Increase In Quantal Content Research Articles

Loss of miR-183/96 Alters Synaptic Strength via Presynaptic and Postsynaptic Mechanisms at a Central Synapse.

A point mutation in miR-96 causes non-syndromic progressive peripheral hearing loss and alters structure and physiology of the central auditory system. To gain further insight into the functions of microRNAs (miRNAs) within the central auditory system, we investigated constitutive Mir-183/96dko mice of both sexes. In this mouse model, the genomically clustered miR-183 and miR-96 are constitutively deleted. It shows significantly and specifically reduced volumes of auditory hindbrain nuclei, because of decreases in cell number and soma size. Electrophysiological analysis of the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) demonstrated strongly altered synaptic transmission in young-adult mice. We observed an increase in quantal content and readily releasable vesicle pool size in the presynapse while the overall morphology of the calyx was unchanged. Detailed analysis of the active zones (AZs) revealed differences in its molecular composition and synaptic vesicle (SV) distribution. Postsynaptically, altered clustering and increased synaptic abundancy of the AMPA receptor subunit GluA1 was observed resulting in an increase in quantal amplitude. Together, these presynaptic and postsynaptic alterations led to a 2-fold increase of the evoked excitatory postsynaptic currents in MNTB neurons. None of these changes were observed in deaf Cldn14ko mice, confirming an on-site role of miR-183 and miR-96 in the auditory hindbrain. Our data suggest that the Mir-183/96 cluster plays a key role for proper synaptic transmission at the calyx of Held and for the development of the auditory hindbrain.SIGNIFICANCE STATEMENT The calyx of Held is the outstanding model system to study basic synaptic physiology. Yet, genetic factors driving its morphologic and functional maturation are largely unknown. Here, we identify the Mir-183/96 cluster as an important factor to regulate its synaptic strength. Presynaptically, Mir-183/96dko calyces show an increase in release-ready synaptic vesicles (SVs), quantal content and abundance of the proteins Bassoon and Piccolo. Postsynaptically, the quantal size as well as number and size of GluA1 puncta were increased. The two microRNAs (miRNAs) are thus attractive candidates for regulation of synaptic maturation and long-term adaptations to sound levels. Moreover, the different phenotypic outcomes of different types of mutations in the Mir-183 cluster corroborate the requirement of mutation-tailored therapies in patients with hearing loss.

Extracellular Protons Mediate Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction

At the vertebrate neuromuscular junction (NMJ), presynaptic homeostatic potentiation (PHP) refers to the upregulation of neurotransmitter release via an increase in quantal content (QC) when the postsynaptic nicotinic acetylcholine receptors (nAChRs) are partially blocked. The mechanism of PHP has not been completely worked out. In particular, the identity of the presumed retrograde signal is still a mystery. We investigated the role of acid-sensing ion channels (ASICs) and extracellular protons in mediating PHP at the mouse NMJ. We found that blocking AISCs using benzamil, psalmotoxin-1 (PcTx1), or mambalgin-3 (Mamb3) prevented PHP. Likewise, extracellular acidification from pH 7.4 to 7.2 triggered a significant, reversable increase in QC and this increase could be prevented by PcTx1. Interestingly, an acidic saline (pH 7.2) also precluded the subsequent induction of PHP. Using immunofluorescence we observed ASIC2a and ASIC1 subunits at the NMJ. Our results indicate that protons and ASIC channels are involved in activating PHP at the mouse NMJ. We speculate that the partial blockade of nAChRs leads to a modest decrease in the pH of the synaptic cleft (∼0.2 pH units) and this activates ASIC channels on the presynaptic nerve terminal.

Target-wide Induction and Synapse Type-Specific Robustness of Presynaptic Homeostasis.

Presynaptic homeostatic plasticity (PHP) is an evolutionarily conserved form of adaptive neuromodulation and is observed at both central and peripheral synapses. In this work, we make several fundamental advances by interrogating the synapse specificity of PHP. We define how PHP remains robust to acute versus long-term neurotransmitter receptor perturbation. We describe a general PHP property that includes global induction and synapse-specific expression mechanisms. Finally, we detail a novel synapse-specific PHP expression mechanism that enables the conversion from short- to long-term PHP expression. If our data can be extended to other systems, including the mammalian central nervous system, they suggest that PHP can be broadly induced and expressed to sustain the function of complex neural circuitry.

Orexin facilitates GABAergic IPSCs via postsynaptic OX1 receptors coupling to the intracellular PKC signalling cascade in the rat cerebral cortex

Orexin has multiple physiological functions including wakefulness, appetite, nicotine intake, and nociception. The cerebral cortex receives abundant orexinergic projections and expresses both orexinergic receptor 1 (OX1R) and 2 (OX2R). However, little is known about orexinergic regulation of GABA-mediated inhibitory synaptic transmission. In the cerebral cortex, there are multiple GABAergic neural subtypes, each of which has its own morphological and physiological characteristics. Therefore, identification of presynaptic GABAergic neural subtypes is critical to understand orexinergic effects on GABAergic connections. We focused on inhibitory synapses at pyramidal neurons (PNs) from fast-spiking GABAergic neurons (FSNs) in the insular cortex by a paired whole-cell patch-clamp technique, and elucidated the mechanisms of orexin-induced IPSC regulation. We found that both orexin A and orexin B enhanced unitary IPSC (uIPSC) amplitude in FSN→PN connections without changing the paired-pulse ratio or failure rate. These effects were blocked by SB-334867, an OX1 receptor (OX1R) antagonist, but not by TCS-OX2-29, an OX2R antagonist. [Ala11, D-Leu15]-orexin B, a selective OX2R agonist, had little effect on uIPSCs. Variance-mean analysis demonstrated an increase in quantal content without a change in release probability or the number of readily releasable pools. Laser photolysis of caged GABA revealed that orexin A enhanced GABA-mediated currents in PNs. Downstream blockade of Gq/11 protein-coupled OX1Rs by IP3 receptor or protein kinase C (PKC) blockers and BAPTA injection into postsynaptic PNs diminished the orexin A-induced uIPSC enhancement. These results suggest that the orexinergic uIPSC enhancement is mediated via postsynaptic OX1Rs, which potentiate GABAA receptors through PKC activation.

3,4-diaminopyridine reverses paralysis in botulinum neurotoxin-intoxicated diaphragms through two functionally distinct mechanisms

Botulinum neurotoxins (BoNTs) are exceedingly potent neurological poisons that prevent neurotransmitter release from peripheral nerve terminals by cleaving presynaptic proteins required for synaptic vesicle fusion. The ensuing neuromuscular paralysis causes death by asphyxiation. Although no antidotal treatments exist to block toxin activity within the nerve terminal, aminopyridine antagonists of voltage-gated potassium channels have been proposed as symptomatic treatments for botulism toxemia. However, clinical evaluation of aminopyridines as symptomatic treatments for botulism has been inconclusive, in part because mechanisms responsible for reversal of paralysis in BoNT-poisoned nerve terminals are not understood. Here we measured the effects of 3,4-diaminopyridine (DAP) on phrenic nerve-elicited diaphragm contraction and end-plate potentials at various times after intoxication with BoNT serotypes A, B, or E. We found that DAP-mediated increases in quantal content promote neurotransmission from intoxicated nerve terminals through two functionally distinguishable mechanisms. First, DAP increases the probability of neurotransmission at non-intoxicated release sites. This mechanism is serotype-independent, becomes less effective as nerve terminals become progressively impaired, and remains susceptible to ongoing intoxication. Second, DAP elicits persistent production of toxin-resistant endplate potentials from nerve terminals fully intoxicated by BoNT/A, but not serotypes B or E. Since this effect appears specific to BoNT/A intoxication, we propose that DAP treatment enables BoNT/A-cleaved SNAP-25 to productively engage in fusogenic release by increasing the opportunity for low-efficiency fusion events. These findings have important implications for DAP as a botulism therapeutic by defining conditions under which DAP may be clinically effective in reversing botulism symptoms.

Presynaptic neuromuscular action of a methanolic extract from the venom of Rhinella schneideri toad

BackgroundRhinella schneideri, previously known as Bufo paracnemis, is a common toad in many regions of Brazil. Its venom exerts important cardiovascular effects on humans and other animals. Although this toad venom has been the subject of intense investigations, little is known about its neuromuscular activity.MethodsThe neurotoxicity of a methanolic extract of R. schneideri venom was tested on mouse phrenic nerve-diaphragm (PND) preparations mounted for conventional twitch tension recording – in response to indirect stimulation – and for electrophysiological measurements.ResultsVenom extract (50 μg/mL) increased the muscle twitch tension in PND preparations but did not significantly alter the resting membrane potential values. Electrophysiological evaluations showed that the extract (50 μg/mL) significantly augmented the frequency of miniature end-plate potential (from 38 ± 3.5 to 88 ± 15 after 60 minutes; n = 5; p < 0.05) and quantal content (from 128 ± 13 to 272 ± 34 after five minutes; n = 5; p < 0.05). Pretreatment with ouabain (1 μg/mL) for five minutes prevented the increase in quantal content (117 ± 18 and 154 ± 33 after five and 60 minutes, respectively).ConclusionThese results indicate that the methanolic extract of R. schneideri venom acts primarily presynaptically to enhance neurotransmitter release in mouse phrenic-diaphragm preparations.

Ral mediates activity-dependent growth of postsynaptic membranes via recruitment of the exocyst

Remodelling neuronal connections by synaptic activity requires membrane trafficking. We present evidence for a signalling pathway by which synaptic activity and its consequent Ca(2+) influx activate the small GTPase Ral and thereby recruit exocyst proteins to postsynaptic zones. In accord with the ability of the exocyst to direct delivery of post-Golgi vesicles, constitutively active Ral expressed in Drosophila muscle causes the exocyst to be concentrated in the region surrounding synaptic boutons and consequently enlarges the membrane folds of the postsynaptic plasma membrane (the subsynaptic reticulum, SSR). SSR growth requires Ral and the exocyst component Sec5 and Ral-induced enlargement of these membrane folds does not occur in sec5(-/-) muscles. Chronic changes in synaptic activity influence the plastic growth of this membrane in a manner consistent with activity-dependent activation of Ral. Thus, Ral regulation of the exocyst represents a control point for postsynaptic plasticity. This pathway may also function in mammals as expression of activated RalA in hippocampal neurons increases dendritic spine density in an exocyst-dependent manner and increases Sec5 in spines.

Involvement of Basal and Calcium-Activated Protein Kinase C in Neurotransmitter Secretion in Mouse Motor Synapses

Blocker of presynaptic protein kinase C isoforms, GF109203X, reduced quantal content of single and rhythmic evoked end-plate potentials. The increase in quantal content of single potentials under the effect of 4- aminopyridine was neutralized by 75% under the effect of L-type Ca(2+)-channel blocker nitrendipine and completely returned to the control level after protein kinase C inhibition with chelerythrine. Neither nitrendipine, nor GF109203X affected the potentiating effect of tetraethylammonium on quantal content of end-plate potentials. Thus, we discovered basal activity of presynaptic protein kinase C under normal conditions that is aimed at the maintenance of quantal content of evoked release. It has been concluded that 4-aminipyridine, but not tetraethylammonium, triggers Ca(2+) entry into the terminal, which activates protein kinase C and enhanced the evoked acetylcholine release.

Facilitation of Neurotransmitter Release in Mouse Motor Synapses in Different Modes of Protein Kinase C Activation

Protein kinase C blocker chelerythrine prevented the increase in quantal content of single and rhythmic evoked end-plate potentials after disinhibition of L-type Ca(2+)-channels with paxillin. Phorbol ester increased quantal content of single end-plate potentials and changed rhythmic activity of mouse motor synapses. The effects of phorbol ester were to a great extent neutralized by L-type Ca(2+)-channel blocker nitrendipine and were completely abolished by K(+)-channel blocker 4-aminopyridine. Thus, we discovered different facilitations of transmission after protein kinase C activation with calcium current through L-type channels and with phorbol ester.

Synaptic changes underlying the strengthening of GABA/glycinergic connections in the developing lateral superior olive

Before hearing onset, the topographic organization of the auditory GABA/glycinergic pathway from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO) is refined by synaptic silencing and strengthening. The synaptic mechanisms underlying the developmental strengthening of maintained MNTB-LSO connections are unknown. Here we address this question using whole-cell recordings from LSO neurons in slices prepared from prehearing mice. Minimal and maximal stimulation techniques demonstrated that during the first two postnatal weeks, individual LSO neurons lose about 55% of their initial presynaptic MNTB partners while maintained single-fiber connections become about 14-fold stronger. Analysis of MNTB-evoked miniature events indicates that this strengthening is accompanied by a 2-fold increase in quantal amplitude. Strengthening is not caused by an increase in the probability of release because paired pulse ratios (PPRs) increased from 0.7 in newborn animals to 0.9 around hearing onset, indicating a developmental decrease rather than increase in release probability. In addition, a possible soma-dendritic relocation of MNTB input seems unlikely to underlie their strengthening as indicated by analysis of the rise times of synaptic currents. Taken together, we conclude that the developmental strengthening of MNTB-LSO connections is achieved by a 2-fold increase in quantal size and an 8-fold increase in quantal content.

Activity-Dependent Regulation of the Binomial Parameters p and n at the Mouse Neuromuscular Junction In Vivo

Block of neurotransmission at the mammalian neuromuscular junction triggers an increase in the number of vesicles released (quantal content). The increase occurs whether nerve and muscle activity are both blocked by placement of a tetrodotoxin (TTX) containing cuff on the nerve or whether muscle activity is selectively blocked by injection of α-bungarotoxin (BTX). We used ANOVA to examine whether the mechanism underlying the increase in quantal content differed between the two types of activity blockade. We found that TTX-induced blockade increased the probability of release (p), whereas BTX-induced blockade increased the number of releasable vesicles (n). The lack of increase in p when postsynaptic activity was blocked with BTX suggests that block of presynaptic activity triggers the increase. To determine whether n is regulated by mismatch of pre- and postsynaptic activity introduced by BTX injection we combined BTX and TTX and still found an increase in n. We conclude that block of acetylcholine binding to acetylcholine receptors during spontaneous release triggers the increase in n.

Mechanism of Inhibition of Acetylcholine Secretion in Newly Formed Mouse Synapses Involving Ca2+-Dependent Kinases and Voltage-Gated K+-Channels

Nifedipine, a blocker of L-type Ca(2+)-channels, increased quantal content of endplate potentials in newly formed nerve-muscle synapses, while R 24571 (calmodulin inhibitor) and KN 62 (inhibitor of calmodulin-dependent kinase II) did not change it. KN 62 suppressed the increase in quantal content of endplate potentials evoked by nifedipine. Similar to nifedipine, chelerythrine and bisindolylmaleimide I (blockers of protein kinase C) increased quantal content of endplate potentials. In the presence of chelerythrine, nifedipine lost its ability to facilitate secretion of neurotransmitter. 4-Aminopyridine, a blocker of voltage-gated potassium channels, increased quantal content of endplate potentials. In the presence of chelerythrine, 4-aminopyridine induced no additional increase in the quantal content of endplate potentials. In its turn, chelerythrine induced no extra facilitation of secretion in the presence of 4-aminopyridine. It is hypothesized that Ca(2+)-dependent inhibition of secretion results from suppression of calmodulin-dependent kinase II and activation of protein kinase C, which potentiates the work of voltage-gated K(+)-channels.

Cooperative regulation of neurotransmitter release by Rab3a and synapsin II

To understand how the presynaptic proteins synapsin and Rab3a may interact in the regulation of the synaptic vesicle cycle and the release process, we derived a double knockout (DKO) mouse lacking both synapsin II and Rab3a. We found that Rab3a deletion rescued epileptic-like seizures typical for synapsin II gene deleted animals (Syn II(−)). Furthermore, action potential evoked release was drastically reduced in DKO synapses, although spontaneous release remained normal. At low Ca 2+ conditions, quantal content was equally reduced in Rab3a(−) and DKO synapses, but as Ca 2+ concentration increased, the increase in quantal content was more prominent in Rab3a(−). Electron microscopy analysis revealed that DKO synapses have a combined phenotype, with docked vesicles being reduced similar to Rab3a(−), and intraterminal vesicles being depleted similar to Syn II(−). Consistently, both Syn II(−) and DKO terminals had increased synaptic depression and incomplete recovery. Taken together, our results suggest that synapsin II and Rab3a have separate roles in maintaining the total store of synaptic vesicles and cooperate in promoting the latest steps of neuronal secretion.

Opposing effects of cannabinoids and vanilloids on evoked quantal release at the frog neuromuscular junction

Cannabinoids and vanilloids are two distinct groups of substances that share some pharmacological targets. Here we report that two cannabinoid type 1 receptor (CB1) agonists, WIN 55212-2 (WIN) and arachidonyl-2′-chloroethylamide (ACEA) have opposing effects on evoked quantal acetylcholine release – WIN decreased quantal content while ACEA increased quantal content. The decrease in quantal content by WIN was blocked by the CB1 antagonist AM 251. The increase in quantal content by ACEA was not blocked by AM 251, indicating it acts through a receptor other than CB1. As ACEA is also an agonist for the vanilloid receptor (TRPV1) we tested the effect of vanilloids on quantal content. Similar to ACEA, the vanilloid agonist capsaicin increased quantal content, and this effect was blocked by capsazepine, a TRPV1 antagonist. Capsazepine also blocked the increase in quantal content by ACEA. Together these data show an inhibitory effect of CB1 activation on evoked acetylcholine release and the first evidence for the presence of a vanilloid receptor at the neuromuscular junction.

Chronic and acute alterations in the functional levels of Frequenins 1 and 2 reveal their roles in synaptic transmission and axon terminal morphology

Frequenin (Frq) and its mammalian homologue, neuronal calcium sensor 1 (NCS-1), are important calcium-binding proteins which enhance neurotransmitter release and facilitation. Here, we report the discovery of a second Frq-encoding gene (frq2) in Drosophila. The temporal and spatial expression patterns of the two genes are very similar, and the proteins they encode, Frq1 and Frq2, are 95% identical in amino acid sequence. Frq1 is more abundant than Frq2, and is most highly expressed in larva. Loss-of-function phenotypes were studied using dominant negative peptides to prevent Frq target binding, RNAi to reduce gene transcription, or both methods. To discriminate chronic from acute loss-of-function effects, we compared the effects of transgenic expression and forward-filling the dominant-negative peptide into presynaptic terminals. In both cases, a 70% reduction in quantal content per bouton occurred, demonstrating that this trait does not result from homeostatic adaptations of the synapse during development. The chronic treatment also produced more synaptic boutons from MNSNb/d-Is motorneurons, but fewer active zones per bouton. By contrast, excess-of-function conditions yielded a 1.4- to 2-fold increase in quantal content and fewer boutons in the same motorneuron. These synaptic effects resulted in behavioural changes in the Buridan locomotion assay, showing that walking speed is dependent on Frq activity in the nervous system. All the effects were identical for both Frqs, and consistent with excess- and loss-of-function genotypes. We conclude that Frqs have two distinct functions: one in neurotransmission, regulating the probability of release per synapse, and another in axonal growth and bouton formation.

NMDA Receptor Subtypes at Autaptic Synapses of Cerebellar Granule Neurons

We studied the action potential-evoked autaptic N-methyl-d-aspartate receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) using solitary cerebellar neurons cultured in microislands from wild-type (+/+), NR2A subunit knockout (NR2A-/-), and NR2C subunit knockout (NR2C-/-) mice. The peak amplitude of autaptic NMDA-EPSCs increased for all genotypes between days in vitro 8 (DIV8) and DIV13. Compared with +/+ cells at DIV13, NR2A-/- cells had smaller and NR2C-/- cells had larger NMDA-EPSCs. The decay time of these currents were all unexpectedly fast, except in NR2A-/- neurons, and showed small but significant shortening with development. Comparison of quantal parameters during development indicated an increase in quantal content in all genotypes. The synaptic portion of NMDA receptors measured using MK-801 blockade was roughly 50% in all genotypes at DIV8, and this percentage became slightly larger in NR2A-/- and NR2C-/- neurons at DIV12. The NR2B-selective antagonists Conantokin G and CP101,606 differed in their blocking actions with development, suggesting the presence of both heterodimeric NR1/NR2B and heterotrimeric NR1/NR2A/NR2B receptors. The most striking result we obtained was the significant increase of NMDA-EPSC peak amplitude and charge transfer in NR2C-/- mice. This was mainly the result of an increase in quantal size as estimated from miniature NMDA-EPSCs. The expression of NR2C subunit containing receptors was supported by the decreased Mg(2+) sensitivity of NMDA receptors at DIV13 in +/+ but not in NR2C-/- cells. Thus solitary cerebellar granule neurons provide a novel model to investigate the role of receptor subtypes in the developmental changes of synaptic NMDA receptors.

Decreased Synaptic Activity Shifts the Calcium Dependence of Release at the Mammalian Neuromuscular JunctionIn Vivo

We examined the mechanism underlying increased quantal content after block of activity at the mouse neuromuscular junction in vivo. We found that, when quantal content was measured in solution containing normal extracellular calcium, block of activity had no effect on either quantal content or the response to repetitive stimulation. However, when quantal content was measured in low extracellular calcium, there was a large increase in quantal content after block of activity. The increase in quantal content was accompanied by increased depression during repetitive stimulation. The explanation for these findings was that there was a shift in the calcium dependence of release after block of activity that manifested as an increase in probability of release in low extracellular calcium. Block of presynaptic P/Q channels eliminated the increase in probability of release. We propose that activity-dependent regulation of presynaptic calcium entry may contribute to homeostatic regulation of quantal content.

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.

Estrogen Receptor Expression in Laryngeal Muscle in Relation to Estrogen-Dependent Increases in Synapse Strength

In Xenopus laevis, the laryngeal neuromuscular synapse is the final effector for sexually differentiated song production. Females have stronger laryngeal synapses than males, and synapse strength is estrogen dependent. Estrogen-induced increases in synaptic strength require at least 3 weeks of exposure, suggesting that the hormone acts via a classical genomic mechanism involving the estrogen receptor (ER). The locus of the sex difference in synapse strength, determined using quantal analysis, is presynaptic, leading to the prediction that estrogen acts directly on vocal motor neurons. However, laryngeal motor neurons do not accumulate estrogen. Estrogen might instead affect motor neuron transmitter release via a retrograde signal from its target muscle. To test this hypothesis, we determined whether laryngeal muscle expresses ER. With RT-PCR using primers that recognize highly conserved domains of the ERα, mRNA products of the predicted size were amplified from laryngeal muscle as well as from other classical target tissues (forebrain and oviduct). Northern blots using a portion of the PCR product as primer revealed the same-sized band in oviduct and laryngeal muscle. Immunocytochemistry and Western blots confirmed the presence of ER protein in laryngeal muscle fibers and revealed several proteins in laryngeal muscle, brain and liver; among these was an approximately 66-kD protein – presumed to be full-length ER – that was the only one found in oviduct. Estrogen treatment of juveniles resulted in an upregulation of the 66-kD ER protein concomitant with an increase in quantal content. Taken together, these experiments strongly suggest that the ER is expressed by laryngeal muscle; this receptor could mediate estrogen-dependent changes in synaptic strength via retrograde signaling.

Analysis of NMDA-independent long-term potentiation induced at CA3-CA1 synapses in rat hippocampus in vitro.

1. Excitatory postsynaptic currents (EPSCs) were evoked at synapses formed by Schaffer collaterals/commissural (CA3) axons with CA1 pyramidal cells using the rat hippocampal slice preparation. Long-term potentiation (LTP) was induced at these synapses using a pairing protocol, with 50 microM d,l-APV present in the artificial cerebrospinal fluid (ACSF). 2. Quantal analysis of the amplitudes of the control and conditioned EPSCs showed that the enhancement of synaptic strength was due entirely to an increase in quantal content of the EPSC. No change occurred in the quantal current. 3. These results were compared with those obtained from a previous quantal analysis of LTP induced in normal ACSF, where both quantal current and quantal content increased. The results suggest that calcium entering via NMDA receptors initiates the signalling cascade that results in enhanced AMPA currents because it is adding to cytoplasmic calcium from other sources to reach a threshold for this signalling pathway, or because calcium entering via NMDA receptors specifically activates this signalling pathway.