Stochastic model of central synapses: slow diffusion of transmitter interacting with spatially distributed receptors and transporters.

A detailed mathematical analysis of the diffusion process of neurotransmitter inside the synaptic cleft is presented and the spatio-temporal concentration profile is calculated. Using information about the experimentally observed time course of glutamate in the cleft the effective diffusion coefficient Dnet is estimated as Dnet approximately 20-50 nm(2) microseconds(-1), implying a strong reduction compared with free diffusion in aqueous solution. The tortuosity of the cleft and interactions with transporter molecules are assumed to affect the transmitter motion. We estimate the transporter density to be 5170 to 8900 micrometer(-2) in the synaptic cleft and its vicinity, using the experimentally observed time constant of glutamate. Furthermore a theoretical model of synaptic transmission is presented, taking the spatial distribution of post-synaptic (AMPA-) receptors into account. The transmitter diffusion and receptor dynamics are modeled by Monte Carlo simulations preserving the typically observed noisy character of post-synaptic responses. Distributions of amplitudes, rise and decay times are calculated and shown to agree well with experiments. Average open probabilities are computed from a novel kinetic model and are shown to agree with averages over many Monte Carlo runs. Our results suggest that post-synaptic currents are only weakly potentiated by clustering of post-synaptic receptors, but increase linearly with the total number of receptors. Distributions of amplitudes and rise times are used to discriminate between different morphologies, e.g. simple and perforated synapses. A skew in the miniature amplitude distribution can be caused by multiple release of pre-synaptic vesicles at perforated synapses.     

A Brownian simulation model of glutamate synaptic diffusion in the femtosecond time scale

 To gain a better understanding of the elementary unit of synaptic communication between hippocampal neurons, we simulated the release of glutamate from a single pre-synaptic vesicle and its diffusion into the synaptic cleft. Diffusion of glutamate was simulated by a Brownian model based on Langevin equations. The model was implemented for parallel computer simulation and tested under different conditions of glutamate release and different geometrical and physical characteristics of the synaptic cleft. All the tested parameters have shown to be important for the synaptic responses. The results show that the synaptic transmission efficacy is influenced by many different geometrical parameters and, as a consequence, the quality of the excitatory post-synaptic response can be very different in the same synapse. The variability in the quantal response found by several authors can also be explained by physical parameters other than by variations in the quantal content of the synaptic vesicle as proposed by these authors. Received: 6 October 1999 / Accepted: 29 February 2000     

Control of neural information transmission by synaptic dynamics.

The computational processing of a neural system is strongly influenced by the dynamical characteristics of the information transmission between neurons. In this work, the control of neural information transmission by synaptic dynamics is investigated by means of a master-equation-based stochastic model of pre-synaptic release of neurotransmitter-containing vesicles. The model incorporates facilitation of vesicle fusion with the pre-synaptic membrane due to intracellular calcium ions and depletion of readily releasable vesicles. The message to be transmitted is coded by the pre-synaptic firing sequence, and the received signal corresponds to the post-synaptic membrane potential response. At the sending end, the stochastic character of the vesicle release contributes to the entropy of the probability distribution of the number of vesicles released and represents noise with respect to information transmission. At the receiving end, the generation of post-synaptic membrane potentials is influenced by the temporal behaviour of ionic currents and membrane charging and is determined by means of a low-dimensional model. The rate and temporal types of neural coding are compatible with limiting cases of the synaptic information transmission as a function of initial vesicle release probability and pre-synaptic firing rate. The effects of the nonlinear dependencies of the vesicle release probability on intracellular calcium concentration and number of available vesicles are analysed. The model is compared with phenomenological and reduced models, a principal advantage being the capability of also determining fluctuations of dynamic variables Copyright 2002 Academic Press.     

Modeling the effect of glutamate diffusion and uptake on NMDA and non-NMDA receptor saturation.

One- and two-dimensional models of glutamate diffusion, uptake, and binding in the synaptic cleft were developed to determine if the release of single vesicles of glutamate would saturate NMDA and non-NMDA receptors. Ranges of parameter values were used in the simulations to determine the conditions when saturation could occur. Single vesicles of glutamate did not saturate NMDA receptors unless diffusion was very slow and the number of glutamate molecules in a vesicle was large. However, the release of eight vesicles at 400 Hz caused NMDA receptor saturation for all parameter values tested. Glutamate uptake was found to reduce NMDA receptor saturation, but the effect was smaller than that of changes in the diffusion coefficient or in the number of glutamate molecules in a vesicle. Non-NMDA receptors were not saturated unless diffusion was very slow and the number of glutamate molecules in a vesicle was large. The release of eight vesicles at 400 Hz caused significant non-NMDA receptor desensitization. The results suggest that NMDA and non-NMDA receptors are not saturated by single vesicles of glutamate under usual conditions, and that tetanic input, of the type typically used to induce long-term potentiation, will increase calcium influx by increasing receptor binding as well as by reducing voltage-dependent block of NMDA receptors.     

Synaptic Efficacy as a Function of Ionotropic Receptor Distribution: A Computational Study

Glutamatergic synapses are the most prevalent functional elements of information processing in the brain. Changes in pre-synaptic activity and in the function of various post-synaptic elements contribute to generate a large variety of synaptic responses. Previous studies have explored postsynaptic factors responsible for regulating synaptic strength variations, but have given far less importance to synaptic geometry, and more specifically to the subcellular distribution of ionotropic receptors. We analyzed the functional effects resulting from changing the subsynaptic localization of ionotropic receptors by using a hippocampal synaptic computational framework. The present study was performed using the EONS (Elementary Objects of the Nervous System) synaptic modeling platform, which was specifically developed to explore the roles of subsynaptic elements as well as their interactions, and that of synaptic geometry. More specifically, we determined the effects of changing the localization of ionotropic receptors relative to the presynaptic glutamate release site, on synaptic efficacy and its variations following single pulse and paired-pulse stimulation protocols. The results indicate that changes in synaptic geometry do have consequences on synaptic efficacy and its dynamics.     

Pre-synaptic and post-synaptic localization of EphA4 and EphB2 in adult mouse forebrain

The ephrin receptors EphA4 and EphB2 have been implicated in synaptogenesis and long-term potentiation in the cerebral cortex and hippocampus, where they are generally viewed as post-synaptic receptors. To determine the precise distribution of EphA4 and EphB2 in mature brain synapses, we used subcellular fractionation and electron microscopy to examine the adult mouse forebrain/midbrain. EphA4 and EphB2 were both enriched in microsomes and synaptosomes. In synaptosomes, they were present in the membrane and the synaptic vesicle fractions. While EphA4 was tightly associated with PSD-95-enriched post-synaptic density fractions, EphB2 was easily extracted with detergents. In contrast, both receptors were found in the pre-synaptic active zone fraction. By electron microscopy, EphA4 was mainly detected in axon terminals, whereas EphB2 was more frequently detected in large dendritic shafts, in the hippocampus and cerebral cortex. However, in the ventrobasal thalamus, EphB2 was detected most frequently in axon terminals and thin dendritic shafts. The localization of EphA4 and EphB2 in multiple compartments of neurons and synaptic junctions suggests that they interact with several distinct scaffolding proteins and play diverse roles at synapses.     

Gephyrin expression and clustering affects the size of glutamatergic synaptic contacts

We have recently shown that disrupting the expression and post-synaptic clustering of gephyrin in cultured hippocampal pyramidal cells, by either gephyrin RNAi (RNA interference) or over-expression of a dominant negative gephyrin-enhanced green fluorescent protein (EGFP) fusion protein, leads to decreased number of post-synaptic gephyrin and GABA_A receptor clusters and to reduced GABAergic innervation of these cells. On the other hand, increasing gephyrin expression led to a small increase in the number of gephyrin and GABA_A receptor clusters and to little or no effect on GABAergic innervation. We are now reporting that altering gephyrin expression and clustering affects the size but not the density of glutamatergic synaptic contacts. Knocking down gephyrin with gephyrin RNAi, or preventing gephyrin clustering by over-expression of the dominant negative gephyrin-enhanced green fluorescent protein fusion protein, leads to larger post-synaptic PSD-95 clusters and larger pre-synaptic glutamatergic terminals. On the other hand, over-expression of gephyrin leads to slightly smaller PSD-95 clusters and pre-synaptic glutamatergic terminals. The change in size of PSD-95 clusters were accompanied by a parallel change in the size of NR2-NMDA receptor clusters. It is concluded that the levels of expression and clustering of gephyrin, a protein that concentrates at the post-synaptic complex of the inhibitory synapses, not only has homotypic effects on GABAergic synaptic contacts, but also has heterotypic effects on glutamatergic synaptic contacts. We are proposing that gephyrin is a counterpart of the post-synaptic glutamatergic scaffold protein PSD-95 in regulating the number and/or size of the excitatory and inhibitory synaptic contacts.     

Functionally opposite receptors on neurones.

According to the second general principle of chemical neurotransmission, formulated by Eccles, a neurotransmitter released from the axonal terminals of a neurone has only one and the same effect on all the follower cells: all the follower cells are either excited or inhibited by it. The evidence reviewed in this paper suggests that there may be some exceptions to this general pattern of synaptic organisation. The co-existence of both excitatory and inhibitory receptors for the same transmitter substance on the same neuronal membrane has been described in invertebrates: ‘opposite receptors’ can occur for acetylcholine, dopamine and glutamate. There is also evidence suggesting the existence of opposite receptors for acetylcholine and nonadrenaline on neurones in the mammalian brain. The activation of functionally opposite receptors results in ‘antagonistic agonism’: the net pharmacological effect is obtained by the algebraic summation of the two opposing component effects. Both the time-course and the polarity of neuronal responses to a given neurotransmitter is influenced by the relationship between the two kinds of receptor. It is suggested that both pre-synaptic and post-synaptic factors may modify this relationship.     

The effects of ethanol on pre-synaptic components of synaptic transmission in a model glutamatergic synapse: the crayfish neuromuscular junction

We have elucidated some of the mechanisms by which ethanol (EtOH) reduces synaptic efficacy at model glutamatergic synapses. The crayfish phasic and tonic neuromuscular junctions are superb models for directly assessing the effects of EtOH on pre-synaptic components of synaptic transmission. The ability to perform quantal analysis of synaptic transmission has allowed us to assess pre-synaptic alterations of release. Using this system, we report that the application of EtOH, within a range observed in intoxicated humans (44 and 88 mM), resulted in a diminution of excitatory post-synaptic potentials (EPSP) amplitudes. Additionally, using focal macro-patch recordings, quantal synaptic currents were recorded to assess the pre-synaptic component as potential target sites for EtOH's action. At the tonic neuromuscular junctions, EtOH (88 mM) reduced the probability of release (p), and in some cases, reduced the number of the release sites (n), but did not alter facilitation index nor did it affect the latency of vesicular release. At the phasic neuromuscular junction, a reduction in synaptic charge occurred during the presence of EtOH. Thus, the observed decrease in synaptic strength is at least partially attributable to a pre-synaptic alteration, specifically the release of fewer vesicles.     

Glutamine uptake by System A transporters maintains neurotransmitter GABA synthesis and inhibitory synaptic transmission

GABA synthesis is necessary to maintain synaptic vesicle filling, and key proteins in its biosynthetic pathways may play a role in regulating inhibitory synaptic stability and strength. GABAergic neurons require a source of precursor glutamate, possibly from glutamine, although it is controversial whether glutamine contributes to the synaptic pool of GABA. Here we report that inhibition of System A glutamine transporters with alpha-(methyl-amino) isobutyric acid rapidly reduced the amplitude of inhibitory post-synaptic currents and miniature inhibitory post-synaptic currents (mIPSCs) recorded in rat hippocampal area cornu ammonis 1 (CA1) pyramidal neurons, indicating that synaptic vesicle content of GABA was reduced. After inhibiting astrocytic glutamine synthesis by either blocking glutamate transporters or the glutamine synthetic enzyme, the effect of alpha-(methyl-amino) isobutyric acid on mIPSC amplitudes was abolished. Exogenous glutamine did not affect mIPSC amplitudes, suggesting that the neuronal transporters are normally saturated. Our findings demonstrate that a constitutive supply of glutamine is provided by astrocytes to inhibitory neurons to maintain vesicle filling. Therefore, glutamine transporters, like those for glutamate, are potential regulators of inhibitory synaptic strength. However, in contrast to glutamate, extracellular glutamine levels are normally high. Therefore, we propose a supportive role for glutamine, even under resting conditions, to maintain GABA vesicle filling.     

Kinetics of Synaptic Transmission at Ribbon Synapses of Rods and Cones

The ribbon synapse is a specialized structure that allows photoreceptors to sustain the continuous release of vesicles for hours upon hours and years upon years but also respond rapidly to momentary changes in illumination. Light responses of cones are faster than those of rods and, mirroring this difference, synaptic transmission from cones is also faster than transmission from rods. This review evaluates the various factors that regulate synaptic kinetics and contribute to kinetic differences between rod and cone synapses. Presynaptically, the release of glutamate-laden synaptic vesicles is regulated by properties of the synaptic proteins involved in exocytosis, influx of calcium through calcium channels, calcium release from intracellular stores, diffusion of calcium to the release site, calcium buffering, and extrusion of calcium from the cytoplasm. The rate of vesicle replenishment also limits the ability of the synapse to follow changes in release. Post-synaptic factors include properties of glutamate receptors, dynamics of glutamate diffusion through the cleft, and glutamate uptake by glutamate transporters. Thus, multiple synaptic mechanisms help to shape the responses of second-order horizontal and bipolar cells.     

Acute ethanol suppresses glutamatergic neurotransmission through endocannabinoids in hippocampal neurons

Ethanol exposure during fetal development is a leading cause of long-term cognitive impairments. Studies suggest that ethanol exposure have deleterious effects on the hippocampus, a brain region that is important for learning and memory. Ethanol exerts its effects, in part, via alterations in glutamatergic neurotransmission, which is critical for the maturation of neuronal circuits during development. The current literature strongly supports the growing evidence that ethanol inhibits glutamate release in the neonatal CA1 hippocampal region. However, the exact molecular mechanism responsible for this effect is not well understood. In this study, we show that ethanol enhances endocannabinoid (EC) levels in cultured hippocampal neurons, possibly through calcium pathways. Acute ethanol depresses miniature post-synaptic current (mEPSC) frequencies without affecting their amplitude. This suggests that ethanol inhibits glutamate release. The CB1 receptors (CB1Rs) present on pre-synaptic neurons are not altered by acute ethanol. The CB1R antagonist SR 141716A reverses ethanol-induced depression of mEPSC frequency. Drugs that are known to enhance the in vivo function of ECs occlude ethanol effects on mEPSC frequency. Chelation of post-synaptic calcium by EGTA antagonizes ethanol-induced depression of mEPSC frequency. The activation of CB1R with the selective agonist WIN55,212-2 also suppresses the mEPSC frequency. This WIN55,212-2 effect is similar to the ethanol effects and is reversed by SR141716A. In addition, tetani-induced excitatory post-synaptic currents (EPSCs) are depressed by acute ethanol. SR141716A significantly reverses ethanol effects on evoked EPSC amplitude in a dual recording preparation. These observations, taken together, suggest the participation of ECs as retrograde messengers in the ethanol-induced depression of synaptic activities.     

Moduler l’action du glutamate dans le cerveau: de nouvelles pistes ouvertes grâce aux récepteurs métabotropiques

The rapid transmission of information in the central nervous system is mostly mediated by glutamate synapses. The control of this system quickly appeared as a way to modulate, and perhaps normalize, a number of brain dysfunctions. However, the central role of glutamate receptors involved in this transmission, the ionotropic AMPA and NMDA receptors, appeared as an obstacle to the development of drugs devoid of side effects. The discovery of a second family of glutamate receptors, more than fifteen years ago, offered new possibilities to act on the glutamate system. These receptors, the metabotropic glutamate (mGlu) receptors which are coupled to G-proteins, modulate excitatory synaptic transmission. Eight mGlu receptors have been identified and localized either on the post-synaptic element, where they can regulate the AMPA and NMDA receptor activity, or on the presynaptic element, where they control the release of glutamate or other neurotransmitters. Recent data highlights the therapeutic potential of drugs acting at these receptors for the treatment of a variety of pathologies including anxiety, schizophrenia and Parkinson’s disease.     

A Monte Carlo model reveals independent signaling at central glutamatergic synapses

We have developed a biophysically realistic model of receptor activation at an idealized central glutamatergic synapse that uses Monte Carlo techniques to simulate the stochastic nature of transmission following release of a single synaptic vesicle. For the a synapse with 80 AMPA and 20 NMDA receptors, a single quantum, with 3000 glutamate molecules, opened approximately 3 NMDARs and 20 AMPARs. The number of open receptors varied directly with the total number of receptors, and the fraction of open receptors did not depend on the ratio of co-localized AMPARs and NMDARs. Variability decreased with increases in either total receptor number or quantal size, and differences between the variability of AMPAR and NMDAR responses were due solely to unequal numbers of receptors at the synapse. Despite NMDARs having a much higher affinity for glutamate than AMPARs, quantal release resulted in similar occupancy levels in both receptor types. Receptor activation increased with number of transmitter molecules released or total receptor number, whereas occupancy levels were only dependent on quantal size. Tortuous diffusion spaces reduced the extent of spillover and the activation of extrasynaptic receptors. These results support the conclusion that signaling is spatially independent within and between central glutamatergic synapses.     

Glycine neurotransmitter transporters: an update

Glycine accomplishes several functions as a transmitter in the central nervous system (CNS). As an inhibitory neurotransmitter, it participates in the processing of motor and sensory information that permits movement, vision, and audition. This action of glycine is mediated by the strychnine-sensitive glycine receptor, whose activation produces inhibitory post-synaptic potentials. In some areas of the CNS, glycine seems to be co-released with GABA, the main inhibitory amino acid neurotransmitter. In addition, glycine modulates excitatory neurotransmission by potentiating the action of glutamate at N-methyl-D-aspartate (NMDA) receptors. It is believed that the termination of the different synaptic actions of glycine is produced by rapid re-uptake through two sodium-and-chloride-coupled transporters, GLYT1 and GLYT2, located in the plasma membrane of glial cells or pre-synaptic terminals, respectively. Glycine transporters may become major targets for therapeutic of pathological alterations in synaptic function. This article reviews recent progress on the study of the molecular heterogeneity, localization, function, structure, regulation and pharmacology of the glycine transporter proteins.     

Glycine neurotransmitter transporters: an update

Glycine accomplishes several functions as a transmitter in the central nervous system(CNS). As an inhibitory neurotransmitter, it participates in the processing of motor and sensory information that permits movement, vision, and audition. This action of glycine is mediated by the strychnine-sensitive glycine receptor, whose activation produces inhibitory post-synaptic potentials. In some areas of the CNS, glycine seems to be co-released with GABA, the main inhibitory amino acid neurotransmitter. In addition, glycine modulates excitatory neurotransmission by potentiating the action of glutamate at N-methyl-D-aspartate (NMDA) receptors. It is believed that the termination of the different synaptic actions of glycine is produced by rapid reuptake through two sodium-and-chloride-coupled transporters, GLYT1 and GLYT2, located in the plasma membrane of glial cells or pre-synaptic terminals, respectively. Glycine transporters may become major targets for therapeutic of pathological alterations in synaptic function. This article reviews recent progress on the study of the molecular heterogeneity, localization, function, structure, regulation and pharmacology of the glycine transporter     

Subcellular compartment-specific molecular diversity of pre- and post-synaptic GABAB-activated GIRK channels in Purkinje cells

Activation of G protein-gated inwardly-rectifying K⁺ (GIRK or Kir3) channels by metabotropic gamma-aminobutyric acid (B) (GABA_B) receptors is an essential signalling pathway controlling neuronal excitability and synaptic transmission in the brain. To investigate the relationship between GIRK channel subunits and GABA_B receptors in cerebellar Purkinje cells at post- and pre-synaptic sites, we used biochemical, functional and immunohistochemical techniques. Co-immunoprecipitation analysis demonstrated that GIRK subunits are co-assembled with GABA_B receptors in the cerebellum. Immunoelectron microscopy showed that the subunit composition of GIRK channels in Purkinje cell spines is compartment-dependent. Thus, at extrasynaptic sites GIRK channels are formed by GIRK1/GIRK2/GIRK3, post-synaptic densities contain GIRK2/GIRK3 and dendritic shafts contain GIRK1/GIRK3. The post-synaptic association of GIRK subunits with GABA_B receptors in Purkinje cells is supported by the subcellular regulation of the ion channel and the receptor in mutant mice. At pre-synaptic sites, GIRK channels localized to parallel fibre terminals are formed by GIRK1/GIRK2/GIRK3 and co-localize with GABA_B receptors. Consistent with this morphological evidence we demonstrate their functional interaction at axon terminals in the cerebellum by showing that GIRK channels play a role in the inhibition of glutamate release by GABA_B receptors. The association of GIRK channels and GABA_B receptors with excitatory synapses at both post- and pre-synaptic sites indicates their intimate involvement in the modulation of glutamatergic neurotransmission in the cerebellum.     

Variability of excitatory currents due to single-channel noise, receptor number and morphological heterogeneity

Patch clamp recordings of excitatory postsynaptic currents (EPSCs) in central neurons reveal large fluctuations in amplitudes and decay times of AMPA-receptor-mediated EPSCs. By using Monte Carlo simulations of synaptic transmission in brainstem interneurons, we tested several hypothesis that could account for the observed variability. The coefficient of variation (CV) of 0.5 for miniature amplitudes cannot be explained by fluctuations in vesicle content or receptor distribution, but is traced to variations in receptor number, which is estimated as 77+/-39 receptors per bouton. As the variability of rise times reflects fluctuations in size of the post-synaptic density and heterogeneity of the receptor distribution, the relatively small CV=0.37 of experimentally determined values points to a homogeneous arrangement of receptors. Within our model the large variability of decay times (CV=0.49) can only be explained by fluctuations in the transmitter time course (mean residence times of 0.4+/-0.13 ms), presumably resulting from heterogeneities in synaptic morphology. Hence, our simulations indicate that different noise sources control the variability of amplitudes, rise and decay times. In particular, the distribution of decay times yields information about the synaptic transmission process, which cannot be obtained from other observables.     

Distinct roles for ephrinB3 in the formation and function of hippocampal synapses

The transmembrane ephrinB ligands and their Eph receptor tyrosine kinases are known to regulate excitatory synaptic functions in the hippocampus. In the CA3-CA1 synapse, ephrinB ligands are localized to the post-synaptic membrane, while their cognate Eph receptors are presumed to be pre-synaptic. Interaction of ephrinB molecules with Eph receptors leads to changes in long-term potentiation (LTP), which has been reported to be mediated by reverse signaling into the post-synaptic membrane. Here, we demonstrate that the cytoplasmic domain of ephrinB3 and hence reverse signaling is not required for ephrinB dependent learning and memory tasks or for LTP of these synapses. Consistent with previous reports, we find that ephrinB3(KO) null mutant mice exhibit a striking reduction in CA3-CA1 LTP that is associated with defective learning and memory tasks. We find the null mutants also show changes in both pre- and post-synaptic proteins including increased levels of synapsin and synaptobrevin and reduced levels of NMDA receptor subunits. These abnormalities are not observed in ephrinB3(lacZ) reverse signaling mutants that specifically delete the ephrinB3 intracellular region, supporting a cytoplasmic domain-independent forward signaling role for ephrinB3 in these processes. We also find that both ephrinB3(KO) and ephrinB3(lacZ) mice show an increased number of excitatory synapses, demonstrating a cytoplasmic-dependent reverse signaling role of ephrinB3 in regulating synapse number. Together, these data suggest that ephrinB3 may act like a receptor to transduce reverse signals to regulate the number of synapses formed in the hippocampus, and that it likely acts to stimulate forward signaling to modulate a number of other proteins involved in synaptic activity and learning/memory.