Effect of Cholecystokinin Octapeptide on Expression of GLT-1 in Glutamate-induced Hippocampal Astrocytes / 华中科技大学学报(医学版)

Objective To investigate the effect of cholecystokinin octapeptide(CCK-8)on glutamate transporter 1(GLT-1)expression in hippocampal astrocytes induced by glutamate(Glu).Methods The mouse hippocampal astrocytes were isolated and the toxicity of CCK-8 at different concentrations on the mouse hippocampal astrocytes was detected.The cells were divided into control group,Glu group,Glu+0.1 μmol/L CCK-8 group,Glu+0.5 μmol/L CCK-8 group and Glu+1.0 μmol/L CCK-8 group.MTT assay was used to detect cell proliferation.Flow cytometry was used to detect cell apoptosis.Biochemical kit was used to detect Glu content in the extracellular supernatant,and qRT-PCR was used to detect the mRNA expression of GLT-1 and glutamate/aspartate transporter(GLAST).The protein expressions of Caspase-3,Bcl-2,GLT-1 and GLAST were detected by Western blotting,and the expression of TNF-α in the cell supernatant was detected by ELISA.Results CCK-8 at different concentrations had no significant effect on the proliferation of mouse hippocampal astrocytes.Compared with the control group,the cell proliferation ability and the expression levels of Bcl-2 protein,GLT-1 and GLAST mRNA and protein in Glu group were significantly decreased(all P＜0.01),the apoptosis rate,extracellular Glu content,Caspase-3 protein expression level in cells and TNF-α level in cell supernatant were significantly increased(all P＜0.01);Compared with the Glu group,the cell proliferation a-bility and the expression levels of Bcl-2 protein,GLT-1 and GLAST mRNA and protein in the Glu+0.5 μmol/L CCK-8 group and Glu+1.0 μmol/L CCK-8 group were significantly increased(all P＜0.05),the apoptosis rate,extracellular Glu content,Caspase-3 protein expression level in cells and TNF-α level in cell supernatant were significantly decreased(all P＜0.01).Con-clusion CCK-8 can inhibit Glu-induced inflammatory response of astrocytes,promote the expression of GLT-1,reduce the con-centration of extracellular Glu,promote cell proliferation and inhibit apoptosis.

Adolescent stress increases depression-like behaviors and alters the excitatory-inhibitory balance in aged mice / 中华医学杂志(英文版)

Background@#Depression affects approximately 5% of elderly people and its etiology might be related to chronic stress exposure during neurodevelopmental periods. In this study, we examined the effects of adolescent chronic social stress in aged mice on depressive behaviors and the excitatory-inhibitory (E/I) balance in stress-sensitive regions of the brain.@*Methods@#Sixty-four adolescent, male C57BL/6 mice were randomly assigned to either the 7-week (from post-natal days 29 to 77) social instability stress (stress group, n = 32) or normal housing conditions (control group, n = 32). At 15 months of age, 16 mice were randomly selected from each group for a series of behavioral tests, including two depression-related tasks (the sucrose preference test and the tail suspension test). Three days following the last behavioral test, eight mice were randomly selected from each group for immunohistochemical analyses to measure the cell density of parvalbumin (PV+)- and calretinin (CR+)-positive gamma-aminobutyric-acid (GABA)ergic inhibitory inter-neurons, and the expression levels of vesicular transporters of glutamate-1 (VGluT1) and vesicular GABA transporter (VGAT) in three stress-sensitive regions of the brain (the medial pre-frontal cortex [mPFC], hippocampus, and amygdala).@*Results@#Behaviorally, compared with the control group, adolescent chronic stress increased depression-like behaviors as shown in decreased sucrose preference (54.96 ± 1.97% vs. 43.11 ± 2.85%, t(22) = 3.417, P = 0.003) and reduced latency to immobility in the tail suspension test (92.77 ± 25.08 s vs. 33.14 ± 5.95 s, t(25) = 2.394, P = 0.025), but did not affect anxiety-like behaviors and pre-pulse inhibition. At the neurobiologic level, adolescent stress down-regulated PV+, not CR+, inter-neuron density in the mPFC (F(1, 39) = 19.30, P < 0.001), and hippocampus (F(1, 42) = 5.823, P = 0.020) and altered the CR+, not PV+, inter-neuron density in the amygdala (F(1, 28) = 23.16, P < 0.001). The VGluT1/VGAT ratio was decreased in all three regions (all F > 10.09, all P < 0.004), which suggests stress-induced hypoexcitability in these regions.@*Conclusions@#Chronic stress during adolescence increased depression-like behaviors in aged mice, which may be associated with the E/I imbalance in stress-sensitive brain regions.

Electroacupuncture Alleviates Motor Symptoms and Up-Regulates Vesicular Glutamatergic Transporter 1 Expression in the Subthalamic Nucleus in a Unilateral 6-Hydroxydopamine-Lesioned Hemi-Parkinsonian Rat Model / 神经科学通报·英文版

Previous studies have shown that electroacupuncture (EA) promotes recovery of motor function in Parkinson's disease (PD). However the mechanisms are not completely understood. Clinically, the subthalamic nucleus (STN) is a critical target for deep brain stimulation treatment of PD, and vesicular glutamate transporter 1 (VGluT1) plays an important role in the modulation of glutamate in the STN derived from the cortex. In this study, a 6-hydroxydopamine (6-OHDA)-lesioned rat model of PD was treated with 100 Hz EA for 4 weeks. Immunohistochemical analysis of tyrosine hydroxylase (TH) showed that EA treatment had no effect on TH expression in the ipsilateral striatum or substantia nigra pars compacta, though it alleviated several of the parkinsonian motor symptoms. Compared with the hemi-parkinsonian rats without EA treatment, the 100 Hz EA treatment significantly decreased apomorphine-induced rotation and increased the latency in the Rotarod test. Notably, the EA treatment reversed the 6-OHDA-induced down-regulation of VGluT1 in the STN. The results demonstrated that EA alleviated motor symptoms and up-regulated VGluT1 in the ipsilateral STN of hemi-parkinsonian rats, suggesting that up-regulation of VGluT1 in the STN may be related to the effects of EA on parkinsonian motor symptoms via restoration of function in the cortico-STN pathway.

The neuroprotective mechanism of ampicillin in a mouse model of transient forebrain ischemia

Ampicillin, a beta-lactam antibiotic, dose-dependently protects neurons against ischemic brain injury. The present study was performed to investigate the neuroprotective mechanism of ampicillin in a mouse model of transient global forebrain ischemia. Male C57BL/6 mice were anesthetized with halothane and subjected to bilateral common carotid artery occlusion for 40 min. Before transient forebrain ischemia, ampicillin (200 mg/kg, intraperitoneally [i.p.]) or penicillin G (6,000 U/kg or 20,000 U/kg, i.p.) was administered daily for 5 days. The pretreatment with ampicillin but not with penicillin G signifi cantly attenuated neuronal damage in the hippocampal CA1 subfield. Mechanistically, the increased activity of matrix metalloproteinases (MMPs) following forebrain ischemia was also attenuated by ampicillin treatment. In addition, the ampicillin treatment reversed increased immunoreactivities to glial fibrillary acidic protein and isolectin B4, markers of astrocytes and microglia, respectively. Furthermore, the ampicillin treatment significantly increased the level of glutamate transporter-1, and dihydrokainic acid (DHK, 10 mg/kg, i.p.), an inhibitor of glutamate transporter-1 (GLT-1), reversed the neuroprotective effect of ampicillin. Taken together, these data indicate that ampicillin provides neuroprotection against ischemia-reperfusion brain injury, possibly through inducing the GLT-1 protein and inhibiting the activity of MMP in the mouse hippocampus.

Effects of fluoxetine on changes of GLT-1 in rat prefrontal cortex after chronic unpredictable stress / 中国药理学通报

Aim To investigate the effects of fluoxe-tine on the changes of of protein levels of GLT-1 in pre-frontal cortex in rat depression model, and to further explore the molecular mechanism of antidepressant ac-tion of fluoxetine. Methods Sixty male SD rats were randomly assigned into three groups: control group, chronic unpredictable stress ( CUS) group, and CUS+fluoxetine group. The rats of CUS group and CUS+flu-oxetine group were subjected to CUS for 2 sessions per day for 35 days. Then, the rats of the CUS+fluoxetine group were given fluoxetine for 28 days. Behavioral changes were assessed by the sucrose preference and open field tests. The GLT-1 protein levels in the pre-frontal cortex were detected by immunohistochemistry and Western blot analysis at the end of the fluoxetine treatment. Results ( 1 ) Compared with the control group,sucrose preference, total traveling distance, ve-locity and frequencies of rearing were reduced in the CUS group ( P < 0. 01 ) . These behavioral changes could be reversed after 28 day fluoxetine treatment. (2 ) Immunohistochemistry assay indicated weak im-munoreactivity for GLT-1 in the prefrontal cortex of CUS group ( versus the control rats: P <0. 01 ); the immunoreactivity for GLT-1 of the fluoxetine-treated rats was significantly up-regulated compared with the CUS group rats ( P<0. 01 ) . ( 3 ) Western blot analy-sis indicated significant reductions of GLT-1 in the pre-frontal cortex of CUS group ( versus the control rats:P<0. 01 ) , and chronic fluoxetine treatment reversed the CUS-induced decrease in GLT-1 levels ( P <0. 01 ) . Conclusions Chronic unpredictable stress ( CUS ) could down-regulate the GLT-1 protein levels in the prefrontal cortex, which is reversed by fluoxe-tine. These results further support the notion that en-hanced expression of the GLT-1 protein could be mo-lecular mechanism of fluoxetine antidepressant effect.

Effects of ceftriaxone on depressive-like behavior and changes of hippocampal glutamate transporter-1 in depression model C57 mice / 中华行为医学与脑科学杂志

Objective To investigate the effects of ceftriaxone on depressive-like behavior and changes of hippocampal glutamate transporter-1 (GLT-1) in C57 mice depression model,and to further explore the molecular mechanism of ceftriaxone on antidepressant action.Methods Thirty male C57 mice were randomly divided into control group(group A,n=10),CUS group(group B,n=10) and CUS+ceftriaxone group(group C,n=10).The mice of the CUS group and the CUS+ceftriaxone group were subjected to chronic unpredictable stress (CUS) for 2 sessions per day for 21 days.Then,the mice of the CUS+ceftriaxone group were given ceftriaxone for 21 days.Behavioral changes were assessed by the sucrose preference test and open field test.The GLT-1 protein levels in the hippocampus were detected by Western blot analysis at the end of the ceftriaxone treatment.Results (1) Compared with the control group,the percentage of sucrose preference,the total traveled distance,the moved velocity,and the frequencies of rearing of the CUS group were significantly decreased(P＜0.05) at the 21 days.However,the percentage of sucrose preference ((78.74 ± 3.54) ％),the total traveled distance ((6818.35 ± 505.14) cm),the moved velocity((12.36±0.89) cm/s),and the frequencies of rearing(58.20±4.05) of the CUS+ceftriaxone group at the end of the ceftriaxone treatment were improved significantly compared with the CUS group ((59.46 ± 2.75) ％,(2931.71±271.89) cm,(5.84±0.42) cm/s,(26.20±2.62),P＜0.05).(2) Western blot analysis indicated significant reductions of the GLT-1 protein levels in the hippocampus of CUS group (versus the control mice:P ＜0.05),and chronic ceftriaxone treatment reversed the CUS-induced decrease in the GLT-1 levels(P＜0.05).Conclusion Ceftriaxone might significantly improve depressive-like behavior in C57 mice depression model.Chronic unpredictable stress (CUS) could down-regulate the GLT-1 protein levels in the hippocampus,which are reversed by ceftriaxone.These results further support the notion enhanced expression of the GLT-1 protcin can be molecular mechanism of ceftriaxone on antidepressant action.

Evaluation of Ampicillin and Ceftriaxone on Ethanol Consumption as Compared to Naltrexone in Alcohol Dependant Rats.

Context: Extracellular glutamate level in reward centre of brain increases during ethanol drinking sessions. Hence, it can be hypothesized that drugs which decrease extracellular glutamate might have deaddictive properties. It has been shown that β-lactam antibiotics are potent stimulators of glutamate transporter 1(GLT1) expression. Previous studies have shown that ceftriaxone decreases ethanol consumption but this has not been compared to standard line of treatment (naltrexone). Also, no study was conducted for testing ampicillin even if in an in-vitro experiment ampicillin has shown to increase GLT1 levels more than ceftriaxone. Hence, our study’s objectives were to compare efficacy of ceftriaxone and ampicillin with naltrexone on ethanol consumption in rats. Methods: Permission of ethics committee was taken. Study was divided into two parts. Part I included standardization of model & Part II included 8 groups of six rats each. Group 1: vehicle control, Group 2: 1mg/kg/d naltrexone, Group 3: 100mg/kg/d ceftriaxone, Group 4: 200mg/kg/d ceftriaxone, Group 5: 100mg/kg/d ampicillin, Group 6: 200mg/kg/d ampicillin were given i.p injections for 15 days and Group 7: 200mg/kg ceftriaxone & Group 8: 200mg/kg ampicillin i.p. single dose. Parameters measured were ethanol & water intake per day for 15 days. Results: Groups 2 to 8 showed statistically significant decrease in ethanol intake as compared to vehicle control. Also, group 3 & 4 showed an increase in water consumption as compared to Group 1. Conclusions: Our study recommends that drugs acting on glutamate pathways like ceftriaxone and ampicillin can be explored for treatment of alcohol dependence.

CHANGES OF EXPRESSION OF VESICULAR GLUTAMATE TRANSPORTER 1 IN THE RAT TRIGEMINAL COMPLEX AFTER MANDIBULAR NERVE TRANSECTION / 解剖学报

Objective The changes of VGluT1-like immunoreactivity(VGluT1-LI) in the trigeminal complex of the rat in different survival time after unilateral mandibular nerve transection were examined. Methods Immunocytochemical staining method and image analysis technique were performed. Results Many VGluT1-LI were observed in the trigeminal complex of the normal rat and were mainly distributed in the terminals.A weak decrease of VGIuT1-LI in the dorsal part of principal trigeminal nucleus(Vp) could be detected at the first week after unilateral transection of mandibular nerve(P