Low-Mg(2+) treatment increases sensitivity of voltage-gated Na(+) channels to Ca(2+)/calmodulin-mediated modulation in cultured hippocampal neurons.

Culture of hippocampal neurons in low-Mg(2+) medium (low-Mg(2+) neurons) results in induction of continuous seizure activity. However, the underlying mechanism of the contribution of low Mg(2+) to hyperexcitability of neurons has not been clarified. Our data, obtained using the patch-clamp technique, show that voltage-gated Na(+) channel (VGSC) activity, which is associated with a persistent, noninactivating Na(+) current (INa,P), was modulated by calmodulin (CaM) in a concentration-dependent manner in normal and low-Mg(2+) neurons, but the channel activity was more sensitive to Ca(2+)/CaM regulation in low-Mg(2+) than normal neurons. The increased sensitivity of VGSCs in low-Mg(2+) neurons was partially retained when CaM12 and CaM34, CaM mutants with disabled binding sites in the N or C lobe, were used but was diminished when CaM1234, a CaM mutant in which all four Ca(2+) sites are disabled, was used, indicating that functional Ca(2+)-binding sites from either lobe of CaM are required for modulation of VGSCs in low-Mg(2+) neurons. Furthermore, the number of neurons exhibiting colocalization of CaM with the VGSC subtypes NaV1.1, NaV1.2, and NaV1.3 was significantly higher in low- Mg(2+) than normal neurons, as shown by immunofluorescence. Our main finding is that low-Mg(2+) treatment increases sensitivity of VGSCs to Ca(2+)/CaM-mediated regulation. Our data reveal that CaM, as a core regulating factor, connects the functional roles of the three main intracellular ions, Na(+), Ca(2+), and Mg(2+), by modulating VGSCs and provides a possible explanation for the seizure discharge observed in low-Mg(2+) neurons.

Evaluation of compressive strength index of the femoral neck in Caucasians and chinese.

Compressive strength index (CSI) of the femoral neck is a parameter that integrates the information of bone mineral density (BMD), femoral neck width (FNW), and body weight. CSI is considered to have the potential to improve the performance of assessment for hip fracture risk. However, studies on CSI have been rare. In particular, few studies have evaluated the performance of CSI, in comparison with BMD, FNW, and bending geometry, for assessment of hip fracture risk. We studied two large populations, including 1683 unrelated U.S. Caucasians and 2758 unrelated Chinese adults. For all the study subjects, CSI, femoral neck BMD (FN_BMD), FNW, and bending geometry (section modulus [Z]) of the samples were obtained from dual-energy X-ray absorptiometry scans. We investigated the age-related trends of these bone phenotypes and potential sex and ethnic differences. We further evaluated the performance of these four phenotypes for assessment of hip fracture risk by logistic regression models. Chinese had significantly lower FN_BMD, FNW, and Z, but higher CSI than sex-matched Caucasians. Logistic regression analysis showed that higher CSI was significantly associated with lower risk of hip fracture, and the significance remained after adjusting for covariates of age, sex, and height. Each standard deviation (SD) increment in CSI was associated with odds ratios of 0.765 (95% confidence interval, 0.634, 0.992) and 0.724 (95% confidence interval, 0.569, 0.921) for hip fracture risk in Caucasians and Chinese, respectively. The higher CSI in Chinese may partially help explain the lower incidence of hip fractures in this population compared to Caucasians. Further studies in larger cohorts and/or longitudinal observations are necessary to confirm our findings.

Zinc transporter 3 immunohistochemical tracing of sprouting mossy fibres.

Zinc transporter 3 (ZNT3) has been shown to transport zinc ions from the cytosol into presynaptic vesicles in the mammalian brain. Several studies have stated that the zinc ion containing synaptic vesicles of zinc-enriched neurons (ZEN) are loaded with ZNT3 proteins in their membranes. This fact makes it possible to trace sprouting mossy fibres in the temporal lobe epileptic hippocampus. In the present study, we examined the expression and distribution patterns of ZNT3 protein and chelatable zinc ions in the mouse hippocampus after pilocarpine treatment. Our results demonstrate that both ZNT3 immunostaining and autometallography reveal identical patterns of sprouting mossy fibres in the inner molecular layer in the mouse hippocampus. Using ZNT3 immuno-electron microscopic analysis we confirmed the presence of ectopic mossy fibre terminals in the inner molecular layer and found additionally by immuno-blotting a significant increase of ZNT3 in the pilocarpine-treated mouse hippocampi compared to age-matched controls. The increase of ZNT3 after pilocarpine treatment was time-dependent. The results support the notion that ZNT3 immunohistochemistry provides an excellent tool for tracing sprouting of ZEN terminals. The progressive increase of ZNT3 immunostaining in the temporal lobe epileptic hippocampus may relate to the increased levels of vesicular zinc ions during seizure.

Voltage-gated sodium channel Nav1.1, Nav1.3 and beta1 subunit were up-regulated in the hippocampus of spontaneously epileptic rat.

The spontaneously epileptic rat (SER), a double mutant (zi/zi, tm/tm), exhibits both tonic convulsions and absence-like seizures from the age of 8 weeks. Since the first point mutation in the voltage-gated sodium channel (VGSC) beta(1) subunit in human generalized epilepsy with febrile seizures plus (GEFS+) was identified, more and more types of genetic epilepsy have been causally suggested to be related to gene changes in VGSC. However, there are no reports that can elucidate the effects of VGSC in SER. The present study was undertaken to detect sodium channel I alpha-isoform (Na(v)1.1), sodium channel III alpha-isoform (Na(v)1.3) and beta(1) subunit from both the level of mRNA and protein in SERs hippocampus compared with control Wistar rats. In this study, the mRNA expressions of Na(v)1.1, Na(v)1.3 and beta(1) subunit in SERs hippocampus were significantly higher than those in control rats hippocampus by real-time RT-PCR; The protein distributions and expressions of Na(v)1.1, Na(v)1.3 and beta(1) subunit in SERs hippocampus were detected by immunofluorescence, immunohistochemistry and western blot, and the protein expressions of Na(v)1.1, Na(v)1.3 and beta(1) subunit were significantly increased. In conclusion, our study suggested for the first time that sodium channel Na(v)1.1, Na(v)1.3 and beta(1) subunit up-regulation at the mRNA and protein levels of SER hippocampus might contribute to the generation of epileptiform activity and underlie the observed seizure phenotype in SER. The results of this study may be of value in revealing components of the molecular mechanisms of hippocampal excitation that are related to genetic epilepsy.

Abnormal expressions of glutamate transporters and metabotropic glutamate receptor 1 in the spontaneously epileptic rat hippocampus.

Excessive glutamatergic neurotransmission is considered an underlying factor of epilepsy. The modulation of the synaptic activity occurs both by the removal of glutamate from the synaptic cleft and by excitatory amino acid transporters (EAATs) and by modulation of glutamate receptors. The spontaneously epileptic rat (SER), a double mutant (zi/zi, tm/tm), exhibits both tonic convulsions and absence-like seizures from the age of 8 weeks. However, there are no reports that can elucidate the effects of EAATs and metabotropic glutamate receptors (mGluRs) in SER. The present study was undertaken to detect EAATs (GLAST, GLT-1 and EAAC-1) and Group I metabotropic glutamate receptors (mGluR1) in SER hippocampus from both the level of mRNA and protein in SERs hippocampus compared with control Wistar rats. In this study, the glutamate concentration in SERs hippocampus was increased compared with that of control rats by high performance liquid chromatography; the mRNA expressions of GLAST and mGluR1 in SERs hippocampus were significantly lower than those in control rats hippocampus, whereas an abundant increase in mRNA for GLT-1 was observed by RT-PCR; EAAC-1 and mGluR1 protein in SERs and control rats were localized widely in the hippocampus including CA1, CA3 and dentate gyrus regions by immunohistochemistry; the number of GLAST and mGluR1-positive cells in the hippocampus of SERs were less than those in control rats, especially for CA3 and DG region; the protein expression of GLT-1 was up-regulated, but the protein expressions of GLAST and mGluR1 were down-regulated in SER hippocampus by western blot. Our data show that epileptogenesis in SER are associated with regulations of glutamate transporters and mGluR1, which might be potential targets for therapy in genetic epilepsy.