Vesicular glutamate filling and AMPA receptor occupancy at the calyx of Held synapse of immature rats

At central glutamatergic synapses, neurotransmitter often saturates postsynaptic AMPA receptors (AMPARs), thereby restricting the dynamic range of synaptic efficacy. Here, using simultaneous pre- and postsynaptic whole-cell recordings, at the calyx of Held synapse of immature rats, we have investigated the mechanism by which transmitter glutamate saturates postsynaptic AMPARs. When we loaded l -glutamate (1–100 m m ) into presynaptic terminals, the quantal EPSC (qEPSC) amplitude changed in a concentration-dependent manner. At physiological temperature (36–37°C), the qEPSC amplitude increased when intraterminal l -glutamate concentration was elevated from 1 m m to 10 m m , but it reached a plateau at 10 m m . This plateau persisted after bath-application of the low affinity AMPAR antagonist kynurenate, suggesting that it was caused by saturation of vesicular filling with glutamate rather than by saturation of postsynaptic AMPARs. In contrast to qEPSCs, action potential-evoked EPSCs remained unchanged by increasing intraterminal l -glutamate from 1 m m to 100 m m , even at room temperature, indicating that multi-quantal glutamate saturated postsynaptic AMPARs. This saturation could be relieved by blocking AMPAR desensitization using cyclothiazide (100 μ m ). The concentration of ambient glutamate in the slice, estimated from NMDA receptor current fluctuations, was 55 n m ; this was far below the concentration required for AMPAR desensitization. We conclude that rapid AMPAR desensitization, caused by glutamate released from multiple vesicles during synaptic transmission, underlies postsynaptic AMPAR saturation at this immature calyceal synapse before the onset of hearing.     

Developmental presence and disappearance of postsynaptically silent synapses on dendritic spines of rat layer 2/3 pyramidal neurons

Silent synapses are synapses whose activation evokes NMDA-type glutamate receptor (NMDAR) but not AMPA-type glutamate receptor (AMPAR) mediated currents. Silent synapses are prominent early in postnatal development and are thought to play a role in the activity- and sensory-dependent refinement of neuronal circuits. The mechanisms that account for their silent nature have been controversial, and both presynaptic and postsynaptic mechanisms have been proposed. Here, we use two-photon laser uncaging of glutamate to directly activate glutamate receptors and measure AMPAR- and NMDAR-dependent currents on individual dendritic spines of rat somatosensory cortical layer 2/3 pyramidal neurons. We find that dendritic spines lacking functional surface AMPARs are commonly found before postnatal day 12 (P12) but are absent in older animals. Furthermore, AMPAR-lacking spines are contacted by release-competent presynaptic terminals. After P12, the AMPAR/NMDAR current ratio at individual spines continues to increase, consistent with continued addition of AMPARs to postsynaptic terminals. Our results confirm the existence of postsynaptically silent synapses and demonstrate that the morphology of the spine is not strongly predictive of its AMPAR content.     

The density of AMPA receptors activated by a transmitter quantum at the climbing fibre-Purkinje cell synapse in immature rats

We aimed to estimate the number of AMPA receptors (AMPARs) bound by the quantal transmitter packet, their single-channel conductance and their density in the postsynaptic membrane at cerebellar Purkinje cell synapses. The synaptic and extrasynaptic AMPARs were examined in Purkinje cells in 2- to 4-day-old rats, when they receive synaptic inputs solely from climbing fibres (CFs). Evoked CF EPSCs and whole-cell AMPA currents displayed roughly linear current-voltage relationships, consistent with the presence of GluR2 subunits in synaptic and extrasynaptic AMPARs. The mean quantal size, estimated from the miniature EPSCs (MEPSCs), was ∼300 pS. Peak-scaled non-stationary fluctuation analysis of spontaneous EPSCs and MEPSCs gave a weighted-mean synaptic channel conductance of ∼5 pS (∼7 pS when corrected for filtering). By applying non-stationary fluctuation analysis to extrasynaptic currents activated by brief glutamate pulses (5 m m ), we also obtained a small single-channel conductance estimate for extrasynaptic AMPARs (∼11 pS). This approach allowed us to obtain a maximum open probability ( P _o,max) value for the extrasynaptic receptors ( P _o,max= 0.72). Directly resolved extrasynaptic channel openings in the continued presence of glutamate exhibited clear multiple-conductance levels. The mean area of the postsynaptic density (PSD) of these synapses was 0.074 μm², measured by reconstructing electron-microscopic (EM) serial sections. Postembedding immunogold labelling by anti-GluR2/3 antibody revealed that AMPARs are localised in PSDs. From these data and by simulating error factors, we estimate that at least 66 AMPARs are bound by a quantal transmitter packet at CF-Purkinje cell synapses, and the receptors are packed at a minimum density of ∼900 μm⁻² in the postsynaptic membrane.     

'Deaf, mute and whispering' silent synapses: their role in synaptic plasticity

Mechanisms of long-term potentiation (LTP) maintenance are discussed in the light of the phenomenon of silent synapses. Evidence that LTP is associated with the insertion of new AMPA receptors (AMPARs) in postsynaptically silent (deaf) synapses expressing only NMDA receptors (NMDARs) before LTP induction has led to the assumption that the debate on pre- versus postsynaptic locus of LTP expression has been resolved in favour of the latter. However, recent data indicate that these synapses are mainly presynaptically silent (mute or whispering), because the probability of glutamate release ( P _r) or glutamate concentration in the cleft is too low to activate AMPARs. In this case LTP could be explained by an increase in P _r or enhanced glutamate concentration to activate low affinity AMPARs. Optical methods to probe calcium transients in dendritic spines have revealed an increase in P _r during LTP with concomitant postsynaptic modifications. A hypothesis is considered that accounts for the differences in both the initial failure rates between AMPAR- and NMDAR-mediated responses, and the LTP-associated decrease in failures of AMPAR-mediated responses. According to this hypothesis, glutamate release is potentiated by the strong postsynaptic depolarization used to identify NMDAR-mediated responses. We suggest that the expression of LTP may depend on coordinated pre- and postsynaptic modifications whose relative contributions vary according to the initial state of the synapse, the experimental protocol and time after induction.     

Activation of postsynaptic Ca(2+) stores modulates glutamate receptor cycling in hippocampal neurons

We show that activation of postsynaptic inositol 1,4,5-tris-phosphate receptors (IP(3)Rs) with the IP(3)R agonist adenophostin A (AdA) produces large increases in AMPA receptor (AMPAR) excitatory postsynaptic current (EPSC) amplitudes at hippocampal CA1 synapses. Co-perfusion of the Ca(2+) chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid strongly inhibited AdA-enhanced increases in EPSC amplitudes. We examined the role of AMPAR insertion/anchoring in basal synaptic transmission. Perfusion of an inhibitor of synaptotagmin-soluble n-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor SNARE-mediated exocytosis depressed basal EPSC amplitudes, whereas a peptide that inhibits GluR2/3 interactions with postsynaptic density-95 (PDZ) domain proteins glutamate receptor interacting protein (GRIP)/protein interacting with C-kinase-1 (PICK1) enhanced basal synaptic transmission. These results suggest that constitutive trafficking and anchoring of AMPARs help maintain basal synaptic transmission. The regulation of postsynaptic AMPAR trafficking involves synaptotagmin-SNARE-mediated vesicle exocytosis and interactions between AMPARs and the PDZ domains in GRIP/PICK1. We show that inhibitors of synaptotagmin-SNARE-mediated exocytosis, or interactions between AMPARs and GRIP/PICK1, attenuated AdA-enhanced increases in EPSC amplitudes. These results suggest that IP(3)R-mediated Ca(2+) release can enhance AMPAR EPSC amplitudes through mechanisms that involve AMPAR-PDZ interactions and/or synaptotagmin-SNARE-mediated receptor trafficking.     

PKC and polyamine modulation of GluR2-deficient AMPA receptors in immature neocortical pyramidal neurons of the rat

AMPA receptors (AMPARs) mediate the bulk of fast synaptic excitation in the CNS. We have recently shown that AMPAR-dependent synaptic transmission in immature neocortical pyramidal neurons is mediated by GluR2-deficient receptors that can be modulated by intra- or extracellular polyamines (PAs). Phosphorylation of AMPARs, e.g. by PKC, can lead to enhanced excitation, and PAs are known to modulate PKC activity. Therefore, PAs and PKC might interact to influence AMPAR function. To test this hypothesis, we made whole cell recordings from immature (P12–14) layer V pyramidal neurons and assayed two measures of PA influence on synaptic AMPAR function – inward rectification and use-dependent unblock (UDU), with the latter assayed by differences in rectification between a pair of EPSCs evoked at short (50 ms) latencies. We have previously shown that EPSCs in immature pyramidal neurons displayed inward rectification, which was enhanced by intracellular spermine, as was UDU. Staurosporin (ST), a PKC inhibitor, reversed the effect of PA on rectification and UDU, suggesting that PKC modulates postsynaptic activation of AMPARs. Similarly, polyamine-dependent rectification of spontaneous EPSCs was reversed by treatment with ST or GFX109203X, a specific PKC inhibitor. Chelating intracellular Ca²⁺ with BAPTA reproduced the effects of ST. In addition, PA immunoreactivity in layer V pyramidal neurons was reduced by PKC inhibition indicating that PKC activity influences PA metabolism. Taken together, these data support the involvement of postsynaptic PKC activation in both the inward rectification and UDU of EPSCs in immature rat cortex, and suggest an important mechanism by which excitatory synaptic transmission can be dynamically modulated by changes in either [Ca²⁺]_i or [PA]_i.     

Changes in synaptic structure underlie the developmental speeding of AMPA receptor-mediated EPSCs

At many excitatory and inhibitory synapses throughout the nervous system, postsynaptic currents become faster as the synapse matures, primarily owing to changes in receptor subunit composition. The origin of the developmental acceleration of AMPA receptor (AMPAR)-mediated excitatory postsynaptic currents (EPSCs) remains elusive. We used patch-clamp recordings, electron microscopic immunogold localization of AMPARs, partial three-dimensional reconstruction of the neuropil and numerical simulations of glutamate diffusion and AMPAR activation to examine the factors underlying the developmental speeding of miniature EPSCs in mouse cerebellar granule cells. We found that the main developmental change that permits submillisecond transmission at mature synapses is an alteration in the glutamate concentration waveform as experienced by AMPARs. This can be accounted for by changes in the synaptic structure and surrounding neuropil, rather than by a change in AMPAR properties. Our findings raise the possibility that structural alterations could be a general mechanism underlying the change in the time course of AMPAR-mediated synaptic transmission.     

Release kinetics, quantal parameters and their modulation during short-term depression at a developing synapse in the rat CNS

We have characterized developmental changes in the kinetics and quantal parameters of action potential (AP)-evoked neurotransmitter release during maturation of the calyx of Held synapse. Quantal size ( q ) and peak amplitudes of evoked EPSCs increased moderately, whereas the fraction of vesicles released by single APs decreased. During synaptic depression induced in postnatal day (P) 5–7 synapses by 10–100 Hz stimulation, q declined rapidly to 40–12% of its initial value. The decrease in q was generally smaller in more mature synapses (P12–14), but quite severe for frequencies ≥ 300 Hz. The stronger decline of q in immature synapses resulted from a slower recovery from desensitization, presumably due to delayed glutamate clearance. Recovery from this desensitization followed an exponential time course with a time constant of ∼480 ms in P5–7 synapses, and sped up > 20-fold during maturation. Deconvolution analysis of EPSCs revealed a significant acceleration of the release time course during development, which was accompanied by a 2-fold increase of the peak release rate. During long 100 Hz trains, more mature synapses were able to sustain average rates of 8–10 quanta s⁻¹ per active zone for phasic release. The rates of asynchronous vesicle release increased transiently > 35-fold immediately after such stimuli and decayed rapidly with an exponential time constant of ∼50 ms to low resting levels of spontaneous release. However, even following extended periods of 100 Hz stimulation, the amount of asynchronous release was relatively minor with peak rates of less than 5% of the average rate of synchronous release measured at steady state during the tetani. Therefore, a multitude of mechanisms seems to converge on the generation of fast, temporally precise and reliable high-frequency transmission at the mature calyx of Held synapse.     

Glutamatergic synapses in the rat nucleus tractus solitarii develop by direct insertion of calcium-impermeable AMPA receptors and without activation of NMDA receptors

Calcium influxes through ionotropic glutamate receptors (AMPA and NMDA receptors, AMPARs and NMDARs) are considered to be critical for the shaping and refinement of neural circuits during synaptogenesis. Using a combined morphological and electrophysiological approach, we evaluated this hypothesis at the level of the nucleus tractus solitarii (NTS), a brainstem structure that is a gateway for many visceral sensory afferent fibres. We confirmed that in the NTS, the first excitatory synapses appeared at embryonic day 18. We next characterized the biophysical properties of NTS AMPARs. Throughout perinatal development, both evoked and miniature EPSCs recorded in the presence of an NMDAR blocker were insensitive to polyamines and had linear current–voltage relationships. This demonstrated that AMPARs at NTS excitatory synapses were calcium-impermeable receptors composed of a majority of GluR₂ subunits. We then investigated the influence of calcium influxes through NMDARs on the development of NTS synaptic transmission. We found that NMDAR expression at synaptic sites did not precede AMPAR expression. Moreover, NMDAR blockade in utero did not prevent the development of AMPAR synaptic currents and the synaptic clustering of GluR₂ subunits. Thus, our data support an alternative model of synaptogenesis that does not depend on calcium influxes through either AMPARs or NMDARs. This model may be particularly relevant to the formation of neural networks devoted to basic behaviours required at birth for survival.     

Receptor saturation controls short-term synaptic plasticity at corticothalamic synapses

Glutamatergic synapses of layer 6 corticothalamic (CT) neurons form a major excitatory input onto thalamic relay cells, allowing neocortex to continuously control sensory information processing in thalamic circuits. CT synapses display both short- and long-term forms of use-dependent synaptic enhancement, mediated at least in part by increases in the probability of transmitter release. At some synapses, such increases in release probability are accompanied by a higher degree of multivesicular release (MVR) and larger glutamate transients at individual release sites, resulting in the saturation of postsynaptic receptors. The extent to which MVR and postsynaptic saturation interact and control short-term plasticity at CT synapses is not known. Here we examined two distinct presynaptic forms of short-term enhancement, facilitation and augmentation, at CT synapses contacting relay neurons in the ventrobasal nucleus of the mouse thalamus. We found that, in the presence of the low-affinity antagonist γ-D-glutamylglycine, to relieve postsynaptic DL-α-amino-3-hydroxy-5-methylisox azole-propionic acid (AMPA) receptor saturation, the magnitude of facilitation and augmentation increased. Whereas receptor saturation was prominent for both AMPA and N-methyl-D-aspartate receptors, desensitization of AMPA receptors did not significantly alter short-term plasticity. Our results suggest that at CT synapses the activity-dependent increase in synaptic strength is controlled by postsynaptic receptor saturation.     

Developmental alterations in the functional properties of excitatory neocortical synapses

In the neocortex, most excitatory, glutamatergic synapses are established during the first 4–5 weeks after birth. During this period profound changes in the properties of synaptic transmission occur. Excitatory postsynaptic potentials (EPSPs) at immature synaptic connections are profoundly and progressively reduced in response to moderate to high frequency (5–100 Hz) stimulation. With maturation, this frequency-dependent depression becomes progressively weaker and may eventually transform into a weak to moderate EPSP facilitation. In parallel to changes in the short-term plasticity, a reduction in the synaptic reliability occurs at most glutamatergic neocortical synapses: immature synapses show a high probability of neurotransmitter release as indicated by their low failure rate and small EPSP amplitude variation. This high reliability is reduced in mature synapses, which show considerably higher failure rates and more variable EPSP amplitudes. During early neocortical development synaptic vesicle pools are not yet fully differentiated and their replenishment may be slow, thus resulting in EPSP amplitude depression. The decrease in the probability of neurotransmitter release may be the result of an altered Ca²⁺ control in the presynaptic terminal with a reduced Ca²⁺ influx and/or a higher Ca²⁺ buffering capacity. This may lead to a lower synaptic reliability and a weaker short-term synaptic depression with maturation.     

Subunit interaction with PICK and GRIP controls Ca2+ permeability of AMPARs at cerebellar synapses

At many excitatory central synapses, activity produces a lasting change in the synaptic response by modifying postsynaptic AMPA receptors (AMPARs). Although much is known about proteins involved in the trafficking of Ca2+-impermeable (GluR2-containing) AMPARs, little is known about protein partners that regulate subunit trafficking and plasticity of Ca2+-permeable (GluR2-lacking) AMPARs. At cerebellar parallel fiber-stellate cell synapses, activity triggers a novel type of plasticity: Ca2+ influx through GluR2-lacking synaptic AMPARs drives incorporation of GluR2-containing AMPARs, generating rapid, lasting changes in excitatory postsynaptic current properties. Here we examine how glutamate receptor interacting protein (GRIP, also known as AMPAR binding protein or ABP) and protein interacting with C-kinase-1 (PICK) regulate subunit trafficking and plasticity. We find that repetitive synaptic activity triggers loss of synaptic GluR2-lacking AMPARs by selectively disrupting their interaction with GRIP and that PICK drives activity-dependent delivery of GluR2-containing receptors. This dynamic regulation of AMPARs provides a feedback mechanism for controlling Ca2+ permeability of synaptic receptors.     

Impaired synaptic scaling in mouse hippocampal neurones expressing NMDA receptors with reduced calcium permeability

NMDA receptors (NMDARs) play a crucial role for the acquisition of functional AMPARs during Hebbian synaptic plasticity at cortical and hippocampal synapses over a short timescale of seconds to minutes. In contrast, homeostatic synaptic plasticity can occur over longer timescales of hours to days. The induction mechanisms of this activity-dependent synaptic scaling are poorly understood but are assumed to be independent of NMDAR signalling in the cortex. Here we investigated in the hippocampus a potential role of NMDAR-mediated Ca²⁺ influx for synaptic scaling of AMPA currents by genetic means. The Ca²⁺ permeability of NMDARs was reduced by selective postnatal expression in principal neurones of mouse forebrain half of the NR1 subunits with an amino acid substitution at the critical channel site (N598R). This genetic manipulation did not reduce the total charge transfer via NMDARs in nucleated patches (somatic) and at synaptic sites. In contrast, the current amplitude and the charge carried through AMPARs were substantially reduced at somatic and synaptic sites in juvenile and adult mutants, indicating persistent downscaling of AMPA responses. Smaller and less frequent AMPA miniature currents in the mutant demonstrated a postsynaptic locus of this down-regulation. Afferent innervation and release probability were unchanged at CA3-to-CA1 synapses of mutants, as judged from input-output and minimal stimulation experiments. Our results indicate that NMDAR-mediated Ca²⁺ signalling is important for synaptic scaling of AMPA currents in the hippocampus in vivo .     

Channel properties reveal differential expression of TARPed and TARPless AMPARs in stargazer neurons

Dynamic regulation of calcium-permeable AMPA receptors (CP-AMPARs) is important for normal synaptic transmission, plasticity and pathological changes. Although the involvement of transmembrane AMPAR regulatory proteins (TARPs) in trafficking of calcium-impermeable AMPARs (CI-AMPARs) has been extensively studied, their role in the surface expression and function of CP-AMPARs remains unclear. We examined AMPAR-mediated currents in cerebellar stellate cells from stargazer mice, which lack the prototypical TARP stargazin (g-2). We found a marked increase in the contribution of CP-AMPARs to synaptic responses, indicating that, unlike CI-AMPARs, these can localize at synapses in the absence of g-2. In contrast with CP-AMPARs in extrasynaptic regions, synaptic CP-AMPARs displayed an unexpectedly low channel conductance and strong block by intracellular spermine, suggesting that they were ‘TARPless’. As a proof of principle that TARP association is not an absolute requirement for AMPAR clustering at synapses, miniature excitatory postsynaptic currents mediated by TARPless AMPARs were readily detected in stargazer granule cells following knockdown of their only other TARP, g-7.     

Postsynaptic conversion of silent synapses during LTP affects synaptic gain and transmission dynamics.

Synaptic transmission relies on both the gain and the dynamics of synapses. Activity-dependent changes in synaptic gain are well-documented at excitatory synapses and may represent a substrate for information storage in the brain. Here we examine the mechanisms of changes in transmission dynamics at excitatory synapses. We show that paired-pulse ratios (PPRs) of AMPAR and NMDAR EPSCs onto dentate gyrus granule cells are often different; this difference is reduced during LTP, reflecting PPR changes of AMPAR but not NMDAR EPSCs. Presynaptic manipulations, however, produce parallel changes in AMPAR and NMDAR EPSCs. LTP at these synapses reflects a reduction in the proportion of silent synapses lacking functional AMPARs. Changes in PPR during LTP therefore reflect the initial difference between PPRs of silent and functional synapses. Functional conversion of silent synapses permits postsynaptic sampling from additional release sites and thereby affects the dynamics and gain of signals conveyed between neurons.     

Persistent decrease in synaptic efficacy induced by nicotine at Schaffer collateral–CA1 synapses in the immature rat hippocampus

Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed within the brain where they contribute to the regulation of higher cognitive functions. The loss of the cholinergic function in Alzheimer's disease patients, along with the well-known memory enhancing effect of nicotine, emphasizes the role of cholinergic signalling in memory functions. The hippocampus, a key structure in learning and memory, is endowed with nAChRs localized at pre- and postsynaptic levels. In previous work on the immature hippocampus we have shown that, at low probability ( P ) synapses, activation of α7 nAChRs by nicotine or by endogenously released acetylcholine persistently enhanced glutamate release and converted 'presynaptically silent' synapses into functional ones. Here we show that in the same preparation, at high P synapses, nicotine induces long-term depression of AMPA- and NMDA-mediated synaptic currents. This effect was mediated by presynaptic α7- and β2-containing receptors and was associated with an increase in the paired pulse ratio and in the coefficient of variation. High P synapses could be converted into low P and vice versa by changing the extracellular Ca²⁺/Mg²⁺ ratio. In these conditions nicotine was able to persistently potentiate or depress synaptic responses depending on the initial P -values. A bi-directional control of synaptic plasticity by nicotine would considerably enhance the computational properties of the network during a critical period of postnatal development thus contributing to sculpt the neuronal circuit.     

Realistic modelling of receptor activation in hippocampal excitatory synapses: analysis of multivesicular release, release location, temperature and synaptic cross-talk

Chemically mediated synaptic transmission results from fusion of synaptic vesicles with the presynaptic plasma membrane, subsequent release of the vesicular content into the cleft and binding to postsynaptic receptors. Previous modelling studies of excitatory neurotransmitter glutamate were based on simplified geometries failing to account for the biologically realistic synaptic environment, in particular, the presence of astrocytes, the geometry of extracellular space, and the neurotransmitter uptake mechanism. Using 3-dimensional reconstructions of hippocampal glutamatergic synapses including the surrounding astrocytic processes we have developed a biologically realistic model to analyse receptor activation in different conditions. We used the finite element method to simulate glutamate release, analyse glutamate diffusion following single and multiple vesicle release and binding at the postsynaptic site to AMPA and NMDA receptors. We demonstrate that: (1) the transmitter diffusion is highly temperature-sensitive; (2) release conditions and geometry more specifically affect AMPARs than NMDARs; (3) the sensitivities of AMPARs and NMDARs to simultaneous vesicular release are different; (4) in the case of multivesicle neurotransmitter release with variable delays, the binding of glutamate to AMPARs is additive up to 1 ms after the release, then becomes independent, but to NMDARs the binding is additive up to 33 ms; (5) the number of AMPARs varies more than the number of NMDRs in response to the input firing patterns; (6) the presence of astrocytes effectively blocks synaptic cross-talk; and (7) synaptic cross-talk, mediated by NMDARs but not AMPARs, is only possible after quasi-simultaneous multivesicular release at physiological temperature (35°C) without intervening astrocytes, but not at 25°C. Our simulations demonstrate the importance of temperature and ultrastructural synaptic environment in synaptic transmission and synaptic cross-talk.     

Stargazin attenuates intracellular polyamine block of calcium-permeable AMPA receptors

Endogenous polyamines profoundly affect the activity of various ion channels, including that of calcium-permeable AMPA-type glutamate receptors (CP-AMPARs). Here we show that stargazin, a transmembrane AMPAR regulatory protein (TARP) known to influence transport, gating and desensitization of AMPARs, greatly reduces block of CP-AMPARs by intracellular polyamines. By decreasing CP-AMPAR affinity for cytoplasmic polyamines, stargazin enhances the charge transfer following single glutamate applications and eliminates the frequency-dependent facilitation seen with repeated applications. In cerebellar stellate cells, which express both synaptic CP-AMPARs and stargazin, we found that the rectification and unitary conductance of channels underlying excitatory postsynaptic currents were matched by those of recombinant AMPARs only when the latter were associated with stargazin. Taken together, our observations establish modulatory actions of stargazin that are specific to CP-AMPARs, and suggest that during synaptic transmission the activity of such receptors, and thus calcium influx, is fundamentally changed by TARPs.     

Properties of excitatory synaptic connections mediated by the corpus callosum in the developing rat neocortex.

Despite the major role of excitatory cortico-cortical connections in mediating neocortical activities, little is known about these synapses at the cellular level. Here we have characterized the synaptic properties of long-range excitatory-to-excitatory contacts between visually identified layer V pyramidal neurons of agranular frontal cortex in callosally connected neocortical slices from postnatal day 13 to 21 (P13-21) rats. Midline stimulation of the corpus callosum with a minimal stimulation paradigm evoked inward excitatory postsynaptic currents (EPSCs) with an averaged peak amplitude of 56.5 +/- 5 pA under conditions of whole cell voltage clamp at -70 mV. EPSCs had fixed latencies from stimulus onset and could follow stimulus trains (1-20 Hz) without changes in kinetic properties. Bath application of 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) abolished these responses completely, indicating that they were mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs). Evoked responses were isolated in picrotoxin to yield purely excitatory PSCs, and a low concentration of NBQX (0.1 microM) was used to partially block AMPARs and prevent epileptiform activity in the tissue. Depolarization of the recorded pyramidal neurons revealed a late, slowly decaying component that reversed at approximately 0 mV and was blocked by D-2-amino-5-phosphonovaleric acid. Thus AMPA and N-methyl-D-aspartate receptors (NMDARs) coexist at callosal synapses and are likely to be activated monosynaptically. The peak amplitudes and decay time constants for EPSCs evoked using minimal stimulation (+/-40 mV) were similar to spontaneously occurring sEPSCs. Typical conductances associated with AMPA and NMDAR-mediated components, deduced from their respective current-voltage (I-V) relationships, were 525 +/- 168 and 966 +/- 281 pS, respectively. AMPAR-mediated responses showed age-dependent changes in the rectification properties of their I-V relationships. While I-Vs from animals >P15 were linear, those in the younger (相似文献   

A delayed response enhancement during hippocampal presynaptic plasticity in mice

High frequency afferent stimulation of chemical synapses often induces short-term increases in synaptic efficacy, due to increased release probability and/or increased supply of readily releasable synaptic vesicles. This may be followed by synaptic depression, often caused by vesicle depletion. We here describe an additional, novel type of delayed and transient response enhancement phase which occurred during prolonged stimulation at 5–20 Hz frequency of excitatory glutamatergic synapses in slices from the adult mouse CA1 hippocampal region. This second enhancement phase, which was most clearly defined at physiological temperatures and essentially absent at 24°C, was dependent on the presence of F-actin filaments and synapsins I and/or II, and could not be ascribed to changes in presynaptic action potentials, inhibitory neurotransmission or glutamate receptor desensitization. Time course studies showed that the delayed response phase interrupted the synaptic decay 3–4 s after stimulus train initiation and continued, when examined at 5–10 Hz frequencies, for approximately 75 stimuli before decay. The novel response enhancement, probably deriving from a restricted pool of synaptic vesicles, may allow maintenance of synaptic efficacy during prolonged periods of excitatory synaptic activity.