| Background: The N-methyl-D-aspartate receptor (NMDAR) plays a pivotal role in the process of glutamate-induced excitotoxicity associated with many neurological disorders including stroke, epilepsy, traumatic brain injury and some neurodegenerative diseases. Thus, a large variety of NMDAR antagonists have been evaluated for potential clinical use. However, most of these drugs produced either little benefit because of a narrow therapeutic time window for administration or significantly adverse effects at effective doses. Establishing a humoral autoimmune response targeting the NR1 subunit of NMDAR may be a novel therapeutic strategy. The previous studies led by our group demonstrated that the extracellular M3-M4 loop of NR1a, which is the dominant subunit of human NMDAR, might be more easily used as a molecular target in immunization intervention to control the activation of NMDAR. So the immune strategy targeting the M3-M4 loop could be expected to be therapeutic potential for excitotoxic brain injuries. Objective: In order to develop potential therapeutic strategy for excitotoxic brain injuries, the patch-clamp technique and acute isolation of hippocampal neurons technique were used to evaluate the effects of monoclonal antibody of NMDAR, MabN1and JHL, on NMDA-activated whole-cell currents in hippocampal neurons. Methods: We selected healthy Sprague–Dawley (S.D.) rats (postnatal days 10±3) and the neurons of the hippocampus were acutely isolated as research objects. Morphological methods and electrophysiological techniques were used to identify the hippocampal neurons. The patch-clamp technique in the whole-cell mode was used to evaluate the effects of MabN1 and JHL on NMDA-activated currents in the hippocampal neurons. In addition, the differences of mAbN1 and JHL from MK-801 control group were observed. Results: 1.Morphological properties: The typical acutely isolated neurons are smooth in the surface of the membrane and the bodies are taper or triangle with two or three dendrite and one long axon which is longer than 200 μ m. The integrated hippocampal neurons can live 4 hours at least in the standard bath solution. 2.Electrophysiological properties: The whole cell model are easily formed in isolated hippocampal neurons, and the success rate is over 90%(n>100). In the Current Clamp model, the rest potential is (- 65.13±5.64) mV(n=35). The membrane capacitance is (16.32±7.68) pF(n=27); the series resistance is (10.69±4.95)MΩ(n=27); the time constant is (163.28±86.65)μs(n=27). 3.The ion channel properties: Most cells (80%, n>100) were sensitive to NMDA(1mM) and glysine(10μM). NMDA can activate an inward current in acuted isolate neurons of the hippocampus. 4.The effects of the antibodies and MK-801 on NMDA-activated whole-cell currents in hippocampal neurons.(1) Monoclonal antibody, mAbN1, can inhibit the NMDA-activated whole-cell currents on hippocampal neurons (p<0.01). (2)Monoclonal antibody, JHL, shows no effect on NMDA-elicited currents (p>0.05). (3)MK-801 significantly ihhibits the NMDA-activated whole-cell currents on hippocampal neurons (p<0.01). (4)The inhibitory effect of 0.2μM mAbN1 is much weaker than that of 40μM MK-801(p<0.01). Conclusion: 1.The results show that monoclonal antibody, mAbN1, can inhibit the NMDA-activated whole-cell currents on hippocampal neurons by combining with NMDAR and inhibiting the ion channel open. Therefore mAbN1 is capable of reducing excitotoxic damage of the hippocampal neuron induced by glutamate. 2.Monoclonal antibody, JHL, is unable to protect the hippocampal neurons from the excitotoxic damage. 3.MK-801 significantly ihhibits the NMDA-activated whole-cell currents on hippocampal neurons. 4.The inhibition effect of 0.2μM mAbN1 is much weaker than that of 40μM MK-801. |
