Calcium regulation of spontaneous and asynchronous neurotransmitter release

The molecular machinery underlying action potential-evoked, synchronous neurotransmitter release, has been intensely studied. It was presumed that two other forms of exocytosis, delayed (asynchronous) and spontaneous transmission, were mediated by the same voltage-activated Ca(2+) channels (VACCs), intracellular Ca(2+) sensors and vesicle pools. However, a recent explosion in the study of spontaneous and asynchronous release has shown these presumptions to be incorrect. Furthermore, the finding that different forms of synaptic transmission may mediate distinct physiological functions emphasizes the importance of identifying the mechanisms by which Ca(2+) regulates spontaneous and asynchronous release. In this article, we will briefly summarize new and published data on the role of Ca(2+) in regulating spontaneous and asynchronous release at a number of different synapses. We will discuss how an increase of extracellular [Ca(2+)] increases spontaneous and asynchronous release, show that VACCs are involved at only some synapses, and identify regulatory roles for other ion channels and G protein-coupled receptors. In particular, we will focus on two novel pathways that play important roles in the regulation of non-synchronous release at two exemplary synapses: one modulated by the Ca(2+)-sensing receptor and the other by transient receptor potential cation channel sub-family V member 1.     

Autonomous function of synaptotagmin 1 in triggering synchronous release independent of asynchronous release

Ca(2+) triggers neurotransmitter release in at least two principal modes, synchronous and asynchronous release. Synaptotagmin 1 functions as a Ca(2+) sensor for synchronous release, but its role in asynchronous release remains unclear. We now show that in cultured cortical neurons stimulated at low frequency (or Hz), deletion of synaptotagmin 1 also alters only synchronous, not asynchronous, release during the stimulus train, but dramatically enhances "delayed asynchronous release" following the stimulus train. Thus synaptotagmin 1 functions as an autonomous Ca(2+) sensor independent of asynchronous release during isolated action potentials and action potential trains, but restricts asynchronous release induced by residual Ca(2+) after action potential trains. We propose that synaptotagmin 1 occupies release "slots" at the active zone, possibly in a Ca(2+)-independent complex with SNARE proteins that are freed when action potential-induced Ca(2+) influx activates synaptotagmin 1.     

Doc2 is a Ca2+ sensor required for asynchronous neurotransmitter release

Synaptic transmission involves a fast synchronous phase and a slower asynchronous phase of neurotransmitter release that are regulated by distinct Ca(2+) sensors. Though the Ca(2+) sensor for rapid exocytosis, synaptotagmin I, has been studied in depth, the sensor for asynchronous release remains unknown. In a screen for neuronal Ca(2+) sensors that respond to changes in [Ca(2+)] with markedly slower kinetics than synaptotagmin I, we observed that Doc2--another Ca(2+), SNARE, and lipid-binding protein--operates on timescales consistent with asynchronous release. Moreover, up- and downregulation of Doc2 expression levels in hippocampal neurons increased or decreased, respectively, the slow phase of synaptic transmission. Synchronous release, when triggered by single action potentials, was unaffected by manipulation of Doc2 but was enhanced during repetitive stimulation in Doc2 knockdown neurons, potentially due to greater vesicle availability. In summary, we propose that Doc2 is a Ca(2+) sensor that is kinetically tuned to regulate asynchronous neurotransmitter release.     

Shunting inhibition controls the gain modulation mediated by asynchronous neurotransmitter release in early development

The sensitivity of a neuron to its input can be modulated in several ways. Changes in the slope of the neuronal input-output curve depend on factors such as shunting inhibition, background noise, frequency-dependent synaptic excitation, and balanced excitation and inhibition. However, in early development GABAergic interneurons are excitatory and other mechanisms such as asynchronous transmitter release might contribute to regulating neuronal sensitivity. We modeled both phasic and asynchronous synaptic transmission in early development to study the impact of activity-dependent noise and short-term plasticity on the synaptic gain. Asynchronous release decreased or increased the gain depending on the membrane conductance. In the high shunt regime, excitatory input due to asynchronous release was divisive, whereas in the low shunt regime it had a nearly multiplicative effect on the firing rate. In addition, sensitivity to correlated inputs was influenced by shunting and asynchronous release in opposite ways. Thus, asynchronous release can regulate the information flow at synapses and its impact can be flexibly modulated by the membrane conductance.     

Humanin derivative,HNG, enhances neurotransmitter release

BackgroundHumanin (HN) is an endogenous 24-residue peptide that was first identified as a protective factor against neuronal death in Alzheimer's disease (AD). We previously demonstrated that the highly potent HN derivative HNG (HN with substitution of Gly for Ser14) ameliorated cognitive impairment in AD mouse models. Despite the accumulating evidence on the antagonizing effects of HN against cognitive deficits, the mechanisms behind these effects remain to be elucidated.MethodsThe extracellular fluid in the hippocampus of wild-type young mice was collected by microdialysis and the amounts of neurotransmitters were measured. The kinetic analysis of exocytosis was performed by amperometry using neuroendocrine cells.ResultsThe hippocampal acetylcholine (ACh) levels were increased by intraperitoneal injection of HNG. HNG did not affect the physical activities of the mice but modestly improved their object memory. In a neuronal cell model, rat pheochromocytoma PC12 cells, HNG enhanced ACh-induced dopamine release. HNG increased ACh-induced secretory events and vesicular quantal size in primary neuroendocrine cells.ConclusionsThese findings suggest that HN directly enhances regulated exocytosis in neurons, which can contribute to the improvement of cognitive functions.General significanceThe regulator of exocytosis is a novel physiological role of HN, which provides a molecular clue for HN's effects on brain functions under health and disease.     

External calcium, intrasynaptosomal free calcium and neurotransmitter release

In a physiological medium the resting membrane potential of synaptosomes from guinea-pig cerebral cortex, estimated from rhodamine 6G fluorescence measurements, was nearly -50mV. This agreed with calculations using the Goldman-Hodgkin-Katz equation. With external [Ca2+] less than or equal to 3 mM veratridine depolarisation (to -30 mV) was accompanied by increases in intrasynaptosomal free calcium concentrations (monitored by entrapped quin2) and parallel increases in total acetylcholine release. With external [Ca2+] greater than 3 mM both intrasynaptosomal free calcium concentrations and transmitter release were paradoxically reduced, providing further evidence for a close correlation between the two events. To support an explanation of these findings based on divalent cation screening of membrane surface charge (increasing the voltage gradient within the membrane and closing voltage-inactivated channels) surface potential measurements were made on synaptic lipid liposomes by using a fluorescent surface-bound pH indicator. These experiments provided evidence for the presence of screenable surface charge on synaptosomes, and it was further shown in depolarised synaptosomes themselves that total external [Ca2+ + Mg2+], and not [Ca2+] alone, set the observed peak in intrasynaptosomal free calcium.     

Calcium control of neurotransmitter release

Upon entering a presynaptic terminal, an action potential opens Ca(2+) channels, and transiently increases the local Ca(2+) concentration at the presynaptic active zone. Ca(2+) then triggers neurotransmitter release within a few hundred microseconds by activating synaptotagmins Ca(2+). Synaptotagmins bind Ca(2+) via two C2-domains, and transduce the Ca(2+) signal into a nanomechanical activation of the membrane fusion machinery; this activation is mediated by the Ca(2+)-dependent interaction of the synaptotagmin C2-domains with phospholipids and SNARE proteins. In triggering exocytosis, synaptotagmins do not act alone, but require an obligatory cofactor called complexin, a small protein that binds to SNARE complexes and simultaneously activates and clamps the SNARE complexes, thereby positioning the SNARE complexes for subsequent synaptotagmin action. The conserved function of synaptotagmins and complexins operates generally in most, if not all, Ca(2+)-regulated forms of exocytosis throughout the body in addition to synaptic vesicle exocytosis, including in the degranulation of mast cells, acrosome exocytosis in sperm cells, hormone secretion from endocrine cells, and neuropeptide release.     

The role of calcium in modulation of the kinetics of synchronous and asynchronous quantal release at the neuromuscular junction

To elucidate the mechanisms of calcium regulation of the kinetics of the evoked neurotransmitter quantal release, we have investigated the temporal parameters of acetylcholine secretion in the mouse neuro-muscular junction at varying extracellular calcium concentration, in the presence of calcium channel blockers or intracellular calcium buffers. Acetylcholine secretion was induced by the motor nerve stimulation at a low frequency, which did not produce facilitation of the neurotransmitter release. The analysis of histograms of synaptic delays of uniquantal endplate currents recorded during 50 ms after the presynaptic action potential revealed three components of the secretion process: early and late periods of synchronous release and a delayed asynchronous release. At reduced extracellular calcium level, the relative number of quanta released during the asynchronous phase of secretion increased, while the rate of quantal release during the early synchronous period decreased. The findings support the hypothesis of participation of low- and high-affinity calcium sensors with different calcium binding kinetics in regulation of, respectively, synchronous and asynchronous release of neurotransmitter quanta.     

Synapsins as regulators of neurotransmitter release

One of the crucial issues in understanding neuronal transmission is to define the role(s) of the numerous proteins that are localized within presynaptic terminals and are thought to participate in the regulation of the synaptic vesicle life cycle. Synapsins are a multigene family of neuron-specific phosphoproteins and are the most abundant proteins on synaptic vesicles. Synapsins are able to interact in vitro with lipid and protein components of synaptic vesicles and with various cytoskeletal proteins, including actin. These and other studies have led to a model in which synapsins, by tethering synaptic vesicles to each other and to an actin-based cytoskeletal meshwork, maintain a reserve pool of vesicles in the vicinity of the active zone. Perturbation of synapsin function in a variety of preparations led to a selective disruption of this reserve pool and to an increase in synaptic depression, suggesting that the synapsin-dependent cluster of vesicles is required to sustain release of neurotransmitter in response to high levels of neuronal activity. In a recent study performed at the squid giant synapse, perturbation of synapsin function resulted in a selective disruption of the reserve pool of vesicles and in addition, led to an inhibition and slowing of the kinetics of neurotransmitter release, indicating a second role for synapsins downstream from vesicle docking. These data suggest that synapsins are involved in two distinct reactions which are crucial for exocytosis in presynaptic nerve terminals. This review describes our current understanding of the molecular mechanisms by which synapsins modulate synaptic transmission, while the increasingly well-documented role of the synapsins in synapse formation and stabilization lies beyond the scope of this review.     

Calcium permeability changes and neurotransmitter release in cultured brain neurons. II. Temporal analysis of neurotransmitter release

The coupling between depolarization-induced calcium entry and neurotransmitter release was studied in rat brain neurons in culture. The endogenous dopamine content of the cells was determined by high performance liquid chromatography utilizing electrochemical detection. The amount of dopamine in unstimulated cells was found to be about 16 ng/mg of protein. Depolarization of the neurons by elevated K+ caused a Ca2+-dependent release of dopamine from the cells. Following 1 min of depolarization, the cellular dopamine content and the amount of [3H]dopamine in cells preloaded with the radioactive transmitter were reduced by 35%. The release of [3H]dopamine by the neurons was measured at 1.5-6-s intervals by a novel rapid dipping technique. Depolarization in the presence of Ca2+ (1.8 mM) enhanced the rate of neurotransmitter release by 90-fold (0.072 +/- 0.003 s-1) over the basal release in the presence of Ca2+. The evoked release consisted of a major rapidly terminating phase (t1/2 = 9.6 s) which comprised about 40% of the neurotransmitter content of the cells and a subsequent slower efflux (t1/2 = 575 s) which was observed during following prolonged depolarization. Predepolarization of the cells in the absence of extracellular Ca2+ did not affect the kinetics of the evoked release. The fast evoked release could be re-elicited in the cells after 20 min "rest" in reference low K+ buffer. The effects of varying the extracellular Ca2+ concentrations on the kinetic parameters of the evoked release were measured. The amount of neurotransmitter released during the fast kinetic phase was very sensitive to the external Ca2+ (from 0% in the absence of Ca2+ to 40% of the neurotransmitter content at Ca2+ 0.3 mM). The rate constant of the fast release did not depend on the extracellular Ca2+, whereas the rate constant of the slow release increased from 0.0004 +/- 0.0001 s-1 at 0.4 mM Ca2+ to 0.0012 +/- 0.0002 s-1 at 0.8 mM Ca2+. The fast evoked release was inhibited by verapamil in a concentration-dependent manner. By contrast, verapamil enhanced the basal and the slow release independent of the presence of Ca2+. Both fast and slow phases of the evoked release were blocked by Co2+. Addition of Co2+ within the first 6 s after the onset of depolarization inhibited the fast release but failed to do so when added later on.(ABSTRACT TRUNCATED AT 400 WORDS)     

How to build fast muscles: synchronous and asynchronous designs

In animals, muscles are the most common effectors that translateneuronal activity into behavior. Nowhere is behavior more restrictedby the limits of muscle performance than at the upper rangeof high-frequency movements. Here, we see new and multiple designsto cope with the demands for speed. Extremely rapid oscillationsin force are required to power cyclic activities such as flightin insects or to produce vibrations for sound. Such behaviorsare seen in a variety of invertebrates and vertebrates, andare powered by both synchronous and asynchronous muscles. Insynchronous muscles, each contraction/relaxation cycle is accompaniedby membrane depolarization and subsequent repolarization, releaseof activator calcium, attachment of cross-bridges and muscleshortening, then removal of activator calcium and cross-bridgedetachment. To enable all of these to occur at extremely highfrequencies a suite of modifications are required, includingprecise neural control, hypertrophy of the calcium handlingmachinery, innovative mechanisms to bind calcium, and molecularmodification of the cross-bridges and regulatory proteins. Sideeffects are low force and power output and low efficiency, butthe benefit of direct, neural control is maintained. Asynchronousmuscles, in which there is not a 1:1 correspondence betweenneural activation and contraction, are a radically differentdesign. Rather than rapid calcium cycling, they rely on delayedactivation and deactivation, and the resonant characteristicsof the wings and exoskeleton to guide their extremely high-frequencycontractions. They thus avoid many of the modifications andattendant trade-offs mentioned above, are more powerful andmore efficient than high-frequency synchronous muscles, butare considerably more restricted in their application.     

Nociceptin and neurotransmitter release in the periphery

Nociceptin exerts a general modulatory effect on transmitter release from sympathetic, parasympathetic, NANC and sensory nerve endings in the peripheral nervous system in various species. This effect occurs at a prejunctional level and is independent from the activation of mu, delta and kappa opioid receptors. Despite the growing evidence describing the peripheral activity of nociceptin since its discovery in 1995, the lack of selective and potent antagonists does not allow us to draw conclusions on the putative physiological role of this peptide at this level.     

Study of asynchronous and synchronous yeast cultures using flow cytofluorometry]

Distribution of Saccharomyces cerevisiae, Candida boidinii and Candida tropicalis cells according to DNA content was investigated using laser flow cytofluorometry. Cells distribution curves according to DNA content possessed two maxima in case the sample belonged to the exponential phase of the asynchronous batch culture, or one maximum in case the sample was from the stationary phase of growth. In synchronous cultures variations of cells distribution curves according to DNA content (age structure of the population) were demonstrated and the curves with one maximum and plateau were observed.     

Receptor-mediated regulation of calcium channels and neurotransmitter release

Ca2+ influx into the nerve terminal is normally the trigger for the release of neurotransmitters. Many neurons possess presynaptic receptors whose activation results in changes in the quantity of neurotransmitter released by an action potential. This paper reviews studies that show that presynaptic receptors can regulate the activity of Ca2+ channels in the nerve terminal, resulting in changes in the influx of Ca2+ and in neurotransmitter release. Neurons possess several different types of voltage-sensitive Ca2+ channels. Ca2+ influx through N-type channels appears to trigger transmitter release in many instances. In other cases Ca2+ influx through L channels can influence transmitter release. Neurotransmitters can inhibit N channels through a G protein-mediated transduction mechanism. The G proteins are frequently pertussis toxin substrates. Inhibition of N channels appears to involve changes in their voltage dependence. Neurotransmitters can also regulate neuronal K+ channels. Activation of these K+ channels can lead to a reduction in Ca2+ influx and neurotransmitter release; these effects are also mediated by G proteins. Thus neurotransmitters may often regulate both presynaptic Ca2+ and K+ channels. These two effects may be synergistic mechanisms for the regulation of Ca2+ influx and neurotransmitter release.     

Survival of bisected rabbit morulae transferred to synchronous and asynchronous recipients

The rabbit was used as a model to test the concept that temporal asynchrony is required to establish physiological synchrony when embryos are bisected to produce demiembryos. In preliminary studies with intact embryos it was confirmed that embryos harvested on days 2, 3, 4, or 5 (day 0 = day of breeding) can be transferred with +/- 1 day of asynchrony to the uteri of recipient rabbits. Three experiments were conducted with bisected embryos. In experiment 1, 192 bisected and 194 control day 3 embryos were transferred to uteri of day 2, 2.5, and 3 recipients (ovulated 0, 12, and 24 h after the donors), with 14% of the bisected and 39% of the intact embryos (P less than .05) resulting in young. Only 4% (2/48) of the day 3 bisected embryos vs. 39% (P less than .05) of the intact day 3 embryos survived in the uteri of day 2 recipients. In experiment 2, day 3 bisected and intact embryos were transferred to the oviducts of day 3, 3.5, or 4 recipients, the speculation being that the oviduct might provide a more neutral environment than the uterus. However, embryo survival was very low, except for the intact embryos transferred to synchronized recipients (42% young born). In experiment 3, 150 intact and 162 (81 pairs) bisected day 3 embryos collected from uteri were transferred to uteri of day 2.5, 3.0, and 3.5 recipient does. Significantly more pregnancies (100% vs. 47%, P less than .01) and young born (56% vs. 19%, P less than .01) resulted from intact embryos than from bisected embryos, irrespective of the uterine age.(ABSTRACT TRUNCATED AT 250 WORDS)