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.

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.

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.

Different glutamate receptor mechanisms in long-term depression induced by different stimulus patterns in the CA1 area of adult rat hippocampus.

Previous reports suggested that a novel stimulus pattern of multi-train stimulus at low-frequency (2-Hz or 5-Hz) could induce stable long-term depression (LTD) in the CA1 area of adult rat hippocampus. In the present study, in order to determine the mechanism in LTD induced by the two novel tetanus patterns, changes in the population spikes (PS) in the hippocampal CA1 area of adult rats following the multi-train stimulus in the presence of AP5 [antagonist of N-methyl-D-aspartate receptors (NMDARs)] or MCPG [antagonist of type I/II metabotropic glutamate receptors (mGluRs)] were recorded. The results showed that both AP5 and MCPG inhibited the LTD induced by 2-Hz multi-train stimulus. The mean amplitude of population spikes (PSA) normalized to the baseline was (96.0±3.5)% after applying AP5 (n=10) and (95.7±4.1)% after applying MCPG (n=8), respectively, measured at 20 min post-tetanus. While 5-Hz multi-train tetanus failed to induce LTD in the presence of MCPG. The mean PSA was (73.6±4.4)% (n=10) and (98.2±8.9)% (n=8) in the presence of AP5 and MCPG, respectively, measured at 35 min post-tetanus. So it is suggested that LTD induced by 2-Hz multi-train tetanus involves co-activation of NMDARs and mGluRs, while LTD induced by 5-Hz multi-train tetanus is only related to activation of mGluRs.

[The expressions and distribution characters of glutamate receptor subunits NR1 and GluR2 in the hippocampal formation of adult rats].

OBJECTIVE: To investigate the expressions and distributions of N-methyl-D-aspartic acid NMDA/ NR1 and alpha-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid AMPA/GluR2 subunits in the hippocampal formation. METHODS: The immunofluorescence histochemical staining was made on paraformaldehyde fixed hippocampal tissue sections for then observed by confocal laser scanning microscopy. RESULTS: There were widely positive expressions and basically same distributions of NR1 and GluR2 in the hippocampal formation of adult rats, especially in pyramidal layer of CA1 and CA3 regions and in the interneurons of hilus fascia dentata. Positive expressions, which mainly existed in the cytomembrane and processes of the neurons, also distributed in molecular layer of CA1 and CA3 regions as well as in granule layer of dentate gyrus. However, the immunoreactive intensity of NMDA/NR1 was stronger than that of AMPA/GluR2 in molecular layer of CA3 area and in polymorphic layer of dentate gyrus. CONCLUSION: The distributions of NMDA/NR1 and AMPA/GluR2 in the hippocampal formation are basically same, this suggests that they may execute cooperatively some important tasks.

Age-specific impairment of morris water maze performance following neonatal exposure to magnetic resonance image in rats.

To investigate the effects of magnetic resonance image (MRI) exposure on CNS development, we examined the effects of neonatal exposure of rats to a MRI magnetic field on their performance in the Morris water maze. After birth, all litters were randomly divided into an MRI-scanning group (experimental group) comprised of 7 mother rats and their offspring, and a control group, which consisted of the other 7 mothers and their pups. Newborn rat pups of MRI-scanning group were exposed to a 1.5 T magnetic resonance image (MRI) magnetic field for 7 days (postnatal day 1-day 7, 10 min/day). And behavioral tests were taken at 1st-, 2nd- and 5th-month after birth. At the age of 2 months postnatal, both male and female rats of the experimental groups made fewer crossings over the target area in the probe trial than did the control group. This result showed that the exposed animals represented a "reference memory" deficit, that is to say, these rats had a deficit ability to use environmental cues to locate the former position in space. No deficits were evident in the 1st- and 5th-month groups. These results demonstrate an age-specific cognitive/behavioral deficit induced by neonatal exposure to an MRI magnetic field. These findings indicate that the safety of MRI exposure must be considered with care and appropriate cautions should be taken.

The tetanus patterns for the induction of long-term depression in the adult rat hippocampus.

Previous reports suggested that a low-frequency stimulus (LFS) of 1~2 Hz (600~900 pulses) induced a homosynaptic long-term depression (LTD) of synaptic efficacy in the hippocampal CA1 area of young rats (< 4-week old). However, these stimulation protocols often failed to induce LTD in the adult CA1 hippocampus. In the present study, we examined the effects of two novel tetanus patterns on LTD induction in adult rat hippocampal slices. We determined that these novel stimulation protocols induced LTD in the adult hippocampus, and that the characteristics of induced LTD were parameter-specific, including latency (period from the end of tetanus to a beginning of LTD) and the amplitude of LTD. These results suggest that LFS with certain patterns can induce LTD in the CA1 area of adult rat hippocampal slices, and that the multi-trains of 2-Hz protocol provided more effective response than the 5-Hz protocol.

[Changes of NMDA receptor expression in rat hippocampal formation after establishment of spatial memory by different training modes].

There is no unambiguous report so far on whether short reinforcing training could establish stable spatial long-term memory and how the NMDA/NR1 expression in the hippocampal formation changes after establishment of short- and long-term memory. In the present study, three spatial memory training modes of Morris water maze were used: spatial long-term memory training mode (LT group), spatial short-term memory training mode (ST group) and short reinforcing training mode (SRT group). The characteristics of those memories established by different training modes were compared. The immunofluorescence histochemical staining technique was used to observe the changes of NMDA/NR1 expression in the hippocampal formation in the three groups. The results showed that, during Morris water maze training, no significant difference was found in the mean latency and the strategies to the target between the LT and SRT groups. The examination of the memories following different trainings indicated that the mean latency and the strategies to the target as well as the times crossing the target in LT group were not significantly different from those in SRT group, except that the mean time remaining in the platform quadrant in LT group was significantly longer than that in SRT group. In addition, no significant difference in the intensity of NMDA/NR1 immunoreactivity in CA1, CA3 and dentate gyrus of the hippocampal formation was observed between the control and ST groups or between LT and SRT groups. However, the intensity of NMDA/NR1 immunoreactivity in CA1 area and dentate gyrus in both LT and SRT groups was significantly increased as compared with that in ST or the control groups. These results suggest that the short reinforcing training mode can establish long-term spatial memory, which is almost the same as that established by the long-term training mode, and that the increase of NMDA/NR1 expression in CA1 area and dentate gyrus of the hippocampal formation may be one of the mechanisms of spatial long-term memory formation.

A remote controlled multimode micro-stimulator for freely moving animals.

This paper presents a remote controlled multimode micro-stimulator based on the chip nRF24E1, which consists mainly of a micro-control unit (MCU) and a radio frequency (RF) transceiver. This micro-stimulator is very compact (18 mmx28 mm two layer printed circuit board) and light (5 g without battery), and can be carried on the back of a small animal to generate electrical stimuli according to the commands sent from a PC 10 meters away. The performance and effectiveness of the micro-stimulator were validated by in vitro experiments on the sciatic nerve (SN) of the frog, where action potentials (APs) as well as artifacts were observed when the SN was stimulated by the micro-stimulator. It was also shown by in vivo behavioral experiments on operant conditioned reflexes in rats which can be trained to obey auditory instruction cues by turning right or left to receive electrical stimulation ('virtual' reward) of the medial forebrain bundle (MFB) in a maze. The correct response for the rats to obey the instructions increased by three times and reached 93.5% in an average of 5 d. This micro-stimulator can not only be used for training small animals to become an 'animal robot', but also provide a new platform for behavioral and neurophysiological experiments.

Changes of synaptic structure after long-lasting LTP induced by high and low frequency tetanus in slices of the rat visual cortex.

Synaptic ultrastructural changes after long-lasting long-term potentiation (L-LTP) induced by 2 and 100 Hz tetanus were investigated by electron microscopic and stereological approach in slices of the developing rat visual cortex (postnatal days 15~21). Both 2 and 100 Hz tetanus-induced L-LTP groups showed significant increases in synaptic interface curvature, synaptic numeric density and postsynaptic density thickness, as well as significant decreases in the cleft width, as compared with the control groups. In addition, the volume density of the active zone (AZ) was increased significantly in the 100 Hz tetanus-induced L-LTP group, but not in the 2 Hz group. The mean lateral area of individual AZ in the 100 Hz group was relatively higher than that in the 2 Hz group. These data suggest that newly formed synapses in the 100 Hz tetanus-induced L-LTP group are larger than those in the 2 Hz group and that 100 Hz tetanus might trigger reorganization or synthesis of postsynaptic cytoskeleton.

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

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

Gender- and age-specific impairment of rat performance in the Morris water maze following prenatal exposure to an MRI magnetic field.

We examined the effects of prenatal exposure (40 min/day, gestation days 12-18) of rats to a magnetic resonance imaging (MRI) magnetic field (MF) on their performance in the Morris water maze. At 2 months of age, female rats showed impaired performance. The animals spent longer time swimming and used inefficient strategies. However, no significant effects on maze performance were observed at 1 and 5 months of age. No evident maze performance deficit was detected in male rats prenatally exposed to the magnetic field. Thus, we conclude that prenatal exposure to MRI magnetic field induces cognitive/behavioral deficits in female rats at a specific age.

Sexual dimorphism in the induction of LTP: critical role of tetanizing stimulation.

Numerous studies have suggested that sexual dimorphism may exist in learning and memory, particularly in types involving the hippocampus. In the present study, we examined the effects of two different tetani on the induction of long-term potentiation in the CA1 region of hippocampal slices from adult female and male rats to determine the sexual differences in their responses to tetanizing stimulation. We found that the induction of LTP is sex-dependent, and that there were clear sexual differences in the responses to different tetanus patterns, but not impulse number or stimulation frequency. Multiple trains of tetani were more effective in the indution of LTP in male rats than in female ones. These findings suggest that male rats can react to a broader range of tetanizing stimulation compared with female rats. Based on our results and the findings of other studies, we propose that the interaction of gonadal hormones with Ca2+/NMDAR and the subsequent regulation of the ERK/MAP kinase pathway are critical mechanisms for sexual dimorphism in the induction of LTP.

Different effects of 2 and 100 Hz tetanus on the expression of long-lasting long-term potentiation in rat visual cortical slices.

Long-term potentiation (LTP) can be induced by various tetanic parameters in the mammalian visual cortex. However, little researches have been done on the relationship between the expression of the long-lasting LTP (late phase LTP or L-LTP) lasting more than 3 h and the tetanic parameters. In the present study, the effects of 2 Hz and 100 Hz tetanic parameters on L-LTP of the field potentials were recorded from the layer II/III of the rat visual cortical slices in response to stimulation of the layer IV. As a result, tetanic parameters that had more than 300 pulses reliably induced L-LTP in the postnatal day 15-21 rats. Obviously different L-LTP expressions were induced by 2 Hz and 100 Hz tetani. There was no difference in L-LTP expression induced by the parameters with the same frequency and different total pulses. These data suggest that L-LTPs induced by different frequency parameters may have different induction and maintenance mechanisms; L-LTPs induced by the parameters with the same frequency may have the same mechanisms.

Morphological alteration of the hippocampal synapses in rats prenatally exposed to magnetic resonance imaging magnetic fields.

The present study investigated the effects of prenatal exposure to the magnetic resonance imaging (MRI) magnetic fields on the synaptic ultrastructure of hippocampal formation of rats at different postnatal development stages. Pregnant rats with gestation of 12-18 days were exposed to the magnetic fields used for MRI clinical applications. When the offspring were 1, 2, or 5-month-old, the synaptic morphologic parameters were measured in female offspring. In the 2-month-old MRI group, the curvature of synaptic interface, the length of active zone and the surface density per unit volume (S(v)) of active zone in the dentate gyrus (DG) decreased significantly, and the width of synaptic cleft increased in the CA1 area. In the 5-month-old MRI group, the width of synaptic cleft increased, the thickness of postsynaptic density and the curvature of synaptic interface decreased significantly in the CA1 region, and the width of synaptic cleft increased in the DG. No significant change was observed in the 1-month-old group. These results suggest that prenatal exposure to the medical magnetic fields causes synaptic ultrastructural changes. The relationship of these changes with behavioral impairments was discussed.

[Microscopic mechanism of excitation-contraction coupling in cardiac myocytes].

Cardiac excitation-contraction coupling (ECC) is, in essence, a communication process between sarcolemmal voltage-dependent L-type calcium channels (LCCs) and ryanodine receptors (RyRs) of sarcoplasmic reticulum (SR) by the mechanism of calcium-induced calcium release (CICR). Recent advances displayed more information about the microscopic signaling between LCCs and RyRs. In calcium release couplons, the calcium influx through the opening of LCCs by membrane depolarization forms calcium sparklets locally which then act on the adjacent SR RyRs. Stochastic activation of RyRs discharges calcium sparks from different calcium couplons, which summate into global calcium transients. Therefore, ignition of calcium sparks by calcium sparklets constitutes the elementary events in ECC. This review focuses on the intermolecular signaling between LCCs and RyRs, to describe the microscopic view of CICR and ECC.

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.

Prolonged reversal of long-term potentiation that is N-methyl-D-aspartate receptor independent: implications for memory formation.

Reversal of long-term potentiation (LTP) by low-frequency stimulation (LFS) is often referred to as depotentiation (DP), a phenomenon that is time-dependent. The present study aimed to determine whether LTP could still be reversed when the stimulation was applied beyond the optimal time window in hippocampal slices from adult rats. Field excitatory postsynaptic potentials (fEPSPs) were recorded from the strata radiatum in CA1, following stimulation of Schaffer collaterals. Theta-burst stimulation (TBS) induced LTP that could be reversed by repeated paired-pulse LFS (PP-LFS) after almost 3 h post-TBS. Only when synapse strength reached a plateau did application of PP-LFS trigger DP. In addition, it was surprising to observe that PP-LFS, which generally induces LTD in adult rats, evoked an LTP-like further strengthening in previously potentiationed synapses, even in the presence of APV, a competitive antagonist of N-methyl-D-aspartate receptors (NMDA-Rs). Our results suggest that LTP can be reversed NMDAR-independently more than 2 h after TBS by PP-LFS in adult hippocampus and that saturation of LTP is effective to promote this process.