torpedo but also those in mammalian tissue. The antigen responsible for the cross reactivity is restricted to the chloroform/methanol-soluble material.The vesicle antiserum labels cholinergic nerve endings in mammalian tissue as well; the relevant antigen in this case is different from the one described above and is likely to be a glycosaminoglycan. The antisera provide valuable markers for cholinergic nerve terminals. In addition, the vesicle antiserum may now be used to study axonal transport and the life cycle of this organelle in the cholinergic neurone.Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - EGTA ethylenebis (oxoethylenenitrilo) tetra-acetic acid - MW apparent molecular weight Enzymes. Na⁺, K⁺-activated ATPase (EC 3.6.1.3); acetylcholine esterase (EC 3.1.1.7); choline acetyl-transferase (EC 2.3.1.6)     

KCl stimulation and ultrastructural responses of tannic acid-stained cholinergic synaptic terminals

The quantal-vesicular hypothesis equates miniature end-plate potentials (MEPPs) with fusions of synaptic vesicles. MEPP production thus predicts vesicle losses, increases in vesicle fusions and increases in terminal plasma membrane. MEPP production and these ultrastructural parameters have been evaluated in the cholinergic presynaptic terminals of skate electric organ following tannic acid saline incubation, known to promote capture and selective staining of dense-core granule fusions, and KCl stimulation, known to elevate MEPP production dramatically in these cholinergic terminals. After pretreatment in tannic acid-elasmobranch saline, KCl stimulation produced MEPPs at 40/s/microm(2)of terminal surface for several minutes with gradual reduction to spontaneous levels by 25-30 min. No loss of vesicles, no vesicle fusions, no expansions of plasma membrane and no tannic acid enhanced staining of vesicles or vacuoles accompanied the generation of 800 MEPPs/microm(3)of terminals having densities of 567 vesicles/microm(3). No ultrastructural footprints were found to support the notion that unnaturally high rates of vesicular exocytosis had occurred.     

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

Summary Synaptogenesis 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 -bungarotoxin, arose previous to the presynaptic antigens, reaching a maximum surface density at 110 mm and then declining in the adult.     

An antiserum specific for cholinergic synaptic vesicles from electric organ

Rabbit antisera to highly purified synaptic vesicles from the electric organ of Narcine brasiliensis, an electric ray, reveal a unique population of synaptic vesicle antigens in addition to a population shared with other electric organ membranes. Synaptic vesicle antigens were detected by binding successively rabbit antivesicle serum and radioactive goat anti-rabbit serum. To remove antibodies directed against antigens common to synaptic vesicles and other electric organ fractions, the antivesicle serum was extensively preadsorbed against an electric organ membrane fraction that was essentially free of synaptic vesicles. The adsorbed serum retained 40% of its ability to bind to synaptic vesicles, suggesting that about half of the antigenic determinants are unique. Vesicle antigens were quantified with a radioimmunoassay (RIA) that utilized precipitation of antibody-antigen complexes with Staphylococcus aureus cells. By this assay, the vesicles, detected by their acetylcholine (ACh) content and the antigens detected by the RIA, have the same buoyant density after isopycnic centrifugation of crude membrane fractions on sucrose and glycerol density gradients. The ratio of ACh to antigenicity was constant across the vesicle peaks and was close to that observed for vesicles purified to homogeneity. Even though the vesicles make up only approximately 0.5% of the material in the original homogenate, the ratio of acetylcholine to vesicle antigenicity could still be measured and also was indistinguishable from that of pure vesicles. We conclude that synaptic vesicles contain unique antigenic determinants not present to any measurable extent in other fractions of the electric organ. Consequently, it is possible to raise a synaptic vesicle- specific antiserum that allows vesicles to be detected and quantified. These findings are consistent with earlier immunohistochemical observations of specific antibody binding to motor nerve terminals.     

EFFECT OF ELECTRICAL STIMULATION ON THE YIELD AND COMPOSITION OF SYNAPTIC VESICLES FROM THE CHOLINERGIC SYNAPSES OF THE ELECTRIC ORGAN OF TORPEDO: A COMBINED BIOCHEMICAL, ELECTROPHYSIOLOGICAL AND MORPHOLOGICAL STUDY

Abstract— The effect of stimulating the electric organ of Torpedo marmorata , anaesthetized with 0.01% Tricaine methane sulphonate, by means of electrical stimulation (5/s) administered via an electrode placed on the electric lobe has been studied electrophysiologically, biochemically and morphologically. The response of the organ declined to about 50 per cent of its initial value after about 500 stimuli, by a further 10 per cent after another 500 stimuli and then to about 12 per cent of the initial value after a further 1000 stimuli. Thereafter the response fell off progressively. However, even when the response was less than 1 per cent of its initial value, the organ had considerable powers of recuperation during a 30-s rest period, to 30–50 per cent of its initial value.
The fall in response was accompanied by a reduction in vesicle size and number, an increase in the area of the presynaptic membrane and a fall in the protein, total nucleotide, ATP and acetylcholine content of the vesicle fraction isolated from the stimulated tissue. However, whereas vesicle numbers and the protein and total nucleotide content of the vesicle fraction fell by only about 50 per cent, vesicular ATP and acetylcholine levels were reduced to about 10 per cent. An analysis of the covariance of vesicular ATP and acetylcholine showed an initial loss of an acetylcholine-rich (relative to ATP) population of vesicles. The early loss of vesicular protein and nucleotide and vesicle numbers as well as the morphological changes seen would be consistent with a loss of vesicles due to fusion with the external membrane. The preferential loss of acetylcholine and ATP from the vesicle fraction indicates that the vesicles surviving the stimulation procedure have been utilized in a number of cycles causing the progressive fall in vesicle volume, and acetylcholine and ATP content.     

Regulation of synaptic vesicles pools within motor nerve terminals during short-term facilitation and neuromodulation.

The reserve pool (RP) and readily releasable pool (RRP) of synaptic vesicles within presynaptic nerve terminals were physiologically differentiated into distinctly separate functional groups. This was accomplished in glutamatergic nerve terminals by blocking the glutamate transporter with dl-threo-beta-benzyloxyaspartate (TBOA; 10 microM) during electrical stimulation with either 40 Hz of 10 pulses within a train or 20- or 50-Hz continuous stimulation. The 50-Hz continuous stimulation decreased the excitatory postsynaptic potential amplitude 60 min faster than for the 20-Hz continuous stimulation in the presence of TBOA (P < 0.05). There was no significant difference between the train stimulation and 20-Hz continuous stimulation in the run-down time in the presence of TBOA. After TBOA-induced synaptic depression, the excitatory postsynaptic potentials were rapidly (<1 min) revitalized by exposure to serotonin (5-HT, 1 microM) in every preparation tested (P < 0.05). At this glutamatergic nerve terminal, 5-HT promotes an increase probability of vesicular docking and fusion. Quantal recordings made directly at nerve terminals revealed smaller quantal sizes with TBOA exposure with a marked increase in quantal size as well as a continual appearance of smaller quanta upon 5-HT treatment after TBOA-induced depression. Thus 5-HT was able to recruit vesicles from the RP that were not rapidly depleted by acute TBOA treatment and electrical stimulation. The results support the notion that the RRP is selectively activated during rapid electrical stimulation sparing the RP; however, the RP can be recruited by the neuromodulator 5-HT. This suggests at least two separate kinetic and distinct regulatory paths for vesicle recycling within the presynaptic nerve terminal.     

Electron microscopic localization of calelectrin, a Mr 36 000 calcium-regulated protein, at the cholinergic electromotor synapse of Torpedo

Calelectrin is a calcium-binding protein of Mr 36 000 which has previously been shown to be associated with membranes of the cholinergic synapse in a calcium-dependent manner. We report here that calelectrin was solubilized from the electric organ of Torpedo marmorata in the absence of calcium together with proteins of Mr 54 000 and Mr 15 000. In cholinergic nerve endings isolated from the electric organ only calelectrin was solubilized in a calcium-dependent manner. A specific antiserum to calelectrin was used to localize the antigen by immunofluorescence microscopy on sections of electric organ and showed that calelectrin is distributed throughout the postsynaptic cell. Calelectrin was also detected in axons and in the cell bodies of the cholinergic neurones where it was concentrated in discrete patches throughout the cells. Electric organ tissue was processed to localize calelectrin with the electron microscope using an immunoperoxidase method. The most intense staining was observed on the cytoplasmic face of the acetylcholine receptor-containing postsynaptic membrane and also associated with the intracellular filaments of the electrocyte. The intensity of staining associated with these structures could be greatly reduced by preincubating the tissue with calcium chelators. In nerve terminals calelectrin was associated with synaptic vesicles in a polarized fashion. Calelectrin was also found on the cytoplasmic face of the synaptosomal plasma membrane and associated with neurofilaments. No extracellular staining was ever observed. Our results strongly support our original hypothesis that calelectrin is a calcium-regulated component of intracellular structure associated both with membranes and filaments.     

DIFFERENT RECOVERY RATES OF THE ELECTROPHYSIOLOGICAL, BIOCHEMICAL AND MORPHOLOGICAL PARAMETERS IN THE CHOLINERGIC SYNAPSES OF THE TORPEDO ELECTRIC ORGAN AFTER STIMULATION

—During stimulation there occurred a decay in electrical response, vesicular acetylcholine, ATP and nucleotide as well as a loss of vesicle number and a decrease in vesicle diameter in the electric organ of Torpedo. These alterations were re-established during a subsequent recovery period. The different parameters recovered at different rates. Firstly, electrical response to single pulses recovered to prestimulation values within about 5 h. Vesicle number and diameter as well as bouton size were found to be re-established fully after 24 h. The newly formed vesicles appeared to be empty as vesicular acetylcholine, ATP and total nucleotide recovered much more slowly and were back to control values after about three days. Acetylcholine reappeared more quickly in the vesicles than ATP. Only after recovery of the vesicular pool of transmitter and ATP did the electric organ regain full stability of the electric discharge pattern on restimulation.     

The isolation, from electromotor neurone perikarya, of messenger RNAs coding for synaptic proteins

Poly(A)-containing mRNA was isolated from the electric lobe, cerebellum and forebrain of Torpedo marmorata and from cholinergic electromotor perikarya isolated from the electric lobe. All the mRNA preparations were translated by a cell-free protein-synthesizing system from rabbit reticulocytes; no brain-specific factors were required. The highest stimulation rate was found with the perikaryal mRNA suggesting that this purely neuronal mRNA is a preferred template in the protein-synthesis system; the molecular basis of this phenomenon remains to be elucidated. The translation products of the perikaryal mRNA were analysed by two-dimensional gel electrophoresis and compared with the proteins of synaptosomes derived from the electromotor nerve terminals. The majority of the synaptosomal proteins comigrated with synthesized products. More than 100 synthesized proteins were detected as individual spots in the gel pattern, among them actin, subunits of neurofilamentous proteins and a protein considered to be a specific component of electromotor synaptic vesicles. Identities were confirmed in some cases by immunochemical methods. The results suggest that protein synthesis in the perikaryon of the electromotor neurone is largely directed to the production of proteins needed to maintain synaptic integrity. A comparison of the translation products of mRNA derived from the highly cholinergic electric lobe and a brain region, the cerebellum, which is non-cholinergic, revealed, as expected, some common translation products and others which appeared to be specific for the brain regions concerned. This approach may lead to the identification of protein specific for neurones of different transmitter types.     

Presence of Vasoactive Intestinal Polypeptide-Like Immunoreactivity in the Cholinergic Electromotor System of Torpedo marmorata

Vasoactive intestinal polypeptide (VIP)-like immunoreactivity was detected in the cholinergic electro-motor system of Torpedo marmorata using a combination of immunohistochemical assays, radioimmunoassay, and HPLC. The immunohistochemical assays revealed that the distribution of VIP-like immunoreactivity in the electric lobes, electromotor nerves, and electric organ is comparable to that of the stable cholinergic synaptic vesicle marker vesicle-specific proteoglycan. Ligation of the electromotor nerves caused a marked accumulation of VIP-like immunoreactivity in the lobes (180%) and the proximal portions of the electromotor nerves (130%) and a decrease in the electric organ (-50%), when measured by radioimmunoassay using synthetic VIP (porcine sequence) as the standard. VIP-like immunoreactivity in extracts of electric lobes electromotor nerves, and electric organ was eluted from a semipreparative reverse-phase HPLC column as a single peak with a retention time similar to that of porcine VIP. Rechromatography at higher resolution on an analytical column indicated diversity between the molecular forms of VIP-like immunoreactivity extracted from electric lobe and electric organ, suggesting the possibility of posttranslational processing.     

Changes in cholinergic synaptic vesicle populations and the ultrastructure of the nerve terminal membranes of Narcine brasiliensis electron organ stimulated to fatigue in vivo

Narcine brasiliensis electric organ was stimulated to fatigue in vivo. Electrical display of organ output and biochemical assay of bound acetylcholine (ACh) and ATP in isolated vesicles were used to assess the state of fatigue relative to denervated control organs of the same fish. A morphometric analysis of the fate of the synaptic vesicle populations in the nerve terminals was carried out. Statistically significant morphological changes in vesicle populations and plasma membranes were observed between control and fatigued electroplaque stacks from individual fish. Pooled data from several fish were used to evaluate the possible role of the different vesicle types in neurotransmission. Fatigue resulted in the loss of 49% of the total vesicle population and a 76% loss of vesicles with bound calcium (Ca). An approximately equivalent increase in the nerve-terminal plasma membrane area was measured. This was predominantly in the form of fingerlike protrusions and/or invaginations of the terminals which were present in the control organs but which were significantly increased by stimulation. Vesicle attachments to the nerve terminal membrane were reduced by 90%. This suggests that the failure in transmission may be due to reduction in the number of vesicles which are loaded with transmitter and can attach to the terminal membrane. The Ca-binding capacity of the lost vesicles was not transferred to the plasma membranes. This result was interpreted as support for the hypothesis that vesicle-bound ATP provides the Ca-binding site.     

Immunological detection of mediatophore in motor end-plates and electric organ subcellular fractions of torpedo marmorata

A rabbit antiserum to mediatophore, a nerve terminal membrane protein involved in calcium dependent ACh release, was raised after immunization with the purified protein. An immunological assay for mediatophore was then developed and the subcellular distribution of this protein in Torpedo electric organ fractions was studied. A good agreement was obtained between the distribution in the different fractions of the antigen and of mediatophore related acetylcholine releasing activity as determined by reconstitution in proteoliposomes. Mediatophore was highly concentrated in presynaptic plasma membranes of electric organ, while very low contents were observed in electric nerves and electric lobes. Although some mediatophore was found in synaptic vesicle fractions, this most probably resulted from presynaptic membrane contamination as evaluated with other presynaptic membrane markers. Nerve terminals of motor end-plates were strongly stained with anti-mediatophore antibodies.     

Reduced Expression of the Vesicular Acetylcholine Transporter and Neurotransmitter Content Affects Synaptic Vesicle Distribution and Shape in Mouse Neuromuscular Junction

In vertebrates, nerve muscle communication is mediated by the release of the neurotransmitter acetylcholine packed inside synaptic vesicles by a specific vesicular acetylcholine transporter (VAChT). Here we used a mouse model (VAChT KD^HOM) with 70% reduction in the expression of VAChT to investigate the morphological and functional consequences of a decreased acetylcholine uptake and release in neuromuscular synapses. Upon hypertonic stimulation, VAChT KD^HOM mice presented a reduction in the amplitude and frequency of miniature endplate potentials, FM 1–43 staining intensity, total number of synaptic vesicles and altered distribution of vesicles within the synaptic terminal. In contrast, under electrical stimulation or no stimulation, VAChT KD^HOM neuromuscular junctions did not differ from WT on total number of vesicles but showed altered distribution. Additionally, motor nerve terminals in VAChT KD^HOM exhibited small and flattened synaptic vesicles similar to that observed in WT mice treated with vesamicol that blocks acetylcholine uptake. Based on these results, we propose that decreased VAChT levels affect synaptic vesicle biogenesis and distribution whereas a lower ACh content affects vesicles shape.     

Identification of a heparan sulphate-containing proteoglycan as a specific core component of cholinergic synaptic vesicles from Torpedo marmorata.

Cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata were found to contain a proteoglycan in their core. The glycosaminoglycan part co-migrates upon thin layer electrophoresis with heparan sulphate and shows a chemical composition characteristic for this carbohydrate. [35S]Sulphate injected into the electric lobes of Torpedo, which contain the perikarya of the electromotor neurons innervating the electric organs, appeared 48 h later in covalently bound form in the synaptic vesicle fraction. The radiolabel had been incorporated into the vesicular heparan sulphate. Upon SDS-polyacrylamide gel electrophoresis fluorography of labelled vesicles a major and a minor band are formed both migrating above a protein standard of mol. wt. 200 000. Similarly, a major peak in the void volume and a minor peak in the included volume are seen upon gel filtration in Ultrogel AcA 34 in the presence of SDS. We interpret the minor fraction as being formed by the loss of glycosaminoglycan from the major fraction. The proteoglycan is located inside the vesicle since antibodies directed against it form immunoprecipitates only with vesicles lysed by detergent treatment. The experiments show that it is possible to label a synaptic organelle specifically by axonal transport.