Quantal analysis of long-term potentiation of combined neuronal postsynaptic potentials on hippocampal slices in vitro

Excitatory postsynaptic potentials (EPSP) were recorded from 14 neurons in guinea pig hippocampal slices (area CAl) after stimulating the stratum radiatum (Schaffer collaterals) and stratum oriens. An increase occurring in EPSP amplitude in 7 units (9 pathways) recorded 15–45 min after tetanic stimulation of Schaffer collaterals is viewed as long-term potentiation (LTP). Statistical analysis conducted according to two sets of quantal theory (histogram and variance methods) showed an increase in mean quantal content (m) during LTP. An increase in quantal size, found only when using the histogram method, did not correlate with LTP level. This increase is thought to be associated with the considerably greater sensitivity of the histogram method to noise level in comparison with the variance method, the latter being more reliable with signals of high noise level. The increase found in m using both methods matches findings previously obtained for the whole brain; it also points to presynaptic location of mechanisms responsible for raised synaptic efficacy during LTP.Institute for Brain Research, All-Union Mental Health Research Center, Academy of Medical Sciences of the USSR, Moscow. Max-Planck Institute of Biophysical Chemistry, Göttingen, West Germany. Institute of Zoology, Jagiellonian University, Cracow, Poland. Translated from Neirofiziologiya, Vol. 22, No. 4, pp. 465–472, July–August, 1990.     

Quantal analysis of long-term posttetanic changes in minimal postsynaptic potentials of hippocampal slices in vitro

Minimal excitatory postsynaptic potentials (EPSP) were investigated in 13 neurons under single or double-pulse near-threshold microstimulation of the radial layer (Schaffer's collaterals) and stratum oriens in surviving hippocampal slices (area CA1) in guinea pigs. The amplitude of 23 EPSP (9 units; 12 pathways) rose after tetanization of Schaffer's collaterals over a 5–55 min period, taken as long-term potentiation (LTP). Statistical analysis conducted using four methods of quantal hypothesis based on a binomial approximation revealed an increase in mean quantal content (m) during LTP. The rise in quantal size was only statistically significant when using data obtained from a section of these methods (mainly for stretches of over 15 min following tetanization) and shows no correlation with intensity of LTP. The pronounced rise in m demonstrated using different methods matches data from experiments on intact animals and indicates a presynaptic location of the mechanisms underlying protracted persistence of residual tetanization lasting some tens of minutes.Institute for Brain Research, All-Union Mental Health Research Center, Academy of Medical Sciences of the USSR, Moscow. Max-Planck Institute of Biophysical Chemistry, Göttingen, Germany. Zoological Institute, Jagiellonian University, Cracow, Poland. Translated from Neirofiziologiya, Vol. 22, No. 6, pp. 752–761, November–December, 1990.     

Expression mechanisms of long-term potentiation in the hippocampus

We have taken a number of different experimental approaches to address whether long-term potentiation (LTP) in hippocampal CA1 pyramidal cells is due primarily to presynaptic or postsynaptic modifications. Examination of miniature EPSCs or EPSCs evoked using minimal stimulation indicate that quantal size increasing during LTP. The conversion of silent to functional synapses may contribute to the LTP-induced changes in mEPSC frequency and failure rate that previously have been attributed to an increase in the probability if transmitter release.     

Joint application of the independent component analysis and the nonstationary fluctuation analysis for the investigation of early stages of long-term potentiation in the rat hippocampus

An invariable interest in mechanisms of synaptic plasticity gave birth to several specific methods of evoked postsynaptic responses analysis: quantal analysis, component analysis, nonstationary fluctuation analysis (NSFA) etc. The major part of these methods are not standardized yet however, that can lead to obtaining different (and even contradictory) results in similar experiments performed by different scientific groups. This paper issues the experiments for revealing pre- or postsynaptic location of the synaptic plasticity mechanisms during the early phases of the long-term potentiation (LTP). On a model we analyse how an estimation of the single-channel current made by the NSFA is influenced by changes in the evoked postsynaptic currents shape variability. A hypothesis is made that the apparent increase in the AMPA-receptor single-channel current, reported in some works for early LTP stages, could be concerned with the increase in the postsynaptic response shape variability rather then with real increase in AMPA-receptor channels conductivity. The shape of the postsynaptic responses can become more variable after LTP-associated unsilencing of the previously silent synapses. A new method of independent component analysis (ICA) is introduced to check this hypothesis first on model and than on physiological data. The results of the experiments in general agree with the hypothesis suggested.     

Determination of Pre- and post-tetanic quantum values in excitatory synapses of CA1 area of hippocampal slices: Application of the deconvolution method

The deconvolution method, i.e., reconstruction of noise-free discrete amplitude distributions of excitatory postsynaptic potentials (EPSPs), has been tested in computer experiments. A generalized quantal model without constraints on distribution of transmitter release probabilities (p) was used. Complex binomial amplitude distributions with a number of discrete components n between 2 and 10 and with different p were simulated. The distance between the discrete components (i.e., the quantum value, v) was determined from simulated distributions with the number of amplitudes N equal to 1000 or 100. For samples with N=100, mean v values were calculated from 10 distributions. The solutions obtained by deconvolution were close to (within ±20% of) the simulated v values when the standard deviation of the noise Sn0.5v. For Sn=0.5–2.0v, v grew in proportion to Sn and was overestimated. A similar correlation was found for v calculated from amplitudes of minimal EPSPs recorded from CA1 area neurons of guinea pig hippocampal slices. However, no significant correlation between v and Sn was found for v>135 µV, i.e., for estimates exceeding the total mean value. A substantial increase in the mean quantal content (m) with a relatively slight increase in v during longterm potentiation (LTP) was confirmed for those cases. Methods of elimination of erroneous v estimates were considered. Rejection of cases with v>2.5Sn (when N=1000) or v>3Sn (when N=100) eliminated erroneous estimates with a high probability but demanded very low noise levels. A new criterion for recognition of correct estimates is suggested. It is based on artificial contamination of the measured amplitudes by the Gaussian noise (noise addition) and on the double-step dependence of v on Sn described above. The solutions selected with this criterion also support a predominant increase of m during LTP with a slight growth of v.Translated from Neirofiziologiya, Vol. 25, No. 2, pp. 84–91, March–April, 1993.     

Hippocampal LTP is accompanied by enhanced F-actin content within the dendritic spine that is essential for late LTP maintenance in vivo

The dendritic spine is an important site of neuronal plasticity and contains extremely high levels of cytoskeletal actin. However, the dynamics of the actin cytoskeleton during synaptic plasticity and its in vivo function remain unclear. Here we used an in vivo dentate gyrus LTP model to show that LTP induction is associated with actin cytoskeletal reorganization characterized by a long-lasting increase in F-actin content within dendritic spines. This increase in F-actin content is dependent on NMDA receptor activation and involves the inactivation of actin depolymerizing factor/cofilin. Inhibition of actin polymerization with latrunculin A impaired late phase of LTP without affecting the initial amplitude and early maintenance of LTP. These observations suggest that mechanisms regulating the spine actin cytoskeleton contribute to the persistence of LTP.     

Effect of dimedrol on the function of the neuromuscular synapse and the generation of action potentials by motor nerve fibers

Microelectrode registration of synaptic potentials in the frog cutaneous-pectoris muscle has shown dimedrol (7.9 X 10(-5) M) to act on synaptic transmission decreasing the quantal content, estimated by mean EPP amplitude to mean miniature EPP amplitude ratio, the quantal content calculated by variation coefficient of EPP amplitude being unaffected. The data suggest possible transmitter release and depletion of mediator stock. The experiments on isolated motor nerve fibers have demonstrated dimedrol to cause the increase in transmitter release probability by widening the action potentials in the terminals and thus enhancing Ca2+ influx.     

Effects of caspase-3 inhibition on long-term potentiation in the hippocampus: analysis of paired pulse facilitation

Incubation of the rat hippocampal slices with caspase-3 inhibitor Z-DEVD-FMK resulted in a time-dependent decrease in long-term potentiation (LTP) magnitude. Analysis of paired pulse facilitation at a 70-msec interval revealed that, after caspase-3 inhibition, the increase in the amplitude of the second response in the pair during LTP that was characteristic for control slices, did not occur. In this situation, the LTP magnitude depended on differences in the amplitudes of the first and second responses before the LTP induction. LTP was absent in slices with initially high efficacy of the afferent stimulation and respective low paired pulse facilitation. The Caspase inhibition seems to prevent structural reorganization during the LTP related to involvement into the response of new synapses and neurons.     

The synaptic bouton acts like a salt shaker

The physiological quantal responses at the neuromuscular junction and the bouton-neuron show two classes based on amplitude such that the larger class is about 10 times that of the smaller class; and, the larger class is composed of the smaller class. The ratio of the two classes changes with synaptogenesis, degeneration, nerve stimulation, and is readily altered with various challenges (ionic, tonicity, pharmacological agents). Statistical analyses demonstrate that each bouton or release site at the neruomuscular junction (NMJ) secretes a standard amount of transmitter (one quantum) with each action potential. The amount of transmitter secreted (quantal size) is frequency dependent. The quantal-vesicular-exocytotic (QVE) hypothesis posits that the packet of secreted transmitter is released from one vesicle by exocytosis. The QVE hypothesis neither explains two quantal classes and subunits nor exocytosis of only one vesicle at each site. The latter observation requires a mechanism to select one vesicle from each array. Our porocytosis hypothesis states that the quantal packet is pulsed from an array of secretory pores. A salt shaker delivers a standard pinch of salt with each shake because salt flows through all openings in the cap. The variation in the pinch of salt or transmitter decreases with an increase in array size. The docked vesicles, paravesicular matrix, and porosomes (pores) of a release site form the secretory unit. In analogy with the sacromere as the functional unit of skeletal muscle, we term the array of docked vesicles and paravesicular grid along with the array of postsynaptic receptors a synaptomere. Pulsed secretion from an array explains the substructure of the postsynaptic response (quantum). The array guarantees a constant amount of secretion with each action potential and permits a given synapse to function in different responses because different frequencies would secrete signature amounts of transmitter. Our porocytosis hypothesis readily explains a change in quantal size during learning and memory with an increase in the number of elements (docked vesicles) composing the array.     

Low intensity tetanic stimulation induces long-term potentiation in hippocampal neurons

A minimal intensity of the stimulation necessary for the induction of long-term potentiation of synaptic transmission (LTP) was investigated by intracellular recording in guinea pig in vitro hippocampal slices. High frequency stimulation of afferent fibres at intensities evoking in CA 1 neurons control excitatory postsynaptic potentials (EPSPs) of amplitudes 1-5 mV, resulted usually in a long-lasting increase in response amplitude. LTP was not observed at lower stimulus strength. The coactivation of a certain, though small number of synaptic contacts is thus necessary for the production of LTP.     

The synaptic bouton acts like a slat shaker

The physiological quantal responses at the neuromuscular junction and the bouton-neuron show two classes based on amplitude such that the larger class is about 10 times that of the smaller class; and, the larger class is composed of the smaller class. The ratio of the two classes changes with synaptogenesis, degeneration, nerve stimulation, and is readily altered with various challenges (ionic, tonicity, pharmacological agents). Statistical analyses demonstrate that each bouton or release site at the neruomuscular junction (NMJ) secretes a standard amount of transmitter (one quantum) with each action potential. The amount of transmitter secreted (quantal size) is frequency dependent. The quantal-vesicular-exocytotic (QVE) hypothesis posits that the packet of secreted transmitter is released from one vesicle by exocytosis. The QVE hypothesis neither explains two quantal classes and subunits nor exocytosis of only one vesicle at each site. The latter observation requires a mechanism to select one vesicle from each array. Our porocytosis hypothesis states that the quantal packet is pulsed from an array of secretory pores. A salt shaker delivers a standard pinch of salt with each shake because salt flows through all openings in the cap. The variation in the pinch of salt or transmitter decreases with an increase in array size. The docked vesicles, paravesicular matrix, and porosomes (pores) of a release site form the secretory unit. In analogy with the sacromere as the functional unit of skeletal muscle, we term the array of docked vesicles and paravesicular grid along with the array of postsynaptic receptors a synaptomere. Pulsed secretion from an array explains the substructure of the postsynaptic response (quantum). The array guarantees a constant amount of secretion with each action potential and permits a given synapse to function in different responses because different frequencies would secrete signature amounts of transmitter. Our porocytosis hypothesis readily explains a change in quantal size during learning and memory with an increase in the number of elements (docked vesicles) composing the array.     

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

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

Homeostatic control of presynaptic release is triggered by postsynaptic membrane depolarization.

Homeostatic mechanisms regulate synaptic function to maintain nerve and muscle excitation within reasonable physiological limits. The mechanisms that initiate homeostasic changes to synaptic function are not known. We specifically impaired cellular depolarization by expressing the Kir2.1 potassium channel in Drosophila muscle. In Kir2.1-expressing muscle there is a persistent outward potassium current ( approximately 10 nA), decreased muscle input resistance (50-fold), and a hyperpolarized resting potential. Despite impaired muscle excitability, synaptic depolarization of muscle achieves wild-type levels. A quantal analysis demonstrates that increased presynaptic release (quantal content), without a change in quantal size (mEPSC amplitude), compensates for altered muscle excitation. Because morphological synaptic growth is normal, we conclude that a homeostatic increase in presynaptic release compensates for impaired muscle excitability. These data demonstrate that a monitor of muscle membrane depolarization is sufficient to initiate synaptic homeostatic compensation.     

Synaptic plasticity at the cerebellum input stage: mechanisms and functional implications

The mf-GrC relay provides the case of a synapse at which elementary neurotransmission mechanisms are particularly well understood allowing a precise investigation of synaptic plasticity. An interesting consequence is that a presynaptic mechanism of LTP could be precisely documented on the basis of quantal analysis. By being presynaptically expressed, LTP becomes instrumental to regulation of short-term synaptic dynamics thereby controlling time-dependent transformations of the incoming mossy fiber input. It is unknown to what extent these considerations could be generalized, but early observations were provided for comparable concepts and mechanisms in neocortical synapses (Tsodyks and Markram, 1997). Although several aspects remain to be investigated, mf-GrC LTP provides a wide substrate for information storage in the cerebellum. In the rat cerebellum, there are 10(11) GrCs and 4 times as many mf-GrC synapses. Mathematical models have suggested that mf-GrC LTP improves mutual information transfer, and that the combination of synaptic and non-synaptic changes improves sparse representation of the mf input (Schweighofer et al., 2000; Philipona et al., 2003). Moreover, mf-GrC LTP could play a key role in regulating neurotransmission dynamics, implementing adaptability in delay lines early envisioned by Breitenberg (1967) and then revisited by Medina and Mauk (2002). These observations challenge the simple view of spatial pattern separation proposed by Marr (1969). The potential consequences of mf-GrC LTP need to be further investigated and confronted with computational models of the cerebellar network.     

Aminoglycoside antibiotics affect hippocampal LTP: a comparative study with the N-type calcium antagonist omega-conotoxin-GVIA.

The in vitro activity of N-type calcium antagonists such as omega-conotoxin-GVIA and the aminoglycoside antibiotics neomycin and streptomycin was studied in rat hippocampal slices. The effects of the drugs were tested on basal CA1 synaptic transmission and on the hippocampal long-term potentiation (LTP) induced by tetanic electrical stimulation and by increasing (4mM) the calcium concentration. Omega-conotoxin-GVIA, neomycin and streptomycin were able to significantly reduce the amplitude of the CA1 population spike at 1 microM, 0.5 mM and 1 mM, respectively. In addition, the drugs affected the induction and maintenance of the CA1 tetanic and calcium-induced LTP at concentrations which did not modify the magnitude of the control CA1 population spike. Omega-conotoxin-GVIA (0.5 microM), neomycin (0.3 mM) and streptomycin (0.7 mM) perfused for 60 min, before inducing LTP, prevented the subsequent increase of the CA1 population spike in all the experiments. The same concentrations of these drugs perfused for 60-min after a previously established LTP significantly reduced the amplitude of the CA1 population spike. The results promote a role for the N-type calcium channels and for the release of neurotransmitters in both the induction and the maintenance of hippocampal LTP.     

Quantal release of ATP in mouse cortex

Transient currents occur at rest in cortical neurones that reflect the quantal release of transmitters such as glutamate and gamma-aminobutyric acid (GABA). We found a bimodal amplitude distribution for spontaneously occurring inward currents recorded from mouse pyramidal neurones in situ, in acutely isolated brain slices superfused with picrotoxin. Larger events were blocked by glutamate receptor (AMPA, kainate) antagonists; smaller events were partially inhibited by P2X receptor antagonists suramin and PPADS. The decay of the larger events was selectively prolonged by cyclothiazide. Stimulation of single intracortical axons elicited quantal glutamate-mediated currents and also quantal currents with amplitudes corresponding to the smaller spontaneous inward currents. It is likely that the lower amplitude spontaneous events reflect packaged ATP release. This occurs with a lower probability than that of glutamate, and evokes unitary currents about half the amplitude of those mediated through AMPA receptors. Furthermore, the packets of ATP appear to be released from vesicle in a subset of glutamate-containing terminals.     

脑发育不同阶段慢性铅暴露对大鼠在体海马齿状回LTP的影响

目的和方法：探讨脑发育不同阶段慢性铅暴露对在体海马ＬＴＰ的影响。应用细胞外微电极记录单脉冲刺激穿通路纤维在海马齿状回诱发的群体锋电位（ＰＳ）,观察母体期、断乳后及出生前后持续性慢性铅暴露大鼠于高频刺激（ＨＦＳ）前后的ＰＳ幅值变化,并与对照组相比较。结果：ＨＦＳ前,基线记录的各铅暴露组ＰＳ平均幅值及峰潜伏期与对照组无显著差异;ＨＦＳ后,各铅暴露组ＬＴＰ发生率虽与对照组无显著差异,但铅暴露组的ＬＴＰ增幅减小,并出现了短时增强转为抑制及ＬＴＤ型反应。统计显示各铅暴露组ＨＦＳ后ＰＳ振幅的平均增强率显著低于对照组：对照组平均增强至基线值的１３８．２％,母体期铅暴露组为基线值的１０８．８％,断乳后铅暴露组为基线值的１０７．８％,持续铅暴露组为基线值的１０４．４％。结论：脑发育任一阶段的慢性铅暴露均可损害海马ＬＴＰ的在体诱导和维持,且以维持过程受损为主;与发育成熟海马相比,未成熟期海马对铅的神经毒性更为敏感,突触可塑性更易受损。     

The development of nerve-muscle junctions in Rana catesbeiana tadpoles

The physiological properties of developing nerve-muscle junctions in Rana catesbeiana tadpoles are described. Developing neurons at different stages of ontogeny formed functional synaptic connections with a section of tail muscle implanted in place of the hind limb bud. Transmission is quantal in nature, sensitive in normal ways to calcium and magnesium concentrations, and conforms to a Poisson distribution. The quantal content is initially low and increases with development. Mepp's occur randomly and have low frequencies which increase slightly with development. The size of a single quantum of transmitter does not change during development. The muscle fibers are multiply innervated, resulting in Epp's with distinct peaks and complex skewed mepp amplitude histograms. No significant increases were observed in the level of differentiation of the developing motor neurons as a result of their having innervated a portion of mature tail muscle. The numbers of developing motor neurons increased in the experimental lateral motor column, and a lag in their maturity was observed relative to motor neurons in the control lateral motor column.     

Long-term potentiation: outstanding questions and attempted synthesis

This article attempts an overview of the mechanism of NMDAR-dependent long-term potentiation (LTP) and its role in hippocampal networks. Efforts are made to integrate information, often in speculative ways, and to identify unresolved issues about the induction, expression and molecular storage processes. The pre/post debate about LTP expression has been particularly difficult to resolve. The following hypothesis attempts to reconcile the available physiological evidence as well as anatomical evidence that LTP increases synapse size. It is proposed that synapses are composed of a variable number of trans-synaptic modules, each having presynaptic release sites and a postsynaptic structure that can be AMPAfied by the addition of a hyperslot assembly that anchors 10-20 AMPA channels. According to a newly developed view of transmission, the quantal response is generated by AMPA channels near the site of vesicle release and so will depend on whether the module where release occurs has been AMPAfied. LTP expression may involve two structurally mediated processes: (i) the AMPAfication of existing modules by addition of hyperslot assemblies: this is a purely postsynaptic process and produces an increase in the probability of an AMPA response, with no change in the NMDA component; and (ii) the addition of new modules: this is a structurally coordinated pre/post process that leads to LTP-induced synapse enlargement and potentiation of the NMDA component owing to an increase in the number of release sites (the number of NMDA channels is assumed to be fixed). The protocol used for LTP induction appears to affect the proportion of these two processes; pairing protocols that involve low-frequency presynaptic stimulation induce only AMPAfication, making LTP purely postsynaptic, whereas high-frequency stimulation evokes both processes, giving rise to a presynaptic component. This model is capable of reconciling much of the seemingly contradictory evidence in the pre/post debate. The structural nature of the postulated changes is relevant to a second debate: whether a CaMKII switch or protein-dependent structural change is the molecular memory mechanism. A possible reconciliation is that a reversible CaMKII switch controls the construction of modules and hyperslot assemblies from newly synthesized proteins.