Long-Term Culture of Astrocytes Attenuates the Readily Releasable Pool of Synaptic Vesicles

The astrocyte is a major glial cell type of the brain, and plays key roles in the formation, maturation, stabilization and elimination of synapses. Thus, changes in astrocyte condition and age can influence information processing at synapses. However, whether and how aging astrocytes affect synaptic function and maturation have not yet been thoroughly investigated. Here, we show the effects of prolonged culture on the ability of astrocytes to induce synapse formation and to modify synaptic transmission, using cultured autaptic neurons. By 9 weeks in culture, astrocytes derived from the mouse cerebral cortex demonstrated increases in β-galactosidase activity and glial fibrillary acidic protein (GFAP) expression, both of which are characteristic of aging and glial activation in vitro. Autaptic hippocampal neurons plated on these aging astrocytes showed a smaller amount of evoked release of the excitatory neurotransmitter glutamate, and a lower frequency of miniature release of glutamate, both of which were attributable to a reduction in the pool of readily releasable synaptic vesicles. Other features of synaptogenesis and synaptic transmission were retained, for example the ability to induce structural synapses, the presynaptic release probability, the fraction of functional presynaptic nerve terminals, and the ability to recruit functional AMPA and NMDA glutamate receptors to synapses. Thus the presence of aging astrocytes affects the efficiency of synaptic transmission. Given that the pool of readily releasable vesicles is also small at immature synapses, our results are consistent with astrocytic aging leading to retarded synapse maturation.     

Pair recordings reveal all-silent synaptic connections and the postsynaptic expression of long-term potentiation

The activation of silent synapses is a proposed mechanism to account for rapid increases in synaptic efficacy such as long-term potentiation (LTP). Using simultaneous recordings from individual pre- and postsynaptic neurons in organotypic hippocampal slices, we show that two CA3 neurons can be connected entirely by silent synapses. Increasing release probability or application of cyclothiazide does not produce responses from these silent synapses. Direct measurement of NMDAR-mediated postsynaptic responses in all-silent synaptic connections before and after LTP induction show no change in failure rate, amplitude, or area. These data do not support hypotheses that synapse silent results from presynaptic factors or that LTP results from increases in presynaptic glutamate release. LTP is also associated with an increase in postsynaptic responsiveness to exogenous AMPA. We conclude that synapse silence, activation, and expression of LTP are postsynaptic.     

Analysis and implications of equivalent uniform approximations of nonuniform unitary synaptic systems

Real synaptic systems consist of a nonuniform population of synapses with a broad spectrum of probability and response distributions varying between synapses, and broad amplitude distributions of postsynaptic unitary responses within a given synapse. A common approach to such systems has been to assume identical synapses and recover apparent quantal parameters by deconvolution procedures from measured evoked (ePSC) and unitary evoked postsynaptic current (uePSC) distributions. Here we explicitly consider nonuniform synaptic systems with both intra (type I) and intersynaptic (type II) response variability and formally define an equivalent system of uniform synapses in which both uePSC and ePSC amplitude distributions best approximate those of the actual nonuniform synaptic system. This equivalent system has the advantage of being fully defined by just four quantal parameters: ?, the number of equivalent synapses;p, the mean probability of quantal release; mu, mean; and sigma(2), variance of the uePSC distribution. We show that these equivalent parameters are weighted averages of intrinsic parameters and can be approximated by apparent quantal parameters, therefore establishing a useful analytical link between the apparent and intrinsic parameters. The present study extends previous work on compound binomial analysis of synaptic transmission by highlighting the importance of the product of p and mu, and the variance of that product. Conditions for a unique deconvolution of apparent uniform synaptic parameters have been derived and justified. Our approach does not require independence of synaptic parameters, such as p and mu from each other, therefore the approach will hold even if feedback (i.e., via retrograde transmission) exists between pre and postsynaptic signals. Using numerical simulations we demonstrate how equivalent parameters are meaningful even when there is considerable variation in intrinsic parameters, including systems where subpopulations of high- and low-release probability synapses are present, therefore even under such conditions the apparent parameters estimated from experiments would be informative.     

The Influence of Synaptic Size on AMPA Receptor Activation: A Monte Carlo Model

Physiological and electron microscope studies have shown that synapses are functionally and morphologically heterogeneous and that variations in size of synaptic junctions are related to characteristics such as release probability and density of postsynaptic AMPA receptors. The present article focuses on how these morphological variations impact synaptic transmission. We based our study on Monte Carlo computational simulations of simplified model synapses whose morphological features have been extracted from hundreds of actual synaptic junctions reconstructed by three-dimensional electron microscopy. We have examined the effects that parameters such as synaptic size or density of AMPA receptors have on the number of receptors that open after release of a single synaptic vesicle. Our results indicate that the maximum number of receptors that will open after the release of a single synaptic vesicle may show a ten-fold variation in the whole population of synapses. When individual synapses are considered, there is also a stochastical variability that is maximal in small synapses with low numbers of receptors. The number of postsynaptic receptors and the size of the synaptic junction are the most influential parameters, while the packing density of receptors or the concentration of extrasynaptic transporters have little or no influence on the opening of AMPA receptors.     

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.     

Independent Regulation of Basal Neurotransmitter Release Efficacy by Variable Ca2+ Influx and Bouton Size at Small Central Synapses

The efficacy of action potential evoked neurotransmitter release varies widely even among synapses supplied by the same axon, and the number of release-ready vesicles at each synapse is a major determinant of this heterogeneity. Here we identify a second, equally important, mechanism for release heterogeneity at small hippocampal synapses, the inter-synaptic variation of the exocytosis probability of release-ready vesicles. Using concurrent measurements of vesicular pool sizes, vesicular exocytosis rates, and presynaptic Ca²⁺ dynamics, in the same small hippocampal boutons, we show that the average fusion probability of release-ready vesicles varies among synapses supplied by the same axon with the size of the spike-evoked Ca²⁺ concentration transient. We further show that synapses with a high vesicular release probability exhibit a lower Ca²⁺ cooperativity, arguing that this is a direct consequence of increased Ca²⁺ influx at the active zone. We conclude that variability of neurotransmitter release under basal conditions at small central synapses is accounted for not only by the number of release-ready vesicles, but also by their fusion probabilities, which are set independently of bouton size by variable spike-evoked presynaptic Ca²⁺ influx.

Author Summary

Synaptic transmission underlies information transfer among neurons in the brain. The probability that a synapse will release neurotransmitter in response to an action potential varies widely, even among synapses supplied by the same axon. The molecular mechanisms underlying this heterogeneity remain poorly understood. At the level of single synapses, release efficacy is determined largely by two factors: (i) the number of neurotransmitter-containing vesicles ready to be released, and (ii) by the fusion probabilities of these vesicles. By using novel imaging techniques at individual hippocampal presynaptic boutons in culture, we distinguish two independent sources of variability of release probability in small central synapses. First, we find differences in the number of releasable vesicles, and second, we find differences in the exocytosis probability of individual vesicles. To our knowledge, this is the first direct experimental demonstration that the fusion probability of release-ready vesicles is variable among synapses supplied by a single axon, and contributes roughly as much to the overall variability in release probability as does the number of release-ready vesicles.     

Modulation of glutamate mobility reveals the mechanism underlying slow-rising AMPAR EPSCs and the diffusion coefficient in the synaptic cleft

Fast- and slow-rising AMPA receptor-mediated EPSCs occur at central synapses. Fast-rising EPSCs are thought to be mediated by rapid local release of glutamate. However, two controversial mechanisms have been proposed to underlie slow-rising EPSCs: prolonged local release of transmitter via a fusion pore, and spillover of transmitter released rapidly from distant sites. We have investigated the mechanism underlying slow-rising EPSCs and the diffusion coefficient of glutamate in the synaptic cleft (Dglut) at cerebellar mossy fiber-granule cell synapses using a combination of diffusion modeling and patch-clamp recording. Simulations show that modulating Dglut has different effects on the peak amplitudes and time courses of EPSCs mediated by these two mechanisms. Slowing diffusion with the macromolecule dextran slowed slow-rising EPSCs and had little effect on their amplitude, indicating that glutamate spillover underlies these currents. Our results also suggest that under control conditions Dglut is approximately 3-fold lower than in free solution.     

SNARE Zippering and Synaptic Strength

Synapses vary widely in the probability of neurotransmitter release. We tested the hypothesis that the zippered state of the trans-SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) complex determines initial release probability. We tested this hypothesis at phasic and tonic synapses which differ by 100-1000-fold in neurotransmitter release probability. We injected, presynaptically, three Clostridial neurotoxins which bind and cleave at different sites on VAMP to determine whether these sites were occluded by the zippering of the SNARE complex or open to proteolytic attack. Under low stimulation conditions, the catalytic light-chain fragment of botulinum B (BoNT/B-LC) inhibited evoked release at both phasic and tonic synapses and cleaved VAMP; however, neither BoNT/D-LC nor tetanus neurotoxin (TeNT-LC) were effective in these conditions. The susceptibility of VAMP to only BoNT/B-LC indicated that SNARE complexes at both phasic and tonic synapses were partially zippered only at the N-terminal end to approximately the zero-layer with the C-terminal end exposed under resting state. Therefore, the existence of the same partially zippered state of the trans-SNARE complex at both phasic and tonic synapses indicates that release probability is not determined solely by the zippered state of the trans-SNARE complex at least to the zero-layer.     

Analysis of factors restricting the use of binomial statistics for studying transmitter release in neuromuscular junctions

Binomial parameters of transmitter secretion were calculated on the basis of analysis of synaptic potentials in the frog sartorius muscle. Negative values of the parameter p were found in some synapses. This happened most often in low Ca²⁺ concentrations and with low amplitude of miniature end-plate potentials. The results were interpreted in terms of a model assuming spatial heterogeneity of probability of transmitter quantum release at different release points. Simulation of transmitter secretion by computer showed that the appearance of negative values of the parameter p and incorrect estimates of n experimentally are connected with the form of distribution of probability of transmitter quantum release in the synapse and with the amplitude of miniature potentials.S. V. Kurashov Kazan' Medical Institute, Ministry of Health of the RSFSR. Translated from Neirofiziologiya, Vol. 16, No. 2, pp. 182–189, March–April, 1984.     

Functional heterogeneity at the frog sensory-motor synapse

This article compares four models of amplitude fluctuations in postsynaptic potentials. The convolution of two binomial distributions and the beta model proved the best fit with experimentally obtained data (as compared with the binomial model). The beta model is based on the assumption that the probability of quantal transmitter release is a random variable with a beta distribution. Numbers of quantal generators as estimated by the beta model were found to resemble numbers of morphological identifiable synaptic boutons. Findings from research using this model showed that the binomial parameter n may be interpreted as the number of transmitter release sites functioning with a probability in excess of 0.2. The findings obtained confirm the postulated functional diversity of release sites at interneuronal synapses.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 21, No. 6, pp. 780–788, November–December, 1989.     

Efficacy of thalamocortical and intracortical synaptic connections: quanta, innervation, and reliability.

Thalamocortical (TC) synapses carry information into the neocortex, but they are far outnumbered by excitatory intracortical (IC) synapses. We measured the synaptic properties that determine the efficacy of TC and IC axons converging onto spiny neurons of layer 4 in the mouse somatosensory cortex. Quantal events from TC and IC synapses were indistinguishable. However, TC axons had, on average, about 3 times more release sites than IC axons, and the mean release probability at TC synapses was about 1.5 times higher than that at IC synapses. Differences of innervation ratio and release probability make the average TC connection several times more effective than the average IC connection, and may allow small numbers of TC axons to dominate the activity of cortical layer 4 cells during sensory inflow.     

Local dendritic activity sets release probability at hippocampal synapses

The arrival of an action potential at a synapse triggers neurotransmitter release with a limited probability, p(r). Although p(r) is a fundamental parameter in defining synaptic efficacy, it is not uniform across all synapses, and the mechanisms by which a given synapse sets its basal release probability are unknown. By measuring p(r) at single presynaptic terminals in connected pairs of hippocampal neurons, we show that neighboring synapses on the same dendritic branch have very similar release probabilities, and p(r) is negatively correlated with the number of synapses on the branch. Increasing dendritic depolarization elicits a homeostatic decrease in p(r), and equalizing activity in the dendrite significantly reduces its variability. Our results indicate that local dendritic activity is the major determinant of basal release probability, and we suggest that this feedback regulation might be required to maintain synapses in their operational range.     

Presynaptic calcium and control of vesicle fusion

Vesicle fusion and transmitter release at synapses is driven by a highly localized Ca2+ signal that rapidly builds up around open Ca2+-channels at and near presynaptic active zones. It has been difficult to estimate the amplitude and the kinetics of this 'microdomain' signal by direct Ca2+-imaging approaches. Recently, Ca2+ uncaging at large CNS synapses, among them the calyx of Held, has shown that the intrinsic cooperativity of Ca2+ in inducing vesicle fusion is high, with 4-5 Ca2+ ions needed to trigger vesicle fusion. Given the Ca2+-sensitivity of vesicle fusion as determined by Ca2+-uncaging, it was found that a surprisingly small (10-25 microM) and brief (<1 ms) local Ca2+ signal is sufficient to achieve the amount, and the kinetics of the physiological transmitter release. The high cooperativity of Ca2+ in inducing vesicle fusion and the non-saturation of the Ca2+-sensor for vesicle fusion renders small changes of the local Ca2+-signal highly effective in changing the release probability; an insight that is important for our understanding of short-term modulation of synaptic strength.     

Exogenous synapsin I promotes functional maturation of developing neuromuscular synapses.

We have investigated the possible role of synapsin I, a nerve terminal-specific protein, in the maturation of neuromuscular synapses in Xenopus cell cultures. Purified synapsin I was loaded into embryonic spinal neurons by injection of the protein into one of the early blastomeres of a Xenopus embryo. At synapses made by synapsin I-loaded neurons, spontaneous synaptic currents occurred with higher frequency and amplitude, and the amplitude exhibited an earlier appearance of a bell-shaped distribution. These characteristics are indicative of more mature quantal secretion. Impulse-evoked synaptic currents also showed a significant increase in amplitude. Using cell manipulation techniques, enhanced transmitter release from synapsin I-loaded neurons was shown to occur at the onset of synaptogenesis, suggesting a presynaptic developmental action of synapsin I prior to synaptic contact. Taken together, these results suggest that endogenous synapsin I may participate in the functional maturation of synapses.     

Selective depression of low-release probability excitatory synapses by sodium channel blockers

Sodium channels (NaChs) play a central role in action potential generation and are uniquely poised to influence the efficacy of transmitter release. We evaluated the effect of partial NaCh blockade on two aspects of synaptic efficacy First, we evaluated whether NaCh blockade accounts for the ability of certain drugs to selectively depress glutamate release. Second, we evaluated the contribution of NaChs to intraneuronal variability in glutamate release probability (p(r)). The antiglutamate drug riluzole nearly completely depresses glutamate excitatory postsynaptic currents (EPSCs) at concentrations that barely affect GABAergic inhibitory postsynaptic currents (IPSCs). NaCh inhibition explains the selective depression. Unlike other presynaptic depressants, partial NaCh blockade increases paired-pulse EPSC depression. This result is explained by selective depression of low-p(r) synapses. We conclude that local variations in the action potential contribute to p(r) variability among excitatory synapses.     

Quantum Characteristics of Neurotransmitter Release in GABA-ergic Synapses of Cultured Neurons of the Rat Spinal Cord

Using the whole-cell patch-clamp technique and stimulation of a single presynaptic terminal, we studied peculiarities of GABA release in inhibitory synapses of cultured neurons of the rat spinal cord. Analyzing the amplitude distributions of evoked inhibitory postsynaptic currents, we estimated the main quantum parameters of transmitter release. It was demonstrated that the minimum transmitter release in GABA-ergic synapses of spinal neurons cultured 9 to 11 days is multiquantum (packets containing at least 2 or 3 quanta). The distribution of the number of released quanta sufficiently agreed with that theoretically calculated according to the Poisson law. It is hypothesized that the minimum simultaneous two (three-)-quantum release of GABA in synapses of spinal neurons can be related to synchronous involvement of two closely adjacent excited terminals, each of which possesses one active zone, or of one terminal with two active zones.     

From the stochasticity of molecular processes to the variability of synaptic transmission

The variability of the postsynaptic response following a single action potential arises from two sources: the neurotransmitter release is probabilistic, and the postsynaptic response to neurotransmitter release has variable timing and amplitude. At individual synapses, the number of molecules of a given type that are involved in these processes is small enough that the stochastic (random) properties of molecular events cannot be neglected. How the stochasticity of molecular processes contributes to the variability of synaptic transmission, its sensitivity and its robustness to molecular fluctuations has important implications for our understanding of the mechanistic basis of synaptic transmission and of synaptic plasticity.     

Impaired synapse function during postnatal development in the absence of CALEB, an EGF-like protein processed by neuronal activity

In an attempt to characterize the molecular components by which electric activity influences the development of synapses, we searched for cell surface proteins modulated by calcium influx and glutamate receptor activity. Here, we report that neuronal depolarization facilitates the conversion of CALEB, which results in a truncated transmembrane form with an exposed EGF domain. To characterize the role of CALEB in synapse development, synaptic features were investigated in slices of the colliculus superior from CALEB-deficient mice. In the absence of CALEB, the number of synapses and their morphological characteristics remained unchanged. However, in CALEB-deficient mice, synapses displayed higher paired-pulse ratios, less depression during prolonged repetitive activation, a lower rate of spontaneous postsynaptic currents, and a lower release probability at early but not mature postnatal stages. Our findings indicate that CALEB provides a molecular basis for maintaining normal release probability at early developmental stages.     

Pre-synaptic and post-synaptic factors influencing the synchronism in the transmitter secretion and the amplitude and temporal parameters of a postsynaptic response in the neuromuscular junction: a model study

Using a model of the frog neuromuscular junction, we studied the influence of pre-synaptic and post-synaptic factors on the amplitude and temporal parameters of end-plate currents (EPC). A nerve terminal (NT) was supposed to include linearly distributed active zones (AZ) that are able to release a transmitter quantum with a definite temporal distribution of the release probability (AZ DRP) after successive activation of these zones by a spreading action potential (AP). An increase in the length of a terminal, distance between AZ, and time constant of the DRP decline, or a decrease in the AP conduction velocity along the NT determines a decrease in the EPS amplitude and prolongation of its rising phase. These effects result from an increased asynchronism in the transmitter release. An expansion of the temporal parameters of minature EPC leads to an increase in the EPC amplitude, i.e., provides minimization of its loss. Various EPC models are compared, and contributions of the examined pre-synaptic and post-synaptic factors in modifications of the amplitude and temporal EPC parameters are evaluated.Neirofiziologiya/Neurophysiology, Vol. 27, No. 3, pp. 163–179, May–June, 1995.