Abstract: These studies were designed to determine whether adult neurogenesis occurs in the dentate gyrus of the tree shrew, an animal phylogenetically between insectivores and primates, and to explore the possibility that this process is regulated by stressful experiences and NMDA receptor activation. We performed immunohistochemistry for cell-specific markers and the thymidine analog bromodeoxyuridine (BrdU), a marker of DNA synthesis that labels proliferating cells and their progeny, on the brains of adult tree shrews subjected to psychosocial stress or NMDA receptor antagonist treatment. Cells that incorporated BrdU in the dentate gyrus of adult tree shrews were primarily located in the subgranular zone, had morphological characteristics of granule neuron precursors, and appeared to divide within 24 hr after BrdU injection. Three weeks after BrdU injection, BrdU-labeled cells had neuronal morphology, expressed the neuronal marker neuron specific enolase, and were incorporated into the granule cell layer. Vimentin-immunoreactive radial glia were observed in the dentate gyrus with cell bodies in the subgranular zone and processes extending into the granule cell layer. Exposure to acute psychosocial stress resulted in a rapid decrease in the number of BrdU-labeled cells in the dentate gyrus. In contrast, blockade of NMDA receptors, with the NMDA receptor antagonist MK-801, resulted in an increase in the number of BrdU-labeled cells in the dentate gyrus. These results indicate that adult neurogenesis occurs in the tree shrew dentate gyrus and is regulated by a stressful experience and NMDA receptor activation. Furthermore, we suggest that these characteristics may be common to most mammalian species.

Abstract: Class I metabotropic glutamate receptors (mGluRs) have been postulated to play a role in synaptic plasticity. To test the involvement of one member of this class, we have recently generated mutant mice that express no mGluR5 but normal levels of other glutamate receptors. The CNS revealed normal development of gross anatomical features. To examine synaptic functions we measured evoked field EPSPs in the hippocampal slice. Measures of presynaptic function, such as paired pulse facilitation in mutant CA1 neurons, were normal. The response of mutant CA1 neurons to low concentrations of (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD) was missing, which suggests that mGluR5 may be the primary high affinity ACPD receptor in these neurons. Long-term potentiation (LTP) in mGluR5 mutants was significantly reduced in the NMDA receptor (NMDAR)-dependent pathways such as the CA1 region and dentate gyrus of the hippocampus, whereas LTP remained intact in the mossy fiber synapses on the CA3 region, an NMDAR-independent pathway. Some of the difference in CA1 LTP could lie at the level of expression, because the reduction of LTP in the mutants was no longer observed 20 min after tetanus in the presence of 2-amino-5-phosphonopentanoate. We propose that mGluR5 plays a key regulatory role in NMDAR-dependent LTP. These mutant mice were also impaired in the acquisition and use of spatial information in both the Morris water maze and contextual information in the fear-conditioning test. This is consistent with the hypothesis that LTP in the CA1 region may underlie spatial learning and memory.

Abstract: The interaction of Ro 25-6981 with N-methyl-D-aspartate (NMDA) receptors was characterized by a variety of different tests in vitro. Ro 25-6981 inhibited 3H-MK-801 binding to rat forebrain membranes in a biphasic manner with IC50 values of 0.003 microM and 149 microM for high- (about 60%) and low-affinity sites, respectively. NMDA receptor subtypes expressed in Xenopus oocytes were blocked with IC50 values of 0.009 microM and 52 microM for the subunit combinations NR1C & NR2B and NR1C & NR2A, respectively, which indicated a >5000-fold selectivity. Like ifenprodil, Ro 25-6981 blocked NMDA receptor subtypes in an activity-dependent manner. Ro 25-6981 protected cultured cortical neurons against glutamate toxicity (16 h exposure to 300 microM glutamate) and combined oxygen and glucose deprivation (60 min followed by 20 h recovery) with IC50 values of 0.4 microM and 0.04 microM, respectively. Ro 25-6981 was more potent than ifenprodil in all of these tests. It showed no protection against kainate toxicity (exposure to 500 microM for 20 h) and only weak activity in blocking Na+ and Ca++ channels, activated by exposure of cortical neurons to veratridine (10 microM) and potassium (50 mM), respectively. These findings demonstrate that Ro 25-6981 is a highly selective, activity-dependent blocker of NMDA receptors that contain the NR2B subunit.

Abstract: Neuropathic pain remains a significant clinical problem. Current understanding implicates the spinal cord dorsal horn N-methyl-d-aspartate (NMDA) receptor apparatus in its pathogenesis. Previous reports have described NMDA antagonist reduction of nerve injury-induced thermal hyperalgesia and formalin injection-related electrical activity. We examined a panel of spinally administered NMDA antagonists in two models: allodynia evoked by tight ligation of the fifth and sixth lumbar spinal nerves (a model of chronic nerve injury pain), and the formalin paw test (a model wherein pretreatment with drug may preempt the development of a pain state). A wide range of efficacies was observed. In the nerve injury model, order of efficacy (expressed as percent of maximum possible effect +/- S.E.), at the maximum dose not yielding motor impairment, was memantine (96 +/- 5%) = AP5 (91 +/- 7%) > dextrorphan (64 +/- 11%) = dextromethorphan (65 +/- 22%) > MK801 (34 +/- 8%) > ketamine (18 +/- 6%). For the formalin test, the order of efficacy was AP5 (86 +/- 9%) > memantine (74 +/- 5%) > or = MK801 (67 +/- 16%) > dextrorphan (47 +/- 16%) > dextromethorphan (31 +/- 12%) > ketamine (17 +/- 15%). In the nerve injury model, no supraspinal action was seen after intracerebroventricular injections of dextromethorphan and ketamine. NMDA antagonists by the spinal route appear to be useful therapeutic agents for chemically induced facilitated pain as well as nerve injury induced tactile allodynia. It is not known what accounts for the wide range of efficacies.

Abstract: Repetitive activation of excitatory synapses in the central nervous system results in a long-lasting increase in synaptic transmission called long-term potentiation (LTP). It is generally believed that this synaptic plasticity may underlie certain forms of learning and memory. LTP at most synapses involves the activation of the NMDA (N-methyl-d-aspartate) subtype of glutamate receptor, but LTP at hippocampal mossy fibre synapses is independent of NMDA receptors and has a component that is induced and expressed presynaptically1. It appears to be triggered by a rise in presynaptic Ca2+(refs 2, 3), and requires the activation of protein kinase A4,5,6, which leads to an increased release of glutamate3,7,8,9,10. Agreat deal is known about the biochemical steps involved in the vesicular release of transmitter11,12,13, but none of these steps has been directly implicated in long-term synaptic plasticity. Here we show that, although a variety of short-term plasticities are normal, LTP at mossy fibre synapses is abolished in mice lacking the synaptic vesicle protein Rab3A.

Abstract: Background:The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is blocked by ketamine, and this action likely contributes to ketamine's anesthetic and analgesic properties. Previous studies suggest that ketamine occludes the open channel by binding to a site located within the channel pore

Abstract: Impulses in primary afferent nerve fibers may produce short- or long-lasting modifications in spinal nociception. Here we have identified a robust long-term depression (LTD) of synaptic transmission in substantia gelatinosa neurons that can be induced by low-frequency stimulation of primary afferent Adelta-fibers. Synaptic transmission between dorsal root afferents and neurons in the substantia gelatinosa of the spinal cord dorsal horn was examined by intracellular recording in a transverse slice dorsal root preparation of rat spinal cord. Conditioning stimulation of dorsal roots with 900 pulses given at 1 Hz (10 V, 0.1 msec) produced LTD of EPSP amplitudes in substantia gelatinosa neurons to 41 +/- 10% of control that lasted for at least 2 hr. When A- and C-fibers were recruited, conditioning stimulation was as effective as A-fiber stimulation alone. After LTD, synaptic strength could be increased to its original level by applying a second, high-frequency tetanic stimulus to the dorsal root, indicating that LTD is reversible and not attributable to damage of individual synapses. Bath application of the GABAA receptor antagonist bicuculline and glycine receptor antagonist strychnine did not affect LTD. When NMDA receptors were blocked by bath application of D-2-amino-5-phosphonovaleric acid, LTD was abolished or strongly reduced. Loading substantia gelatinosa neurons with Ca2+ chelator BAPTA also blocked or reduced LTD. After incubation of slices with calyculin A, a selective and membrane permeable inhibitor of protein phosphatases 1 and 2A, LTD was not attenuated. We propose that this form of LTD may be relevant for long-lasting segmental antinociception after afferent stimulation.

Abstract: Animals vary in their sensitivity to ethanol, a trait at least partly determined by genetic factors. In order to identify possible responsible genes, mice lacking Fyn, a non-receptor type tyrosine kinase, were investigated. These mice were hypersensitive to the hypnotic effect of ethanol. The administration of ethanol enhanced tyrosine phosphorylation of the N-methyl-D-aspartate receptor (NMDAR) in the hippocampus of control mice but not in Fyn-deficient mice. An acute tolerance to ethanol inhibition of NMDAR-mediated excitatory postsynaptic potentials in hippocampal slices developed in control mice but not in Fyn-deficient mice. These results indicate that Fyn affects behavioral, biochemical, and physiological responses to ethanol.

Abstract: Neurotoxicity induced by overstimulation of N-methyl-d-aspartate (NMDA) receptors is due, in part, to a sustained rise in intracellular Ca2+; however, little is known about the ensuing intracellular events that ultimately result in cell death. Here we show that overstimulation of NMDA receptors by relatively low concentrations of glutamate induces apoptosis of cultured cerebellar granule neurons (CGNs) and that CGNs do not require new RNA or protein synthesis. Glutamate-induced apoptosis of CGNs is, however, associated with a concentration- and time-dependent activation of the interleukin 1β-converting enzyme (ICE)/CED-3-related protease, CPP32/Yama/apopain (now designated caspase 3). Further, the time course of caspase 3 activation after glutamate exposure of CGNs parallels the development of apoptosis. Moreover, glutamate-induced apoptosis of CGNs is almost completely blocked by the selective cell permeable tetrapeptide inhibitor of caspase 3, Ac-DEVD-CHO but not by the ICE (caspase 1) inhibitor, Ac-YVAD-CHO. Western blots of cytosolic extracts from glutamate-exposed CGNs reveal both cleavage of the caspase 3 substrate, poly(ADP-ribose) polymerase, as well as proteolytic processing of pro-caspase 3 to active subunits. Our data demonstrate that glutamate-induced apoptosis of CGNs is mediated by a posttranslational activation of the ICE/CED-3-related cysteine protease caspase 3.

Abstract: This study examined the role of metabotropic glutamate receptors (mGluRs) in hippocampal long-term depression (LTD) in vivo. The group 1 mGluR antagonist (S)4-carboxyphenylglycine (4CPG), group 1/2 antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG), and group 2 antagonists (RS)-alpha-methylserine-O-phos-phate monophenyl ester (MSOPPE) and (2S)-alpha-ethylglutamic acid (EGLU) were used. The NMDA receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid (AP5) was used to examine the NMDA receptor contribution to the observed LTD. Adult male Wistar rats underwent implantation of stimulating and recording electrodes into the Schaffer collaterals and CA1 stratum radiatum, respectively. After recovery of 5-7 d, the field EPSP was measured from freely moving animals. Drugs were applied either before or after 1 Hz low-frequency train (LFT) or 100 Hz stimulation via a cannula implanted in the lateral cerebral ventricle. Nine hundred pulses at 1 Hz produced an LTD that was marked and long-lasting. This LTD was completely inhibited by pre-LFT application of AP5. MCPG inhibited LTD from 2 hr post-LFT. 4CPG partially impaired LTD. MSOPPE and EGLU completely blocked induction of LTD, although short-term depression remained intact. MSOPPE did not block long-term potentiation (LTP) induced by 100 Hz stimulation, whereas 4CPG produced a significant inhibition. When MSOPPE was present, LTD could not be induced either before or after LTP induction, whereas LTD could be induced in an identical protocol in vehicle-injected animals. These results suggest a differential role for mGluRs in NMDA receptor-dependent hippocampal LTD in vivo. Group 1 mGluRs may play a role in both LTD and LTP, whereas group 2 mGluRs may be critically involved only in LTD induction.

Abstract: The distribution of NMDA receptor subunit (NR1, NR2B) and glia-bound glutamate transporter (GLT1) mRNAs was investigated in postmortem brains of Huntington's disease (HD) patients and controls by means of in situ hybridization using radiolabeled deoxyoligonucleotides. In the neostriatum of HD, NR1, NR2B and GLT1mRNA decreased in correlation to disease severity. GLT1mRNA was not as low as NR1/NR2BmRNA. Losses were more prominent in putamen than in the distinctly atrophied caudate. NR1/NR2BmRNA decreased corresponding to neuronal loss, GLT1mRNA due to reduced cellular expression. The number of GLT1mRNA expressing cells identified as astrocytes increased in the neostriatum (astrogliosis). In contrast to controls, most of these astrocytes contained glial fibrillary acidic protein. NR1/NR2B and GLT1mRNA expression was not homogeneously lower in the neostriatum; zones with stronger hybridization signals corresponded to the matrix compartment and consisted of a larger number of cells with high mRNA levels. Early in the disease, cellular NR1/ NR2BmRNA levels were higher in these zones than in controls. These findings indicate a loss of neurons with NMDA receptors in the neostriatum of HD. A concomitant proliferation of astrocytes with GLT1 transcripts may represent a compensatory mechanism protecting neostriatal neurons from glutamate excitotoxicity.

Abstract: We demonstrate that the NMDA receptor is involved in taste learning in the insular cortex of the behaving rat and describe two facets of this involvement. Blockage of the NMDA receptor in the insular cortex by the reversible antagonist APV during training in a conditioned taste aversion (CTA) paradigm impaired CTA memory, whereas blockage of the NMDA receptor in an adjacent cortex or before a retrieval test had no effect. When rats sampled an unfamiliar taste and hence learned about it, either incidentally or in the context of CTA training, the tyrosine phosphorylation of the NMDA receptor subunit 2B (NR2B) in the insular cortex was specifically increased. The level of tyrosine phosphorylation on NR2B was a function of the novelty of the taste stimulus and the quantity of the taste substance consumed, properties that also determined the efficacy of the taste stimulus as a conditioned stimulus in CTA; however, blockage of the NMDA receptor by APV during training did not prevent tyrosine phosphorylation of NR2B. We suggest that tyrosine phosphorylation of NR2B subserves encoding of saliency in the insular cortex during the first hours after an unfamiliar taste is sampled and that this encoding is independent of another, necessary role of NMDA receptors in triggering experience-dependent modifications in the insular cortex during taste learning. Because a substantial fraction of the NR2B protein in the insular cortex seems to be expressed in interneurons, saliency and the tyrosine phosphorylation of NR2B correlated with it may modulate inhibition in cortex.

Abstract: The cellular mechanisms underlying the neuroprotective action of the immunosuppressant FK506 in experimental stroke remain uncertain, although in vitro studies have implicated an antiexcitotoxic action involving nitric oxide and calcineurin. The present in vivo study demonstrates that intraperitoneal pretreatment with 1 and 10 mg/kg FK506, doses that reduced the volume of ischemic cortical damage by 56-58%, did not decrease excitotoxic damage induced by quinolinate, NMDA, and AMPA. Similarly, intravenous FK506 did not reduce the volume of striatal quinolinate lesions at a dose (1 mg/kg) that decreased ischemic cortical damage by 63%. The temporal window for FK506 neuroprotection was defined in studies demonstrating efficacy using intravenous administration at 120 min, but not 180 min, after middle cerebral artery occlusion. The noncompetitive NMDA receptor antagonist MK801 reduced both ischemic and excitotoxic damage. Histopathological data concerning striatal quinolinate lesions were replicated in neurochemical experiments. MK801, but not FK506, attenuated the loss of glutamate decarboxylase and choline acetyltransferase activity induced by intrastriatal injection of quinolinate. The contrasting efficacy of FK506 in ischemic and excitotoxic lesion models cannot be explained by drug pharmacokinetics, because brain FK506 content rose rapidly using both treatment protocols and was sustained at a neuroprotective level for 3 d. Although these data indicate that an antiexcitotoxic mechanism is unlikely to mediate the neuroprotective action of FK506 in focal cerebral ischemia, the finding that intravenous cyclosporin A (20 mg/kg) reduced ischemic cortical damage is consistent with the proposed role of calcineurin.

Abstract: 1. The actions of opioids on synaptic transmission in rat periaqueductal grey (PAG) neurones were examined using whole-cell patch-clamp recordings in brain slices. 2. Methionine enkephalin (ME; 10 microM) inhibited evoked GABAergic inhibitory postsynaptic currents (IPSCs) by 57%, non-NMDA excitatory postsynaptic currents (EPSCs) by 60%, and NMDA EPSCs by 43% in PAG neurones. This inhibition was associated with an increase in paired-pulse facilitation, was mimicked by the mu-agonist DAMGO (1-3 microM) and abolished by naloxone (1 microM). Neither the kappa-agonist U69593 (1-3 microM), nor the delta-agonist DPDPE (3-10 microM) had any specific actions on evoked PSCs. 3. ME decreased the frequency of spontaneous miniature, action potential-independent postsynaptic currents (mIPSCs by 65%, mEPSCs by 54%) in all PAG neurones, but had no effect on their amplitude distributions. The reduction in mIPSC frequency persisted in nominally Ca(2+)-free, high-Mg2+ (10 mM) solutions, which also contained Cd2+ (100 microM), or Ba2+ (10 mM). Opioid inhibition of mIPSC frequency is unlikely to be mediated by presynaptic Ca2+ or K+ conductances which are sensitive to extracellular Cd2+ or Ba2+. 4. In a subpopulation of PAG neurones, ME increased a Ba(2+)-sensitive K+ conductance at potentials below -97 mV. Opioids inhibited both GABAergic and glutamatergic synaptic transmission in all PAG neurones, independent of any postsynaptic opioid sensitivity. 5. These observations are consistent with, but only partially support, the opioid disinhibition model of PAG-induced analgesia. mu-Opioids also have the potential to modulate the behavioural and autonomic functions of the PAG via modulation of both inhibitory and excitatory presynaptic mechanisms, as well as postsynaptic mechanisms.

Abstract: It is well established that behavioral sensitization to psychomotor stimulants is associated with adaptations in the mesoaccumbens dopamine (DA) system. We showed previously that the responsiveness of ventral tegmental area (VTA) DA neurons to glutamate was significantly enhanced in amphetamine- and cocaine-pretreated rats tested after 3 days of withdrawal, which suggests that adaptations in excitatory amino acid transmission also contribute to sensitization. The purpose of the present study was to determine the subtype of excitatory amino acid receptor responsible for this effect and to examine its persistence during withdrawal. Extracellular single cell recording and microiontophoresis were used to investigate possible alterations in the ability of glutamate agonists [(S)-alpha-amino-3- hydroxy-5-methyl-4-isoxazole propionate (AMPA), N-methyl-D-aspartate (NMDA), and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-t-ACPD)] to stimulate the firing of VTA DA neurons after 3 days of withdrawal from repeated administration of saline, cocaine or amphetamine. Current-response curves showed that responses to iontophoretic AMPA, but not NMDA or 1S,3R-t-ACPD were significantly enhanced in cocaine- or amphetamine-pretreated rats in that neurons entered into a state of apparent depolarization block at significantly lower iontophoretic currents. When rats were tested for responsiveness to iontophoretic glutamate after 14 days of withdrawal, there was no significant difference between cocaine- or amphetamine- and saline-pretreated rats with respect to glutamate current-response curves. These results suggest that increased responsiveness of AMPA receptors on VTA DA neurons may contribute to sensitization at early withdrawal times, but that this alteration, like others described within the VTA, is transient.

Abstract: Zinc has been shown to be present in synaptic vesicles of a subset of glutamatergic boutons and is believed to be coreleased with glutamate at these synapses. A variety of studies have suggested that zinc might play a role in modulation of excitatory transmission, as well as excitotoxicity, by inhibitingN-methyl-d-aspartate (NMDA)-type glutamate receptors. To further investigate the modulatory effects of zinc on NMDA receptors of different subunit compositions, we coexpressed the recombinant subunit NR1 with NR2A and/or NR2B in HEK 293 cells. In whole-cell patch-clamp recordings from these transfected cells, zinc inhibited peak glutamate-evoked current responses in a noncompetitive manner, but there were significant differences between the receptor subtypes in sensitivity to zinc inhibition. For NR1/NR2A, ∼40% of the peak current was inhibited by zinc in a voltage-independent manner with an IC50 value of 5.0 ± 1.6 nm and at a VH value of −60 mV; the remainder was blocked at a second, voltage-dependent site with an IC50 value of 79 ± 18 μm. In contrast, NR1/NR2B currents showed nearly complete inhibition at a voltage-independent site with an IC50 value of 9.5 ± 3.3 μm. Cells cotransfected with NR1, NR2A, and NR2B showed zinc sensitivity intermediate between that characteristic of NR1/NR2A and that of NR1/NR2B. Furthermore, zinc accelerated the macroscopic desensitization of both NR1/NR2A and NR1/NR2B in a dose-dependent manner, apparently independently of glycine-sensitive desensitization and Ca2+-dependent inactivation; maximal effects were to decrease desensitization time constants for NR1/NR2A by ∼75% and for NR1/NR2B by ∼90%. Differential modulation of NR1/NR2A and NR1/NR2B currents by zinc may play a role in regulating NMDA receptor-induced synaptic plasticity and neurotoxicity.

Abstract: A series of novel tricyclic pyrido-phthalazine-dione derivatives was tested for antagonistic effects at the strychnine-insensitive modulatory site of the N-methyl-D-aspartate (NMDA) receptor (glycineB). All compounds displaced [3H]MDL-105,519 binding to rat cortical membranes with IC50 values of between 90 nM and 3.6 microM. In patch-clamp experiments, steady-state inward current responses of cultured hippocampal neurons to NMDA (200 microM, glycine 1 microM) were antagonized by these same compounds with IC50 values of 0.14 to 13.8 microM. The antagonism observed was typical for glycineB antagonists, i.e., they induced desensitization and their effects were not use or voltage dependent. Moreover, increasing concentrations of glycine were able to decrease their apparent potency. Much higher concentrations (>100 microM) were required to antagonize alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced currents. They were potent, systemically active NMDA receptor antagonists in vivo against responses of single neurons in the rat spinal cord to microelectrophoretic application of NMDA with ID50 values in the low milligram per kilogram i.v. range. They also inhibited pentylenetetrazol-, NMDA- and maximal electroshock-induced convulsions in mice with ED50 values ranging from 8 to 100 mg/kg i.p. The duration of anticonvulsive action was rather short but was prolonged by the organic acid transport inhibitor probenecid (200 mg/kg). The agents tested represent a novel class of systemically active glycineB antagonists with greatly improved bioavailability.