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OBJECTIVE There are serious hazards in depression,and the precise mechanism underlying the delayed onset of clinical antidepressants remains unclear.The purpose of this study was to investigate the regular pattern of the speed-limiting role of excitation/inhibition(E/I)function balance in the mechanism of antidepressant action.METHODS Based on the previous study,we focused on glutamatergic pyramidal neurons in the medial prefrontal cortex(mPFC)here and used its excitability to represent the establishment of a new E/I functional balance.We studied the changes in the firing activity of glutamatergic pyramidal neuron in the mPFC at different administration times for five types of antidepressants that act on different pharmacological targets and different onset times,including fluoxetine(SSRI),duloxetine(SNRI),vilazodone[serotonin 1A receptor(5-HT1A)ago-nist and SSRI],ketamine[N-methyl-D-aspartate(NMDA)receptor antagonist],and hypidone hydrochloride(YL-0919,new antidepressant with sigma-1 receptor ago-nist and SSRI).We first examined the initial onset time of activation of pyramidal neurons using multichannel elec-trophysiological recordings and tested the antidepressant behavioral effects using the FST.We then selected three antidepressants(fluoxetine,ketamine,and vilazodone)to explore its effects on the BDNF-mTOR pathway by West-ern blotting.In addition,we disrupted the E/I function bal-ance using chemogenetics to investigate the antidepres-sant-like effects of YL-0919 and ketamine in the FST and TST.RESULTS We found that treatment with fluoxetine for 17 days significantly increased the firing activity of pyramidal neurons and decreased the immobility duration in the FST.Similarly,it took duloxetine for 10 d,vilazodone for 4 d,YL-0919 for 3 d and ketamine for 24 h,to exert such effects.Meanwhile,Western blotting results sug-gested that the expression of BDNF and phosphorylation of mTOR in the mPFC significantly increased.How-ever,haloperidol,a classic antipsychotic(without antide-pressant effects),exerted no such effects on the firing activities of pyramidal neurons.In addition,disrupting the E/I function balance(via activating the GABA neurons and inhibiting the glutamate neurons)blocks out the antidepressant-like effects of YL-0919 and ketamine in the FST and TST.CONCLUSION Taken together,our findings suggest that the commencement of antide-pressant effects may be accompanied by the increase in the firing activity of pyramidal neurons and the activation of the BDNF-mTOR pathway,which may be a necessary and rate-limiting process.The re-establishment of the E/I balance may be a landmark event for the onset of antide-pressant effects.
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Objective To evaluate the relationship between the hippocampal neuron-protective mechanism of hydrogen in a rat model of oxygen-glucose deprivation and restoration(OGD/R)and mito-chondrial autophagy.Methods Hippocampal neurons isolated from healthy Sprague-Dawley rats(24 h af-ter birth)were cultured in vitro,seeded in polylysine-coated 6-well plates at a density of 7×105 cells/well and then divided into 5 groups(n＝30 each)using a random number table method: control group(C group),OGD/R group,OGD/R+H2 group,OGD/R plus 3-methyladenine(3-MA)group(OGD/R+3-MA group),and OGD/R plus H2 plus 3-MA group(OGD/R+H2+3-MA group).The cells were cultured for 24 h in normal culture atmosphere(75％N2-20％O2-5％CO2)in group C,and cells were subjected to oxygen-glucose deprivation for 2 h followed by O2-glucose supply for 24 h to establish the model of OGD/R injury in OGD/R,OGD/R+H2,OGD/R+3-MA and OGD/R+H2+3-MA groups.The cells were cultured for 24 h in a hydrogen-rich incubator(60％H2-10％O2-5％CO2-25％N2)after establishing the model in group OGD/R+H2.Autophagy inhibitor 3-MA 10 mmol/L was added,and then cultured for 24 h in normal culture atmosphere after establishing the model in group OGD/R+3-MA.Autophagy inhibitor 3-MA 10 mmol/L was added,and then cultured for 24 h in hydrogen-rich incubator after establishing the model in group OGD/R+H2+3-MA.The cell survival rate was measured using MTT assay.DCFH-DA fluorescent probe was applied for determination of reactive oxygen species(ROS)activity.The mitochondrial membrane potential was measured using a JC-10 assay kit.The neuronal apoptosis was detected by flow cytometry,and apoptosis rate was calculated.The expression of mitophagy-related protein microtubule-associated protein 1 light chain 3(LC3),PINK1 and Parkin was determined by Western blot,and LC3Ⅱ/LC3Ⅰ ratio was calculated.Results Compared with group C,the cell survival rate and MMP were significantly decreased,the apop-tosis rate and ROS activity were increased,and the expression of PINK1 and Parkin and LC3Ⅱ/LC3Ⅰrati-o were increased in OGD/R and OGD/R+H2 groups(P＜0.05).Compared with group OGD/R,the cell survival rate and MMP were significantly increased,the apoptosis rate and ROS activity were decreased,and the expression of PINK1 and Parkin and LC3Ⅱ/LC3Ⅰ ratio were increased in group OGD/R+H2(P＜0.05),and the cell survival rate and MMP were significantly decreased,the apoptosis rate and ROS activ-ity were increased,and the expression of PINK1 and Parkin and LC3Ⅱ/LC3Ⅰ ratio were decreased in group OGD/R+3-MA(P＜0.05).Compared with group OGD/R+H2,the cell survival rate and MMP were significantly decreased,the apoptosis rate and ROS activity were increased,and the expression of PINK1 and Parkin and LC3Ⅱ/LC3Ⅰ ratio were decreased in OGD/R+3-MA and OGD/R+H2+3-MA groups(P＜0.05).Conclusion Hippocampal neuron-protective mechanism of hydrogen against OGDR injury is relat-ed to promoting mitochondrial autophagy in rats.
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Objective:To observe the effects of Chinese herbal compound'Jisuikang'on the phagocytosis of microglia and the regeneration of injured neurons in co-culture system.Methods: Prepared drug serum of 'Jisuikang′ and isolated and identified the primary neuron and microglia.The neuron cells were induced apoptosis by glutamic acid and the microglia cells were predisposed by drug serum of'Jisuikang'.Then,the co-culture system of injured neurons and microglia cells was established.24 h and 96 h after co-culture,engulfment of neuron debris by microglia cells and regeneration of injured neurons were observed by immunofluorescence double labeling method.Results: 24 h after co-culture,middle and high dose of'Jisuikang' showed greater phagocytic percentage and phagocytic index than that of control.In comparison of LPS,high dose of'Jisuikang' showed no significant difference.96 h after co-culture,first grade of neuritis of middle and high dose of'Jisuikang' were more than that of control,and there were no significant difference in comparison of LPS.Neuritis' mean length per cell of middle and high dose of'Jisuikang' were larger than that of con-trol.Neuritis' mean length per cell of high dose of'Jisuikang' showed significant difference in comparison of LPS.Conclusion:Traditional Chinese medicine compound'Jisuikang'may enhance engulfment of neuron debris by microglia to improve local microenvi-ronment,which promote the repair and regeneration of injured neurons.
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Aim To investigate the protective effect of noble dendrobium polysaccharides ( NDP ) on lipopo-lysaccharide ( LPS)-induced neuron injuries in newborn rat cerebral cortex glial cells and neuron mixed cul-tures.Methods The primary cultures of newborn rat cortical neurons and glial cells were established and the existence of the neurons , astrocytes and microglia was verified respectively .NDP was given to LPS-induced mixed cultures , the mRNA levels of IL-1β, TNF-αand COX-2 were assayed by real time PCR .Results NDP reduced the glial cell activation and neuron dam-age after it was given to LPS-induced mixed cultures . The mRNA levels of IL-1β, TNF-α, COX-2 were re-duced .Conclusion NDP protects against LPS-in-duced neuron-inflammation in neurons and glial cells cultures.
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Aim To provide an electrophysiological basis for hippocampus involved emotional changes caused by tinnitus, the effects of salicylate on electro-physiological characteristics of the voltage-gated calci-um channels in hippocampal neurons were performed. Method The effects of salicylate on voltage-gated cal-cium channels in rat hippocampal neurons were stud-ied, using the whole-cell voltage clamp method. Re-sults Salicylate blocked the voltage-gated calcium channels ( ICa ) in a concentration-dependent manner (0.1~10 mmol · L-1 ) . The half-inhibition concen-tration ( IC50 ) values for the blocking action of salicy-late on ICa were 1.64 mmol·L-1 . With 1 mmol·L-1 salicylate applicalted into bath solution, the steady-state activation curve of calcium channel was shifted by about 9 mV in the hyperpolarizing direction, and its steady-state inactivation curve was not changed. Con-clusion Salicylate inhibits ICa in rat hippocampal neu-rons and significantly affects the activation kinetics of ICa ,which could be related to emotional changes caused by tinnitus.
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Aim To investigate the effects of 17β-es-tradiol on the apoptosis induced by ketamine in primary cultured cortical neurons. Methods Primary cultured cortical neurons were treated with different concentra-tions of ketamine or 17β-estradiol respectively. 24 hours after various treatments, neuron viability was measured by MTT assay. The structure of neurons was analyzed using microscope. Apoptotic neurons were de-termined by the TUNEL assay. The level of pAkt ex-pression was analyzed by Western blot. ResultsCompared with the control group, ketamine decreased neuron viability in a dose-dependent manner. Com-pared with ketamine group, 17β-estradiol increased neuron viability in a dose-dependent manner. Lack of three-dimensional sense,faded color,uncleared outline were observed, and fractured neuron axons or neurons death were also observed in neurons treated by 100μmol · L-1 ketamine. 100 μmol · L-1 ketamine in-creased the number of apoptotic neurons and decreased the expression of pAkt. 0.1 μmol · L-1 17β-estradiol decreased the number of apoptotic neurons and in-creased the expression of pAkt. LY294002 inhibited the protective effects of 17β-estradiol, the number of apoptotic neurons increased, and the level of pAkt de-creased significantly. Conclusion 17β-estradiol ex-erts the neuroprotective effects against ketamine-in-duced neuroapoptosis by activating the PI3 K/Akt sig-naling pathway.