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Counting quanta: Direct measurements of transmitter release at a central synapse
Abstract
Contradictory hypotheses regarding the nature of synaptic transmission in the CNS have arisen from indirect methods of quantal analysis. In this study, we directly count the quanta released following nerve stimulation to examine synaptic transmission at a fast glutamatergic synapse in the mammalian auditory brainstem. Our results demonstrate the relationship between spontaneous and nerve-evoked synaptic events, indicate that asynchronous transmitter release governs the time course of evoked transmission, and show that the stochastic quantal release process, as originally proposed at the neuromuscular junction, is highly conserved at this central synapse.
... This is the smallest unit of neurotransmission, which is known as a "quantum" (Fatt and Katz, 1952;Del Castillo and Katz, 1954). A synaptic bouton may contain multiple active zones or release sites (Korn et al., 1987;Korn and Faber, 1991;Maass and Zador, 1999), each of which are capable of probabilistically secreting a single quantum of neurotransmitter in response to an action potential (Isaacson and Walmsley, 1995;Korn and Faber, 1998;Maass and Zador, 1999). Although the release of multiple quanta has been documented many times (Tong and Jahr, 1994;Auger et al., 1998;Oertner et al., 2002;Lisman, 2009;Jensen et al., 2019), evoked responses are typically assumed to be due to the linear summation of single quanta released across multiple sites. ...
... Through their seminal recordings of the amphibian neuromuscular junction, it was observed that evoked potentials in a muscle fiber randomly fluctuate between integer multiples of the spontaneous miniature potential or basic quantal unit, q (Del Castillo and Katz, 1954;Faber, 1991, 1998). This finding has since been replicated at other synapse types (Redman and Walmsley, 1983;Korn et al., 1987;Isaacson and Walmsley, 1995). Quantal analysis relies on the pattern of fluctuations in evoked responses to calculate presynaptic factors influencing neurotransmitter release and postsynaptic factors influencing synaptic responsiveness, thereby allowing the locus of plasticity expression to be determined (Malinow and Tsien, 1990;Redman, 1990;Isaac et al., 1996;Reid and Clements, 1999;Enoki et al., 2009). ...
... However, some debate still remains surrounding this definition (Scheuss and Neher, 2001). For example, n has alternatively been proposed to represent the maximum number of quanta available for evoked release at a given synapse (Redman, 1990;Isaacson and Walmsley, 1995), i.e., the number of docked vesicles or the size of the readily releasable pool (Kaeser and Regehr, 2017). Here, we are adhering to the more common view that n corresponds to the number of release sites. ...
Long-term synaptic plasticity is widely believed to underlie learning and memory in the brain. Whether plasticity is primarily expressed pre- or postsynaptically has been the subject of considerable debate for many decades. More recently, it is generally agreed that the locus of plasticity depends on a number of factors, such as developmental stage, induction protocol, and synapse type. Since presynaptic expression alters not just the gain but also the short-term dynamics of a synapse, whereas postsynaptic expression only modifies the gain, the locus has fundamental implications for circuits dynamics and computations in the brain. It therefore remains crucial for our understanding of neuronal circuits to know the locus of expression of long-term plasticity. One classical method for elucidating whether plasticity is pre- or postsynaptically expressed is based on analysis of the coefficient of variation (CV), which serves as a measure of noise levels of synaptic neurotransmission. Here, we provide a practical guide to using CV analysis for the purposes of exploring the locus of expression of long-term plasticity, primarily aimed at beginners in the field. We provide relatively simple intuitive background to an otherwise theoretically complex approach as well as simple mathematical derivations for key parametric relationships. We list important pitfalls of the method, accompanied by accessible computer simulations to better illustrate the problems (downloadable from GitHub), and we provide straightforward solutions for these issues.
... Four parameters are thought to be particularly important in determining trial-to-trial EPSC variability: the time course and probability of quantal release, the number of release sites, and the quantal size, which is the postsynaptic response to a single vesicle (Katz, 1969;Sun et al., 2002). Previous studies have quantified the determinants of the mean EPSC waveform by using deconvolution methods to estimate the time course of vesicular release (Diamond and Jahr, 1995;Isaacson and Walmsley, 1995;Borst and Sakmann, 1996;Geiger et al., 1997;Schneggenburger and Neher, 2000). However, quantifying the origins and assessing the impact of EPSC variability has been complicated by uncertainty about whether multiple vesicles [multivesicular release (Wadiche and Jahr, 2001;Oertner et al., 2002)] are released per synaptic contact per action potential (AP), because this can cause postsynaptic saturation [full receptor occupancy (Foster et al., 2002;Harrison and Jahr, 2003)] and thus introduce nonlinear quantal summation. ...
... We used competitive antagonist techniques to probe the linearity of quantal summation at MF connections (Wadiche and Jahr, 2001;Silver et al., 2003). By combining multiple-probability fluctuation analysis (MPFA) (Silver et al., 1998;Silver, 2003) and deconvolution (Diamond and Jahr, 1995;Isaacson and Walmsley, 1995;Borst and Sakmann, 1996;Geiger et al., 1997), we determined the time course and probability of vesicular release, the number of release sites, the quantal size, and the properties of spillover currents at each connection. By constructing stochastic models of transmission for individual MG-GC connections, we show that rapid vesicular release, quantal variability, and spillover currents introduce considerable EPSP variability and influence precision and reliability of MF-GC synaptic signaling. ...
... If quantal events sum linearly, the mean EPSC waveform is the convolution of the quantal current waveform (impulse response in linear systems analysis) and the vesicular release time course. Under these conditions, deconvolution analysis (Cohen et al., 1981) can therefore be used to calculate the time course of quantal release from the mean EPSC and the quantal EPSC waveforms ( Van der Kloot, 1988;Borst et al., 1995;Diamond and Jahr, 1995;Isaacson and Walmsley, 1995;Chen and Regehr, 1999;Schneggenburger and Neher, 2000) (but, for a nonlinear approach, see Neher and Sakaba, 2001). Before applying deconvolution to the multisite connections, we first tested whether it could be used to estimate reliably the time course of release under our experimental conditions. ...
... The endbulb of Held contains hundreds of synaptic specializations and forms a powerful glutamatergic connection with the soma of bushy cells in the anteroventral cochlear nucleus (Lenn & Reese, 1966;Cant & Morest, 1979;Ryugo & Fekete, 1982;Liberman, 1991;Ryugo & Sento, 1991). Our previous electrophysiological studies have demonstrated that nerve stimulation at the endbulb-bushy cell connection results in a large, brief excitatory postsynaptic current (EPSC), which rapidly depresses on repetitive stimulation (Isaacson & Walmsley, 1995a,b, 1996Bellingham & Walmsley, 1999). We have used the general kinase inhibitor H7 and phorbol ester activators and inhibitors of protein kinase C to investigate which aspects of transmission may be modulated by phosphorylation at this synapse. ...
... Parasaggital slices (150 ìm) were made of the anterior ventral cochlear nucleus (AVCN) of 10-to 12-day-old Wistar rats, following decapitation in accordance with local ethical guidelines (Isaacson & Walmsley, 1995a,b, 1996Bellingham & Walmsley, 1999). Wholecell patch electrode recordings were performed at room temperature (22-25°C) from bushy cells visualized in thin slices using infra-red differential interference contrast (DIC) optics. ...
... Trains of stimuli consisted of 10 pulses at 100 Hz, 1 min apart. The evoked EPSCs were identified as endbulb AMPA receptor-mediated currents by their amplitude, fast kinetics and all-or-none response to graded stimulation intensities (Isaacson & Walmsley, 1995a). The synaptic currents were recorded and filtered at 10 kHz with an Axopatch 200B amplifier (Axon Instruments) before being digitized at 20 kHz. ...
1The role of phosphorylation in synaptic transmission was investigated at a large glutamatergic terminal, the endbulb of Held, on bushy cells in the rat anteroventral cochlear nucleus (AVCN).2Whole-cell recordings of excitatory postsynaptic currents (EPSCs) were used to examine the effects of kinase inhibitors and activators on low-frequency (baseline) evoked release, spontaneous release, paired-pulse facilitation (PPF) or depression (PPD), repetitive stimuli and recovery from depression.3Application of the kinase inhibitor H7 (100 μm) reduced low-frequency evoked EPSC amplitude (by 15 %) and simultaneously increased PPF (or reduced PPD), with no significant change in other aspects of transmission. H7 did not affect the amplitude or frequency of spontaneous miniature EPSCs.4Phorbol esters increased EPSC amplitude (by 50 %) with a concomitant decrease in PPF (or increase in PPD), and reduced the final EPSC amplitude during repetitive stimuli. The effect of phorbol esters was due exclusively to protein kinase C (PKC) activation, as the specific PKC inhibitor bis-indolylmaleimide (Bis) completely blocked the potentiating effect of phorbol esters on EPSC amplitude.5Significantly, phorbol esters did not increase the evoked EPSC amplitude at connections in which release was maximized using high extracellular calcium concentrations (4-6 mm).6Phorbol esters increased the frequency of spontaneous miniature EPSCs in physiological calcium (by 275 %), and in high extracellular calcium (by 210 %) when phorbol esters did not increase the evoked EPSC amplitude.7Our results are most consistent with the actions of H7 to decrease low-frequency release probability and phorbol esters to increase low-frequency release probability at the endbulb-bushy cell synaptic connection in the AVCN. The effects of H7 and phorbol esters on paired-pulse responses and tetanic depression appear to be largely consequential to these changes in low-frequency release probability.
... The discrepancy between evoked and miniature EPSCs might not be too surprising, as eEPSCs represent summations of quantal events where some part of the variance is explained by differences in timing between quantal events. Thus, even slightly asynchronous summation of quantal events likely impacts the decay phase of the eEPSCs in addition to the deactivation kinetics of AMPAR currents (Diamond and Jahr, 1995;Isaacson and Walmsley, 1995). Additionally (partial) saturation of iGluSnFR copies at higher glutamate concentrations following evoked release may reduce the relative effect of iGluSnFR on glutamate dynamics within the cleft. ...
Introduction
Recently developed fluorescent neurotransmitter indicators have enabled direct measurements of neurotransmitter in the synaptic cleft. Precise optical measurements of neurotransmitter release may be used to make inferences about presynaptic function independent of electrophysiological measurements.
Methods
Here, we express iGluSnFR, a genetically encoded glutamate reporter in mouse spiral ganglion neurons to compare electrophysiological and optical readouts of presynaptic function and short-term synaptic plasticity at the endbulb of Held synapse.
Results
We show iGluSnFR robustly and approximately linearly reports glutamate release from the endbulb of Held during synaptic transmission and allows assessment of short-term plasticity during high-frequency train stimuli. Furthermore, we show that iGluSnFR expression slightly alters the time course of spontaneous postsynaptic currents, but is unlikely to impact measurements of evoked synchronous release of many synaptic vesicles.
Discussion
We conclude that monitoring glutamate with optical sensors at fast and large central synapses like the endbulb of Held is feasible and allows robust quantification of some, but not all aspects of glutamate release.
... where M and CV 2 are the mean and the coefficients of variation of the 1st eEPSCs in pairs under paired-pulse stimulation; q and cv 2 are the mean and the coefficients of variation of miniature synaptic currents (mEPSCs). The glutamatergic mEPSCs were measured in extracellular low Ca 2+ /high Mg 2+ solution (Isaacson and Walmsley, 1995) containing 0,5 mM Ca 2+ , 10 mM Mg 2+ and 0,25 µM TTX (Fedulova et al., 1999). ...
CITATION Shypshyna M, Kolesnyk O, Fedulova S and Veselovsky N (2023) Insulin modulates the paired-pulse plasticity at glutamatergic synapses of hippocampal neurons under hypoinsulinemia. Hypoinsulinemia is a pathological consequence of diabetes mellitus that can cause a number of complications of the central and peripheral nervous system. Dysfunction of signaling cascades of insulin receptors under insulin deficiency can contribute to the development of cognitive disorders associated with impaired synaptic plasticity properties. Earlier we have shown that hypoinsulinemia causes a shift of short-term plasticity in glutamatergic hippocampal synapses from facilitation to depression and apparently involves mechanisms of glutamate release probability reduction. Here we used the whole cell patch-clamp recording of evoked glutamatergic excitatory postsynaptic currents (eEPSCs) and the method of local extracellular electrical stimulation of a single presynaptic axon to investigate the effect of insulin (100 nM) on the paired-pulse plasticity at glutamatergic synapses of cultured hippocampal neurons under hypoinsulinemia. Our data indicate that under normoinsulinemia additional insulin enhances the paired-pulse facilitation (PPF) of eEPSCs in hippocampal neurons by stimulating the glutamate release in their synapses. Under hypoinsulinemia, insulin did not have a significant effect on the parameters of paired-pulse plasticity on neurons of PPF subgroup, which may indicate the development of insulin resistance, while the effect of insulin on PPD neurons indicates its ability to recover the form normoinsulinemia, including the increasing probability of plasticity to the control level in of glutamate release in their synapses.
... TBI altered several electrophysiological parameters, including sEPSC amplitude, input resistance, FI gain, spike threshold, and burst size. TBI/Veh neurons exhibited increased sEPSC amplitude (but not frequency) compared with Sham/Veh neurons, suggesting increased synaptic excitability following TBI (Isaacson and Walmsley 1995). EC degradation inhibitors JZL184, JZL195, and URB97, but not MJN110, significantly attenuated the TBI-induced increase in sEPSC amplitude. ...
Our previous work showed that lateral fluid percussion injury to the sensorimotor cortex (SMC) of anesthetized rats increased neuronal synaptic hyperexcitability in layer 5 (L5) neurons in ex vivo brain slices 10 days post-injury. Furthermore, endocannabinoid (EC) degradation inhibition via intraperitoneal JZL184 injection 30 min post-injury attenuated synaptic hyperexcitability. This study tested the hypothesis that traumatic brain injury (TBI) induces synaptic and intrinsic neuronal alterations of L5 SMC pyramidal neurons and that these alterations are significantly attenuated by in vivopost-TBI treatment with EC degradation inhibitors. We tested the effects of systemically administered EC degradation enzyme inhibitors (JZL184, MJN110, URB597, or JZL195) with differential selectivity for fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) on electrophysiological parameters in SMC neurons of TBI and sham-treated rats 10 days post-TBI. We recorded intrinsic neuronal properties, including resting membrane voltage, input resistance, spike threshold, spiking responses to current input, voltage "SAG" (rebound response to hyperpolarization-activated inward current), and burst firing. We also measured the frequency and amplitude of spontaneous excitatory postsynaptic currents. We then used the aggregate parameter sets (intrinsic + synaptic properties) to apply a machine learning classification algorithm to quantitatively compare neural population responses from each experimental group. Collectively, our electrophysiological and computational results indicate that Sham neurons are the most distinguishable from TBI neurons. Administration of EC degradation inhibitors post-TBI exerted varying degrees of rescue, approximating the neuronal phenotype of Sham neurons, with neurons from TBI/JZL195 (a dual MAGL/FAAH inhibitor) being most similar to neurons from Sham rats.
... In quantal analysis, it is generally assumed that quanta summate linearly (Reid and Clements, 1999;Clements and Silver, 2000;Humeau et al., 2007), such that the peak of an evoked EPSC represents the number of released quanta times the mean amplitude of the synaptic response evoked by the release of one quantum. Because vesicle release is not perfectly synchronous, the contribution of individual quanta to the peak amplitude of the evoked EPSC will be somewhat smaller than expected (Isaacson and Walmsley, 1995;Bellingham et al., 1998), leading to an underestimate of mean quantal size. Furthermore, dendritic filtering alters the amplitude and time course of synaptic responses that originate at a distance from the recording site (Bekkers and Stevens, 1990), making the contribution to the evoked EPSC of quanta released further from the recording site smaller. ...
Background:
The strength of synaptic transmission onto a neuron depends on the number of functional vesicle release sites (N), the probability of vesicle release (Pr), and the quantal size (Q). Statistical tools based on the quantal model of synaptic transmission can be used to acquire information on which of these parameters is the source of plasticity. However, quantal analysis depends on assumptions that may not be met at central synapses.
New method:
We examined the merit of quantal analysis to extract the mechanisms underlying synaptic plasticity by applying binomial statistics on the variance in amplitude of postsynaptic currents evoked at Schaffer collateral-CA1 (Sc-CA1) synapses in mouse hippocampal slices. We extend this analysis by combining the conventional inverse square of the coefficient of variation (1/CV2) with the variance-to-mean ratio (VMR).
Results:
This method can be used to assess the relative, but not absolute, contribution of N, Pr and Q to synaptic plasticity. The changes in 1/CV2 and VMR values correctly reflect experimental modifications of N, Pr and Q at Sc-CA1 synapses.
Comparison with existing methods:
While the 1/CV2 depends on N and Pr, but is independent of Q, the VMR is dependent on Pr and Q, but not on N. Combining both allows for a rapid assessment of the mechanism underlying synaptic plasticity without the need for additional electrophysiological experiments.
Conclusion:
Combining the 1/CV2 with the VMR allows for a reliable prediction of the relative contribution of changes in N, Pr and Q to synaptic plasticity.
... The charge corresponding to the first 25 ms of the eIPSC was used as an estimation of synchronous release to normalize asynchronous release. The estimation of the synchronous charge in this time frame (25 ms) avoids any possible contamination from the asynchronous release component since a correlation exists between the extent of asynchronous release and the decay phase of the eIPSC (Isaacson and Walmsley 1995;Iremonger and Bains 2016). The asynchronous release was measured as the charge in the 10 s following the train (2s@40Hz) for the extracellular stimulation experiments and was normalized, for each individual neuron, to the first 25 ms charge of the mean eIPSC average of 10 eIPSCs recorded at the same stimulation intensity before applying the train. ...
Neurotransmitters can be released either synchronously or asynchronously with respect to action potential timing. Synapsins (Syns) are a family of synaptic vesicle (SV) phosphoproteins that assist gamma-aminobutyric acid (GABA) release and allow a physiological excitation/inhibition balance. Consistently, deletion of either or both Syn1 and Syn2 genes is epileptogenic. In this work, we have characterized the effect of SynI knockout (KO) in the regulation of GABA release dynamics. Using patch-clamp recordings in hippocampal slices, we demonstrate that the lack of SynI impairs synchronous GABA release via a reduction of the readily releasable SVs and, in parallel, increases asynchronous GABA release. The effects of SynI deletion on synchronous GABA release were occluded by ω-AgatoxinIVA, indicating the involvement of P/Q-type Ca2+channel-expressing neurons. Using in situ hybridization, we show that SynI is more expressed in parvalbumin (PV) interneurons, characterized by synchronous release, than in cholecystokinin or SOM interneurons, characterized by a more asynchronous release. Optogenetic activation of PV and SOM interneurons revealed a specific reduction of synchronous release in PV/SynIKO interneurons associated with an increased asynchronous release in SOM/SynIKO interneurons. The results demonstrate that SynI is differentially expressed in interneuron subpopulations, where it boosts synchronous and limits asynchronous GABA release.
... Endbulbs are synapses modified to be able to release a big number of vesicles simultaneously. For this, each endbulb contains 100 or more active zones capable to release several vesicles after a single action potential (Nicol and Walmsley, 2002) producing a large synaptic current (up to 10 nA; Isaacson and Walmsley, 1995) necessary to produce a post-synaptic potential always above the action potential threshold against the low membrane input resistance of the bushy cell. These currents are mediated by AMPA receptors GluA3 and GluA4 in the flop configuration, producing currents with a fast kinetics and a big unitary conductance (Geiger et al., 1995;Gardner et al., 1999Gardner et al., , 2001Petralia et al., 2000), being the GluA3 the most abundant (Rubio et al., 2017). ...
The auditory part of the brainstem is composed of several nuclei specialized in the computation of the different spectral and temporal features of the sound before it reaches the higher auditory regions. There are a high diversity of neuronal types in these nuclei, many with remarkable electrophysiological and synaptic properties unique to these structures. This diversity reflects specializations necessary to process the different auditory signals in order to extract precisely the acoustic information necessary for the auditory perception by the animal. Low threshold Kv1 channels and HCN channels are expressed in neurons that use timing clues for auditory processing, like bushy and octopus cells, in order to restrict action potential firing and reduce input resistance and membrane time constant. Kv3 channels allow principal neurons of the MNTB and pyramidal DCN neurons to fire fast trains of action potentials. Calcium channels on cartwheel DCN neurons produce complex spikes characteristic of these neurons. Calyceal synapses compensate the low input resistance of bushy and principal neurons of the MNTB by releasing hundreds of glutamate vesicles resulting in large EPSCs acting in fast ionotropic glutamate receptors, in order to reduce temporal summation of synaptic potentials, allowing more precise correspondence of pre- and post-synaptic potentials, and phase-locking. Pre-synaptic calyceal sodium channels have fast recovery from inactivation allowing extremely fast trains of action potential firing, and persistent sodium channels produce spontaneous activity of fusiform neurons at rest, which expands the dynamic range of these neurons. The unique combinations of different ion channels, ionotropic receptors and synaptic structures create a unique functional diversity of neurons extremely adapted to their complex functions in the auditory processing.
... Apart from a scaling factor according to release probability or to previous synaptic activity, synaptic latencies have classically been considered constant 7,8 . Accordingly, only modest changes of latency distributions have been reported in the calyx of Held 6,9 and endbulb of Held 10 during train stimulations. ...
It is often assumed that only stably docked synaptic vesicles can fuse following presynaptic action potential stimulation. However, during action potential trains docking sites are increasingly depleted, raising the question of the source of synaptic vesicles during sustained release. We have recently developed methods to reliably measure release latencies during high frequency trains at single synapses between parallel fibers and molecular layer interneurons. The latency distribution exhibits a single fast component at train onset but contains both a fast and a slow component later in the train. The contribution of the slow component increases with stimulation frequency and with release probability and decreases when blocking the docking step with latrunculin. These results suggest that the slow component reflects sequential docking and release in immediate succession. The transition from fast to slow component, as well as a later transition to asynchronous release, appear as successive adaptations of the synapse to maintain fidelity at the expense of time accuracy.
... This is an interesting hypothesis, which needs the following considerations: 1) to what degree a ribbon can act as a diffusional barrier for Ca 2ϩ needs to be further addressed (Ca 2ϩ imaging experiments show no exclusion of Ca 2ϩ from the ribbon volume, at least not in mammalian IHCs upon longer stimulation; Refs. 110,262,402), and 2) at conventional synapses (81,157) temporal jitter in the process of exocytosis occurs downstream of Ca 2ϩ entry and binding to molecular sensors. Conflicting observations made at mammalian versus frog hair cell ribbon synapse as well as retinal ribbon synapses raise a general, and important, question of whether there is a prototypical ribbon synapse. ...
Calcium influx through voltage-gated Ca (Ca V) channels is the first step in synaptic transmission. This review concerns Ca V channels at ribbon synapses in primary sense organs and their specialization for efficient coding of stimuli in the physical environment. Specifically, we describe molecular, biochemical, and biophysical properties of the Ca V channels in sensory receptor cells of the retina, cochlea, and vestibular apparatus, and we consider how such properties might change over the course of development and contribute to synaptic plasticity. We pay particular attention to factors affecting the spatial arrangement of Ca V channels at presynaptic, ribbon-type active zones, because the spatial relationship between Ca V channels and release sites has been shown to affect synapse function critically in a number of systems. Finally, we review identified synaptopathies affecting sensory systems and arising from dysfunction of L-type, Ca V 1.3, and Ca V 1.4 channels or their protein modulatory elements.
... Classified as excitatory or inhibitory, chemical synapses can generate various modulatory signals. A number of controversies regarding the nature of synaptic transmission (Isaacson and Walmsley, 1995), in addition to a dynamic shift between inhibition and excitation, have strongly limited the applicability of this principle (Heiss et al., 2008;Zuo et al., 1999). The inhibitory neurotransmitter GABAA (Luscher et al., 2011) can become excitatory in case of an increased Cl¯ concentration inside the cell (Taketo and Yoshioka, 2000). ...
... This jitter is illustrated for all of the individual events across all trials in Fig. 4F. The variable release latency for each site is drawn from a log-normal distribution according to the measurement of individual release events under conditions of low release probability (Isaacson and Walmsley, 1995). In addition, a time-dependent shift in the mean latency (visible in Fig. 4F) can be included in the model, to mimic a similar shift seen experimentally. ...
Models of the auditory brainstem have been an invaluable tool for testing hypotheses about auditory information processing and for highlighting the most important gaps in the experimental literature. Due to the complexity of the auditory brainstem, and indeed most brain circuits, the dynamic behavior of the system may be difficult to predict without a detailed, biologically realistic computational model. Despite the sensitivity of models to their exact construction and parameters, most prior models of the cochlear nucleus have incorporated only a small subset of the known biological properties. This confounds the interpretation of modelling results and also limits the potential future uses of these models, which require a large effort to develop. To address these issues, we have developed a general purpose, biophysically detailed model of the cochlear nucleus for use both in testing hypotheses about cochlear nucleus function and also as an input to models of downstream auditory nuclei. The model implements conductance-based Hodgkin-Huxley representations of cells using a Python-based interface to the NEURON simulator. Our model incorporates most of the quantitatively characterized intrinsic cell properties, synaptic properties, and connectivity available in the literature, and also aims to reproduce the known response properties of the canonical cochlear nucleus cell types. Although we currently lack the empirical data to completely constrain this model, our intent is for the model to continue to incorporate new experimental results as they become available.
... In the quantal theory of neurotransmitter release, a change in amplitude of miniature synaptic currents is considered to reflect an alteration in post-synaptic neurotransmitter receptor activity, while a change in the frequency of miniature synaptic currents indicates a change in action potential-independent pre-synaptic release probability (Redman, 1990;Stevens, 1993;Isaacson and Walmsley, 1995). Because capsaicin increased mEPSC frequency without altering mESPC amplitude, we interpret this as an enhancement of action potential-independent release probability. ...
We investigated whether capsaicin modulated synaptic transmission to hypoglossal motor neurons (HMNs) by acting on transient receptor potential vanilloid type 1 (TRPV1) receptors. Using whole-cell patch clamp recording from neonatal rat HMNs, we found that capsaicin increased spontaneous excitatory post-synaptic current (sEPSC) frequency and amplitude. Interestingly, the only effect of capsaicin on spontaneous inhibitory post-synaptic currents (sIPSCs) was a significant decrease in sIPSC amplitude without altering frequency, indicating a post-synaptic mechanism of action. The frequency of miniature excitatory post-synaptic currents (mEPSCs), recorded in the presence of tetrodotoxin (TTX), was also increased by capsaicin, but capsaicin did not alter mEPSC amplitude, consistent with a pre-synaptic mechanism of action. A negative shift in membrane current (Iholding) was elicited by capsaicin under both recording conditions. The effect of capsaicin on excitatory synaptic transmission remained unchanged in the presence of the TRPV1 antagonists, capsazepine or SB366791, suggesting that capsaicin acts to modulate EPSCs via a mechanism which does not require TRPV1 activation. Capsaicin, however, did not alter evoked excitatory post-synaptic currents (eEPSCs) or the paired-pulse ratio (PPR) of eEPSCs. Repetitive action potential (AP) firing in HMNs was also unaltered by capsaicin, indicating that capsaicin does not change HMN intrinsic excitability. We have demonstrated that capsaicin modulates glutamatergic excitatory, as well as glycinergic inhibitory, synaptic transmission in HMNs by differing pre- and post-synaptic mechanisms. These results expand our understanding regarding the extent to which capsaicin can modulate synaptic transmission to central neurons.
... A higher efficiency of release may be achieved by an increased Ca 2+ sensitivity which will result in a higher P v . When Ca 2+ influx into the presynaptic terminal is reduced, this is accompanied by increased fluctuations in both PSC amplitude and latency (Isaacson & Walmsley, 1995). ...
Key points:
Auditory brainstem neurons involved in sound source localization are equipped with several morphological and molecular features that enable them to compute interaural level and time differences. As sound source localization works continually, synaptic transmission between these neurons should be reliable and temporally precise, even during sustained periods of high-frequency activity. Using patch-clamp recordings in acute brain slices, we compared synaptic reliability and temporal precision in the seconds-minute range between auditory and two types of hippocampal synapses; the latter are less confronted with temporally precise high-frequency transmission than the auditory ones. We found striking differences in synaptic properties (e.g. continually high quantal content) that allow auditory synapses to reliably release vesicles at much higher rate than their hippocampal counterparts. Thus, they are indefatigable and also in a position to transfer information with exquisite temporal precision and their performance appears to be supported by very efficient replenishment mechanisms.
Abstract:
At early stations of the auditory pathway, information is encoded by precise signal timing and rate. Auditory synapses must maintain the relative timing of events with submillisecond precision even during sustained and high-frequency stimulation. In non-auditory brain regions, e.g. telencephalic ones, synapses are activated at considerably lower frequencies. Central to understanding the heterogeneity of synaptic systems is the elucidation of the physical, chemical and biological factors that determine synapse performance. In this study, we used slice recordings from three synapse types in the mouse auditory brainstem and hippocampus. Whereas the auditory brainstem nuclei experience high-frequency activity in vivo, the hippocampal circuits are activated at much lower frequencies. We challenged the synapses with sustained high-frequency stimulation (up to 200 Hz for 60 s) and found significant performance differences. Our results show that auditory brainstem synapses differ considerably from their hippocampal counterparts in several aspects, namely resistance to synaptic fatigue, low failure rate and exquisite temporal precision. Their high-fidelity performance supports the functional demands and appears to be due to the large size of the readily releasable pool and a high release probability, which together result in a high quantal content. In conjunction with very efficient vesicle replenishment mechanisms, these properties provide extremely rapid and temporally precise signalling required for neuronal communication at early stations of the auditory system, even during sustained activation in the minute range.
... Concentrations of glutamate released into the synaptic cleft have been shown to be equivalent at either receptor type in the synapse (Clements, 1996). Differences in the glutamate concentration possibly indicated by vesicle size at LOT and ASSN synapses (Bekkers et al., 1990, Schikorski and Stevens, 1999, Andrasfalvy and Magee, 2001 or glutamate time course induced by asynchronous release of transmitter may contribute to functional differences in receptor currents (Diamond andJahr, 1995, Isaacson andWalmsley, 1995). AMPAR at LOT synapses are developmentally recruited to the synapses by NMDAR activation in a manner that is not observed at ASSN synapses (Franks and Isaacson, 2005). ...
The influence of anatomical, developmental and degenerative factors on the function of synaptically expressed ionotropic glutamate receptors was assessed in murine models. Recordings were obtained in whole cell configuration from principal cells in layer II of acutely prepared slices of anterior piriform cortex (APC). Synaptic currents mediated by AMPARs and NMDARs were elicited by evoked stimulation and isolated on the basis of differences in pharmacological and kinetic characteristics of each receptor type. The relative current contribution in synaptic populations was assessed by an NMDA/AMPA ratio calculated from measurement of evoked currents. AMPAR currents were characterized at single synapses by mEPSCs and response to minimal stimulation.
Afferent and intrinsic axonal fiber tracts were stimulated to elicit currents at lateral olfactory tract (LOT) and association (ASSN) synapses, two anatomically and physiologically distinct populations of synapses. Paired pulse responses at 50 ms ISI revealed differences in the amount of facilitation between both synaptic populations. Synaptic transmission mediated by AMPAR function was assessed by minimal stimulation and determined to be equivalent in amplitude between LOT and ASSN synapses, although differences in AMPAR kinetic characteristics were detected between pathways. The NMDA/AMPA ratio was decreased at LOT compared to ASSN synapses. Differences found in the relative NMDAR and AMPAR complement and similarities in AMPAR function suggest differences in NMDAR function between LOT and ASSN synapses. Kinetic differences detected in AMPAR-mediated currents suggest different AMPAR complements are also expressed at both pathways.
Synaptic receptor function was characterized in a mouse model for developmental intellectual disability, the Fmr1-KO. Synaptic NMDAR and AMPAR function was assessed at ASSN synapses in 3-6 month old Fmr1-KO and WT littermates. The NMDA/AMPA ratio was reduced at ASSN synapses of the Fmr1-KO and similar amplitudes in AMPAR-mediated mEPSCs were observed in both groups. No differences were observed in voltage sensitivities or kinetic characteristics of either NMDAR or AMPAR currents. These findings suggest a reduction in NMDAR function at these synapses in the Fmr1-KO compared to WT.
The effect of aging on NMDAR and AMPAR function was assessed at LOT and ASSN synapses in 3-28 month old mice. A significant reduction in AMPAR-mediated mEPSC amplitude was observed in 24-28 month old mice. No age related difference was detected in the NMDA/AMPA ratio or paired pulse ratio. These findings suggest that concomitant downregulation of AMPAR and NMDAR function occurs at both LOT and ASSN synapses in aged mice.
The relative and absolute function of NMDARs and AMPARs at LOT and ASSN synapses were found to be differentially affected in all three comparisons. Reduction of currents mediated by one or both synaptic receptor types were observed in all conditions. Hypofunction of one or both receptor type and the relative ratios thereof may explain specific characteristics of LTP induction and expression in APC, and learned behaviors mediated by this synaptic plasticity, in anatomical and etiological conditions.
... Furthermore, early observations reporting occurrence of asynchronous release from GCs (Isaacson and Walmsley 1995;Schoppa et al. 1998) and ...
... TBI altered several electrophysiological parameters, including sEPSC amplitude, input resistance, FI gain, spike threshold, and burst size. TBI/Veh neurons exhibited increased sEPSC amplitude (but not frequency) compared with Sham/Veh neurons, suggesting increased synaptic excitability following TBI (Isaacson and Walmsley 1995). EC degradation inhibitors JZL184, JZL195, and URB97, but not MJN110, significantly attenuated the TBI-induced increase in sEPSC amplitude. ...
Traumatic brain injury (TBI) is an increasingly prevalent condition affecting soldiers, athletes, and motor vehicle accident victims. Unfortunately, it currently lacks effective therapeutic interventions. TBI is defined as a primary mechanical insult followed by a secondary cascade involving inflammation, apoptosis, release of reactive oxygen species, and excitotoxicity, all of which can cause synaptic changes, altered neuronal signaling, and ultimately, behavioral changes. Previously we showed that preventing degradation of the endocannabinoid (EC) 2-acylglycerol (2-AG) with JZL184 following mild TBI attenuated neuroinflammation and improved recovery of neurobehavioral function during the early 24 h post-TBI period. The aim of this study was to extend the timeline of observations to two weeks post-injury and to investigate JZL184’s impact on synaptic transmission, which we view as potential mechanism for TBI-induced cellular and behavioral pathology. Adult male rats were subjected to mild TBI (mTBI) followed by a single intraperitoneal injection of JZL184 or vehicle thirty minutes post-injury. JZL184 administered-TBI animals showed improved neurobehavioral recovery compared to vehicle-injected TBI animals beginning 24 hours post-injury and persisting for two weeks. JZL184-treated animals had significantly diminished gray and white matter astrocyte activation when compared to vehicle-treated animals at day 7 post-TBI. JZL184 administration significantly attenuated the increased pGluR1S845/GluR1 and pERK 1/2 / ERK and the increases in miniature excitatory postsynaptic potential (mEPSC) frequency and amplitude observed in layer 5 pyramidal neurons at 10 days post-TBI. These results suggest a neuroprotective role for ECs in ameliorating the TBI-induced neurobehavioral, neuroinflammatory and glutamate dyshomeostasis from mTBI. Further studies elucidating the cellular mechanisms involved are warranted.
... Our model formally resembled a model recently described by but differed in some of its underlying assumptions, and in using Poissonian rather than binomial statistics. The model assumed that excitatory synapses in CA3 are spontaneously active at a rate that fluctuates with a Poissonian distribution around a mean instantaneous rate µ (Fatt and Katz, 1952;Rotshenker and Rahamimoff, 1970;Isaacson and Walmsley, 1995). It further assumed that µ falls to 0 immediately after an nIB and then recovers exponentially, with a time constant τ, to a steady-state level µ ss . ...
... Diese Synapsen setzten den Transmitter Glutamat frei (z.B. Hackney et al., 1996;Isaacson and Walmsley, 1995) und sind exzitatorisch. Neben den exzitatorichen Eingängen werden die SBCs auch durch glycinerge und GABAerge Synapsen sowie Synapsen, die Vesikel mit beiden Transmittern enthalten (Altschuler et al., 1993;Juiz et al., 1996) inhibitorisch innerviert. ...
... These changes were most pronounced in cool non-responders but were obvious in all neurons by 15 o C (Figure 3.5). Non-NMDA receptor-mediated glutamatergic transmission is highly sensitive to temperature, with a Q 10 > 3 (Q 10 is the change in rate of activity resulting from a 10 o C increase in temperature) (Isaacson & Walmsley, 1995). A reduction in temperature therefore led to a decrease in miniature EPSC rate and amplitude, and a slowing of kinetics (rise times and decay constants), similar to that observed in other brain regions (Tong & Jahr, 1994;Zhang & Trussell, 1994). ...
... It has been shown that the application of TPMPA, a low affinity GABA A competitor (Jones et al., 2001;Barberis et al., 2005;Szabadics et al., 2007), decreases the time constants of the IPSCs suggesting that receptors located further away from the release site might be affected in a more prominent manner than those sitting directly at the postsynaptic site (Szabadics et al., 2007). In agreement with the literature, the Previous studies have shown that lowering release probability reduces the level of interaction of transmitter released from neighboring release sites and that this can accelerate the decay of synaptic currents at GABAergic (Roepstorff and Lambert, 1994;Overstreet and Westbrook, 2003) and glutamatergic (Trussell et al., 1993;Otis and Trussell, 1996;Silver et al., 1996;Diamond and Jahr, 1995;Isaacson and Walmsley, 1995;DiGregorio et al., 2002) synapses. ...
... Asychronous release is often shown as the individual quantal events that can be resolved following the synchronous evoked response (Chen and Regehr 1999; Isaacson and Walmsley 1995). At the immature retinogeniculate synapse, we were unable to reliably isolate such events for several reasons: 1) the peak quantal amplitude at young ages is small (7 pA); 2) both retinogeniculate and corticothalamic contacts contribute to a relatively high frequency of spontaneous miniature EPSCs; and 3) spillover leads to extensive AMPAR desensitization that may mask individual single asynchronous events (Chen et al. 2002;Chen and Regehr 2000;Liu and Chen 2008 Isaacson and Walmsley 1995;Lu and Trussell 2000) and has been shown to influence synaptic integration and postsynaptic firing patterns (Crowley et al. 2009;Iremonger and Bains 2007;Rudolph et al. 2011). We find that the developmental decrease in EPSC time course corresponds to the acceleration of the presynaptic calcium transient. ...
The retinogeniculate synapse, the connection between retinal ganglion cells (RGC) and thalamic relay neurons, undergoes robust changes in connectivity over development. This process of synapse elimination and strengthening of remaining inputs is thought to require synapse specificity. Here we show that glutamate spillover and asynchronous release are prominent features of retinogeniculate synaptic transmission during this period. The immature EPSCs exhibit a slow decay timecourse that is sensitive to low affinity glutamate receptor antagonists and extracellular calcium concentrations, consistent with glutamate spillover. Furthermore, we uncover and characterize a novel, purely spillover-mediated AMPA receptor current from immature relay neurons. The isolation of this current strongly supports the presence of spillover between boutons of different RGCs. In addition, fluorescence measurements of presynaptic calcium transients suggest that prolonged residual calcium contributes to both glutamate spillover and asynchronous release. These data indicate that during development, far more RGCs contribute to relay neuron firing than would be expected based on predictions from anatomy alone.
... A number of these are considered below. Further details can be found elsewhere (Prior et al., 1993;Isaacson and Walmsley, 1995). ...
An important function of all nervous systems is to control muscle contraction so that movements of the body are appropriate to promote survival. In most species, specialized motor neurons convey the signals that represent the neural commands for contraction from the central nervous system to the muscle fibers. This chapter describes the process of neuromuscular transmission. The chapter emphasizes on how the properties of the neuro muscular junctions (NMJs) ensure the reliability of transmission in a wide range of biological circumstances and how changes to those properties may lead to impaired transmission in disease. The main features of neuromuscular transmission, in addition to the essential presynaptic and postsynaptic aspects of neuromuscular transmission, are also described in the chapter. The chapter describes the measurement of safety factors and some of the modulating influences on the safety factor during normal use. The chapter accounts for several important aspects of the “biology” and adaptive plasticity of the NMJ, triggered by trauma or intoxication that promotes maintenance of the effective neural control of muscle. The principles of the functional organization of the NMJ can inform efforts to understand impaired neuromuscular transmission in disease. This account of neuromuscular transmission and its reliability is based on studies of a small number of laboratory species. The emphasis is on mammals, including humans. While neuromuscular transmission in different species has much in common, there are also many important differences. It is therefore always necessary to use caution when trying to interpret findings in one species on the basis of knowledge of another. The ability of the NMJ to recover from a wide variety of mechanical and chemical assaults indicates the evolutionary importance of effective and reliable neuromuscular transmission.
... Independently fusing vesicles are likely to form the two rapid kinetic phases of release The synchronous phase is realized by stepping to 0 mV and is proposed to originate from the fusion of vesicles in less than perfect unison (Isaacson and Walmsley, 1995), as can be inferred from the small spikes decorating the burst phase as it rises, peaks, and then falls (Fig. 3 A, D,H ). The synchrony was disrupted by stepping to less depolarizing voltages for 30 ms ; and at the other extreme, stepping to ϩ40 mV for 0.5 ms elicited small amplitude, single events that were tightly synchronized to the brief tail currents (Fig. 3 I, K ). ...
The neurotransmitter glutamate is used by most neurons in the brain to activate a multitude of different types of glutamate receptors and transporters involved in fast and relatively slower signaling. Synaptic ribbons are large presynaptic structures found in neurons involved in vision, balance, and hearing, which use a large number of glutamate-filled synaptic vesicles to meet their signaling demands. To directly measure synaptic vesicle release events, the ribbon-type presynaptic terminals of goldfish retinal bipolar cells were coaxed to release a false transmitter that could be monitored with amperometry by placing the carbon fiber directly on the larger synaptic terminal. Spontaneous secretion events formed a unimodal charge distribution, but single spike properties were heterogeneous. Larger events rose exponentially without interruption (τ ∼ 30 μs), and smaller events exhibited a stammer in their rising phase that is interpreted as a brief pause in pore dilation, a characteristic commonly associated with large dense core granule fusion pores. These events were entirely Ca(2+)-dependent. Holding the cells at -60 mV halted spontaneous release; and when the voltage was stepped to >-40 mV, secretion ensued. When stepping the voltage to 0 mV, novel kinetic phases of vesicle recruitment were revealed. Approximately 14 vesicles were released per ribbon in two kinetic phases with time constants of 1.5 and 16 ms, which are proposed to represent different primed states within the population of docked vesicles.
... and 3.67 ± 0.58 ms, P ¼ 0.516; t slow : 22.01 ± 3.33 and 25.40 ± 3.20 ms, P ¼ 0.48; n ¼ 9 neurons/4 mice and n ¼ 13 neurons/6 mice for WT and KO neurons, respectively) were modified between genotypes, suggesting a normal postsynaptic function in KO mice (Fig. 1d,e). The slower decay kinetics of evoked responses with respect to the miniature currents in WT neurons has been previously attributed to higher level of asynchronous vesicle release 28 . Thus, these data suggest that the absence of Syn II favours synchronous release at dentate gyrus inhibitory synapses. ...
In the central nervous system, most synapses show a fast mode of neurotransmitter release known as synchronous release followed by a phase of asynchronous release, which extends over tens of milliseconds to seconds. Synapsin II (SYN2) is a member of the multigene synapsin family (SYN1/2/3) of synaptic vesicle phosphoproteins that modulate synaptic transmission and plasticity, and are mutated in epileptic patients. Here we report that inhibitory synapses of the dentate gyrus of Syn II knockout mice display an upregulation of synchronous neurotransmitter release and a concomitant loss of delayed asynchronous release. Syn II promotes γ-aminobutyric acid asynchronous release in a Ca(2+)-dependent manner by a functional interaction with presynaptic Ca(2+) channels, revealing a new role in synaptic transmission for synapsins.
... However, experimental conditions differed between the two recording paradigms, and their potential impact on the interpretation of the data warrants discussion, (i) Temperature — The in vivo recordings of nerve activity were done at body temperature (41°C), $15°C warmer than the temperature of the in vitro capacitance measurements. Although not known for the cochlear hair cell-afferent fiber synapse, the Q 10 of release kinetics at other synapses is $3 (Katz and Miledi 1965; Barrett and Stevens 1972; Isaacson and Walmsley 1995) and would predict as much as a fivefold increase in the hair cell exocytic kinetics in vivo. (ii) Endogenous calcium buffer — Our ''control'' intracellular buffer condition was 0.2 mM EGTA. ...
... The applicability of this model at many central synapses has been complicated by the fact that synaptic events at different locations suffer from variable amounts of electrotonic attenuation, can be small in comparison to background noise, and exhibit large variability in P v and Q P across and within boutons. As a result, release at small central synapses has been better described by non-uniform statistical models ((Walmsley et al., 1987); see also (Isaacson and Walmsley, 1995)). Our results provide a mechanistic basis for the heterogeneity of release within and across terminals, and suggest it might be due to the presence of few functional Ca V s sparse within the active zone. ...
Fast synaptic transmission requires tight colocalization of Ca(2+) channels and neurotransmitter vesicles. It is generally thought that Ca(2+) channels are expressed abundantly in presynaptic active zones, that vesicles within the same active zone have similar release properties, and that significant vesicle depletion only occurs at synapses with high release probability. Here we show, at excitatory CA3→CA1 synapses in mouse hippocampus, that release from individual vesicles is generally triggered by only one Ca(2+) channel and that only few functional Ca(2+) channels may be spread in the active zone at variable distances to neighboring neurotransmitter vesicles. Using morphologically realistic Monte Carlo simulations, we show that this arrangement leads to a widely heterogeneous distribution of release probability across the vesicles docked at the active zone, and that depletion of the vesicles closest to Ca(2+) channels can account for the Ca(2+) dependence of short-term plasticity at these synapses. These findings challenge the prevailing view that efficient synaptic transmission requires numerous presynaptic Ca(2+) channels in the active zone, and indicate that the relative arrangement of Ca(2+) channels and vesicles contributes to the heterogeneity of release probability within and across synapses and to vesicle depletion at small central synapses with low average release probability.
... The decay-time constant of Sr 2+ -asynchronized EPSCs, however, did not increase in response to PP activation (n = 8, P = 0.19; Fig. 6D). The mean decay-time constant of the asynchronized EPSC (1.49 ± 0.16 ms, n = 8) seemed to be slightly smaller than the τ fast value of the first EPSCs evoked by electrical stimulation in normal ACSF (1.92 ± 0.14 ms, n = 11, P = 0.055), possibly reflecting the contribution of less synchronous vesicular release and/or multivesicular release (MVR) to the evoked EPSC (Diamond & Jahr, 1995;Isaacson & Walmsley, 1995). Overall, the results of these experiments clearly rule out the possibility that repetitive GC stimulation changed the properties of postsynaptic AMPARs. ...
Key points
Paired‐pulse facilitation (PPF) is a widely observed form of presynaptically originated short‐term plasticity.
Here, we report that, in rat cerebellar cortex, paired‐pulse activation of glutamatergic granule cell axon fibres causes not only a facilitation in the peak amplitude (PPF amp ) but also a prolongation in the decay‐time constant (PPP decay ) of the EPSCs recorded from molecular‐layer interneurones (INs).
PPF amp is elicited, in part, by an increase in the number of released vesicles (that is, multivesicular release), whereas PPP decay results from extrasynaptic spillover of the transmitter glutamate and its intersynaptic pooling among active synapses, as well as from delayed release as has been explained.
PPF amp and PPP decay play unique roles in determining excitability of the INs.
These findings help us understand the mechanisms underlying encoding and processing of the neuronal information in the cerebellar cortex.
Abstract A simple form of presynaptic plasticity, paired‐pulse facilitation (PPF), has been explained as a transient increase in the probability of vesicular release. Using the whole‐cell patch‐clamp technique to record synaptic activity in rat cerebellar slices, we found different forms of presynaptically originated short‐term plasticity during glutamatergic excitatory neurotransmission from granule cells (GCs) to molecular‐layer interneurones (INs). Paired‐pulse activation of GC axons at short intervals (30–100 ms) elicited not only a facilitation in the peak amplitude (PPF amp ), but also a prolongation in the decay‐time constant (PPP decay ) of the EPSCs recorded from INs. The results of pharmacological tests and kinetics analyses suggest that the mechanisms underlying the respective types of short‐term plasticity were different. PPF amp was elicited by a transient increase in the number of released vesicles. On the other hand, PPP decay was caused not only by delayed release as has been reported but also by extrasynaptic spillover of the GC transmitter and the subsequent intersynaptic pooling. Both PPF amp and PPP decay closely rely on repetitive‐activation‐induced multivesicular release. Using a dynamic clamp technique, we further examined the physiological significance of different presynaptic plasticity, and found that PPF amp and PPP decay can differentially encode and process neuronal information by influencing the total synaptic charge transferred to postsynaptic INs to reflect activation frequency of the presynaptic GCs.
... The physiological responses to current injection and synaptic stimulation and the morphology of such juvenile rat GP neurones are very similar to those of adult rat GP (Ogura & Kita, 2000) and the binding of [ 3 H]CGS21680 is saturated in rat GP at 10-15 days postnatal (Johansson et al. 1997). To investigate the locus of the A 2A receptor-mediated regulation, we have examined the effects of CGS21680 on the PPF, a presynaptic process (Isaacson & Walmsley 1995), and analysed the mIPSCs. The enhancement of evoked IPSCs induced by CGS21680 was accompanied by a reduction in the PPF ratio (Fig. 5). ...
• The actions of adenosine A2A receptor agonists were examined on GABAergic synaptic transmission in the globus pallidus (GP) in rat brain slices using whole-cell patch-clamp recording. GP neurones were characterized into two major groups, type I and type II, according to the degree of time-dependent hyperpolarization-activated inward rectification and the size of input resistance. • The A2A receptor agonist 2-[p-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamido- adenosine (CGS21680; 0.3-3 m) enhanced IPSCs evoked by stimulation within the GP. The actions of CGS21680 were blocked by the A2A antagonists (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF17837) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385). • The CGS21680-induced increase in IPSCs was associated with a reduction in paired-pulse facilitation. CGS21680 (0.3 m) increased the frequency of miniature IPSCs (mIPSCs) without affecting mIPSC amplitude. These observations demonstrated that the enhancement of IPSCs in the GP was attributable to presynaptic, but not postsynaptic, A2A receptors. • The results suggest that A2A receptors in the GP serve to inhibit GP neuronal activity, thereby disinhibiting subthalamic nucleus neurone activity. Thus, the A2A receptor-mediated presynaptic regulation in the GP, together with the A2A receptor-mediated intrastriatal presynaptic control of GABAergic neurotransmission described previously, may play a crucial role in controlling the neuronal functions of basal ganglia. This A2A receptor-mediated presynaptic dual control in the striatopallidal pathway could also afford the mode of action of A2A antagonists for ameliorating the symptoms of Parkinson's disease in an animal model.
Escaping from imminent danger is an instinctive behaviour fundamental for survival that requires classifying sensory stimuli as harmless or threatening. The absence of threat allows animals to forage for essential resources, but as the level of threat and potential for harm increases, they have to decide whether or not to seek safety ¹ . Despite previous work on instinctive defensive behaviours in rodents 2–13 , little is known about how the brain computes the threat level for initiating escape. Here we show that the probability and vigour of escape in mice scale with the intensity of innate threats, and are well described by a theoretical model that computes the distance between threat level and an escape threshold. Calcium imaging and optogenetics in the midbrain of freely behaving mice show that the activity of excitatory VGluT2+ neurons in the deep layers of the medial superior colliculus (mSC) represents the threat stimulus intensity and is predictive of escape, whereas dorsal periaqueductal gray (dPAG) VGluT2 ⁺ neurons encode exclusively the escape choice and control escape vigour. We demonstrate a feed-forward monosynaptic excitatory connection from mSC to dPAG neurons that is weak and unreliable, yet necessary for escape behaviour, and which we suggest provides a synaptic threshold for dPAG activation and the initiation of escape. This threshold can be overcome by high mSC network activity because of short-term synaptic facilitation and recurrent excitation within the mSC, which amplifies and sustains synaptic drive to the dPAG. Thus, dPAG VGluT2 ⁺ neurons compute escape decisions and vigour using a synaptic mechanism to threshold threat information received from the mSC, and provide a biophysical model of how the brain performs a critical behavioural computation.
Escaping from imminent danger is an instinctive behaviour that is fundamental for survival, and requires the classification of sensory stimuli as harmless or threatening. The absence of threat enables animals to forage for essential resources, but as the level of threat and potential for harm increases, they have to decide whether or not to seek safety 1 . Despite previous work on instinctive defensive behaviours in rodents2-11, little is known about how the brain computes the threat level for initiating escape. Here we show that the probability and vigour of escape in mice scale with the saliency of innate threats, and are well described by a model that computes the distance between the threat level and an escape threshold. Calcium imaging and optogenetics in the midbrain of freely behaving mice show that the activity of excitatory neurons in the deep layers of the medial superior colliculus (mSC) represents the saliency of the threat stimulus and is predictive of escape, whereas glutamatergic neurons of the dorsal periaqueductal grey (dPAG) encode exclusively the choice to escape and control escape vigour. We demonstrate a feed-forward monosynaptic excitatory connection from mSC to dPAG neurons, which is weak and unreliable-yet required for escape behaviour-and provides a synaptic threshold for dPAG activation and the initiation of escape. This threshold can be overcome by high mSC network activity because of short-term synaptic facilitation and recurrent excitation within the mSC, which amplifies and sustains synaptic drive to the dPAG. Therefore, dPAG glutamatergic neurons compute escape decisions and escape vigour using a synaptic mechanism to threshold threat information received from the mSC, and provide a biophysical model of how the brain performs a critical behavioural computation.
Glycinergic synapses predominate in brainstem and spinal cord where they modulate motor and sensory processing. Their postsynaptic mechanisms have been considered rather simple because they lack a large variety of glycine receptor isoforms and have relatively simple postsynaptic densities at the ultrastructural level. However, this simplicity is misleading being their postsynaptic regions regulated by a variety of complex mechanisms controlling the efficacy of synaptic inhibition. Early studies suggested that glycinergic inhibitory strength and dynamics depend largely on structural features rather than on molecular complexity. These include regulation of the number of postsynaptic glycine receptors, their localization and the amount of co-localized GABAA receptors and GABA-glycine co-transmission. These properties we now know are under the control of gephyrin. Gephyrin is the first postsynaptic scaffolding protein ever discovered and it was recently found to display a large degree of variation and regulation by splice variants, posttranslational modifications, intracellular trafficking and interactions with the underlying cytoskeleton. Many of these mechanisms are governed by converging excitatory activity and regulate gephyrin oligomerization and receptor binding, the architecture of the postsynaptic density (and by extension the whole synaptic complex), receptor retention and stability. These newly uncovered molecular mechanisms define the size and number of gephyrin postsynaptic regions and the numbers and proportions of glycine and GABAA receptors contained within. All together, they control the emergence of glycinergic synapses of different strength and temporal properties to best match the excitatory drive received by each individual neuron or local dendritic compartment.
RIMs and RIM-binding proteins (RBPs) are evolutionary conserved multidomain proteins of presynaptic active zones that are known to recruit Ca(2+) channels; in addition, RIMs perform well-recognized functions in tethering and priming synaptic vesicles for exocytosis. However, deletions of RIMs or RBPs in mice cause only partial impairments in various active zone functions and have no effect on active zone structure, as visualized by electron micrographs, suggesting that their contribution to active zone functions is limited. Here, we show in synapses of the calyx of Held in vivo and hippocampal neurons in culture that combined, but not individual, deletions of RIMs and RBPs eliminate tethering and priming of synaptic vesicles, deplete presynaptic Ca(2+) channels, and ablate active zone complexes, as analyzed by electron microscopy of chemically fixed synapses. Thus, RBPs perform unexpectedly broad roles at the active zone that together with those of RIMs are essential for all active zone functions.
The Superior Paraolivary Nucleus (SPON) is a prominent structure in the mammalian auditory brainstem with a proposed role in encoding transient broadband sounds such as vocalized utterances. Currently, the source of excitatory pathways that project to the SPON and how these inputs contribute to SPON function are poorly understood. To shed light on the nature of these inputs, we measured evoked excitatory postsynaptic currents (EPSCs) in the SPON originating from the intermediate acoustic stria and compared them with the properties of EPSCs in the Lateral Superior Olive (LSO) originating from the ventral acoustic stria during auditory development from postnatal day 5 to 22 in mice. Before hearing onset, EPSCs in the SPON and LSO are very similar in size and kinetics. After the onset of hearing, SPON excitation is refined to extremely few (2:1) fibers, with each strengthened by an increase in release probability, yielding fast and strong EPSCs. LSO excitation is recruited from more fibers (5:1), resulting in strong EPSCs with a comparatively broader stimulus-response range after hearing onset. Evoked SPON excitation is comparatively weaker than evoked LSO excitation, likely due to a larger fraction of postsynaptic GluR2-containing Ca2+-impermeable AMPA receptors after hearing onset. Taken together, SPON excitation develops synaptic properties that are suited for transmitting single events with high temporal reliability and the strong, dynamic LSO excitation is compatible with high rate level-sensitivity. Thus, the excitatory input pathways to the SPON and LSO mature to support different decoding strategies of respective coarse temporal and sound intensity information at the brainstem level.
Unlabelled:
Although synapsins regulate GABA release, it is unclear which synapsin isoforms are involved. We identified the synapsin isoforms that regulate GABA release via rescue experiments in cultured hippocampal neurons from synapsin I, II, and III triple knock-out (TKO) mice. In situ hybridization indicated that five different synapsin isoforms are expressed in hippocampal interneurons. Evoked IPSC amplitude was reduced in TKO neurons compared with triple wild-type neurons and was rescued by introducing any of the five synapsin isoforms. This contrasts with hippocampal glutamatergic terminals, where only synapsin IIa rescues the TKO phenotype. Deconvolution analysis indicated that the duration of GABA release was prolonged in TKO neurons and this defect in release kinetics was rescued by each synapsin isoform, aside from synapsin IIIa. Because release kinetics remained slow, whereas peak release rate was rescued, there was a 2-fold increase in GABA release in TKO neurons expressing synapsin IIIa. TKO neurons expressing individual synapsin isoforms showed normal depression kinetics aside from more rapid depression in neurons expressing synapsin IIIa. Measurements of the cumulative amount of GABA released during repetitive stimulation revealed that the rate of mobilization of vesicles from the reserve pool to the readily releasable pool and the size of the readily releasable pool of GABAergic vesicles were unaffected by synapsins. Instead, synapsins regulate release of GABA from the readily releasable pool, with all isoforms aside from synapsin IIIa controlling release synchrony. These results indicate that synapsins play fundamentally distinct roles at different types of presynaptic terminals.
Significance statement:
Synapsins are a family of proteins that regulate synaptic vesicle (SV) trafficking within nerve terminals. Here, we demonstrate that release of the inhibitory neurotransmitter GABA is supported by many different synapsin types. This contrasts with the release of other neurotransmitters, which typically is supported by only one type of synapsin. We also found that synapsins serve to synchronize the release of GABA in response to presynaptic action potentials, which is different from the synapsin-dependent trafficking of SVs in other nerve terminals. Our results establish that different synapsins play fundamentally different roles at nerve terminals releasing different types of neurotransmitters. This is an important clue to understanding how neurons release their neurotransmitters, a process essential for normal brain function.
Unlabelled:
Many central glutamatergic synapses contain a single presynaptic active zone and a single postsynaptic density. However, the basic functional properties of such "simple synapses" remain unclear. One important step toward understanding simple synapse function is to analyze the number of synaptic vesicles released in such structures per action potential, but this goal has remained elusive until now. Here, we describe procedures that allow reliable vesicular release counting at simple synapses between parallel fibers and molecular layer interneurons of rat cerebellar slices. Our analysis involves local extracellular stimulation of single parallel fibers and deconvolution of resulting EPSCs using quantal signals as template. We observed a reduction of quantal amplitudes (amplitude occlusion) in pairs of consecutive EPSCs due to receptor saturation. This effect is larger (62%) than previously reported and primarily reflects receptor activation rather than desensitization. In addition to activation-driven amplitude occlusion, each EPSC reduces amplitudes of subsequent events by an estimated 3% due to cumulative desensitization. Vesicular release counts at simple synapses follow binomial statistics with a maximum that varies from 2 to 10 among experiments. This maximum presumably reflects the number of docking sites at a given synapse. These results show striking similarities, as well as significant quantitative differences, with respect to previous results at simple GABAergic synapses.
Significance statement:
It is generally accepted that the output signal of individual central synapses saturates at high release probability, but it remains unclear whether the source of saturation is presynaptic, postsynaptic, or both presynaptic and postsynaptic. To clarify this and other issues concerning the function of synapses, we have developed new recording and analysis methods at single central glutamatergic synapses. We find that individual release events engage a high proportion of postsynaptic receptors (62%), revealing a larger component of postsynaptic saturation than anticipated. Conversely, we also find that the number of released synaptic vesicles is limited at each active zone. Altogether, our results argue for both presynaptic and postsynaptic contributions to signal saturation at single glutamatergic synapses.
The structure and function of fast excitatory synapses in the central nervous system often represent compromises between several physiological imperatives. To achieve a short delay between the pre- and postsynaptic signal, the transmitter glutamate directly gates ion channels, leading to rapid depolarization and initiation of action potentials. However, the need to integrate signals from thousands of different inputs and to perform miniature computations on the signals from small groups of inputs requires that synapses be distributed over a complex dendritic tree, which necessarily slows the onset of excitation. In the auditory system, the need for speed outweighs all others, and it is there that giant, somatic synapses are found. In recent years, these calyceal and endbulb synapses have been rediscovered by biophysicists because of technical advantages the synapses offer for the study of basic features of glutamatergic transmission in the central nervous system, such as better voltage control for measuring the synaptic current and electrical and physical access to the presynaptic terminal. In the course of these studies, we have learned that the demands of acoustic processing have not only selected for the unique morphology of the giant synapses but also for biophysical specializations which compromise (or make more interesting) their service as “model synapses”. In this chapter, we will look at the control of synaptic strength at calyceal/endbulb synapses, with particular, but not exclusive, emphasis on post-synaptic issues.
According to the modern conceptions of the processes of synaptic transmission of excitation there are two forms of quantal neurotransmitter release evoked by the neural stimulus–phasic synchronous and delayed asynchronous release differentiated by the intensity and temporal parameters of quanta secretion. This review is dedicated to the analysis of temporal characteristics of evoked synchronous and delayed asynchronous release of neurotransmitter quanta at chemical synapses. The data indicative of different mechanisms of realization and modulation of these types of the evoked quantal secretion are discussed. The importance of temporal parameters of neuronal secretion for maintenance of effective synaptic transmission of excitation and alteration of these parameters in some pathologies is considered.
This chapter summarizes classical knowledge and recent advances in the release of neurotransmitters, emphasizing evidence supporting present concepts, rather than a recitation of dogma. Topics covered include:. •Structural organization of chemical synapses•Quantal nature of transmission; identification of the synaptic vesicle as the physical quantum•Vesicle membrane and transmitter cycles•Biophysics of excitation-secretion coupling; secretory trigger properties•Calcium microdomains in triggering exocytosis•Mobilizing vesicles to docking sites and priming vesicles for release•Presynaptic protein identification by vesicle purification and genetics•SNARE proteins: assembly, disassembly, and vesicle fusion•Synaptotagmin: calcium sensor and secretory trigger•Docking and priming proteins•Molecular mechanisms of endocytosis and vesicle refilling•Quantal analysis at neuromuscular junctions and central synapses•Poisson and binomial models; meaning of statistical parameters•Limitations of the standard Katz model•Single and multivesicular release at active zones•Spontaneous vs. evoked release•Estimating and manipulating statistical parameters•Quantal analysis of release modulation.
Complexins (Cplxs) are small synaptic proteins that cooperate with SNARE-complexes in the control of synaptic vesicle (SV) fusion. Studies involving genetic mutation, knock-down, or knock-out indicated two key functions of Cplx that are not mutually exclusive but cannot easily be reconciled, one in facilitating SV fusion, and one in "clamping" SVs to prevent premature fusion. Most studies on the role of Cplxs in mammalian synapse function have relied on cultured neurons, heterologous expression systems, or membrane fusion assays in vitro, whereas little is known about the function of Cplxs in native synapses. We therefore studied consequences of genetic ablation of Cplx1 in the mouse calyx of Held synapse, and discovered a developmentally exacerbating phenotype of reduced spontaneous and evoked transmission but excessive asynchronous release after stimulation, compatible with combined facilitating and clamping functions of Cplx1. Because action potential waveforms, Ca(2+) influx, readily releasable SV pool size, and quantal size were unaltered, the reduced synaptic strength in the absence of Cplx1 is most likely a consequence of a decreased release probability, which is caused, in part, by less tight coupling between Ca(2+) channels and docked SV. We found further that the excessive asynchronous release in Cplx1-deficient calyces triggered aberrant action potentials in their target neurons, and slowed-down the recovery of EPSCs after depleting stimuli. The augmented asynchronous release had a delayed onset and lasted hundreds of milliseconds, indicating that it predominantly represents fusion of newly recruited SVs, which remain unstable and prone to premature fusion in the absence of Cplx1.
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We studied the effects of GABAB receptor activation on either glycine or GABAA receptor-mediated synaptic transmission to hypoglossal motoneurons (HMs, P8-13) using a rat brainstem slice preparation. Activation of GABAB receptors with baclofen, a GABAB receptor agonist, inhibited the amplitude of evoked glycine and GABAA receptor-mediated inhibitory postsynaptic currents. Additionally, with blockade of postsynaptic GABAB receptors baclofen decreased the frequency of both glycine and GABAA receptor-mediated spontaneous miniature inhibitory postsynaptic currents (mIPSCs), indicating a presynaptic site of action. Conversely, the GABAB receptor antagonist CGP 35348 increased the frequency of glycine receptor-mediated mIPSCs. Application of the GABA transport blocker SKF 89976A decreased the frequency of glycinergic mIPSCs. Lastly, we compared the effects of baclofen on the frequency of glycine and GABAA receptor-mediated mIPSC during HM development. At increased postnatal ages (P8-13 versus P1-3) mIPSC frequency was more strongly reduced by baclofen. These results show that presynaptic GABAB receptors inhibits glycinergic and GABAergic synaptic transmission to HMs, and the presynaptic sensitivity to baclofen is increased in P8-13 versus P1-3 HMs. Further, endogenous GABA is capable of modulating inhibitory synaptic transmission to HMs.
Synaptic transmission alters the strength of the postsynaptic potential, through a process called short-term synaptic plasticity (STP). In this study, endplate potentials (EPPs) from the frog neuromuscular junction were used to resolve and quantify the presynaptic components involved in enhancement and depression of transmitter release during repetitive stimulation under normal quantal release conditions (2 mM Ca2+, 1mM Mg2+). During trains of stimulation given between 10 - 200 Hz, the amplitude of the EPPs first increased then decreased; a maximum increase of 77% was produced after 2-4 stimuli. EPP amplitudes began to increase at ~ 20 Hz, were maximal at ~ 55 Hz, and thereafter, decreased as the rate of stimulation increased. The integrated total release after 25 stimuli was little changed across frequencies between 10 - 100 Hz. EPPs ran down in two phases: a fast phase, attributed to the depletion of a readily releasable pool (RRP) of synaptic vesicles, followed by a slow phase, attributed to the depletion of vesicles from a depot pool (DP). Depletion of the readily releasable pool of synaptic vesicles (RRP) was determined by quantifying release under the fast and slow time rundowns and subtracting the number of vesicles associated with mobilization to the RRP from the total number of vesicles released during stimulation trains of 50 impulses. Impulses were delivered at 12 different rates ranging from 50 to 200 /s. Estimates of the number of vesicles released from the RRP increased with frequency of stimulation until maximal depletion levels of 5500 - 6000 vesicles were reached at stimulation rates between 90-130/s, assuming a control quantal content of 200 vesicles released per impulse. Depletion was less at lower frequencies when the number of stimuli delivered was identical. When the RRP maximally depleted, release was inversely related to stimulation rate, as would be expected if mobilization from the depot pool was the sole determinate of release during the slow phase. An equation constructed from four known components of enhancement and two components of depression - the depletion of vesicles from a readily releasable pool (RRP) and from the depot pool (DP) that refills the RRP, was used to fit and then simulate EPPs obtained during trains using different patterns of stimulation and varying amounts of extracellular Ca2+; the decay time constant parameters of enhancement, numerically derived from the observed data, were fixed at tau ~ 46, 220, 1600, and 20000 ms. The number of components of enhancement necessary to approximate the data decreased, from four in low (0.14 - 0.2mM) extracellular Ca2+, to one (tau ~ 46 ms) in 2.0 mM extracellular Ca2+, but four components of enhancement were necessary to fit the data when the amplitude of the EPP was not depressed below the control amplitude. This model was able to predict within ~ 3 % EPP amplitudes over a 10-fold range of frequency and Ca2+ concentration.
The duration of the synaptic current is a key element in determining the overall function of glutamatergic synapses. The duration of the postsynaptic response is of importance in determining the consequence of repetitive synaptic activity. Temporal summation—a fundamental component in the integration of synaptic signals—is prominent when excitatory postsynaptic potentials are long enough to overlap. Conversely, precise neural coincidence detection requires the minimization of temporal summation so that only near-simultaneous, narrow synaptic events are able to drive a cell to action potential threshold. In addition to mediating electrical computation, glutamatergic synaptic events drive long-term changes in the biochemical state of postsynaptic cells. Studies in the past years have identified many of the major parameters that influence the duration of the synaptic current. These include synaptic geometry, receptor-binding affinity and channel gating kinetics, glutamate transporters, and quantal release kinetics. Synaptic transmission mediated by glutamate and glutamate receptors draws upon a wide variety of factors to determine the duration of the synaptic current. Because these factors can be varied independently, glutamate synapses can, with appropriate parallel variation in postsynaptic cable properties, be specialized for integration, coincidence detection, or the passive relay of signals.
Numerous experimental data suggest that simultaneously or sequentially activated assemblies of neurons play a key role in the storage and computational use of long-term memory in the brain. However, a model that elucidates how these memory traces could emerge through spike-timing-dependent plasticity (STDP) has been missing. We show here that stimulus-specific assemblies of neurons emerge automatically through STDP in a simple cortical microcircuit model. The model that we consider is a randomly connected network of well known microcircuit motifs: pyramidal cells with lateral inhibition. We show that the emergent assembly codes for repeatedly occurring spatiotemporal input patterns tend to fire in some loose, sequential manner that is reminiscent of experimentally observed stereotypical trajectories of network states. We also show that the emergent assembly codes add an important computational capability to standard models for online computations in cortical microcircuits: the capability to integrate information from long-term memory with information from novel spike inputs.
During the last decade, advances in experimental techniques and quantitative modelling have resulted in the development of the calyx of Held as one of the best preparations in which to study synaptic transmission. Here we review some of these advances, including simultaneous recording of pre- and postsynaptic currents, measuring the Ca2+ sensitivity of transmitter release, reconstructing the 3-D anatomy at the electron microscope (EM) level, and modelling the buffered diffusion of Ca2+ in the nerve terminal. An important outcome of these studies is an improved understanding of the Ca2+ signal that controls phasic transmitter release. This article illustrates the spatial and temporal aspects of the three main steps in the presynaptic signalling cascade: Ca2+ influx through voltage-gated calcium channels, buffered Ca2+ diffusion from the channels to releasable vesicles, and activation of the Ca2+ sensor for release. Particular emphasis is placed on how presynaptic Ca2+ buffers affect the Ca2+ signal and thus the amplitude and time course of the release probability. Since many aspects of the signalling cascade were first conceived with reference to the squid giant presynaptic terminal, we include comparisons with the squid model and revisit some of its implications. Whilst the characteristics of buffered Ca2+ diffusion presented here are based on the calyx of Held, we demonstrate the circumstances under which they may be valid for other nerve terminals at mammalian CNS synapses.
The acoustic environment contains biologically relevant information on timescales from microseconds to tens of seconds. The auditory brainstem nuclei process this temporal information through parallel pathways that originate in the cochlear nucleus from different classes of cells. Although the roles of ion channels and excitatory synapses in temporal processing have been well studied, the contribution of inhibition is less well understood. Here, we show in CBA/CaJ mice that the two major projection neurons of the ventral cochlear nucleus, the bushy and T-stellate cells, receive glycinergic inhibition with different synaptic conductance time courses. Bushy cells, which provide precisely timed spike trains used in sound localization and pitch identification, receive slow inhibitory inputs. In contrast, T-stellate cells, which encode slower envelope information, receive inhibition that is eightfold faster. Both types of inhibition improved the precision of spike timing but engage different cellular mechanisms and operate on different timescales. Computer models reveal that slow IPSCs in bushy cells can improve spike timing on the scale of tens of microseconds. Although fast and slow IPSCs in T-stellate cells improve spike timing on the scale of milliseconds, only fast IPSCs can enhance the detection of narrowband acoustic signals in a complex background. Our results suggest that target-specific IPSC kinetics are critical for the segregated parallel processing of temporal information from the sensory environment.
The input synapses of cerebellar Purkinje cells (PCs) have been extensively studied and much has been learned about their dynamics, plasticity and functionality. In contrast there is limited information available about PC output synapses. This study uses dual cell recording methods to investigate synaptic dynamics and plasticity at individual PC synapses onto neighboring PCs in in vitro preparations of the mormyrid cerebellum. This synaptic connectivity may be strong or weak. For strong connections, inhibitory postsynaptic potentials (IPSPs) or currents (IPSCs) are synchronized with the action potentials of the presynaptic cell. For weak connections, however, the pre- and postsynaptic potentials are no longer synchronized, and presynaptic burst firing at intraburst rates of ∼50Hz or higher is required to reliably induce the postsynaptic inhibition. A depression of this postsynaptic inhibition was observed for both types of connectivity following repeated presynaptic bursts, which was subsequently largely reversed following pairings of the presynaptic burst-induced IPSPs/IPSCs with evoked burst firing of the postsynaptic cell. Moreover, the original postsynaptic depression was found to be either augmented or reversed depending on the temporal order of each pair of additional pre- and postsynaptic cell activations, hence demonstrating a reversible and spike timing-dependent plasticity (STDP) at this synapse.
Recent studies of the mechanism of quantal neurotransmitter release have assumed that the number of quanta released at each stimulation is binomially distributed and have sought to estimate the binomial parameters n and p. Mathematical analysis and computer simulations show that temporal variation in the number of eligible or filled release sites and either spatial or temporal variation in the probability of release at a site can drastically bias such estimates, while the experimental histograms remain statistically indistinguishable from those predicted by the binomial law. Interpretation of the estimates n and p in terms of ultrastructural or physiological characteristics of the nerve terminal is liable to significant error if departures from the binomial assumptions are not suitably assessed.
Simultaneous intracellular recordings were obtained from the goldfish Mauthner cell (M-cell) and adjacent identifiable inhibitory interneurons. Amplitude fluctuations of unitary inhibitory postsynaptic potentials (IPSPs) produced by directly evoked presynaptic impulses were subjected to quantal analysis. The release parameters were correlated with histological features of the pe-synpatic cells, which were systematically iontophoretically injected with horseradish peroxidase (HRP) and reconstructed. A computational procedure was developed that provided the probability density function (PDF) for a given set of responses and, by minimizing the effects of background noise through a deconvolution process, allowed optimal fits of the data according to the predictions of both Poisson and binomial equations. These two models were further compared on the basis of their likelihood criteria. The binomial relation always gave a more adequate description of the PDFs than did the Poisson, this conclusion being substantiated by statistical tests. A striking equivalence was found between the binomial term n and the number of stained presynaptic boutons. This identity was verified for 18 cells that met the mathematical requirement for the analysis (namely, a mean quantal content less than 7), with binomial n ranging from 3 to 28. The probability of release parameter, p, was consistently high, averaging 0.37 which further favors the adoption of the bimodial model. There was a possible tendency for p to be inversely related to n. In the analysis of data from six additional cells for which an elevated np product required that the binomial n be fixed to the value of its histological counterpart, the average p was similar, suggesting that the validity of the model extends to cells with an even larger number of terminal boutons. The binomial quantal size, q, was compared for all cells studied by expressing its respective computed amplitudes relative to that of the full-sized collateral IPSP, which is itself a constant fraction of the driving force. In this manner q was remarkably constant at about 1.1% of the full-sized response. Calculations taking into account the value of q, the resting membrane conductance, and the conductance increase during a unitary IPSP, provided support for the postulate that the action of a single quantum (and, therefore, of one releasing terminal) is functionally similar to that of the contents of a single synaptic vesicle. The method used for this study was applied to the synaptic depression observed during high-frequency stimulation. Despite a limited sample, evidence was obtained that even under those conditions every synaptic bouton continues to function as an independent all-or-none releasing unit; the reduction in IPSP amplitudes could be solely attributed to a lower probability of release, p.
Nissl-stained tissue from brain slice preparations of the anteroventral cochlear nucleus of the mouse resembles tissue fixed in situ. Multipolar, spherical, globular, and granule cells can be distinguished after intracellular injection with horseradish peroxidase (HRP). Stellate cells have relatively large dendritic fields; their axons have collaterals which terminate within the cochlear nuclear complex. Bushy cells have smaller dendritic fields; where they can be seen, axons have no collaterals. Granule cells have few short dendrites; their very fine axons branch close to the cell body and could be followed only for short distances. Intracellular recordings from six stellate cells labeled by intracellular injection of HRP revealed that they have linear current-voltage relationships around the resting potential and that they respond to suprathreshold depolarization with large, regularly firing action potentials. Intracellular recordings from four bushy cells, also labeled by injection of HRP, showed that these cells have nonlinear current-voltage relationships around the resting potential and that they respond to suprathreshold depolarizations with only one or two small action potentials. The anatomical and physiological features of bushy cells reduce summing in time and space and make bushy cells well suited to preserve the firing patterns of auditory nerve inputs. The anatomical and physiological features of stellate cells, in contrast, allow summing in time and space.
1. We have studied the statistical properties of excitatory post-synaptic currents (EPSCs) measured at small numbers of synaptic contacts between pairs of hippocampal neurons maintained in dissociated cell culture. Synaptic transmission at few synapses was enabled by microperfusion of a small region of the postsynaptic cell with Ca-containing solution, while blocking transmission at all other synaptic boutons by bathing them in low-Ca solution. Frequency histograms of the amplitudes of EPSCs recorded in this way showed no clear quantization. Numbers of active synapses, estimated immunohistochemically with the use of light microscopy, ranged from 4 to 14 in different experiments. 2. Miniature EPSCs (mEPSCs), originating in the same small population of synapses as produced the evoked EPSCs, were elicited by microperfusion of bath solution made hypertonic by the addition of sucrose. These "sucrose-evoked" mEPSCs appeared to be identical to "spontaneous" mEPSCs in every respect except control over their frequency and site of origin. Sucrose-evoked mEPSCs originating in few synapses still exhibited a broad amplitude distribution. Thus, if mEPSCs constitute the postsynaptic response to a single quantum of neurotransmitter (the "quantal amplitude"), their broad amplitude distribution would tend to obliterate evidence of quantization in evoked EPSC amplitudes, even if evoked release was, indeed, quantal. 3. This idea, which is a corollary of the Katz model of quantal transmission, was tested quantitatively by assuming 1) neurotransmitter release obeys uniform binomial statistics, and 2) the quantal amplitude has a distribution given by the observed distribution of sucrose-evoked mEPSCs. The expected distribution, calculated on the basis of these two assumptions, was fitted to the observed distribution of evoked EPSC amplitudes by varying two free parameters, the binomial parameters N and p. In five cells out of six that were fully analyzed, the Poisson limit of the binomial model (N large, p small) provided a very good fit to the data. This and other evidence suggests that the release probability at a single presynaptic terminal is low. In two out of the six cells, the binomial model, with N constrained to the histochemically determined bouton count, yielded acceptable fits; for the remaining cells the constrained binomial model could be rejected. 4. It is concluded that the Katz model of quantized release of neurotransmitter gives an adequate description of excitatory synaptic transmission in hippocampal cultures, when one assumes the broad distribution of mEPSC amplitudes reflects the distribution of the postsynaptic effect of a single quantum of transmitter.
After the arrival of a presynaptic nerve impulse at an excitatory synapse in hippocampal neurons, the rate of neurotransmitter release increases rapidly and then returns to low levels with a biphasic decay. The two kinetically distinct components are differentially affected when Sr2+ is substituted for Ca2+ ions. Our findings are comparable to those of the classical studies for the frog neuromuscular junction, and thus the basic aspects of Ca(2+)-activated transmitter release machinery appear to be conserved in central synapses. The method we have used, in addition, permits us to estimate the average neurotransmitter release rate for a single bouton. The observation of differential Ca2+/Sr2+ sensitivity is consistent with a release mechanism mediated by two Ca2+ sensors with distinct Ca2+ affinities: the low-affinity Ca2+ sensor facilitates the fast synchronous phase of release, whereas the high-affinity sensor sustains the slow asynchronous phase of release.
A distinctive characteristic of the AMPA subset of glutamate receptor channels is their remarkably rapid desensitization. A family of compounds, the benzothiadiazides, is described here that potently inhibit rapid glutamate receptor desensitization. The structure-activity relationships of these compounds are examined and the actions of cyclothiazide (CYZ), the most potent of these compounds, are described in detail. At the macroscopic level CYZ reduced rapid desensitization, enhancing the steady-state and peak current produced by 1 mM quisqualate with EC50 values of 14 and 12 microM, respectively, and shifted the quisqualate peak current concentration-response relation to the left. The slight outward rectification of the steady-state quisqualate current-voltage relationship was reduced by CYZ. At the microscopic level CYZ caused glutamate to induce long bursts of channel openings, and greatly increased the number of repeated openings. At 10 microM CYZ did not have measurable effects on the fast component of deactivation nor did it have statistically significant effects on the distribution of the faster components of glutamate-induced burst duration. In contrast, 10 microM CYZ increased the amplitude and significantly prolonged the duration of the spontaneous miniature EPSCs. The identification and characterization of this new family of gating modifiers may further facilitate the investigation into the mechanisms underlying rapid glutamate receptor desensitization and the physiological roles that it may serve.
The bushy cells in the anterior division of the anteroventral cochlear nucleus of the cat were studied with the electron microscope. In the anterior part of the anterior division, profiles of bushy cells and their processes are easily identified, since few cells of other types are found in this region. In the posterior and posterodorsal parts of the anterior division, the bushy cells are intermingled with stellate and small cells but can be identified on the basis of light-microscopic descriptions and comparisons with the results from the anterior part. Bushy cells are large, spherical cells with a centrally located nucleus enveloped by sheets of rough endoplasmic reticulum. Thin proximal dendrites jut abruptly from the cell body and contain a relatively pale cytoplasm. The distal dendrites contain few organelles other than numerous, very large mitochondria. The cell soma and proximal dendrites, as well as the axon hillock, receive numerous synaptic terminals, but the distal dendritic processes are contacted by relatively few endings. At least four types of terminals form synaptic contacts with the bushy cells. Very large terminals, containing large, spherical synaptic vesicles and forming multiple asymmetrical contacts, correspond to the end-bulbs of Held from the cochlea. These terminals disappear after cochlear ablations, but the other three types remain. The most numerous of these is a large terminal that contains flattened synaptic vesicles and forms long, nearly symmetrical contacts with the soma and dendrites of bushy cells. The second type of non-cochlear terminal is smaller and contains small, pleiomorphic synaptic vesicles that are not flattened. The third type occurs mainly on bushy cell dendrites, contains small, spherical synaptic vesicles, and forms moderately asymmetrical contacts.
The anterior division of the anteroventral cochlear nucleus of the cat was studied in the light microscope. Criteria were developed to distinguish neurons in the Nissl-stained anteroventral cochlear nucleus which could then be correlated with those types found in Golgi preparations. Based on the patterns of distribution of the neuronal types, their size and shape, and the number of primary dendrites, the bushy cells (Golgi) are shown to correspond to the spherical cells (Nissl), whereas the stellate and small cells (Golgi) correspond to the ovoid cells (Nissl).The present results provide a background for a detailed study of the synaptic organization of the different cell types of the anterior division with the electron microscope and electrophysiological methods.
1. The origins of fluctuations in charge transfer during the generation of Ia e.p.s.p.s have been investigated. The discrete components which make up the fluctuating e.p.s.p. have been separated. 2. Some e.p.s.p.s fluctuate between two different amplitudes and time courses. These fluctuations have been analysed to show that charge transmission always occurs at one synaptic location, but not always at a second synaptic location. 3. The failures in transmission were study by stimulating the afferent fibre at different frequencies. Although different probabilities of failure were obtained at different frequencies, there was no systematic change in probability with increasing frequency. 4. Single afferents were tetanized and histograms of charge transfer computed during post-tetanic potentiation (p.t.p.). Only half of the units studied showed any p.t.p. In those that did, evidence was found for a decrease in the probability of failure during potentiation. 5. The results could not be used to distinguish between failure of the impulse to always propagate into the terminals, and failure of the terminals to release transmitter following adequate depolarization. 6. The fluctuations in transmission at a single synapse can be described by a binomial process with n = 1 and p less than or equal to 1. Junctional mechanisms consistent with this description are discussed. Alternative mechanisms which associate failures with failure of impulse transmission at afferent fibre branch points are also suggested.
Binomial distributions of amplitudes of excitatory postsynaptic potentials (EPSPs) mixed with Gaussian noise were simulated. The objective of Monte Carlo simulations was, firstly, to study influences of sampling size (N) and noise standard deviation (Sn) on estimates of mean quantal content (m), quantal size (v) and binomial parameters (n and p) by four methods of quantal analysis (histogram, variance, failures and combined method) based on the binomial model and, secondly, to modify these methods on the basis of comparison of estimated with simulated parameters. Reliable estimates (within +/- 10% of the simulated values) were obtained for large sample sizes (N = 500-1000) with Sn less than or equal to v by the histogram (deconvolution) method and with Sn less than or equal to 2v by the other three methods. Similar results were obtained by averages from about 10 simulations if smaller samples were used (N = 50-200). In electrophysiological experiments on slices, "minimal" EPSPs were recorded from CA1 pyramidal cells after low-intensity stimuli to stratum radiatum or stratum oriens. Amplitudes of minimal EPSPs fluctuated in a manner predicted by the quantum hypothesis. Amplitude distributions of EPSPs in the non-facilitated state were adequately described either by binomial statistics with an average p equal to about 0.4 (a range of 0.3-0.7) and an average n of about 3 (range 2-6) or by Poisson statistics with m of about 1. The quantal analysis suggests that typical values of m and v for a single activated fibre in stratum radiatum might be about 0.5-1 and 300-400 microV, respectively, with low p (0.1-0.3) and n (2-4). However, the estimates of binomial parameters should be considered as coarse approximations in view of the simulation results and a possible nonuniformity of parameter p. The comparison of results of various methods based on the binomial model, in both simulation and physiological experiments, indicates the reliability of estimates of basic quantal parameters (m and v) under realistic conditions of physiological experiments. The methods are considered to be sufficiently sensitive to make use of them for studies on mechanisms of long-term synaptic plasticity.
The quantal hypothesis proposes that chemical synaptic transmission involves the probabilistic release of multimolecular packets of transmitter. Analysis of the resulting trial-to-trial fluctuations in postsynaptic response can provide estimates both of the number of quanta released and of the size of their postsynaptic effect. This in turn permits the quantification of the relative contributions of pre- and postsynaptic factors to the strength of a given synapse. Quantal analysis of excitatory synapses in the hippocampus has proved difficult and has led to contradictory conclusions when applied to long-term potentiation. Here we report the use of a combination of quantal analysis procedures to provide evidence that both pre- and postsynaptic changes can contribute substantially to the maintenance of long-term potentiation in the CA1 region of the hippocampus. The initial setting of the presynaptic release mechanism seems to determine their relative importance.
The peak concentration and rate of clearance of neurotransmitter from the synaptic cleft are important determinants of synaptic function, yet the neurotransmitter concentration time course is unknown at synapses in the brain. The time course of free glutamate in the cleft was estimated by kinetic analysis of the displacement of a rapidly dissociating competitive antagonist from N-methyl-D-aspartate (NMDA) receptors during synaptic transmission. Glutamate peaked at 1.1 millimolar and decayed with a time constant of 1.2 milliseconds at cultured hippocampal synapses. This time course implies that transmitter saturates postsynaptic NMDA receptors. However, glutamate dissociates much more rapidly from alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Thus, the time course of free glutamate predicts that dissociation contributes to the decay of the AMPA receptor-mediated postsynaptic current.
Neurotransmission from mossy fibre terminals onto cerebellar granule cells is almost certainly mediated by L-glutamate. By taking advantage of the small soma size, limited number of processes and short dendrite length of granule cells, we have obtained high-resolution recordings of spontaneous miniature excitatory postsynaptic currents (m.e.p.s.cs) and evoked currents in thin cerebellar slices. Miniature currents have a similar time-course and pharmacology to evoked currents and consist of an exceptionally fast non-NMDA (N-methyl-D-aspartate) component (measured rise-time, 200 microseconds; estimated pre-filtered rise-time less than 100 microseconds; decay time constant, tau = 1.0 ms), followed by 50 pS NMDA channel openings that are directly resolvable. We could find no evidence for the recent proposal that miniature currents in granule cells are mediated solely by NMDA channels with a novel time course. The non-NMDA receptor component of m.e.p.s.cs has a skewed amplitude distribution, which suggests potential complications for quantal analysis. The difference in time course between the m.e.p.s.cs reported here and other synaptic currents in the brain could reflect differences in synaptic function or electrotonic filtering; the relative contribution of these possibilities has yet to be established.
Brief glutamate applications to membrane patches, excised from neurons in the rat visual cortex, were used to assess the role of desensitization in determining the AMPA/kainate receptor-mediated excitatory postsynaptic current (EPSC) time course. A brief (1 ms) application of glutamate (1-10 mM) produced a response that mimicked the time course of miniature EPSCs (mEPSCs). Direct evidence is presented that the rate of onset of desensitization is much slower than the decay rate of the response to a brief application of glutamate, implying that the decay of mEPSCs reflects channel closure into a state readily available for reactivation. Rapid application of glutamate combined with nonstationary variance analysis provided an estimate of the single-channel conductance and open probability, allowing an approximation of the number of available channels at a single synaptic site.
Quantal analysis of synaptic transmission at connections between neurons in the CNS has provided insights concerning the structural constraints on transmitter release and postsynaptic responsiveness. However, it has proven difficult in many cases to resolve the size and variability of a single quantum or to distinguish clear peaks in amplitude histograms of evoked responses, due in part to the superposition of background instrumental and biological noise. These limitations raise questions about recent attempts to use direct or indirect methods of quantal analysis in order to distinguish between pre- and postsynaptic loci of the modifications underlying long-term potentiation, particularly since the interpretations are model-dependent and the statistical treatments and experimental techniques employed incorporate simplifying assumptions not yet proven.
It is now possible to relate the intrinsic electrical properties of particular cells in the cochlear nuclei of mammals with their biological function. In the layered dorsal cochlear nucleus, information concerning the location of a sound source seems to be contained in the spatial pattern of activation of a population of neurons. In the unlayered, ventral cochlear nucleus, however, neurons carry information in their temporal firing patterns. The voltage-sensitive conductances that make responses to synaptic current brief enable bushy cells to convey signals from the auditory nerve to the superior olivary complex with a temporal precision of at least 120 microseconds.
This report focuses on a class of large synaptic endings, the endbulbs of Held. These endings are located in the anteroventral cochlear nucleus and arise from the axons of type I spiral ganglion neurons. Axons were stained with horseradish peroxidase (HRP) using intracellular injections of single fibers or extracellular injections into the auditory nerve. Individual endbulbs or pairs of endbulbs that converged onto the same spherical bushy cell were examined with the aid of a light microscope and subjected to morphometric analyses. Endbulbs of fibers having low spontaneous discharge rates (SR, ≤ 18 spikes/sec) have a more complex shape than those of high SR fibers (> 18 s/s), a feature represented by systematic differences in endbulb silhouette perimeter without differences in endbulb silhouette area. Consequently, the ratio, silhouette area divided by silhouette perimeter, yields a “form factor” separating endbulbs of high SR from those of low SR. High SR fibers had ratios > 0.52 (mean = 0.63 ± 0.09), whereas low SR fibers had ratios <.52 (mean = 0.45 ± 0.06). Pairs of endbulbs with unknown physiological properties had similar form factor values, despite the wide range of values observed in the endbulb population. These data imply that endbulbs converging upon the cell body of a spherical bushy cell arise from fibers of the same SR group.
There is considerable evidence supporting the all-or-none hypothesis for the quantal synaptic potential at synapses in the CNS; a constant amplitude synaptic potential is generated at a single synaptic site each time transmitter is released at that site. The evidence is indirect: the definitive experiment of recording postsynaptically, or focally, from a single release site has not yet been achieved. It is not known if the release of more than one vesicle of transmitter from the same site can be triggered by a single impulse. The evidence for lack of significant variability in the quantal amplitude at release sites arising from the same axon and located at the same electronic location is again indirect but compelling, especially for synapses on DSCT neurons. Quantal size and variability have been measured directly for IPSPs in M cells, and the results confirm earlier assumptions that the variability is small compared with the recording noise. The best explanation for the lack of variability of quantal size is that a quantum of transmitter opens all, or almost all, of the subsynaptic channels. Variations in quantal size for synapses located at different electrotonic locations on dendrites but arising from the same axon might be expected and would be unresolvable using present methods of recording and analysis. The morphology of the connections made by 1a axons with motoneurons indicates that many dendritic connections involve only a small number of contacts at a localized region of the dendrites, and, under these circumstances, any variation in quantal size will not be caused by electrotonic factors. The invariance of quantal size measured at the soma suggests that the quantal EPSP generated at dendritic synapses results from a larger quantal current than that which occurs at somatic synapses.
1. Excitatory postsynaptic potentials (EPSPs) evoked by impulses in single group I muscle afferents were recorded in dorsal spinocerebellar tract (DSCT) neurons in the spinal cords of anesthetized cats. Fluctuations in the amplitude of these single-fiber EPSPs were determined from measurements of EPSP peak amplitude and contaminating noise (800-4600 trials). 2. In a previous study at this connection, we found that these single-fiber EPSPs fluctuated in amplitude between approximately equal, or quantal, increments. However, these quantal fluctuations could not be described by simple binomial statistics (39). In the present study we have applied further analysis procedures to the same single-fiber EPSPs to formulate a more appropriate probabilistic model of transmission at this connection. 3. In the first stage we have demonstrated that each single-fiber EPSP is composed of the sum of a number (3-30) of uniform quantal events, and that there is extremely little variability in the amplitude of the single quantal event. 4. In a further procedure, we have demonstrated that these quantal fluctuations can be described by a compound binomial model in which each underlying quantal event is associated with a particular, but independent, release probability. The results of this analysis indicate that the probability of transmitter release varies considerably between release sites at this connection. (The use of such a compound binomial model reemphasized previous warnings concerning the interpretation of the results of all statistical models of quantal release. Problems regarding the non-unique nature of N, the total population of quantal events, and other such difficulties are discussed.) 5. A model of transmission at this connection is proposed, in which there are a number of "active" release sites, exhibiting generally high release probabilities, and a number of "reserve" release sites, with zero, or close to zero, release probability. The physiological consequences of such a scheme are discussed.
1. The preceding paper (Van der Kloot, 1988) described a method for estimating the timing of quantal releases during an end-plate current. This period of elevated quantal release is called the early release period or ERP (Barrett & Stevens, 1972b). In the present paper, this deconvolution method is used to study the effects of varying quantal output by extracellular ions, stimulus patterns and drugs. 2. The data were obtained by voltage clamping end-plates in low-Ca2+ high-Mg2+ solutions, or in solutions containing tubocurarine (measuring the decay of the miniature end-plate currents (MEPCs) before curarization and assuming a value for MEPC amplitude after curarization). Data were also obtained by extracellular recording in Ca2+-free solution, using a recording pipette filled with CaCl2 and regulating Ca2+ release with a bias current. The three approaches led to similar conclusions. 3. Quantal release rose during the ERP along a sigmoid curve and reached a maximum after about 1.4 ms at 10 degrees C. This is called the time to peak. Quantal release then fell, following an exponential time course with a time constant of about 1.2 ms (10 degrees C). This is called the time constant for decline. 4. The ERP was followed by further, elevated quantal release, at a much lower rate, which declined over a longer time course. This is called late release. The magnitude of late release appears to be almost independent of the magnitude of release during the ERP, although the deconvolution method is a poor one for determining late release. The remainder of the results therefore focus on the ERP. 5. Increasing [Ca2+]o increased quantal output, and the rate of quantal output. It did not change the time to peak or the time constant of decline. Similarly, replacing Ca2+ with Sr2+ did not alter the time course of the ERP. 6. Two-pulse facilitation increased quantal output without changing the time to peak or the time constant of decline. 7. Quantal output was enhanced still more following a brief series of repetitive nerve stimulations. There was a lengthening of the time to peak; there was no change in the decline. The depression produced by longer series of repetitive stimulations did not change the time course of the ERP. 8. 4-Aminopyridine (4-AP) and dimethylsulphoxide (DMSO) increased quantal output and lengthened the time to peak, without altering the time constant for decline. 9. Adenosine decreased quantal output without altering the time course of the ERP.(ABSTRACT TRUNCATED AT 400 WORDS)
1. Transmitter release at excitatory junctions on the opener muscle of the crayfish dactyl was studied by recording junctional potentials with extracellular micro‐electrodes.
2. At low temperatures, evoked release was dispersed sufficiently in time for potentials produced by individual quanta to be counted, and the mean ( m ) and variance (σ ² ) of the quantum content distribution for a series of trials measured directly. These values were used to calculate the average probability of quantal release ( p ), assuming a binomial distribution.
3. For all values of m and p , the observed release pattern (number of 0, 1, 2, 3,... quantal releases during a series of trials) was approximated closely by the corresponding binomial distribution. However, Poisson predictions differed significantly from the observed quantal distribution for values of p > 0·2.
1. Fluctuations in the latency of focally recorded end‐plate currents were analysed to determine the time course of the probabilistic presynaptic process underlying quantal release evoked after single nerve stimuli at the frog neuromuscular junction.
2. The early falling phase of the presynaptic probability function can be fitted by a single exponential over two orders of magnitude of quantal release rate. The time constant of the early falling phase is about 0·5 msec at 11° C, and increases with decreasing temperature with a Q 10 of at least 4 over the range 1–12° C.
3. After this early exponential fall, quantal release probability returns to control levels with a much slower time course.
4. Conditioning nerve stimuli increase the magnitude and slightly prolong the early time course of release evoked by a test stimulus. When facilitation is calculated for matched time intervals following the conditioning and testing stimuli, it is found that the magnitude of the small, late residual tail of release is facilitated by a greater percentage than the magnitude of larger, early portions of release.
5. These results are discussed in terms of the hypothesis (Katz & Miledi, 1968) that evoked release and facilitation are mediated by a common presynaptic factor which activates release in a non‐linear manner.
1. The transient increase in secretion of quanta of acetylcholine (phasic secretion) produced by an action potential or brief depolarizing current pulse in mouse phrenic nerve terminals was examined. 2. Following an activating stimulus, there was a brief delay (minimum latency) followed by a sigmoidal increase in secretion which then decayed exponentially. 3. The minimum latency, rise time and rate of decay of phasic secretion, whether elicited by action potentials or electrotonic depolarization, were all extremely sensitive to temperature, with Q10s as high as 4 at temperatures of 5-15 degrees C. Arrhenius plots of results showed a change in slope with temperature, the change appearing most marked at 20-25 degrees C. 4. Phasic secretion in response to action potentials prolonged by inhibitors of K conductance (4-aminopyridine, uranyl, tetraethylammonium or Zn ions) showed an increase in minimum latency but no other change in time course. 5. Depolarizing pulses of varying width (0.2-2 msec) applied to nerve terminals (in the presence of tetrodotoxin and 4-aminopyridine) affected minimum latency, but had no great effect on the time course of phasic secretion. 6. Neither an increase in extracellular K ion concentration nor a decrease in pH had any effect on the time course of phasic secretion nor was any change produced by ethanol or octanol. 7. Variations in extracellular Ca concentration, substitution of Sr ions for Ca ions and repetitive stimulation, while producing changes in the magnitude of secretion, produced no change in the time course of phasic secretion.
The values of quantal content (m) and quantal amplitude (q) of excitatory postsynaptic potentials (EPSPs) elicited in CA3 neurons by activation of granule cells were estimated in thin hippocampal sections maintained in vitro. For this purpose, DL-homocysteate was administered to granule cells, and trains of EPSPs that were typical for single granule cell activation were recorded from individual CA3 neurons. The amplitudes of the first and second EPSPs in each train were measured. Fron the mean and variance of the amplitude of the EPSPs, the values of q and m were calculated. The values of m and q for the first EPSPs were estimated at 8.3 and 0.28 mV, respectively, on the average. Potentiation of the second EPSPs was accompanied by a two-fold increase in the values of m without changes in the values of q. Therefore, frequency potentiation in synapses between mossy fibers and CA3 neurons may be explained by an increase in number of released quanta. Amplitudes of EPSPs were found to fluctuate in a manner described by Poisson's law.
We have measured the time intervals (latencies) between the delivery of paired stimuli and the appearance of the extracellularly recorded nerve terminal action potentials (n.a.p.s) and end-plate currents (e.p.c.s) at different temperatures in the sciatic nerve, sartorius muscle preparation of the frog. When the interstimulus interval was 70 ms, the n.a.p. and e.p.c. latencies following the second stimulus of the pair were shorter than following the first at 10°C and 15°C, and facilitation of the e.p.c. was observed. The shortened latency was also observed at 20° C by Barton and Cohen (1982), who suggested that this may be related to facilitation. At 5° C, however, the n.a.p. and e.p.c. latencies following the second stimulus were longer than following the first, and facilitation was still observed. When a 30 ms interstimulus interval was used, the changes in latency were even greater at the temperatures studied.
The changes in e.p.c. latency were due to changes in the conduction velocity of the n.a.p., not changes in synaptic delay. At all temperatures and interstimulus intervals studied, the larger amplitude n.a.p.s were conducted more slowly, however, facilitation was always observed. We conclude that changes in the amplitude and conduction velocity of the nerve action potential do not obviously affect facilitation at the frog neuromuscular junction.
Excitatory postsynaptic currents in neurones of the central nervous system have a dual-component time course that results from the co-activation of AMPA/kainate-type and NMDA-type glutamate receptors. New approaches in electrophysiology and molecular biology have provided a better understanding of the factors that determine the kinetics of excitatory postsynaptic currents. Recent studies suggest that the time course of neurotransmitter concentration in the synaptic cleft, the gating properties of the native channels, and the glutamate receptor subunit composition all appear to be important factors.
A fragile balance between excitation and inhibition maintains the normal functioning of the CNS. The dominant inhibitory neurotransmitter of the mammalian brain is GABA, which acts mainly through GABAA and GABAB receptors. Small changes in GABA-mediated inhibition can alter neuronal excitability profoundly and, therefore, a wide range of compounds that clearly modify GABAA-receptor function are used clinically as anesthetics or for the treatment of various nervous system disorders. Recent findings have started to unravel the operation of central GABA synapses where inhibitory events appear to result from the synchronous opening of only tens of GABAA receptors activated by a saturating concentration of GABA. Such properties of GABA synapses impose certain constraints on the physiological and pharmacological modulation of inhibition in the brain.
At some synaptic connections in the central nervous system, amplitude distributions of evoked synaptic currents exhibit surprisingly sharp and regularly spaced peaks. At these connections, detailed analysis of the peaks has led to the proposal that the 'quantal' synaptic current displays very little variability, not only at a release site, but also between release sites. In this study the latter hypothesis has been tested using simulations of evoked transmission. In contrast with previous conclusions, these simulations demonstrate that the experimental observation of regularly spaced peaks in amplitude distributions of synaptic currents is compatible with large underlying differences in the synaptic current amplitudes between release sites. The simulations also reveal that quantal analysis based entirely on the observation and analysis of regularly spaced peaks in evoked synaptic current amplitude distributions, cannot be used with confidence to estimate presynaptic release probabilities, 'quantal' current amplitudes at each release site, or the total number of available release sites. This problem may be a confounding factor in determining whether pre- or postsynaptic changes underlie alterations in synaptic efficacy, such as occurs during long term potentiation.
1. The anteroventral cochlear nucleus (AVCN) contains two principal cell types that receive input from the auditory nerve. Stellate cells receive conventional synapses on their dendrites, and bushy cells of the AVCN receive axosomatic input via large, calyceal terminals (the end bulbs of Held). We have used whole cell patch-clamp recording techniques to study excitatory postsynaptic currents (EPSCs) in these two principal cells of the rat AVCN. 2. EPSCs evoked in stellate cells by stimulation of the auditory nerve were graded with stimulus strength, indicating a high degree of convergence of input to these cells. At depolarized membrane potentials, EPSCs evoked in stellate neurons had a dual-component time course. The slow component was blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV), and the fast component was abolished by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). 3. EPSCs evoked in bushy cells by auditory nerve stimulation were large (50 nS average conductance) and all-or-none at the threshold stimulus level. At -70 mV, the time course of the EPSC was very brief (average time constant of decay 700 microseconds at room temperature). Membrane depolarization revealed a slow component to the EPSC. The fast and slow components were mediated by non-NMDA and NMDA receptors, respectively. The switch-off of end bulb NMDA EPSCs by voltage jumps to the EPSC reversal potential was very rapid, suggesting that the NMDA component arises from sites on or close to the soma. 4. Miniature EPSCs, recorded in the presence of tetrodotoxin (TTX) at depolarized potentials, also had a dual-component time course. The fast and slow components of the miniature EPSCs were blocked by CNQX and APV, respectively. This result indicates that NMDA and non-NMDA receptors can be co-localized at the same, presumably end bulb, release sites. 5. The relative contribution of the slow, NMDA component to the end bulb EPSC declined significantly with age (postnatal days 11-22). 6. These results indicate that both NMDA and non-NMDA receptors underlie excitatory synaptic transmission in the AVCN of young rats. The end bulb synapse onto bushy cells generates a non-NMDA receptor-mediated EPSC with very fast kinetics. NMDA receptors can also mediate synaptic transmission at the end bulb synapse, but their contribution becomes less as the auditory system matures. This finding suggests that NMDA receptors may play an important role in the development of this synapse.
We have investigated the role of AMPA receptor desensitization during transmission at a calyceal synapse. Cyclothiazide blocked the rapid desensitization of AMPA receptors and markedly prolonged the decay time of the evoked excitatory postsynaptic current (PSC). This effect was greater than what would be expected from a simple prolongation of channel open time. Additionally, the drug reduced the depression of PSCs evoked at brief intervals. The effects of cyclothiazide on the PSC were reduced when the level of transmitter release was lowered. These data indicate that AMPA receptors are desensitized by neurotransmitter and that this desensitization depends on the number of quanta in the synaptic cleft. We propose that release of transmitter from many closely spaced sites prolongs the time of receptor-transmitter contact and thereby promotes desensitization. Desensitization may therefore contribute to synaptic depression and prevent the interaction of transmitter quanta within the synaptic cleft.
1. The properties of evoked excitatory postsynaptic currents (EPSCs) and spontaneous miniature excitatory postsynaptic currents (mEPSCs) have been studied in neurons of the nucleus magnocellularis (nMAG), one of the avian cochlear nuclei which receive somatic, calyceal innervation from auditory nerve fibres. Whole-cell patch clamp techniques were used to voltage clamp visually identified neurons in brain slices. 2. EPSCs resulting from activation of single axonal inputs were on average -5.3 nA at a driving force of -25 mV. Current-voltage relationships for the peak of the EPSC were linear with a peak conductance of 211 nS. The rate of EPSC decay showed a linear increase with temperature, with a temperature coefficient (Q10) of 2.2 between 25 and 35 degrees C; in vivo (41 degrees C) the EPSC would decay in 0.2 ms. 3. The EPSC was composed of two pharmacologically and kinetically distinct components: an early phase due to non-NMDA (N-methyl-D-aspartate) receptors and a late phase resulting from NMDA receptors. Both components reversed near 0 mV. While both subtypes of glutamate receptor were activated by transmitter, NMDA receptors had a peak conductance at positive potentials which was only 11% of the peak non-NMDA receptor component. 4. EPSCs during trains of stimuli exhibited a progressive decrease in amplitude. The extent of depression increased with the frequency of stimulation and was reduced by drugs which prevent receptor desensitization, indicating that, in part, postsynaptic factors limit synaptic strength during repetitive synaptic activity. Additionally, the coefficient of variation of the EPSC amplitude increased during trains, consistent with presynaptic depression. 5. mEPSCs occurred randomly in the presence of tetrodotoxin and presumably correspond to transmitter quanta. These synaptic events rose (10-90%) within 100 microseconds and decayed with an exponential of 180 microseconds at 29-32 degrees C. Despite the somatic location of the synapse, mEPSCs varied widely in amplitude, suggesting differences in the quantal synaptic current at each synaptic site. The ratio of the average peak conductance of the EPSC and mEPSC gave an estimated quantal content of 103.
1. To determine the quantal size at retinogeniculate synapses, spontaneous and evoked excitatory postsynaptic currents (EPSCs) were recorded in twelve neurones of the dorsal lateral geniculate nucleus in guinea-pig thalamic slices using the whole-cell patch-clamp technique. We limited our study to the fast non-N-methyl-D-aspartate (NMDA) component of the EPSCs by adding the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid to the perfusion medium. 2. Spontaneous EPSCs occurred at a frequency between 0.5 and 6.6 Hz (mean 2.5 Hz). The modal value of the peak conductance change of spontaneous excitatory events varied between cells from 102 to 179 pS. 3. EPSCs were evoked by electrical stimulation in the optic tract. The peak conductance change of EPSCs evoked by stimulation of a putative single input fibre ranged from 0.6 to 3.4 nS (mean 1.7 nS). 4. To resolve the quantal components of evoked EPSCs the external Ca2+ concentration was reduced and the external Mg2+ concentration increased for four cells. In this condition failures occurred and the amplitude histograms were multimodal with approximately equidistant peaks. 5. These multimodal histograms could be fitted by a sum of Gaussian functions with mean values corresponding to integer multiples of the modal value of the spontaneous EPSCs for the same cell. Thus, the quantal size of evoked EPSCs was the same as the modal value of spontaneous EPSCs. The mean of the apparent quantal conductance change was 138 pS. The estimated number of quanta released by stimulating a putative single input fibre in the control condition ranged from 4 to 27 (mean 13).(ABSTRACT TRUNCATED AT 250 WORDS)
A change in the probability of neurotransmitter release (Pr) is an important mechanism underlying synaptic plasticity. Although Pr is often assumed to be the same for all terminals at a single synapse, this assumption is difficult to reconcile with the nonuniform size and structure of synaptic terminals in the central nervous system. Release probability was measured at excitatory synapses on cultured hippocampal neurons by analysis of the progressive block of N-methyl-D-aspartate receptor-mediated synaptic currents by the irreversible open channel blocker MK-801. Release probability was nonuniform (range of 0.09 to 0.54) for terminals arising from a single axon, the majority of which had a low Pr. However, terminals with high Pr are more likely to be affected by the activity-dependent modulation that occurs in long-term potentiation.
When an action potential reaches a synaptic terminal, fusion of a transmitter-containing vesicle with the presynaptic membrane occurs with a probability (pr) of less than one. Despite the fundamental importance of this parameter, pr has not been directly measured in the central nervous system. Here we describe a novel approach to determine pr, monitoring the decrement of NMDA (N-methyl-D-aspartate)-receptor mediated synaptic currents in the presence of the use-dependent channel blocker MK-801 (ref. 2). On a single postsynaptic CA1 hippocampal slice neuron, two classes of synapses with a sixfold difference in pr are resolved. Synapses with low pr contribute to over half of transmission and are more sensitive to drugs enhancing transmitter release. Switching between these two classes of synapses provides the potential for large changes in synaptic efficacy and could underlie forms of activity-dependent plasticity.
1. Excitatory postsynaptic currents (EPSCs) were recorded in CA3 pyramidal cells of hippocampal slices of 15- to 24-day-old rats (22°C) using the whole-cell configuration of the patch clamp technique. 2. Composite EPSCs were evoked by extracellular stimulation of the mossy fibre tract. Using the selective blockers 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D-2-amino-5-phosphonopentanoic acid (APV), a major α-amino-3-hydroxy-5-methylisoxazole-4-propionate AMPA/kainate receptor-mediated component and a minor NMDA receptor-mediated component with slower time course were distinguished. For the AMPA/kainate receptor-mediated component, the peak current-voltage (I-V) relation was linear, with a reversal potential close to 0 mV. The half-maximal blocking concentration of CNQX was 353 nM. 3. Unitary EPSCs of the mossy fibre terminal (MF)-CA3 pyramidal cell synapse were evoked at membrane potentials of -70 to -90 mV by low-intensity extracellular stimulation of granule cell somata using fine-tipped pipettes. The EPSC peak amplitude as a function of stimulus intensity showed all-or-none behaviour. The region of low threshold was restricted to a few micrometres. This suggests that extracellular stimulation was focal, and that the stimulus-evoked EPSCs were unitary. 4. Latency and rise time histograms of EPSCs evoked by granule cell stimulation showed narrow unimodal distributions within each experiment.
In the molecular layer of the cerebellar cortex, Purkinje cells and interneurons receive a common excitatory input from parallel fibers. The AMPA/kainate receptor-mediated parallel fiber excitatory postsynaptic current (EPSC) recorded in Purkinje cells decays much more slowly than that recorded in interneurons. We show that this slowness of decay does not result from dendritic filtering and that it is unlikely to reflect the deactivation kinetics of the postsynaptic receptors. Agents blocking glutamate uptake prolong the EPSC in Purkinje cells. We conclude that the slow EPSC decay results from the continued presence of transmitter glutamate. This may be due to retarded transmitter diffusion around spines or to cross-talk between neighboring active synapses.
In the past year, important advances have been made in the understanding of quantal neurotransmission at central synapses. These include new statistical tests for the significance of quantal peaks in synaptic amplitude histograms, greater understanding of possible sources of quantal variance, and new attempts to undertake a rigorous quantal analysis of neurotransmission.
We have studied the effects of blockers of glutamate transporters on excitatory synaptic transmission to determine whether transporters increase the clearance rate of transmitter from the synaptic cleft on the millisecond time scale. The transporter blockers Li+ and THA increased the amplitude, but not the decay time, of spontaneous miniature AMPA receptor EPSCs recorded at 34 degrees C but not 24 degrees C. Evoked AMPA receptor EPSCs were similarly affected by THA. The rapidly dissociating AMPA receptor competitive antagonist PDA inhibited evoked AMPA receptor EPSCs less in the presence of THA at both temperatures, implying that transporter blockade slows clearance. We suggest that transporters speed glutamate clearance mainly by binding glutamate and that AMPA receptors are not saturated by synaptically released glutamate at 34 degrees C.
Miniature excitatory postsynaptic currents (mEPSCs) were elicited from small numbers of release sites after brief microperfusion of Ba2+ and K+ onto proximal dendritic processes of hippocampal neurons in culture. Temporal summation of closely timed mEPSCs deviated significantly from linearity. The number of instances of closely timed mEPSCs that were also closely matched in terms of peak amplitudes was significantly greater than that expected by chance. Amplitude pairing became statistically more significant after prolongation of mEPSC duration and inhibition of glutamate receptor desensitization with cyclothiazide. These results are best explained by postsynaptic receptors that approach saturation after quantal release of transmitter.
This review focuses on two aspects of synaptic transmission that have recently attracted considerable interest: the quantal release of neurotransmitter at synapses, and a phenomenon, hippocampal long-term potentiation (LTP), that is widely accepted as the substrate for certain forms of memory. The discussion is divided into two main parts. The first presents 'standard views' of synaptic transmission and of LTP, and the second deals with challenges to and uncertainties about these views. The standard views presented are not my own - indeed, I would dispute a variety of their assumptions - but rather they represent classic or popular notions that serve as a framework for the critical discussion in the second part of the presentation. Such a short review of so vast a subject cannot, of course, be comprehensive, and I have selected for discussion several specific topics that currently are being most actively investigated and debated. Each of the sections can be read independently of the others.