Quantal Neurotransmitter Release Research Articles

The Presence of Septin Proteins in the Neuromuscular Junction of Somatic Muscle in the Earthworm Lumbricus terrestris

Using fluorescent confocal microscopy it has been shown that proteins belonging to the septin family such as Septin 2, 3, 5, 7, and 9 are present in the somatic muscle of the earthworm Lumbricus terrestris. Septins 2 and 9 are associated with the lack of their specific concentrations in a cholinergic synapse, while septins 3, 5, and especially 7 levels correlate with quantitatively expressed binding in their localization to the motor end plate. It is assumed that Septins 3, 5 and, mostly, 7 can be involved in the mechanisms of modulation of quantal release of neurotransmitters.

BDNF prodomain inhibits neurotransmitter quantal release in mouse motor synapses with the necessary participation of sortilin and adenosine A<sub>1</sub>-receptors

Brain neurotrophin (BDNF) is synthesized by proteolysis of proneurotrophin to form mature BDNF and the prodomain, whose regulatory activity on neuromuscular transmission is just beginning to be studied. At motor synapses, the BDNF prodomain has an inhibitory effect, stimulating GIRK potassium channels via activation of p75 receptors. The aim of this work was to study was to study the initiation and implementation of the mechanism of inhibitory action of the BDNF prodomain in mature motor synapses of the mouse diaphragm. Microelectrodes were used to record spontaneous (miniature) and multiquantal endplate potentials evoked by stimulation of motor axons (MEPP and EPP, respectively). Using selective antagonists, it was revealed that the inhibitory effect of the prodomain on synaptic transmission requires the participation of sortilin, but not TrkB receptors. Stimulation of GIRK induced by the prodomain requires the participation of synaptic metabotropic receptors, which ensure the action of βγ-subunits of Gi proteins on GIRK. Using selective inhibitors, it was found that M2 cholinergic receptors and P2Y13 purinoceptors negatively regulate presynaptic L-type calcium channels, but these metabotropic receptors are not functionally related to the action of the BDNF prodomain. It turned out that the inhibition of quantal release of acetylcholine in motor synapses caused by BDNF prodomain requires the activity of the adenosine A1-receptors only. In addition, when pannexin 1 was pharmacologically blocked by probenecid, the BDNF prodomain lost its inhibitory effect on neuromuscular transmission. Thus, BDNF prodomain-induced inhibition of quantal neurotransmitter release in mouse motor synapses requires the participation of sortilin and endogenous activation of adenosine A1-receptors, which requires the functioning of pannexins 1, which most likely provide an additional source of synaptic ATP to the vesicular one.

Vesicular release probability sets the strength of individual Schaffer collateral synapses

Information processing in the brain is controlled by quantal release of neurotransmitters, a tightly regulated process. From ultrastructural analysis, it is known that presynaptic boutons along single axons differ in the number of vesicles docked at the active zone. It is not clear whether the probability of these vesicles to get released (pves) is homogenous or also varies between individual boutons. Here, we optically measure evoked transmitter release at individual Schaffer collateral synapses at different calcium concentrations, using the genetically encoded glutamate sensor iGluSnFR. Fitting a binomial model to measured response amplitude distributions allowed us to extract the quantal parameters N, pves, and q. We find that Schaffer collateral boutons typically release single vesicles under low pves conditions and switch to multivesicular release in high calcium saline. The potency of individual boutons is highly correlated with their vesicular release probability while the number of releasable vesicles affects synaptic output only under high pves conditions.

Tuning the Size of Large Dense-Core Vesicles and Quantal Neurotransmitter Release via Secretogranin II Liquid-Liquid Phase Separation.

Large dense‐core vesicles (LDCVs) are larger in volume than synaptic vesicles, and are filled with multiple neuropeptides, hormones, and neurotransmitters that participate in various physiological processes. However, little is known about the mechanism determining the size of LDCVs. Here, it is reported that secretogranin II (SgII), a vesicle matrix protein, contributes to LDCV size regulation through its liquid–liquid phase separation in neuroendocrine cells. First, SgII undergoes pH‐dependent polymerization and the polymerized SgII forms phase droplets with Ca2+ in vitro and in vivo. Further, the Ca2+‐induced SgII droplets recruit reconstituted bio‐lipids, mimicking the LDCVs biogenesis. In addition, SgII knockdown leads to significant decrease of the quantal neurotransmitter release by affecting LDCV size, which is differently rescued by SgII truncations with different degrees of phase separation. In conclusion, it is shown that SgII is a unique intravesicular matrix protein undergoing liquid–liquid phase separation, and present novel insights into how SgII determines LDCV size and the quantal neurotransmitter release.

Measurement of intact quantal packet of transmitters released from single nerve terminal by loose-patch amperometry

Neuronal information is majorly encoded chemically at synapses and the elementary unit of synaptic transmission is the contents of neurotransmitter released from single vesicle. However, the contents of quantal neurotransmitter have never been precisely estimated at synapses, which largely prevent our understanding the nature of quantal neurotransmitter release and its impact on neuronal information processing. In order to break through the technical bottleneck of precisely counting quantal neurotransmitter molecules, we developed a new approach in combination of electrophysiology and electrochemistry to measure intact quantal content of single vesicles. An etched submicro-carbon fiber electrode for electrochemical detection was designed to be enclosed in an electrophysiologically used glass pipette. The glass pipette allowed the electrochemical electrode to access the release site, and amperometric recordings were made within the enclosed space at the electrophysiological loose-patch mode. Our study showed that the intact quantal release could be successfully detected at the dopaminergic varicosities by this loose-patch amperometric measurement in real time with negligible leakage.

Selective Knockout of the Vesicular Monoamine Transporter 2 (Vmat2) Gene in Calbindin2/Calretinin-Positive Neurons Results in Profound Changes in Behavior and Response to Drugs of Abuse.

The vesicular monoamine transporter 2 (VMAT2) has a range of functions in the central nervous system, from sequestering toxins to providing conditions for the quantal release of monoaminergic neurotransmitters. Monoamine signaling regulates diverse functions from arousal to mood, movement, and motivation, and dysregulation of VMAT2 function is implicated in various neuropsychiatric diseases. While all monoamine-releasing neurons express the Vmat2 gene, only a subset is positive for the calcium-binding protein Calbindin 2 (Calb2; aka Calretinin, 29 kDa Calbindin). We recently showed that about half of the dopamine neurons in the mouse midbrain are positive for Calb2 and that Calb2 is an early developmental marker of midbrain dopamine cells. Calb2-positive neurons have also been identified in other monoaminergic areas, yet the role of Calb2-positive monoaminergic neurons is poorly understood. To selectively address the impact of Calb2-positive monoaminergic neurons in behavioral regulation, we took advantage of the Cre-LoxP system to create a new conditional knockout (cKO) mouse line in which Vmat2 expression is deleted selectively in Calb2-Cre-positive neurons. In this Vmat2lox/lox;Calb2−Cre cKO mouse line, gene targeting of Vmat2 was observed in several distinct monoaminergic areas. By comparing control and cKO mice in a series of behavioral tests, specific dissimilarities were identified. In particular, cKO mice were smaller than control mice and showed heightened sensitivity to the stereotypy-inducing effects of amphetamine and slight reductions in preference toward sucrose and ethanol, as well as a blunted response in the elevated plus maze test. These data uncover new knowledge about the role of genetically defined subtypes of neurons in the brain’s monoaminergic systems.

Synchronous and asynchronous quantal release at synapses

According to the modern conceptions of the processes of synaptic transmission of excitation there are two forms of quantal neurotransmitter release evoked by the neural stimulus–phasic synchronous and delayed asynchronous release differentiated by the intensity and temporal parameters of quanta secretion. This review is dedicated to the analysis of temporal characteristics of evoked synchronous and delayed asynchronous release of neurotransmitter quanta at chemical synapses. The data indicative of different mechanisms of realization and modulation of these types of the evoked quantal secretion are discussed. The importance of temporal parameters of neuronal secretion for maintenance of effective synaptic transmission of excitation and alteration of these parameters in some pathologies is considered.

Muscarinic inhibition of nicotinic transmission in rat sympathetic neurons and adrenal chromaffin cells.

Little is known about the interactions between nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Here we report that methacholine (MCh), a selective agonist of mAChRs, inhibited up to 80% of nicotine-induced nAChR currents in sympathetic superior cervical ganglion neurons and adrenal chromaffin cells. The muscarine-induced inhibition (MiI) substantially reduced ACh-induced membrane currents through nAChRs and quantal neurotransmitter release. The MiI was time- and temperature-dependent. The slow recovery of nAChR current after washout of MCh, as well as the high value of Q10 (3.2), suggested, instead of a direct open-channel blockade, an intracellular metabotropic process. The effects of GTP-γ-S, GDP-β-S and pertussis toxin suggested that MiI was mediated by G-protein signalling. Inhibitors of protein kinase C (bisindolymaleimide-Bis), protein kinase A (H89) and PIP2 depletion attenuated the MiI, indicating that a second messenger pathway is involved in this process. Taken together, these data suggest that mAChRs negatively modulated nAChRs via a G-protein-mediated second messenger pathway. The time dependence suggests that MiI may provide a novel mechanism for post-synaptic adaptation in all cells/neurons and synapses expressing both types of AChRs.

RIM1α SUMOylation Is Required for Fast Synaptic Vesicle Exocytosis

SummaryThe rapid, activity-dependent quantal presynaptic release of neurotransmitter is vital for brain function. The complex process of vesicle priming, fusion, and retrieval is very precisely controlled and requires the spatiotemporal coordination of multiple protein-protein interactions. Here, we show that posttranslational modification of the active zone protein Rab3-interacting molecule 1α (RIM1α) by the small ubiquitin-like modifier 1 (SUMO-1) functions as a molecular switch to direct these interactions and is essential for fast synaptic vesicle exocytosis. RIM1α SUMOylation at lysine residue K502 facilitates the clustering of CaV2.1 calcium channels and enhances the Ca2+ influx necessary for vesicular release, whereas non-SUMOylated RIM1α participates in the docking/priming of synaptic vesicles and maintenance of active zone structure. These results demonstrate that SUMOylation of RIM1α is a key determinant of rapid, synchronous neurotransmitter release, and the SUMO-mediated “switching” of RIM1α between binding proteins provides insight into the mechanisms underpinning synaptic function and dysfunction.

Astrocyte calcium microdomains are inhibited by Bafilomycin A1 and cannot be replicated by low-level Schaffer collateral stimulation in situ

Astrocyte Gq GPCR and IP3 receptor-dependent Ca2+ elevations occur spontaneously in situ and in vivo. These events vary considerably in size, often remaining confined to small territories of astrocyte processes called “microdomains” and sometimes propagating over longer distances that can include the soma. It has remained unclear whether these events are driven by constitutive (basal) GPCR signaling activity, neuronal action potential-dependent or quantal vesicular release, or some combination of these mechanisms. Here, we applied manipulations to increase or inhibit neuronal vesicular neurotransmitter release together with low-level stimulation of Schaffer collaterals in acute mouse hippocampal slices in an effort to determine the mechanisms underlying spontaneous astrocyte Ca2+ events. We found no significant change in spontaneous microdomain astrocyte Ca2+ elevations when neuronal action potentials were significantly enhanced or blocked. The astrocyte Ca2+ activity was also not affected by inhibitors of group I mGluRs. However, blockade of miniature neurotransmitter release using Bafilomycin A1 significantly reduced the frequency of microdomain astrocyte Ca2+ elevations. We then tested whether astrocyte Ca2+ microdomains can be evoked by low intensity SC stimulation. Importantly, microdomains could not be reproduced even using single, low intensity pulses to the SCs at a minimum distance from the astrocyte. Evoked astrocyte Ca2+ responses most often included the cell soma, were reduced by group I mGluR antagonists, and were larger in size compared to spontaneous Ca2+ microdomains. Overall, our findings suggest that spontaneous microdomain astrocyte Ca2+ elevations are not driven by neuronal action potentials but require quantal release of neurotransmitter which cannot be replicated by stimulation of Schaffer collaterals.

HDAC activity is required for BDNF to increase quantal neurotransmitter release and dendritic spine density in CA1 pyramidal neurons

Molecular mechanisms involved in the strengthening and formation of synapses include the activation and repression of specific genes or subsets of genes by epigenetic modifications that do not alter the genetic code itself. Chromatin modifications mediated by histone acetylation have been shown to be critical for synaptic plasticity at hippocampal excitatory synapses and hippocampal-dependent memory formation. Considering that brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity and behavioral adaptations, it is not surprising that regulation of this gene is subject to histone acetylation changes during synaptic plasticity and hippocampal-dependent memory formation. Whether the effects of BDNF on dendritic spines and quantal transmitter release require histone modifications remains less known. By using two different inhibitors of histone deacetylases (HDACs), we describe here that their activity is required for BDNF to increase dendritic spine density and excitatory quantal transmitter release onto CA1 pyramidal neurons in hippocampal slice cultures. These results suggest that histone acetylation/deacetylation is a critical step in the modulation of hippocampal synapses by BDNF. Thus, mechanisms of epigenetic modulation of synapse formation and function are novel targets to consider for the amelioration of symptoms of intellectual disabilities and neurodegenerative disorders associated with cognitive and memory deficits.

Age-dependent remodelling of ionotropic signalling in cortical astroglia

Cortical astrocytes express fast ionotropic receptors for glutamate and ATP, although their role in neurone-glia communication remains controversial. Stimulation of neuronal afferents in mice neocortex triggers complex glial synaptic currents (GSCs) mediated by NMDA, P2X and AMPA receptors and glutamate transporters. In addition, astrocytes demonstrate spontaneous 'miniature' GSCs resulting from quantal release of neurotransmitters. Here, we demonstrate that maturation and aging of the brain of mice (from 1 to 21 months) affect the density of ionotropic receptors in astrocytes and their role in GSCs generation. The AMPA-receptor-mediated component is the largest in young animals and progressively declines with age. The P2X and NMDA components of GSC are smallest in young, maximal in adult (3 and 6 months old) and once more decrease in old mice, probably reflecting the remodelling of neuronal-glial circuitry. Our results demonstrate that fast synaptic transmission between neurones and astrocytes in neocortex that may be involved in information processing in neuronal-glial networks undergoes remodelling during brain maturation and aging.

Local Presynaptic Activity Gates Homeostatic Changes in Presynaptic Function Driven by Dendritic BDNF Synthesis

Homeostatic synaptic plasticity is important for maintaining stability of neuronal function, but heterogeneous expression mechanisms suggest that distinct facets of neuronal activity may shape the manner in which compensatory synaptic changes are implemented. Here, we demonstrate that local presynaptic activity gates a retrograde form of homeostatic plasticity induced by blockade of AMPA receptors (AMPARs) in cultured hippocampal neurons. We show that AMPAR blockade produces rapid (<3 hr) protein synthesis-dependent increases in both presynaptic and postsynaptic function and that the induction of presynaptic, but not postsynaptic, changes requires coincident local activity in presynaptic terminals. This "state-dependent" modulation of presynaptic function requires postsynaptic release of brain-derived neurotrophic factor (BDNF) as a retrograde messenger, which is locally synthesized in dendrites in response to AMPAR blockade. Taken together, our results reveal a local crosstalk between active presynaptic terminals and postsynaptic signaling that dictates the manner by which homeostatic plasticity is implemented at synapses.

Possible specialization of motoneuron axonal compartments in synthesis of particular proteins

Spontaneous quantal neurotransmitter release and its modulation was studied on neuromuscular preparations of rat soleus from intact animals and from animals in which colchicine had been applied to the sciatic nerve to block the axonal transport. After six days of colchicine application, neither the spontaneous quantal secretion nor its reaction to potassium-induced membrane depolarization or to activation of the presynaptic acetylcholine receptors with carbachol were disturbed in any way. Keeping in mind the relatively short half-life of proteins that take part in exocytosis and its regulation, it may be concluded that the functioning of the terminal neurosecretory apparatus does not depend on the state of axonal transport. These data are consistent with the earlier hypothesis that some proteins performing their functions in nerve terminals are synthesized directly at the site of their operation, rather than in the perikaryon as traditionally assumed.

Neuro-exocytosis: botulinum toxins as inhibitory probes and versatile therapeutics☆

For the fundamental process of quantal neurotransmitter release, a consensus is being reached on the recycling pathways for transmitter-containing, small synaptic vesicles (SSVs), and major inroads have been made into deciphering the multiple steps of regulated exocytosis. These advances arose from the identification of approximately 80 proteins in SSVs, elucidation of the structures of pertinent macromolecular complexes, utilisation of different serotypes (A-G) of botulinum neurotoxin (BoNT) together with transgenic mice lacking key genes. Hence, converging evidence continues to emerge for the sequential formation of complexes between the three SNAREs (SNAP-25, syntaxin and VAMP) and their regulatory proteins (complexins, Munc18), as well as for the Ca2+ triggering of membrane fusion/exocytosis via its sensor, synaptotagmin. Moreover, molecular data gained on BoNTs have been translated into Clinical Medicine with type A now being applied worldwide for effectively treating >100 human conditions due to overactivity of nerves supplying various muscles or glands. A recent advance is the successful engineering of a chimera from two BoNTs to acquire the capability of re-targeting a more active moiety to sensory neurons, with resultant inhibition of the release of a pain mediator. Encouragingly, this novel recombinant protein blocks the exocytotic response triggered by a stimulant (capsaicin) of nociceptive C fibres that activates their vanilloid receptors, a feat not possible for either parental toxin. Reaching this landmark has generated optimism for designing further variants of such a versatile therapeutic for normalising the hyper-activity of particular cell types, especially those underlying the many cases of chronic pain that do not respond to existing drugs.

Evoking and Resolving Quantal Neurotransmitter Release on a Microchip

A microchip that facilitates in-vitro electrical and electrochemical measurements of individual cells and cell clusters was fabricated using surface micromachining and thick film technologies. In the present study, the device was applied towards the detection of exocytotic events from electrically stimulated rat pheochromocytoma (PC12) cells. Using device microfluidics, cells were positioned in a recording chamber over a 5 μm × 10 μm gold working electrode (WE). Channel dimensions (10 μm deep × 10 μm wide) ensured a tight fit for the ∼12 μm diameter PC12 cells in the chamber resulting in direct contact of the cells with the WE. This proximity allowed for quantal resolution of catecholamine release events from the cells and corresponding analysis of release kinetics and quantal size. Cells were stimulated through the application of sinusoidal voltage waveforms across axially-positioned, extracellular electrodes. In this manner, patterned extracellular gradients were generated across the cell thereby resulting in membrane depolarization. To facilitate interpretation of the stimulating electric field in relation to the cell and subsequent dopamine release, quasi-static electromagnetic FEM models were generated using COMSOL Multiphysics software. Upon depolarization, simultaneous chronoamperometric recordings at the WE confirmed stimulus-triggered dopamine release from the cells with a small subset of cells exhibiting release that modulated with the depolarizing cycle of the sinusoidal stimulus. It is anticipated that such a chip could provide a semi-automated alternative to the conventional, labor-intensive carbon fiber electrode (CFE) approach to neurotransmitter measurement.This work was supported in part by the National Institutes of Health, NIDCD R01 DC04928 and by National Science Foundation, IGERT NSF DGE-9987616.

Synapsin II and calcium regulate vesicle docking and the cross‐talk between vesicle pools at the mouse motor terminals

The synapsins, an abundant and highly conserved family of proteins that associate with synaptic vesicles, have been implicated in regulating the synaptic vesicle cycle. However, it has not been determined whether synapsin directly regulates the number of docked vesicles. Here we document that reducing Ca(2+) concentration [Ca(2+)](o) in the extracellular medium from 2 to 0.5 mm led to an approximately 40% decrease in both docked and undocked synaptic vesicles in wild-type nerve terminals of the mouse diaphragm. The same treatment reduced the number of undocked vesicles in nerve terminals derived from synapsin II gene deleted animals, but surprisingly it did not decrease vesicle docking, indicating that synapsin II inhibits docking of synaptic vesicles at reduced [Ca(2+)](o). In accordance with the morphological findings, at reduced [Ca(2+)](o) synapsin II (-) terminals had a higher rate of quantal neurotransmitter release. Microinjection of a recombinant synapsin II protein into synapsin II (-) terminals reduced vesicular docking and inhibited quantal release, indicating a direct and selective synapsin II effect for regulating vesicle docking and, in turn, quantal release. To understand why [Ca(2+)](o) has a prominent effect on synapsin function, we investigated the effect of [Ca(2+)](o) on the distribution of synaptic vesicles and on the concentration of intraterminal Ca(2+). We found that reduced [Ca(2+)](o) conditions produce a decrease in intracellular Ca(2+) and overall vesicle depletion. To explore why at these conditions the role of synapsin II in vesicle docking becomes more prominent, we developed a quantitative model of the vesicle cycle, with a two step synapsin action in stabilizing the vesicle store and regulating vesicle docking. The results of the modelling were in a good agreement with the observed dependence of vesicle distribution on synapsin II and calcium deficiency.

Modeling of quantal neurotransmitter release kinetics in the presence of fixed and mobile calcium buffers

The local calcium concentration in the active zone of secretion determines the number and kinetics of neurotransmitter quanta released after the arrival of a nerve action potential in chemical synapses. The small size of mammalian neuromuscular junctions does not allow direct measurement of the correlation between calcium influx, the state of endogenous calcium buffers determining the local concentration of calcium and the time course of quanta exocytosis. In this work, we used computer modeling of quanta release kinetics with various levels of calcium influx and in the presence of endogenous calcium buffers with varying mobilities. The results of this modeling revealed the desynchronization of quanta release under low calcium influx in the presence of an endogenous fixed calcium buffer, with a diffusion coefficient much smaller than that of free Ca(2+), and synchronization occurred upon adding a mobile buffer. This corresponds to changes in secretion time course parameters found experimentally (Samigullin et al., Physiol Res 54:129-132, 2005; Bukharaeva et al., J Neurochem 100:939-949, 2007).

Adaptation Reduces Spike-Count Reliability, But Not Spike-Timing Precision, of Auditory Nerve Responses

Sensory systems use adaptive coding mechanisms to filter redundant information from the environment to efficiently represent the external world. One such mechanism found in most sensory neurons is rate adaptation, defined as a reduction in firing rate in response to a constant stimulus. In auditory nerve, this form of adaptation is likely mediated by exhaustion of release-ready synaptic vesicles in the cochlear hair cell. To better understand how specific synaptic mechanisms limit neural coding strategies, we examined the trial-to-trial variability of auditory nerve responses during short-term rate-adaptation by measuring spike-timing precision and spike-count reliability. After adaptation, precision remained unchanged, whereas for all but the lowest-frequency fibers, reliability decreased. Modeling statistical properties of the hair cell-afferent fiber synapse suggested that the ability of one or a few vesicles to elicit an action potential reduces the inherent response variability expected from quantal neurotransmitter release, and thereby confers the observed count reliability at sound onset. However, with adaptation, depletion of the readily releasable pool of vesicles diminishes quantal content and antagonizes the postsynaptic enhancement of reliability. These findings imply that during the course of short-term adaptation, coding strategies that employ a rate code are constrained by increased neural noise because of vesicle depletion, whereas those that employ a temporal code are not.

Bernard Katz, quantal transmitter release and the foundations of presynaptic physiology

Bernard Katz, quantal transmitter release and the foundations of presynaptic physiology