A metabotropic glutamate receptor regulates transmitter release from cone presynaptic terminals in carp retinal slices.

The role of group III metabotropic glutamate receptors (mGluRs) in photoreceptor-H1 horizontal cell (HC) synaptic transmission was investigated by analyzing the rate of occurrence and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in H1 HCs uncoupled by dopamine in carp retinal slices. Red light steps or the application of 100 microM cobalt reduced the sEPSC rate without affecting their peak amplitude, which is consistent with hyperpolarization or the suppression of Ca(2+) entry into cone synaptic terminals reducing vesicular transmitter release. Conversely, postsynaptic blockade of H1 HC AMPA receptors by 500 nM CNQX reduced the amplitude of sEPSCs without affecting their rate. This analysis of sEPSCs represents a novel methodology for distinguishing between presynaptic and postsynaptic sites of action. The selective agonist for group III mGluRs, l-2-amino-4-phosphonobutyrate (L-APB or L-AP4; 20 microM), reduced the sEPSC rate with a slight reduction in amplitude, which is consistent with a presynaptic action on cone synaptic terminals to reduce transmitter release. During L-APB application, recovery of sEPSC rate occurred with 500 microM (s)-2-methyl-2-amino-4-phosphonobutyrate (MAP4), a selective antagonist of group III mGluR, and with 200 microM 4-aminopyridine (4-AP), a blocker of voltage-dependent potassium channels. Whole-cell recordings from cones in the retinal slice showed no effect of L-APB on voltage-activated Ca(2+) conductance. These results suggest that the activation of group III mGluRs suppresses transmitter release from cone presynaptic terminals via a 4-AP-sensitive pathway. Negative feedback, operating via mGluR autoreceptors, may limit excessive glutamate release from cone synaptic terminals.     

Presynaptic calcium stores and synaptic transmission

Following the gradual recognition of the importance of intracellular calcium stores for somatodendritic signaling in the mammalian brain, recent reports have also indicated a significant role of presynaptic calcium stores. Ryanodine-sensitive stores generate local, random calcium signals that shape spontaneous transmitter release. They amplify spike-driven calcium signals in presynaptic terminals, and consequently enhance the efficacy of transmitter release. They appear to be recruited by an association with certain types of calcium-permeant ion channels, and they induce specific forms of synaptic plasticity. Recent research also indicates a role of inositoltrisphosphate-sensitive presynaptic calcium stores in synaptic plasticity.     

Interactions between proteins implicated in exocytosis and voltage-gated calcium channels

Neurotransmitter release from synaptic vesicles is triggered by voltage-gated calcium influx through P/Q-type or N-type calcium channels. Purification of N-type channels from rat brain synaptosomes initially suggested molecular interactions between calcium channels and two key proteins implicated in exocytosis: synaptotagmin I and syntaxin 1. Co-immunoprecipitation experiments were consistent with the hypothesis that both N- and P/Q-type calcium channels, but not L-type channels, are associated with the 7S complex containing syntaxin 1, SNAP-25, VAMP and synaptotagmin I or II. Immunofluorescence confocal microscopy at the frog neuromuscular junction confirmed that calcium channels, syntaxin 1 and SNAP-25 are co-localized at active zones of the presynaptic plasma membrane where transmitter release occurs. Experiments with recombinant proteins were performed to map synaptic protein interaction sites on the alpha 1A subunit, which forms the pore of the P/Q-type calcium channel. In vitro-translated 35S-synaptotagmin I bound to a site located on the cytoplasmic loop linking homologous domains II and III of the alpha 1A subunit. This direct link would target synaptotagmin, a putative calcium sensor for exocytosis, to a microdomain of calcium influx close to the channel mouth. Cysteine string proteins (CSPs) contain a J-domain characteristic of molecular chaperones that cooperate with Hsp70. They are located on synaptic vesicles and thought to be involved in modulating the activity of presynaptic calcium channels. CSPs were found to bind to the same domain of the calcium channel as synaptotagmin, and also to associate with VAMP. CSPs may act as molecular chaperones in association with Hsp70 to direct assembly or dissociation of multiprotein complexes at the calcium channel.     

Can presynaptic depolarization release transmitter without calcium influx?

Recent experimental evidence suggesting that presynaptic depolarization can evoke transmitter release without calcium influx has been re-examined. The presynaptic terminal of the squid giant synapse can be depolarized by variable amounts while recording presynaptic calcium current under voltage clamp and postsynaptic responses. Small depolarizations open few calcium channels with large single channel currents. Large depolarizations approaching the calcium equilibrium potential open many channels with small single channel currents. When responses to small and large depolarizations eliciting similar total macroscopic calcium currents are compared, the large pulses evoke more transmitter release. This apparent voltage-dependence of transmitter release may be explained by the greater overlap of calcium concentration domains surrounding single open calcium channels when many closely apposed channels open at large depolarizations. This channel domain overlap leads to higher calcium concentrations at transmitter release sites and more release for large depolarizations than for small depolarizations which open few widely dispersed channels. At neuromuscular junctions, a subthreshold depolarizing pulse to motor nerve terminals may release over a thousand times as much transmitter if it follows a brief train of presynaptic action potentials than if it occurs in isolation. This huge synaptic facilitation has been taken as indicative of a direct effect of voltage which is manifest only when prior activity raises presynaptic resting calcium levels. This large facilitation is actually due to a post-tetanic supernormal excitability in motor nerve terminals, causing the previously subthreshold test pulse to become suprathreshold and elicit a presynaptic action potential. When motor nerve terminals are depolarized by two pulses, as the first pulse increases above a certain level it evokes more transmitter release but less facilitation of the response to the second pulse.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bilateral processing in chemical synapses with electrical 'ephaptic' feedback: a theoretical model

I have developed a detailed biophysical model of the chemical synapse which hosts voltage-dependent presynaptic ion channels and takes into account the capacitance of synaptic membranes. I find that at synapses with a relatively large cleft resistance (e.g., mossy fiber or giant calyx synapse) the rising postsynaptic current could activate, within the synaptic cleft, electrochemical phenomena that induce rapid widening of the presynaptic action potential (AP). This mechanism could boost fast Ca(2+) entry into the terminal thus increasing the probability of subsequent synaptic releases. The predicted difference in the AP waveforms generated inside and outside the synapse can explain the previously unexplained fast capacitance transient recorded in the postsynaptic cell at the giant calyx synapse. I propose therefore the mechanism of positive ephaptic feedback that acts between the postsynaptic and presynaptic cell contributing to the basal synaptic transmission at large central synapses. This mechanism could also explain the supralinear voltage dependence of EPSCs recorded at hyperpolarizing membrane potentials in low extracellular calcium concentration.     

Presynaptic N-type calcium channels regulate synaptic growth

Voltage-gated calcium channels couple changes in membrane potential to neuronal functions regulated by calcium, including neurotransmitter release. Here we report that presynaptic N-type calcium channels not only control neurotransmitter release but also regulate synaptic growth at Drosophila neuromuscular junctions. In a screen for behavioral mutants that disrupt synaptic transmission, an allele of the N-type calcium channel locus (Dmca1A) was identified that caused synaptic undergrowth. The underlying molecular defect was identified as a neutralization of a charged residue in the third S4 voltage sensor. RNA interference reduction of N-type calcium channel expression also reduced synaptic growth. Hypomorphic mutations in syntaxin-1A or n-synaptobrevin, which also disrupt neurotransmitter release, did not affect synapse proliferation at the neuromuscular junction, suggesting calcium entry through presynaptic N-type calcium channels, not neurotransmitter release per se, is important for synaptic growth. The reduced synapse proliferation in Dmca1A mutants is not due to increased synapse retraction but instead reflects a role for calcium influx in synaptic growth mechanisms. These results suggest N-type channels participate in synaptic growth through signaling pathways that are distinct from those that mediate neurotransmitter release. Linking presynaptic voltage-gated calcium entry to downstream calcium-sensitive synaptic growth regulators provides an efficient activity-dependent mechanism for modifying synaptic strength.     

How does calcium trigger neurotransmitter release?

Recent work has established that different geometric arrangements of calcium channels are found at different presynaptic terminals, leading to a wide spectrum of calcium signals for triggering neurotransmitter release. These calcium signals are apparently transduced by synaptotagmins - calcium-binding proteins found in synaptic vesicles. New biochemical results indicate that all synaptotagmins undergo calcium-dependent interactions with membrane lipids and a number of other presynaptic proteins, but which of these interactions is responsible for calcium-triggered transmitter release remains unclear.     

The maintenance of LTP at hippocampal mossy fiber synapses is independent of sustained presynaptic calcium.

We have examined the role of presynaptic residual calcium in maintaining long-term changes in synaptic efficacy observed at mossy fiber synapses between hippocampal dentate granule cells and CA3 pyramidal cells. Calcium concentrations in individual mossy fiber terminals in hippocampal slice were optically measured with the calcium indicator fura-2 while stimulating the mossy fiber pathway and recording excitatory postsynaptic potentials extracellularly. Short-term synaptic enhancement was accompanied by increased presynaptic residual calcium concentration. A 2-fold enhancement of transmitter release was accompanied by a 10-30 nM increase in residual calcium. Following induction of mossy fiber LTP, transiently elevated presynaptic calcium decayed to prestimulus levels, whereas enhancement of synaptic transmission persisted. Our results demonstrate that, despite an apparent strong sensitivity of synaptic enhancement to presynaptic residual calcium levels, sustained increases in presynaptic residual calcium levels are not responsible for the maintained synaptic enhancement observed during mossy fiber LTP.     

Ion channels in nerve ending

The modern data about the structure and function of the nerve ending ion channels are generalized and systematized. Ion channels of nerve endings provide the forming of the rest membrane potential, excitability, generation of action potential, regulate the intracellular concentration of calcium ions, take part in exocytosis of synaptic vesicules, participate in short-term and long-term synaptic plasticity, ensure the modulation of presynaptic functions. Methods of investigation of ion channels and data about their localization in central and peripheral nerve systems are represented. The review gives the functional characteristics, molecular structure and mechanisms of regulation of the known voltage- and ligand-dependent ion channels, the role of the certain types of ion channels in the machinery of transmitter release.     

Calcium channel structural determinants of synaptic transmission between identified invertebrate neurons

We report here that unlike what was suggested for many vertebrate neurons, synaptic transmission in Lymnaea stagnalis occurs independent of a physical interaction between presynaptic calcium channels and a functional complement of SNARE proteins. Instead, synaptic transmission in Lymnaea requires the expression of a C-terminal splice variant of the Lymnaea homolog to mammalian N- and P/Q-type calcium channels. We show that the alternately spliced region physically interacts with the scaffolding proteins Mint1 and CASK, and that synaptic transmission is abolished following RNA interference knockdown of CASK or after the injection of peptide sequences designed to disrupt the calcium channel-Mint1 interactions. Our data suggest that Mint1 and CASK may serve to localize the non-L-type channels at the active zone and that synaptic transmission in invertebrate neurons utilizes a mechanism for optimizing calcium entry, which occurs independently of a physical association between calcium channels and SNARE proteins.     

Multitude of ion channels in the regulation of transmitter release

The presynaptic nerve terminal is of key importance in communication in the nervous system. Its primary role is to release transmitter quanta on the arrival of an appropriate stimulus. The structural basis of these transmitter quanta are the synaptic vesicles that fuse with the surface membrane of the nerve terminal, to release their content of neurotransmitter molecules and other vesicular components. We subdivide the control of quantal release into two major classes: the processes that take place before the fusion of the synaptic vesicle with the surface membrane (the pre-fusion control) and the processes that occur after the fusion of the vesicle (the post-fusion control). The pre-fusion control is the main determinant of transmitter release. It is achieved by a wide variety of cellular components, among them the ion channels. There are reports of several hundred different ion channel molecules at the surface membrane of the nerve terminal, that for convenience can be grouped into eight major categories. They are the voltage-dependent calcium channels, the potassium channels, the calcium-gated potassium channels, the sodium channels, the chloride channels, the non-selective channels, the ligand gated channels and the stretch-activated channels. There are several categories of intracellular channels in the mitochondria, endoplasmic reticulum and the synaptic vesicles. We speculate that the vesicle channels may be of an importance in the post-fusion control of transmitter release.     

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.     

An emerging view of presynaptic structure from electron microscopic studies

In response to calcium influx, some of the synaptic vesicles in presynaptic terminals fuse rapidly with the presynaptic membrane, allowing fast synaptic transmission. The regulated recycling of synaptic vesicles at the terminals is required for a sustained release of neurotransmitters. Localization of 'ready to be released' vesicles in close vicinities to voltage-gated calcium channels enables the rapid release of neurotransmitters. Thus, recycling vesicles must translocate from the sites of endocytosis to these release sites. However, the sub-cellular organization that supports this local vesicular traffic remains poorly understood. We will review the results of various electron microscopy studies, which have begun to unveil the structure of presynaptic terminals.     

Mitochondrial regulation of synaptic plasticity in the hippocampus

Synaptic mechanisms of plasticity are calcium-dependent processes that are affected by dysfunction of mitochondrial calcium buffering. Recently, we observed that mice deficient in mitochondrial voltage-dependent anion channels, the outer component of the mitochondrial permeability transition pore, have impairments in learning and hippocampal synaptic plasticity, suggesting that the mitochondrial permeability transition pore is involved in hippocampal synaptic plasticity. In this study, we examined the effect on synaptic transmission and plasticity of blocking the permeability transition pore with low doses of cyclosporin A and found a deficit in synaptic plasticity and an increase in base-line synaptic transmission. Calcium imaging of presynaptic terminals revealed a transient increase in the resting calcium concentration immediately upon incubation with cyclosporin A that correlated with the changes in synaptic transmission and plasticity. The effect of cyclosporin A on presynaptic calcium was abolished when mitochondria were depolarized prior to cyclosporin A exposure, and the effects of cyclosporin A and mitochondrial depolarization on presynaptic resting calcium were similar, suggesting a mitochondrial locus of action of cyclosporin A. To further characterize the calcium dynamics of the mitochondrial permeability transition pore, we used an in vitro assay of calcium handling by isolated brain mitochondria. Cyclosporin A-exposed mitochondria buffered calcium more rapidly and subsequently triggered a more rapid mitochondrial depolarization. Similarly, mitochondria lacking the voltage-dependent anion channel 1 isoform depolarized more readily than littermate controls. The data suggest a role for the mitochondrial permeability transition pore and voltage-dependent anion channels in mitochondrial synaptic calcium buffering and in hippocampal synaptic plasticity.     

Association of neuronal calcium channels with modular adaptor proteins.

Presynaptic voltage-gated calcium (Ca(2+)) channels mediate Ca(2+) influx into the presynaptic terminal that triggers synaptic vesicle fusion and neurotransmitter release. The immediate proximity of Ca(2+) channels to the synaptic vesicle release apparatus is critical for rapid and efficient synaptic transmission. In a series of biochemical experiments, we demonstrate a specific association of the cytosolic carboxyl terminus of the N-type Ca(2+) channel pore-forming alpha(1B) subunit with the modular adaptor proteins Mint1 and CASK. The carboxyl termini of alpha(1B) bind to the first PDZ domain of Mint1 (Mint1-1). The proline-rich region present in the carboxyl termini of alpha(1B) binds to the SH3 domain of CASK. Mint1-1 is specific for the E/D-X-W-C/S-COOH consensus, which defines a novel class of PDZ domains (class III). The Mint1-1 PDZ domain-binding motif is present only in the "long" carboxyl-terminal splice variants of N-type (alpha(1B)) and P/Q-type (alpha(1A)) Ca(2+) channels, but not in R-type (alpha(1E)) or L-type (alpha(1C)) Ca(2+) channels. Our results directly link presynaptic Ca(2+) channels to a macromolecular complex formed by modular adaptor proteins at synaptic junction and advance our understanding of coupling between cell adhesion and synaptic vesicle exocytosis.     

Effects of 4-aminopyridine on the action potential and the after-hyperpolarization of cat spinal motoneurons

In cats under pentobarbital anaesthesia, intramotoneuronal administrations of 4-aminopyridine significantly prolong the falling phase of the antidromic action potential but have much less effect on the orthodromic action potential. 4-aminopyridine probably blocks the fast K channels involved in the repolarization of the membrane and indirectly activates ionic channels through enhancement of synaptic transmission, also suggested by the potentiation of excitatory postsynaptic potentials. In many cells, 4-aminopyridine depresses the amplitude and prolongs the time course of the after-hyperpolarization; therefore 4-aminopyridine may also partly block Ca2+-activated K+ channels.     

Regulation of transmitter release from retinal bipolar cells.

Mb1 bipolar cells (ON-type cells) of the goldfish retina have exceptionally large (approximately 10 microns in diameter) presynaptic terminals, and thus, are suitable for investigating presynaptic mechanisms for transmitter release. Using enzymatically dissociated Mb1 bipolar cells under whole-cell voltage clamp, we measured the Ca2+ current (ICa), the intracellular free Ca2+ concentration ([Ca2+]i), and membrane capacitance changes associated with exocytosis and endocytosis. Release of transmitter (glutamate) was monitored electrophysiologically by a glutamate receptor-rich neuron as a probe. L-type Ca2+ channels were localized at the presynaptic terminals. The presynaptic [Ca2+]i was strongly regulated by cytoplasmic Ca2+ buffers, the Na(+)-Ca2+ exchanger and the Ca2+ pump in the plasma membrane. Once ICa was activated, a steep Ca2+ gradient was created around Ca2+ channels; [Ca2+]i increased to approximately 100 microM at the fusion sites of synaptic vesicles whereas up to approximately 1 microM at the cytoplasm. The short delay (approximately 1 ms) of exocytosis and the lack of prominent asynchronous release after the termination of ICa suggested a low-affinity Ca2+ fusion sensor for exocytosis. Depending on the rate of Ca2+ influx, glutamate was released in a rapid phasic mode as well as a tonic mode. Multiple pools of synaptic vesicles as well as vesicle cycling seemed to support continuous glutamate release. Activation of protein kinase C increased the size of synaptic vesicle pool, resulting in the potentiation of glutamate release. Goldfish Mb1 bipolar cells may still be an important model system for understanding the molecular mechanisms of transmitter release.     

Calcium stores in hippocampal synaptic boutons mediate short-term plasticity, store-operated Ca2+ entry, and spontaneous transmitter release

Evoked transmitter release depends upon calcium influx into synaptic boutons, but mechanisms regulating bouton calcium levels and spontaneous transmitter release are obscure. To understand these processes better, we monitored calcium transients in axons and presynaptic terminals of pyramidal neurons in hippocampal slice cultures. Action potentials reliably evoke calcium transients in axons and boutons. Calcium-induced calcium release (CICR) from internal stores contributes to the transients in boutons and to paired-pulse facilitation of EPSPs. Store depletion activates store-operated calcium channels, influencing the frequency of spontaneous transmitter release. Boutons display spontaneous Ca2+ transients; blocking CICR reduces the frequency of these transients and of spontaneous miniature synaptic events. Thus, spontaneous transmitter release is largely calcium mediated, driven by Ca2+ release from internal stores. Bouton store release is important for short-term synaptic plasticity and may also contribute to long-term plasticity.     

Presynaptic facilitation at the crayfish neuromuscular junction. Role of calcium-activated potassium conductance

Membrane potential was recorded intracellularly near presynaptic terminals of the excitor axon of the crayfish opener neuromuscular junction (NMJ), while transmitter release was recorded postsynaptically. This study focused on the effects of a presynaptic calcium-activated potassium conductance, gK(Ca), on the transmitter release evoked by single and paired depolarizing current pulses. Blocking gK(Ca) by adding tetraethylammonium ion (TEA; 5-20 mM) to a solution containing tetrodotoxin and aminopyridines caused the relation between presynaptic potential and transmitter release to steepen and shift to less depolarized potentials. When two depolarizing current pulses were applied at 20-ms intervals with gK(Ca) not blocked, the presynaptic voltage change to the second (test) pulse was inversely related to the amplitude of the first (conditioning) pulse. This effect of the conditioning prepulse on the response to the test pulse was eliminated by 20 mM TEA and by solutions containing 0 mM Ca2+/1 mM EGTA, suggesting that the reduction in the amplitude of the test pulse was due to activation of gK(Ca) by calcium remaining from the conditioning pulse. In the absence of TEA, facilitation of transmitter release evoked by a test pulse increased as the conditioning pulse grew from -40 to -20 mV, but then decreased with further increase in the conditioning depolarization. A similar nonmonotonic relationship between facilitation and the amplitude of the conditioning depolarization was reported in previous studies using extracellular recording, and interpreted as supporting an additional voltage-dependent step in the activation of transmitter release. We suggest that this result was due instead to activation of a gK(Ca) by the conditioning depolarization, since facilitation of transmitter release increased monotonically with the amplitude of the conditioning depolarization, and the early time course of the decay of facilitation was prolonged when gK(Ca) was blocked. The different time courses for decay of the presynaptic potential (20 ms) and facilitation (greater than 50 ms) suggest either that residual free calcium does not account for facilitation at the crayfish NMJ or that the transmitter release mechanism has a markedly higher affinity or stoichiometry for internal free calcium than does gK(Ca). Finally, our data suggest that the calcium channels responsible for transmitter release at the crayfish NMJ are not of the L, N, or T type.     

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.