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A single sub-anesthetic dose of ketamine evokes rapid and long-lasting beneficial effects in patients with a major depressive disorder. However, the mechanisms underlying this effect are unknown. It has been proposed that astrocyte dysregulation of extracellular K + concentration ([K + ] o ) alters neuronal excitability, thus contributing to depression. We examined how ketamine affects inwardly rectifying K + channel Kir4.1, the principal regulator of K + buffering and neuronal excitability in the brain. Cultured rat cortical astrocytes were transfected with plasmid-encoding fluorescently tagged Kir4.1 (Kir4.1-EGFP) to monitor the mobility of Kir4.1-EGFP vesicles at rest and after ketamine treatment (2.5 or 25 µM). Short-term (30 min) ketamine treatment reduced the mobility of Kir4.1-EGFP vesicles compared with the vehicle-treated controls ( p < 0.05). Astrocyte treatment (24 h) with dbcAMP (dibutyryl cyclic adenosine 5'-monophosphate, 1 mM) or [K + ] o (15 mM), which increases intracellular cAMP, mimicked the ketamine-evoked reduction of mobility. Live cell immunolabelling and patch-clamp measurements in cultured mouse astrocytes revealed that short-term ketamine treatment reduced the surface density of Kir4.1 and inhibited voltage-activated currents similar to Ba 2+ (300 µM), a Kir4.1 blocker. Thus, ketamine attenuates Kir4.1 vesicle mobility, likely via a cAMP-dependent mechanism, reduces Kir4.1 surface density, and inhibits voltage-activated currents similar to Ba 2+ , known to block Kir4.1 channels.

Regulated exocytosis consists of the fusion between vesicles and the plasma membranes, leading to the formation of a narrow fusion pore through which secretions exit the vesicle lumen into the extracellular space. An increase in the cytosolic concentration of free Ca 2+ ([Ca 2+ ] i ) is considered the stimulus of this process. However, whether this mechanism can be preserved in a simplified system of membrane lawns with docked secretory vesicles, devoid of cellular components, is poorly understood. Here, we studied peptide discharge from individual secretory vesicles docked at the plasma membrane, prepared from primary endocrine pituitary cells (the lactotrophs), releasing hormone prolactin. To label secretory vesicles, we transfected lactotrophs to express the fluorescent atrial natriuretic peptide (ANP.emd), previously shown to be expressed in and released from prolactin-containing vesicles. We used stimulating solutions containing different [Ca 2+ ] to evoke vesicle peptide discharge, which appeared similar in membrane lawns and in intact stimulated lactotrophs. All vesicles examined discharged peptides in a subquantal manner, either exhibiting a unitary or sequential time course. In the membrane lawns, the unitary vesicle peptide discharge was predominant and slightly slower than that recorded in intact cells, but with a shorter delay with respect to the stimulation onset. This study revealed directly that Ca 2+ triggers peptide discharge from docked single vesicles in the membrane lawns with a half-maximal response of ∼8 µM [Ca 2+ ], consistent with previous whole-cell patch-clamp studies in endocrine cells where the rapid component of exocytosis, interpreted to represent docked vesicles, was fully activated at <10 µM [Ca 2+ ]. Interestingly, the sequential subquantal peptide vesicle discharge indicates that fluctuations between constricted and dilated fusion pore states are preserved in membrane lawns and that fusion pore regulation appears to be an autonomously controlled process., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Interactive mechanical forces between pairs of individual SNARE proteins synaptobrevin 2 (Sb2) and syntaxin 1A (Sx1A) may be sufficient to mediate vesicle docking. This notion, based on force spectroscopy single molecule measurements probing recombinant Sx1A an Sb2 in silico, questioned a predominant view of docking via the ternary SNARE complex formation, which includes an assembly of the intermediate cis binary complex between Sx1A and SNAP25 on the plasma membrane to engage Sb2 on the vesicle. However, whether a trans binary Sx1A-Sb2 complex alone could mediate vesicle docking in a cellular environment remains unclear. To address this issue, we used atomic force microscopy (AFM) in the force spectroscopy mode combined with fluorescence imaging. Using AFM tips functionalized with the full Sx1A cytosolic domain, we probed native Sb2 studding the membrane of secretory vesicles docked at the plasma membrane patches, referred to as "inside-out lawns", identified based on fluorescence stains and prepared from primary culture of lactotrophs. We recorded single molecule Sx1A-Sb2 mechanical interactions and obtained measurements of force (∼183 pN) and extension (∼21.6 nm) necessary to take apart Sx1A-Sb2 binding interactions formed at tip-vesicle contact. Measured interactive force between a single pair of Sx1A-Sb2 molecules is sufficient to hold a single secretory vesicle docked at the plasma membrane within distances up to that of the measured extension. This finding further advances a notion that native vesicle docking can be mediated by a single trans binary Sx1A-Sb2 complex in the absence of SNAP25., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In some lysosomal storage diseases (LSD) cholesterol accumulates in vesicles. Whether increased vesicle cholesterol affects vesicle fusion with the plasmalemma, where the fusion pore, a channel between the vesicle lumen and the extracellular space, is formed, is unknown. Super-resolution microscopy revealed that after stimulation of exocytosis, pituitary lactotroph vesicles discharge cholesterol which transfers to the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes, both exhibiting Ca 2+ -dependent exocytosis regulated by distinct Ca 2+ sources, evokes vesicle secretion. Although this treatment enhanced cytosolic levels of Ca 2+ in lactotrophs but decreased it in astrocytes, this indicates that cholesterol may well directly define the fusion pore. In an attempt to explain this mechanism, a new model of cholesterol-dependent fusion pore regulation is proposed. High-resolution membrane capacitance measurements, used to monitor fusion pore conductance, a parameter related to fusion pore diameter, confirm that at resting conditions reducing cholesterol increases, while enrichment with cholesterol decreases the conductance of the fusion pore. In resting fibroblasts, lacking the Npc1 protein, a cellular model of LSD in which cholesterol accumulates in vesicles, the fusion pore conductance is smaller than in controls, showing that vesicle cholesterol controls fusion pore and is relevant for pathophysiology of LSD., (Copyright © 2021. Published by Elsevier Ltd.)

The plasticity of astrocytes is fundamental for their principal function, maintaining homeostasis of the central nervous system throughout life, and is associated with diverse exposomal challenges. Here, we used cultured astrocytes to investigate at subcellular level basic cell processes under controlled environmental conditions. We compared astroglial functional and signaling plasticity in standard serum-containing growth medium, a condition mimicking pathologic conditions, and in medium without serum, favoring the acquisition of arborized morphology. Using opto-/electrophysiologic techniques, we examined cell viability, expression of astroglial markers, vesicle dynamics, and cytosolic Ca 2+ and cAMP signaling. The results revealed altered vesicle dynamics in arborized astrocytes that was associated with increased resting [Ca 2+ ] i and increased subcellular heterogeneity in [Ca 2+ ] i , whereas [cAMP] i subcellular dynamics remained stable in both cultures, indicating that cAMP signaling is less prone to plastic remodeling than Ca 2+ signaling, possibly also in in vivo contexts., (© 2021 The Authors. GLIA published by Wiley Periodicals LLC.)

Major depressive disorders (MDD) a worldwide psychiatric disease, is yet to be adequately controlled by therapies; while the mechanisms of action of antidepressants are yet to be fully characterised. In the last two decades, an increasing number of studies have demonstrated the role of astrocytes in the pathophysiology and therapy of MDD. Selective serotonin reuptake inhibitors (SSRIs) are the most widely used antidepressants. It is generally acknowledged that SSRIs increase serotonin levels in the central nervous system by inhibiting serotonin transporters, although the SSRIs action is not ideal. The SSRIs antidepressant effect develops with considerable delay; their efficacy is low and frequent relapses are common. Neither cellular nor molecular pharmacological mechanisms of SSRIs are fully characterised; in particular their action on astrocytes remain underappreciated. In this paper we overview potential therapeutic mechanisms of SSRIs associated with astroglia and report the results of meta-analysis of studies dedicated to MDD, SSRIs and astrocytes. In particular, we argue that fluoxetine, the representative SSRI, improves depressive-like behaviours in animals treated with chronic mild stress and reverses depression-associated decrease in astrocytic glial fibrillary acidic protein (GFAP) expression. In addition, fluoxetine upregulates astrocytic mRNA expression of 5-hydroxytriptamin/serotonin 2B receptors (5-HT 2B R). In summary, we infer that SSRIs exert their anti-depressant effect by regulating several molecular and signalling pathways in astrocytes., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

Ketamine, a non-competitive N -methyl-d-aspartate receptor (NMDAR) antagonist, exerts a rapid, potent and long-lasting antidepressant effect, although the cellular and molecular mechanisms of this action are yet to be clarified. In addition to targeting neuronal NMDARs fundamental for synaptic transmission, ketamine also affects the function of astrocytes, the key homeostatic cells of the central nervous system that contribute to pathophysiology of major depressive disorder. Here, I review studies revealing that (sub)anesthetic doses of ketamine elevate intracellular cAMP concentration ([cAMP] i ) in astrocytes, attenuate stimulus-evoked astrocyte calcium signaling, which regulates exocytotic secretion of gliosignaling molecules, and stabilize the vesicle fusion pore in a narrow configuration, possibly hindering cargo discharge or vesicle recycling. Next, I discuss how ketamine affects astrocyte capacity to control extracellular K + by reducing vesicular delivery of the inward rectifying potassium channel (K ir 4.1) to the plasmalemma that reduces the surface density of Kir4.1. Modified astroglial K + buffering impacts upon neuronal firing pattern as demonstrated in lateral habenula in a rat model of depression. Finally, I highlight the discovery that ketamine rapidly redistributes cholesterol in the astrocyte plasmalemma, which may alter the flux of cholesterol to neurons. This structural modification may further modulate a host of processes that synergistically contribute to ketamine's rapid antidepressant action.

Ketamine, a non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist, exerts rapid, potent and long-lasting antidepressant effect already after a single administration of a low dose into depressed individuals. Apart from targeting neuronal NMDARs essential for synaptic transmission, ketamine also interacts with astrocytes, the principal homoeostatic cells of the central nervous system. The cellular mechanisms underlying astrocyte-based rapid antidepressant effect are incompletely understood. Here we overview recent data that describe ketamine-dependent changes in astrocyte cytosolic cAMP activity ([cAMP] i ) and ketamine-induced modifications of stimulus-evoked Ca 2+ signalling. The latter regulates exocytotic release of gliosignalling molecules and stabilizes the vesicle fusion pore in a narrow configuration that obstructs cargo discharge or vesicle membrane recycling. Ketamine also instigates rapid redistribution of cholesterol in the astrocyte plasmalemma that may alter flux of cholesterol to neurones, where it is required for changes in synaptic plasticity. Finally, ketamine attenuates mobility of vesicles carrying the inward rectifying potassium channel (K ir 4.1) and reduces the surface density of K ir 4.1 channels that control extracellular K + concentration, which tunes the pattern of action potential firing in neurones of lateral habenula as demonstrated in a rat model of depression. Thus, diverse, but not mutually exclusive, mechanisms act synergistically to evoke changes in synaptic plasticity leading to sustained strengthening of excitatory synapses necessary for rapid antidepressant effect of ketamine., (© 2021. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Ketamine, a general anaesthetic and psychotomimetic drug, exerts rapid, potent and long-lasting antidepressant effect, albeit the cellular and molecular mechanisms of this action are yet to be discovered. Besides targeting neuronal NMDARs fundamental for synaptic transmission, ketamine affects the function of astroglia the key homeostatic cells of the central nervous system that contribute to pathophysiology of psychiatric diseases including depression. Here we review studies revealing that (sub)anaesthetic doses of ketamine elevate intracellular cAMP concentration ([cAMP] i ) in astrocytes, attenuate stimulus-evoked astrocyte calcium signalling, which regulates exocytotic secretion of gliosignalling molecules, and stabilize the vesicle fusion pore in a narrow configuration possibly hindering cargo discharge or vesicle recycling. Next we discuss how ketamine affects astroglial capacity to control extracellular K + by reducing cytoplasmic mobility of vesicles delivering the inward rectifying potassium channel (Kir4.1) to the plasmalemma. Modified astroglial K + buffering impacts upon neuronal excitability as demonstrated in the lateral habenula rat model of depression. Finally, we highlight the recent discovery that ketamine rapidly redistributes cholesterol in the plasmalemma of astrocytes, but not in fibroblasts nor in neuronal cells. This alteration of membrane structure may modulate a host of processes that synergistically contribute to ketamine's rapid and prominent antidepressant action., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Astrocytes are the key homeostatic cells in the central nervous system; initiation of reactive astrogliosis contributes to neuroinflammation. Pro-inflammatory cytokine interferon γ (IFNγ) induces the expression of the major histocompatibility complex class II (MHCII) molecules, involved in antigen presentation in reactive astrocytes. The pathway for MHCII delivery to the astrocyte plasma membrane, where MHCII present antigens, is unknown. Rat astrocytes in culture and in organotypic slices were exposed to IFNγ to induce reactive astrogliosis. Astrocytes were probed with optophysiologic tools to investigate subcellular localization of immunolabeled MHCII, and with electrophysiology to characterize interactions of single vesicles with the plasmalemma. In culture and in organotypic slices, IFNγ augmented the astrocytic expression of MHCII, which prominently co-localized with lysosomal marker LAMP1-EGFP, modestly co-localized with Rab7, and did not co-localize with endosomal markers Rab4A, EEA1, and TPC1. MHCII lysosomal localization was corroborated by treatment with the lysosomolytic agent glycyl-L-phenylalanine-β-naphthylamide, which reduced the number of MHCII-positive vesicles. The surface presence of MHCII was revealed by immunolabeling of live non-permeabilized cells. In IFNγ-treated astrocytes, an increased fraction of large-diameter exocytotic vesicles (lysosome-like vesicles) with prolonged fusion pore dwell time and larger pore conductance was recorded, whereas the rate of endocytosis was decreased. Stimulation with ATP, which triggers cytosolic calcium signaling, increased the frequency of exocytotic events, whereas the frequency of full endocytosis was further reduced. In IFNγ-treated astrocytes, MHCII-linked antigen surface presentation is mediated by increased lysosomal exocytosis, whereas surface retention of antigens is prolonged by concomitant inhibition of endocytosis.

Aim: Astrocytes play a homeostatic role in the central nervous system and influence numerous aspects of neurophysiology via intracellular trafficking of vesicles. Intermediate filaments (IFs), also known as nanofilaments, regulate a number of cellular processes including organelle trafficking and adult hippocampal neurogenesis. We have recently demonstrated that the IF protein nestin, a marker of neural stem cells and immature and reactive astrocytes, is also expressed in some astrocytes in the unchallenged hippocampus and regulates neurogenesis through Notch signalling from astrocytes to neural stem cells, possibly via altered trafficking of vesicles containing the Notch ligand Jagged-1., Methods: We thus investigated whether nestin affects vesicle dynamics in astrocytes by examining single vesicle interactions with the plasmalemma and vesicle trafficking with high-resolution cell-attached membrane capacitance measurements and confocal microscopy. We used cell cultures of astrocytes from nestin-deficient (Nes -/- ) and wild-type (wt) mice, and fluorescent dextran and Fluo-2 to examine vesicle mobility and intracellular Ca 2+ concentration respectively., Results: Nes -/- astrocytes exhibited altered sizes of vesicles undergoing full fission and transient fusion, altered vesicle fusion pore geometry and kinetics, decreased spontaneous vesicle mobility and altered ATP-evoked mobility. Purinergic stimulation evoked Ca 2+ signalling that was slightly attenuated in Nes -/- astrocytes, which exhibited more oscillatory Ca 2+ responses than wt astrocytes., Conclusion: These results demonstrate at the single vesicle level that nestin regulates vesicle interactions with the plasmalemma and vesicle trafficking, indicating its potential role in astrocyte vesicle-based communication., (© 2019 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

The finding that ketamine, an anaesthetic, can elicit a rapid antidepressant effect at low doses that lasts for weeks in patients with depression is arguably a major achievement in psychiatry in the last decades. However, the mechanisms of action are unclear. The glutamatergic hypothesis of ketamine action posits that ketamine is a N-methyl-D-aspartate receptor (NMDAR) antagonist modulating downstream cytoplasmic events in neurons. In addition to targeting NMDARs in synaptic transmission, ketamine may modulate the function of astroglia, key homeostasis-providing cells in the central nervous system, also playing a role in many neurologic diseases including depression, which affects to 20% of the population globally. We first review studies on astroglia revealing that (sub)anaesthetic doses of ketamine attenuate stimulus-evoked calcium signalling, a process of astroglial cytoplasmic excitability, regulating the exocytotic release of gliosignalling molecules. Then we address how ketamine alters the fusion pore activity of secretory vesicles, and how ketamine affects extracellular glutamate and K + homeostasis, both considered pivotal in depression. Finally, we also provide evidence indicating reduced cytoplasmic mobility of astroglial vesicles carrying the inward rectifying potassium channel (Kir4.1), which may regulate the density of Kir4.1 at the plasma membrane. These results indicate that the astroglial capacity to control extracellular K + concentration may be altered by ketamine and thus indirectly affect the action potential firing of neurons, as is the case in lateral habenula in a rat disease model of depression. Hence, ketamine-altered functions of astroglia extend beyond neuronal NMDAR antagonism and provide a basis for its antidepressant action through glia.

The intermediate filament (nanofilament) protein nestin is a marker of neural stem cells, but its role in neurogenesis, including adult neurogenesis, remains unclear. Here, we investigated the role of nestin in neurogenesis in adult nestin-deficient (Nes-/-) mice. We found that the proliferation of Nes-/- neural stem cells was not altered, but neurogenesis in the hippocampal dentate gyrus of Nes-/- mice was increased. Surprisingly, the proneurogenic effect of nestin deficiency was mediated by its function in the astrocyte niche. Through its role in Notch signaling from astrocytes to neural stem cells, nestin negatively regulates neuronal differentiation and survival; however, its expression in neural stem cells is not required for normal neurogenesis. In behavioral studies, nestin deficiency in mice did not affect associative learning but was associated with impaired long-term memory., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Astroglia, the primary homeostatic cells of the central nervous system, play an important role in neuroinflammation. They act as facultative immunocompetent antigen-presenting cells (APCs), expressing major histocompatibility complex (MHC) class II antigens upon activation with interferon (IFN)-γ and possibly other proinflammatory cytokines that are upregulated in disease states, including multiple sclerosis (MS). We characterized the anti-inflammatory effects of fingolimod (FTY720), an established drug for MS, and its phosphorylated metabolite (FTY720-P) in IFN-γ-activated cultured rat astrocytes. The expression of MHC class II compartments, β 2 adrenergic receptor (ADR-β 2 ), and nuclear factor kappa-light-chain enhancer of activated B cells subunit p65 (NF-κB p65) was quantified in immunofluorescence images acquired by laser scanning confocal microscopy. In addition, MHC class II-enriched endocytotic vesicles were labeled by fluorescent dextran and their mobility analyzed in astrocytes subjected to different treatments. FTY720 and FTY720-P treatment significantly reduced the number of IFN-γ-induced MHC class II compartments and substantially increased ADR-β 2 expression, which is otherwise small or absent in astrocytes in MS. These effects could be partially attributed to the observed decrease in NF-κB p65 expression, because the NF-κB signaling cascade is activated in inflammatory processes. We also found attenuated trafficking and secretion from dextran-labeled endo-/lysosomes that may hinder efficient delivery of MHC class II molecules to the plasma membrane. Our data suggest that FTY720 and FTY720-P at submicromolar concentrations mediate anti-inflammatory effects on astrocytes by suppressing their action as APCs, which may further downregulate the inflammatory process in the brain, constituting the therapeutic effect of fingolimod in MS.

Ketamine is an antidepressant with rapid therapeutic onset and long-lasting effect, although the underlying mechanism(s) remain unknown. Using FRET-based nanosensors we found that ketamine increases [cAMP] i in astrocytes. Membrane capacitance recordings, however, reveal fundamentally distinct mechanisms of effects of ketamine and [cAMP] i on vesicular secretion: a rise in [cAMP] i facilitated, whereas ketamine inhibited exocytosis. By directly monitoring cholesterol-rich membrane domains with a fluorescently tagged cholesterol-specific membrane binding domain (D4) of toxin perfringolysin O, we demonstrated that ketamine induced cholesterol redistribution in the plasmalemma in astrocytes, but neither in fibroblasts nor in PC 12 cells. This novel mechanism posits that ketamine affects density and distribution of cholesterol in the astrocytic plasmalemma, consequently modulating a host of processes that may contribute to ketamine's rapid antidepressant action.

Once infected by HIV-1, microglia abundantly produce accessory protein Nef that enhances virus production and infectivity, but little is known about its intracellular compartmentalization, trafficking mode(s), and release from microglia. Here, we transfected immortalized human microglia with a plasmid encoding Nef tagged with green fluorescent protein (Nef.GFP) to biochemically and microscopically identify Nef.GFP-associated cellular compartments and examine their mobility and Nef release from cultured cells. Immunoblotting revealed that Nef.GFP confined to subcellular fractions with a buoyant density similar to organelles positive for lysosomal-associated membrane protein 1 (LAMP1) but structurally segregated from dextran-laden and LysoTracker-laden endo-/lysosomes in live cells. As revealed by confocal microscopy, Nef.GFP-positive vesicle-like structures were smaller than dextran-laden vesicles and displayed slow and non-directional mobility, in contrast to the faster and directional mobility of dextran-laden vesicles. Ionomycin-evoked elevation in intracellular free Ca 2+ concentration ([Ca 2+ ] i ) negligibly affected mobility of Nef.GFP structures but strongly and irrecoverably attenuated mobility of dextran-laden vesicles. A slow time-dependent decrease in the number of Nef.GFP-positive structures was observed in non-stimulated controls (5 ± 1 structures/min), but not in ionomycin-stimulated cells (0 ± 2 structures/min; P < 0.05), indicating that elevated [Ca 2+ ] i inhibits the release of Nef.GFP structures. The latter significantly co-localized with membrane sites immunopositive for the tetraspanins CD9 (36 ± 4%) and CD81 (22 ± 1%). This is the first report to demonstrate that microglial CD9- and CD81-positive plasma membrane-derived compartments are associated with biogenesis and Nef release.

PKH lipophilic dyes are highly fluorescent and stain membranes by intercalating their aliphatic portion into the exposed lipid bilayer. They have established use in labeling and tracking of cells in vivo and in vitro. Despite wide use of PKH-labeled extracellular vesicles (EVs) in cell targeting and functional studies, nonEV-associated fluorescent structures have never been examined systematically, nor was their internalization by cells. Here, we have characterized PKH26-positive particles in lymphoblastoid B exosome samples and exosome-free controls stained by ultracentrifugation, filtration, and sucrose-cushion-based and sucrose-gradient-based procedures, using confocal imaging and asymmetric-flow field-flow fractionation coupled to multi-angle light-scattering detector analysis. We show for the first time that numerous PKH26 nanoparticles (nine out of ten PKH26-positive particles) are formed during ultracentrifugation-based exosome staining, which are almost indistinguishable from PKH26-labeled exosomes in terms of size, surface area, and fluorescence intensity. When PKH26-labeled exosomes were purified through sucrose, PKH26 nanoparticles were differentiated from PKH26-labeled exosomes based on their reduced size. However, PKH26 nanoparticles were only physically removed from PKH26-labeled exosomes when separated on a sucrose gradient, and at the expense of low PKH26-labeled exosome recovery. Overall, low PKH26-positive particle recovery is characteristic of filtration-based exosome staining. Importantly, PKH26 nanoparticles are internalized by primary astrocytes into similar subcellular compartments as PKH26-labeled exosomes. Altogether, PKH26 nanoparticles can result in false-positive signals for stained EVs that can compromise the interpretation of EV internalization. Thus, for use in EV uptake and functional studies, sucrose-gradient-based isolation should be the method of choice to obtain PKH26-labeled exosomes devoid of PKH26 nanoparticles., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In the past, vesicle content release was thought to occur immediately and completely after triggering of exocytosis. However, vesicles may merge with the plasma membrane to form an Ångstrom diameter fusion pore that prevents the exit of secretions from the vesicle lumen. The advantage of such a narrow pore is to minimize the delay between the trigger and the release. Instead of stimulating a sequence of processes, leading to vesicle merger with the plasma membrane and a formation of a fusion pore, the stimulus only widens the pre-established fusion pore. The fusion pore may be stable and may exhibit repetitive opening of the vesicle lumen to the cell exterior accompanied by a content discharge. Such release of vesicle content is partial (subquantal), and depends on fusion pore open time, diameter and the diffusibility of the cargo. Such transient mode of fusion pore opening was not confirmed until the development of the membrane capacitance patch-clamp technique, which enables high-resolution measurement of changes in membrane surface area. It allows millisecond dwell-time measurements of fusion pores with subnanometer diameters. Currently, the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) proteins are considered to be key entities in end-stage exocytosis, and the SNARE complex assembly/disassembly may regulate the fusion pore. Moreover, lipids or other membrane constituents with anisotropic (non-axisymmetric) geometry may also favour the establishment of stable narrow fusion pores, if positioned in the neck of the fusion pore., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Dynamin is a multidomain GTPase exhibiting mechanochemical and catalytic properties involved in vesicle scission from the plasmalemma during endocytosis. New evidence indicates that dynamin is also involved in exocytotic release of catecholamines, suggesting the existence of a dynamin-regulated structure that couples endo- to exocytosis., Methods: Thus we here employed high-resolution cell-attached capacitance measurements and super-resolution structured illumination microscopy to directly examine single vesicle interactions with the plasmalemma in cultured rat astrocytes treated with distinct pharmacological modulators of dynamin activity. Fluorescent dextrans and the lipophilic plasmalemmal marker DiD were utilized to monitor uptake and distribution of vesicles in the peri-plasmalemmal space and in the cell cytosol., Results: Dynamin inhibition with Dynole™-34-2 and Dyngo™-4a prevented vesicle internalization into the cytosol and decreased fusion pore conductance of vesicles that remained attached to the plasmalemma via a narrow fusion pore that lapsed into a state of repetitive opening and closing - flickering. In contrast, the dynamin activator Ryngo™-1-23 promoted vesicle internalization and favored fusion pore closure by prolonging closed and shortening open fusion pore dwell times. Immunocytochemical staining revealed dextran uptake into dynamin-positive vesicles and increased dextran uptake into Syt4- and VAMP2-positive vesicles after dynamin inhibition, indicating prolonged retention of these vesicles at the plasmalemma., Conclusions: Our results have provided direct evidence for a role of dynamin in regulation of fusion pore geometry and kinetics of endo- and exocytotic vesicles, indicating that both share a common dynamin-regulated structural intermediate, the fusion pore., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the brain, astrocytes signal to neighboring cells via regulated exocytotic release of gliosignaling molecules, such as brain-derived neurotrophic factor (BDNF). Recent studies uncovered a role of ketamine, an anesthetic and antidepressant, in the regulation of BDNF expression and in the disruption of astrocytic Ca 2+ signaling, but it is unclear whether it affects astroglial BDNF release. We investigated whether ketamine affects ATP-evoked Ca 2+ signaling and exocytotic release of BDNF at the single-vesicle level in cultured rat astrocytes. Cells were transfected with a plasmid encoding preproBDNF tagged with the pH-sensitive fluorescent protein superecliptic pHluorin, (BDNF-pHse) to load vesicles and measure the release of BDNF-pHse when the exocytotic fusion pore opens and alkalinizes the luminal pH. In addition, cell-attached membrane capacitance changes were recorded to monitor unitary vesicle interaction with the plasma membrane. Intracellular Ca 2+ activity was monitored with Fluo-4 and confocal microscopy, which was also used to immunocytochemically characterize BDNF-pHse-laden vesicles. As revealed by double-fluorescent micrographs, BDNF-pHse localized to vesicles positive for the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins, vesicle-associated membrane protein 2 (VAMP2), VAMP3, and synaptotagmin IV. Ketamine treatment decreased the number of ATP-evoked BDNF-pHse fusion/secretion events (P < 0.05), the frequency of ATP-evoked transient (P < 0.001) and full-fusion exocytotic (P < 0.05) events, along with a reduction in the ATP-evoked increase in intracellular Ca 2+ activity in astrocytes by ~70 % (P < 0.001). The results show that ketamine treatment suppresses ATP-triggered vesicle fusion and BDNF secretion by increasing the probability of a narrow fusion pore open state and/or by reducing astrocytic Ca 2+ excitability.

Astrocytes, the most heterogeneous glial cells in the central nervous system, contribute to brain homeostasis, by regulating a myriad of functions, including the clearance of extracellular debris. When cells are damaged, cytoplasmic proteins may exit into the extracellular space. One such protein is S100B, which may exert toxic effects on neighboring cells unless it is removed from the extracellular space, but the mechanisms of this clearance are poorly understood. By using time-lapse confocal microscopy and fluorescently labeled S100B (S100B-Alexa 488 ) and fluorescent dextran (Dextran 546 ), a fluid phase uptake marker, we examined the uptake of fluorescently labeled S100B-Alexa 488 from extracellular space and monitored trafficking of vesicles that internalized S100B-Alexa 488 . Initially, S100B-Alexa 488 and Dextran 546 internalized with distinct rates into different endocytotic vesicles; S100B-Alexa 488 internalized into smaller vesicles than Dextran 546 . At a later stage, S100B-Alexa 488 -positive vesicles substantially co-localized with Dextran 546 -positive endolysosomes and with acidic LysoTracker-positive vesicles. Cell treatment with anti-receptor for advanced glycation end products (RAGE) antibody, which binds to RAGE, a 'scavenger receptor', partially inhibited uptake of S100B-Alexa 488 , but not of Dextran 546 . The dynamin inhibitor dynole 34-2 inhibited internalization of both fluorescent probes. Directional mobility of S100B-Alexa 488 -positive vesicles increased over time and was inhibited by ATP stimulation, an agent that increases cytosolic free calcium concentration ([Ca 2+ ] i ). We conclude that astrocytes exhibit RAGE- and dynamin-dependent vesicular mechanism to efficiently remove S100B from the extracellular space. If a similar process occurs in vivo, astroglia may mitigate the toxic effects of extracellular S100B by this process under pathophysiologic conditions. This study reveals the vesicular clearance mechanism of extracellular S100B in astrocytes. Initially, fluorescent S100B internalizes into smaller endocytotic vesicles than dextran molecules. At a later stage, both probes co-localize within endolysosomes. S100B internalization is both dynamin- and RAGE-dependent, whereas dextran internalization is dependent on dynamin. Vesicle internalization likely mitigates the toxic effects of extracellular S100B and other waste products., (© 2016 International Society for Neurochemistry.)

Ketamine is an anesthetic that exhibits analgesic, psychotomimetic, and rapid antidepressant effects that are of particular neuropharmacological interest. Recent studies revealed astrocytic Ca(2+) signaling and regulated exocytosis as ketamine-targeted processes. Thus high-resolution cell-attached membrane capacitance measurements were performed to examine the influence of ketamine on individual vesicle interactions with the plasma membrane in cultured rat astrocytes. Ketamine evoked long-lasting bursts of repetitive opening and closing of the fusion pore that were both time- and concentration-dependent. Moreover, acute application and subanesthetic doses of ketamine elicited a significant increase in the occurrence of bursts that were characterized by a decreased fusion pore conductance, indicating that the fusion pore was stabilized in a narrow configuration. The time- and concentration-dependent increase in burst occurrence was correlated with a decrease in full fission events. This study has demonstrated a novel effect of ketamine manifested as stabilization of a fusion pore incapable of transiting to full vesicle fission, suggestive of an inhibitory effect on vesicle retrieval. This until now unrecognized effect of ketamine on the vesicle fusion pore might play a role in astroglial release and (re)uptake of molecules, modulating synaptic activity. This study demonstrates a novel effect of ketamine on the fusion pore. High-resolution cell-attached membrane capacitance measurements revealed that ketamine evokes long-lasting flickering of a narrow fusion pore that is incapable of transiting to full fission. Astrocytic vesicle fusion/retrieval modified by subanesthetic ketamine doses most likely affects gliotransmission and indicates a non-neuronal mechanism of ketamine action that may contribute to its behavioral effects., (© 2016 International Society for Neurochemistry.)

In the brain, astrocytes provide metabolic and trophic support to neurones. Failure in executing astroglial homeostatic functions may contribute to the initiation and propagation of diseases, including Alzheimer disease (AD), characterized by a progressive loss of neurones over years. Here, we examined whether astrocytes from a mice model of AD isolated in the presymptomatic phase of the disease exhibit alterations in vesicle traffic, vesicular peptide release and purinergic calcium signaling. In cultured astrocytes isolated from a newborn wild-type (wt) and 3xTg-AD mouse, secretory vesicles and acidic endosomes/lysosomes were labeled by transfection with plasmid encoding atrial natriuretic peptide tagged with mutant green fluorescent protein (ANP.emd) and by LysoTracker, respectively. The intracellular Ca(2+) concentration ([Ca(2+)]i) was monitored with Fluo-2 and visualized by confocal microscopy. In comparison with controls, spontaneous mobility of ANP- and LysoTracker-labeled vesicles was diminished in 3xTg-AD astrocytes; the track length (TL), maximal displacement (MD) and directionality index (DI) were all reduced in peptidergic vesicles and in endosomes/lysosomes (P < 0.001), as was the ATP-evoked attenuation of vesicle mobility. Similar impairment of peptidergic vesicle trafficking was observed in wt rat astrocytes transfected to express mutated presenilin 1 (PS1M146V). The ATP-evoked ANP discharge from single vesicles was less efficient in 3xTg-AD and PS1M146V-expressing astrocytes than in respective wt controls (P < 0.05). Purinergic stimulation evoked biphasic and oscillatory [Ca(2+)]i responses; the latter were less frequent (P < 0.001) in 3xTg-AD astrocytes. Expression of PS1M146V in astrocytes impairs vesicle dynamics and reduces evoked secretion of the signaling molecule ANP; both may contribute to the development of AD., (© 2015 Wiley Periodicals, Inc.)

Astrocytes are considered essential in the etiopathogenesis of amyotrophic lateral sclerosis (ALS). We have demonstrated previously that immunoglobulins G (IgG) isolated from patients with ALS enhance the mobility of acidic vesicles in cultured astrocytes in a Ca(2+)-dependent manner. Here we directly examined the impact of purified sporadic ALS IgG on cytosolic [Ca(2+)] ([Ca(2+)]i) in astrocytes. Confocal time-lapse images were acquired and fluorescence of a non-ratiometric Ca(2+) indicator was recorded before and after the application of IgG. ALS IgG (0.1 mg/ml) from 7 patients evoked transient increases in [Ca(2+)]i in ~50% of tested astrocytes. The probability of observing a response was independent of extracellular Ca(2+). The peak increase in [Ca(2+)]i developed ~3 times faster and the time integral of evoked transients was ~2-fold larger; the peak amplitude itself was not affected by extracellular Ca(2+). Application of pharmacological inhibitors revealed that activation of inositol-1,4,5-triphosphate receptors is necessary and sufficient to initiate transients in [Ca(2+)]i; the Ca(2+) influx through store-operated calcium entry prolongs the transient increase in [Ca(2+)]i. Thus, ALS IgG acutely affect [Ca(2+)]i by mobilizing both, intra- and extracellular Ca(2+) into the cytosol of cultured astrocytes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Aquaporin 4 (AQP4) is the predominant water channel in the brain, expressed mainly in astrocytes and involved in water transport in physiologic and pathologic conditions. Besides the classical isoforms M1 (a) and M23 (c), additional ones may be present at the plasma membrane, such as the recently described AQP4b, d, e, and f. Water permeability regulation by AQP4 isoforms may involve several processes, such as channel conformational changes, the extent and arrangement of channels at the plasma membrane, and the dynamics of channel trafficking to/from the plasma membrane. To test whether vesicular trafficking affects the abundance of AQP4 channel at the plasma membrane, we studied the subcellular localization of AQP4 in correlation with vesicle mobility of AQP4e, one of the newly discovered AQP4 isoforms. In cultured rat astrocytes, recombinant AQP4e acquired plasma membrane localization, which resembled that of the antibody labeled endogenous AQP4 localization. Under conditions mimicking reactivation of astrocytes (increase in cytosolic cAMP) and brain edema, an increase in the AQP4 plasma membrane localization was observed. The cytoskeleton remained unaffected with the exception of rearranged actin filaments in the model of reactive astrocytes and vimentin meshwork depolymerization in hypoosmotic conditions. AQP4e vesicle mobility correlated with changes in the plasma membrane localization of AQP4 in all stimulated conditions. Hypoosmotic stimulation triggered a transient reduction in AQP4e vesicle mobility mirrored by the transient changes in AQP4 plasma membrane localization. We suggest that regulation of AQP4 surface expression in pathologic conditions is associated with the mobility of AQP4-carrying vesicles., (Copyright © 2013 Wiley Periodicals, Inc.)

Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i) intercellular communication by gliotransmitters (glutamate, adenosine 5'-triphosphate, atrial natriuretic peptide), (ii) plasma membrane exchange of transporters and receptors (EAAT2, MHC-II), and (iii) the involvement of vesicle mobility carrying aquaporins (AQP4) in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions.

In this study we used live-cell immunocytochemistry and confocal microscopy to study the release from a single vesicle in a simplified system called membrane lawns. The lawns were prepared by exposing differentiated pituitary prolactin (PRL)-secreting cells to a hypoosmotic shear stress. The density of the immunolabeled ternary soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) complexes that bind complexin was approximately 10 times lower than the PRL-positive, lawn-resident vesicles; this indicates that some but not all vesicles are associated with ternary SNARE complexes. However, lawn-resident PRL vesicles colocalized relatively well with particular SNARE proteins: synaptobrevin 2 (35%), syntaxin 1 (22%), and 25-kDa synaptosome associated protein (6%). To study vesicle discharge, we prepared lawn-resident vesicles, derived from atrial natriuretic peptide tagged with emerald fluorescent protein (ANP.emd)-transfected cells, which label vesicles. These maintained the structural passage to the exterior because approximately 40% of ANP.emd-loaded vesicles were labeled by extracellular PRL antibodies. Cargo release from the lawn-resident vesicles, monitored by the decline in the ANP.emd fluorescence intensity, was similar to that in intact cells. It is likely that SNARE proteins are required for calcium-dependent release from these vesicles. This is because the expression of the dominant-negative SNARE peptide, which interferes with SNARE complex formation, reduced the number of PRL-positive spots per cell (PRL antibodies placed extracellularly) significantly, from 58 ± 9 to 4 ± 2. In dominant-negative SNARE-treated cells, the PRL-positive area was reduced from 0.259 ± 0.013 to 0.123 ± 0.014 μm(2), which is consistent with a hindered vesicle luminal access for extracellular PRL antibodies. These results indicate that vesicle discharge is regulated by SNARE-mediated fusion pore widening.

Adult neurogenesis is regulated by a number of cellular players within the neurogenic niche. Astrocytes participate actively in brain development, regulation of the mature central nervous system (CNS), and brain plasticity. They are important regulators of the local environment in adult neurogenic niches through the secretion of diffusible morphogenic factors, such as Wnts. Astrocytes control the neurogenic niche also through membrane-associated factors, however, the identity of these factors and the mechanisms involved are largely unknown. In this study, we sought to determine the mechanisms underlying our earlier finding of increased neuronal differentiation of neural progenitor cells when cocultured with astrocytes lacking glial fibrillary acidic protein (GFAP) and vimentin (GFAP(-/-) Vim(-/-) ). We used primary astrocyte and neurosphere cocultures to demonstrate that astrocytes inhibit neuronal differentiation through a cell-cell contact. GFAP(-/-) Vim(-/-) astrocytes showed reduced endocytosis of Notch ligand Jagged1, reduced Notch signaling, and increased neuronal differentiation of neurosphere cultures. This effect of GFAP(-/-) Vim(-/-) astrocytes was abrogated in the presence of immobilized Jagged1 in a manner dependent on the activity of γ-secretase. Finally, we used GFAP(-/-) Vim(-/-) mice to show that in the absence of GFAP and vimentin, hippocampal neurogenesis under basal conditions as well as after injury is increased. We conclude that astrocytes negatively regulate neurogenesis through the Notch pathway, and endocytosis of Notch ligand Jagged1 in astrocytes and Notch signaling from astrocytes to neural stem/progenitor cells depends on the intermediate filament proteins GFAP and vimentin., (Copyright © 2012 AlphaMed Press.)

In the brain, astrocytes signal to the neighboring cells by the release of chemical messengers (gliotransmitters) via regulated exocytosis. Recent studies uncovered a potential role of signaling lipids in modulation of exocytosis. Hence, we investigated whether sphingosine and the structural analog fingolimod/FTY720, a recently introduced therapeutic for multiple sclerosis, affect (i) intracellular vesicle mobility and (ii) vesicle cargo discharge from cultured rat astrocytes. Distinct types of vesicles, peptidergic, glutamatergic, and endosomes/lysosomes, were fluorescently prelabeled by cell transfection with plasmids encoding atrial natriuretic peptide tagged with mutant green fluorescent protein and vesicular glutamate transporter tagged with enhanced green fluorescent protein or by LysoTracker staining, respectively. The confocal and total internal reflection fluorescence microscopies were used to monitor vesicle mobility in the cytoplasm and near the basal plasma membrane, respectively. Sphingosine and FTY720, but not the membrane impermeable lipid analogs, dose-dependently attenuated vesicle mobility in the subcellular regions studied, and significantly inhibited stimulated exocytotic peptide and glutamate release. We conclude that in astrocytes, cell permeable sphingosine-like lipids affect regulated exocytosis by attenuating vesicle mobility, thereby preventing effective vesicle access/interaction with the plasma membrane docking/release sites., (Copyright © 2012 Wiley Periodicals, Inc.)

The view of how astrocytes, a type of glial cells, contribute to the functioning of the central nervous system (CNS) has changed greatly in the last decade. Although glial cells outnumber neurons in the mammalian brain, it was considered for over a century that they played a subservient role to neurons. This view changed. Functions thought to be exclusively present in neurons, i.e. excitability mediated release of chemical messengers, has also been demonstrated in astrocytes. In this process, following an increase in cytosolic calcium activity, membrane bound vesicles, storing chemical messengers (gliotransmitters), fuse with the plasma membrane, a process known as exocytosis, permitting the exit of vesicle cargo into the extracellular space. Vesicles are delivered to and are removed from the site of exocytosis by an amazingly complex set of processes that we have only started to learn about recently. In this paper we review vesicle traffic, which is subject to physiological regulation and may be changed under pathological conditions.

Intermediate filament (IF) proteins upregulation is a hallmark of astrocyte activation and reactive gliosis, but its pathophysiological implications remain incompletely understood. A recently reported association between IFs and directional mobility of peptidergic vesicles allows us to hypothesize that IFs affect vesicle dynamics and exocytosis-mediated astrocyte communication with neighboring cells. Here, we ask whether the trafficking of recycling vesicles (i.e., those fused to and then retrieved from the plasma membrane) and endosomes/lysosomes depends on IFs. Recycling vesicles were labeled by antibodies against vesicle glutamate transporter 1 (VGLUT1) and atrial natriuretic peptide (ANP), respectively, and by lysotracker, which labels endosomes/lysosomes. Quantitative fluorescence microscopy was used to monitor the mobility of labeled vesicles in astrocytes, derived from either wild-type (WT) mice or mice deficient in glial fibrillary acidic protein and vimentin (GFAP(-/-)Vim(-/-)), the latter lacking astrocyte IFs. Stimulation with ionomycin or ATP enhanced the mobility of VGLUT1-positive vesicles and reduced the mobility of ANP-positive vesicles in WT astrocytes. In GFAP(-/-)Vim(-/-) astrocytes, both vesicle types responded to stimulation, but the relative increase in mobility of VGLUT1-positive vesicles was more prominent compared with nonstimulated cells, whereas the stimulation-dependent attenuation of ANP-positive vesicles mobility was reduced compared with nonstimulated cells. The mobility of endosomes/lysosomes decreased following stimulation in WT astrocytes. However, in GFAP(-/-)Vim(-/-) astrocytes, a small increase in the mobility of endosomes/lysosomes was observed. These findings show that astrocyte IFs differentially affect the stimulation-dependent mobility of vesicles. We propose that upregulation of IFs in pathologic states may alter the function of astrocytes by deregulating vesicle trafficking., ((c) 2010 Wiley-Liss, Inc.)

Prolonged exposure to aluminium may impact health. Aluminium's deleterious effects are mostly attributed to its selective accumulation in particular organs and cell types. Occupational exposure to aluminium is allied with a reduced level of serum prolactin, a stress peptide hormone mainly synthesised and secreted by the anterior pituitary lactotrophs. Our aim was to study the effect of aluminium on the viability of rat lactotrophs in primary suspension cultures where multicellular aggregates tend to form, comprising approximately two thirds of the total cell population as confirmed by confocal microscopy. Flow cytometric light scattering of calcein acetoxymethyl ester and ethidium homodimer-1 labelled cells was used to define subpopulations of live and dead cells in heterogeneous suspensions comprised of single cells and multicellular aggregates of distinct size. Concentration-dependent effects of AlCl(3) were observed on aggregate size and cell survival. After 24-h exposure to 3 mM AlCl(3), viability of single cells declined from 5% to 3%, while in multicellular aggregates, viability declined from 23% to 20%. The proportion of single cells increased from 30% to 42% within the same concentration range, while in large aggregates, the proportion remained approximately constant representing 35% of the cell suspension. In large aggregates, cell viability (75%) remained unaltered after exposure to AlCl(3) concentrations up to 300 microM, while in single cells, viability was halved at 30 microM. In conclusion, our finding indicates that prolonged exposure to aluminium may lead to significant loss of pituitary cells.

It is believed that in regulated exocytosis the vesicle membrane fuses with the plasma membrane in response to a physiological stimulus. However, in the absence of stimulation, repetitive transient fusion events are also observed, reflecting a stable state. The mechanisms by which the initial fusion pore attains stability are poorly understood. We modelled energetic stability of the fusion pore by taking into account the anisotropic, intrinsic shape of the membrane constituents and their in-plane ordering in the local curvature of the membrane. We used cell-attached membrane capacitance techniques to monitor the appearance and conductance of single fusion pore events in cultured rat lactotrophs. The results revealed a bell-shaped distribution of the fusion pore conductance with a modal value of 25 pS. The experimentally observed increase of the fusion pore stability with decreasing fusion pore radius agrees well with the theoretical predictions. Moreover, the results revealed a correlation between the amplitude of transient capacitance increases and the fusion pore conductance, indicating that larger vesicles may attain a stable fusion pore with larger fusion pore diameters.

Exocytosis, the merger of the vesicle membrane with the plasma membrane, is thought to mediate the release of hormones and neurotransmitters from secretory vesicles. The work of Bernard Katz and colleagues decades ago considered that vesicle cargo discharge initially requires the delivery of secretory vesicles to the plasma membrane where vesicles dock and are primed for fusion with the plasma membrane. Then, upon stimulation, the vesicle and the plasma membranes fuse to form a transient fusion pore through which cargo molecules diffuse out of the vesicle lumen into the extracellular space. Katz and colleagues considered this process to occur in an all-or-none fashion. However, recent studies show that this may not be so simple. The aim of this overview is to highlight the novel findings that indicate that fusion pores are subject to regulations, which affect the release competence of a single vesicle. Here we discuss the elementary properties of spontaneous and stimulated peptidergic vesicle discharge, which appears to be modulated, at least in pituitary lactotrophs, by fusion pore conductance (pore diameter) and fusion pore gating (kinetics).

Neurotransmitter and hormone release from vesicles involves fusion between the vesicle and the plasma membranes, a process termed exocytosis. Recently we reported that most of the spontaneous and stimulated exocytotic events in pituitary lactotrophs are transient and repetitive, appearing in bursts lasting more than 100 s. However, whether this is also the case in compound vesicle-to-vesicle exocytosis is unknown. Here we investigated compound exocytotic events in resting and stimulated lactotrophs by using optical and cell-attached patch-clamp capacitance measurements. Elementary compound exocytotic events were characterized by multiple-amplitude on-steps in synaptopHluorin fluorescence and in membrane capacitance signals. Multiple-amplitude on-steps appeared either as a relatively large upward step, indicating that vesicles were fused with each other prior to fusion of the vesicle membrane with the plasma membrane (multivesicular exocytosis), or as a time-dependent stepwise signal increase, indicating sequential fusion of two or more vesicles with the plasma membrane (sequential exocytosis). In the majority of membrane capacitance recordings (>90%), multiple-amplitude on-steps terminated as multiple-amplitude off-steps. These complex amplitude events were repetitive, indicating that transient fusion pore openings reflect repetitive interactions of a single vesicle or vesicles in a cluster with the plasma membrane. Out of many mechanisms, these interactions may enable the diffusion of fusion proteins from the plasma membrane to the membrane of the primary fused vesicles, consequently enabling vesicle-to-vesicle fusion. The incidence of compound exocytotic events increased by 33% after stimulation, which is consistent with the enhanced efficiency of hormone secretion after the stimulus.