Cd2+- and K+-evoked ACh release induce different synaptophysin and synaptobrevin immunolabelling at the frog neuromuscular junction

Summary Synaptophysin and synaptobrevin, two integral proteins of synaptic vesicles, have been used as immunocytochemical markers of the synaptic vesicle membrane during Cd²⁺- or K⁺-induced ACh release at the frog neuromuscular junction. ACh release was stimulated in cutaneous pectoris nerve-muscle preparations by: (1) 1 mM Cd²⁺ in Ca²⁺-free medium for a period of 3 h, (2) 25 or 40mM K⁺ in normal Ringer's solution. Synaptophysin and synaptobrevin were immunolabelled in single fibres teased from fixed muscles using rabbit antisera raised against synaptophysin and synaptobrevin revealed with fluoresceinconjugated IgG. The postsynaptic ACh receptors were simultaneously labelled with rhodaminated -bungarotoxin. Unstimulated and K⁺-stimulated preparations showed synaptophysin and synaptobrevin immunolabelling only after membrane permeabilization with 0.1% Triton X-100. In preparations stimulated with Cd²⁺ in Ca²⁺-free medium, the immunofluorescence was also observed in non Triton X-100 treated muscle fibres. Confocal laser scanning microscopy analysis revealed that in unstimulated and K⁺-stimulated preparations, synaptophysin and synaptobrevin immunofluorescence appears as bands regularly spaced along the permeabilized nerve terminals and that their distribution corresponds to clusters of synaptic vesicles. After Cd²⁺ stimulation in Ca²⁺-free medium, labelling for both proteins is irregularly distributed, being more intense at the lateral margins of swollen nerve terminals, suggesting a translocation of synaptic vesicle proteins to the axolemma. At the electron microscopic level, Cd²⁺ stimulation in Ca²⁺-free medium produces nerve terminal swelling and synaptic vesicle depletion. The results show that when ACh release is stimulated under an impairment of synaptic vesicle recycling, which leads to synaptic vesicle depletion, synaptophysin and synaptobrevin translocation occurs. These findings are in favour of a permanent incorporation of synaptic vesicle membrane into the axolemma. In contrast, after K⁺ stimulation, the immunofluorescence and the normal synaptic vesicle population observed, suggest that a double process of synaptic vesicle exo-endocytosis rapidly occurs, without incorporation of synaptic vesicle components into the axolemma.     

Double mode of action of black widow spider venom on frog neuromuscular junction

Summary Black widow spider venom (BWSV) contains a toxin, -latrotoxin, which is capable of stimulating vesicle release, resulting eventually in depletion of vesicles and block of neuromuscular transmission at the frog neuromuscular junction. Since it has been shown that -latrotoxin very markedly increases the cation conductance of artificial lipid bilayers, it was postulated that BWSV stimulates release by opening channels permeable to Ca²⁺ and, in the case of Ca²⁺-free Ringer's, to Na⁺ which would release Ca²⁺ from intracellular stores. To test this hypothesis we chose as a sodium substitute, glucosamine, which is impermeable to the venom-induced channels in the lipid bilayers and to the postsynaptic membrane of the frog neuromuscular junction. Electron microscopical analysis showed that up to 75 min perfusion in Na⁺ and Ca²⁺-free medium did not alter the ultrastructure of the nerve terminals. However when BWSV was applied in this medium a significant depletion was noticeable within 15 min and after 60 min the terminals were depleted of vesicles whereas the mitochondria were unchanged in number and structure. If BWSV is applied for 60 min in glucosamine Ringer's containing 1.8 mM Ca²⁺, most of the nerve terminals still have synaptic vesicles scattered in the cytoplasm or clustered around amorphous structures and the mitochondria are swollen. Application of large doses of BWSV in low Ca²⁺ Ringer's leads to damage of the mitochondria and to very pronounced swelling of the nerve endings, whereas this is not observed if the dose of venom is applied in Na⁺-free and Ca²⁺-free Ringer's. Electrophysiological recording showed that neuromuscular transmission is already blocked after 15 min treatment with BWSV in glucosamme-Ringer's. From these results we conclude that BWSV increases the conductance of the nerve terminal membrane to cations such as Na⁺ and Ca²⁺ and stimulates release by a mechanism which may not involve its ionophore property.     

Formation, stabilisation and fusion of the readily releasable pool of secretory vesicles

Calcium-triggered exocytosis of neurotransmitter or hormone-filled vesicles has developed as the main mechanism for cell-to-cell communication in animals. Consequently, in the course of evolution this form of exocytosis has been optimized for speed. Since many of the maturation processes of vesicles are intrinsically slow, the solution has been to develop a pool of vesicles that are fully matured and can be fused very rapidly upon stimulation. Vesicles in this readily releasable pool are characterized by very low release rate constants at the resting cytosolic [Ca²⁺] ([Ca²⁺]_i) and very high release rate constants at stimulated [Ca²⁺]_i. Here I review the kinetic and molecular requirements for the existence of such a pool of vesicles, focusing on chromaffin cells of the adrenal medulla. I discuss how the use of assay methods with different time resolution may lead to fundamentally different conclusions about the role of proteins in exocytosis. Finally, I review recent evidence that the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, formed between proteins residing in the vesicle and the plasma membrane, is involved in formation and stabilization of the readily releasable vesicle pool, whereas synaptotagmin, a Ca²⁺- and phospholipid-binding vesicular protein, is involved in setting the Ca²⁺ dependence of the fusion process itself. Future studies are likely to focus on the interaction between these two classes of proteins.     

Incorporation of vesicular antigens into the presynaptic membrane during exocytosis at the frog neuromuscular junction: a light and electron microscopy immunochemical study

We have studied the incorporation of vesicular membrane antigens into the presynaptic membrane during exocytosis of neurotransmitter at the frog neuromuscular junction. In a preliminary series of experiments, we first confirmed by electron microscopy that the synaptic vesicles are labelled following incubation with rabbit antisynaptic vesicle antibody of neuromuscular junction cross sections (cytoplasm and organelles reached by the antibodies). In a second series of experiments, intact neuromuscular junctions were stimulated with black widow spider venom and fixed with paraformaldehyde. The presence or absence of vesicular antigens in the presynaptic membrane was monitored with rabbit antisynaptic vesicle antibody and revealed with a second antibody coupled to peroxidase. In light microscopy, the labelled neuromuscular junctions are almost completely restricted to muscles stimulated with black widow spider venom and incubated with rabbit antisynaptic vesicle antibody. Only a few control muscles (not stimulated with black widow spider venom, but incubated with rabbit antisynaptic vesicle antibody) had labelled neuromuscular junctions. All control neuromuscular junctions, not incubated with rabbit antisynaptic vesicle antibody were unlabelled. Electron microscopy indicated that it is the presynaptic membrane of intact stimulated neuromuscular junctions which is labelled. In these intact neuromuscular junctions, the synaptic vesicles are usually unlabelled. Electron microscopy also indicated that the presynaptic membrane of only one type of control junctions (not stimulated with black widow spider venom, but incubated with rabbit antisynaptic vesicle antibody) is rarely and weakly labelled. Other types of controls (not incubated with rabbit antisynaptic vesicle antibody) are never labelled. Therefore our results are consistent with the incorporation of vesicular antigens into the plasma membrane during exocytosis produced by the black widow spider venom. The low level of labelling of unstimulated neuromuscular junctions suggest a rather complete retrieval of the vesicular proteins during endocytosis.     

Synaptic vesicle recycling at the neuromuscular junction in the presence of a presynaptic membrane marker

Staining of the presynaptic axonal membrane of the neuromuscular junction with horseradish peroxidase-labeled α-bungarotoxin was utilized as a marker for observing directly the fate of this membrane during the process of synaptic vesicle release and recycling. The neuromuscular junctions of frog sartorius-sciatic nerve preparations were stained with horseradish peroxidase-α-bungarotoxin and stimulated by electrical stimulation of the nerve, high concentration of external potassium ions, and black widow spider venom. Some preparations were stimulated in the presence of exogenous horseradish peroxidase tracer after incubation in the conjugate and were found to contain horseradish peroxidase within many synaptic vesicles, indicating that the conjugate did not affect the process of synaptic vesicle recycling. Stimulation was followed by depletion of synaptic vesicles and appearance of axolemmal infoldings and membranous cisternae. With rest after electrical and potassium stimulation, synaptic vesicles were reconstituted and terminals assumed a more normal appearance. Membrane staining after stimulation occurred in the axolemmal infoldings, some of the intra-axonal cisternae, and in a few coated vesicles. However, all synaptic vesicles were unreactive, in either rested or unrested terminals. Thus, axonal membrane labeled with horseradish peroxidase-α-bungarotoxin did not become incorporated into new synaptic vesicles.These observations support a mechanism of recycling of synaptic vesicles by specific retrieval of vesicle membrane or constituents from the axolemma.     

Calcium dependence of neurotransmitter release

Neurotransmitter is released from synaptic terminals by rapid and highly targeted fusion of synaptic vesicles with the presynaptic membrane. Several lines of evidence suggest that the trigger for vesicle fusion is the large increase in internal [Ca²⁺] (up to hundreds of micromolar) achieved within the submicroscopic domain of elevated calcium near open calcium channels. The rapid rise and fall of [Ca²⁺] in this microdomain, together with the fast kinetics of the calcium-triggered fusion machinery, account for the speed of synaptic exocytosis.     

The effects of chronic stimulation on the morphology of the frog neuromuscular junction

Summary A quantitative study was made of the effects of 24 h continuous stimulation on the morphology of the frog neuromuscular junction. The synaptic vesicle concentration in the nerve endings of frog sartorius muscles stimulatedin vitro for 24 h at 2 Hz was the same as that in controls stimulated for only 0.3 h at 2 Hz. The control preparations were either freshly dissected or maintained at restin vitro for 23 h prior to stimulation. Chronically stimulated terminals differed from their controls only in having more cisternae and fewer dense cored vesicles. Varying the lengths of the nerves to both chronically stimulated andin vitro control muscles had little effect on the morphology of the nerve endings.Continuous recording of muscle twitch tension demonstrated that neurotransmission was effective throughout the 24 h period of stimulation. Additional evidence that nerve failure or degeneration was not a factor in the results came from a second set of control and chronically stimulated preparations that were tetanized at 30 Hz for 0.3 h before fixation. Changes attributable to rapid stimulation were evident in 87 to 100% of their nerve terminals.Although the distribution of membrane among various membrane organelles differed from one treatment group to another, the total amount of measurable membrane in the nerve terminals was the same in all of the treatment groups; that is, the total amount of membrane was not altered by maintenancein vitro, chronic stimulation at 2 Hz, rapid stimulation at 30 Hz, reduced nerve length, or any tested combination of these treatments. This conservation of total membrane suggests that membrane exchange between axon and nerve terminal occurs at a relatively slow rate which is unaffected by synaptic activity, and that the local mechanism for recycling synaptic vesicle membrane in frog neuromuscular junctions is more autonomous and durable than has been suspected.     

Membrane cycling after the excess retrieval mode of rapid endocytosis in mouse chromaffin cells

Aim: After exocytosis, neuroendocrine cells and neurones keep constant the plasma membrane and the releasable vesicle pools by performing endocytosis and vesicular cycling. Patch‐clamp capacitance measurements on chromaffin cells showed that strong Ca⁺² entry activates excess retrieval: a rapid endocytosis process that retrieves more membrane than the one fused by preceding exocytosis. The main purpose of the present experiments was to study the recycling pathway that follows excess retrieval, which is unknown. Methods: Membrane recycling after exocytosis–endocytosis can be studied by fluorescence imaging assays with FM1‐43 (Perez Bay et al. Am J Physiol Cell Physiol 2007; 293, C1509). In this work, we used this assay in combination with fluorescent dextrans and specific organelle‐targeted antibodies to study the membrane recycling after excess retrieval in mouse chromaffin cells. Results: Excess retrieval was observed after the application of high‐K⁺ or cholinergic agonists during 15 or 30 s in the presence of FM1‐43. We found that the excess retrieval membrane pool (defined as endocytosis–exocytosis) was associated with the generation of a non‐releasable fraction of membrane (up to 30% of plasma membrane surface) colocalizing with the lysosomal compartment. The excess retrieval membrane pool followed a saturable cytosolic Ca²⁺ dependency, and it was suppressed by inhibitors of L‐type Ca²⁺ channels, endoplasmic reticulum Ca²⁺ release and PKC. Conclusion: Excess retrieval is not associated with the cycling of releasable vesicles, but it is related to the formation of non‐releasable endosomes. This process is activated by a concerted contribution of Ca²⁺ entry through L‐channels and Ca²⁺ release from endoplasmic reticulum.     

Amyotrophic lateral sclerosis immunoglobulins G enhance the mobility of Lysotracker-labelled vesicles in cultured rat astrocytes

Aim: We examined the effect of purified immunoglobulins G (IgG) from patients with amyotrophic lateral sclerosis (ALS) on the mobility and exocytotic release from Lysotracker‐stained vesicles in cultured rat astrocytes. Methods: Time‐lapse confocal images were acquired, and vesicle mobility was analysed before and after the application of ALS IgG. The vesicle counts were obtained to assess cargo exocytosis from stained organelles. Results: At rest, when mobility was monitored for 2 min in bath with Ca²⁺, two vesicle populations were discovered: (1) non‐mobile vesicles (6.1%) with total track length (TL) < 1 μm, averaging at 0.33 ± 0.01 μm (n = 1305) and (2) mobile vesicles (93.9%) with TL > 1 μm, averaging at 3.03 ± 0.01 μm (n = 20 200). ALS IgG (0.1 mg mL^?1) from 12 of 13 patients increased the TL of mobile vesicles by approx. 24% and maximal displacement (MD) by approx. 26% within 4 min, while the IgG from control group did not alter the vesicle mobility. The mobility enhancement by ALS IgG was reduced in extracellular solution devoid of Ca²⁺, indicating that ALS IgG vesicle mobility enhancement involves changes in Ca²⁺ homeostasis. To examine whether enhanced mobility relates to elevated Ca²⁺ activity, cells were stimulated by 1 mm ATP, a cytosolic Ca²⁺ increasing agent, in the presence (2 mm ) and in the absence of extracellular Ca²⁺. ATP stimulation triggered an increase in TL by approx. 7% and 12% and a decrease in MD by approx. 11% and 1%, within 4 min respectively. Interestingly, none of the stimuli triggered the release of vesicle cargo. Conclusion: Amyotrophic lateral sclerosis‐IgG‐enhanced vesicle mobility in astrocytes engages changes in calcium homeostasis.     

Action of brown widow spider venom and botulinum toxin on the frog neuromuscular junction examined with the freeze-fracture technique.

1. Structural changes which normally accompany transmitter release at frog neuromuscular junctions are visualized with the freeze-fracture technique. The effects of brown widow spider venom and botulinum toxin were evaluated in terms of their ability to block or produce these structural changes. Changes produced by these neuropoisons were correlated with their known effects on neurotransmitter release. 3. Fusion of synaptic vesicles with the presynaptic plasmalemma, normally evoked by electrical stimulation, was abolished at neuromuscular junctions from frogs treated with botulinum toxin. 3. The concentration of large intramembranous particles in the presynaptic plasmalemma, an indication of the excess of synaptic vesicle fusion over recovery of synaptic vesicle membrane, was increased by treatment with brown widow spider venom, even in the presence of botulinum toxin. 4. When external calcium was present, sites of vesicle fusion induced by brown widow spider venom, as well as by electrical stimulation, were located mainly in the active zone. In the absence of external calcium, many plasmalemmal deformations, also though to be sites of vesicle fusion, were more evenly dispersed over the presynaptic surface of nerve terminals. 5. Botulinum toxin decreased the number of vesicle fusion sites in the active zone induced by spider venom in the presence of external calcium but had little effect on the number of fusion sites induced by spider venom in the absence of external calcium. 6. Nerve terminals soaked in a sodium-free Ringer solution were partially depleted of vesicles. Addition of spider venom to this Ringer did not cause additional depletion of vesicles. 7. Formation of cation-permeable channels in the presynaptic membrane could account for these effects of spider venom on the frog neuromuscular junction. Botulinum toxin blocks vesicle fusion by some means which is not yet understood.     

A comparison of synaptic protein localization in hippocampal mossy fiber terminals and neurosecretory endings of the neurohypophysis using the cryo-immunogold technique

In central synapses synaptic vesicle docking and exocytosis occurs at morphologically specialized sites (active zones) and requires the interaction of specific proteins in the formation of a SNARE complex. In contrast, neurosecretory terminals lack active zones. Using the cryo-immunogold technique we analyzed the localization of synaptic vesicle proteins and of proteins of the docking complex at active zones. This was compared to the localization of the identical proteins in neurosecretory terminals. In addition we compared the vesicular and granular localization of the proteins investigated. Synaptic vesicles in rat hippocampal mossy fiber synapses and microvesicles in the neurosecretory terminals of the neurohypophysis contained in common the proteins VAMP II (a v-SNARE), SV2, rab3A, and N-type Ca²⁺ channels. Only minor immunolabeling for these proteins was observed at neurosecretory granules. These results support the notion of a close functional identity of microvesicles from neurosecretory endings of the neurohypophysis and of synaptic vesicles. The vesicular pool of N-type Ca²⁺ channels may serve their stimulation-induced translocation into the plasma membrane. We find increased labeling for VAMP II, SNAP-25, N-type Ca²⁺ channels and of rab3A at the active zones of mossy fiber synapses. Labeling at release sites is by far highest for Bassoon, a high molecular weight protein of the active zone. The labeling pattern implies an association of Bassoon with presynaptic dense projections. Bassoon is absent from neurosecretory terminals and VAMP II, SNAP-25, rab3A, and N-type Ca²⁺ channels reveal a scattered distribution over the plasma membrane. The competence of the presynaptic active zone for selective vesicle docking may not primarily result from its contents in SNARE proteins but rather from the preformation of presynaptic dense projections as structural guides for vesicle exocytosis.     

The effects of nerve stimulation and hemicholinium on synaptic vesicles at the mammalian neuromuscular junction

1. Electron micrographs of nerve terminals in rat phrenic nerve—diaphragm preparations have been studied. This has been done before and after prolonged nerve stimulation. The effectiveness of nerve stimulation has been monitored by intracellular micro-electrode recordings from the muscle cells.

2. Characteristic changes in the form and distribution of the nerve terminal mitochondria were noted after nerve stimulation.

3. Synaptic vesicle numbers in the region of nerve terminal less than 1800 Å from the synaptic cleft were significantly greater in tissue taken 2 and 3 min after nerve stimulation, than in unstimulated preparations.

4. The long and short diameters of the synaptic vesicle profiles less than 1800 Å from the synaptic cleft were measured. Analysis of the distribution of the diameters indicated synaptic vesicles to be basically spherical structures. Estimates of synaptic vesicle volume were made from the measurements. Synaptic vesicle volume was significantly reduced in tissue taken 2 and 4 min following nerve stimulation.

5. If hemicholinium, a compound which inhibits acetylcholine synthesis, was present during the period of nerve stimulation, much greater reductions in synaptic vesicle volume occurred. Synaptic vesicle numbers in the region of nerve terminal less than 1800 Å from the synaptic cleft were also reduced, compared with unstimulated control preparations.

6. These results are regarded as support for the hypothesis that the synaptic vesicles in nerve terminals at the mammalian neuromuscular junction represent stores of the transmitter substance, acetylcholine.

     

Recycling and biogenesis of synaptic vesicles

Synaptic vesicles are specialized organelles used for the fast, point-to-point signaling typical of the nervous system. Like secretory granules of the classical regulated secretory pathway, synaptic vesicles undergo exocytosis in a Ca²⁺-dependent fashion. However, synaptic vesicles are also closely related to vesicles involved in the constitutive recycling of membrane components between the plasmalemma and the early endosome. Here we review available information and open questions regarding the biogenesis of synaptic vesicles, their traffic within nerve terminals and their relationship to vesicles of other vesicular pathways to the plasmalemma.     

Mitochondria and chromaffin cell function

Chromaffin cells are an excellent model for stimulus?Csecretion coupling. Ca²⁺ entry through plasma membrane voltage-operated Ca²⁺ channels (VOCC) is the trigger for secretion, but the intracellular organelles contribute subtle nuances to the Ca²⁺ signal. The endoplasmic reticulum amplifies the cytosolic Ca²⁺ ([Ca²⁺]_C) signal by Ca²⁺-induced Ca²⁺ release (CICR) and helps generation of microdomains with high [Ca²⁺]_C (HCMD) at the subplasmalemmal region. These HCMD induce exocytosis of the docked secretory vesicles. Mitochondria close to VOCC take up large amounts of Ca²⁺ from HCMD and stop progression of the Ca²⁺ wave towards the cell core. On the other hand, the increase of [Ca²⁺] at the mitochondrial matrix stimulates respiration and tunes energy production to the increased needs of the exocytic activity. At the end of stimulation, [Ca²⁺]_C decreases rapidly and mitochondria release the Ca²⁺ accumulated in the matrix through the Na⁺/Ca²⁺ exchanger. VOCC, CICR sites and nearby mitochondria form functional triads that co-localize at the subplasmalemmal area, where secretory vesicles wait ready for exocytosis. These triads optimize stimulus?Csecretion coupling while avoiding propagation of the Ca²⁺ signal to the cell core. Perturbation of their functioning in neurons may contribute to the genesis of excitotoxicity, ageing mental retardation and/or neurodegenerative disorders.     

Characterization of ouabain-induced noradrenaline and acetylcholine release from in situ cardiac autonomic nerve endings

Aim: Although ouabain modulates autonomic nerve ending function, it is uncertain whether ouabain‐induced releasing mechanism differs between in vivo sympathetic and parasympathetic nerve endings. Using cardiac dialysis, we examined how ouabain induces neurotransmitter release from autonomic nerve ending. Methods: Dialysis probe was implanted in left ventricle, and dialysate noradrenaline (NA) or acetylcholine (ACh) levels in the anaesthetized cats were measured as indices of neurotransmitter release from post‐ganglionic autonomic nerve endings. Results: Locally applied ouabain (100 μm ) increased in dialysate NA or ACh levels. The ouabain‐induced increases in NA levels remained unaffected by cardiac sympathetic denervation and tetrodotoxin (Na⁺ channel blocker, TTX), but the ouabain‐induced increases in ACh levels were attenuated by TTX. The ouabain‐induced increases in NA levels were suppressed by pretreatment with desipramine (NA transport blocker) and augmented by reserpine (vesicle NA transport blocker). In contrast, the ouabain‐induced increases in ACh levels remained unaffected by pretreatment with hemicholinium‐3 (choline transport blocker) but suppressed by vesamicol (vesicle ACh transport blocker). The ouabain‐induced increases in NA levels were suppressed by pretreatment with ω‐conotoxin GVIA (N‐type Ca²⁺ channel blocker), verapamil (L‐type Ca²⁺ channel blocker) and TMB‐8 (intracellular Ca²⁺ antagonist). The ouabain‐induced increases in ACh levels were suppressed by pretreatment with ω‐conotoxin MVIIC (P/Q‐type Ca²⁺ channel blocker), and TMB‐8. Conclusions: Ouabain‐induced NA release is attributable to the mechanisms of regional exocytosis and/or carrier‐mediated outward transport of NA, from stored NA vesicle and/or axoplasma, respectively, while the ouabain‐induced ACh release is attributable to the mechanism of exocytosis, which is triggered by regional depolarization. At both sympathetic and parasympathetic nerve endings, the regional exocytosis is because of opening of calcium channels and intracellular calcium mobilization.     

Empty synaptic vesicles recycle and undergo exocytosis at vesamicol-treated motor nerve terminals.

We investigated whether recycled cholinergic synaptic vesicles, which were not refilled with ACh, would join other synaptic vesicles in the readily releasable store near active zones, dock, and continue to undergo exocytosis during prolonged stimulation. Snake nerve-muscle preparations were treated with 5 microM vesamicol to inhibit the vesicular ACh transporter and then were exposed to an elevated potassium solution, 35 mM potassium propionate (35 KP), to release all preformed quanta of ACh. At vesamicol-treated endplates, miniature endplate current (MEPC) frequency increased initially from 0.4 to >300 s-1 in 35 KP but then declined to <1 s-1 by 90 min. The decrease in frequency was not accompanied by a decrease in MEPC average amplitude. Nerve terminals accumulated the activity-dependent dye FM1-43 when exposed to the dye for the final 6 min of a 120-min exposure to 35 KP. Thus synaptic membrane endocytosis continued at a high rate, although MEPCs occurred infrequently. After a 120-min exposure in 35 KP, nerve terminals accumulated FM1-43 and then destained, confirming that exocytosis also still occurred at a high rate. These results demonstrate that recycled cholinergic synaptic vesicles that were not refilled with ACh continued to dock and undergo exocytosis after membrane retrieval. Thus transport of ACh into recycled cholinergic vesicles is not a requirement for repeated cycles of exocytosis and retrieval of synaptic vesicle membrane during prolonged stimulation of motor nerve terminals.     

The effect of α-latrotoxin on the neurosecretory PC12 cell line: Electron microscopy and cytotoxicity studies

Neurosecretory PC 12 cells were exposed in a variety of experimental conditions to nanomolar concentrations of α-latrotoxin purified from the venom of the black widow spider. When applied in a modified Ringer medium containing millimolar Ca²⁺ the toxin rapidly elicited a marked stimulation of exocytosis, as indicated by the appearance of typical images of granule-plasmalemma interaction and by the decreased density (number/unit area) of secretion granules in the cytoplasm. Without Ca²⁺ in the medium this early toxin effect was delayed and evolved less rapidly, but was still clearly appreciable. These morphological results appear in good quantitative agreement with the biochemical data on dopamine release reported in the preceding article.²⁸ The stimulation of exocytosis was followed after a short delay by a stimulation of endocytosis, as revealed by an increased accumulation of the extracellular tracer, [¹⁴C]sucrose, within the toxin-treated cells. At later times after the application of α-latrotoxin other effects appeared, but only in the presence of Ca²⁺: these included changes in cell shape; focal alterations of the mitochondrial matrix (clear discrete areas and dense precipitates) and frank signs of cytotoxicity (rupture of the plasmalemma, clearing of the cytoplasmatic matrix). The toxin-induced cell death was studied quantitatively by using trypan blue exclusion as well as the⁵¹Cr test, and was found to be dependent on α-latrotoxin concentration, temperature of incubation and Ca²⁺ concentration in the medium. Ionic substitutions concerning anions as well as cations other than Ca²⁺ had minor or no consequences.Thus, the early effect of α-latrotoxin in PC12 cells (stimulation of exocytosis, at least partially Ca²⁺-independent) can be dissociated from the late ‘toxic’ effect (strictly Ca²⁺-dependent).     

Motor responses of the urinary bladder and skeletal muscle in Botulinum intoxicated rats

1. Type A or type D botulinum toxin administered to rats did not produce a generalized paralysis of skeletal muscles at the time of ventilatory arrest. However, if survival was extended by artificial ventilation complete blockade of neuromuscular transmission developed 6.5 hr after 100 MLD of type D and 5 hr after 1000 MLD of type A toxin. The onset of paralysis of a muscle was shortened by repetitive stimulation of the motor nerves.2. There was no consistent blockade of parasympathetically innervated viscera in animals dying after type A toxin. Animals given type D toxin displayed mydriasis and urinary retention before death.3. Motor responses to electrical stimulation, of bladder preparations in vitro were more vulnerable to type D than to type A toxin. When somatic paralysis was complete in animals treated with type A or type D toxin the excised bladders produced pressure elevations 45 and 25%, respectively, of control preparations.4. During electrical stimulation of bladder preparations nearly paralysed by either toxin, the ACh release was significantly diminished from controls. In the rat bladder botulinum toxin specifically disrupted the liberation of mediator from post-ganglionic nerve endings.     

Calcium binding activity by chick intestinal brush-border membrane vesicles

Uptake of Ca²⁺ by vesicle preparation of chick intestinal brush-border membranes was rapid and extensive. With tracer quantities of Ca²⁺ uptake was complete in 10 min whereas with 2.0 mM Ca²⁺ maximum uptake by the vesicles occurred after one hour incubation. The maximum concentration of Ca²⁺ found in the vesicles was four times greater than the external Ca²⁺ concentration showing that the majority of the Ca²⁺ was membrane bound.The Ca²⁺ taken up by the vesicles was probably bound to the vesicle's interior since it was not replaced by exposure of loaded vesicles to La³⁺ (5 mM). The uptake of Ca²⁺ by the vesicles at different Ca²⁺ concentrations was analyzed and a high affinity Ca²⁺ binding site was found with an association constant for Ca²⁺ of 5×10^–5 M. More of these sites were found in the duodenum than the ileum and vitamin D increases the number of these sites.     

Fast exocytosis and endocytosis triggered by depolarisation in single adrenal chromaffin cells before rapid Ca2+ current run-down

The kinetics of exocytosis and membrane retrieval (endocytosis) were examined in bovine chromaffin cells using membrane capacitance measurement during whole-cell recording. At early times after breakthrough to the whole-cell recording mode, depolarisation for 1 s resulted in a fast (600 vesicles per s) exocytotic response and efficient membrane retrieval with a time constant of 25 s. The ability to activate fast exocytosis and retrieval was lost during intracellular dialysis, with a time constant of 40 s. At later times, a slow exocytotic response could be elicited with no membrane retrieval following single depolarisations. The wash-out of the responses appeared to be due to a rapid loss of a portion of the Ca²⁺ current. Trains of depolarisation at late times after breakthrough could elicit a fast (time constant 4 s) retrieval. These data show that in addition to a previously studied slow Ca²⁺-independent retrieval mechanism, chromaffin cells also possess an efficient and rapid retrieval pathway coupled to exocytosis that can be activated following depolarisation. The fast endocytosis appears to have a higher threshold for activation than exocytosis, probably due to a higher Ca²⁺ requirement. Rapid membrane retrieval appears to occur via a clathrin-independent pathway in chromaffin cells.