Postsynaptic Receptor Saturation Research Articles

Developmental Easing of Short-Term Depression in “Winner” Climbing Fibers

The postnatal development of cerebellar climbing fiber (CF) to Purkinje neuron (PN) synapses is characterized by a substantial pruning during the first 3 weeks after birth, switching from multiple- to single-CF innervation. Previous studies suggested that CF maturation is governed by bidirectional changes of synaptic plasticity. The strengthening of surviving “winner” CFs, which translocate from the PN soma to the dendrite, is thought to be guided by long-term potentiation (LTP), while weakening of to-be-eliminated “loser” CFs, which remain on the soma, was proposed to be due to long-term depression (LTD). However, there are conflicting results from previous studies, whether or not strengthening of winner and weakening of loser CFs during postnatal development is accompanied by changes in short-term plasticity and, thus, whether pre- or postsynaptic forms of LTD and LTP are operational. We, therefore, analyzed the developmental profile of paired-pulse depression (PPD) in “weak” and “strong” CFs in 3–21-day old Igsf9-eGFP mice, which allow visual identification of GFP-labeled CFs. We found that in 3–8-day old mice strong CFs are marked by a stronger PPD compared to weak CFs. Surprisingly, PPD of strong CFs eases during maturation, while PPD in weak CFs remains unchanged. This easing of PPD is neither due to changes in presynaptic influx-release coupling nor to an increased saturation of postsynaptic receptors. Thus, our results imply that synaptic contacts of CFs show distinct features of PPD depending on their affiliation to winner or loser CFs and depending on their somatic or dendritic location.

Extracting quantal properties of transmission at central synapses.

Chemical synapses enable neurons to communicate rapidly, process and filter signals and to store information. However, studying their functional properties is difficult because synaptic connections typically consist of multiple synaptic contacts that release vesicles stochastically and exhibit time-dependent behavior. Moreover, most central synapses are small and inaccessible to direct measurements. Estimation of synaptic properties from responses recorded at the soma is complicated by the presence of nonuniform release probability and nonuniform quantal properties. The presence of multivesicular release and postsynaptic receptor saturation at some synapses can also complicate the interpretation of quantal parameters. Multiple-probability fluctuation analysis (MPFA; also known as variance-mean analysis) is a method that has been developed for estimating synaptic parameters from the variance and mean amplitude of synaptic responses recorded at different release probabilities. This statistical approach, which incorporates nonuniform synaptic properties, has become widely used for studying synaptic transmission. In this chapter, we describe the statistical models used to extract quantal parameters and discuss their interpretation when applying MPFA.

Complexin: Masterpiece in Vesicle Cycling and Release

Synaptic vesicle pools and cycling are crucial for neurotransmitter release and short-term plasticity. At the last step of vesicle fusion, the SNARE-complex binding protein Complexin clamps spontaneous fusion and promotes calcium-triggered release. In addition, the availability of the releasable vesicle pool is thought to be modulated by Complexin. However, the role of Complexin in vesicle cycling and release is still under investigation. Here we scrutinize the regulatory function of Complexin in the releasable pools and cycling at Drosophila neuromuscular junction. Nerve-evoked synchronous/asynchronous release is enhanced by Complexin and calcium. In addition, Complexin reduces the strontium evoked release sensitivity. The delayed asynchronous release induced by high-frequency nerve-stimulation is suppressed by Complexin and EGTA-AM. Moreover, delayed release is prolonged and early activated in Complexin null. These results indicates that Complexin suppress the asynchronous release activation either by strontium or by tetanic stimulation. Desensitization experiments discard the post synaptic receptor saturation during delayed release. Deconvolution analysis during trains of stimulation indicates that the synchronous, asynchronous and delayed releases are bidirectionally regulated by complexin. The time-course for depletion and the fluorescent dye unloading are consistent with reduced vesicle priming in Complexin null.

Vesicular Release Statistics and Unitary Postsynaptic Current at Single GABAergic Synapses

The existence of vesicular docking sites in central synapses is supported by morphological and biochemical evidence, but their functional role remains elusive. To investigate this role we have studied single depressing GABAergic synapses where multivesicular release and postsynaptic receptor saturation have been documented. We used failure/success patterns to estimate the number of vesicular docking sites, which varied from one to six among synapses. Variations of docking site numbers account for differences in release probability, as well as in the amplitude and decay kinetics of unitary postsynaptic currents. Upon repetitive stimulation, decreasing docking site occupancy likewise accounts for changes both in presynaptic and postsynaptic parameters. Finally steady-state docking site occupancy during train stimulations can be modulated by applying subthreshold presynaptic conditioning potential steps. The results suggest that differences in docking site numbers determine intersynaptic variability and that docking site occupancy is a key parameter controlling single synapse signaling.

Calcium-Dependent PKC Isoforms Have Specialized Roles in Short-Term Synaptic Plasticity

Posttetanic potentiation (PTP) is a widely observed form of short-term plasticity lasting for tens of seconds after high-frequency stimulation. Here we show that although protein kinase C (PKC) mediates PTP at the calyx of Held synapse in the auditory brainstem before and after hearing onset, PTP is produced primarily by an increased probability of release (p) before hearing onset, and by an increased readily releasable pool of vesicles (RRP) thereafter. We find that these mechanistic differences, which have distinct functional consequences, reflect unexpected differential actions of closely related calcium-dependent PKC isoforms. Prior to hearing onset, when PKCγ and PKCβ are both present, PKCγ mediates PTP by increasing p and partially suppressing PKCβ actions. After hearing onset, PKCγ is absent and PKCβ produces PTP by increasing RRP. In hearing animals, virally expressed PKCγ overrides PKCβ to produce PTP by increasing p. Thus, two similar PKC isoforms mediate PTP in distinctly different ways.

Receptor saturation controls short-term synaptic plasticity at corticothalamic synapses

Glutamatergic synapses of layer 6 corticothalamic (CT) neurons form a major excitatory input onto thalamic relay cells, allowing neocortex to continuously control sensory information processing in thalamic circuits. CT synapses display both short- and long-term forms of use-dependent synaptic enhancement, mediated at least in part by increases in the probability of transmitter release. At some synapses, such increases in release probability are accompanied by a higher degree of multivesicular release (MVR) and larger glutamate transients at individual release sites, resulting in the saturation of postsynaptic receptors. The extent to which MVR and postsynaptic saturation interact and control short-term plasticity at CT synapses is not known. Here we examined two distinct presynaptic forms of short-term enhancement, facilitation and augmentation, at CT synapses contacting relay neurons in the ventrobasal nucleus of the mouse thalamus. We found that, in the presence of the low-affinity antagonist γ-D-glutamylglycine, to relieve postsynaptic DL-α-amino-3-hydroxy-5-methylisox azole-propionic acid (AMPA) receptor saturation, the magnitude of facilitation and augmentation increased. Whereas receptor saturation was prominent for both AMPA and N-methyl-D-aspartate receptors, desensitization of AMPA receptors did not significantly alter short-term plasticity. Our results suggest that at CT synapses the activity-dependent increase in synaptic strength is controlled by postsynaptic receptor saturation.

Multivesicular Release Differentiates the Reliability of Synaptic Transmission between the Visual Cortex and the Somatosensory Cortex

Neurons in layer 4 (L4) of the cortex play an important role in transferring signals from thalamus to other layers of the cortex. Understanding the fundamental properties of synaptic transmission between L4 neurons helps us gain a clear picture of how the neuronal network in L4 cooperates to process sensory information. In the present study, we have determined the underlying parameters that govern synaptic strength, such as quantal size, size of readily releasable vesicle pool, and release probability (Pr) of excitatory synaptic connections within L4 of the visual cortex (V1) and the somatosensory cortex (S1) in mice. Although only a single vesicle is released per release site under physiological conditions at V1 synapses, multivesicular release (MVR) is observed at S1 synapses. In addition, we observed a saturation of postsynaptic receptors at S1 synapses. Other synaptic properties are similar in both cortices. Dynamic clamp experiments suggest that higher Pr and MVR at S1 synapses lower the requirement of the number of synaptic inputs to generate postsynaptic action potentials. In addition, the slower decay of synaptic current and the intrinsic membrane properties of the postsynaptic neuron also contribute to the reliable transmission between S1 neurons.

Cholinergic modulation of multivesicular release regulates striatal synaptic potency and integration

The pleiotropic actions of neuromodulators on pre- and postsynaptic targets present challenges to disentangling the mechanisms underlying regulation of synaptic transmission. Within the striatum, acetylcholine modulates glutamate release via activation of muscarinic receptors (mAchRs), although the consequences for postsynaptic signaling are unclear. Using 2-photon microscopy and glutamate uncaging to examine individual synapses in the rat striatum, we find that glutamatergic afferents exhibit a high degree of multivesicular release (MVR) in the absence of postsynaptic receptor saturation. We show that mAchR activation decreases both the probability of release and the concentration of glutamate in the synaptic cleft. The corresponding decrease in synaptic potency reduces the duration of synaptic potentials and limits temporal summation of afferent inputs. These findings reveal a mechanism by which a combination of basal MVR and low receptor saturation allow the presynaptic actions of a neuromodulator to control the engagement of postsynaptic nonlinearities and regulate synaptic integration.

Noradrenergic Control of Associative Synaptic Plasticity by Selective Modulation of Instructive Signals

Synapses throughout the brain are modified through associative mechanisms in which one input provides an instructive signal for changes in the strength of a second coactivated input. In cerebellar Purkinje cells, climbing fiber synapses provide an instructive signal for plasticity at parallel fiber synapses. Here, we show that noradrenaline activates alpha2-adrenergic receptors to control short-term and long-term associative plasticity of parallel fiber synapses. This regulation of plasticity does not reflect a conventional direct modulation of the postsynaptic Purkinje cell or presynaptic parallel fibers. Instead, noradrenaline reduces associative plasticity by selectively decreasing the probability of release at the climbing fiber synapse, which in turn decreases climbing fiber-evoked dendritic calcium signals. These findings raise the possibility that targeted presynaptic modulation of instructive synapses could provide a general mechanism for dynamic context-dependent modulation of associative plasticity.

Frequency-Independent Synaptic Transmission Supports a Linear Vestibular Behavior

The vestibular system is responsible for transforming head motion into precise eye, head, and body movements that rapidly stabilize gaze and posture. How do central excitatory synapses mediate behavioral outputs accurately matched to sensory inputs over a wide dynamic range? Here we demonstrate that vestibular afferent synapses in vitro express frequency-independent transmission that spans their in vivo dynamic range (5-150 spikes/s). As a result, the synaptic charge transfer per unit time is linearly related to vestibular afferent activity in both projection and intrinsic neurons of the vestibular nuclei. Neither postsynaptic glutamate receptor desensitization nor saturation affect the relative amplitude or frequency-independence of steady-state transmission. Finally, we show that vestibular nucleus neurons can transduce synaptic inputs into linear changes in firing rate output without relying on one-to-one calyceal transmission. These data provide a physiological basis for the remarkable linearity of vestibular reflexes.

Multivesicular release and saturation of glutamatergic signalling at retinal ribbon synapses

Pronounced multivesicular release (MVR) occurs at the ribbon synapses of sensory neurones that signal via graded potential changes. As MVR increases the likelihood of postsynaptic receptor saturation, it is of interest to consider how sensory synapses overcome this problem and use MVR to encode signals of widely varying intensities. Here, I discuss three postsynaptic mechanisms that permit three different retinal synapses to utilize MVR.

Activity-dependent regulation of vesicular glutamate and GABA transporters: A means to scale quantal size

The functional balance of glutamatergic and GABAergic signaling in neuronal cortical circuits is under homeostatic control. That is, prolonged alterations of global network activity leads to opposite changes in quantal amplitude at glutamatergic and GABAergic synapses. Such scaling of excitatory and inhibitory transmission within cortical circuits serves to restore and maintain a constant spontaneous firing rate of pyramidal neurons. Our recent work shows that this includes alterations in the levels of expression of vesicular glutamate (VGLUT1 and VGLUT2) and GABA (VIAAT) transporters. Other vesicle markers, such as synaptophysin or synapsin, are not regulated in this way. Endogenous regulation at the level of mRNA and synaptic protein controls the number of transporters per vesicle and hence, the level of vesicle filling with transmitter. Bidirectional and opposite activity-dependent regulation of VGLUT1 and VIAAT expression would serve to adjust the balance of glutamate and GABA release and therefore the level of postsynaptic receptor saturation. In some excitatory neurons and synapses, co-expression of VGLUT1 and VGLUT2 occurs. Bidirectional and opposite changes in the levels of two excitatory vesicular transporters would enable individual neocortical neurons to scale up or scale down the level of vesicular glutamate storage, and thus, the amount available for release at individual synapses. Regulated vesicular transmitter storage and release via selective changes in the level of expression of vesicular glutamate and GABA transporters indicates that homeostatic plasticity of synaptic strength at cortical synapses includes presynaptic elements.

The Influence of Multivesicular Release and Postsynaptic Receptor Saturation on Transmission at Granule Cell to Purkinje Cell Synapses

The properties of a synapse are crucially dependent on whether an action potential can trigger the release of multiple vesicles at an individual release site [multivesicular release (MVR)] and whether fusion of a single vesicle leads to receptor saturation. MVR and receptor saturation both occur at some high p synapses, but it is not known whether they also occur at low p synapses. Here we examine this issue at the low p synapse between parallel fibers and Purkinje cells using the low-affinity antagonist DGG (gamma-D-glutamylglycine) to relieve AMPA receptor saturation. We find that the presence of MVR and receptor saturation at this synapse alters the calcium dependence of synaptic transmission and reduces the extent of facilitation. These findings establish that MVR and postsynaptic receptor saturation can influence transmission even at synapses with a low initial probability of release and suggest that these properties may be common at synapses in the mammalian brain.

Reluctant Vesicles Contribute to the Total Readily Releasable Pool in Glutamatergic Hippocampal Neurons

The size of the readily releasable pool (RRP) of vesicles is critically important for determining the size of postsynaptic currents generated in response to action potentials. However, discrepancies in RRP estimates exist among methods designed to measure RRP size. In glutamatergic hippocampal neurons, we found that hypertonic sucrose application yielded RRP size estimates approximately fivefold larger than values obtained with high-frequency action potential trains commonly assumed to deplete the RRP. This discrepancy was specific for glutamatergic neurons, because no difference was found between sucrose and train estimates of RRP size in GABAergic neurons. A small component of the difference in excitatory neurons was accounted for by postsynaptic receptor saturation. Train estimates of vesicle pool size obtained using more stimuli revealed that action potential-elicited EPSCs did not truly reach a steady state during shorter trains, and RRP estimates were closer to sucrose estimates made in the same neurons. This suggested that reluctant vesicles may contribute to the total available pool. Two additional lines of evidence supported this hypothesis. First, RRP estimates from strongly depolarizing hyperkalemic solutions closely matched those obtained with sucrose. Second, when Ca2+ influx was enhanced during trains, train estimates of pool size matched those obtained with sucrose. These data suggest that glutamatergic hippocampal neurons maintain a heterogeneous population of vesicles that can be differentially released with varying Ca2+ influx, thereby increasing the range of potential synaptic responses.

Variance-Mean Analysis in the Presence of a Rapid Antagonist Indicates Vesicle Depletion Underlies Depression at the Climbing Fiber Synapse

Many types of synapses throughout the nervous system are transiently depressed during high-frequency stimulation. Several mechanisms have been proposed to account for this depression, including depletion of release-ready vesicles. However, numerous studies have raised doubts about the importance of depletion in depression of central synapses and have implicated alternative mechanisms, such as decreased release probability. We use variance-mean analysis to determine the mechanism of depression at the climbing fiber to Purkinje cell synapse. We find that postsynaptic receptor saturation makes it difficult to distinguish between a decrease in available vesicles and a reduction in release probability. When AMPA receptor saturation is relieved with a low-affinity antagonist, variance-mean analysis reveals that depression arises from a decrease in the number of release-ready vesicles. Vesicle depletion is prominent, despite numerous docked vesicles at each release site, due to multivesicular release. We conclude that vesicle depletion can contribute significantly to depression of central synapses.

Interaction of Postsynaptic Receptor Saturation with Presynaptic Mechanisms Produces a Reliable Synapse

Synapses that reliably activate their postsynaptic targets typically release neurotransmitter with high probability, are not very sensitive to changes in calcium entry, and depress. We have determined the mechanisms that give rise to these characteristic features at the climbing fiber to Purkinje cell synapse. We find that saturation of presynaptic calcium entry, of presynaptic release, and of postsynaptic receptors combine to produce a postsynaptic response that is near maximal. Postsynaptic receptor saturation also accelerates recovery from depression, in part by accentuating a rapid calcium-dependent recovery phase. Thus, postsynaptic receptor saturation interacts with presynaptic mechanisms to produce highly reliable synapses that can effectively drive their targets even during sustained activation.

Estimation of quantal parameters at the calyx of Held synapse.

The calyx of Held has recently emerged as a convenient model system to study CNS synapses. In order to understand the mechanisms of synaptic transmission and short-term synaptic plasticity, quantal parameters and their changes should be estimated precisely. For this purpose, various methods have been applied to the calyx of Held synapse. The results confirm many aspects of the early findings on transmission at the neuromuscular junction. On the other hand, the simplest quantal hypothesis does not work at the calyx of Held, because of additional factors such as heterogeneous release probability of synaptic vesicles, intra- and intersite quantal variability, an overlap of facilitation and depression of transmitter release, changes in quantal sizes due to desensitization and saturation of postsynaptic receptors, and delayed clearance of transmitter from the synaptic cleft. These factors should always be taken into account for fully understanding the mechanisms of synaptic transmission and plasticity.

Separation of Presynaptic and Postsynaptic Contributions to Depression by Covariance Analysis of Successive EPSCs at the Calyx of Held Synapse

Synaptic short-term plasticity is considered to result from multiple cellular mechanisms, which may include presynaptic and postsynaptic contributions. We have recently developed a nonstationary EPSC fluctuation analysis (Scheuss and Neher, 2001) to estimate synaptic parameters and their transient changes during short-term synaptic plasticity. Extending the classical variance-mean approach, a short train of stimuli is applied repetitively, and the resulting EPSCs are analyzed for means, variances, and covariances. This provides estimates of the quantal size and quantal content for each EPSC in the train, and furthermore, an estimate of the number of release sites. The latter is less sensitive to heterogeneity in the release probability than that of the variance-mean approach. Here, we applied this analysis to the calyx of Held synapse in brainstem slices of young rats (postnatal day 8-10). We found significant negative covariance in the amplitude of successive EPSCs in a train. The analysis showed that the 10-fold depression in the EPSC amplitude during 100 Hz trains at elevated extracellular Ca(2+) concentration resulted from a 2.5-fold reduction in quantal size caused by postsynaptic AMPA receptor desensitization and saturation, and a fourfold reduction in quantal content, which was partially relieved by application of cyclothiazide. The number of release sites estimated by covariance analysis was approximately 2000 and significantly larger than estimates from variance-mean parabolas.

Estimation of Quantal Size and Number of Functional Active Zones at the Calyx of Held Synapse by Nonstationary EPSC Variance Analysis

At the large excitatory calyx of Held synapse, the quantal size during an evoked EPSC and the number of active zones contributing to transmission are not known. We developed a nonstationary variant of EPSC fluctuation analysis to determine these quantal parameters. AMPA receptor-mediated EPSCs were recorded in slices of young (postnatal 8-10 d) rats after afferent fiber stimulation, delivered in trains to induce synaptic depression. The means and the variances of EPSC amplitudes were calculated across trains for each stimulus number. During 10 Hz trains at 2 mm Ca(2+) concentration ([Ca(2+)]), we found linear EPSC variance-mean relationships, with a slope that was in good agreement with the quantal size obtained from amplitude distributions of spontaneous miniature EPSCs. At high release probability with 10 or 15 mm [Ca(2+)], competitive antagonists were used to partially block EPSCs. Under these conditions, the EPSC variance-mean plots could be fitted with parabolas, giving estimates of quantal size and of the binomial parameter N. With the rapidly dissociating antagonist kynurenic acid, quantal sizes were larger than with a slowly dissociating antagonist, suggesting that the effective glutamate concentration was increased at high release probability. Considering the possibility of multivesicular release and moderate saturation of postsynaptic AMPA receptors, we conclude that the binomial parameter N (637 +/- 117; mean +/- SEM) represents an upper limit estimate of the number of functional active zones. We estimate that during normal synaptic transmission, the probability of vesicle fusion at single active zones is in the range of 0.25-0.4.

Estimating Synaptic Parameters from Mean, Variance, and Covariance in Trains of Synaptic Responses

Fluctuation analysis of synaptic transmission using the variance–mean approach has been restricted in the past to steady-state responses. Here we extend this method to short repetitive trains of synaptic responses, during which the response amplitudes are not stationary. We consider intervals between trains, long enough so that the system is in the same average state at the beginning of each train. This allows analysis of ensemble means and variances for each response in a train separately. Thus, modifications in synaptic efficacy during short-term plasticity can be attributed to changes in synaptic parameters. In addition, we provide practical guidelines for the analysis of the covariance between successive responses in trains. Explicit algorithms to estimate synaptic parameters are derived and tested by Monte Carlo simulations on the basis of a binomial model of synaptic transmission, allowing for quantal variability, heterogeneity in the release probability, and postsynaptic receptor saturation and desensitization. We find that the combined analysis of variance and covariance is advantageous in yielding an estimate for the number of release sites, which is independent of heterogeneity in the release probability under certain conditions. Furthermore, it allows one to calculate the apparent quantal size for each response in a sequence of stimuli.