Non-negative Matrix Factorization as a Tool to Distinguish Between Synaptic Vesicles in Different Functional States

Synaptic vesicles (SVs) undergo multiple steps of functional maturation (priming) before being fusion competent. We present an analysis technique, which decomposes the time course of quantal release during repetitive stimulation as a sum of contributions of SVs, which existed in distinct functional states prior to stimulation. Such states may represent different degrees of maturation in priming or relate to different molecular composition of the release apparatus. We apply the method to rat calyx of Held synapses. These synapses display a high degree of variability, both with respect to synaptic strength and short-term plasticity during high-frequency stimulus trains. The method successfully describes time courses of quantal release at individual synapses as linear combinations of three components, representing contributions from functionally distinct SV subpools, with variability among synapses largely covered by differences in subpool sizes. Assuming that SVs transit in sequence through at least two priming steps before being released by an action potential (AP) we interpret the components as representing SVs which had been ‘fully primed’, ‘incompletely primed’ or undocked prior to stimulation. Given these assumptions, the analysis reports an initial release probability of 0.43 for SVs that were fully primed prior to stimulation. Release probability of that component was found to increase during high-frequency stimulation, leading to rapid depletion of that subpool. SVs that were incompletely primed at rest rapidly obtain fusion-competence during repetitive stimulation and contribute the majority of release after 3–5 stimuli

Superpriming of synaptic vesicles as a common basis for intersynapse variability and modulation of synaptic strength.

Glutamatergic synapses show large variations in strength and short-term plasticity (STP). We show here that synapses displaying an increased strength either after posttetanic potentiation (PTP) or through activation of the phospholipase-C-diacylglycerol pathway share characteristic properties with intrinsically strong synapses, such as (i) pronounced short-term depression (STD) during high-frequency stimulation; (ii) a conversion of that STD into a sequence of facilitation followed by STD after a few conditioning stimuli at low frequency; (iii) an equalizing effect of such conditioning stimulation, which reduces differences among synapses and abolishes potentiation; and (iv) a requirement of long periods of rest for reconstitution of the original STP pattern. These phenomena are quantitatively described by assuming that a small fraction of "superprimed" synaptic vesicles are in a state of elevated release probability (p ∼ 0.5). This fraction is variable in size among synapses (typically about 30%), but increases after application of phorbol ester or during PTP. The majority of vesicles, released during repetitive stimulation, have low release probability (p ∼ 0.1), are relatively uniform in number across synapses, and are rapidly recruited. In contrast, superprimed vesicles need several seconds to be regenerated. They mediate enhanced synaptic strength at the onset of burst-like activity, the impact of which is subject to modulation by slow modulatory transmitter systems

A sequential two-step priming scheme reproduces diversity in synaptic strength and short-term plasticity

Glutamatergic synapses display variable strength and diverse short-term plasticity (STP), even for a given type of connection. Using nonnegative tensor factorization and conventional state modeling, we demonstrate that a kinetic scheme consisting of two sequential and reversible steps of release–machinery assembly and a final step of synaptic vesicle (SV) fusion reproduces STP and its diversity among synapses. Analyzing transmission at the calyx of Held synapses reveals that differences in synaptic strength and STP are not primarily caused by variable fusion probability (pfusion) but are determined by the fraction of docked synaptic vesicles equipped with a mature release machinery. Our simulations show that traditional quantal analysis methods do not necessarily report pfusion of SVs with a mature release machinery but reflect both pfusion and the distribution between mature and immature priming states at rest. Thus, the approach holds promise for a better mechanistic dissection of the roles of presynaptic proteins in the sequence of SV docking, two-step priming, and fusion. It suggests a mechanism for activity-induced redistribution of synaptic efficacy

Mental representations of partner task cause interference in picture naming

Interference in picture naming occurs from representing a partner's preparations to speak (Gambi, van de Cavey, & Pickering, 2015). We tested the origins of this interference using a simple non-communicative joint naming task based on Gambi et al. (2015), where response latencies indexed interference from partner task and partner speech content, and eye fixations to partner objects indexed overt attention. Experiment 1 contrasted a partner-present condition with a control partner-absent condition to establish the role of the partner in eliciting interference. For latencies, we observed interference from the partner's task and speech content, with interference increasing due to partner task in the partner-present condition. Eye-tracking measures showed that interference in naming was not due to overt attention to partner stimuli but to broad expectations about likely utterances. Experiment 2 examined whether an equivalent non-verbal task also elicited interference, as predicted from a language as joint action framework. We replicated the finding of interference due to partner task and again found no relationship between overt attention and interference. These results support Gambi et al. (2015). Individuals co-represent a partner's task while speaking, and doing so does not require overt attention to partner stimuli

Duality Based A Posteriori Error Estimation for Quasi-Periodic Solutions Using Time Averages

We propose an a posteriori error estimation technique for the computation of average functionals of solutions for nonlinear time dependent problems based on duality techniques. The exact solution is assumed to have a periodic or quasi-periodic behavior favoring a fixed mesh strategy in time. We show how to circumvent the need of solving time dependent dual problems. The estimator consists of an averaged residual weighted by sensitivity factors coming from a stationary dual problem and an additional averaging error term coming from nonlinearities of the operator considered. In order to illustrate this technique the resulting adaptive algorithm is applied to several model problems: a linear scalar parabolic problem with known exact solution, the nonsteady Navier–Stokes equations with known exact solution, and finally to the well-known benchmark problem for Navier–Stokes (flow behind a cylinder) in order to verify the modeling assumptions

Mental representations of partner task cause interference in picture naming

Interference in picture naming occurs from representing a partner's preparations to speak (Gambi, van de Cavey, & Pickering, 2015). We tested the origins of this interference using a simple non-communicative joint naming task based on Gambi et al. (2015), where response latencies indexed interference from partner task and partner speech content, and eye fixations to partner objects indexed overt attention. Experiment 1 contrasted a partner-present condition with a control partner-absent condition to establish the role of the partner in eliciting interference. For latencies, we observed interference from the partner's task and speech content, with interference increasing due to partner task in the partner-present condition. Eye-tracking measures showed that interference in naming was not due to overt attention to partner stimuli but to broad expectations about likely utterances. Experiment 2 examined whether an equivalent non-verbal task also elicited interference, as predicted from a language as joint action framework. We replicated the finding of interference due to partner task and again found no relationship between overt attention and interference. These results support Gambi et al. (2015). Individuals co-represent a partner's task while speaking, and doing so does not require overt attention to partner stimuli

Speech modifications in interactive speech: Effects of age, sex and noise type

When attempting to maintain conversations in noisy communicative settings, talkers typically modify their speech to make themselves understood by the listener. In this study, we investigated the impact of background interference type and talker age on speech adaptations, vocal effort and communicative success. We measured speech acoustics (articulation rate, mid-frequency energy, fundamental frequency), vocal effort (correlation between mid-frequency energy and fundamental frequency) and task completion time in 114 participants aged 8–80 years carrying out an interactive problem-solving task in good and noisy listening conditions (quiet, non-speech noise, background speech). We found greater changes in fundamental frequency and mid-frequency energy in non-speech noise than in background speech and similar reductions in articulation rate in both. However, older participants (50+ years) increased vocal effort in both background interference types, whereas younger children (less than 13 years) increased vocal effort only in background speech. The presence of background interference did not lead to longer task completion times. These results suggest that when the background interference involves a higher cognitive load, as in the case of other speech of other talkers, children and older talkers need to exert more vocal effort to ensure successful communication. We discuss these findings within the communication effort framework. This article is part of the theme issue ‘Voice modulation: from origin and mechanism to social impact (Part II)’

Acute complexin knockout abates spontaneous and evoked transmitter release

SNARE-mediated synaptic vesicle (SV) fusion is controlled by multiple regulatory proteins that determine neurotransmitter release efficiency. Complexins are essential SNARE regulators whose mode of action is unclear, as available evidence indicates positive SV fusion facilitation and negative 'fusion clamp'-like activities, with the latter occurring only in certain contexts. Because these contradictory findings likely originate in part from different experimental perturbation strategies, we attempted to resolve them by examining a conditional complexin-knockout mouse line as the most stringent genetic perturbation model available. We found that acute complexin loss after synaptogenesis in autaptic and mass-cultured hippocampal neurons reduces SV fusion probability and thus abates the rates of spontaneous, synchronous, asynchronous, and delayed transmitter release but does not affect SV priming or cause 'unclamping' of spontaneous SV fusion. Thus, complexins act as facilitators of SV fusion but are dispensable for 'fusion clamping' in mammalian forebrain neurons

Elevated alpha-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells

We have assessed the impact of α-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinson´s disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of α-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson´s disease progression, particularly in the context of bioenergetic dysfunction.Fil: Oliveira, L. M. A.. Max-Planck-Institut für biophysikalische Chemie; AlemaniaFil: Falomir Lockhart, Lisandro Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentina. Max-Planck-Institut für biophysikalische Chemie; AlemaniaFil: Botelho, M. G.. Max-Planck-Institut für biophysikalische Chemie; Alemania. Universidade Federal do Rio de Janeiro; BrasilFil: Lin, K. H.. Max-Planck-Institut für biophysikalische Chemie; AlemaniaFil: Wales, P.. Universität Göttingen; AlemaniaFil: Koch, J. C.. Universität Göttingen; AlemaniaFil: Gerhardt, Elizabeth. Universität Göttingen; AlemaniaFil: Taschenberger, H.. Max-Planck-Institut für biophysikalische Chemie; AlemaniaFil: Outeiro, T. F.. Universität Göttingen; AlemaniaFil: Lingor, P.. Universität Göttingen; AlemaniaFil: Schüele, B.. The Parkinson’s Institute; Estados UnidosFil: Arndt Jovin, D. J.. Max-Planck-Institut für biophysikalische Chemie; AlemaniaFil: Jovin, T. M.. Max-Planck-Institut für biophysikalische Chemie; Alemani

Munc13-1 is a Ca2+-phospholipid-dependent vesicle priming hub that shapes synaptic short-term plasticity and enables sustained neurotransmission

During ongoing presynaptic action potential (AP) firing, transmitter release is limited by the availability of release-ready synaptic vesicles (SVs). The rate of SV recruitment (SVR) to release sites is strongly upregu- lated at high AP frequencies to balance SV consumption. We show that Munc13-1—an essential SV priming protein—regulates SVR via a Ca2+-phospholipid-dependent mechanism. Using knockin mouse lines with point mutations in the Ca2+-phospholipid-binding C2B domain of Munc13-1, we demonstrate that abolishing Ca2+-phospholipid binding increases synaptic depression, slows recovery of synaptic strength after SV pool depletion, and reduces temporal fidelity of synaptic transmission, while increased Ca2+-phospholipid binding has the opposite effects. Thus, Ca2+-phospholipid binding to the Munc13-1-C2B domain accelerates SVR, reduces short-term synaptic depression, and increases the endurance and temporal fidelity of neurotrans- mission, demonstrating that Munc13-1 is a core vesicle priming hub that adjusts SV re-supply to demand