[Developmental changes in synaptic and extrasynaptic N-methyl-D-aspartate receptors in cultured rat hippocampal neurons].

OBJECTIVE: To investigate changes in synaptic and extrasynaptic N-methyl-D-aspartate receptors (NMDAR) during the development of cultured rat hippocampal neurons. METHODS: Synaptic and extrasynaptic NMDAR channel currents were recorded from 1-day-old rat hippocampal neurons cultured for 1 and 2 weeks with patch-clamp technique in whole-cell configuration and outside-out configuration, respectively. RESULTS: The amplitude of NMDAR-mediated miniature excited postsynaptic current (Meps(CNMDA)) decreased in neurons cultured for 2 weeks as compared with that recorded in neurons cultured for 1 week, and the 2-week neurons showed also much lowered sensitivity to selective NR2B blocker ifenprodil. The amplitude and open probability of extrasynaptic NMDAR in the 2-week neurons were significantly higher than those in the 1-week neurons, but the neurons differred little in conduction and reverse potential. Ifenprodil decreased the high conductance and open probability in both neurons, but the effect was more potent in the 2-week ones. CONCLUSIONS: There can be developmental changes in synaptic and extrasynaptic NMDAR channel currents in cultured rat hippocampal neurons, indicating that different NMDAR subtypes are expressed in the synaptic and extrasynaptic regions during the development of the hippocampal neurons. In 1-week neurons, NR2B are predominant both in synaptic and extrasynaptic regions, and at 2 weeks, synaptic NR2B are replaced by NR2A but NR2B still remains the predominant subtypes outside the synapses.

Contribution of downregulation of L-type calcium currents to delayed neuronal death in rat hippocampus after global cerebral ischemia and reperfusion.

Transient forebrain ischemia induces delayed, selective neuronal death in the CA1 region of the hippocampus. The underlying molecular mechanisms are as yet unclear, but it is known that activation of L-type Ca2+ channels specifically increases the expression of a group of genes required for neuronal survival. Accordingly, we examined temporal changes in L-type calcium-channel activity in CA1 and CA3 pyramidal neurons of rat hippocampus after transient forebrain ischemia by patch-clamp techniques. In vulnerable CA1 neurons, L-type Ca2+-channel activity was persistently downregulated after ischemic insult, whereas in invulnerable CA3 neurons, no change occurred. Downregulation of L-type calcium channels was partially caused by oxidation modulation in postischemic channels. Furthermore, L-type but neither N-type nor P/Q-type Ca2+-channel antagonists alone significantly inhibited the survival of cultured hippocampal neurons. In contrast, specific L-type calcium-channel agonist remarkably reduced neuronal cell death and restored the inhibited channels induced by nitric oxide donor. More importantly, L-type calcium-channel agonist applied after reoxygenation or reperfusion significantly decreased neuronal injury in in vitro oxygen-glucose deprivation ischemic model and in animals subjected to forebrain ischemia-reperfusion. Together, the present results suggest that ischemia-induced inhibition of L-type calcium currents may give rise to delayed death of neurons in the CA1 region, possibly via oxidation mechanisms. Our findings may lead to a new perspective on neuronal death after ischemic insult and suggest that a novel therapeutic approach, activation of L-type calcium channels, could be tested at late stages of reperfusion for stroke treatment.

[Neuroprotection of chloride channel blockers against NMDA-induced apoptosis of cultured rat hippocampal neurons].

Activation of N-methyl-d-aspartic acid (NMDA) receptor plays an important role in neuronal apoptosis induced by cerebral ischemia but the underlying mechanisms are still unclear. The present study examined the neuroprotection of three chloride blockers in an in vitro cell model of cerebral ischemia established by treatment of cultured rat hippocampal neurons with NMDA. Hoechst 33258 staining and MTT assay were used to detect neuronal apoptosis and cell viability, respectively. The neuroprotective effects of chloride channel blockers on the cell viability and neuronal apoptosis were only observed when the blockers were applied before NMDA exposure. In comparison with DIDS, SITS showed more potent protective effect in a dose-dependent manner, whereas NPPB showed no significant neuroprotective effect. The results demonstrate that pretreatment with both SITS and DIDS have protective effect against neuronal apoptosis, which is achieved by blocking both NMDA receptor and chloride channel.

[Differential expression of L-type calcium channel alpha1 subunits on adult rat hippocampal neurons].

OBJECTIVE: To study the localization of L-type calcium channel alpha1 subunits CaV1.2alpha1C and CaV1.3alpha1D in CA1 and CA3 regions of adult rat hippocampus. METHODS: Immunohistochemical staining was employed for specific labeling of alpha1 subunits CaV1.2alpha1C and CaV1.3alpha1D. RESULTS: CaV1.2alpha1C subunit was mainly located in the apical and basal dendrites in the CA1 area and the CA3 area, neuronal soma, basal dendrites and distal apical dendrites were all positively stained. In contrast to CaV1.2alpha1C, CaV1.3alpha1D displayed no obvious difference in immunostaining between CA1 and CA3, and was distributed mainly in the neuronal soma and proximal dendrites. At cellular level, CaV1.2alpha1C distribution showed a distinct clustered pattern while a uniform distribution was observed for CaV1.3alpha1D subunit. CONCLUSION: Different alpha1 subunits of L-type calcium channel have differential expression in adult hippocampal neurons.

[Protective effects of liquid extract of Lishi No.5 formula against excitatory amino acid damage of neurons].

OBJECTIVE: To investigate the effect of liquid extract of Lishi No.5 formula in protecting the neurons from excitatory amino acid injury. METHODS: Cultured neonatal SD rat hippocampal neurons were treated with 300 micromol/L NMDA and 5 micromol/L glycine for 10 min, and the conditioned culture medium was replaced by normal culture medium. After cell culture for 18 h, MTT assay and Hoechst33258 staining were performed to examine the cell survival rate and cell apoptosis, respectively. Intracellular free calcium concentration was assayed by image analysis of the calcium signals with confocal microscope and Fluo-3/AM indicator. RESULTS: The survival rate of the cultured cell was significantly decreased in response to treatment with 300 micromol/L NMDA and 5 micromol/L glycine, but the effects were obviously reversed by treatment with 0.5 mg/ml liquid extract from Lishi No.5 formula. Intracellular free calcium concentration was significantly increased by 10 micromol/L NMDA, which was remarkably inhibited by the liquid extract. The effects of NMDA on intracellular free calcium was abolished by pretreatment of the cells with MK-801 for 10 min, whereas the liquid extract significantly lowered the antagonizing effect of MK-801. CONCLUSION: Lishi No.5 formula protects the neurons from toxicity by excitatory amino acids possibly by alleviating intracellular free calcium overload induced by these amino acids.