Objective To evaluate whether the thermotaxis tracking model is suitable for assessing long-term memory （LTM） in the nematode Caenorhabditis elegans. Methods Animals were trained at 20℃ overnight in presence of foo...Objective To evaluate whether the thermotaxis tracking model is suitable for assessing long-term memory （LTM） in the nematode Caenorhabditis elegans. Methods Animals were trained at 20℃ overnight in presence of food. The percentage of animals performing isothermal tracking （IT） behavior was measured at different time intervals after the training. Results The percentage of animals performing IT behavior, the numbers of body bends inside and outside the training temperature, and the expression patterns of AFD and AIY neurons were similar to those in control animals at 36 and 48 h after training; whereas when extending to 60, 72, and 84 h, locomotory behavior defects were observed in the assayed animals, suggesting that this thermal tracking model is feasible for analyzing LTM at 36 and 48 h after training. Moreover, the percent-age of animals performing IT behavior was reduced at 18, 36, and 48 h after training in neuronal calcium sensor-1 gene （nsc-1） mutant animals compared with that in wild-type N2 animals. In addition, exposure to plumbum （Pb） significantly repressed the LTM at 18, 36, and 48 h after training in both wild-type N2 and ncs-1 mutant animals. Conclusion The thermotaxis tracking model is suitable for evaluating the LTM regulated by NCS-1, and can be employed for elucidating regulatory functions of specific genes or effects of stimuli on memory in C. elegans.展开更多
To investigate the intracellular mechanism that interleukin 1β (IL 1β) facilitates epileptic seizure and neuronal damage, the effect of IL 1β alone or IL 1β plus glutamate (Glu) on the intracellular free calci...To investigate the intracellular mechanism that interleukin 1β (IL 1β) facilitates epileptic seizure and neuronal damage, the effect of IL 1β alone or IL 1β plus glutamate (Glu) on the intracellular free calcium ([Ca 2+ ] i) of single cultured hippocampal neuron was examined by using EPC 9 light electricity measurement system. The results showed that IL 1β of different concentrations (5×10 3 U/L, 10×10 3 U/L, 20×10 3 U/L, 30×10 3 U/L, 50×10 3 U/L, 100×10 3 U/L) failed to affect the neuronal [Ca 2+ ] i, but IL 1β could facilitate the augmentation of neuronal [Ca 2+ ] i induced by Glu in a dose dependent pattern. MK 801 inhibited the effect of Glu on [Ca 2+ ] i, and also inhibited the effect of IL 1β on [Ca 2+ ] i induced by Glu, while verapamil did not influence the effect of Glu or IL 1β. It is concluded that IL 1β, as a neuromodulator, can facilitate the activation of NMDA receptor by Glu, induce the increase of intracellular calcium, which enhances the excitement of neuron.展开更多
Activation of metabotropic glutamate receptor la aggravates traumatic brain injury. The constitutively expressed protein Homerlb/c participates in delivering and anchoring metabotropic glutamate receptors in neurons. ...Activation of metabotropic glutamate receptor la aggravates traumatic brain injury. The constitutively expressed protein Homerlb/c participates in delivering and anchoring metabotropic glutamate receptors in neurons. Here, we aimed to verify whether down-regulation of Homerlb/c by RNA interference could protect cultured rat cortical neurons from traumatic injury. We showed that 36 hours after transfection of Homerlb/c small interfering RNA, metabotropic glutamate receptor la was present only in the neuronal cytoplasm, but not in the dendrites. Calcium fluorescence intensity was also decreased significantly. Moreover, lactate dehydrogenase concentration was significantly decreased in Homerlb/c small interfering RNA-transfected cells compared with that in untransfected and control small interfering RNA-transfected cells 24 hours after traumatic neuronal injury. Our findings indicate that down-regulation of Homerlb/c could reduce metabotropic glutamate receptor la transfer from the cell body to the dendrite, relieve calcium overload, and protect neurons from traumatic injury.展开更多
Activation of cannabinoid receptor type 1 on presynaptic neurons is postulated to suppress neu- ~ ~ ~ 2＋ ~ ~ 2＋ rotransmlsslon by decreasing Ca reflux through high voltage-gated Ca channels. However, recent studies...Activation of cannabinoid receptor type 1 on presynaptic neurons is postulated to suppress neu- ~ ~ ~ 2＋ ~ ~ 2＋ rotransmlsslon by decreasing Ca reflux through high voltage-gated Ca channels. However, recent studies suggest that cannabinoids which activate cannabinoid receptor type 1 can increase neurotransmitter release by enhancing Ca2＋ influx in vitro. The aim of the present study was to investigate the modulation of intracellular Ca2＋ concentration by the cannabinoid receptor type 1 agonist anandamide, and its underlying mechanisms. Using whole cell voltage-damp and calcium imaging in cultured trigeminal ganglion neurons, we found that anandamide directly caused Ca2＋ influx in a dose-dependent manner, which then triggered an increase of intracellular Ca2＋ concentration. The cyclic adenosine and guanosine monophosphate-dependent protein kinase systems, but not the protein kinase C system, were involved in the increased intracellular Ca2＋concentration by anandamide. This result showed that anandamide increased intracellu- lar Ca2＋ concentration and inhibited high voltage-gated Ca2＋ channels through different signal transduction pathways.展开更多
Objective Presynaptic voltage-gated Ca^2＋ channels mediate rapid Ca^2＋ influx into the synaptic terminal which triggers synaptic vesicle exocytosis and neurotransmitter release. The FM 1-43 dye was firstly introduce...Objective Presynaptic voltage-gated Ca^2＋ channels mediate rapid Ca^2＋ influx into the synaptic terminal which triggers synaptic vesicle exocytosis and neurotransmitter release. The FM 1-43 dye was firstly introduced as a fluorescence probe by Betz and his colleagues in 1992, and has been used to monitor exocytosis, endocytosis and endosomal traffic in a variety of cell types. The present study aims to investigate the feasibility of applying the FM 1-43 dye in the functional analysis of calcium channel-mediated exocytosis in synaptic terminals. Methods The hippocampi were isolated from embryos of pregnant rats, and hippocampal neurons were then transfected with Ds-Red conjugated plasmid. The neurons were then loaded with 8 μmol/L FM 1-43 and 47 mmol/L KCl for 90 s after transfection. After that, 90 mmol/L KCI was employed to induce FM dye destaining, which was recorded by FM imaging system. Results The neuron synaptic terminals of rat hippocampus could be effectively stained by the FM 1-43 dye. Besides, the destaining of the labeled neuron terminals was in accordance with the transmitter release, which could be blocked by the application of nifedipine （inhibitor for L-type calcium channel）. Conclusion The FM imaging technique is an advanced and effective method for analyzing synaptic vesicle exocytosis and neurotransmitter release, and can be applied in various synaptic functional studies.展开更多
Objective Glycine acts as a co-agonist for the activation of N-methyl-D-aspartate receptors （NMDARs） by binding to glycine sites, thus potentiating glutamate-elicited responses and inhibiting NMDAR desensitization i...Objective Glycine acts as a co-agonist for the activation of N-methyl-D-aspartate receptors （NMDARs） by binding to glycine sites, thus potentiating glutamate-elicited responses and inhibiting NMDAR desensitization in a dose-dependent manner. The present study aimed to characterize the glycine-dependent inactivation of NMDARs and to explore its pathophysiological significance. Methods Primary hippocampal cell cultures from embryonic days 17-18 rats were treated with NMDA or NMDA plus glycine. Patch-clamp recording and intracellular Ca 2＋ imaging were performed to test the effects of glycine on NMDA-activated currents and increase of intracellular free Ca 2＋ respectively. Immunofluorescence staining was conducted to examine NR1 internalization. Cell damage was tested with MTT method and lactate dehydrogenase leakage. Results Glycine reduced the peak current and Ca 2＋ influx elicited by NMDA application at concentrations ≥300 μmol/L. This is a novel suppressive influence of glycine on NMDAR function, since it occurs via the NMDAR glycine-binding site, in contrast to the classic suppression, which occurs through the binding of glycine to glycine receptors. The level of membrane NMDARs was measured to evaluate whether internalization was involved. Immunohistochemical labeling showed that incubation with high concentrations of NMDA plus glycine did not change the expression of NMDARs on the cell surface when compared to the expression without glycine; hence the possibility of NMDAR internalization primed by glycine binding was excluded. Conclusion In summary, the novel suppressive effect of glycine on NMDARs was mediated via binding to the glycine site of the NMDAR and not by activation of the strychnine-sensitive glycine-receptor-gated chloride channel or by the internalization of NMDARs. The inhibitory influence of glycine on NMDARs adds a new insight to our knowledge of the complexity of synaptic transmission.展开更多
目的探讨N-甲基-D-天门冬氨酸(NMDA)诱导的小脑颗粒神经元(CGNs)钙超载与NMDA受体(NMDAR)及细胞内钙库受体三磷酸肌醇受体(IP3R)和兰尼定受体(RyR)之间的关系。方法取出生后8 d SD大鼠小脑进行颗粒神经元体外培养。用NMDA(100μmol/L)...目的探讨N-甲基-D-天门冬氨酸(NMDA)诱导的小脑颗粒神经元(CGNs)钙超载与NMDA受体(NMDAR)及细胞内钙库受体三磷酸肌醇受体(IP3R)和兰尼定受体(RyR)之间的关系。方法取出生后8 d SD大鼠小脑进行颗粒神经元体外培养。用NMDA(100μmol/L)急性损伤神经元,在激光扫描共聚焦显微镜(LSCM)扫描开始前30 min,或扫描开始后4,9,14 min分别加入NMDAR、IP3R、RyR拮抗剂MK-801、2-APB和DAN,检测神经元内Ca2+浓度的动态变化。结果 MK-801预孵育神经元经NMDA急性刺激后,神经元内Ca2+的荧光强度不再升高,NMDA刺激后加入MK-801,上升的Ca2+水平立即下降,最终下降至基线水平;NMDA急性刺激2-APB预孵育神经元,神经元内Ca2+的荧光强度升高,但升高幅度明显低于未经NMDA刺激组,NMDA刺激后加入2-APB,细胞内升高的Ca2+水平急剧下降,最终下降至接近基线水平;DAN预孵育的神经元经NMDA急性刺激后,胞内Ca2+的荧光强度急剧升高,达到NMDA刺激组水平,NMDA刺激后加入DAN,神经元内Ca2+水平无明显下降。结论 NMDA诱导的神经元Ca2+超载,主要由细胞膜钙通道NMDAR和细胞内钙释放通道IP3R介导,而细胞内钙释放通道RyR不起主导作用。展开更多
文摘Objective To evaluate whether the thermotaxis tracking model is suitable for assessing long-term memory （LTM） in the nematode Caenorhabditis elegans. Methods Animals were trained at 20℃ overnight in presence of food. The percentage of animals performing isothermal tracking （IT） behavior was measured at different time intervals after the training. Results The percentage of animals performing IT behavior, the numbers of body bends inside and outside the training temperature, and the expression patterns of AFD and AIY neurons were similar to those in control animals at 36 and 48 h after training; whereas when extending to 60, 72, and 84 h, locomotory behavior defects were observed in the assayed animals, suggesting that this thermal tracking model is feasible for analyzing LTM at 36 and 48 h after training. Moreover, the percent-age of animals performing IT behavior was reduced at 18, 36, and 48 h after training in neuronal calcium sensor-1 gene （nsc-1） mutant animals compared with that in wild-type N2 animals. In addition, exposure to plumbum （Pb） significantly repressed the LTM at 18, 36, and 48 h after training in both wild-type N2 and ncs-1 mutant animals. Conclusion The thermotaxis tracking model is suitable for evaluating the LTM regulated by NCS-1, and can be employed for elucidating regulatory functions of specific genes or effects of stimuli on memory in C. elegans.
文摘To investigate the intracellular mechanism that interleukin 1β (IL 1β) facilitates epileptic seizure and neuronal damage, the effect of IL 1β alone or IL 1β plus glutamate (Glu) on the intracellular free calcium ([Ca 2+ ] i) of single cultured hippocampal neuron was examined by using EPC 9 light electricity measurement system. The results showed that IL 1β of different concentrations (5×10 3 U/L, 10×10 3 U/L, 20×10 3 U/L, 30×10 3 U/L, 50×10 3 U/L, 100×10 3 U/L) failed to affect the neuronal [Ca 2+ ] i, but IL 1β could facilitate the augmentation of neuronal [Ca 2+ ] i induced by Glu in a dose dependent pattern. MK 801 inhibited the effect of Glu on [Ca 2+ ] i, and also inhibited the effect of IL 1β on [Ca 2+ ] i induced by Glu, while verapamil did not influence the effect of Glu or IL 1β. It is concluded that IL 1β, as a neuromodulator, can facilitate the activation of NMDA receptor by Glu, induce the increase of intracellular calcium, which enhances the excitement of neuron.
基金supported by the National Natural Science Foundation of China, No. 30930093the Natural Science Foundation of Shaanxi Province, No. 2005C_245
文摘Activation of metabotropic glutamate receptor la aggravates traumatic brain injury. The constitutively expressed protein Homerlb/c participates in delivering and anchoring metabotropic glutamate receptors in neurons. Here, we aimed to verify whether down-regulation of Homerlb/c by RNA interference could protect cultured rat cortical neurons from traumatic injury. We showed that 36 hours after transfection of Homerlb/c small interfering RNA, metabotropic glutamate receptor la was present only in the neuronal cytoplasm, but not in the dendrites. Calcium fluorescence intensity was also decreased significantly. Moreover, lactate dehydrogenase concentration was significantly decreased in Homerlb/c small interfering RNA-transfected cells compared with that in untransfected and control small interfering RNA-transfected cells 24 hours after traumatic neuronal injury. Our findings indicate that down-regulation of Homerlb/c could reduce metabotropic glutamate receptor la transfer from the cell body to the dendrite, relieve calcium overload, and protect neurons from traumatic injury.
基金supported by NIH,grant No.GM-63577NNSF,grant No.30571537,No.30271500+1 种基金the National Natural Science Foundation of China,No.30271500,30571537 and 813702462010 National Clinical Key Disciplines Construction Grant from the Ministry of Health of the People’s Republic of China
文摘Activation of cannabinoid receptor type 1 on presynaptic neurons is postulated to suppress neu- ~ ~ ~ 2＋ ~ ~ 2＋ rotransmlsslon by decreasing Ca reflux through high voltage-gated Ca channels. However, recent studies suggest that cannabinoids which activate cannabinoid receptor type 1 can increase neurotransmitter release by enhancing Ca2＋ influx in vitro. The aim of the present study was to investigate the modulation of intracellular Ca2＋ concentration by the cannabinoid receptor type 1 agonist anandamide, and its underlying mechanisms. Using whole cell voltage-damp and calcium imaging in cultured trigeminal ganglion neurons, we found that anandamide directly caused Ca2＋ influx in a dose-dependent manner, which then triggered an increase of intracellular Ca2＋ concentration. The cyclic adenosine and guanosine monophosphate-dependent protein kinase systems, but not the protein kinase C system, were involved in the increased intracellular Ca2＋concentration by anandamide. This result showed that anandamide increased intracellu- lar Ca2＋ concentration and inhibited high voltage-gated Ca2＋ channels through different signal transduction pathways.
基金supported by National Basic Research Development Program of China (No.2006CB705600)National Natural Science Foundation of China(No. 30700253+2 种基金No. 30800355)the Scientific Research Foundation for the Returned Overseas Chinese Scholars, Ministry of Education, China (No. 2008101)Program of Changjiang scholar and innovative research team in University of China(No. IRT 0734)
文摘Objective Presynaptic voltage-gated Ca^2＋ channels mediate rapid Ca^2＋ influx into the synaptic terminal which triggers synaptic vesicle exocytosis and neurotransmitter release. The FM 1-43 dye was firstly introduced as a fluorescence probe by Betz and his colleagues in 1992, and has been used to monitor exocytosis, endocytosis and endosomal traffic in a variety of cell types. The present study aims to investigate the feasibility of applying the FM 1-43 dye in the functional analysis of calcium channel-mediated exocytosis in synaptic terminals. Methods The hippocampi were isolated from embryos of pregnant rats, and hippocampal neurons were then transfected with Ds-Red conjugated plasmid. The neurons were then loaded with 8 μmol/L FM 1-43 and 47 mmol/L KCl for 90 s after transfection. After that, 90 mmol/L KCI was employed to induce FM dye destaining, which was recorded by FM imaging system. Results The neuron synaptic terminals of rat hippocampus could be effectively stained by the FM 1-43 dye. Besides, the destaining of the labeled neuron terminals was in accordance with the transmitter release, which could be blocked by the application of nifedipine （inhibitor for L-type calcium channel）. Conclusion The FM imaging technique is an advanced and effective method for analyzing synaptic vesicle exocytosis and neurotransmitter release, and can be applied in various synaptic functional studies.
基金supported by Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions
文摘Objective Glycine acts as a co-agonist for the activation of N-methyl-D-aspartate receptors （NMDARs） by binding to glycine sites, thus potentiating glutamate-elicited responses and inhibiting NMDAR desensitization in a dose-dependent manner. The present study aimed to characterize the glycine-dependent inactivation of NMDARs and to explore its pathophysiological significance. Methods Primary hippocampal cell cultures from embryonic days 17-18 rats were treated with NMDA or NMDA plus glycine. Patch-clamp recording and intracellular Ca 2＋ imaging were performed to test the effects of glycine on NMDA-activated currents and increase of intracellular free Ca 2＋ respectively. Immunofluorescence staining was conducted to examine NR1 internalization. Cell damage was tested with MTT method and lactate dehydrogenase leakage. Results Glycine reduced the peak current and Ca 2＋ influx elicited by NMDA application at concentrations ≥300 μmol/L. This is a novel suppressive influence of glycine on NMDAR function, since it occurs via the NMDAR glycine-binding site, in contrast to the classic suppression, which occurs through the binding of glycine to glycine receptors. The level of membrane NMDARs was measured to evaluate whether internalization was involved. Immunohistochemical labeling showed that incubation with high concentrations of NMDA plus glycine did not change the expression of NMDARs on the cell surface when compared to the expression without glycine; hence the possibility of NMDAR internalization primed by glycine binding was excluded. Conclusion In summary, the novel suppressive effect of glycine on NMDARs was mediated via binding to the glycine site of the NMDAR and not by activation of the strychnine-sensitive glycine-receptor-gated chloride channel or by the internalization of NMDARs. The inhibitory influence of glycine on NMDARs adds a new insight to our knowledge of the complexity of synaptic transmission.
