Tai-Zhen Han

Prenatal exposure to heroin in mice elicits memory deficits that can be attributed to neuronal apoptosis.

Maternal heroin abuse has been shown to result in teratogenic neurobehavioral defects in the offspring, but the underlying mechanisms remain largely unknown. This study was designed to explore the role of neuronal apoptosis in the heroin-induced neurobehavioral defects of learning and memory. Pregnant BALB/c mice were treated with either heroin or saline. The animals in the heroin group received heroin subcutaneously at a dosage of 10 mg/kg/day on embryonic days (E) 9-18, while those in the saline group were treated as drug-naive. Offspring were grouped as prenatal heroin exposure (HER), prenatal saline exposure (SAL), and control (CON) groups, according to the maternal treatment regimen. Some of the mice were killed and their hippocampus harvested on postnatal day (P) 14, and the tissue subjected to reverse transcription polymerase chain reaction, Western blotting, and immunohistochemistry to reveal the mRNA and protein expressions of caspase-3, Bcl-2, and Bax. The Morris water maze was applied to assess the learning and memory capability of the mice at P30; poor maze performances were observed for the animals in the HER group. The results also showed that the mRNA and protein expressions of caspase-3 and Bax were significantly increased, while that of Bcl-2 was markedly decreased in the HER group compared with both the SAL and CON groups. The immunohistochemistry revealed significant caspase-3 immunoreactivity in the dentate gyrus and cornu ammonis (CA) 1 subareas of the hippocampal formation, whereas, no significant changes were seen in subarea CA3. These findings suggest that prenatal heroin exposure during the E9-18 period enhances neuronal apoptosis by altering the expressions of caspase-3, Bcl-2, and Bax in the mouse hippocampus, and leads to impairment in hippocampus-dependent learning and memory.

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.

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.

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.

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.