Rate Of Spontaneous Release Research Articles

Frequency of Spontaneous Neurotransmission at Individual Boutons Corresponds to the Size of the Readily Releasable Pool of Vesicles.

Synapses maintain two forms of neurotransmitter release to support communication in the brain. First, evoked neurotransmitter release is triggered by the invasion of an action potential (AP) across en passant boutons that form along axons. The probability of evoked release (Pr) varies substantially across boutons, even within a single axon. Such heterogeneity is the result of differences in the probability of a single synaptic vesicle (SV) fusing (Pv) and in the number of vesicles available for immediate release, known as the readily releasable pool (RRP). Spontaneous release (also known as a mini) is an important form of neurotransmission that occurs in the absence of APs. Because it cannot be triggered with electrical stimulation, much less is known about potential heterogeneity in the frequency of spontaneous release between boutons. We utilized a photostable and bright fluorescent indicator of glutamate release (iGluSnFR3) to quantify both spontaneous and evoked release at individual glutamatergic boutons. We found that the rate of spontaneous release is quite heterogenous at the level of individual boutons. Interestingly, when measuring both evoked and spontaneous release at single synapses, we found that boutons with the highest rates of spontaneous release also displayed the largest evoked responses. Using a new optical method to measure RRP at individual boutons, we found that this heterogeneity in spontaneous release was strongly correlated with the size of the RRP, but not related to Pv. We conclude that the RRP is a critical and dynamic aspect of synaptic strength that contributes to both evoked and spontaneous vesicle release.

Astrocyte Calcium Signaling Shifts the Polarity of Presynaptic Plasticity

Astrocytes have been increasingly acknowledged to play active roles in regulating synaptic transmission and plasticity. Through a variety of metabotropic and ionotropic receptors expressed on their surface, astrocytes detect extracellular neurotransmitters, and in turn, release gliotransmitters to modify synaptic strength, while they can also alter neuronal membrane excitability by modulating extracellular ionic milieu. Given the seemingly large repertoire of synaptic modulation, when, where and how astrocytes interact with synapses remain to be fully understood. Previously, we have identified a role for astrocyte NMDA receptor and L-VGCCs signaling in heterosynaptic presynaptic plasticity and promoting the heterogeneity of presynaptic strengths at hippocampal synapses. Here, we have sought to further clarify the mode by which astrocytes regulate presynaptic plasticity by exploiting a reduced culture system to globally evoke NMDA receptor-dependent presynaptic plasticity. Recording from a postsynaptic neuron intracellularly loaded with BAPTA, briefly bath applying NMDA and glycine induces a stable decrease in the rate of spontaneous glutamate release, which requires the presence of astrocytes and the activation of A1 adenosine receptors. Upon preventing astrocyte calcium signaling or blocking L-VGCCs, NMDA + glycine application triggers an increase, rather than a decrease, in the rate of spontaneous glutamate release, thereby shifting the presynaptic plasticity to promote an increase in strength. Our findings point to a crucial and surprising role of astrocytes in controlling the polarity of NMDA receptor and adenosine-dependent presynaptic plasticity. Such a pivotal mechanism unveils the power of astrocytes in regulating computations performed by neural circuits and is expected to profoundly impact cognitive processes.

Prognostic factors associated with the course of vitreomacular traction in eyes with age-related macular degeneration

Clinical relevanceVitreomacular traction(VMT) is a clinical syndrome that can cause decreased vision and may affect the treatment response in cases of age-related macular degeneration(AMD). Factors affecting the course of VMT in AMD cases will guide the clinician in terms of patient management. BackgroundThe aim of this study was to determine the prevalence of VMT in patients with AMD, to evaluate the natural course of VMT, and to investigate factors associated with the prognosis of VMT in eyes with AMD. MethodsThis retrospective case series was conducted with 55 eyes of 46 patients who were diagnosed as having AMD accompanying with VMT. Demographic data, complete ophthalmologic examination findings, type of AMD, receiving an intravitreal injection(IVI), number of IVIs, and the presence of complete spontaneous release were obtained from the medical records of the patients. The horizontal length of VMT(HLVMT), central macular thickness(CMT), the horizontal length of choroidal neovascularization(HLCNV) were evaluated from spectral-domain optical coherence tomography(SD-OCT) images. ResultsSpontaneous release was observed in 7(28%) eyes of the exudative AMD group and 10(33.3%) eyes of the nonexudative AMD group. On the last visit, the HLVMT was increased in 22(40%) of the eyes and a decrease in HLVMT was observed in 8(14.5%) of the eyes. In the remaining 12(21.8%) eyes had unchanged HLVMT. In all eyes with CNV, the area of VMT corresponded in 100% with localization of the CNV complex. No significant difference was found between the eyes with spontaneous release and persistent traction in terms of the type of AMD, IVI, HLVMT, age, gender, and crystalline lens status. ConclusionIn this study, VMT was observed at higher rates in eyes with exudative AMD compared to the eyes with nonexudative AMD. However, spontaneous release rates were found close to those with idiopathic VMT independently of the type of AMD, HLVMT, and IVI.

Polybasic Patches in Both C2 Domains of Synaptotagmin-1 Are Required for Evoked Neurotransmitter Release.

Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release, composed of a single-pass transmembrane domain linked to two C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is poorly understood. Calcium binding to C2B is critical for synchronous release, and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here, we asked what critical feature of C2A may be responsible for its functional role and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1K189-192A and Syt1K326-327A) side by side to determine their relative contributions to Syt1 function in cultured cortical neurons from mice of either sex and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain-membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The size of the readily releasable vesicle pool and the rate of spontaneous release were unaffected, so both patches are likely required specifically for synchronization of release. We suggest these patches contribute to cooperative membrane binding, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release.SIGNIFICANCE STATEMENT Synaptotagmin-1 is a vesicular calcium sensor required for synchronous neurotransmitter release. Its tandem cytosolic C2 domains (C2A and C2B) bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. How calcium binding to Synaptotagmin-1 leads to release and the relative contributions of the C2 domains are unclear. Combining electrophysiological recordings from cultured neurons and optical tweezers measurements of single C2 domain-membrane interactions, we show that conserved polybasic regions in both domains contribute to membrane binding cooperatively, and both are required for evoked release, likely by increasing the overall affinity of Synaptotagmin-1 for acidic membranes.

Idiopathic Vitreomacular Traction Managed with Initial Observation: Clinical Course and Outcomes

To review the clinical course and outcomes of patients with idiopathic vitreomacular traction (VMT) managed initially by observation. Retrospective chart review including patients with idiopathic VMT based on clinical symptoms and findings on OCT between January 1, 2015, and February 15,2021. The study included 436 eyes of 317 patients with a mean age of 72.2 years ± 8.9 at initial visit and mean follow-up time of 34 months ± 19.2. Vitreomacular traction severity grade was ascribed to each patient using previously published grading criteria. Grade 1 denoted incomplete cortical vitreous separation with attachment at the fovea and visible distortion of the foveal surface. Grade 2 included intraretinal cysts or clefts along with grade 1 findings. Grade 3 included subfoveal fluid along with grade 2 traits. The rate of spontaneous release, grade at baseline compared with grade at final follow-up, and outcomes of interventions, if performed. At baseline, mean best corrected visual acuity (BCVA) was 20/40. Baseline OCT demonstrated grade 1 VMT in 212 eyes (48.6%), grade 2 VMT in 172 eyes (39.4%), and grade 3 VMT in 52 eyes (11.9%). Among eyes that were initially grade 1, 25.0% had spontaneous release of VMT (median, 290.0 days; mean, 404.5 days ± 323.9), 50.9% remained stable, and 10.4% worsened. Among eyes that were initially grade 2, 14.5% had spontaneous release of VMT (median, 570.0 days; mean, 692.9 days ± 477.5), 55.2% remained stable, 4.7% improved, and 2.3% worsened. Among eyes that were initially grade 3, 5.8% had spontaneous release of VMT (median, 790.0 days; mean, 839.3 days ± 246.7), 28.8% remained stable, and 5.8% improved. Of the 436 eyes, macular hole development occurred in 42 eyes (9.6%). Pars plana vitrectomy was performed in 94 of 436 eyes (21.6%) with mean BCVA before pars plana vitrectomy of 20/78 and final follow-up BCVA of 20/55. This study demonstrates the generally stable clinical course of VMT when managed initially by observation. Stable VMT grade was the most frequent outcome, and eyes with grade 1 VMT were more likely to undergo spontaneous release than eyes with grade 2 or3.

Presynaptic mechanisms underlying GABAB-receptor-mediated inhibition of spontaneous neurotransmitter release.

SUMMARYInhibition of neurotransmitter release by neurotransmitter substances constitutes a fundamental means of neuromodulation. In contrast to well-delineated mechanisms that underlie inhibition of evoked release via suppression of voltage-gated Ca2+ channels, processes that underlie neuromodulatory inhibition of spontaneous release remain unclear. Here, we interrogated inhibition of spontaneous glutamate and GABA release by presynaptic metabotropic GABAB receptors. Our findings show that this inhibition relies on Gβγ subunit action at the membrane, and it is largely independent of presynaptic Ca2+ signaling for both forms of release. In the case of spontaneous glutamate release, inhibition requires Gβγ interaction with the C terminus of the key fusion machinery component SNAP25, and it is modulated by synaptotagmin-1. Inhibition of spontaneous GABA release, on the other hand, is independent of these pathways and likely requires alternative Gβγ targets at the presynaptic terminal.

The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF

Ariadne-1 (Ari-1) is an E3 ubiquitin-ligase essential for neuronal development, but whose neuronal substrates are yet to be identified. To search for putative Ari-1 substrates, we used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics of Drosophila heads. We identified 16 candidates that met the established criteria: a significant change of at least twofold increase on ubiquitination, with at least two unique peptides identified. Among those candidates, we identified Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF), which is involved in neurotransmitter release. Using a pull-down approach that relies on the overexpression and stringent isolation of a GFP-fused construct, we validate Comt/NSF to be an ubiquitination substrate of Ari-1 in fly neurons, resulting in the preferential monoubiquitination of Comt/NSF. We tested the possible functional relevance of this modification using Ari-1 loss-of-function mutants, which displayed a lower rate of spontaneous neurotransmitter release due to failures at the presynaptic side. By contrast, evoked release in Ari-1 mutants was enhanced compared with controls in a Ca2+-dependent manner without modifications in the number of active zones, indicating that the probability of release per synapse is increased in these mutants. This phenotype distinction between spontaneous and evoked release suggests that NSF activity may discriminate between these two types of vesicle fusion. Our results thus provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitination.

Resting and stimulated mouse rod photoreceptors show distinct patterns of vesicle release at ribbon synapses.

The vertebrate visual system can detect and transmit signals from single photons. To understand how single-photon responses are transmitted, we characterized voltage-dependent properties of glutamate release in mouse rods. We measured presynaptic glutamate transporter anion current and found that rates of synaptic vesicle release increased with voltage-dependent Ca2+ current. Ca2+ influx and release rate also rose with temperature, attaining a rate of ∼11 vesicles/s/ribbon at -40 mV (35°C). By contrast, spontaneous release events at hyperpolarized potentials (-60 to -70 mV) were univesicular and occurred at random intervals. However, when rods were voltage clamped at -40 mV for many seconds to simulate maintained darkness, release occurred in coordinated bursts of 17 ± 7 quanta (mean ± SD; n = 22). Like fast release evoked by brief depolarizing stimuli, these bursts involved vesicles in the readily releasable pool of vesicles and were triggered by the opening of nearby ribbon-associated Ca2+ channels. Spontaneous release rates were elevated and bursts were absent after genetic elimination of the Ca2+ sensor synaptotagmin 1 (Syt1). This study shows that at the resting potential in darkness, rods release glutamate-filled vesicles from a pool at the base of synaptic ribbons at low rates but in Syt1-dependent bursts. The absence of bursting in cones suggests that this behavior may have a role in transmitting scotopic responses.

Syntaxin 1B regulates synaptic GABA release and extracellular GABA concentration, and is associated with temperature-dependent seizures.

De novo heterozygous mutations in the STX1B gene, encoding syntaxin 1B, cause a familial, fever-associated epilepsy syndrome. Syntaxin 1B is an essential component of the pre-synaptic neurotransmitter release machinery as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein that regulates the exocytosis of synaptic vesicles. It is also involved in regulating the functions of the SLC6 family of neurotransmitter transporters that reuptake neurotransmitters, including inhibitory neurotransmitters, such as γ-aminobutyric acid (GABA) and glycine. The purpose of the present study was to elucidate the molecular mechanisms underlying the development of febrile seizures by examining the effects of syntaxin 1B haploinsufficiency on inhibitory synaptic transmission during hyperthermia in a mouse model. Stx1b gene heterozygous knockout (Stx1b+/- ) mice showed increased susceptibility to febrile seizures and drug-induced seizures. In cultured hippocampal neurons, we examined the temperature-dependent properties of neurotransmitter release and reuptake by GABA transporter-1 (GAT-1) at GABAergic neurons using whole-cell patch-clamp recordings. The rate of spontaneous quantal GABA release was reduced in Stx1b+/- mice. The hyperthermic temperature increased the tonic GABAA current in wild-type (WT) synapses, but not in Stx1b+/- synapses. In WT neurons, recurrent bursting activities were reduced in a GABA-dependent manner at hyperthermic temperature; however, this was abolished in Stx1b+/- neurons. The blockade of GAT-1 increased the tonic GABAA current and suppressed recurrent bursting activities in Stx1b+/- neurons at the hyperthermic temperature. These data suggest that functional abnormalities associated with GABA release and reuptake in the pre-synaptic terminals of GABAergic neurons may increase the excitability of the neural circuit with hyperthermia.

Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons.

The success of comparative cell biology for determining protein function relies on quality disruption techniques. Long-lived proteins, in postmitotic cells, are particularly difficult to eliminate. Moreover, cellular processes are notoriously adaptive; for example, neuronal synapses exhibit a high degree of plasticity. Ideally, protein disruption techniques should be both rapid and complete. Here, we describe knockoff, a generalizable method for the druggable control of membrane protein stability. We developed knockoff for neuronal use but show it also works in other cell types. Applying knockoff to synaptotagmin 1 (SYT1) results in acute disruption of this protein, resulting in loss of synchronous neurotransmitter release with a concomitant increase in the spontaneous release rate, measured optically. Thus, SYT1 is not only the proximal Ca2+ sensor for fast neurotransmitter release but also serves to clamp spontaneous release. Additionally, knockoff can be applied to protein domains as we show for another synaptic vesicle protein, synaptophysin 1.

Doc2 Proteins Are Not Required for the Increased Spontaneous Release Rate in Synaptotagmin-1-Deficient Neurons.

Regulated secretion is controlled by Ca2+ sensors with different affinities and subcellular distributions. Inactivation of Syt1 (synaptotagmin-1), the main Ca2+ sensor for synchronous neurotransmission in many neurons, enhances asynchronous and spontaneous release rates, suggesting that Syt1 inhibits other sensors with higher Ca2+ affinities and/or lower cooperativities. Such sensors could include Doc2a and Doc2b, which have been implicated in spontaneous and asynchronous neurotransmitter release and compete with Syt1 for binding SNARE complexes. Here, we tested this hypothesis using triple-knock-out mice. Inactivation of Doc2a and Doc2b in Syt1-deficient neurons did not reduce the high spontaneous release rate. Overexpression of Doc2b variants in triple-knock-out neurons reduced spontaneous release but did not rescue synchronous release. A chimeric construct in which the C2AB domain of Syt1 was substituted by that of Doc2b did not support synchronous release either. Conversely, the soluble C2AB domain of Syt1 did not affect spontaneous release. We conclude that the high spontaneous release rate in synaptotagmin-deficient neurons does not involve the binding of Doc2 proteins to Syt1 binding sites in the SNARE complex. Instead, our results suggest that the C2AB domains of Syt1 and Doc2b specifically support synchronous and spontaneous release by separate mechanisms. (Both male and female neurons were studied without sex determination.)SIGNIFICANCE STATEMENT Neurotransmission in the brain is regulated by presynaptic Ca2+ concentrations. Multiple Ca2+ sensor proteins contribute to synchronous (Syt1, Syt2), asynchronous (Syt7), and spontaneous (Doc2a/Doc2b) phases of neurotransmitter release. Genetic ablation of synchronous release was previously shown to affect other release phases, suggesting that multiple sensors may compete for similar release sites, together encoding stimulus-secretion coupling over a large range of synaptic Ca2+ concentrations. Here, we investigated the extent of functional overlap between Syt1, Doc2a, and Doc2b by reintroducing wild-type and mutant proteins in triple-knock-out neurons, and conclude that the sensors are highly specialized for different phases of release.

Two types of magnetite-containing liposomes for magnetocontrolled drug release

The paper is dedicated to a comparative study of pharmaceutical properties of magnetite containing liposomes. We have investigated two types of liposomes: 1) magnetic shell liposomes containing magnetic nanoparticles in the coating lipid bilayer and 2) magnetic core liposomes containing the nanoparticles in the internal volume (water phase). Both types of liposomes were obtained by the thin film hydration method from phosphatidylcholine and cholesterol. Fluorescent dye 5,6-carboxyfluorescein was used as a marker substance to indicate release. The dimensional characteristics, the degree of dye release under the influence of an alternating magnetic field, the speed of spontaneous release and the stability of liposomes during storage were investigated. It has been shown that liposomes with nanoparticles in the internal space are more stable, have low rate of spontaneous release of the incorporated substance, but the rate of release under the influence of an alternating field is also low. Liposomes with magnetic nanoparticles inside the lipid bilayer have high release degree influenced by alternating magnetic field, but they are less stable during storage and more prone to spontaneous loss of the active substance.

5-HT3R–sourced calcium enhances glutamate release from a distinct vesicle pool

The serotonin 3 receptor (5-HT3R) is a calcium-permeant channel heterogeneously expressed in solitary tract (ST) afferents. ST afferents synapse in the nucleus of the solitary tract (NTS) and rely on a mix of voltage-dependent calcium channels (CaVs) to control synchronous glutamate release (ST-EPSCs). CaV activation triggers additional, delayed release of glutamate (asynchronous EPSCs) that trails after the ST-EPSCs but only from afferents expressing the calcium-permeable, transient receptor potential vanilloid type 1 receptor (TRPV1). Most afferents express TRPV1 and have high rates of spontaneous glutamate release (sEPSCs) that is independent of CaVs. Here, we tested whether 5-HT3R-sourced calcium contributes to these different forms of glutamate release in horizontal NTS slices from rats. The 5-HT3R selective agonist, m-chlorophenyl biguanide hydrochloride (PBG), enhanced sEPSCs and/or delayed the arrival times of ST-EPSCs (i.e. increased latency). The specific 5-HT3R antagonist, ondansetron, attenuated these effects consistent with direct activation of 5-HT3Rs. PBG did not alter ST-EPSC amplitude or asynchronous EPSCs. These independent actions suggest two distinct 5-HT3R locations; axonal expression that impedes conduction and terminal expression that mobilizes a spontaneous vesicle pool. Calcium chelation with EGTA-AM attenuated the frequency of 5-HT3R-activated sEPSCs by half. The mixture of chelation-sensitive and resistant sEPSCs suggests that 5-HT3R-activated vesicles span calcium diffusion distances that are both distal (micro-) and proximal (nanodomains) to the channel. Our results demonstrate that the calcium domains of 5-HT3Rs do not overlap other calcium sources or their respective vesicle pools. 5-HT3Rs add a unique calcium source on ST afferents as part of multiple independent synaptic signaling mechanisms.

Disruption of Otoferlin Alters the Mode of Exocytosis at the Mouse Inner Hair Cell Ribbon Synapse.

Sound encoding relies on Ca2+-mediated exocytosis at the ribbon synapse between cochlear inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs). Otoferlin, a multi-C2 domain protein, is proposed to regulate Ca2+-triggered exocytosis at this synapse, but the precise mechanisms of otoferlin function remain to be elucidated. Here, performing whole-cell voltage-clamp recordings of excitatory postsynaptic currents (EPSCs) from SGNs in otoferlin mutant mice, we investigated the impact of Otof disruption at individual synapses with single release event resolution. Otof deletion decreased the spontaneous release rate and abolished the stimulus-secretion coupling. This was evident from failure of potassium-induced IHC depolarization to stimulate release and supports the proposed role of otoferlin in Ca2+ sensing for fusion. A missense mutation in the Otof gene (pachanga), in which otoferlin level at the IHC plasma membrane was lowered without changing its Ca2+ binding, also reduced the spontaneous release rate but spared the stimulus-secretion coupling. The slowed stimulated release rate supports the hypothesis that a sufficient abundance of otoferlin at the plasma membrane is crucial for the vesicle supply. Large-sized monophasic EPSCs remained present upon Otof deletion despite the drastic reduction of the rate of exocytosis. However, EPSC amplitude, on average, was modestly decreased. Moreover, a reduced contribution of multiphasic EPSC was observed in both Otof mutants. We argue that the presence of large monophasic EPSCs despite the exocytic defect upon Otof deletion supports the uniquantal hypothesis of transmitter release at the IHC ribbon synapse. Based upon the reduced contribution of multiphasic EPSC, we propose a role of otoferlin in regulating the mode of exocytosis in IHCs.

CAST/ELKS Proteins Control Voltage-Gated Ca2+ Channel Density and Synaptic Release Probability at a Mammalian Central Synapse

SUMMARYIn the presynaptic terminal, the magnitude and location of Ca2+ entry through voltage-gated Ca2+ channels (VGCCs) regulate the efficacy of neurotransmitter release. However, how presynaptic active zone proteins control mammalian VGCC levels and organization is unclear. To address this, we deleted the CAST/ELKS protein family at the calyx of Held, a CaV2.1 channel-exclusive presynaptic terminal. We found that loss of CAST/ELKS reduces the CaV2.1 current density with concomitant reductions in CaV2.1 channel numbers and clusters. Surprisingly, deletion of CAST/ELKS increases release probability while decreasing the readily releasable pool, with no change in active zone ultrastructure. In addition, Ca2+ channel coupling is unchanged, but spontaneous release rates are elevated. Thus, our data identify distinct roles for CAST/ELKS as positive regulators of CaV2.1 channel density and suggest that they regulate release probability through a post-priming step that controls synaptic vesicle fusogenicity.

Subfoveal Lucency after Treatment of Vitreomacular Traction without Macular Hole in the Phase 3 Trials of Ocriplasmin Vitreolysis

To describe the clinical impact of subfoveal lucency (SFL) seen on OCT before and after treatment of vitreomacular traction (VMT) or symptomatic vitreomacular adhesion (VMA) without macular hole (MH) in the prospective phase 3 trials of ocriplasmin vitreolysis. Randomized double-blind placebo-controlled multicenter study. Four hundred ninety-nine eyes with VMT or VMA without MH. Eyes were randomized to either a single intravitreal injection of ocriplasmin or placebo treatment and were followed up for 6 months. Eyes were analyzed for presence, dimensions, and course of SFL on OCT, release of VMT, and their effect on visual acuity (VA). Among eyes without baseline SFL, new SFL was more frequent after ocriplasmin than placebo at week 1 (23.1% vs. 6.54%; P= 0.0002) and week 2 (19.7% vs. 3.85%; P= 0.0001), but was similar in frequency by week 4 (8.37% vs. 7.69%; P= 0.83). Six-month VA for eyes demonstrating SFL after ocriplasmin during the first 2 weeks was comparable with those without SFL at any point in the study (P= 0.12). In placebo-treated eyes but not ocriplasmin-treated eyes, SFL was associated with worse VA at all visits. The increase in SFL width from baseline to week 2 was significantly greater with ocriplasmin than placebo (P= 0.029). Among ocriplasmin-treated eyes at month 6, those with SFL and VMT release had better VA than those with SFL and persistent adhesion (P= 0.037) and similar VA to those with persistent adhesion without SFL (P= 0.17). In the placebo group, those with baseline SFL showed low rates of spontaneous VMT release at month 6 compared with those without baseline SFL (3/21 eyes vs. 33/99 eyes; P= 0.045). Compared with placebo, SFL occurred more frequently only in the initial weeks after treatment with ocriplasmin. When SFL developed after ocriplasmin administration, VA was not impacted at the 6-month visit. Persistent SFL in ocriplasmin-treated eyes at month 6 was associated with poorer vision if VMT had not released. Eyes with SFL at baseline had low rates of spontaneous VMT release and may need treatment.

Receptor Level Dissection of Common Versus Discrete Vesicle Release Pathways from Primary Vagal Afferent Terminals

Primary vagal afferent neurons form strong excitatory glutamatergic synapses onto second-order neurons in the nucleus of the solitary tract and initiate key autonomic and homeostatic reflex pathways. Vesicle fusion and release from vagal central terminals occurs via three distinct pathways including synchronous, asynchronous, and spontaneous. While synchronous and asynchronous release mechanisms require presynaptic action-potential depolarization; spontaneous release is ongoing and independent of action potentials. Evidence suggests these forms of release can be controlled independently indicating discrete and potentially separate vesicle pools and release sites. However, the presence and activation of transient receptor potential (TRP) channels (including TRPV1) at the central terminals dramatically increases the rate of spontaneous vesicle release and eventually diminish action-potential driven synchronous release; consistent with a common releasable pool. Confounding this interpretation is the observation that TRP channel activation also diminishes voltage activated sodium channel signaling. As such the decrease in synchronous release may be a result of action potential failure, rather than depleted vesicle pools. Given that synchronous and spontaneously release glutamate activates both AMPA and NMDA receptors postsynaptically we utilized ligand dependent post-synaptic NMDA receptor blockade (MK-801) to overcome this confound. We found that treatment with MK-801 during exclusively spontaneous release blocked the NMDA component of spontaneous events and subsequent action-potential driven synchronous release. This result demonstrates that glutamate released via spontaneous vesicle release is activating the same population of NMDA receptors as glutamate released synchronously. This finding is consistent with a common release site for synchronous and spontaneous release at vagal afferent terminals.

Ménage-à-Trois: The Ratio of Bicarbonate to CO2 and the pH Regulate the Capacity of Neutrophils to Form NETs.

In this study, we identified and characterized the potential of a high ratio of bicarbonate to CO2 and a moderately alkaline pH to render neutrophils prone to undergo neutrophil extracellular trap (NET) formation. Both experimental settings increased the rate of spontaneous NET release and potentiated the NET-inducing capacity of phorbol esters (phorbol-2-myristate-13-acetate), ionomycin, monosodium urate, and LPS. In contrast, an acidic environment impaired NET formation both spontaneous and induced. Our findings indicate that intracellular alkalinization of neutrophils in response to an alkaline environment leads to an increase of intracellular calcium and neutrophil activation. We further found that the anion channel blocker DIDS strongly reduced NET formation induced by bicarbonate. This finding suggests that the effects observed are due to a molecular program that renders neutrophils susceptible to NET formation. Inflammatory foci may be characterized by an acidic environment. Our data indicate that NET formation is favored by the higher pH at the border regions of inflamed areas. Moreover, our findings highlight the necessity for strict pH control during assays of NET formation.

Effects of aconitine, mesaconitine and hypaconitine on calcium release in isolated adult rat cardiac myocytes

Objective To investigate the effects of aconitine, mesaconitine and hypaconitine on calcium release in isolated adult rat cardiac myocytes. Methods The left ventricular cardiac myocytes isolated from adult Sprague-Dawley rats were perfused with acnitine, mesaconitine and hypaconitine at 0.3 μmol/L, 1 μmol/L, 3 μmol/L for 12 min. The spontaneous calcium release (SCR) rate, the end-diastolic [Ca2+] (F0) and the calcium transient amplitude (ΔF) were detected 4 min, 8 min and 12 min after the perfusion. 12 min after the perfusion with acnitine, mesaconitine and hypaconitine at 0.3 μmol/L, the changes of systolic dynamics and calcium transient were detected for the positive inotropic effect. Results Any of aconitine, mesaconitine and hypaconitine induced SCR, mesconitine- induced SCR rate was highest at low concentration (0.3 μmol/L), and aconitine-induced SCR rate highest at high concentration (3 μmol/L). Compared with the control, 12 min after the perfusion with acnitine, mesaconitine and hypaconitine at 3 μmol/L elevated F0 (1.459 ± 0.379, 1.585 ± 0.493, 1.213 ± 0.254 vs. 1.079 ± 0.108, all P<0.05) and ΔF(1.615 ± 0.455, 2.210 ± 0.756, 1.528 ± 0.422 vs. 1.036 ± 0.125, all P<0.05), mesaconitine with ΔF higher than aconitine and hypaconitine. At low concentration (0.3 μmol/L), compared with control, aconitine, mesaconitine and hypaconitine increased ΔF (0.409 ± 0.127, 0.423 ± 0.107, 0.414 ± 0.118 vs. 0.260 ± 0.065; P<0.05 or P<0.01) and contraction amplitudes (5.464% ± 2.239%, 7.449% ± 2.548%, 5.524% ± 1.645% vs. 3.428% ± 0.911%; P<0.05 or P<0.01), prolonged the time to peak of calcium transient (0.041 ± 0.016 s, 0.039 ± 0.009 s, 0.038 ± 0.011 s vs. 0.032 ± 0.007 s; P<0.05 or P<0.01); compared with aconitine, mesaconitine and hypaconitine decreased calcium transient time constant (0.301 ± 0.054 s, 0.324 ± 0.064 s vs. 0.361 ± 0.076 s; P<0.05 or P<0.01) and diastolic t50 (0.124 ± 0.035 s, 0.126 ± 0.040 s vs. 0.157 ± 0.056 s; P<0.05 or P<0.01). Conclusions Aconitine, mesaconitine and hypaconitine reveal the positive inotropic effects couple with the toxic effects. Increased [Ca2+] in cardiac myocytes is the key factor for the positive inotropic effects, but also the risk factor for SCR. Key words: Calcium; Myocytes, cardiac; Drugs, Chinese herbal; Aconitine; Rats; Mesaconitine; Hypaconitine

Differential Expression and Release of Activin A and Follistatin in Chronic Rhinosinusitis with and without Nasal Polyps.

BackgroundChronic rhinosinusitis with (CRSwNP) and without nasal polyps (CRSsNP) should be regarded as distinct clinical entities based on differential inflammatory mediator and remodeling profiles. Activin A, a member of the TGF-β superfamily, plays an important role in inflammation and remodeling in the lower airways, although its expression and release in the upper airways remain undescribed.ObjectiveTo investigate the expression of activin A and its inhibitor follistatin in nasal tissue samples from CRSsNP and CRSwNP patients, and to monitor the spontaneous release of these molecules in a human mucosal model.MethodsProtein levels were determined using ELISA for activin A, follistatin, TGF-β1 and indicator proteins (IL-5, ECP, IFNγ) in 13 CRSsNP, 23 CRSwNP, and 10 control samples. The spontaneous release rate and the release ratios of activin A, follistatin and TGF-β1 were determined in 9 CRSsNP and 7 CRSwNP tissue fragments cultured ex-vivo. The induction of activin A and TGF-β1 by one another was studied in 7 CRSsNP tissue fragments cultured ex-vivo.ResultsSignificantly higher concentrations of activin A, follistatin, TGF-β1, and IFNγ were observed in CRSsNP compared with CRSwNP samples, whereas the concentrations of IL-5 and ECP were significantly lower. Follistatin was positively and linearly correlated with activin A in CRSsNP and CRSwNP. Activin A, follistatin and TGF-β1 were all spontaneously released by the samples, although the relative ratios released by tissue fragments from CRSsNP and CRSwNP samples were significantly different, with a higher follistatin/activin A-ratio and a follistatin/TGFß1-ratio (with less overall TGF-β1) in CRSwNP than in CRSsNP. Furthermore, TGF-β1 enhanced activin A secretion in CRSsNP tissue fragments cultured ex-vivo.ConclusionThe differences in tissue concentrations and spontaneous release rates for activin A and follistatin in different CRS samples support the hypothesis that CRSsNP and CRSwNP are two distinct disease entities with respect to remodeling patterns.