A single packet of transmitter does not saturate postsynaptic glutamate receptors

Neurotransmitter is stored in synaptic vesicles and released by exocytosis into the synaptic cleft. One of the fundamental questions in central synaptic transmission is whether a quantal packet of transmitter saturates postsynaptic receptors. To address this question, we loaded the excitatory transmitter L-glutamate via whole-cell recording pipettes into the giant nerve terminal, the calyx of Held, in rat brainstem slices. This caused marked potentiations of both quantal and action potential-evoked EPSCs mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptors. These results directly demonstrate that neither AMPA nor NMDA receptors are saturated by a single packet of transmitter, and indicate that vesicular transmitter content is an important determinant of synaptic efficacy.     

Distinct quantal features of AMPA and NMDA synaptic currents in hippocampal neurons: implication of glutamate spillover and receptor saturation

Excitatory postsynaptic currents (EPSCs) were studied in the CA1 pyramidal cells of rat hippocampal slices. Components mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) and by N-methyl-D-aspartate (NMDA) receptors were separated pharmacologically. Quantal parameters of AMPA and NMDA receptor-mediated EPSCs were obtained using both maximal likelihood and autocorrelation techniques. Enhancement of transmitter release with 4-aminopyridine caused a significant increase in quantal size of NMDA EPSC. This was accompanied by a slowing of the EPSC decay. The maximal number of quanta in the NMDA current was unchanged, while the probability of quantal event dramatically enhanced. In contrast, neither the quantal size nor the kinetics of AMPA EPSC was altered by 4-aminopyridine, while the maximal number of quanta increased. These changes in the quantal parameters are consistent with a transition to multivesicular release of the neurotransmitter. Spillover of excessive glutamate on the nonsynaptic areas of dendritic spines causes an increase in the quantal size of NMDA synaptic current. The difference in quantal behavior of AMPA and NMDA EPSCs implies that different mechanisms underlie their quantization: the additive response of nonsaturated AMPA receptors contrasts with the variable involvement of saturated intrasynaptic and nonsaturated extrasynaptic NMDA receptors.     

Multivesicular release at climbing fiber-Purkinje cell synapses.

Synapses driven by action potentials are thought to release transmitter in an all-or-none fashion; either one synaptic vesicle undergoes exocytosis, or there is no release. We have estimated the glutamate concentration transient at climbing fiber synapses on Purkinje cells by measuring the inhibition of excitatory postsynaptic currents (EPSCs) produced by a low-affinity competitive antagonist of AMPA receptors, gamma-DGG. The results, together with simulations using a kinetic model of the AMPA receptor, suggest that the peak glutamate concentration at this synapse is dependent on release probability but is not affected by pooling of transmitter released from neighboring synapses. We propose that the mechanism responsible for the elevated glutamate concentration at this synapse is the simultaneous release of multiple vesicles per site.     

Fusion pore modulation as a presynaptic mechanism contributing to expression of long-term potentiation

Working on the idea that postsynaptic and presynaptic mechanisms of long-term potentiation (LTP) expression are not inherently mutually exclusive, we have looked for the existence and functionality of presynaptic mechanisms for augmenting transmitter release in hippocampal slices. Specifically, we asked if changes in glutamate release might contribute to the conversion of 'silent synapses' that show N-methyl-D-aspartate (NMDA) responses but no detectable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) responses, to ones that exhibit both. Here, we review experiments where NMDA receptor responses provided a bioassay of cleft glutamate concentration, using opposition between peak [glu](cleft )and a rapidly reversible antagonist, L-AP5. We discuss findings of a dramatic increase in peak [glu](cleft) upon expression of pairing-induced LTP (Choi). We present simulations with a quantitative model of glutamatergic synaptic transmission that includes modulation of the presynaptic fusion pore, realistic cleft geometry and a distributed array of postsynaptic receptors and glutamate transporters. The modelling supports the idea that changes in the dynamics of glutamate release can contribute to synaptic unsilencing. We review direct evidence from Renger et al., in accord with the modelling, that trading off the strength and duration of the glutamate transient can markedly alter AMPA receptor responses with little effect on NMDA receptor responses. An array of additional findings relevant to fusion pore modulation and its proposed contribution to LTP expression are considered.     

Variability of neurotransmitter concentration and nonsaturation of postsynaptic AMPA receptors at synapses in hippocampal cultures and slices

To understand the elementary unit of synaptic communication between CNS neurons, one must know what causes the variability of quantal postsynaptic currents and whether unitary packets of transmitter saturate postsynaptic receptors. We studied single excitatory synapses between hippocampal neurons in culture. Focal glutamate application at individual postsynaptic sites evoked currents (I(glu)) with little variability compared with quantal excitatory postsynaptic currents (EPSCs). The maximal I(glu) was >2-fold larger than the median EPSC. Thus, variations in [glu]cleft are the main source of variability in EPSC size, and glutamate receptors are generally far from saturation during quantal transmission. This conclusion was verified by molecular antagonism experiments in hippocampal cultures and slices. The general lack of glutamate receptor saturation leaves room for increases in [glu]cleft as a mechanism for synaptic plasticity.     

Extracellular glutamate diffusion determines the occupancy of glutamate receptors at CA1 synapses in the hippocampus

Following exocytosis at excitatory synapses in the brain, glutamate binds to several subtypes of postsynaptic receptors. The degree of occupancy of AMPA and NMDA receptors at hippocampal synapses is, however, not known. One approach to estimate receptor occupancy is to examine quantal amplitude fluctuations of postsynaptic signals in hippocampal neurons studied in vitro. The results of such experiments suggest that NMDA receptors at CA1 synapses are activated not only by glutamate released from the immediately apposed presynaptic terminals, but also by glutamate spillover from neighbouring terminals. Numerical simulations point to the extracellular diffusion coefficient as a critical parameter that determines the extent of activation of receptors positioned at different distances from the release site. We have shown that raising the viscosity of the extracellular medium can modulate the diffusion coefficient, providing an experimental tool to investigate the role of diffusion in activation of synaptic and extrasynaptic receptors. Whether intersynaptic cross-talk mediated by NMDA receptors occurs in vivo remains to be determined. The theoretical and experimental approaches described here also promise to shed light on the roles of metabotropic and kainate receptors, which often occur in an extrasynaptic distribution, and are therefore positioned to sense glutamate escaping from the synaptic cleft.     

Presynaptic control of quantal size: kinetic mechanisms and implications for synaptic transmission and plasticity

Although the strength of quantal synaptic transmission is jointly controlled by pre- and post-synaptic mechanisms, the presynaptic mechanisms remain substantially less well characterized. Recent studies reveal that a single package of neurotransmitter is generally insufficient to activate all available postsynaptic receptors, whereas the sum of transmitter from multiple vesicles can result in receptor saturation. Thus, depending upon the number of vesicles released, a given synaptic pathway might be either 'reliable' or 'unreliable'. A lack of receptor saturation in turn makes it possible to modify quantal size by altering the flux of transmitter through the synaptic cleft. Studies are now illuminating several new mechanisms behind the regulation of this transmitter flux--characteristics that control how transmitter is loaded into vesicles, how it is released and the manner by which it interacts with postsynaptic receptors.     

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.     

The effects of geometrical parameters on synaptic transmission: a Monte Carlo simulation study

Monte Carlo simulations of transmitter diffusion and its interactions with postsynaptic receptors have been used to study properties of quantal responses at central synapses. Fast synaptic responses characteristic of those recorded at glycinergic junctions on the teleost Mauthner cell (time to peak approximately 0.3-0.4 ms and decay time constant approximately 3-6 ms) served as the initial reference, and smaller contacts with fewer postsynaptic receptors were also modeled. Consistent with experimental findings, diffusion, simulated using a random walk algorithm and assuming a diffusion coefficient of 0.5-1.0 x 10(-5) cm2 s(-1), was sufficiently fast to account for transmitter removal from the synaptic cleft. Transmitter-receptor interactions were modeled as a two-step binding process, with the double-bound state having opened and closed conformations. Addition of a third binding step only slightly decreased response amplitude but significantly slowed both its rising and decay phases. The model allowed us to assess the sources of response variability and the likelihood of postsynaptic saturation as functions of multiple kinetic and spatial parameters. The method of nonstationary fluctuation analysis, typically used to estimate the number of functional channels at a synapse and single channel current, proved unreliable, presumably because the receptors in the postsynaptic matrix are not uniformly exposed to the same profile of transmitter concentration. Thus, the time course of the probability of channel opening most likely varies among receptors. Finally, possible substrates for phenomena of synaptic plasticity, such as long-term potentiation, were explored, including the diameter of the contact zone, defined by the region of pre- and postsynaptic apposition, the number and distribution of the receptors, and the degree of vesicle filling. Surprisingly, response amplitude is quite sensitive to the size of the receptor-free annulus surrounding the receptor cluster, such that expansion of the contact zone could produce an appreciable increase in quantal size, normally attributed to either the presence of more receptors or the release of more transmitter molecules.     

Transmitter concentration profiles in the synaptic cleft: an analytical model of release and diffusion.

A three-dimensional model for release and diffusion of glutamate in the synaptic cleft was developed and solved analytically. The model consists of a source function describing transmitter release from the vesicle and a diffusion function describing the spread of transmitter in the cleft. Concentration profiles of transmitter at the postsynaptic side were calculated for different transmitter concentrations in a vesicle, release scenarios, and diffusion coefficients. From the concentration profiles the receptor occupancy could be determined using alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor kinetics. It turned out that saturation of receptors and sufficiently fast currents could only be obtained if the diffusion coefficient was one order of magnitude lower than generally assumed, and if the postsynaptic receptors formed clusters with a diameter of roughly 100 nm directly opposite the release sites. Under these circumstances the gradient of the transmitter concentration at the postsynaptic membrane outside the receptor clusters was steep, with minimal cross-talk among neighboring receptor clusters. These findings suggest that for each release site a corresponding receptor aggregate exists, subdividing an individual synapse into independent functional subunits without the need for specific lateral diffusion barriers.     

Spillover of glutamate onto synaptic AMPA receptors enhances fast transmission at a cerebellar synapse

Diffusion of glutamate from the synaptic cleft can activate high-affinity receptors, but is not thought to contribute to fast AMPA receptor-mediated transmission. Here, we show that single AMPA receptor EPSCs at the cerebellar mossy fiber-granule cell connection are mediated by both direct release of glutamate and rapid diffusion of glutamate from neighboring synapses. Immunogold localization revealed that AMPA receptors are located exclusively in postsynaptic densities, indicating that spillover of glutamate occurs between synaptic contacts. Spillover currents contributed half the synaptic charge and exhibited little trial-to-trial variability. We propose that spillover of glutamate improves transmission efficacy by both increasing the amplitude and duration of the EPSP and reducing fluctuations arising from the probabilistic nature of transmitter release.     

AMPA receptor trafficking at excitatory synapses

Excitatory synapses in the CNS release glutamate, which acts primarily on two sides of ionotropic receptors: AMPA receptors and NMDA receptors. AMPA receptors mediate the postsynaptic depolarization that initiates neuronal firing, whereas NMDA receptors initiate synaptic plasticity. Recent studies have emphasized that distinct mechanisms control synaptic expression of these two receptor classes. Whereas NMDA receptor proteins are relatively fixed, AMPA receptors cycle synaptic membranes on and off. A large family of interacting proteins regulates AMPA receptor turnover at synapses and thereby influences synaptic strength. Furthermore, neuronal activity controls synaptic AMPA receptor trafficking, and this dynamic process plays a key role in the synaptic plasticity that is thought to underlie aspects of learning and memory.     

AMPA receptor-dependent clustering of synaptic NMDA receptors is mediated by Stargazin and NR2A/B in spinal neurons and hippocampal interneurons

Under standard conditions, cultured ventral spinal neurons cluster AMPA- but not NMDA-type glutamate receptors at excitatory synapses on their dendritic shafts in spite of abundant expression of the ubiquitous NMDA receptor subunit NR1. We demonstrate here that the NMDA receptor subunits NR2A and NR2B are not routinely expressed in cultured spinal neurons and that transfection with NR2A or NR2B reconstitutes the synaptic targeting of NMDA receptors and confers on exogenous application of the immediate early gene product Narp the ability to cluster both AMPA and NMDA receptors. The use of dominant-negative mutants of GluR2 further showed that the synaptic targeting of NMDA receptors is dependent on the presence of synaptic AMPA receptors and that synaptic AMPA and NMDA receptors are linked by Stargazin and a MAGUK protein. This system of AMPA receptor-dependent synaptic NMDA receptor localization was preserved in hippocampal interneurons but reversed in hippocampal pyramidal neurons.     

The neurotransmitter cycle and quantal size

Changes in the response to release of a single synaptic vesicle have generally been attributed to postsynaptic modification of receptor sensitivity, but considerable evidence now demonstrates that alterations in vesicle filling also contribute to changes in quantal size. Receptors are not saturated at many synapses, and changes in the amount of transmitter per vesicle contribute to the physiological regulation of release. On the other hand, the presynaptic factors that determine quantal size remain poorly understood. Aside from regulation of the fusion pore, these mechanisms fall into two general categories: those that affect the accumulation of transmitter inside a vesicle and those that affect vesicle size. This review will summarize current understanding of the neurotransmitter cycle and indicate basic, unanswered questions about the presynaptic regulation of quantal size.     

CPG2: a brain- and synapse-specific protein that regulates the endocytosis of glutamate receptors

Long-term maintenance and modification of synaptic strength involve the turnover of neurotransmitter receptors. Glutamate receptors are constitutively and acutely internalized, presumptively through clathrin-mediated receptor endocytosis. Here, we show that cpg2 is a brain-specific splice variant of the syne-1 gene that encodes a protein specifically localized to a postsynaptic endocytotic zone of excitatory synapses. RNAi-mediated CPG2 knockdown increases the number of postsynaptic clathrin-coated vesicles, some of which traffic NMDA receptors, disrupts the constitutive internalization of glutamate receptors, and inhibits the activity-induced internalization of synaptic AMPA receptors. Manipulating CPG2 levels also affects dendritic spine size, further supporting a function in regulating membrane transport. Our results suggest that CPG2 is a key component of a specialized postsynaptic endocytic mechanism devoted to the internalization of synaptic proteins, including glutamate receptors. The activity dependence and distribution of cpg2 expression further suggest that it contributes to the capacity for postsynaptic plasticity inherent to excitatory synapses.     

A developmental switch in neurotransmitter flux enhances synaptic efficacy by affecting AMPA receptor activation

Formation of glutamatergic synapses entails development of "silent" immature contacts into mature functional synapses. To determine how this transformation occurs, we investigated the development of neurotransmission at single synapses in vitro. Maturation of presynaptic function, assayed with endocytotic markers, followed accumulation of synapsin I. During this period, synaptic transmission was primarily mediated by activation of NMDA receptors, suggesting that most synapses were functionally silent. However, local glutamate application to silent synapses indicated that these synapses contained functional AMPA receptors, suggesting a possible presynaptic locus for silent transmission. Interference with presynaptic vesicle fusion by exposure to tetanus toxin reverted functional to silent transmission, implicating SNARE-mediated fusion as a determinant of the ratio of NMDA:AMPA receptor activation. This work reveals that functional maturation of synaptic transmission involves transformation of presynaptic silent secretion into mature synaptic transmitter release.