Acute destruction of the synaptic ribbon reveals a role for the ribbon in vesicle priming

In vision, balance and hearing, sensory receptor cells translate sensory stimuli into electrical signals whose amplitude is graded with stimulus intensity. The output synapses of these sensory neurons must provide fast signaling to follow rapidly changing stimuli while also transmitting graded information covering a wide range of stimulus intensity and must be able to sustain this signaling for long time periods. To meet these demands, specialized machinery for transmitter release, the synaptic ribbon, has evolved at the synaptic outputs of these neurons. We found that acute disruption of synaptic ribbons by photodamage to the ribbon markedly reduced both sustained and transient components of neurotransmitter release in mouse bipolar cells and salamander cones without affecting the ultrastructure of the ribbon or its ability to localize synaptic vesicles to the active zone. Our results indicate that ribbons mediate both slow and fast signaling at sensory synapses and support an additional role for the synaptic ribbon in priming vesicles for exocytosis at active zones.     

Temperature enhances exocytosis efficiency at the mouse inner hair cell ribbon synapse

Hearing relies on fast and sustained neurotransmitter release from inner hair cells (IHCs) onto the afferent auditory nerve fibres. The temperature dependence of Ca²⁺ current and transmitter release at the IHCs ribbon synapse has not been investigated thus far. To assess the influence of temperature on calcium-triggered exocytosis, patch-clamp recordings of voltage-gated L-type Ca²⁺ influx and exocytic membrane capacitance changes were performed at room (25°C) and physiological (35–37°C) temperatures. An increase in temperature within this range increased the L-type Ca²⁺ current amplitude of IHCs ( Q ₁₀= 1.3) and accelerates the activation kinetics. Fast exocytosis, probed by 20 ms depolarization, was enhanced at physiological temperature with a Q ₁₀ of 2.1. The amplitude of fast release was elevated disproportionately to the increase in Ca²⁺ influx. In contrast, the rate of sustained exocytosis (exocytic rate between 20 and 100 ms of depolarization) did not show a significant increase at physiological temperature. Altogether, these data indicate that the efficiency of fast exocytosis is higher at physiological temperature than at room temperature and suggest that the number of readily releasable vesicles available at the active zone is higher at physiological temperature.     

Effects of cholesterol oxidation products on exocytosis

Increase in levels of oxysterols or cholesterol oxidation products have been detected in brain areas undergoing neuroinflammation after excitotoxic injury, and the present study was carried out to elucidate possible effects of these products on exocytosis in rat pheochromocytoma-12 (PC12) cells. An increase in vesicle fusion with the cell membrane indicating exocytosis was observed by total internal reflection microscopy (TIRFM), and confirmed by capacitance measurements, after addition of 7 ketocholesterol, 24 hydroxycholesterol or cholesterol 5, 6 beta epoxide. 7 ketocholesterol induced exocytosis was attenuated by pretreatment with a disruptor of cholesterol-rich domains or “lipid rafts”, methyl-β-cyclodextrin (MβCD) as demonstrated by capacitance and amperometry measurements of neurotransmitter release. Moreover, treatment of cells with thapsigargin to deplete intracellular calcium, or treatment of cells with lanthanum chloride to block calcium channels resulted in attenuation of 7 ketocholesterol induced exocytosis. Fura-2 imaging showed that 7 ketocholesterol induced rapid and sustained increases in intracellular calcium concentration, and that this effect was attenuated in cells that were pre-treated with MβCD, thapsigargin or lanthanum chloride. Together, the results suggest that neurotransmitter release triggered by 7 ketocholesterol is dependent on the integrity of cholesterol rich lipid domains on cellular membranes and a rise in intracellular calcium, either through release from internal stores or influx via calcium channels. Increased cholesterol oxidation product concentrations in brain areas undergoing neuroinflammation may enhance exocytosis and neurotransmitter release, thereby aggravating excitotoxicity.     

Calcium influx through N-methyl-D-aspartate receptors triggers GABA release at interneuron-Purkinje cell synapse in rat cerebellum

Ca(2+)-dependent neurotransmitter release was originally thought to occur only following activation of presynaptic voltage-gated calcium channels after a presynaptic action potential. Recent evidence suggests that not only opening of voltage-gated but also ligand-gated ion channels, such as neurotransmitter receptors, can trigger exocytosis, as well as Ca(2+) release from intracellular Ca(2+) stores. It was shown that activation of N-methyl-d-aspartate (NMDA) receptors on presynaptic interneurons led to increases in GABA release from these neurons onto postsynaptic Purkinje cells in rat cerebellum in the presence of tetrodotoxin (TTX), suggesting a presynaptic location for the underlying NMDA receptors. However, the mechanism for the NMDA-induced increase in GABA release remained unclear. The present study addresses the question whether Ca(2+) influx through presynaptic NMDA receptors alone is sufficient to trigger presynaptic GABA release at this synapse or whether activation of presynaptic NMDA receptors leads to opening of voltage-gated Ca(2+) channels, thereby increasing exocytosis. The results suggest that the NMDA-induced increase in presynaptic GABA release neither requires activation of presynaptic voltage-gated Ca(2+) channels nor Ca(2+) release from presynaptic Ca(2+) stores. It is concluded that Ca(2+) influx through the NMDA receptor alone is sufficient to drive presynaptic GABA release at the rat interneuron-Purkinje cell synapse.     

Dopamine neurons release transmitter via a flickering fusion pore

A key question in understanding mechanisms of neurotransmitter release is whether the fusion pore of a synaptic vesicle regulates the amount of transmitter released during exocytosis. We measured dopamine release from small synaptic vesicles of rat cultured ventral midbrain neurons using carbon fiber amperometry. Our data indicate that small synaptic vesicle fusion pores flicker either once or multiple times in rapid succession, with each flicker releasing approximately 25-30% of vesicular dopamine. The incidence of events with multiple flickers was reciprocally regulated by phorbol esters and staurosporine. Thus, dopamine neurons regulate the amount of neurotransmitter released by small synaptic vesicles by controlling the number of fusion pore flickers per exocytotic event. This mode of exocytosis is a potential mechanism whereby neurons can rapidly reuse vesicles without undergoing the comparatively slow process of recycling.     

Genetic ablation of the t-SNARE SNAP-25 distinguishes mechanisms of neuroexocytosis.

Axon outgrowth during development and neurotransmitter release depends on exocytotic mechanisms, although what protein machinery is common to or differentiates these processes remains unclear. Here we show that the neural t-SNARE (target-membrane-associated-soluble N-ethylmaleimide fusion protein attachment protein (SNAP) receptor) SNAP-25 is not required for nerve growth or stimulus-independent neurotransmitter release, but is essential for evoked synaptic transmission at neuromuscular junctions and central synapses. These results demonstrate that the development of neurotransmission requires the recruitment of a specialized SNARE core complex to meet the demands of regulated exocytosis.     

Large releasable pool of synaptic vesicles in chick cochlear hair cells

Hearing requires the hair cell synapse to maintain notable temporal fidelity (< or =1 ms) while sustaining neurotransmitter release for prolonged periods of time (minutes). Here we probed the properties and possible anatomical substrate of prolonged neurotransmitter release by using electrical measures of cell surface area as a proxy for neurotransmitter release to study hair cell exocytosis evoked by repetitive stimuli. We observed marked depression of exocytosis by chick tall hair cells. This exocytic depression cannot be explained by calcium current inactivation, presynaptic autoinhibition by metabotropic glutamate receptors, or postsynaptic receptor desensitization. Rather, cochlear hair cell exocytic depression resulted from the exhaustion of a functional vesicle pool. This releasable vesicle pool is large, totaling approximately 8,000 vesicles, and is nearly 10 times greater than the number of vesicles tethered to synaptic ribbons. Such a large functional pool suggests the recruitment of cytoplasmic vesicles to sustain exocytosis, important for maintaining prolonged, high rates of neural activity needed to encode sound.     

Role of brain-derived neurotrophic factor and presynaptic proteins in passive avoidance learning in day-old domestic chicks.

The consolidation of a one-trial passive avoidance learning task in the day-old chick involves a number of transient and longer-term biochemical processes, including increased release of glutamate. This study demonstrated that brain-derived neurotrophic factor, a proposed modulator of synaptic transmission and neurotransmitter release, is involved in the cascade associated with memory consolidation in the chick and that its actions were linked to modulation of expression of SNAP-25, syntaxin and synaptophysin, required for exocytosis. Intracerebral injections of 5 microl of antibodies to brain-derived neurotrophic factor into the left and right intermediate medial hyperstriatum ventrale resulted in a dose-dependent reduction in avoidance of an "aversive" bead by 3 h after training. Neurotrophin antibodies (0.5 microg/chick) administered between 1 h before, and up to 30 min after, training induced amnesia by 3 h which was sustained for at least 24 h. Injections of recombinant brain-derived neurotrophic factor (50 microg/ml; 0.5 microg/chick) just before training maintained avoidance in birds trained with a weaker aversant (10% methylanthranilate), such that chicks showed enhanced recall at times (24 h) beyond that when shorter-term forms of memory have decayed. In lysed synaptosomal membranes prepared from chicks injected with antibodies to brain-derived neurotrophic factor there was a decrease in expression of SNAP-25 and syntaxin in the left, but not the right, intermediate medial hyperstriatum ventrale, a region known to be associated with memory formation, which correlated with the decrease in neurotrophin concentration. Thus, these data indicate that brain-derived neurotrophic factor is involved in the formation of a long-term memory for an aversive stimulus and may function as a modulator of presynaptic proteins associated with exocytosis, enabling increases in neurotransmitter release.     

Potassium ion- and nitric oxide-induced exocytosis from populations of hippocampal synapses during synaptic maturation in vitro

The development of mechanisms of neurotransmitter release is an important component in the formation of functional synaptic connections. Synaptic neurotransmitter release can be modulated by nitric oxide, a compound shown to have a variety of physiologic functions in the nervous system. The goal of this study was to determine whether, during synaptic maturation, nitric oxide is capable of affecting exocytosis of synaptic vesicles, and to compare its effects with those elicited by strongly depolarizing stimuli. To address these questions we examined vesicle release from large numbers of individual synapses of hippocampal neurons between five and 13 days in culture. Synaptic vesicles were labelled by uptake of the styrylpyridinium dye N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43) and their release was monitored by fluorescence imaging. Across populations of developing synapses, there was a good correspondence between FM1-43 staining and synapsin immunocytochemistry. A marked heterogeneity was observed in the ability to release vesicles both after potassium and nitric oxide stimulation. In less mature populations of synapses, the rate of potassium- and nitric oxide-induced exocytosis gradually increased, while at later stages nitric oxide-induced responses levelled off and potassium-induced responses continued to rise. Application of nitric oxide donors did not trigger any detectable changes in intracellular calcium. Combined immunocytochemical analysis of cultured hippocampal neurons for neuronal nitric oxide synthase and synapsin revealed that nitric oxide synthase was present within neurites of cultured hippocampal neurons, largely distributed in a bead-like pattern which partially overlapped presynaptic sites. Stimulation of the N-methyl-d-aspartate receptor while blocking propagation of action potentials with tetrodotoxin resulted in exocytosis from numerous individually resolved sites. Preincubation of neurons with an nitric oxide synthase inhibitor or addition of an nitric oxide scavenger eliminated these responses indicating a role for nitric oxide in N-methyl-d-aspartate-stimulated exocytosis.Using fluorescence imaging of individually resolved synaptic sites, we provide direct evidence for an effect of nitric oxide on vesicular neurotransmitter release in intact neurons. Nitric oxide is capable to produce this effect at all stages of synaptic development and acts independently of calcium influx. We show that nitric oxide synthase is present at synaptic sites and endogenously produced nitric oxide is sufficient to cause exocytosis.Taken together, these experiments suggest a possible role for nitric oxide in calcium-independent transmitter release in populations of synapses at all stages of maturation.     

Two sites of action for synapsin domain E in regulating neurotransmitter release

Synapsins, a family of synaptic vesicle proteins, have been shown to regulate neurotransmitter release; the mechanism(s) by which they act are not fully understood. Here we have studied the role of domain E of synapsins in neurotransmitter release at the squid giant synapse. Two squid synapsin isoforms were cloned and found to contain a carboxy (C)-terminal domain homologous to domain E of the vertebrate a-type synapsin isoforms. Presynaptic injection of a peptide fragment of domain E greatly reduced the number of synaptic vesicles in the periphery of the active zone, and increased the rate and extent of synaptic depression, suggesting that domain E is essential for synapsins to regulate a reserve pool of synaptic vesicles. Domain E peptide had no effect on the number of docked synaptic vesicles, yet reversibly inhibited and slowed the kinetics of neurotransmitter release, indicating a second role for synapsins that is more intimately associated with the release process itself. Thus, synapsin domain E is involved in at least two distinct reactions that are crucial for exocytosis in presynaptic terminals.     

Visualization of transmitter release with zinc fluorescence detection at the mouse hippocampal mossy fibre synapse

Exocytosis of synaptic vesicle contents defines the quantal nature of neurotransmitter release. Here we developed a technique to directly assess exocytosis by measuring vesicular zinc release with the zinc-sensitive dye FluoZin-3 at the hippocampal mossy fibre (MF) synapse. Using a photodiode, we were able to clearly resolve the zinc fluorescence transient ([Zn²⁺]_t) with a train of five action potentials in mouse hippocampal brain slices. The vesicular origin of [Zn²⁺]_t was verified by the lack of zinc signal in vesicular zinc transporter Znt3-deficient mice. Manipulating release probability with the application of neuromodulators such as DCG IV, 4-aminopyridine and forskolin as well as a paired train stimulation protocol altered both the [Zn²⁺]_t and the field excitatory postsynaptic potential (fEPSP) coordinately, strongly indicating that zinc is co-released with glutamate during exocytosis. Since zinc ions colocalize with glutamate in small clear vesicles and modulate postsynaptic excitability at NMDA and GABA receptors, the findings establish zinc as a cotransmitter during physiological signalling at the mossy fibre synapse. The ability to directly visualize release dynamics with zinc imaging will facilitate the exploration of the molecular pharmacology and plasticity of exocytosis at MF synapses.     

T-type channel-mediated neurotransmitter release

Besides controlling a wide variety of cell functions, T-type channels have been shown to regulate neurotransmitter release in peripheral and central synapses and neuroendocrine cells. Growing evidence over the last 10 years suggests a key role of Cav3.2 and Cav3.1 channels in controlling basal neurosecretion near resting conditions and sustained release during mild stimulations. In some cases, the contribution of low-voltage-activated (LVA) channels is not directly evident but requires either the activation of coupled presynaptic receptors, block of ion channels, or chelation of metal ions. Concerning the coupling to the secretory machinery, T-type channels appear loosely coupled to neurotransmitter and hormone release. In neurons, Cav3.2 and Cav3.1 channels mainly control the asynchronous appearance of “minis” [miniature inhibitory postsynaptic currents (mIPSCs) and miniature excitatory postsynaptic currents (mEPSCs)]. The same loose coupling is evident from membrane capacity and amperometric recordings in chromaffin cells and melanotropes where the low-threshold-driven exocytosis possesses the same linear Ca²⁺ dependence of the other voltage-gated Ca²⁺ channels (Cav1 and Cav2) that is strongly attenuated by slow calcium buffers. The intriguing issue is that, despite not expressing a consensus “synprint” site, Cav3.2 channels do interact with syntaxin 1A and SNAP-25 and, thus, may form nanodomains with secretory vesicles that can be regulated at low voltages. In this review, we discuss all the past and recent issues related to T-type channel-secretion coupling in neurons and neuroendocrine cells.     

Differential modulation of short-term synaptic dynamics by long-term potentiation at mouse hippocampal mossy fibre synapses

After exocytosis, synaptic vesicle components are selectively retrieved by clathrin-mediated endocytosis and then re-used in future rounds of transmitter release. Under some conditions, synaptic terminals in addition perform bulk endocytosis of large membranous sacs. Bulk endocytosis is less selective than clathrin-mediated endocytosis and probably internalizes components normally targeted to the plasma membrane. Nonetheless, this process plays a major role in some tonic ribbon-type synapses, which release neurotransmitter for prolonged periods of time. We show here, that large endosomes formed after strong and prolonged stimulation undergo stimulated exocytosis in retinal bipolar neurons. The result suggests how cells might return erroneously internalized components to the plasma membrane, and also demonstrates that synaptic vesicles are not the only neuronal organelle that stains with styryl dyes and undergoes stimulated exocytosis.     

Group IIA secretory phospholipase A2 stimulates exocytosis and neurotransmitter release in pheochromocytoma-12 cells and cultured rat hippocampal neurons

Recent evidence shows that secretory phospholipase A2 (sPLA2) may play a role in membrane fusion and fission, and may thus affect neurotransmission. The present study therefore aimed to elucidate the effects of sPLA2 on vesicle exocytosis. External application of group IIA sPLA2 (purified crotoxin subunit B or purified human synovial sPLA2) caused an immediate increase in exocytosis and neurotransmitter release in pheochromocytoma-12 (PC12) cells, detected by carbon fiber electrodes placed near the cells, or by changes in membrane capacitance of the cells. EGTA and a specific inhibitor of sPLA2 activity, 12-epi-scalaradial, abolished the increase in neurotransmitter release, indicating that the effect of sPLA2 was dependent on calcium and sPLA2 enzymatic activity. A similar increase in neurotransmitter release was also observed in hippocampal neurons after external application of sPLA2, as detected by changes in membrane capacitance of the neurons. In contrast to external application, internal application of sPLA2 to PC12 cells and neurons produced blockade of neurotransmitter release. Our recent studies showed high levels of sPLA2 activity in the normal rat hippocampus, medulla oblongata and cerebral neocortex. The sPLA2 activity in the hippocampus was significantly increased, after kainate-induced neuronal injury. The observed effects of sPLA2 on neurotransmitter release in this study may therefore have a physiological, as well as a pathological role.     

Investigation of the modulation of glutamate release by sodium channels using neurotoxins

The modulation of neurotransmitter release by calcium channels is well established, yet, sodium channels were regarded mainly as charge carriers. Many lines of evidence suggest a more fine-tuning role played by sodium channels. Using rat cerebrocortical isolated nerve endings (synaptosomes) and two toxins that have separate sites of action on sodium channels and provoke distinct changes in channel kinetics, we were able to show that depending on the rate of increase in channel conductance, the outcome in terms of neurotransmitter release and calcium channel types coupled to that event are different. Mainly, our study focused on veratridine, an alkaloid from lilaceous plants that binds to sodium channel toxin site 2, and tityustoxin, a toxin purified from the venom of the Brazilian yellow scorpion Tityus serrulatus that binds to site 3. Veratridine induces a slower increase in intrasynaptosomal sodium and calcium concentrations, slower depolarization, delayed exocytosis and a slower and predominantly calcium-independent glutamate release, when compared to tityustoxin.Thus, we have used these two toxins to investigate the events that start with sodium entry and culminate with the release of glutamate in isolated nerve endings (synaptosomes) from rat cerebral cortex. With that in mind we measured intrasynaptosomal free sodium concentration [Na(+)](i), intrasynaptosomal free calcium concentration [Ca(2+)](i), membrane potential, exocytosis and glutamate release using fluorescent probes.     

The D1 dopamine receptor agonist SKF-38393 stimulates the release of glutamate in the hippocampus

The present study was undertaken to better assess the role of dopamine on exocytosis. Since direct activation of adenylate cyclase (e.g., with forskolin) enhances neurotransmitter release it was of interest to see whether the activation of D1-type dopamine receptors, which are positively coupled to adenylate cyclase, could also modulate the molecular machinery underlying the fusion of synaptic vesicles and the release of neurotransmitter. To answer this question we have looked at the effect of the D1-type dopamine receptor agonist SKF-38393 on the spontaneous release of glutamate from cultured rat hippocampal neurons. SKF-38393 enhanced the frequency but not the amplitude of tetrodotoxin-resistant excitatory postsynaptic currents which argues for a presynaptic locus of D1 action. This effect was blocked by the D1-dopaminergic receptor antagonist SCH-23390 and the protein kinase A inhibitors H-7 and Rp-cAMP whereas pertussis toxin failed to affect the dopaminergic response. In addition, carbachol and Ruthenium Red also stimulated exocytosis but did not occlude the SKF-38393-induced modulation. These results indicate that SKF-38393 presynaptically enhances the release of glutamate via a pertussis toxin-insensitive and protein kinase A-dependent mechanism, which most likely involves D1-type dopamine receptors. Our results underline the importance of protein kinase A as potent modulator of synaptic transmission and suggest that high concentrations of dopamine can greatly enhance the release of glutamate in the hippocampus.     

Activity-dependent changes in partial VAMP complexes during neurotransmitter release

The temporal sequence of SNARE protein interactions that cause exocytosis is unknown. Blockade of synaptic neurotransmitter release through cleavage of VAMP/synaptobrevin by tetanus toxin light chain (TeNT-LC) was accelerated by nerve stimulation. Botulinum/B neurotoxin light chain (BoNT/B-LC), which cleaves VAMP at the same site as TeNT-LC, did not require stimulation. Because TeNT-LC requires the N-terminal coil domain of VAMP for binding but BoNT/B-LC requires the C-terminal coil domain, it seems that, before nerve activity, the N-terminal domain is shielded in a protein complex, but the C-terminal domain is exposed. This N-terminal complex lasts until nerve activity occurs and may serve to cock synaptic vesicles for immediate exocytosis upon Ca2+ entry.     

Liprin-α is involved in exocytosis and cell spreading in mast cells

The active zone is a specialized region of the presynaptic plasma membrane where the neurotransmitter release occurs by exocytosis. Mast cells also release inflammatory mediators by exocytosis resulting in induction of allergic responses. In our previous reports, we found that active zone proteins, Munc13-1 and ELKS regulates exocytosis of mast cell positively. In this study, we investigated the involvement of liprin-α, another active zone protein, in exocytosis in mast cells. We found that three isoforms of liprin-α, liprin-α1, -α2 and -α3 were expressed. Immunocytochemical experiments revealed that liprin-α1 resided both in the cytoplasm and on the plasma membrane. Upon stimulation with antigen, the area of a cell increased remarkably due to cell spreading and the distribution of liprin-α1 became punctuated. Interestingly, knockdown of liprin-α1 caused decrease in exocytotic release and cell spreading. These results suggest that liprin-α1 facilitates exocytosis and cell spreading, and these events might have correlated each other in mast cells.