Postsynaptic GluN2B-containing NMDA receptors contribute to long-term depression induction in medial vestibular nucleus neurons of juvenile rats.

Medial vestibular nucleus (MVN) neurons are involved in the regulation of eye movements to endure the stability of the image during head movement, and play a critical role in plasticity of the vestibulo-ocular reflex (VOR) during the juvenile period. We have previously shown that the long-term depression (LTD) of synaptic transmission was induced by high frequency stimulation (HFS) and blocked by N-methyl-D-aspartate (NMDA) receptor antagonist D-APV at the vestibular afferent synapses of type-B MVN neurons. In the present study, we used whole-cell patch-clamp recordings in vitro to investigate the subunit composition of these NMDA receptors in the induction of LTD in MVN slices from postnatal 13-16 day rats. We found that LTD induced in type-B neurons of the rat MVN with HFS was blocked by Ro 25-6981, a specific antagonist for GluN2B-containing NMDA receptors. Moreover, the other selective GluN2B-containing NMDA receptor antagonist (ifenprodil) also prevented the induction of LTD. However, bath application of the GluN2A-containing NMDA receptor antagonists (Zn2+ and TCN 201) had no influence on the induction of LTD. Similar results were obtained by exogenously applied two GluN2C/GluN2D-preferring NMDA receptor antagonists (PPDA and UBP 141). Furthermore, presynaptic NMDA receptor subunits are not necessary for vestibular LTD. These results suggest that the induction of LTD by HFS in vestibular afferent synapses of type-B MVN neurons requires postsynaptic GluN2B-containing NMDA receptors, but not GluN2A-containing NMDA receptors or GluN2C/GluN2D-containing NMDA receptors.

Co-activation of both 5-HT1A and 5-HT7 receptors induced attenuation of glutamatergic synaptic transmission in the rat visual cortex.

It has been suggested that functional interactions between the 5-HT receptor subtypes may modulate glutamatergic synaptic transmission. In this study, we used whole-cell patch-clamp recordings to test the role of 5-HT receptors in mediating the AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) in layer II/III pyramidal neurons of the rat visual cortex. We found that the AMPA receptor-mediated component of mEPSCs could be inhibited by exogenously applied 5-HT. 5-HT significantly reduced the glutamatergic mEPSC amplitude and increased the inter-event interval of glutamatergic mEPSCs. Bath application of 5-CT or 8-OH-DPAT (the 5-HT1A and 5-HT7 receptor agonist) mimicked 5-HT in its effect on mEPSCs. Additionally, a selective antagonist for the 5-HT7 receptor, SB-269970, displayed no influence on the inhibition of glutamatergic synaptic transmission by 5-CT or 8-OH-DPAT. Similar results were obtained by exogenously applied WAY-100135, the selective 5-HT1A receptor antagonist. However, the inhibition of glutamatergic synaptic transmission by 5-CT or 8-OH-DPAT was completely blocked by co-application of WAY-100135 and SB-269970. Altogether, our results indicated that 5-HT suppressed glutamatergic synaptic transmission by co-activation of synaptic 5-HT1A receptors and 5-HT7 receptors in layer II/III pyramidal neurons of the rat visual cortex.

Activation of 5-HT7 receptors reverses NMDA-R-dependent LTD by activating PKA in medial vestibular neurons.

The medial vestibular nucleus (MVN) is a major output station for neurons that project to the vestibulo-spinal pathway. MVN neurons show capacity for long-term depression (LTD) during the juvenile period. We investigated LTD of MVN neurons using whole-cell patch-clamp recordings. High frequency stimulation (HFS) robustly induced LTD in 90% of type B neurons in the MVN, while only 10% of type A neurons were responsive, indicating that type B neurons are the major contributors to LTD in the MVN. The neuromodulator serotonin (5-HT) is known to modulate LTD in neural circuits of the cerebral cortex and the hippocampus. We therefore aim to determine the action of 5-HT on the LTD of type B MVN neurons and elucidate the relevant 5-HT receptor subtypes responsible for its action. Using specific agonists and antagonists of 5-HT receptors, we found that selective activation of 5-HT7 receptor in type B neurons in the MVN of juvenile (P13-16) rats completely abolished NMDA-receptor-mediated LTD in a protein kinase A (PKA)-dependent manner. Our finding that 5-HT restricts plasticity of type B MVN neurons via 5-HT7 receptors offers a mechanism whereby vestibular tuning contributes to the maturation of the vestibulo-spinal circuit and highlights the role of 5-HT in postural control.

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-HT_1A 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-HT_1A 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-HT_1A receptors. These observations might be clinically relevant to schizophrenia and Alzheimer's disease (AD), where enhancing NMDA receptor-mediated neurotransmission is considered to be a promising strategy for treatment of these diseases.

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.

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.

Chronic administration of Liu Wei Dihuang protects rat's brain against D-galactose-induced impairment of cholinergic system.

This study was aimed to investigate the protective effect of Liu Wei Dihuang (LWDH) against D-galactose (D-gal)-induced brain injury in rats and the existence of sex-dependent differences in LWDH protection. Sixty-four rats evenly composed of males and females were randomly assigned into 4 groups (n = 8): normal saline (NS) + NS (N + N), NS + LWDH (N + L), D-gal + NS (D + N) and D-gal + LWDH (D + L) groups. Rats in D + N and D + L groups received daily injection of D-gal (100 mg/kg, s.c.) for six weeks to establish the aging model, while rats in N + N and N + L groups were injected with the same volume of NS. From the third week, rats in N + L and D + L groups were orally administered with a decoction of LWDH for subsequent six weeks. Rats in N + N and D + N groups were orally administered just with the same volume of NS simultaneously. Morris water maze test was employed to evaluate the ability of learning and memory of the rats in all the groups. Acetylcholine (ACh) content, activities of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in visual cortex were assayed. Hematoxylin and eosin (HE) staining were used to observe the morphologic injury in hippocampus and visual cortex, and immunohistochemistry was performed to evaluate ChAT and AChE expression levels in the visual cortex. The results showed that the rats in D + N groups exhibited a longer escape latency to platform, lower swimming speed, less percent of target quadrant search time and platform crossings, compared with N + N groups, suggesting the establishment of aging model, while LWDH improved these indexes in D-gal-treated rats. Compared with D + N groups, LWDH increased ACh content and ChAT activity, and decreased AChE activity in visual cortex. Remarkable loss of neurons was found in hippocampus and visual cortex of aging rats, and the injury was significantly attenuated by LWDH. Immunohistochemistry showed D-gal-induced decreases of ChAT and AChE expressions were restored by LWDH. Furthermore, under the neural protection of LWDH, the improvement on platform crossings in male aging rats was better than that in female ones, while in ChAT expression and neuron density in visual cortex, female aging rats obtained more amelioration. These results suggest LWDH can markedly reverse the D-gal-induced cognitive impairments and neuronal damage in both hippocampus and visual cortex, which are achieved at least partly through restoring cholinergic system in central nervous system. Moreover, there is some sex difference in protective effects of LWDH against D-gal-induced impairment.

[The expressions of AMPAR/GluR2 in hippocampal CA1 area of rats before and after late-phase long-term potentiation reversal].

Late-phase long-term potentiation (L-LTP) plays a very important role in the maintenance of long-term memory in hippocampus. However, studies have shown that L-LTP can be reversed by subsequent neuronal activity. The aim of the present study is to investigate whether the presynaptic mechanism and the change of AMPARs expressions are involved in the reversal of L-LTP in hippocampal CA1 area. Standard extracellular recording technique was used to record the potential change in the stratum radiatum of CA1 area of adult rat hippocampal slices. Two hours after LTP induction, which was induced by high-frequency stimulation (HFS), two episodes of high-intensity paired-pulse low-frequency stimulation (HI-PP-LFS) were delivered to induce L-LTP reversal. Paired-pulse ratios (PPR) were obtained before LTP induction, 2 h after LTP induction and 30 min after LTP reversal. On the other hand, immunofluorescence histochemistry was used to detect AMPARs expressions before and after L-LTP reversal. The results showed that, after 2 h of induction, L-LTP was partially reversed by two episodes of HI-PP-LFS, and the percentage of depotentiation was 61.79%+/-14.51%. PPR obtained before and after LTP induction, and as well that after LTP reversal, are all more than 1, showing paired-pulse facilitation (PPF). Multiple comparison indicated PPR before LTP induction was the greatest one, and PPR after LTP induction was the smallest. In addition, no significant difference was observed in the intensity of AMPAR/GluR2 immunoreactivity in CA1 area among control group, LTP group and LTP reversal group. These results suggest that the presynaptic mechanism is involved in both the maintenance and reversal of L-LTP and there is no change in AMPAR/GluR2 expression before and after the reversal of L-LTP.

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.

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 Zn(2+) (30 nm), a voltage-independent NR2A-NMDAR antagonist. However, NR2B-NMDAR antagonists (Ro 25-6981, 1 mum) 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 microm) and cycloheximide (60 microm) 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.

Presynaptic NR2B-containing NMDA autoreceptors mediate gluta-matergic 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.

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.

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.

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.

Glycine binding sites of presynaptic NMDA receptors may tonically regulate glutamate release in the rat visual cortex.

In the CNS, activation of N-methyl-D-aspartate receptor (NMDA-R) glycine binding sites is a prerequisite for activation of postsynaptic NMDA-Rs by the excitatory neurotransmitter glutamate. Here we provide electrophysiological evidence that the glycine binding sites of presynaptic NMDA-Rs regulate glutamate release in layer II/III pyramidal neurons of the rat visual cortex. Specifically, our results reveal that the frequency of miniature excitatory postsynaptic currents is significantly reduced by 7-chloro-kynurenic acid (7-Cl KYNA), a NMDA-R glycine binding site antagonist, and glycine or D-serine reverses this effect. Similar results are obtained when the open-channel NMDA receptor blocker, MK-801, is included in the recording pipette. Our data indicate that the glycine binding site of postsynaptic NMDA-Rs is not saturated. Moreover, they suggest that presynaptic NMDA-Rs are located in layer II/III pyramidal neurons of the rat visual cortex and that the glycine binding site of presynaptic NMDA-Rs tonically regulates glutamate release.