The actin cytoskeleton and neurotransmitter release: an overview

Here we review evidence that actin and its binding partners are involved in the release of neurotransmitters at synapses. The spatial and temporal characteristics of neurotransmitter release are determined by the distribution of synaptic vesicles at the active zones, presynaptic sites of secretion. Synaptic vesicles accumulate near active zones in a readily releasable pool that is docked at the plasma membrane and ready to fuse in response to calcium entry and a secondary, reserve pool that is in the interior of the presynaptic terminal. A network of actin filaments associated with synaptic vesicles might play an important role in maintaining synaptic vesicles within the reserve pool. Actin and myosin also have been implicated in the translocation of vesicles from the reserve pool to the presynaptic plasma membrane. Refilling of the readily releasable vesicle pool during intense stimulation of neurotransmitter release also implicates synapsins as reversible links between synaptic vesicles and actin filaments. The diversity of actin binding partners in nerve terminals suggests that actin might have presynaptic functions beyond synaptic vesicle tethering or movement. Because most of these actin-binding proteins are regulated by calcium, actin might be a pivotal participant in calcium signaling inside presynaptic nerve terminals. However, there is no evidence that actin participates in fusion of synaptic vesicles.     

Immunolabelling of the presynaptic membrane of Torpedo electric organ nerve terminals with an antiserum towards the acetylcholine releasing protein mediatophore

Mediatophore is a nerve terminal membrane protein purified from Torpedo electric organ on its ability to translocate acetylcholine upon calcium action. An antiserum able to immunoprecipitate mediatophore activity was used to study the subcellular distribution of this protein. The presynaptic membrane exhibited a strong and discontinuous immunogold labelling, especially at the active zone where ACh is thought to be released. Two antigens were recognized on immunoblots of synaptosomal membranes: the 15-kDa subunit of mediatophore and a 14-kDa membrane protein that has a wide non-neuronal distribution. Antibodies purified from the serum on native mediatophore and monospecific towards the 15-kDa antigen still gave a high presynaptic membrane localized labelling. In addition, a few 14-kDa protein sites were present at the active zone. The Schwann cell finger interposed between the presynaptic membrane and the postsynaptic arch also exhibited the 14-kDa antigen raising the question of a possible interaction of mediatophore with the 14-kDa protein originating from the Schwann cell.     

Unwebbing the presynaptic web

The release of neurotransmitter from nerve terminals occurs at a specialized region of the presynaptic plasma membrane called the active zone. A dense matrix of proteins associated with the active zone, called the presynaptic web, is thought to play a fundamental role in defining these neurotransmitter release sites. In this issue of Neuron, Phillips et al. have identified conditions for the biochemical purification of the presynaptic web and show that the web is comprised of proteins involved in the docking, fusion, and recycling of synaptic vesicles.     

An ultrastructural comparison of neuromuscular junctions in normal and developmentally arrested Rana pipiens larvae: limited maturation in the absence of metamorphosis

Neuromuscular junctions in the rectus abdominis muscles of normal and developmentally arrested Rana pipiens larvae were studied with freeze fracture and conventional electron microscopy to determine whether structural aspects of junctional maturation depend on metamorphosis. Comparison was made between junctions in premetamorphic larvae 1-3 months old and junctions in larvae that had remained in premetamorphosis for more than a year (more than four times as long as normal). In most respects, junctions from the two groups of larvae were similar. Unlike adult junctions, nerve-muscle contacts in both larval groups were pleomorphic and often involved more than one neuronal process; Schwann cell processes very rarely extended between nerve and muscle. Active zone structure ranged from total disorganization to an adult pattern of highly ordered double rows of particles aligned over junctional folds. Only quantitative analysis revealed differences between junctions in old and young larvae. The older larvae had fewer nerve-muscle contact sites involving multiple neuronal elements and a higher ratio of active zone length to presynaptic membrane area, although the mean active zone length was the same in the two groups. The results indicate that the maturation of junctional shape, the branching pattern of the axons, and the relationship of presynaptic axons to Schwann cells must be directly or indirectly dependent on the hormonal or behavioral changes associated with metamorphosis.     

Synaptic vesicle recruitment for release explored by Monte Carlo stimulation at the crayfish neuromuscular junction

Neurotransmission at chemically transmitting synapses requires calcium-mediated fusion of synaptic vesicles with the presynaptic membrane. Utilizing ultrastructural information available for the crustacean excitatory neuromuscular junction, we developed a model that employs the Monte Carlo simulation technique to follow the entry and movement of Ca2+ ions at a presynaptic active zone, where synaptic vesicles are preferentially docked for release. The model includes interaction of Ca2+ with an intracellular buffer, and variable separation between calcium channels and vesicle-associated Ca(2+)-binding targets that react with Ca2+ to trigger vesicle fusion. The end point for vesicle recruitment for release was binding of four Ca2+ ions to the target controlling release. The results of the modeling experiments showed that intracellular structures that interfere with Ca2+ diffusion (in particular synaptic vesicles) influence recruitment or priming of vesicles for release. Vesicular recruitment is strongly influenced by the separation distance between an opened calcium channel and the target controlling release, and by the concentration and binding properties of the intracellular buffers, as in previous models. When a single opened calcium channel is very close to the target, a single synaptic vesicle can be recruited. However, many of the single-channel openings actuated by a nerve impulse are likely to be ineffective for release, although they contribute to the buildup of total intracellular Ca2+. Thus, the overall effectiveness of single calcium channels in causing vesicles to undergo exocytosis is likely quite low.     

Probabilistic secretion of quanta and the synaptosecretosome hypothesis: evoked release at active zones of varicosities, boutons, and endplates.

A quantum of transmitter may be released upon the arrival of a nerve impulse if the influx of calcium ions through a nearby voltage-dependent calcium channel is sufficient to activate the vesicle-associated calcium sensor protein that triggers exocytosis. A synaptic vesicle, together with its calcium sensor protein, is often found complexed with the calcium channel in active zones to form what will be called a "synaptosecretosome." In the present work, a stochastic analysis is given of the conditions under which a quantum is released from the synaptosecretosome by a nerve impulse. The theoretical treatment considers the rise of calcium at the synaptosecretosome after the stochastic opening of a calcium channel at some time during the impulse, followed by the stochastic binding of calcium to the vesicle-associated protein and the probability of this leading to exocytosis. This allows determination of the probabilities that an impulse will release 0, 1, 2,... quanta from an active zone, whether this is in a varicosity, a bouton, or a motor endplate. A number of experimental observations of the release of transmitter at the active zones of sympathetic varicosities and boutons as well as somatic motor endplates are described by this analysis. These include the likelihood of the secretion of only one quantum at an active zone of endplates and of more than one quantum at an active zone of a sympathetic varicosity. The fourth-power relationship between the probability of transmitter release at the active zones of sympathetic varicosities and motor endplates and the external calcium concentration is also explained by this approach. So, too, is the fact that the time course of the increased rate of quantal secretion from a somatic active zone after an impulse is invariant with changes in the amount of calcium that enters through its calcium channel, whether due to changes consequent on the actions of autoreceptor agents such as adenosine or to facilitation. The increased probability of quantal release that occurs during F1 facilitation at the active zones of motor endplates and sympathetic boutons is predicted by the residual binding of calcium to a high-affinity site on the vesicle-associated protein. The concept of the stochastic operation of a synaptosecretosome can accommodate most phenomena involving the release of transmitter quanta at these synapses.     

Osmotically-induced trapping of terbium ions in axonal membrane vesicles

Using terbium ions as fluorescence probes of calcium-binding sites and osmotic shock to induce trapping of Tb3+ in the vesicle interior, direct binding assays have been developed to study the competition between calcium and local anesthetics for binding sites at the cytoplasmic surface of axonal membrane vesicles. Pharmacologically active concentrations of the membrane-permeable local anesthetic, lidocaine, competitively displace bound Tb3+ in the vesicles, while QX-314, a quaternary ammonium analog of lidocaine that has poor access to the vesicle interior, exhibits no significant displacement of osmotically-loaded, internally-bound Tb3+. These experiments support the hypothesis that local anesthetics may function by displacing Ca2+ from a functionally specific binding site in nerve membranes.     

Innervation and membrane specialization at neuromuscular junctions in submaxillaris muscle of the frog

The submaxillaris muscle of the frog after zinc iodide-osmium staining reveals the presence of polyneural innervation. Cholinesterase staining shows that the longer terminals have postsynaptic folds whereas the smaller terminals (up to 5 micron) lack them. Thin-section electron microscopy shows that muscle fibers with or without an M line have terminals with and without postsynaptic folds. The terminals with postsynaptic folds have presynaptic membrane outpocketings above folds. These outpocketings are rudimentary or absent in the terminals without postsynaptic folds. In longer junctions, the P face of the presynaptic membrane has double rows of paired particles on active zone ridges perpendicular to the axis of the muscle. In smaller junctions active zone ridges are rudimentary or absent and double rows of particles form various patterns. Postsynaptic active zones in longer junctions consist of clusters of particles leaving gaps in between, whereas in the smaller junctions they lack gaps. The polyneural innervation and different deployment of membrane particles at neuromuscluar junctions could be a factor responsible for different physiological properties of this muscle.     

The binding of calcium to fibrinogen: some structural features.

Experiments are described which suggest that structural features are related to the existence of three high affinity calcium-binding sites in the fibrinogen molecule. The circular dichroism spectra analysis shows that the binding of calcium to this protein does not entail an overall conformational change. However several calcium-induced protective effects may be observed: 1. At pH 5.0 calcium-free fibrinogen is slightly acid-denatured. This denaturation is counteracted by the presence of calcium, whereas magnesium ions have no effect. 2. A temperature transition shift of 3 degrees C is measured in the presence of bound calcium during thermal denaturation, whereas magnesium ions have no effect. 3. Resistance to proteolysis by plasmin is observed when calcium is bound to fibrinogen. The velocity of the splitting of the earliest plasmin-succeptible bonds is reduced in the presence of calcium, whereas magnesium ions have no effect. It can be concluded from these results that the calcium binding centers are located in a more or less flexible zone of the molecule probably involving the C-terminal part of the Aalpha chain. And that the calcium divalent cation stabilizes a more compact structure of the fibrinogen molecule.     

René Couteaux (1909-1999) and the morphological identification of synapses

During a historical research, we realized that René Couteaux (1909-1999) was the first histologist who stained the postsynaptic structure of the neuromuscular junction. By means of Janus Green B dye, he revealed the membranous 'subneural apparatus' related to the 'synaptic gutter'. Hence, he justified the use of the physiological term 'synapse' in histology. A few years later, Couteaux localized acetylcholinesterase activity on this subneural apparatus with a histo-enzymatic method. The histochemical staining confirmed his histological observations that the subneural apparatus of the muscle cells was distinct from the nerve terminal. Thus Couteaux not only supported Cajal's neuron theory, but also gave decisive morphological basis to the concept of the synapse, thought to be up to that time a physiological entity. These observations were performed several years before the first electron microscopic data obtained in the U.S.A. When the electron microscopy became available in Europe, Couteaux studied the ultrastructural details of the myoneural synapse. He found evidence for the presynaptic 'active zone' as a restricted zone of the thickened presynaptic membrane flanked on both sides with synaptic vesicles, some of which were observed to be opening into the synaptic cleft by exocytosis. Furthermore, the synaptic vesicles were shown to be displayed in two rows along the linearly prolonged active zone which is situated just above the 'trench-like' junctional folds of the muscle membrane. The discontinuity of the synaptic structure was confirmed by the three-dimensional observation of the intraneuronal network of neurofibrils with the first prototype of high-voltage electron microscope.     

α-Latrotoxin: preparation and effects on calcium fluxes

Abstract A toxin that causes a massive presynaptic activation of transmitter release from nerve terminals is α-latrotoxin, isolated from Latrodectus tredecimguttatus spider venom. This toxin has been highly purified, utilizing as a biological assay a toxin-dependent increase in ⁴⁵Ca²⁺-accumulation by PC12 cells. The purification protocol includes an ion-exchange step and a gel-filtration column, by fast-flow liquid chromatography. The resulting toxin is a polypeptide of about 125 kDa in molecular mass. At nmol concentrations it specifically activates calcium influx and transmitter secretion after interacting with neuronal acceptors of the presynaptic membrane. The inhibitory effect of trivalent ions (which may develop as degradation product of ⁴⁵Ca²⁺) on toxin-dependent calcium accumulation by PC12 cells is described. The results obtained suggest that calcium fluxes directly involved in the neurosecretory event, may occur through newly formed toxin-dependent channels.     

Interactions between Presynaptic Calcium Channels and Proteins Implicated in Synaptic Vesicle Trafficking and Exocytosis

Monoclonal antibodies were generated by immunizing mice with chick brain synaptic membranes and screening for immunoprecipitation of solubilized conotoxin GVIA receptors (N-type calcium channels). Antibodies against two synaptic proteins (p35--syntaxin 1 and p58--synaptotagmin) were produced and used to purify and characterize a ternary complex containing N-type channels associated with these two proteins. These results provided the first evidence for a specific interaction between presynaptic calcium channels and SNARE proteins involved in synaptic vesicle docking and calcium-dependent exocytosis. Immunoprecipitation experiments supported the conclusion that syntaxin 1/SNAP-25/VAMP/synaptotagmin I or II complexes associate with N-type, P/Q-type, but not L-type calcium channels from rat brain nerve terminals. Immunofluorescent confocal microscopy at the frog neuromuscular junction was consistent with the co-localization of syntaxin 1, SNAP-25, and calcium channels, all of which are predominantly expressed at active zones of the presynaptic plasma membrane facing post-synaptic folds rich in acetylcholine receptors. The interaction of proteins implicated in calcium-dependent exocytosis with presynaptic calcium channels may locate the sensor(s) that trigger vesicle fusion within a microdomain of calcium entry.     

Polyhedral Protein Cages Encase Synaptic Vesicles and Participate in Their Attachment to the Active Zone

In an effort to elucidate the interactions between synaptic vesicles and the membrane of the active zone, we have investigated the structure of interneuronal asymmetric synapses in the neocortex of adult rats using thin-sectioning, freeze-fracture, and negative staining electron microscopy. We identified three subtypes of spherical synaptic vesicles. Type I were agranular vesicles of 47.5 ± 3.8 nm (mean SD,n = 24) in diameter usually seen aggregated in clusters in the presynaptic bouton. Type II synaptic vesicles were composed of a ∼45-nm-diameter lipid bilayer sphere encased in a cage 77 ± 4.6 nm (mean SD,n = 42) in diameter. The cage was composed of open-faced pentamers 20–22 nm/side arranged as a regular polyhedron. Type II caged vesicles were found in clusters at the boutons, adhered to the active zone, and were also present in axons. Type III synaptic vesicles appeared as electron-dense spheres 60–75 nm in diameter abutted to the membrane of the active zone. Clathrin-coated vesicles and pits of 116.6 ± 9 nm (mean SD,n = 14) in diameter were also present in both the pre- and postsynaptic sides. Freeze-fracture showed that some intrinsic membrane proteins in the active zone were arranged as pentamers exhibiting the same dimension of those forming cages (∼22 nm/side). From these data, we concluded that: (a) the presynaptic bouton contains a heterogeneous population of “caged” and “plain” synaptic vesicles and (b) type II synaptic vesicles bind to receptors in the active zone. Therefore, current models of transmitter release should take into account the substantial heterogeneity of the vesicle population and the binding of vesicular cages to the membrane of the active zone.     

Presynaptic calcium concentration microdomains and transmitter release.

n-Aequorin J, a luminescent protein which responds to calcium concentration changes in the order of several hundred micromoles, was injected into the preterminal fiber in the squid giant synapse. The activation of the presynaptic terminal leading to release of transmitter was accompanied by light emission at well-defined sites at the active zone in the presynaptic terminal. Location of these light emission sites was very much the same from one stimulus to the next, indicating that light emission was triggered by the inward calcium current occurring at specific and invariant locations. The distribution, size and number of these QEDs (quantum emission domains) coincides well with the location and number of active zones in the presynaptic terminal. The results imply that transmitter release is triggered by very well-localized calcium concentration changes that may be as high as several hundred micromoles.     

Synaptic vesicles and microtubules in frog motor endplates.

Motor endplates of the cutaneous pectoris skeletal muscle of the frog have been examined by electron microscopy using a new technique. This involves pretreatment with an albumin solution, followed by fixation with 4% unbuffered tetroxide. A small proportion of the endplate axonal ramifications show microtubules clothed in synaptic vesicles and focused on the presynaptic membrane, in particular on the active zones. The microtubules run in the presynaptic cytoplasm either parallel to or across the active zones. These two sets of microtubules cross each other at the active zones, which lie opposite the dips in the post-junctional folds. The possibility that the microtubules are involved in the translocation of synaptic vesicles to the active zone is discussed.     

Presynaptic calcium diffusion from various arrays of single channels. Implications for transmitter release and synaptic facilitation.

A one-dimensional model of presynaptic calcium diffusion away from the membrane, with cytoplasmic binding, extrusion by a surface pump, and influx during action potentials, can account for the rapid decay of phasic transmitter release and the slower decay of synaptic facilitation following one spike, as well as the very slow decline in total free calcium observed experimentally. However, simulations using this model, and alternative versions in which calcium uptake into organelles and saturable binding are included, fail to preserve phasic transmitter release to spikes in a long tetanus. A three-dimensional diffusion model was developed, in which calcium enters through discrete membrane channels and acts to release transmitter within 50 nm of entry points. Analytic solutions of the equations of this model, in which calcium channels were distributed in active zone patches based on ultrastructural observations, were successful in predicting synaptic facilitation, phasic release to tetanic spikes, and the accumulation of total free calcium. The effects of varying calcium buffering, pump rate, and channel number and distribution were explored. Versions appropriate to squid giant synapses and frog neuromuscular junctions were simulated. Limitations of key assumptions, particularly rapid nonsaturable binding, are discussed.     

Regeneration of the active zone at the frog neuromuscular junction

The active zone is a unique specialization of the presynaptic membrane and is believed to be the site of transmitter release. The formation of the active zone and the relationship of this process to transmitter release were studied at reinnervated neuromuscular junctions in the frog. At different times after a nerve crush, the cutaneous pectoris muscles were examined with intracellular recording recording and freeze-fracture electron microscopy. The P face of a normal active zone typically consists of two double rows of particles lined up in a continuous segment located opposite a junctional fold. In the initial stage of reinnervation, clusters of large intramembrane particles surrounding membrane elevations appeared on the P face of nerve terminals. Like normal active zones, these clusters were aligned with junctional folds. Vesicle openings, which indicate transmitter release, were seen at these primitive active zones, even though intramembrane particles were not yet organized into the normal pattern of two double rows. The length of active zones at this stage was only approximately 15% of normal. During the secondary stage, every junction was reinnervated and most active zones had begun to organize into the normal pattern with normal orientation. Unlike normal, there were often two or more discontinuous short segments of active zone aligned with the same junctional fold. The total length of active zone per junctional fold increased to one-third of normal, mainly because of the greater number of segments. In the third stage, the number of active zone segments per junctional fold showed almost no change when compared with the secondary stage. However, individual segments elongated and increased the total length of all active zone segments per junctional fold to about two-thirds of the normal length. The dynamic process culminated in the final stage, during which elongating active zones appeared to join together and the number of active zone segments per junctional fold decreased to normal. Thus, in most regions, regeneration of the active zones was complete. These results suggest that the normal organization of two double rows is not necessary for the active zone to be functional. Furthermore, localization of regenerating active zones is related to junctional folds and/or their associated structures.     

Molecular organization and plasticity of the cytomatrix at the active zone

Regulated neurotransmitter release from presynaptic boutons is crucial for the functioning of chemical synapses, what in turn governs the functional performance of the nervous system. Release occurs at the active zone (AZ), a specialized region of the presynaptic plasma membrane that is defined by a unique and complex meshwork of proteins--the cytomatrix at the AZ (CAZ). Important functions of CAZ proteins include recruitment, docking and priming of synaptic vesicles as well as appropriate localization of voltage-gated calcium channels near vesicle docking sites. We will discuss recent progress in the understanding of the topological localization and the molecular functions of characteristic CAZ proteins as well as emerging molecular mechanisms underlying presynaptic plasticity that involve significant structural CAZ remodeling.     

Molecular Mechanism of Active Zone Organization at Vertebrate Neuromuscular Junctions

Organization of presynaptic active zones is essential for development, plasticity, and pathology of the nervous system. Recent studies indicate a trans-synaptic molecular mechanism that organizes the active zones by connecting the pre- and the postsynaptic specialization. The presynaptic component of this trans-synaptic mechanism is comprised of cytosolic active zone proteins bound to the cytosolic domains of voltage-dependent calcium channels (P/Q-, N-, and L-type) on the presynaptic membrane. The postsynaptic component of this mechanism is the synapse organizer (laminin β2) that is expressed by the postsynaptic cell and accumulates specifically on top of the postsynaptic specialization. The pre- and the postsynaptic components interact directly between the extracellular domains of calcium channels and laminin β2 to anchor the presynaptic protein complex in front of the postsynaptic specialization. Hence, the presynaptic calcium channel functions as a scaffolding protein for active zone organization and as an ion-conducting channel for synaptic transmission. In contrast to the requirement of calcium influx for synaptic transmission, the formation of the active zone does not require the calcium influx through the calcium channels. Importantly, the active zones of adult synapses are not stable structures and require maintenance for their integrity. Furthermore, aging or diseases of the central and peripheral nervous system impair the active zones. This review will focus on the molecular mechanisms that organize the presynaptic active zones and summarize recent findings at the neuromuscular junctions and other synapses.