Glutamate (Glu) is the major afferent excitatory neurotransmitter in the auditory system, and excessive Glu may play an important role in cochlear dysfunction. It is unclear how excessive Glu plays roles in cochlear dysfunction in cochlear organotypic cultures. In this study neonatal rat cochlear organotypic cultures were prepared, and then the cochlear tissues were incubated with a new medium containing specific concentrations of Glu (0.1, 0.5, 1, 10 or 20 mmol/L) for 24 h, or incubated with the medium containing a concentration of 20 mmol/L Glu for 6, 12, 24 or 72 h, respectively. It was found that when the cochlear tissues were cultured for 24 h, the inner hair cells (IHCs) were damaged at the concentration of 0.5 mmol/L Glu, and with the increases of the concentrations, the injury was gradually aggravated, and 20 mmol/L Glu resulted in the significant loss of IHCs. In the 20 mmol/L Glu groups, the stereocilia bundles were missing or disarrayed on a few IHCs after culture for 6 h and the damage effect was time-dependent. The missing of IHCs was more significant in the basal turn of the cochlea than in the middle turn of the cochlea under the same concentration of Glu exposure. These results suggest that excessive exogenous Glu affects the morphology of IHCs, but not affects the outer hair cells (OHCs) in cochlear organotypic cultures, and the excitotoxic effects are different on IHCs of different parts of the cochlea under the same concentration of Glu exposure.
Animals ; Cochlea ; drug effects ; Dose-Response Relationship, Drug ; Glutamic Acid ; toxicity ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of lead-exposed astrocyte conditioned medium (ACM) on the synaptic formation of neurons and to provide reference for the mechanism of lead neurotoxicity.

METHODSAstrocytes were cultured in the medium containing 50, 100, 200, 400, and 800 µmol/L lead acetate for 72 h. Alamar Blue was used to assess the cell viability of astrocytes, and then ACM was collected. Primarily cultured neurons were divided into six groups: pure culture group, non-glutamic acid (Glu)-induced ACM treatment group, Glu-induced lead-free ACM treatment group, and Glu-induced 50, 100, and 200 µmol/L lead acetate-exposed ACM treatment groups. Neurons were collected after being cultured in ACM for 24, 48, or 72 h. The content of synaptophysin (SYP) in neurons was determined by Western blot. The SYP expression in neurons was measured by immunofluorescence after being cultured in ACMfor 72 h.

RESULTSIn all lead-exposed groups, the cell viability of astrocytes declined with increasing concentration of lead (P < 0.05). The Western blot showed that compared with the pure culture group, the non-Glu-induced ACM treatment group and Glu-induced lead- free ACM treatment group had significantly increased content of SYP in neurons (P < 0.01); compared with the non-Glu-induced ACM treatment group, the Glu-induced ACM treatment groups had significantly reduced SYP expression in neurons (P < 0.05); compared with the Glu-induced lead-free ACM treatment group, all lead-exposed ACM treatment groups had the content of SYP in neurons significantly reduced with increasing concentration of lead after 72-h culture (P < 0.01), the 200 µmol/L lead-exposed ACM treatment group had significantly reduced content of SYP in neurons after 48-h culture (P < 0.01), and all lead-exposed ACM treatment groups showed no significant changes in the content of SYP in neurons after 24-h culture. Double-labeling immunofluorescence of SYP showed that all lead-exposed ACM treatment groups had a significant decrease in the number of SYP-fluorescent particles after 72-h culture (P < 0.05).

CONCLUSIONAstrocytes promote synaptic formation of neurons, which may be inhibited during lead exposure.

Astrocytes ; drug effects ; physiology ; Cell Survival ; drug effects ; Cells, Cultured ; Culture Media, Conditioned ; metabolism ; Glutamic Acid ; metabolism ; Lead ; toxicity ; Neurons ; drug effects ; Synapses ; drug effects ; physiology

Yi-Gan San (YGS), a traditional Chinese medicine for dementia-related symptoms, was previously fractionated. One active fraction, YGS40 exhibited a neuroprotective effect against glutamate-induced cytotoxicity. In the present study, high-performance liquid chromatography, coupled with diode-array detection and quadrupole time-of-flight mass spectrometry, was applied for the identification of its chemical constituents and for quantification studies. The chemical profile of YGS40 consisted of sixty-four identified or tentatively characterized compounds. The levels of the major marker compounds increased significantly in the mixed decoction compared with those in the single plant decoction. The results suggest the high precision of the analyses of most of the constituents in YGS40 and establish the quantitative variations of the major marker compounds between the single and mixed decoction processes.
Chromatography, Liquid ; Cytotoxins ; Drugs, Chinese Herbal ; analysis ; Glutamic Acid ; toxicity ; Mass Spectrometry ; Neuroprotective Agents ; analysis ; therapeutic use

OBJECTIVETo study the protective effect of cerebrospinal fluid containing Qingxin Kaiqiao recipe on PC12 cell injury induced by glutamate (Glu), in order to provide basis for the conical application of the recipe.

METHODSD rats were orally administered with decoction of Qingxin Kaiqiao recipe (7.9 g x kg(-1)) for three and a half days, 2 times a day, in order to prepare cerebrospinal fluid containing Qingxin Kaiqiao recipe. PC cells were divided into the normal group, the model group, the nimodipine group, the 10% normal CSF group, the 10% medicated CSF group, the 20% normal CSF group, the 20% medicated CSF group. Except for the normal group, other groups were cultured with PC12 cells and Glu with the final concentration of 20 mmol x L(-1) to establish the nerve cell injury model. Apart from the model group and the normal group, other groups were intervened with nimodipine, normal cerebrospinal fluid, and 10% and 20% medicated CSF. RT-PCR was used to detect the expression level of Bax mRNA, Bcl-2 mRNA and Caspase-3 mRNA, and MTT method was used to detect the activity of PC12 cells.

RESULTThe activity of PC12 cells of all of medicated CSF groups was higher than that of the model group, with the decrease in the expression of Bax mRNA and Caspase-3 mRNA and the increase in the expression of Bcl-2 mRNA. They showed a significant different with the model group (P < 0.01). The 20% medicated CSF group was superior than the 10% medicated CSF group (P < 0.01).

CONCLUSIONQingxin Kaiqiao recipe shows an apparent protective effect on PC12 cells injured by Glu.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; genetics ; Cerebrospinal Fluid ; Glutamic Acid ; toxicity ; Male ; Medicine, Chinese Traditional ; Neuroprotective Agents ; pharmacology ; PC12 Cells ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; Rats ; Rats, Sprague-Dawley

BACKGROUNDMonosodium L-glutamate (MSG) is a food flavour enhancer and its potential harmfulness to the heart remains controversial. We investigated whether MSG could induce cardiac arrhythmias and apoptosis via the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor.

METHODSMyocardial infarction (MI) was created by ligating the coronary artery and ventricular arrhythmias were monitored by electrocardiogram in the rat in vivo. Neonatal rat cardiomyocytes were isolated and cultured. Cell viability was estimated by 3-(4,5)-dimethylthiahiazo(-z-yl)-3,5-di-phenytetrazoliumromide (MTT) assay. Calcium mobilization was monitored by confocal microscopy. Cardiomyocyte apoptosis was evaluated by acridine orange staining, flow cytometry, DNA laddering, reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.

RESULTSMSG (i.v.) decreased the heart rate at 0.5 g/kg and serious bradycardia at 1.5 g/kg, but could not induce ventricular tachyarrhythmias in normal rats in vivo. In rats with acute MI in vivo, however, MSG (1.5 g/kg, i.v.) induced ventricular tachyarrhythmias and these arrhythmias could be prevented by blocking the AMPA and N-methyl-d-aspartate (NMDA) receptors. Selectively activating the AMPA or NMDA receptor induced ventricular tachyarrhythmias in MI rats. At the cellular level, AMPA induced calcium mobilization, oxidative stress, mitochondrial dysfunction and apoptosis in cultured cardiomyocytes, especially when the AMPA receptor desensitization were blocked by cyclothiazide. The above toxic cellular effects of AMPA were abolished by AMPA receptor blockade or by H2O2 scavengers.

CONCLUSIONSMSG induces bradycardia in normal rats, but triggers lethal tachyarrhythmias in myocardial infarcted rats probably by hindering AMPA receptors. AMPA receptor overstimulation also induces cardiomyocyte apoptosis, which may facilitate arrhythmia.

Animals ; Apoptosis ; drug effects ; Arrhythmias, Cardiac ; chemically induced ; Calcium ; metabolism ; Cell Survival ; drug effects ; Cells, Cultured ; DNA Fragmentation ; drug effects ; Glutamic Acid ; toxicity ; Male ; Microscopy, Confocal ; Myocardial Infarction ; chemically induced ; Rats ; Rats, Wistar ; Receptors, AMPA ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sodium Glutamate ; toxicity ; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid ; toxicity

OBJECTIVETo probe the effect of sodium para-aminosalicylate (PAS-Na) on concentration of amino acid neurotransmitters including glutamate (Glu), glutamine (Gln), glycine (Gly) and gamma-aminobutyric acid (GABA) in basal ganglia of subacute manganese (Mn)-exposed rats.

METHODSForty Sprague-Dawley male rats were randomly divided into the control, Mn-exposed, low dose PAS-Na (L-PAS) and high dose PAS-Na (H-PAS) groups. Rats in experiment groups received daily intraperitoneally injections of manganese chloride (MnCl₂ · 4H₂O, 15 mg/kg), while rats in control group received daily intraperitoneally injections of normal saline (NS), all at 5 days/week for 4 weeks. Then the rats in PAS groups followed by a daily subcutaneously dose of PAS-Na (100 and 200 mg/kg as the L-PAS and H-PAS groups, respectively) for another 3 and 6 weeks; while the rats in Mn-exposed and control group received NS. The concentrations of Glu, Gln, Gly and GABA in basal ganglia of rat was detected by the high performance liquid chromatography fluorescence detection technique.

RESULTSAfter treating with PAS-Na for 3 weeks, the concentration of Gly in the Mn-exposed rats decreased to (0.165 ± 0.022) µmol/L (control = (0.271 ± 0.074) µmol/L, Mn vs control, t = 4.65, P < 0.05). After the further 6-week therapy with PAS-Na, the concentrations of Glu, Gln, Gly in the Mn-exposed rats were lower than those of the control rats ((0.942 ± 0.121), (0.377 ± 0.070), (0.142 ± 0.048), (1.590 ± 0.302), (0.563 ± 0.040), (0.247 ± 0.084) µmol/L; t = 7.72, 5.85, 4.30, P < 0.05); and also lower than in L-PAS and H-PAS groups, whose concentrations were separately (1.268 ± 0.124), (1.465 ± 0.196), (0.497 ± 0.050), (0.514 ± 0.103), (0.219 ± 0.034) µmol/L (L-PAS Glu and Gln vs Mn, t = 3.87, 3.77, P < 0.05; H-PAS Glu, Gln and Gly vs Mn, t = 6.78, 4.70, 3.42, P < 0.05).

CONCLUSIONThe toxic effect of manganese on Glu, Gln and Gly in basal ganglia of Mn-exposed rats is obvious, especially appears earlier on Gly. The toxic effect still continues to develop when relieved from the exposure. PAS-Na may play an antagonism role in toxic effect of manganese on concentration of Glu, Gln and Gly in basal ganglia of Mn-exposed rats.

Amino Acids ; metabolism ; Animals ; Basal Ganglia ; drug effects ; metabolism ; Glutamic Acid ; metabolism ; Male ; Manganese ; toxicity ; Neurotransmitter Agents ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sodium Salicylate ; pharmacology ; gamma-Aminobutyric Acid ; metabolism

OBJECTIVETo explore the effects of 1,2-dichloroethane (1,2-DCE) on the behavior and the brain neurotransmitter levels in mice.

METHODSThirty mice were randomly divided into four groups, which were control group and groups of low, middle and high exposure (225, 450 and 900 mg/m3) to 1,2-DCE for 10 days (3.5 h a day) by inhalation. After the last exposure, the open field test was performed immediately. After exposure all mice were killed and the brain tissues were taken up rapidly. The levels of aspartate (Asp), glutamate (Glu) and gamma-aminobutyric acid (GABA) in the brain were detected by high performance liquid chromatography (HPLC).

RESULTSLevels of Asp and Glu in all exposure groups increased with doses. As compared to the control group, levels of Glu in all exposure groups increased significantly (P < 0.05). Levels of GABA in the low exposure group were significantly lower than those in control group, but those in the high exposure group were significantly higher than those in control group. The results of the open field test showed that effect of low exposure to 1,2-DCE on the behavior was stimulant, but the high exposure to 1,2-DCE inhibited behavior of exploration, excitement and sport.

CONCLUSIONSSubacute exposure to 1,2-DCE could result in the change of amino acid neurotransmitter content and ratio in the brain, thereby change the behavior of mice appeared, which might be the mechanism of neurotoxicity caused by 1,2-DCE in part.

Animals ; Aspartic Acid ; analysis ; Behavior, Animal ; drug effects ; Brain ; metabolism ; Ethylene Dichlorides ; toxicity ; Female ; Glutamic Acid ; analysis ; Mice ; Mice, Inbred Strains ; Neurotransmitter Agents ; metabolism ; gamma-Aminobutyric Acid ; analysis

OBJECTIVETo investigate the effects of formaldehyde inhalation on the morphological damage, and Glu, GABA and NOS contents in olfactory bulb and hippocampus of rats.

METHODSTwenty SD rats were equally divided into two groups: rats in the control group inhaled fresh air, while the animals in experimental group were exposed to the air containing formaldehyde (12.5 mg/m(3), 4 h/d) for 7 days. Then rats were sacrificed and frozen sections of olfactory bulb and hippocampus were prepared. The morphological changes were examined and the Glu, GABA and NOS contents were detected using Nissl-staining, immunohistochemistry and Western blot, respectively.

RESULTCompared with the control group, there was a significant confusion and shrink of neuron morphology in experimental group, the number and staining intensity of Glu and NOS positive cells and protein contents were reduced. The protein expression of GABA was also decreased in the formaldehyde group.

CONCLUSIONFormaldehyde inhalation can cause a severe morphological damage of olfactory bulb and hippocampus in SD rats,which may further impair memory and learning ability through the reduction of Glu, GABA and NOS expression.

Animals ; Formaldehyde ; toxicity ; Glutamic Acid ; metabolism ; Hippocampus ; drug effects ; metabolism ; pathology ; Inhalation Exposure ; Learning ; drug effects ; Neurons ; drug effects ; metabolism ; pathology ; Nitric Oxide Synthase ; metabolism ; Olfactory Bulb ; drug effects ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; gamma-Aminobutyric Acid ; metabolism

OBJECTIVETo observe the protective effect of serum with Tongqiao Huoxue decoction (TQHXD) on PC12 cells damaged by glutamate(Glu) and provide clinical proof of the formulae.

METHODSprague Dawley rats underwent intragastric administration of Tongqiao Huoxue decoction twice a day for five days, the administration dose for rats was 4 g x kg(-1). Thus the serum containing TQHXD was prepared. The model of neurocytes damaged by Glu had been established by adding 12.5 mmol x L(-1) Glu to culture medium of PC12 cells. Photic microscope had been used to observe the morphologic changes of cells, the proliferation and activity of PC12 cells had been detected by MTT. Cell membrane permeability had been investigated by the methods of the coloration of trypan blue and AO/EB, the activities of LDH and the contents of NO in the supernatant of PC12 cells had been detected after 5%, 10%, 20% TQHXD being added to the culture medium of PC12 cells damaged by Glu.

RESULTThe morphologic changes in cells were observed under the inverted microscope. Normal PC12 cells in control group were full and bright, but the cells exposed to Glu were shrank; moreover, some cells were disrupted, configuration of the cells in the TQHXD group was closed to that of normal group. Compared with model group, the ratio of living cells in the group being treated by TQHXD after trypan blue staining had a significant increase. After AO/EB staining, cells in TQHXD groups were less being stained by EB. The absorbance (A) values of TQHXD group increased largely. LDH and NO in cell culture supernatant had decreased, it had shown the marked difference.

CONCLUSIONTQHXD showed an apparent protective effect on neurocytes damaged by Glu on proliferation activity and membrane permeability.

Animals ; Cells, Cultured ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Glutamic Acid ; toxicity ; Male ; Neurons ; drug effects ; Neuroprotective Agents ; administration & dosage ; pharmacology ; Neurotoxins ; toxicity ; PC12 Cells ; Rats ; Rats, Sprague-Dawley ; Serum ; chemistry

OBJECTIVEto investigate the changes of γ-aminobutyric acid (GABA) and glutamate (Glu) in the cerebral cortex following acute bromoxynil intoxication in mice and the protective effect of sodium dimercaptopropane sulfonate (Na-DMPS).

METHODS30 ICR mice were randomly divided into blank control group (10), exposure group (10) and Na-DMPS protection group (10). The levels of GABA and Glu in the cerebral cortex were measured by RP-HPLC. The glutamine (Gln) level and the glutamine synthetase (GS), glutamate decarboxylation enzyme (GAD), γ-aminobutyric acid transaminase (GABA-T) activity in the cerebral cortex were determined by UV colorimetric.

RESULTScompared with the control group [GABA: (3.41 ± 0.12) micromol/g, Glu (14.00 ± 0.16) micromol/g, Gln (1.25 ± 0.19) micromol/g, GAD (13.50 ± 0.25) micromol × g(-1) × h(-1), GABA-T (25.51 ± 0.21) micromol × g(-1) × h(-1), GS(142.19 ± 1.31) U/mg pro], the level of GABA [(3.14 ± 0.14) micromol/g] was decreased (P < 0.05), whereas the level of Glu [(17.54 ± 0.40) micromol/g] and Gln [(3.35 ± 0.27) micromol/g] were increased (P < 0.05), the activity of GAD [(11.93 ± 0.15 micromol × g(-1) × h(-1)], GABA-T [(24.15 ± 0.22) micromol × g(-1) × h(-1)], GS [(140.75 ± 1.01) U/mg pro] was decreased (P < 0.05) in acute intoxication group; Compared with the acute intoxication group, the level of GABA [(3.52 ± 0.30) micromol/g] was increased (P < 0.05), whereas the level of Glu [(14.20 ± 0.32) micromol/g] and Gln [(1.32 ± 0.17) micromol/g] were decreased (P < 0.05), the activity of GAD [(13.01 ± 0.45 micromol × g(-1) × h(-1)], GABA-T [(25.19 ± 0.26) micromol × g(-1) × h(-1), GS [(142.35 ± 1.20) U/mg pro] was increased (P < 0.05); In contrast, the levels of GABA, Glu, Gln and the activity of GAD, GABA-T, and GS in Na-DMPS protection group were not significantly different in comparison with control group (P > 0.05).

CONCLUSIONthe central toxic effects of mice with acute bromoxynil intoxication may be related to the changes of GABA and Glu content in the cerebral cortex;Na-DMPS can protect mice from bromoxynil-induced central toxic effects and GABA and Glu abnormal change in the cerebral cortex.

Animals ; Cerebral Cortex ; drug effects ; metabolism ; Female ; Glutamic Acid ; metabolism ; Male ; Mice ; Mice, Inbred ICR ; Nitriles ; poisoning ; Toxicity Tests, Acute ; Unithiol ; pharmacology ; gamma-Aminobutyric Acid ; metabolism