OBJECTIVEThe present study was undertaken to design antisense oligodeoxynucleotides (AS-ODNs) of glial glutamate transporter-la (GLT-1a) and to evaluate the effectiveness of the designed AS-ODNs on the expression of GLT-1a.

METHODSFive sequences of GLT-1a AS-ODNs were designed according to the C terminus specific sequences of GLT-1a mRNA using antisense design software of IDT Com- pany. Western blot analysis was used to evaluate the inhibition effects of the five GLT-1a AS-ODNs on the expression of GLT-la.

RESULTSThe sequence of GLT-1a AS-ODNs with sequence of 5'-GGTTCTTCCTCAACACTGCA-3' could specifically inhibit the expression of GLT-1a in the hippocampal CA1 subfield of rats, while it had no effect on the expression of GLT-1b. This sequence showed similar inhibition on the expression of GLT-la in sham and ceftriaxone (Cef)-treated rats. It could also significantly inhibit the cerebral ischemic preconditioning (CIP)-induced up-regulation in the expression of GLT-1a. The magnitude of the inhibition in sham, Cef- or CIP-treated rats was similar by more than 60%.

CONCLUSIONFrom the designed five sequences of GLT-1a AS-ODNs, we obtained an effective sequence which can specifically inhibit the expression of GLT-1a.

Animals ; CA1 Region, Hippocampal ; metabolism ; Excitatory Amino Acid Transporter 2 ; antagonists & inhibitors ; metabolism ; Ischemic Preconditioning ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; Rats ; Up-Regulation

The purpose of this study was to investigate the expression of c-fos in the spinal cord during the development of allodynia, induced by peripheral nerve injury. Following tight ligation of the left L5 and L6 spinal nerves of Sprague- Dawley rat, the lumbar spinal cord was postfixed following perfusion. Frontal frozen sections of 40nm were immunostained according to the peroxidase-antiperoxidase method. The allodynic threshold was checked with 8 calibrated von Frey filaments. MK-801 (0.3 mg/kg), morphine (3 mg/kg) and saline (as a placebo) were administered subcutaneously 30 min before, and 24 and 48 hrs after surgery. The tactile threshold decreased below 3 g since 2 days after surgery in the saline and morphine groups, but delayed a little in the MK-801 group. In the superficial layer the number of Fos-like immunoreactive neurones (Fos-LI) peaked at 2 hours and decreased thereafter, and reached normal levels 24 hrs following operation, for all groups. In the deep layer they were biphasic, - peaking at 2 and 24 hrs - in the saline group, but were suppressed in the morphine and MK-801 groups until 7 days following operation. The above discrepancy between the number of Fos-LI and the allodynic threshold showed that central sensitizations are not critically involved in the development of nerve injury induced tactile allodynia.
Analgesics, Opioid/*pharmacology ; Animal ; Dizocilpine Maleate/*pharmacology ; Excitatory Amino Acid Antagonists/*pharmacology ; Hyperesthesia/etiology/*metabolism ; Ligation ; Male ; Morphine/*pharmacology ; Proto-Oncogene Proteins c-fos/*metabolism ; Rats ; Rats, Sprague-Dawley ; Spinal Cord/*metabolism ; Spinal Nerves/injuries ; Wounds and Injuries/complications

Animals ; Animals, Newborn ; Brain ; drug effects ; metabolism ; pathology ; Disease Models, Animal ; Excitatory Amino Acid Antagonists ; pharmacology ; therapeutic use ; Female ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; Ketamine ; pharmacology ; therapeutic use ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Treatment Outcome

OBJECTIVEAnimal trials have demonstrated that memantine has neuroprotective effects on hypoxic-ischemic (HI) brain damage. Whether memantine can improve the long-term prognosis of rats with HI brain damage has not been reported. This study was designed to investigate the long-term effect of memantine therapy on neonatal rats with HI brain damage.

METHODSSixty postnatal 7-day-old newborn rats were randomly assigned into Normal control, HI and Memantine treated groups. Memantine (20 mg/kg) was administered immediately after HI in the Memantine-treated group. All subjects received a 5-day training of Morris water maze test from 23 days old. The escape latency (EL) was recorded at 28 and 35 days old.

RESULTSThe EL values of the Normal control, HI and Memantine-treated groups at 28 days old were 23.1 +/- 21.8, 35.1 +/- 5.3, and 20.6 +/- 3.4 seconds, respectively. There was a significant difference in the EL value between the HI and the Normal control groups (P < 0.05). The EL value of the Normal control, HI and Memantine-treated groups at 35 days old were 19.7 +/- 16.7, 35.6 +/- 32.3, and 16.3 +/- 13.2 seconds, respectively. A prolonged EL induced by HI still existed (P < 0.05 vs Normal controls) but memantine treatment shortened the EL (P < 0.01 vs HI group) at 35 days old.

CONCLUSIONSAdministering memantine immediately after HI can markedly increase the abilities of spatial discrimination, learning and memory and improve the long-term prognosis in rats with HI brain damage.

Animals ; Animals, Newborn ; Avoidance Learning ; drug effects ; Brain ; metabolism ; Excitatory Amino Acid Antagonists ; therapeutic use ; Female ; HSP70 Heat-Shock Proteins ; genetics ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; psychology ; Male ; Maze Learning ; drug effects ; Memantine ; therapeutic use ; Rats ; Rats, Sprague-Dawley

Spinal gabapentin has been known to show the antinociceptive effect. Although several assumptions have been suggested, mechanisms of action of gabapentin have not been clearly established. The present study was undertaken to examine the action mechanisms of gabapentin at the spinal level. Male SD rats were prepared for intrathecal catheterization. The effect of gabapentin was assessed in the formalin test. After pretreatment with many classes of drugs, changes of effect of gabapentin were examined. General behaviors were also observed. Intrathecal gabapentin produced a suppression of the phase 2 flinching, but not phase 1 in the formalin test. The antinociceptive action of intrathecal gabapentin was reversed by intrathecal NMDA, AMPA, D-serine, CGS 15943, atropine, and naloxone. No antagonism was seen following administration of bicuculline, saclofen, prazosin, yohimbine, mecamylamine, L-leucine, dihydroergocristine, or thapsigargin. Taken together, intrathecal gabapentin attenuated only the facilitated state. At the spinal level, NMDA receptor, AMPA receptor, nonstrychnine site of NMDA receptor, adenosine receptor, muscarinic receptor, and opioid receptor may be involved in the antinociception of gabapentin, but GABA receptor, L-amino acid transporter, adrenergic receptor, nicotinic receptor, serotonin receptor, or calcium may not be involved.
Acetic Acids/administration & dosage ; Acetic Acids/metabolism ; Acetic Acids/pharmacology* ; Adrenergic Antagonists/metabolism ; Adrenergic alpha-Antagonists/metabolism ; Analgesics/administration & dosage ; Analgesics/metabolism ; Analgesics/pharmacology* ; Animals ; Atropine/metabolism ; Dihydroergocristine/metabolism ; Enzyme Inhibitors/metabolism ; Excitatory Amino Acid Agonists/metabolism ; GABA Antagonists/metabolism ; Injections, Spinal ; Leucine/metabolism ; Male ; Mecamylamine/metabolism ; Muscarinic Antagonists/metabolism ; N-Methylaspartate/metabolism ; Naloxone/metabolism ; Narcotic Antagonists/metabolism ; Nicotinic Antagonists/metabolism ; Pain Measurement ; Quinazolines/metabolism ; Rats ; Rats, Sprague-Dawley ; Serine/metabolism ; Spinal Cord/drug effects* ; Thapsigargin/metabolism ; Triazoles/metabolism ; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/metabolism

Animals ; Animals, Newborn ; Drugs, Chinese Herbal ; therapeutic use ; Excitatory Amino Acid Antagonists ; therapeutic use ; Hypoxia-Ischemia, Brain ; metabolism ; prevention & control ; Memantine ; therapeutic use ; Platelet Activating Factor ; analysis ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of D-AP5 (D-2-amino-5-phosphonopentanoate, a specific NMDA-antagonist) on the increase of intracellular free Ca2+ concentration ([Ca2+]i) induced by glutamate in isolated cochlear inner hair cells (IHCs), and to detect the autoreceptors of the IHC membrane.

METHODSWhen a laser scanning confocal microscope (LSCM) was used, the exogenous glutamate (Glu)-induced changes in [Ca2+]i of isolated IHCs and OHCs of guinea pig cochlea were observed with fluo-3, a fluorescent probe for [Ca2+]i. After D-AP5 or CNQX (6--cyano--7--nitroguinoxaline--2, 3--dione, a specific AMPA- antagonist) was administered, the exogenous glutamate (Glu)-induced changes in [Ca2+]i of isolated IHCs were recorded.

RESULTSIn the presence of a low concentration Glu (3.85 mumol/L), there was an increase of [Ca2+]i in IHCs, whereas there was no change in OHCs. When 50 mumol/L of D-AP5 was administrated in advance, Glu did not induce a corresponding increase in [Ca2+]i in IHCs, and 50 mumol/L of CNQX did not completely block the increase of [Ca2+]i in IHCs.

CONCLUSIONSThese results suggest that the autoreceptors existing in the IHC membrane are mainly of NMDA type, while there are relatively few AMPA receptors. Exogenous Glu is capable of increasing [Ca2+]i in IHCs by acting on the NMDA autoreceptor of IHCs in a positive feedback manner.

2-Amino-5-phosphonovalerate ; pharmacology ; 6-Cyano-7-nitroquinoxaline-2,3-dione ; pharmacology ; Animals ; Calcium ; metabolism ; Excitatory Amino Acid Antagonists ; pharmacology ; Glutamic Acid ; pharmacology ; Guinea Pigs ; Hair Cells, Auditory, Inner ; drug effects ; metabolism ; In Vitro Techniques ; Receptors, N-Methyl-D-Aspartate ; antagonists & inhibitors

This study explored the effect of the excitatory amino acid receptor antagonists on the impairment of learning-memory and the hyperphosphorylation of Tau protein induced by electroconvulsive shock (ECT) in depressed rats, in order to provide experimental evidence for the study on neuropsychological mechanisms improving learning and memory impairment and the clinical intervention treatment. The analysis of variance of factorial design set up two intervention factors which were the electroconvulsive shock (two level: no disposition; a course of ECT) and the excitatory amino acid receptor antagonists (three level: iv saline; iv NMDA receptor antagonist MK-801; iv AMPA receptor antagonist DNQX). Forty-eight adult Wistar-Kyoto (WKY) rats (an animal model for depressive behavior) were randomly divided into six experimental groups (n = 8 in each group): saline (iv 2 mL saline through the tail veins of WKY rats ); MK-801 (iv 2 mL 5 mg/kg MK-801 through the tail veins of WKY rats) ; DNQX (iv 2 mL 5 mg/kg DNQX through the tail veins of WKY rats ); saline + ECT (iv 2 mL saline through the tail veins of WKY rats and giving a course of ECT); MK-801 + ECT (iv 2 mL 5 mg/kg MK-801 through the tail veins of WKY rats and giving a course of ECT); DNQX + ECT (iv 2 mL 5 mg/kg DNQX through the tail veins of WKY rats and giving a course of ECT). The Morris water maze test started within 1 day after the finish of the course of ECT to evaluate learning and memory. The hippocampus was removed from rats within 1 day after the finish of Morris water maze test. The content of glutamate in the hippocampus of rats was detected by high performance liquid chromatography. The contents of Tau protein which included Tau5 (total Tau protein), p-PHF1(Ser396/404), p-AT8(Ser199/202) and p-12E8(Ser262) in the hippocampus of rats were detected by immunohistochemistry staining (SP) and Western blot. The results showed that ECT and the glutamate ionic receptor blockers (NMDA receptor antagonist MK-801 and AMPA receptor antagonist DNQX) induced the impairment of learning and memory in depressed rats with extended evasive latency time and shortened space exploration time. And the two factors presented a subtractive effect. ECT significantly up-regulated the content of glutamate in the hippocampus of depressed rats which were not affected by the glutamate ionic receptor blockers. ECT and the glutamate ionic receptor blockers did not affect the total Tau protein in the hippocampus of rats. ECT up-regulated the hyperphosphorylation of Tau protein in the hippocampus of depressed rats, while the glutamate ionic receptor blockers down-regulated it, and combination of the two factors presented a subtractive effect. Our results indicate that ECT up-regulates the content of glutamate in the hippocampus of depressed rats, which up-regulates the hyperphosphorylation of Tau protein resulting in the impairment of learning and memory in depressed rats.
Animals ; Disease Models, Animal ; Dizocilpine Maleate ; pharmacology ; Electroshock ; adverse effects ; Excitatory Amino Acid Antagonists ; pharmacology ; Glutamic Acid ; metabolism ; Hippocampus ; metabolism ; Learning ; Memory ; Memory Disorders ; Phosphorylation ; Quinoxalines ; pharmacology ; Rats ; Rats, Inbred WKY ; Rats, Sprague-Dawley ; Receptors, AMPA ; antagonists & inhibitors ; Receptors, N-Methyl-D-Aspartate ; antagonists & inhibitors ; tau Proteins ; metabolism

Mounting evidence supports an important role of chemokines, produced by spinal cord astrocytes, in promoting central sensitization and chronic pain. In particular, CCL2 (C-C motif chemokine ligand 2) has been shown to enhance N-methyl-D-aspartate (NMDA)-induced currents in spinal outer lamina II (IIo) neurons. However, the exact molecular, synaptic, and cellular mechanisms by which CCL2 modulates central sensitization are still unclear. We found that spinal injection of the CCR2 antagonist RS504393 attenuated CCL2- and inflammation-induced hyperalgesia. Single-cell RT-PCR revealed CCR2 expression in excitatory vesicular glutamate transporter subtype 2-positive (VGLUT2) neurons. CCL2 increased NMDA-induced currents in CCR2/VGLUT2 neurons in lamina IIo; it also enhanced the synaptic NMDA currents evoked by dorsal root stimulation; and furthermore, it increased the total and synaptic NMDA currents in somatostatin-expressing excitatory neurons. Finally, intrathecal RS504393 reversed the long-term potentiation evoked in the spinal cord by C-fiber stimulation. Our findings suggest that CCL2 directly modulates synaptic plasticity in CCR2-expressing excitatory neurons in spinal lamina IIo, and this underlies the generation of central sensitization in pathological pain.
Animals ; Benzoxazines ; pharmacology ; therapeutic use ; Chemokine CCL2 ; antagonists & inhibitors ; genetics ; metabolism ; pharmacology ; Excitatory Amino Acid Agents ; pharmacology ; Excitatory Amino Acid Agonists ; pharmacology ; Female ; Freund's Adjuvant ; toxicity ; Hyperalgesia ; chemically induced ; metabolism ; prevention & control ; Long-Term Potentiation ; drug effects ; physiology ; Luminescent Proteins ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Myelitis ; chemically induced ; drug therapy ; metabolism ; Neurons ; drug effects ; Pain Management ; Somatostatin ; genetics ; metabolism ; Spinal Cord ; cytology ; Spiro Compounds ; pharmacology ; therapeutic use ; Vesicular Glutamate Transport Protein 2 ; genetics ; metabolism ; Vesicular Inhibitory Amino Acid Transport Proteins ; genetics ; metabolism

OBJECTIVETo study the protective effect and mechanism of salvianolic acid B (Sal B) on glutamate-induced excito-toxicity.

METHODGlutamate-induced PC12 cell injury model was established to detect the cell survival rate by MTT, the leakage rate of lactic dehydrogenases using LDH, and the cell apoptosis by using AO/EB double staining for fluorescence microscope and PI single staining flow cytometry which was also used to detect the content of intracellular reactive oxygen species. The expression of Caspase-3 protein was also detected by the Western blotting method.

RESULTSal B is proved to inhibit glutamate-induced PC12 cells from injury and prevent them from releasing LDH within the range from 50 micromol x L(-1) to 200 micromol x L(-1). Meanwhile, Sal B has an effect on significantly reducing the expression of inhibit glutamate-induced active Caspase-3 protein, inhibiting accumulated glutamate-induced ROS and decreasing PC12 cell apoptosis rate within the range from 50 micromol x L(-1) to 200 micromol x L(-1).

CONCLUSIONThe study proves that Sal B prevented against glutamate-induced cell injury via inhibiting ROS formation and Caspase-3 pathway-dependent apoptosis in PC12 cells.

Action Potentials ; drug effects ; Animals ; Apoptosis ; drug effects ; Benzofurans ; pharmacology ; Caspase 3 ; metabolism ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Excitatory Amino Acid Antagonists ; pharmacology ; Glutamic Acid ; adverse effects ; Lactate Dehydrogenases ; metabolism ; PC12 Cells ; Pheochromocytoma ; metabolism ; Rats ; Reactive Oxygen Species ; metabolism