Step In Synaptic Transmission Research Articles

Real-time three-dimensional tracking of single synaptic vesicles reveals that synaptic vesicles undergoing kiss-and-run fusion remain close to their original fusion site before reuse

The release of neurotransmitters via the fusion between synaptic vesicles and the presynaptic membrane is an essential step in synaptic transmission. Synaptic vesicles generally undergo two distinct modes of exocytosis called full-collapse fusion and kiss-and-run fusion. In kiss-and-run fusion, the fusion pore of the synaptic vesicle opens transiently without the vesicle collapsing fully into the plasma membrane; thus, each synaptic vesicle can be used multiple times to release neurotransmitters. Despite considerable research, the detailed mechanisms that underlie kiss-and-run fusion remain elusive, particularly the location of synaptic vesicles after kiss-and-run events. To address this question, we performed real-time three-dimensional tracking of single synaptic vesicles labeled with a single quantum dot in the presynaptic terminal of cultured hippocampal neurons and analyzed the three-dimensional trajectories of these vesicles undergoing kiss-and-run fusion. We found that the majority of these synaptic vesicles underwent another exocytosis event within 120 nm of their original fusion site and underwent a second exocytosis event within 10 s of the first fusion event. These results indicate that after kiss-and-run fusion, synaptic vesicles remain relatively close to their original fusion site and can release repeatedly at brief intervals, allowing neurons to maintain neurotransmitter release during bursting activity.

Imaging and Analysis of Presynaptic Calcium Influx in Cultured Neurons Using synGCaMP6f.

Presynaptic Ca2+ influx through voltage-gated calcium channels (VGCCs) is a key step in synaptic transmission that links action potential (AP)-derived depolarization to vesicle release. However, investigation of presynaptic Ca2+ influx by patch clamp recordings is difficult due to the small size of the majority of synaptic boutons along thin axons that hamper clamp control. Genetically encoded calcium indicators (GECIs) in combination with live cell imaging provide an alternative method to study Ca2+ transients in individual presynaptic terminals. The indicator GCaMP6f was developed for fast speed and high sensitivity in detecting Ca2+ transients even in subcellular compartments. We fused GCaMP6f to synaptophysin (synGCaMP6f) to enrich the calcium indicator in presynaptic boutons of transfected primary hippocampal neurons to study presynaptic Ca2+ changes in response to individual APs or short bursts. Changes in fluorescence intensity were evaluated by normalization to control level or, alternatively, by normalization to maximal fluorescence using the calcium ionophore ionomycin. Measurements revealed robust Ca2+ transients with amplitudes that depend on parameters like the number of APs, stimulation frequency or external calcium concentration. Our findings indicate an appropriate sensitivity of synGCaMP6f for studying total presynaptic Ca2+ transients induced by single APs or short bursts that showed little rundown of the response after repeated bursts. Moreover, these recordings are fast enough to even study short-term plasticity like paired pulse facilitation (PPF) and frequency dependence of Ca2+ transients. In addition, synGCaMP6f could be used to dissect the contribution of different subtypes of VGCCs to presynaptic Ca2+ influx. Our results demonstrate that synGCaMP6f allows the reliable analysis of changes in presynaptic calcium concentration at many individual synaptic boutons in parallel and provides the possibility to study the regulation of this important step in synaptic transmission.

Voltage-Gated Calcium Channels: Key Players in Sensory Coding in the Retina and the Inner Ear.

Calcium influx through voltage-gated Ca (CaV) channels is the first step in synaptic transmission. This review concerns CaV channels at ribbon synapses in primary sense organs and their specialization for efficient coding of stimuli in the physical environment. Specifically, we describe molecular, biochemical, and biophysical properties of the CaV channels in sensory receptor cells of the retina, cochlea, and vestibular apparatus, and we consider how such properties might change over the course of development and contribute to synaptic plasticity. We pay particular attention to factors affecting the spatial arrangement of CaV channels at presynaptic, ribbon-type active zones, because the spatial relationship between CaV channels and release sites has been shown to affect synapse function critically in a number of systems. Finally, we review identified synaptopathies affecting sensory systems and arising from dysfunction of L-type, CaV1.3, and CaV1.4 channels or their protein modulatory elements.

A three-pool model dissecting readily releasable pool replenishment at the calyx of held.

Although vesicle replenishment is critical in maintaining exo-endocytosis recycling, the underlying mechanisms are not well understood. Previous studies have shown that both rapid and slow endocytosis recycle into a very large recycling pool instead of within the readily releasable pool (RRP), and the time course of RRP replenishment is slowed down by more intense stimulation. This finding contradicts the calcium/calmodulin-dependence of RRP replenishment. Here we address this issue and report a three-pool model for RRP replenishment at a central synapse. Both rapid and slow endocytosis provide vesicles to a large reserve pool (RP) ~42.3 times the RRP size. When moving from the RP to the RRP, vesicles entered an intermediate pool (IP) ~2.7 times the RRP size with slow RP-IP kinetics and fast IP-RRP kinetics, which was responsible for the well-established slow and rapid components of RRP replenishment. Depletion of the IP caused the slower RRP replenishment observed after intense stimulation. These results establish, for the first time, a realistic cycling model with all parameters measured, revealing the contribution of each cycling step in synaptic transmission. The results call for modification of the current view of the vesicle recycling steps and their roles.

Presynaptic K+ Channels, Vesicular Ca2+/H+ Antiport—Synaptotagmin, and Acetylcholinesterase, Three Mechanisms Cutting Short the Cholinergic Signal at Neuromuscular and Nerve–Electroplaque Junctions

In neuromuscular and nerve-electroplaque junctions, nerve impulses can be transmitted at high frequencies. This implies that transmission of individual impulses must be very brief. We describe three mechanisms which curtail the time course of individual impulses at these synapses: (1) opening of presynaptic K(+) channels (delayed rectifier) efficiently curtails the presynaptic action potential. Inhibition of K(+) channel by aminopyridines transforms the normally brief postsynaptic potential (2-3ms) to a long-lasting "giant" potential (exceeding half a second); (2) a low-affinity Ca(2+)/H(+) antiport ensures rapid Ca(2+) sequestration into synaptic vesicles, curtailing the calcium signal and thereby the duration of transmitter release. Indeed vesicular Ca(2+)/H(+) antiport inhibition by bafilomycin or Sr(2+) prolongs the duration of the postsynaptic potential. We recently showed that synaptotagmin-1 is required for this antiport activity; thus the vesicular Ca(2+)/H(+) antiport might be synaptotagmin itself, or regulated by it; and (3) it is recalled that, in these junctions, acetylcholinesterase is highly concentrated in the synaptic cleft and that anticholinesterases lengthen the endplate time course. Therefore, at three different steps of synaptic transmission, an efficient mechanism curtails the local synaptic signal. When one of these three mechanisms is inhibited, the duration of individual impulses is prolonged, but the synapse loses its faculty to fire at high frequencies.

Corticosterone targets distinct steps of synaptic transmission via concentration specific activation of mineralocorticoid and glucocorticoid receptors

Hippocampal neurons are affected by chronic stress and have a high density of cytoplasmic mineralocorticoid and glucocorticoid receptors (MR and GR). Detailed studies on the genomic effects of the stress hormone corticosterone at physiologically relevant concentrations on different steps in synaptic transmission are limited. In this study, we tried to delineate how activation of MR and GR by different concentrations of corticosterone affects synaptic transmission at various levels. The effect of 3-h corticosterone (25, 50, and 100 nM) treatment on depolarization-mediated calcium influx, vesicular release and properties of miniature excitatory post-synaptic currents (mEPSCs) were studied in cultured hippocampal neurons. Activation of MR with 25 nM corticosterone treatment resulted in enhanced depolarization-mediated calcium influx via a transcription-dependent process and increased frequency of mEPSCs with larger amplitude. On the other hand, activation of GR upon 100 nM corticosterone treatment resulted in increase in the rate of vesicular release via the genomic actions of GR. Furthermore, GR activation led to significant increase in the frequency of mEPSCs with larger amplitude and faster decay. Our studies indicate that differential activation of the dual receptor system of MR and GR by corticosterone targets the steps in synaptic transmission differently.

Protein Kinase A-Mediated Synapsin I Phosphorylation Is a Central Modulator of Ca2+-Dependent Synaptic Activity

Protein kinase A (PKA) modulates several steps of synaptic transmission. However, the identification of the mediators of these effects is as yet incomplete. Synapsins are synaptic vesicle (SV)-associated phosphoproteins that represent the major presynaptic targets of PKA. We show that, in hippocampal neurons, cAMP-dependent pathways affect SV exocytosis and that this effect is primarily brought about through synapsin I phosphorylation. Phosphorylation by PKA, by promoting dissociation of synapsin I from SVs, enhances the rate of SV exocytosis on stimulation. This effect becomes relevant when neurons are challenged with sustained stimulation, because it appears to counteract synaptic depression and accelerate recovery from depression by fostering the supply of SVs from the reserve pool to the readily releasable pool. In contrast, synapsin phosphorylation appears to be dispensable for the effects of cAMP on the frequency and amplitude of spontaneous synaptic currents and on the amplitude of evoked synaptic currents. The modulation of depolarization-evoked SV exocytosis by PKA phosphorylation of synapsin I is primarily caused by calmodulin (CaM)-dependent activation of cAMP pathways rather than by direct activation of CaM kinases. These data define a hierarchical crosstalk between cAMP- and CaM-dependent cascades and point to synapsin as a major effector of PKA in the modulation of activity-dependent SV exocytosis.

Principal pathway coupling agonist binding to channel gating in nicotinic receptors

Synaptic receptors respond to neurotransmitters by opening an intrinsic ion channel in the final step in synaptic transmission. How binding of the neurotransmitter is conveyed over the long distance to the channel remains a central question in neurobiology. Here we delineate a principal pathway that links neurotransmitter binding to channel gating by using a structural model of the Torpedo acetylcholine receptor at 4-A resolution, recordings of currents through single receptor channels and determinations of energetic coupling between pairs of residues. We show that a pair of invariant arginine and glutamate residues in each receptor alpha-subunit electrostatically links peripheral and inner beta-sheets from the binding domain and positions them to engage with the channel. The key glutamate and flanking valine residues energetically couple to conserved proline and serine residues emerging from the top of the channel-forming alpha-helix, suggesting that this is the point at which the binding domain triggers opening of the channel. The series of interresidue couplings identified here constitutes a primary allosteric pathway that links neurotransmitter binding to channel gating.

Elementary steps in synaptic transmission revealed by currents through single ion channels.

An account is presented of how the molecular basis of synaptic transmission at peripheral and central synapses is elucidated by combining patch clamp and recombinant DNA techniques.

Elementary Steps in Synaptic Transmission Revealed by Currents through Single Ion Channels (Nobel Lecture)

A new era in cell biology has been heralded by the prize-winning research of Erwin Neher and Bert Sakmann. These laureates measured the picoampere currents through individual ion channels (see A, below; ACh = acetylcholine). The breakthrough in suppressing the background noise was achieved by applying a slight suction to the pipette used for the current measurement. This improved the crucial pipette-to-membrane seal by two orders of magnitude. In B, below, such a “patch pipette” is shown on the end-plate membrane of denervated frog muscle fiber. Today the patch-clamp technique is used routinely with other methods such as DNA recombination or fluorimetric techniques, for example, to study the synaptic transfer in its molecular detail.

Nobel Lecture. Elementary steps in synaptic transmission revealed by currents through single ion channels.

Nobel Lecture. Elementary steps in synaptic transmission revealed by currents through single ion channels.

Elementary steps in synaptic transmission revealed by currents through single ion channels.

The plasma membrane of a cell separates its interior from the extracellular environment and from other cells and acts both as a diffusional barrier and as an electrical insulator. This allows differentiation of cells with specialized functions. Coordinated behavior of multicellular organisms requires exchange of signals between individual cells. Because the signal must be transferred from one cell to another, it must occur by a mechanism that allows it to traverse the insulating cell membrane. Signaling occurs in various ways via specific receptors on the receiving cells and subsequent generation of a transmembrane signal. The nervous system connects cells in a very specific way, and signal transmission between individual cells takes place at contacts, called synapses, that are anatomically and functionally highly specialized.

Elementary steps in synaptic transmission revealed by currents through single ion channels

The plasma membrane of a cell separates its interior from the extracellular environment and from other cells and acts both as a diffusional barrier and as an electrical insulator. This allows differentiation of cells with specialized functions. Coordinated behavior of multicellular organisms requires exchange of signals between individual cells. Because the signal must be transferred from one cell to another, it must occur by a mechanism that allows it to traverse the insulating cell membrane. Signaling occurs in various ways via specific receptors on the receiving cells and subsequent generation of a transmembrane signal. The nervous system connects cells in a very specific way, and signal transmission between individual cells takes place at contacts, called synapses, that are anatomically and functionally highly specialized.

Retroviral transfer of a human tyrosine hydroxylase cDNA in various cell lines: regulated release of dopamine in mouse anterior pituitary AtT-20 cells.

Little is known about the molecular events mediating neurotransmitter release, a crucial step in synaptic transmission. In this paper, the biosynthesis and release of L-beta-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine were analyzed in three heterologous cell lines after retroviral-mediated gene transfer of tyrosine hydroxylase (EC 1.14.16.2), the rate-limiting enzyme in catecholamine synthesis. A recombinant retrovirus encoding human tyrosine hydroxylase type I as well as neomycin-resistance gene was used to infect a fibroblast (NIH 3T3), a neuroblastoma (NS20 Y), and a neuroendocrine (AtT-20) cell line. After selection in the presence of neomycin and in tyrosine-free medium, high levels of exogenous tyrosine hydroxylase activity were detected in extracts of the three cell lines. High-performance liquid chromatography of cell extracts and culture supernatants confirmed that the three cell lines hydroxylated tyrosine to form L-DOPA and released this metabolite into the culture medium. Interestingly, the neuroendocrine cell line AtT-20 synthesized not only L-DOPA but also dopamine. Evoked secretion studies established that AtT-20 cells released the transmitter upon depolarization in a regulated, calcium-dependent way. We discuss the implication of this approach for the analyses of neurotransmitter release as well as in the context of degenerative disorders such as Parkinson disease.

Possible roles of protein kinase C in neurotransmission.

Protein kinase C (PKC) is an ubiquitous Ca2+-activated phospholipiddependent protein kinase abundant in the central nervous system(1–4). This enzyme is activated by 1,2-diacylglycerol which transiently appears in the cell membrane, as a consequence of receptor-mediated and voltage-dependent hydrolysis of inositol phospholipids. The link of the phosphoinositide system to PKC seems to be that of a major second messenger system in the central nervous system and the possible involvement in significant steps in synaptic transmission have to be given attention. Until recently, PKC was thought to be a single entity, however, molecular cloning analysis has shown that there is a family of PKC-related genes(5–11). At least four subspecies of PKC (α, βI, βII and γ) are expresssed in the mammalian brain. Enzymatically, three major isozymes of PKC (types I, II and III) have been resolved from rat brains, using hydroxyapatite column chromatography(12). All subtypes were activated by diacylglycerol as well as tumor-promoting phorbol esters in the presence of Ca2+ and phosphatidylserine. They bound radioactive phorbol ester and showed similar kinetic properties, although the enzyme having the β-sequence was active at very low concentrations of Ca (8,13). The amino acid sequence of these three types has been identified by comparison with the enzyme separately expressed in the COS cells transfected by respective cDNAs-containing plasmids (14). Type I corresponds to the enzyme encoded with the γ-sequence. Type II is a mixture of βI- and βII-subspecies derived from alternative splicing of a single gene. Type III corresponds to α-subspecies. The αI-and βII-subspecies differ by only about 50 amino acid residues in the carboxyl-terminal regions, show similar kinetic properties and are not separable by conventional enzyme purification procedures. To characterize the tissue specificity of these subspecies, a specific antibody against each subspecies should be a pertinent tool. We present here the cell specific expression of each subspecies of PKC in the rat brain, determined using subspecies specific antibodies.

Progress on LTP at hippocampal synapses: a post-synaptic Ca 2+ trigger for memory storage?

Progress on LTP at hippocampal synapses: a post-synaptic Ca 2+ trigger for memory storage?

The fusogenic substance dimethyl sulfoxide enhances exocytosis in motor nerve endings

Fusion of synaptic vesicles with the surface membrane of the nerve terminal is a key step in synaptic transmission, which normally requires the entry of calcium ions into the cell. We report that this fusion and the subsequent liberation of transmitter can also be induced by the fusogenic substances DMSO (dimethyl sulfoxide) and PEG (poly(ethylene glycol)). Calcium ions and DMSO exhibit a synergistic effect in the fusion of synaptic vesicles with the axolemma, resembling their action on fusion phenomena in liposomes.