Yan-Hai Li

NR2A-containing NMDA receptors are required for L-LTP induction and depotentiation in CA1 region of hippocampal slices.

Long‐term potentiation (LTP) is a well‐characterized form of synaptic plasticity that fulfills many of the criteria for the neural correlate of memory. LTP reversal (or depotentiation, DP) is thought to correlate with prevention or elimination of memory storage. LTP during and immediately after induction can be easily reversed by afferent stimulation, when applied within the optimal time window. The aim of the present study was to determine whether later‐phase LTP (L‐LTP) could be reversed by special patterned stimulation applied at 2 h after LTP induction, as well as to characterize the receptor mechanisms underlying this reversal. Field excitatory postsynaptic potentials evoked by Schaffer collateral stimulation were recorded from the CA1 subfield of adult rat hippocampal slices. Results demonstrated that stable LTP, which was induced by six theta‐burst stimulations, was mediated by NR2A‐containing N‐methyl‐d‐aspartate receptors (NMDARs). This L‐LTP was partially reversed by high‐intensity paired‐pulse low‐frequency stimulation (HI‐PP‐LFS) and was inhibited by Zn2+ (30 nm), a voltage‐independent NR2A‐NMDAR antagonist. However, NR2B‐NMDAR antagonists (Ro 25‐6981, 1 μm) displayed no effect on L‐LTP reversal. L‐LTP partial reversal was also induced by HI‐PP‐LFS, when the protein synthesis inhibitors anisomycin (25 μm) and cycloheximide (60 μm) were applied following LTP induction. These results suggested that NR2A‐containing NMDARs are required for L‐LTP induction and DP in the hippocampal CA1 area of adult rats. Moreover, HI‐PP‐LFS was an effective stimulation pattern to induce DP.

Tonic facilitation of glutamate release by glycine binding sites on presynaptic NR2B-containing NMDA autoreceptors in the rat visual cortex

We have previously shown that glycine binding sites on presynaptic NMDA receptors (NMDA-Rs) can tonically regulate glutamate release in the rat visual cortex. In the present study, we investigated the subunit composition of these presynaptic NMDA-Rs. We recorded miniature a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (mEPSCs) using whole-cell voltage clamp in layer II/III pyramidal neurons of the rat visual cortex with the open-channel NMDA receptor blocker, MK-801, in the recording pipette. We found that the frequency of mEPSCs is significantly reduced by 7-chloro-kynurenic acid (7-Cl KYNA) an NMDA-R glycine binding site antagonist, and glycine reverses this effect. Using a specific antagonist for NR2B-NMDA-Rs, Ro 25-6981 [(alphaR,betaS)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidinepropanol hydrochloride], instead of 7-Cl KYNA, we found that the frequency of mEPSCs is also significantly reduced but glycine cannot reverse this effect. Moreover, Zn(2+), an NR2A-NMDA-R antagonist, did not affect mEPSC frequency. These results suggest that presynaptic NR2B-containing NMDA-Rs are located in layer II/III pyramidal neurons of the rat visual cortex, and that the glycine binding site of these type NMDA-Rs tonically regulates glutamate release.

NR2A-containing NMDA receptors are required for LTP induction in rat dorsolateral striatum in vitro

N-methyl-D-aspartate receptors (NMDARs) have been implicated in various forms of synaptic plasticity. In recent years, studies have been shown that NMDA receptor subunits play different roles in several forms of NMDAR-dependent synaptic plasticity. However, the contribution of NR2A and NR2B subunits in the induction of long-term potentiation (LTP) in the corticostriatal pathway remains unclear. The present study used patch-clamp recordings to study the role of NR2A-containing and NR2B-containing NMDARs in LTP induction in corticostriatal slices from 13-14-day old rats. High-frequency stimulation (HFS) of the corticostriatal pathway readily induced LTP of excitatory postsynaptic currents (EPSCs), and D-APV, a selective NMDAR antagonist, blocked LTP. Moreover, NR2B-containing NMDAR antagonists (Ro 25-6981 and ifenprodil) displayed no influence on LTP induction. However, LTP was not inducible in the presence of Zn(2+), an NR2A-containing NMDAR antagonist. These results suggest that the induction of LTP by HFS in the dorsolateral striatum is NMDAR-dependent and requires NR2A-containing NMDARs, not NR2B-containing NMDARs.

Presynaptic NR2B-Containing NMDA Autoreceptors Mediate Glutamatergic Synaptic Transmission in the Rat Visual Cortex

N-methyl-D-aspartate (NMDA) receptors (NMDA-Rs) have different modulatory effects on excitatory synaptic transmission depending on the receptor subtypes involved. The present study investigated the subunit composition of the presynaptic NMDA-Rs in layer II/III pyramidal neurons of the rat visual cortex. We recorded evoked a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (eEPSCs) using whole-cell voltage clamp with the open-channel NMDA receptor (NMDA-R) blocker, (+)-5-Methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine hydrogen maleate (MK-801), in the recording pipette. We found that the paired-pulse ratio (PPR) by two successive stimuli with inter-pulse intervals of 50 ms was significantly increased by D-APV, a selective NMDA-R antagonist. Using a specific antagonist for NR2B-NMDA-Rs, (alphaR,betaS)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidinepropanol hydrochloride (Ro 25-6981), instead of d-2-amino-5-phosphonovalerate (D-APV), we found that the PPR of eEPSCs was also significantly increased. Moreover, Zn(2+), an NR2A-NMDA-R antagonist, did not influence on the PPR. These results suggest that presynaptic NR2B-containing NMDA-Rs are located in layer II/III pyramidal neurons of the rat visual cortex, and that presynaptic NR2B-containing NMDA autoreceptors but not NR2A-containing NMDA autoreceptors mediate glutamate release in the rat visual cortex. Moreover, these findings may be clinically relevant to schizophrenia, where enhancing NMDA-R function is considered to be a promising strategy for treatment of the disease.

Glycine modulates synaptic NR2A- and NR2B-containing NMDA receptor-mediated responses in the rat visual cortex

In the central nervous system, activation of N-methyl-d-aspartate receptor (NMDA-R) glycine binding sites is a prerequisite for activation of synaptic NMDA-Rs by the excitatory neurotransmitter glutamate. Here we used patch-clamp recordings in transverse slice preparations to study whether the glycine binding site of the NMDA-R saturates and to determine their subunit composition in layer II/III pyramidal neurons of the rat visual cortex. We found that the NMDA-R-mediated component of miniature excitatory postsynaptic currents (mEPSCs) could be potentiated by exogenously applied glycine. Similar results were obtained by exogenously applied d-serine. A specific antagonist for NR2B-NMDA-Rs, Ro 25-6981, reduced NMDA-R-mediated mEPSCs, and glycine with Ro 25-6981 enhanced NMDA-R-mediated mEPSCs. Moreover, Zn2+, an NR2A-NMDA-R antagonist, also reduced NMDA-mediated mEPSCs and glycine with Zn2+ enhanced the NMDA-mediated mEPSCs. Our data indicate that the glycine binding site of synaptic NR2A-containing and NR2B-containing NMDA-Rs does not saturate and that glycine may act as a modulator of NMDA-R-mediated transmission in layer II/III pyramidal neurons of the rat visual cortex.

Membrane insertion of new AMPA receptors and LTP induced by glycine is prevented by blocking NR2A-containing NMDA receptors in the rat visual cortex in vitro.

N-methyl-D-aspartate receptors (NMDA-Rs) activation has been implicated in various forms of synaptic plasticity depending on the receptor subtypes involved. However, the contribution of NR2A and NR2B subunits in glycine-induced long-term potentiation (LTP) of miniature excitatory postsynaptic currents (mEPSCs) in layer II/III pyramidal neurons of the rat visual cortex remains unclear. The present study used whole-cell patch-clamp recordings in vitro to investigate the role of NR2A-containing and NR2B-containing NMDA-Rs in glycine-induced LTP in visual cortical slices from 13-15 day old rats. We found that glycine-induced LTP of mEPSCs was readily induced in layer II/III pyramidal neurons of the rat visual cortex with glycine. D-APV, a selective NMDA-R antagonist, blocked the glycineinduced LTP. Moreover, the selective NR2B-containing NMDA-R antagonists (Ro 25-6981) displayed no influence on the glycine-induced LTP. However, Zn2+, a voltage-independent NR2A-containing NMDA-R antagonist, prevented glycine-induced LTP. These results suggest that the glycine-induced LTP in layer II/III pyramidal neurons of the rat visual cortex is NMDA-R-dependent and requires NR2A-containing NMDA-Rs, not NR2B-containing NMDA-Rs.

Ca2+-permeable AMPA receptors mediate induction of test pulse depression of naive synapses in rat visual cortical slices at early postnatal stage.

Synaptic depression in the hippocampus at early postnatal stage can be induced by test pulse stimulation (<1 Hz). However, the receptor mechanism for induction of this synaptic depression is unclear. In the present study, we used whole-cell patch clamp recording in vitro to investigate how excitatory and inhibitory synapses onto layer II/III pyramidal neurons of the primary visual cortex adapt to test pulse activation from a previously non-activated (naive) state. We found that excitatory postsynaptic currents (EPSCs) of pyramidal neurons were rapidly depressed by 0.1 Hz stimulation in acutely prepared slices from rats at 11-12 postnatal days, while this phenomena disappeared in slices from young adolescent rats (23-24 postnatal days). By contrast, inhibitory postsynaptic currents (IPSCs) were relatively stable following 0.1 Hz stimulation of rat slices at the same early postnatal stage. Moreover, the test pulse depression of EPSCs was associated with a decrease in 1/coefficient of variation (CV)(2) and no change in the paired-pulse ratio. These data imply silencing of synapses and no significant change either in postsynaptic receptor density or presynaptic terminal release probability. This synaptic depression was unaffected by the competitive NMDA receptor antagonist D-APV. Ca(2+)-permeable AMPA receptor selective antagonists, Naspm or IEM-1460, prevented the induction of the test pulse depression. These data suggest that EPSCs, but not IPSCs, were rapidly depressed by test pulse stimulation in rats at early postnatal stage via a Ca(2+)-permeable AMPA receptor-dependent mechanism.

Increased Expression of Phospho-Cofilin in CA1 and Subiculum Areas after Theta-Burst Stimulation of Schaffer Collateral-Commissural Fibers in Rat Hippocampal Slices

Activity-dependent structural plasticity of dendritic spines of pyramidal neurons in the central neuron system has been proposed to be a cellular basis of learning and memory. Long-term potentiation (LTP) is accompanied by changes in synaptic morphology and structural remodeling of dendritic spines. However, there is considerable uncertainty as to the nature of the adjustment. The present study tested whether immunoreactive phospho-cofilin, an index of altered actin filament assembly, could be increased by theta-burst stimulations (TBS), which is an effective stimulation pattern for inducing LTP in the hippocampus. The slope of fEPSPs evoked by TBS to Schaffer collateral-commissural fibers in hippocampal slices was measured, and p-cofilin expression was examined using immunofluorescence techniques. Results indicated that saturated L-LTP was produced by multiple TBS episodes to Schaffer collateral-commissural fibers in the hippocampal CA1 area, and TBSs also increased immunoreactive p-cofilin expression in the stratum radiatum of the hippocampal CA1 area and pyramidal layer of the subiculum. D-2-amino-5-phosphonovalerate (D-APV) prevented LTP and expression of p-cofilin immunoreactive induced by multiple TBS episodes in the stratum radiatum of the hippocampal CA1 area. Two paired-pulse low-frequency stimulation (PP-LFS) episodes to Schaffer collateral-commissural fibers induced long-term depression (LTD), and did not affect p-cofilin expression in the stratum radiatum of the hippocampal CA1 area. These results suggest that LTP induction is associated with altered actin filament assembly. Moreover, the CA1 and subiculum areas of the hippocampal formation possibly cooperate with each other in important physiological functions, such as learning and memory, or in pathological diseases, such as epilepsy.

Selective 5-HT7 Receptor Activation May Enhance Synaptic Plasticity Through N-methyl-D-aspartate (NMDA) Receptor Activity in the Visual Cortex.

Serotonin (5-hydroxytryptamine, 5-HT) is an important neurotransmitter that modulates N-methyl-D-aspartate (NMDA) receptor activity by binding to several different 5-HT receptor subtypes. In the present study, we used whole-cell patch-clamp recordings in transverse slice preparations to test the role of 5-HT receptors in modulating the NMDA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) in layer II/III pyramidal neurons of the rat visual cortex. We found that the NMDA receptor-mediated component of mEPSCs could be potentiated by exogenously applied 5-HT. Similar results were obtained by exogenously applied 5-CT or 8-OH-DPAT (the 5-HT1A and 5-HT7 receptor agonist). A specific antagonist for the 5-HT7 receptor, SB-269970, completely blocked the increase in NMDA receptor-mediated component of mEPSCs by 5-CT or 8- OH-DPAT. Moreover, the selective 5-HT1A receptor antagonist, WAY-100135, displayed no influence on the enhancement in NMDA receptor-mediated component of mEPSCs by 5-CT or 8-OHDPAT. These results indicated that the increase in NMDA receptor-mediated component of mEPSCs by 5-HT in layer II/III pyramidal neurons of the young rat visual cortex requires activation of 5-HT7 receptors, but not 5-HT1A receptors. These observations might be clinically relevant to schizophrenia and Alzheimers disease (AD), where enhancing NMDA receptor-mediated neurotransmission is considered to be a promising strategy for treatment of these diseases.

Involvement of synaptic NR2B-containing NMDA receptors in long-term depression induction in the young rat visual cortex in vitro.

Activation of N-methyl-D-aspartate receptors (NMDARs) has been implicated in various forms of synaptic plasticity depending on the receptor subtypes involved. However, the contribution of NR2A and NR2B subunits in the induction of long-term depression (LTD) of excitatory postsynaptic currents (EPSCs) in layer II/III pyramidal neurons of the young rat visual cortex remains unclear. The present study used whole-cell patch-clamp recordings in vitro to investigate the role of NR2A- and NR2B-containing NMDARs in the induction of LTD in visual cortical slices from 12- to 15-day old rats. We found that LTD was readily induced in layer II/III pyramidal neurons of the rat visual cortex with 10-min 1-Hz stimulation paired with postsynaptic depolarization. D-APV, a selective NMDAR antagonist, blocked the induction of LTD. Moreover, the selective NR2B-containing NMDAR antagonists (Ro 25-6981 and ifenprodil) also prevented the induction of LTD. However, Zn2+, a voltage-independent NR2A-containing NMDAR antagonist, displayed no influence on the induction of LTD. These results suggest that the induction of LTD in layer II/III pyramidal neurons of the young rat visual cortex is NMDAR-dependent and requires NR2B-containing NMDARs, not NR2A-containing NMDARs.