Probability Of Transmitter Release Research Articles

Reluctant Vesicles Coaxed into the Limelight

Synapses respond to brief, repetitive stimulation with synaptic depression when initial transmitter release probability is high. Vesicle depletion has been a long-standing hypothesis for depression, but results unexplained by the depletion hypothesis have been nagging. In this issue of Neuron, Xu and Wu show that, under some conditions, calcium current inactivation explains stimulus-dependent depression at the calyx of Held.

Adenosine Mediation of Presynaptic Feedback Inhibition of Glutamate Release

Conditions of increased metabolic demand relative to metabolite availability are associated with increased extracellular adenosine in CNS tissue. Synaptic activation of postsynaptic NMDA receptors on neurons of the cholinergic brainstem arousal center can increase sufficient extracellular adenosine to act on presynaptic A1 adenosine receptors (A1ADRs) of glutamate terminals, reducing release from the readily releasable pool. The time course of the adenosine response to an increase in glutamate release is slow (tau > 10 min), consistent with the role of adenosine as a fatigue factor that inhibits the activity of cholinergic arousal centers to reduce arousal.

Fast neurotransmission in the rat medial preoptic nucleus

The functional properties of neurotransmission in the medial preoptic nucleus (MPN) were studied in a brain slice preparation from young male rats. The aims were to evaluate the thin slice preparation for studying evoked synaptic responses in MPN neurons, to characterize the fast responses triggered by activation of presynaptic nerve fibers in the MPN, and to identify the involved receptor types. Presynaptic stimulation within the MPN evoked postsynaptic voltage and current responses that were blocked by 200 μM Cd 2+ or by 2.0 μM tetrodotoxin and were attributed to action potential-evoked transmitter release. The relation to stimulus strength and comparison with spontaneous synaptic currents suggested that in many cases only one presynaptic nerve fiber was excited by the stimulus. Furthermore, the transmission was probabilistic in nature, with frequent failures. Thus, response probability, most likely reflecting transmitter release probability, could be evaluated in the thin slice preparation. Evoked excitatory postsynaptic currents recorded under voltage-clamp conditions were, due to kinetics, I– V relation, and pharmacological properties, attributed to AMPA/kainate receptors and NMDA receptors, whereas inhibitory currents were attributed to GABA A receptors. No responses that could be attributed to glycine or other types of primary transmitters were detected. Although serotonin (5-HT) did not appear to function as a primary transmitter, glutamate- as well as GABA-mediated transmission was suppressed by 500 μM 5-HT, with a clear reduction in response probability observed. 5-HT also reduced the frequency, but not the amplitude, of spontaneous postsynaptic currents and was therefore ascribed a presynaptic site of action.

Stabilizing effects of extracellular ATP on synaptic efficacy and plasticity in hippocampal pyramidal neurons

The role of adenosine triphosphate (ATP) as a neurotransmitter and extracellular diffusible messenger has recently received considerable attention because of its possible participation in the regulation of synaptic plasticity. However, the possible contribution of extracellular ATP in maintaining and regulating synaptic efficacy during intracellular ATP depletion is understudied. We tested the effects of extracellular ATP on excitatory postsynaptic currents (EPSCs) evoked in CA1 pyramidal neurons by Schaffer collateral stimulation. In the absence of intracellular ATP, EPSC rundown was neutralized when a low concentration of ATP (1 microm) was added to the extracellular solution. Adenosine and ATP analogues did not prevent the EPSC rundown. The P(2) antagonists piridoxal-5'-phosphate-azophenyl 2',4'-disulphonate (PPADS) and reactive blue-2, and the P(1) adenosine receptor antagonist 8-cyclopentyltheophylline (CPT) had no detectable effects in cells depleted of ATP. However, the protective action of extracellular ATP on synaptic efficacy was blocked by extracellular application of the protein kinase inhibitors K252b and staurosporine. In contrast, K252b and staurosporine per se did not interfere with synaptic transmission in ATP loaded cells. Without intracellular ATP, bath-applied caffeine induced a transient (< 35 min) EPSC potentiation that was transformed into a persistent long-term potentiation (> 80 min) when 1 microm ATP was added extracellularly. An increased probability of transmitter release paralleled the long-term potentiation induced by caffeine, suggesting that it originated presynaptically. Therefore, we conclude that extracellular ATP may operate to maintain and regulate synaptic efficacy and plasticity in conditions of abnormal intracellular ATP depletion by phosphorylation of a surface protein substrate via activation of ecto-protein kinases.

Activity‐dependent maturation of excitatory synaptic connections in solitary neuron cultures of mouse neocortex

Activity plays important roles in the formation and maturation of synaptic connections. We examined these roles using solitary neocortical excitatory neurons, receiving only self-generated synaptic inputs, cultured in a microisland with and without spontaneous spike activity. The amplitude of excitatory postsynaptic currents (EPSCs), evoked by applying brief depolarizing voltage pulses to the cell soma, continued to increase from 7 to 14 days in culture. Short-term depression of EPSCs in response to paired-pulse or 10-train-pulse stimulation decreased with time in culture. These developmental changes were prevented when neurons were cultured in a solution containing tetrodotoxin (TTX). The number of functional synapses estimated by recycled synaptic vesicles with FM4-64 was significantly smaller in TTX-treated than control neurons. However, the miniature EPSC amplitude remained unchanged during development, irrespective of activity. Transmitter release probability, assessed by use-dependent blockade of N-methyl-D-aspartate receptor-mediated EPSCs with MK-801, was higher in TTX-treated than control neurons. Therefore, the activity-dependent increase in EPSC amplitude was mainly ascribed to the increase in synapse number, while activity-dependent alleviation of short-term depression was mostly ascribed to the decrease in release probability. The effect of activity blockade on short-term depression, but not EPSC amplitude, was reversed after 4 days of TTX removal, indicating that synapse number and release probability are controlled by activity in very different ways. These results demonstrate that activity regulates the conversion of immature synapses transmitting low-frequency input signals preferentially to mature synapses transmitting both low- and high-frequency signals effectively, which may be necessary for information processing in mature cortex.

Stressor‐related impairment of synaptic transmission in hippocampal slices from α‐synuclein knockout mice

The role of alpha-synuclein (alpha-Syn) has recently received considerable attention because it seems to play a role in Parkinson's disease (PD). Missense mutations in the alpha-Syn gene were found in autosomal dominant PD and alpha-Syn was shown to be a major constituent of protein aggregates in sporadic PD and other synucleinopathies. Under normal conditions, alpha-Syn protein is found exclusively in synaptic terminals. However, the potential participation of alpha-synuclein in maintaining and regulating synaptic efficacy is unknown. We have investigated the excitatory synaptic modulation of alpha-synuclein in CA1 pyramidal neurons, using the in vitro hippocampal slice technique. The 4-aminopyridine-induced increase of both spontaneous excitatory postsynaptic current (EPSC) frequency and amplitude was significantly higher in alpha-Syn wild-type than knockout mice, whereas basal spontaneous EPSC frequency and amplitude was similar in both animals. As the spontaneous synaptic activity was abolished by tetrodotoxin, which indicates that it was a result of action potential-mediated transmitter release from presynaptic terminals, spontaneous EPSC changes observed in alpha-Syn knockout mice suggest that these animals present a modification of synaptic transmission with a presynaptic origin. Presynaptic depression of evoked EPSCs by hypoxia or adenosine was significantly larger in alpha-Syn knockout than in wild-type mice, further supporting the hypothesis of regulation of synaptic transmission by alpha-Syn. Together, these observations indicate that the loss of alpha-Syn reduces synaptic efficacy when the probability of transmitter release is modified. We conclude that alpha-Syn might have important actions on the maintenance of the functional integrity of synaptic transmission and its regulation in hippocampus.

Metaplastic Facilitation and Ultrastructural Changes in Synaptic Properties Are Associated with Long-Term Modulation of the Lamprey Locomotor Network

The neuropeptide substance P evokes a long-term protein synthesis-dependent increase in the cycle frequency of locomotor network activity in the lamprey. Although cellular and synaptic mechanisms that could induce this effect have been identified, nothing is known of the underlying maintenance mechanisms. These mechanisms have been examined here. Substance P potentiates low-frequency-evoked EPSPs from excitatory network interneurons. It also converts the depression of the EPSP during spike trains into facilitation, an example of metaplasticity. The metaplasticity was associated with a reduction of the transmitter release probability but an increase in the number of release sites. Although the potentiation of low-frequency-evoked EPSPs recovered within 1 hr, the metaplastic facilitation had not recovered 3-4 hr after substance P application. The metaplasticity thus extended into the protein synthesis-dependent maintenance phase of the network modulation, making it the only identified cellular or synaptic effect of substance P to last this long. It also had the same induction and maintenance features as the network burst frequency modulation, further suggesting that the two effects were related. Long-term changes in synaptic properties are often associated with changes in synaptic organization. We have thus also examined the effects of substance P on synaptic ultrastructure up to 5 hr after substance P application. Substance P had several significant effects. These included an increase in the number of docked vesicles and a reduction of the synaptic gap. Substance P thus has long-term effects on synaptic organization and function. The relevance of these effects to the long-term locomotor network modulation is discussed.

Dehydration-induced synaptic plasticity in magnocellular neurons of the hypothalamic supraoptic nucleus.

Norepinephrine plays a critical role in the regulation of hypothalamic neuroendocrine function, in large part through modulation of synaptic glutamate and gamma-aminobutyric acid (GABA) release. Hypothalamic magnocellular neuroendocrine cells undergo dramatic changes in synaptic organization under conditions of increased hormone release, including increased numbers of glutamatergic, GABAergic and noradrenergic synapses. We studied the functional plasticity of magnocellular neurons of the rat supraoptic nucleus induced by chronic dehydration using whole-cell recordings in hypothalamic slices. Dehydrated rats showed increases in glutamate and GABA release onto magnocellular neurons, as evidenced by an increase in the frequency of spontaneous excitatory (29%) and inhibitory (33%) postsynaptic currents. The change in glutamate release was likely due to increased numbers of release sites because paired-pulse facilitation analysis did not reveal a change in the probability of transmitter release. In untreated rats, norepinephrine facilitates glutamate release and attenuates GABA release onto magnocellular neurons. Dehydration resulted in a marked enhancement of norepinephrine's actions, doubling both the norepinephrine-induced increase in glutamate release and decrease in GABA release. The norepinephrine dose-response curve was shifted to the left with dehydration, revealing an increase in norepinephrine sensitivity. Thus, dehydration leads to an increase in glutamate and GABA release onto supraoptic magnocellular neurons as well as a marked enhancement of the facilitatory effect of norepinephrine on glutamate release and inhibitory effect on GABA release. This synaptic plasticity would be expected to increase the excitability of the magnocellular neurons and support the enhanced bursting capacity and facilitated hormone secretion observed in vivo with chronic dehydration.

Changes in the paired-pulse ratio after long-term potentiation induced by 2- and 100-Hz tetanus in rat visual cortical slices

The effects of 2- and 100-Hz tetanus on long-term potentiation (LTP) of field potentials recorded from layers II/III and induced in layer IV in rat visual cortical slices were examined. Paired-pulse stimulation was used to probe the different mechanisms of LTP induced by 2- and 100-Hz tetanus. The paired-pulse ratio (PPR) decreased after the LTP induced by 2-Hz tetanus, with the changes in PPR being correlated with LTP amplitude. However, in the LTP induced by 100-Hz tetanus, the changes in PPR were not correlated with LTP expression. These experiments suggest that an enhanced probability of presynaptic transmitter release underlies LTP induced by 2-Hz tetanus, but not LTP induced by 100-Hz tetanus.

The effect of N‐ethylmaleimide on transmitter release from the skeletal neuromuscular junction of Bufo marinus

N-ethylmaleimide (NEM) has been used extensively in biochemical assays as an inhibitor of the NEM sensitive fusion protein (NSF). However, examination of the effect of NEM on transmitter release in more physiologically relevant preparations has proved inconclusive. In the present study, we have examined the effect of low concentrations of NEM on synaptic transmission in intact nerve-muscle preparations from toads (Bufo marinus). Under conditions of low transmitter release probability (0.3 mM calcium, 1 mM magnesium), treatment with NEM (10 microM) caused a significant increase in the amplitude of stimulus-evoked endplate potentials (EPPs) and a significant increase in the frequency of spontaneously occurring miniature EPPS (MEPPS) without affecting the amplitude of MEPPs. When the calcium concentration in the bath was raised to 4 mM, 10 microM NEM had no effect on EPP amplitude. Under these conditions, NEM treatment reduced paired pulse facilitation and increased depression during stimulus trains. Treatment with NEM also resulted in a significant decrease in the synaptic delay. The effects of NEM on transmitter release in the present study were not due to inactivation of G-proteins. The results of the present study show a calcium-dependent facilitation of stimulus-evoked transmitter release by NEM. These results are discussed in terms of the possible sites of NEM action leading to the observed changes in transmitter release.

GABA B-receptor modulation of short-term synaptic depression at an excitatory input to murine hippocampal CA3 pyramidal neurons

GABA B agonists inhibit excitatory transmission to hippocampal CA3 neurons during low frequency stimulation. We examined whether GABA B receptor activation can also enhance synaptic efficacy, when investigated at an input with high initial release probability. Short-term depression of field excitatory postsynaptic potential (EPSP) amplitude was observed during trains of stimuli applied to associational/commissural inputs (10–50 Hz; 22 °C). Baclofen (10 μM) reduced the amplitude of initial EPSPs in a train, and also reduced the degree of short-term depression. EPSPs recorded late in a train were significantly larger in baclofen than those recorded in control solution. These dual effects were mimicked by another selective GABA B agonist (SKF 97541, 10 μM), and abolished by a GABA B-selective antagonist (SCH 50911, 20 μM). The effects of baclofen were similar at a higher recording temperature (32 °C), where short-term depression was observed at higher stimulation frequencies. These results are consistent with the idea that a reduction of transmitter release probability could increase the fidelity of high-frequency transmission at this input, an effect that could help account for excitatory effects of GABA B agonists in some seizure models.

Plastic Elimination of Functional Glutamate Release Sites by Depolarization

To examine persisting effects of depolarizing rises in extracellular potassium concentration ([K +] o) on synapses, we depolarized cells to simulate ischemia-like rises in [K +] o. Elevated [K +] o for 1–16 hr severely depressed glutamate signaling, while mildly depressing GABA transmission. The glutamate-specific changes were plastic over several hours and involved a decrease in the size of the pool of releasable vesicles. Rather than a reduction of the number of vesicles per release site, the change involved functional elimination of release sites. This change was clearly dissociable from a second effect, depressed probability of transmitter release, which was common to both glutamate and GABA transmission. Thus, while other recent evidence links alteration of the releasable pool size with changes in p r, our results suggest the two can be independently manipulated. Selective depression of glutamate release may provide an adaptive mechanism by which neurons limit excitotoxicity.

BK potassium channels control transmitter release at CA3-CA3 synapses in the rat hippocampus.

Large conductance calcium- and voltage-activated potassium channels (BK channels) activate in response to calcium influx during action potentials and contribute to the spike repolarization and fast afterhyperpolarization. BK channels targeted to active zones in presynaptic nerve terminals have been shown to limit calcium entry and transmitter release by reducing the duration of the presynaptic spike at neurosecretory nerve terminals and at the frog neuromuscular junction. However, their functional role in central synapses is still uncertain. In the hippocampus, BK channels have been proposed to act as an 'emergency brake' that would control transmitter release only under conditions of excessive depolarization and accumulation of intracellular calcium. Here we demonstrate that in the CA3 region of hippocampal slice cultures, under basal experimental conditions, the selective BK channel blockers paxilline (10 microM) and iberiotoxin (100 nM) increase the frequency, but not the amplitude, of spontaneously occurring action potential-dependent EPSCs. These drugs did not affect miniature currents recorded in the presence of tetrodotoxin, suggesting that their action was dependent on action potential firing. Moreover, in double patch-clamp recordings from monosynaptically interconnected CA3 pyramidal neurones, blockade of BK channels enhanced the probability of transmitter release, as revealed by the increase in success rate, EPSC amplitude and the concomitant decrease in paired-pulse ratio in response to pairs of presynaptic action potentials delivered at a frequency of 0.05 Hz. BK channel blockers also enhanced the appearance of delayed responses, particularly following the second action potential in the paired-pulse protocol. These results are consistent with the hypothesis that BK channels are powerful modulators of transmitter release and synaptic efficacy in central neurones.

Optical quantal analysis indicates that long-term potentiation at single hippocampal mossy fiber synapses is expressed through increased release probability, recruitment of new release sites, and activation of silent synapses.

It is generally believed that long-term potentiation (LTP) at hippocampal mossy fiber synapses between dentate granule and CA3 pyramidal cells is expressed through presynaptic mechanisms leading to an increase in quantal content. The source of this increase has remained undefined but could include enhanced probability of transmitter release at existing functional release sites or increases in the number of active release sites. We performed optical quantal analyses of transmission at individual mossy fiber synapses in cultured hippocampal slices, using confocal microscopy and intracellular fluorescent Ca(2+) indicators. Our results indicate that LTP is expressed at functional synapses by both increased probability of transmitter release and recruitment of new release sites, including the activation of previously silent synapses here visualized for the first time.

Valproate reduced excitatory postsynaptic currents in hippocampal CA1 pyramidal neurons

Valproate (VPA) is one of the most widely used antiepileptic drugs, and it is also increasingly used for the treatment of neuropsychological disorders and neuropathic pain, as well as migraine prophylaxis. However, the underlying cellular mechanisms of VPA on the synaptic physiology remain unclear. We investigated the effects of VPA on synaptic transmission using the in vitro rat hippocampal slice technique and whole-cell patch clamp recordings from CA1 pyramidal neurons. Perfusion with VPA, at therapeutically attainable concentrations, decreased the amplitude of excitatory postsynaptic currents (EPSCs) evoked by Schaffer collateral stimulation, without modifying inhibitory postsynaptic currents (IPSCs). Furthermore, VPA induced a significant reduction of the non-NMDA EPSC (non-NMDA EPSC) component, without modifying the NMDA EPSC (NMDA EPSC) component. Paired pulse facilitation and EPSC variance were not significantly affected by VPA, indicating that VPA did not decrease transmitter release probability, which suggests a postsynaptic mechanism of action. We therefore conclude that VPA decreases excitatory synaptic activity through the modulation of postsynaptic non-NMDA receptors, without modifying synaptic inhibition, and that this reduction of excitation is, at least in part, responsible for the effects of VPA.

Correlation of non-uniform protein expression with variation in transmitter release probability

The strength of synaptic transmission is highly variable between different synapses. The present study examined some factors that may contribute to this variation in the strength of neurotransmission in sympathetic varicosities of the mouse vas deferens. Transmitter release was measured using a focal macropatch electrode placed over pairs of visualised varicosities. By regulating the calcium concentration of the solutions inside the recording electrode and in the bath independently of each other, transmitter release was restricted to one or two surface varicosities at each recording site. Using this technique, transmitter release probability was shown to be highly variable, even between adjacent varicosities on single axon branches. Very little variation was observed in the calcium influx following single impulse nerve stimulation between adjacent Oregon Green BAPTA-1 loaded varicosities. However, the staining intensities of three vesicular proteins, SV2, synaptophysin, and synaptotagmin 1, showed considerable variation between adjacent varicosities on single axon branches. This variation in staining intensity may be partly explained by variation in the density of synaptic vesicles. However, double staining experiments using two vesicular antigens showed some varicosities staining for one vesicular antigen, but not for the second, suggesting that the expression of these release machinery proteins is regulated locally within the varicosities. The results of the present study strengthen suggestions that synaptic strength is at least in part, regulated by variation in the expression of vesicular proteins.

NMDA Currents and Receptor Protein Are Downregulated in the Amygdala during Maintenance of Fear Memory

The amygdala plays a critical role in fear conditioning, a model of emotional learning and cue-induced anxiety. In the lateral amygdala, fear conditioning is associated with an enduring increase in synaptic strength mediated through AMPA receptors and with a reduction in paired-pulse facilitation, reflecting an increased probability of neurotransmitter release. Here we show that NMDA-mediated transmission in the thalamic-to-lateral amygdala pathway is not facilitated after fear conditioning, although probability of transmitter release is enhanced. Rather, the EC50 for NMDA receptor (NR)-mediated current is shifted threefold to fourfold to the right in fear-conditioned animals, suggesting a postsynaptic alteration in NMDA receptors in the maintenance phase of fear memory. Furthermore, the ability of nonselective and subunit-selective antagonists of NMDA receptors to block NMDA receptor-mediated EPSCs is reduced in lateral amygdala neurons from fear-conditioned animals, suggesting a reduction in NMDA receptors at thalamolateral amygdala synapses. In addition, Western blots show a reduction in phosphorylated-NR1, NR2A, and NR2B subunit protein expression in amygdalas from fear-conditioned animals. These data indicate that postsynaptic mechanisms are involved in synaptic plasticity in the thalamoamygdala pathway in fear conditioning and raise the possibility that: (1) downregulation of the NMDA receptor may protect against excitotoxicity of unchecked NMDA receptor recruitment during induction and consolidation of fear memories, (2) reduced NMDA current and protein may allow persistence of the "capacity to reactivate" amygdala pathways in NMDA receptor-dependent fear memories, or (3) a persistent long-term depression of NMDA transmission may occur after fear learning.

Adenosine gates synaptic plasticity at hippocampal mossy fiber synapses.

The release properties of synapses in the central nervous system vary greatly, not only across anatomically distinct types of synapses but also among the same class of synapse. This variation manifests itself in large part by differences in the probability of transmitter release, which affects such activity-dependent presynaptic forms of plasticity as paired-pulse facilitation and frequency facilitation. This heterogeneity in presynaptic function reflects differences in the intrinsic properties of the synaptic terminal and the activation of presynaptic neurotransmitter receptors. Here we show that the unique presynaptic properties of the hippocampal mossy fiber synapse are largely imparted onto the synapse by the continuous local action of extracellular adenosine at presynaptic A1 adenosine receptors, which maintains a low basal probability of transmitter release.

Synaptic dynamics on different time scales in a parallel fiber feedback pathway of the weakly electric fish.

Synaptic dynamics comprise a variety of interacting processes acting on a wide range of time scales. This enables a synapse to perform a large array of computations, from temporal and spatial filtering to associative learning. In this study, we describe how changing synaptic gain via long-term plasticity can act to shape the temporal filtering of a synapse through modulation of short-term plasticity. In the weakly electric fish, parallel fibers from cerebellar granule cells provide massive feedback inputs to the pyramidal neurons of the electrosensory lateral line lobe. We demonstrate a long-term synaptic enhancement (LTE) of these synapses that is biochemically similar to the presynaptic long-term potentiation expressed by parallel fibers in the mammalian cerebellum. Using a novel stimulation protocol and a simple modeling paradigm, we then quantify the changes in short-term plasticity during the induction of LTE and show that these changes can be explained by gradual changes in only one model parameter, that which is associated with the baseline probability of transmitter release. These changes lead to a shift in the spike frequency preference of the synapse, suggesting that long-term plasticity is not only involved in controlling the gain of the parallel fiber synapse, but also provides a means of controlling synaptic filtering over multiple time scales.

Factors underlying bursting behavior in a network of cultured hippocampal neurons exposed to zero magnesium.

Factors contributing to reduced magnesium-induced neuronal action potential bursting were investigated in primary hippocampal cell culture at high and low culture density. In nominally zero external magnesium medium, pyramidal neurons from high-density cultures produced recurrent spontaneous action potential bursts superimposed on prolonged depolarizations. These bursts were partially attenuated by the NMDA receptor antagonist d-APV. Pharmacological analysis of miniature excitatory postsynaptic currents (EPSCs) revealed 2 components: one sensitive to d-APV and another to the AMPA receptor antagonist DNQX. The components were kinetically distinct. Participation of NMDA receptors in reduced magnesium-induced synaptic events was supported by the localization of the NR1 subunit of the NMDA receptor with the presynaptic vesicular protein synaptophysin. Presynaptically, zero magnesium induced a significant increase in EPSC frequency likely attributable to increased neuronal hyperexcitability induced by reduced membrane surface charge screening. Mean quantal content was significantly increased in zero magnesium. Cells from low-density cultures did not exhibit action potential bursting in zero magnesium but did show increased EPSC frequency. Low-density neurons had less synaptophysin immunofluorescence and fewer active synapses as determined by FM1-43 analysis. These results demonstrate that multiple factors are involved in network bursting. Increased probability of transmitter release presynaptically, enhanced NMDA receptor-mediated excitability postsynaptically, and extent of neuronal interconnectivity contribute to initiation and maintenance of elevated network excitability.