6-cyano-7-nitroquinoxaline-2-3-dione and 1-amino-1-3-dicarboxycyclopentane

The six-layered mammalian neocortex evolved from the three-layered paleocortex, which is retained in present-day reptiles such as the turtle. Thus the turtle offers an opportunity to examine which cellular and circuit properties are fundamental to cortical function. We characterized the dendritic properties of pyramidal neurons in different cortical regions of mature turtles, Pseudemys scripta elegans, using whole cell recordings and calcium imaging from the axon, soma, and dendrites in a slice preparation. The firing properties, in response to intrasomatic depolarization, resembled those previously recorded with sharp electrodes in this preparation. Somatic spikes led to active backpropagating high-amplitude dendritic action potentials and intracellular calcium ion concentration ([Ca2+]i) changes at all dendritic locations, suggesting that both backpropagation and dendritic voltage-gated Ca2+ channels are primitive traits. We found no indication that Ca2+ spikes could be evoked in the dendrites, but fast Na+ spikes could be initiated there following intradendritic stimulation. Several lines of evidence indicate that fast, smaller-amplitude somatic spikes ("prepotentials") that are easily recorded in this preparation are generated in the axon. Most synaptically activated [Ca2+]i changes resulted from Ca2+ entry through voltage-gated channels. In some cells synaptic stimulation evoked a delayed Ca2+ wave due to release from internal stores following activation of metabotropic glutamate receptors. With some small differences these properties resemble those of pyramidal neurons in mammalian species. We conclude that spike backpropagation, dendritic Ca2+ channels, and synaptically activated Ca2+ release are primitive and conserved features of cortical pyramidal cells, and therefore likely fundamental to cortical function.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Brain; Calcium; Cycloleucine; Dendrites; Dose-Response Relationship, Radiation; Electric Stimulation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; In Vitro Techniques; Patch-Clamp Techniques; Pyramidal Cells; Sodium; Turtles

We compared the contribution of metabotropic glutamate receptors (mGluRs) to the generation and modulation of synaptic responses elicited in intracellularly recorded L5 motoneurons from neonatal rats by segmental and descending fibers. Dorsal root (DR) stimulation at high intensity (C-fiber strength) evoked long latency (2-5-s) depolarization in addition to early monosynaptic and polysynaptic responses. Stimulation of the descending ventrolateral funiculus (VLF) failed to evoke a late response in the same motoneuron. The mGluR antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG; 0.4 mM) selectively blocked the long latency DR response. This mGluR-mediated response persisted in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate or N-methyl-d-aspartate (NMDA) antagonists, but not both, suggesting that glutamate transmission (either AMPA/kainate or NMDA) is required for mGluR-mediated inputs from small diameter sensory afferents to affect the motoneuron. Although MCPG inhibited the long latency DR response, it induced moderate facilitation of monosynaptic DR and VLF responses. The mGluR agonist 1s3r-ACPD induced motoneuron depolarization and depressed the monosynaptic DR and VLF responses. MCPG also facilitated the neurotrophin-3 and brain-derived neurotrophic factor induced strengthening of the monosynaptic DR responses (but only before P6, since neurotrophins are ineffective later at DR synapses and never at VLF synapses after birth). Our results suggest that mGluRs are involved in synaptic pathways to motoneurons made by DR but not VLF fibers. MCPG-induced facilitation of monosynaptic AMPA/kainate DR and VLF responses suggests the possibility of tonic mGluR-mediated inhibition of DR and VLF responses. We speculate that MCPG facilitates neurotrophin-induced strengthening of monosynaptic DR responses by reducing this tonic inhibition.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Benzoates; Brain-Derived Neurotrophic Factor; Cycloleucine; Electric Stimulation; Electrophysiology; Excitatory Amino Acid Antagonists; Glycine; Membrane Potentials; Motor Neurons; Neurotrophin 3; Patch-Clamp Techniques; Phosphinic Acids; Rats; Receptors, Metabotropic Glutamate; Spinal Cord; Spinal Nerve Roots

Tonic impulse flow in the septohippocampal GABAergic pathway is essential for normal cognitive functioning and is sustained, in part, by acetylcholine (ACh) that is released locally via axon collaterals of septohippocampal cholinergic neurons. Septohippocampal cholinergic neurons degenerate in Alzheimer's disease and other neurodegenerative disorders. While the importance of the muscarinic effects of ACh on septohippocampal GABAergic neurons is well recognized, the nicotinic effects of ACh remain unstudied despite the reported benefits of nicotine on cognitive functioning. In the present study, using electrophysiological recordings in a rat brain slice preparation, rapid applications of nicotine excited 90% of retrogradely labelled septohippocampal GABA-type neurons with an EC50 of 17 microm and increased the frequency of spontaneously occurring, impulse-dependent fast GABAergic and glutamatergic synaptic currents via the alpha4beta2-nicotinic receptor. Interestingly, tetrodotoxin blocked all effects of nicotine on septohippocampal GABAergic type neurons, suggesting involvement of indirect mechanisms. We demonstrate that the effects of nicotine on septohippocampal GABA-type neurons involve recruitment of a novel, local glutamatergic circuitry as (i). Group I metabotropic glutamatergic receptor antagonists reduced the effects of nicotine; (ii). the number of nicotine responsive neurons was significantly reduced in recordings from slices that had been trimmed so as to reduce the number of glutamate-containing neurons within the slice preparation; (iii). in light and ultrastructural double immunocytochemical labelling studies vesicular glutamate 2 transporter immunoreactive terminals made synaptic contacts with parvalbumin-immunoreactive septohippocampal GABAergic neurons. The discovery of a local glutamatergic circuit within the septum may provide another avenue for restoring septohippocampal GABAergic functions in neurodegenerative disorders associated with a loss of septohippocampal cholinergic neurons.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Aconitine; Acyclovir; Animals; Animals, Newborn; Atropine; Bicuculline; Bungarotoxins; Carrier Proteins; Cell Count; Choline; Chromones; Cycloleucine; Dose-Response Relationship, Drug; Drug Administration Routes; Drug Interactions; Electric Conductivity; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; gamma-Aminobutyric Acid; Glycine; Hippocampus; Immunohistochemistry; In Vitro Techniques; Male; Membrane Potentials; Membrane Transport Proteins; Microscopy, Electron; Muscarine; Muscarinic Agonists; Muscarinic Antagonists; Neurons; Neuroprotective Agents; Nicotine; Nicotinic Agonists; Nicotinic Antagonists; Parvalbumins; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Resorcinols; Septum of Brain; Synapses; Tetrodotoxin; Tubocurarine; Valine; Vesicular Glutamate Transport Protein 2; Vesicular Transport Proteins

By monitoring changes in the cytosolic [Ca2+](i) and rates of juvenile hormone (JH) synthesis in response to L-glutamate agonists and antagonists, we identified and characterized glutamate receptor subtypes in corpus allatum (CA) cells of the cockroach, Diploptera punctata. During the first ovarian cycle, corpora allata exhibited a cycle of changes in sensitivity to L-glutamate correlated to cyclic changes in rates of JH synthesis. When exposed to 60 microM L-glutamate in vitro, the active corpora allata of day-4 mated females produced 60% more JH, while inactive corpora allata at other ages showed 10-20% stimulatory response. Pharmacological characterization using various L-glutamate receptor agonists and antagonists indicated that several ionotropic subtypes of L-glutamate receptors were present in the CA. The CA showed an increase in rates of JH synthesis in response to NMDA, kainate, and quisqualate, but not to AMPA in both L-15 medium and minimum incubation medium. In contrast, applications of the metabotropic receptor-specific agonist trans-ACPD failed to elicit a change in the cytosolic [Ca2+](i) and JH production. An elevation of cytosolic calcium concentration, followed by 20-30% rise in JH production, was observed when active CA cells were exposed to 10-40 microM kainate. Kainate had no stimulatory effect on JH synthesis in calcium-free medium. The kainate-induced JH synthesis was blocked by 20 microM CNQX but was not affected by 20 microM NBQX. Kainate-stimulated JH production was not suppressed by MK-801 (a specific blocker of NMDA-receptor channel), nor was NMDA-stimulated JH production affected by CNQX (a specific antagonist of kainate receptor). These data suggest that active CA cells are stimulated to synthesize more JH by a glutamate-induced calcium rise via NMDA-, kainate- and/or quisqualate-sensitive subtypes of ionotropic L-glutamate receptors. The metabotropic-subtype and ionotropic AMPA-subtype L-glutamate receptors are unlikely to be present on active CA cells.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Calcium; Cockroaches; Corpora Allata; Cycloleucine; Cytosol; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamates; Juvenile Hormones; Kainic Acid; Quinoxalines; Quisqualic Acid; Receptors, Glutamate; Receptors, Metabotropic Glutamate

In neocortex glutamate activates ionotropic and metabotropic receptors (mGluRs). Whole-cell current-clamp recordings in the in vitro rat auditory cortex at 32 degrees C were used to explore the role that mGluRs have in regulation of AMPA/kainate, NMDA, and GABA receptor-mediated synaptic transmission. Our findings are: (a) The fast EPSP (AMPA/kainate), slow EPSP (NMDA), and IPSPs (GABAA, GABAB), elicited in pyramidal neurons are reduced in the presence of (1S,3R)-ACPD (mGluR agonist) with greatest effect on the slow IPSP>fast IPSP>>fast EPSP. The effect is likely the result of ACPD acting at presynaptic mGluRs because the probability of release of glutamate and GABA is reduced in the presence of ACPD, intracellular infusion of a G protein antagonist (GDPPS) did not block the effect of ACPD, nor were iontophoretic kainic acid or NMDA-induced depolarizations reduced by ACPD. (b) The slow EPSP is enhanced following washout of ACPD and enhancement is not due to disinhibition because it is present in the absence of IPSPs, but if IPSPs are present, its magnitude can be influenced. Iontophoretic NMDA responses are enhanced in the presence of ACPD, an effect blocked by GDPbetaS and heparin (intracellular inositol 1,4,5-trisphosphate receptor antagonist). Taken together, this evidence suggests that enhancement is a result of group I postsynaptic mGluR activation. (c) In fast-spiking cells ACPD reduces the EPSP (AMPA/kainate and NMDA-mediated). This action is likely presynaptic because it persists when GDPbetaS is in the cells. (d) The rate of spike discharge recorded from fast-spiking cells is accelerated in ACPD but does not change in the presence of GDPbetaS, suggesting a postsynaptic effect. Our data indicate that mGluRs can influence neocortical synaptic transmission in complex ways by acting presynaptically and postsynaptically.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Auditory Cortex; Cycloleucine; Excitatory Postsynaptic Potentials; GluK2 Kainate Receptor; Guanosine Diphosphate; Interneurons; Male; Neocortex; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Receptors, Kainic Acid; Receptors, Metabotropic Glutamate; Synaptic Transmission; Thionucleotides

We investigated whether tetanic-stimulation and activation of metabotropic glutamate receptors (mGluRs) can modify field-synaptic-potentials and protein kinase activity in rat auditory cortex, specifically protein kinase A (PKA) and protein kinase C (PKC). Tetanic stimulation (50 Hz, 1 s) increases PKA and PKC activity only if the CNQX-sensitive field-EPSP (f-EPSP) is also potentiated. If the f-EPSP is unchanged, then PKA and PKC activity remains unchanged. Tetanic stimulation decreases a bicuculline-sensitive field-IPSP (f-IPSP), and this occurs whether the f-EPSP is potentiated or not. Potentiation of the f-EPSP is blocked by antagonists of mGluRs (MCPG) and PKC (calphostin-C, tamoxifen), suggesting that the potentiation of the f-EPSP is dependent on mGluRs and PKC. PKC antagonists block the rise in PKC and PKA activity, which suggests that these may be coupled. In contrast, ACPD (agonist at mGluRs) decreases both the f-EPSP and the f-IPSP, but increases PKC and PKA activity. Quisqualate (group I mGluR agonist), decreases the f-IPSP, and increases PKA activity, suggesting that the increase in PKA activity is a result of activation of group I mGluRs. Additionally, the increase in PKC and PKA activity appears to be independent of the decrease of the f-EPSP and f-IPSP, because PKC antagonists block the increase in PKC and PKA activity levels but do not block ACPD's effect on the f-EPSP or f-IPSP. These data suggest that group I mGluRs are involved in potentiating the f-EPSP by a PKC and possibly PKA dependent mechanism which is separate from the mechanism that decreases the f-EPSP and f-IPSP.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Auditory Cortex; Cyclic AMP-Dependent Protein Kinases; Cycloleucine; Electric Stimulation; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Long-Term Potentiation; Male; Naphthalenes; Neural Inhibition; Neurons; Neuroprotective Agents; Phosphorylation; Protein Kinase C; Quisqualic Acid; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Synaptic Transmission

We investigated expression of Na pump isoforms in cultured cerebellar granule cells and measured in situ ion pump activities of the isoforms, to elucidate functions of Na pump isoforms in neurons. The cells expressed three Na pump isoforms (alpha1, alpha2 and alpha3 isoforms), however the alpha1 isoform acted as a main ion pump under basal conditions. The ion pump activity of the alpha3/ alpha2 isoforms increased remarkably after stimulation of the neurons with glutamate, therefore the alpha3/alpha2 isoforms as well as the alpha1 isoform acted as ion pumps after the stimulation. The glutamate effects were mainly mediated by non-NMDA receptors. These results suggest that alpha1 isoform and alpha3/alpha2 isoforms are functionally important under basal conditions and after neuronal excitation, respectively.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Biological Transport; Cerebellum; Cycloleucine; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Isoenzymes; Kainic Acid; N-Methylaspartate; Neurons; Neuroprotective Agents; Potassium Radioisotopes; Rats; Rats, Sprague-Dawley; Sodium-Potassium-Exchanging ATPase

Muscarinic acetylcholine receptors (mAChR) in the central nervous system are involved in learning and memory, epileptic seizures, and processing the amyloid precursor protein. The M(1) receptor is the predominant mAChR subtype in the cortex and hippocampus. Although the five mAChR fall into two broad functional groups, all five subtypes, when expressed in recombinant systems, can activate the mitogen-activated protein kinase (MAPK) pathway. The MAPK pathway has been implicated in learning and memory, amyloid protein processing, and neuronal plasticity. We used M(1) knock-out mice to determine the role of this receptor subtype in signal transduction in the mouse forebrain. In primary cortical cultures from mice lacking the M(1) mAChR, agonist-stimulated phosphoinositide hydrolysis was reduced by more than 60% compared with cultures from wild type mice. Although muscarinic agonists induced robust activation of MAPK in cortical cultures from wild type mice, mAChR-mediated activation of MAPK was virtually absent in cultures from M(1)-deficient mice. These results indicate that the M(1) mAChR is the major subtype that mediates activation of phospholipase C and MAPK in mouse forebrain.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Carbachol; Cells, Cultured; Cerebral Cortex; Cycloleucine; Enzyme Activation; Hippocampus; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; Muscarinic Agonists; Neurons; Phosphatidylinositols; Receptor, Muscarinic M1; Receptors, Muscarinic

IP(3) signaling in Purkinje cells is involved in the regulation of cell functions including LTD. We have used a GFP-tagged pleckstrin homology domain to visualize IP(3) dynamics in Purkinje cells. Surprisingly, IP(3) production was observed in response not only to mGluR activation, but also to AMPA receptor activation in Purkinje cells in culture. AMPA-induced IP(3) production was mediated by depolarization-induced Ca(2+) influx because it was mimicked by depolarization and was blocked by inhibition of the P-type Ca(2+) channel. Furthermore, trains of complex spikes, elicited by climbing fiber stimulation (1 Hz), induced IP(3) production in Purkinje cells in cerebellar slices. These results revealed a novel IP(3) signaling pathway in Purkinje cells that can be elicited by synaptic inputs from climbing fibers.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Benzoates; Blood Proteins; Calcium; Calcium Channels; Cell Membrane; Cells, Cultured; Cycloleucine; Cytoplasm; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Genetic Vectors; Glutamic Acid; Glycine; Green Fluorescent Proteins; Indicators and Reagents; Inositol 1,4,5-Trisphosphate; Luminescent Proteins; Membrane Potentials; Mice; Mice, Inbred ICR; Neuroprotective Agents; Patch-Clamp Techniques; Phosphoproteins; Purkinje Cells; Receptors, AMPA; Receptors, Metabotropic Glutamate; Sindbis Virus

Intracellular Ca(2+) ([Ca(2+)](i)) was fluorometrically measured with fura-2 in lumbar motoneurons of acutely isolated spinal cord slices from embryonic rats. In ester-loaded cells, bath-applied glutamate (3 microM to 1 mM) evoked a [Ca(2+)](i) increase by up to 250 nM that was abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) plus 2-amino-5-phosphonovalerate (APV). CNQX or APV alone reduced the response by 82 and 25%, respectively. The glutamatergic agonists kainate (KA), quisqualate (QUI), and S-alpha-amino-3-hydroxy-5-methyl-4-isoxalone (S-AMPA) evoked a similar [Ca(2+)](i) transient as glutamate. N-methyl-D-aspartate (NMDA) was only effective to increase [Ca(2+)](i) in Mg(2+)-free saline, whereas [1S,3R]-1-aminocyclopentane-1,3-dicarboxylic acid ([1S,3R]-ACPD) had no effect. The glutamate-induced [Ca(2+)](i) rise was suppressed in Ca(2+)-free superfusate. Depletion of Ca(2+) stores with cyclopiazonic acid (CPA) did not affect the response. Thirty-six percent of the [Ca(2+)](i) increase in response to membrane depolarization induced by a 50 mM K(+) solution persisted on combined application of the voltage-gated Ca(2+) channel blockers nifedipine, omega-conotoxin-GVIA and omega-agatoxin-IVA. In fura-2 dialyzed motoneurons, the glutamate-induced [Ca(2+)](i) increase was attenuated by approximately 70% after changing from current to voltage clamp. Forty percent of the remaining [Ca(2+)](i) transient and 20% of the concomitant inward current of 0.3 nA were blocked by Joro spider toxin-3 (JSTX). The results show that voltage-gated Ca(2+) channels, including a major portion of R-type channels, constitute the predominant component of glutamate-induced [Ca(2+)](i) rises. NMDA and Ca(2+)-permeable KA/AMPA receptors contribute about equally to the remaining component of the Ca(2+) rise. The results substantiate previous assumptions that Ca(2+) influx through JSTX-sensitive KA/AMPA receptors is involved in (trophic) signaling in developing motoneurons.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Calcium; Calcium Channel Blockers; Cycloleucine; Embryo, Mammalian; Excitatory Amino Acid Antagonists; Glutamic Acid; In Vitro Techniques; Kainic Acid; Lumbosacral Region; Motor Neurons; Neurotoxins; Patch-Clamp Techniques; Rats; Rats, Wistar; Receptors, AMPA; Receptors, Kainic Acid; Spider Venoms; Spinal Cord

Research has provided evidence about the role of excitotoxicity in the pathophysiology of sporadic amyotrophic lateral sclerosis and suggests that AMPA/kainate receptor activation contributes greatly in mediating glutamate injury to motor neurons. The recent finding of variable expression of metabotropic glutamate (mGlu) receptor subtypes in adult rat spinal cord has prompted us to investigate their contribution to the excitotoxic process. We report here that stimulation of mGlu receptors efficiently prevents motor neuron degeneration induced by kainate. The application of kainate to lumbar spinal cord slices from adult rats induced a massive degeneration of motor neurons which became shrunken, dark and TUNEL-positive. On the contrary, no significant neurotoxicity was observed after NMDA application. A blockade of ionotropic non-NMDA receptors by CNQX, and mGlu receptor stimulation, efficiently counteracted kainate-mediated cell death. Among the various agonists for mGlu receptors, we tested 3-hydroxyphenylglycine (3HPG), which selectively stimulates group I mGlu receptors. In addition, we tested 2-(carboxycyclopropyl)glycine (L-CCG-I) and 4-carboxy-3-hydroxyphenylglycine (4C3HPG), two selective agonists for group II receptors, as well as L-amino-4-phosphonobutyrate (L-AP4), a preferential agonist for group III. The results suggest that all three groups of mGlu receptors are involved in inhibiting excitotoxic phenomena mediated by kainate on spinal cord motor neurons. This was despite being localized differently and, possibly, activating different neuroprotective pathways.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Benzoates; Cell Survival; Choline O-Acetyltransferase; Cycloleucine; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glycine; In Situ Nick-End Labeling; In Vitro Techniques; Kainic Acid; Male; Motor Neurons; N-Methylaspartate; Nerve Degeneration; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Spinal Cord

1. Whole-cell patch recording and puff pipette techniques were used to identify glutamate receptor mechanisms on bipolar cell (BC) dendrites in the zebrafish retinal slice. Recorded neurons were stained with Lucifer Yellow, to correlate glutamate responses with BC morphology. 2. BC axon terminals (ATs) consisted of swellings or varicosities along the axon, as well as at its end. AT stratification patterns identified three regions in the inner plexiform layer (IPL): a thick sublamina a, with three bands of ATs, a narrow terminal-free zone in the mid-IPL, and a thin sublamina b, with two bands of ATs. BCs occurred with ATs restricted to sublamina a(Group a), sublamina b(Group b) or with ATs in both sublaminae (Group a/b). 3. OFF-BCs belonged to Group a or Group a/b. These cells responded to glutamate or kainate with a CNQX-sensitive conductance increase. Reversal potential (Erev) ranged from -0.6 to +18 mV. Bipolar cells stimulated sequentially with both kainate and glutamate revealed a population of glutamate-insensitive, kainate-sensitive cells in addition to cells sensitive to both agonists. 4. ON-BCs responded to glutamate via one of three mechanisms: (a) a conductance decrease with Erev approximately 0 mV, mimicked by L-(+)-2-amino-4-phosphonobutyric acid (APB) or trans-1-amino-1, 3-cyclopentanedicarboxylic acid (trans-ACPD), (b) a glutamate-gated chloride conductance increase (IGlu-like) characterized by Erev >= ECl (where ECl is the chloride equilibrium potential) and partial blockade by extracellular Li+/Na+ substitution or (c) the activation of both APB and chloride mechanisms simultaneously to produce a response with outward currents at all holding potentials. APB-like responses were found only among BCs in Group b, with a single AT ramifying deep within sublamina b; whereas, cells expressing IGlu-like currents had one or more ATs, and occurred within Groups b or a/b. 5. Multistratified cells (Group a/b) were common and occurred with either ON- or OFF-BC physiology. OFF-BCs typically had one or more ATs in sublamina a and only one AT in sublamina b. In contrast, multistratified ON-BCs had one or more ATs in sublamina b and a single AT ramifying deep in sublamina a. Multistratified ON-BCs expressed the IGlu-like mechanism only. 6. Visual processing in the zebrafish retina involves at least 13 BC types. Some of these BCs have ATs in both the ON- and OFF-sublaminae, suggesting a significant role for ON- and OFF-inputs throughout the IPL.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Aminobutyrates; Animals; Axons; Chlorides; Cycloleucine; Dendrites; Excitatory Amino Acid Antagonists; Fluorescent Dyes; Glutamic Acid; In Vitro Techniques; Isoquinolines; Kainic Acid; Neurons; Picrotoxin; Retina; Strychnine; Zebrafish

Metabotropic glutamate receptors (mGluRs) have been proposed to be involved in oscillatory rhythmic activity in the hippocampus. However, the subtypes of mGluRs involved and their precise distribution in different populations of interneurons is unclear. In this study, we combined functional analysis of mGluR-mediated inward currents in CA1 oriens-alveus interneurons with anatomical and immunocytochemical identification of these interneurons and expression analysis of group I mGluR using single-cell reverse transcription-PCR (RT-PCR). Four major interneuron subtypes could be distinguished based on the mGluR-mediated inward current induced by the application of 100 microm trans-(1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid (ACPD) under voltage-clamp conditions and the action potential firing pattern under current-clamp conditions. Type I interneurons responded with a large inward current of approximately 224 pA, were positive for somatostatin, and the majority expressed both mGluR1 and mGluR5. Type II interneurons responded with an inward current of approximately 80 pA, contained calbindin, and expressed mainly mGluR1. Type III interneurons responded with an inward current of approximately 60 pA. These interneurons were fast-spiking, contained parvalbumin, and expressed mainly mGluR5. Type IV interneurons did not respond with an inward current upon application of ACPD, yet they expressed group I mGluRs. Activation of group I mGluRs under current-clamp conditions increased spike frequency and resulted in rhythmic firing activity in type I and II, but not in type III and IV, interneurons. RT-PCR results suggest that activation of mGluR1 in the subsets of GABAergic interneurons, classified here as type I and II, may play an important role in mediating synchronous activity.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Benzoates; Cycloleucine; Excitatory Amino Acid Antagonists; GABA Antagonists; Gene Expression; Glycine; Hippocampus; Interneurons; Neuroprotective Agents; Patch-Clamp Techniques; Periodicity; Picrotoxin; Rats; Rats, Wistar; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Reverse Transcriptase Polymerase Chain Reaction; Tetrodotoxin

Activation of metabotropic glutamate receptors (mGluRs) has diverse effects on the functioning of vertebrate synapses. The cellular mechanisms that underlie these changes, however, are largely unknown. The role of presynaptic mGluRs in modulating Ca(2+) dynamics and regulating neurotransmitter release was investigated at the vestibulospinal-reticulospinal (VS-RS) synapse in the lamprey brain stem. Application of the specific Group I mGluRs antagonist 7-(hydroxyimino) cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) reduced the amplitude of consecutive high-frequency evoked excitatory postsynaptic currents (EPSCs). A series of experiments using techniques of electrophysiology and calcium imaging were carried out to determine the cellular mechanisms by which this phenomenon occurs. Concentration-dependent increases in the pre- and postsynaptic [Ca(2+)](i) were seen with the application of mGluR agonists. Similarly, high-frequency stimulation of axons caused a Group I mGluR-dependent enhancement in presynaptic Ca(2+) transients. Application of mGluR agonist caused a depolarization of the presynaptic elements, while thapsigargin decreased the high-frequency stimulus- and agonist-induced rises in [Ca(2+)](i). These data suggest that both membrane depolarization and the release of Ca(2+) from intracellular stores potentially play a role in mGluR-induced Ca(2+) signaling. To determine the effect of this modulation of Ca(2+) dynamics on spontaneous glutamate release, miniature EPSCs were recorded from postsynaptic reticulospinal neurons. A potent Group I mGluR agonist, (S)-homoquisqualic acid, caused a large increase in the frequency of events. These results demonstrate the presence of presynaptic Group I mGluRs at the VS-RS synapse. Activation of these receptors leads to a rise in [Ca(2+)](i) and enhances the spontaneous and evoked release of glutamate. Taken together, these studies highlight the importance of synaptic activation of these facilitatory autoreceptors in both short-term plasticity and synaptic transmission.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Axons; Brain Stem; Calcium; Chromones; Cycloleucine; Dendrites; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Fluorometry; Glutamic Acid; Glycine Agents; GTP-Binding Proteins; In Vitro Techniques; Lampreys; Larva; Membrane Potentials; N-Methylaspartate; Neuronal Plasticity; Neuroprotective Agents; Patch-Clamp Techniques; Presynaptic Terminals; Quisqualic Acid; Receptors, Metabotropic Glutamate; Strychnine; Tetrodotoxin

We studied N-methyl-D-aspartate-induced cell death in organotypic hippocampal slices from seven-day-old Wistar rat pups cultured for 12-14 days in a medium containing no added glutamate. Propidium iodide fluorescence intensity was used as an indicator of cell death measured with the help of confocal microscopy. Exposure of slices for 2h to L-glutamate (1-500 microM) prior to the N-methyl-D-aspartate challenge significantly reduced N-methyl-D-aspartate-induced cell death. Glutamate at 10 and 500 microM concentrations was highly protective against N-methyl-D-aspartate-induced cell death, but was less protective at the 1 microM concentration. The protection was not blocked by the Na(+) channel blocker tetrodotoxin (1 microM), the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonopentanoic acid (20 microM) or the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (20 microM). 1S, 3R-1-Aminocyclopentane-trans-1,3-dicarboxylic acid, an agonist at metabotropic glutamate receptor types 1, 2/3 and 5, was protective at 100 microM but not at 50 microM. In contrast, the ionotropic glutamate receptor agonist aspartate (250 microM) facilitated N-methyl-D-aspartate toxicity. Treatment of slices with the protein kinase C inhibitor staurosporine (0.2 microM) or antisense oligonucleotide (10nM, 72 h) that selectively inhibits metabotropic glutamate receptor type 5 synthesis significantly reduced glutamate protection. These results suggest that ambient glutamate may reduce nerve cell susceptibility to injury caused by excessive N-methyl-D-aspartate receptor activation by acting at metabotropic glutamate receptors linked to protein kinase C.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Aspartic Acid; Cell Death; Cycloleucine; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; N-Methylaspartate; Neurons; Neuroprotective Agents; Oligodeoxyribonucleotides, Antisense; Organ Culture Techniques; Rats; Rats, Wistar; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Tetrodotoxin

We have studied the activation of phospholipase D (PLD) by glutamate in rat cultured astrocytes by measuring the PLD-catalyzed formation of [32P]phosphatidylbutanol in [32P]Pi-prelabeled cells, stimulated in the presence of butanol. Glutamate elicited the activation of PLD in cortical astrocytes but not in cortical neurons, whereas similar glutamate activation of phosphoinositide phospholipase C was found in both astrocytes and neurons. The extent of PLD stimulation by glutamate was similar in astrocytes from brain cortex and hippocampus, but no effect was found in cerebellar astrocytes. In cortical astrocytes, the glutamate response was insensitive to antagonists of ionotropic glutamate receptors and was reproduced by agonists of metabotropic glutamate receptors (mGluRs) with a rank order of agonist potency similar to that reported for group I mGluR-mediated phosphoinositide phospholipase activation [quisqualate > (S)-3,5-dihydroxyphenylglycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid]. The response to (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid was inhibited by the mGluR antagonist (S)-alpha-methyl-4-carboxyphenylglycine and, less potently, by 1-aminoindan-1,5-dicarboxylic acid and 4-carboxyphenylglycine, two antagonists of group I mGluRs that display higher potency on mGluR1 than on mGluR5. The mGluR5-selective agonist (RS)-2-chloro-5-hydroxyphenylglycine also activated PLD in astrocytes. These findings indicate the involvement of group I mGluRs, most likely mGluR5, in the glutamate activation of PLD in cultured rat cortical astrocytes.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Astrocytes; Benzoates; Carcinogens; Cerebellum; Cerebral Cortex; Cycloleucine; Dose-Response Relationship, Drug; Enzyme Activation; Excitatory Amino Acid Antagonists; Glutamic Acid; Glycine; Hippocampus; Neurons; Neuroprotective Agents; Norepinephrine; Phospholipase D; Phosphorus Radioisotopes; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Sympathomimetics; Tetradecanoylphorbol Acetate; Type C Phospholipases

We have examined the development of expression of group I and II metabotropic glutamate receptors (mGluRs) in pure rat spinal cord astrocyte cultures, using immunocytological and calcium imaging techniques. mGluR1alpha and mGluR2/3 antibodies were found to label roughly 10% of the total astrocyte population at all time points examined, whereas mGluR5 was poorly expressed in our culture system. Results from intracellular Ca2+ imaging experiments, measured using fura-2 ratio imaging, suggest that 20% of these cultured astrocytes express functional group I mGluRs (mGluR1 and/or 5). Our results contrast with previously published work in cultured cortical astrocytes where mGluR5 and not mGluR1 is expressed, suggesting that cultured astrocytes from different parts of the CNS exhibit different patterns of mGluR expression.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Astrocytes; Calcium; Cells, Cultured; Cycloleucine; Excitatory Amino Acid Antagonists; Fura-2; Gene Expression Regulation; Rats; Rats, Wistar; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Spinal Cord

The cerebellar long-term depression (LTD) is the long-lasting reduction of transmission efficacy at the granule neuron-Purkinje neuron (G-P) synapses and is a candidate mechanism for the motor learning. Despite extensive studies on its induction and expression mechanisms, it has not been known how long the LTD lasts. The LTD is accompanied by the decrease in the postsynaptic responsiveness to glutamate, the transmitter at G-P synapses. Therefore, during the LTD, the amplitude of miniature EPSCs (mEPSCs) at G-P synapses should decrease. We studied the depression of mEPSC amplitudes (DME) as a possible contributing factor for the LTD and found that the conditioning treatment of cultured cerebellar neurons with 50 mM K(+) and 100 microM glutamate, an analogous condition used to induce the LTD, induced the long-lasting DME. The mEPSC amplitudes recovered to the original level 48 hr after the 5 min conditioning treatment. Changing the duration of the conditioning revealed that the DME consisted of two distinct phases: the early phase lasting for a few hours and the late phase for >1 d. The latter was distinguished from the former by its requirement of prolonged conditioning treatment and syntheses of mRNA and protein for the induction. There were critical periods for mRNA and protein syntheses. The critical period for protein synthesis was much longer than that for mRNA synthesis. These results demonstrate that the DME lasts for 1-2 d and that it consists of two phases, whose induction and maintenance mechanisms are distinct.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Cells, Cultured; Cerebellum; Cycloleucine; Evoked Potentials; Fetus; Kinetics; Neuroprotective Agents; Patch-Clamp Techniques; Potassium; Purkinje Cells; Rats; Rats, Wistar; Reaction Time

Metabotropic glutamate receptors modulate neuronal excitability via a multitude of mechanisms, and they have been implicated in the pathogenesis of neurodegenerative processes. Here we investigated the responses mediated by group I metabotropic glutamate receptors (mGluRs) in dopamine neurons of the rat substantia nigra pars compacta, using whole cell patch-clamp recordings in combination with microfluorometric measurements of [Ca(2+)](i) and [Na(+)](i). The selective group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (3,5-DHPG) was bath-applied (20 microM, 30 s to 2 min) or applied locally by means of short-lasting (2-4 s) pressure pulses, delivered through an agonist-containing pipette positioned close to the cell body of the neuron. 3,5-DHPG evoked an inward current characterized by a transient and a sustained component, the latter of which was uncovered only with long-lasting agonist applications. The fast component coincided with a transient elevation of [Ca(2+)](i), whereas the total current was associated with a rise in [Na(+)](i). These responses were not affected either by the superfusion of ionotropic excitatory amino acid antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D-2-amino-5-phosphono-pentanoic acid (D-APV), nor by the sodium channel blocker tetrodotoxin (TTX). (S)-alpha-methyl-4-carboxyphenylglycine (S-MCPG) and the more selective mGluR1 antagonist 7(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate (CPCCOEt) depressed both 3,5-DHPG-induced inward current components and, although less effectively, the associated [Ca(2+)](i) elevations. On repeated agonist applications the inward current and the calcium transients both desensitized. The time constant of recovery from desensitization differed significantly between these two responses, being 67.4+/-4.4 s for the inward current and 28.6+/-2.7 s for the calcium response. Bathing the tissue in a calcium-free/EGTA medium or adding thapsigargin (1 microM) to the extracellular medium prevented the generation of the [Ca(2+)](i) transient, but did not prevent the activation of the inward current. These electrophysiological and fluorometric results show that the 3, 5-DHPG-induced inward current and the [Ca(2+)](i) elevations are mediated by independent pathways downstream the activation of mGluR1.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calcium; Chromones; Cycloleucine; Egtazic Acid; Excitatory Amino Acid Antagonists; Glycine; In Vitro Techniques; Microscopy, Fluorescence; Neurons; Patch-Clamp Techniques; Rats; Receptors, Metabotropic Glutamate; Resorcinols; Substantia Nigra; Tetrodotoxin; Thapsigargin

Midbrain non-dopaminergic neurons of the substantia nigra pars reticulata play an important role in the basal ganglia circuitry. The regulation of their electrical activity by excitatory amino acid (EAA) inputs was investigated using in vivo electrophysiological methods in chloral hydrate-anaesthetized rats. We first determined the subtypes of EAA receptors present on reticulata neurons, using microiontophoretic application of selective agonists: kainic acid (KA), (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-D-aspartic acid (NMDA), and trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD). Each agonist activated reticulata neurons and the apparent rank order of efficacy was: KA> or =AMPA=NMDA>trans-ACPD. Using pressure or iontophoretic microejections of ionotropic and metabotropic receptor antagonists, we then investigated EAA receptor subtypes involved in the spontaneous firing rate of reticulata neurons. Kynurenic acid and (+/-)-2-amino-5-phosphonopentanoic acid (AP-5) markedly decreased the spontaneous firing rate of reticulata neurons, while 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) was much less effective. The metabotropic receptor antagonist (R,S)-alpha-methyl-4-carboxyphenylglycine (MCPG) failed to affect the spontaneous electrical activity. In contrast to CNQX, microapplications of AP-5 sometimes produced total inhibition. This powerful effect may reflect the potential importance of NMDA receptors in regulating the activity of some reticulata neurons. These results indicate that both functional ionotropic (NMDA and non-NMDA) and metabotropic EAA receptors are present on non-dopaminergic substantia nigra pars reticulata neurons. Moreover, in the anaesthetized animal, the spontaneous firing rate of these neurons, mediated by EAA inputs, seems mainly due to the tonic activation of ionotropic, but not metabotropic, receptors.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Anesthesia; Animals; Cycloleucine; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Iontophoresis; Kainic Acid; Male; N-Methylaspartate; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Substantia Nigra

Doc2alpha is a synaptic vesicle-associated Ca2 + -binding protein. To study the role of Doc2alpha in synaptic transmission and modulation, we generated homozygous null Doc2alpha mutant mice. In the CA1 region of hippocampal slices in the mutant mice, excitatory synaptic responses evoked with prolonged 5 Hz stimulation showed a significantly larger frequency facilitation followed by a steeper depression than those in wild-type mice, whereas there was no difference in synaptic transmission at lower frequencies or in paired-pulse facilitation. These results suggest that Doc2alpha regulates synaptic transmission when high Ca2 + concentrations in the presynaptic terminal are sustained. Furthermore, the mutant mice showed impairment in long-term potentiation and passive avoidance task. Thus, Doc2alpha may regulate transmitter release during repetitive synaptic activation, thereby contributing to memory formation.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Behavior, Animal; Calcium-Binding Proteins; Cells, Cultured; Cycloleucine; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Gene Targeting; Hippocampus; Long-Term Potentiation; Mice; Mice, Inbred C57BL; Mice, Neurologic Mutants; Mutagenesis, Site-Directed; Nerve Tissue Proteins; Neuronal Plasticity; Neuroprotective Agents; Protein Isoforms; Receptors, Metabotropic Glutamate; Synapses; Synaptic Transmission

We studied synaptic transmission in the granule cells in the olfactory bulb of the homozygous Fyn (a nonreceptor type tyrosine kinase)-deficient (fynz/fynz) and heterozygous Fyn-deficient (+/fynz) mice by using slice preparations from the olfactory bulb. Stimulation to the lateral olfactory tract and/or centrifugal fibers to the olfactory bulb evoked field excitatory postsynaptic potentials (fEPSPs) in the granule cells. In +/fynz mice, fEPSPs were augmented by bicuculline, a gamma-aminobutyric acid (GABAA) antagonist and picrotoxin, whereas fEPSPs in fynz/fynz mice were much less sensitive to bicuculline and picrotoxin. Application of D-2-amino-5-phosphonopentanoic acid had no effect but 6-cyano-7-nitroquinoxaline-2,3-dione produced almost complete block of fEPSPs in both +/fynz mice and fynz/fynz mice. (1S,3R)-1-aminocyclo-pentane-1.3-dicarboxylate, an agonist of metabotropic glutamate receptors caused a similar depression of fEPSPs in both +/fynz and fynz/fynz mice. In +/fynz mice tetanic stimulation to the lateral olfactory tract and/or centrifugal fibers induced N-methyl-D-aspartate (NMDA)-dependent long-term potentiation (LTP) of fEPSPs, whereas LTP was impaired in fynz/fynz mice. Our results demonstrate altered functions of GABAA and NMDA receptors in the olfactory system of Fyn-deficient mice.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Bicuculline; Crosses, Genetic; Cycloleucine; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA-A Receptor Antagonists; Heterozygote; In Vitro Techniques; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Knockout; Neurons; Neuroprotective Agents; Olfactory Bulb; Picrotoxin; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fyn; Receptors, Glutamate; Synaptic Transmission

On-line in vivo microdialysis was used to determine the effects of a 16-min handling period on release of dopamine (DA) in the nucleus accumbens and of DA and noradrenaline (NA) in the medial prefrontal cortex of awake, freely moving rats. DA and NA were determined in one HPLC run. Handling resulted in an immediate and strong increase of both catecholamines in the prefrontal cortex. Maximal values for DA were 295%, and for NA 225%, of controls. DA in the nucleus accumbens was also increased (to 135% of controls) but only after a short delay. Local inhibition of ionotropic glutamate receptors by continuous reversed dialysis of the drugs 6-cyano-7-nitroquinoxaline, D-2-amino-5-phosphonopentanoic acid, or dizocilpine did not significantly affect handling-induced increases in cortical DA and NA release. Neither did the agonist of metabotropic glutamate receptors, trans-(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), or the GABA-B agonist baclofen. Reversed dialysis of dizocilpine in the nucleus accumbens was equally ineffective, but ACPD inhibited the increase in DA release in this area. Stimulation of metabotropic glutamate receptors in the nucleus accumbens was previously reported to inhibit activation of DA release in that area after stimulation of glutamatergic or dopaminergic afferents. It is concluded that metabotropic receptors in the nucleus accumbens are important for the control of activation of DA release in the accumbens by physiological stimuli but that a similar mechanism is lacking in the prefrontal cortex.

Topics: 3,4-Dihydroxyphenylacetic Acid; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Baclofen; Cycloleucine; Dizocilpine Maleate; Dopamine; Excitatory Amino Acid Antagonists; GABA Agonists; Homovanillic Acid; Hydroxyindoleacetic Acid; Male; Microdialysis; Neuroprotective Agents; Norepinephrine; Nucleus Accumbens; Prefrontal Cortex; Rats; Rats, Wistar; Receptors, GABA-B; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; Stress, Physiological

Expression of GAP-43 in the cerebellum and selected regions of the brain has been shown to be developmentally regulated. Localization of GAP-43 mRNA within granule cells of the immature and mature rat cerebellum has been demonstrated by in situ hybridization. Higher levels are detected in the neonate compared to the adult. To determine if the cerebellar neurotransmitters, GABA (gamma-amino-butyric acid) and glutamate are involved in the modulation of GAP-43 expression, cultured cerebellar granule cells were exposed to these transmitters. Cultures were treated with glutamate, GABA, or the agonists/antagonists to their receptors in serum-free media for 5-7 days. Analysis of the levels of GAP-43 mRNA by in situ hybridization indicated that a 7-day exposure to GABA (25 and 50 microM) significantly lowered levels of granule cell GAP-43 mRNA. Specific agonists to the GABAA (muscimol) and GABAB (baclofen) receptors produced a decrease similar to that observed for GABA. Results from these studies also indicated that exposure to non-NMDA (CNQX) and NMDA (CPP, MK-801) glutamate receptor antagonists, and a metabotropic receptor glutamate agonist (ACPD), decreased the level of GAP-43 mRNA. The involvement of GABA and glutamate in the modulation of GAP-43 expression was corroborated by Northern hybridization. These studies revealed that a 5-day exposure to GABA decreased the cellular content of GAP-43 mRNA by 21% whereas exposure to glutamate resulted in a 37% increase. Findings from the studies reported here, using an in vitro cerebellar granule cell model, suggest that levels of GAP-43 mRNA, in vivo, are modulated by input from both excitatory glutamatergic mossy fibers and inhibitory GABAergic Golgi interneurons. Thus, modulation of GAP-43 mRNA by these neurotransmitters may influence granule cell maturation during development in the neonate and neuroplasticity in the adult, possibly at the parallel fiber-Purkinje cell synapse.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Baclofen; Blotting, Northern; Cells, Cultured; Cerebellum; Cycloleucine; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; GABA Agonists; gamma-Aminobutyric Acid; GAP-43 Protein; Gene Expression Regulation, Developmental; Glial Fibrillary Acidic Protein; Glutamic Acid; Immunohistochemistry; In Situ Hybridization; Microtubule-Associated Proteins; Muscimol; N-Methylaspartate; Neurons; Neuroprotective Agents; Piperazines; Rats; Rats, Sprague-Dawley; RNA, Messenger

Hippocampal inhibitory cells are diverse. It is supposed that they fall into functionally distinct subsets defined by a similar morphology and physiology. Switching between functions could be accomplished by activating receptors for modulating transmitters expressed selectively by different subsets of interneurons. We tested this hypothesis by comparing morphology, physiology, and neurotransmitter receptor expression for CA1 hippocampal interneurons. We distinguished 16 distinct morphological phenotypes and 3 different modes of discharge. Subsets of inhibitory cells were excited or inhibited by agonists at receptors for noradrenaline, muscarine, serotonin, and mGluRs. Most cells responded to 2 or 3 agonists, and 25 different response combinations were detected. Subsets defined by morphology, physiology, and receptor expression did not coincide, suggesting that hippocampal interneurons cannot easily be segregated into a few well-defined groups.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Axons; Cell Size; Cycloleucine; Dendrites; Excitatory Amino Acid Antagonists; Hippocampus; Interneurons; Muscarine; Muscarinic Agonists; Neural Inhibition; Neuroprotective Agents; Neurotransmitter Agents; Norepinephrine; Rats; Receptors, Metabotropic Glutamate; Serotonin; Sympathomimetics; Tetrodotoxin

Kindling induced by 13 intraperitoneal injections of 40 mg/kg pentylenetetrazole (PTZ) over a period of 4 weeks resulted in a significant long-lasting increase in both the convulsive susceptibility of animals to the convulsant and the density of the specific [3H]-L-glutamate binding sites in the hippocampus. The quisqualate- and kainate-sensitive [3H]-L-glutamate binding sites were increased 24 h after the final PTZ injection, whereas the N-methyl-D-aspartate (NMDA)-sensitive sites had only a tendency to be enhanced. Furthermore, we investigated [3H]-L-glutamate binding on metabotropic receptors and found a significant increase in the hippocampus following PTZ kindling. In addition, in hippocampal tissue of kindled rats (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD)-stimulated inositol phosphate formation is increased. It can be concluded that the increase in metabotropic glutamate receptor (mGluR) density may be the expression of a specific enhancement in susceptibility of the glutamatergic systems to this excitatory amino acid developing in the course of PTZ-induced kindling.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Brain Chemistry; Convulsants; Cycloleucine; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; Inositol Phosphates; Kindling, Neurologic; Male; Neuroprotective Agents; Pentylenetetrazole; Radioligand Assay; Rats; Rats, Wistar; Receptors, AMPA; Receptors, Glutamate; Receptors, Kainic Acid; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; Tritium

The development of receptor function at corticothalamic synapses during the first 20 days of postnatal development is described. Whole cell excitatory postsynaptic currents (EPSCs) were evoked in relay neurons of the ventral posterior nucleus (VP) by stimulation of corticothalamic fibers in in vitro slices of mouse brain from postnatal day 1 (P1). During P1-P12, excitatory postsynaptic conductances showed strong voltage dependence at peak current and at 100 ms after the stimulus and were almost completely antagonized by -2-amino-5-phosphonopentoic acid (APV), indicating that N-methyl--aspartate (NMDA) receptor-mediated currents dominate corticothalamic EPSCs at this time. After P12, in 42% of cells, excitatory postsynaptic conductances showed no voltage-dependence at peak current but still showed voltage-dependence 100-ms poststimulus. This voltage-dependent conductance was antagonized by APV. The nonvoltage-dependent component was APV resistant, showed fast decay, and was antagonized by the nonNMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In the remaining 58% of cells after P12, excitatory postsynaptic conductances showed moderate voltage dependence at peak conductance and strong voltage dependence 100 ms after the stimulus. Analysis of EPSCs before and after APV showed a significant increase in the relative contribution of the non-NMDA conductance after the second postnatal week. From P1 to P16, there was a significant decrease in the time constant of decay of the NMDA EPSC but no change in the voltage dependence of the NMDA response. After P8, slow EPSPs, 1.5-30 s in duration and mediated by metabotropic glutamate receptors (mGluRs), could be evoked by high-frequency stimulation of corticothalamic fibers in the presence of APV and CNQX. Similar slow depolarizations could be evoked by local application of the mGluR agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) but from P0. Both conductances were blocked by the mGluR antagonist, (RS)-alpha-methyl-4-carboxyphenylglycine. Hence functional mGluR receptors are present on VP cells from birth, but their synaptic activation at corticothalamic synapses can only be detected after P8. In voltage clamp, the extrapolated reversal potential of the t-ACPD current, with potassium gluconate-based internal solution, was +12 +/- 10 (SE) mV, and the measured reversal potential with cesium gluconate-based internal solution was 1.5 +/- 9.9 mV, suggesting that the mGluR-mediated depola

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Aging; Animals; Animals, Newborn; Benzoates; Cerebral Cortex; Cycloleucine; Evoked Potentials; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glycine; Mice; Mice, Inbred ICR; Nerve Fibers; Neurons; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; Synapses; Thalamus

Glutamate receptors underlying synaptic excitation in the rat's lateral superior olive were studied by whole-cell patch clamp recordings in a brain slice preparation. Recordings from two morphological types of cells, bipolar and multipolar, identified by intracellular labeling with biocytin, showed that there were no obvious differences in responses mediated or modulated by ionotropic and metabotropic receptors between these two types of neurons. The excitatory postsynaptic potentials (EPSPs) elicited by ipsilateral stimulation of the trapezoid body consisted of two components. An earlier component, which had faster rise time constant and decay time constant, was mediated by non-NMDA receptors. A later component, which had slower rise time and decay time constants, was mediated by NMDA receptors. Suprathreshold responses (action potentials), which arose from the early component, were always abolished by the non-NMDA antagonist, CNQX, but not by the NMDA antagonist, APV. These results suggest that both non-NMDA and NMDA receptors are present in LSO neurons, and that fast excitatory transmission in LSO is primarily mediated by non-NMDA receptors. The metabotropic glutamate receptor agonists, t-ACPD and L-AP4, reduced the size of EPSPs evoked by stimulation of the ipsilateral trapezoid body in LSO neurons; the reductive action of t-ACPD was reversed by the antagonist, MCPG, indicating that metabotropic glutamate receptors, probably group II and III subtypes, can modulate excitatory synaptic transmission in LSO.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Aminobutyrates; Animals; Cycloleucine; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; In Vitro Techniques; Neuroprotective Agents; Olivary Nucleus; Patch-Clamp Techniques; Rats; Rats, Wistar; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; Synaptic Transmission

We studied how metabotropic glutamate receptor (mGluR) activation modifies the synaptic and intrinsic membrane properties of neonatal rat trigeminal motoneurons using the broad-spectrum mGluR agonist (1S,3R)-1-amino-1,3-cyclopentane-dicarboxylic acid [(1S,3R)-ACPD], group I/II antagonist (+/-)-alpha-methyl-4-carboxy-phenylglycine (MCPG), and group III agonist L-2-amino-4-phosphonobutanoate (L-AP4). (1S,3R)-ACPD depressed excitatory transmission to trigeminal motoneurons presynaptically and postsynaptically via presynaptic inhibition and by reducing the currents carried by ionotropic glutamate receptors selective for AMPA. (1S,3R)-ACPD also depolarized trigeminal motoneurons and increased input resistance by suppressing a Ba2+-sensitive leakage K+ current. These effects were not mimicked by L-AP4 (100-200 microM). High-threshold Ca2+ currents were also suppressed by (1S,3R)-ACPD. Repetitive stimulation of excitatory premotoneurons mimicked the postsynaptic effects of (1S, 3R)-ACPD. The postsynaptic effects of (1S,3R)-ACPD and repetitive stimulation were both antagonized by MCPG, suggesting that mGluRs were similarly activated in both experiments. We conclude that mGluRs can be recruited endogenously by glutamatergic premotoneurons and that mGluR-mediated depression of excitatory transmission, combined with increased postsynaptic excitability, enhances the signal-to-noise ratio of oral-related synaptic input to trigeminal motoneurons during rhythmical jaw movements.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Animals, Newborn; Benzoates; Cycloleucine; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glycine; Membrane Potentials; Motor Neurons; N-Methylaspartate; Neuroprotective Agents; Patch-Clamp Techniques; Rats; Receptors, Metabotropic Glutamate; Synaptic Transmission; Tetrodotoxin; Trigeminal Nuclei

We tested the hypothesis that stimulation of metabotropic glutamate receptors (mGluRs) increases nitric oxide (NO) production in the hippocampus in vivo. Microdialysis probes were placed bilaterally into the CA3 region of the hippocampus of adult Sprague-Dawley rats under pentobarbital anesthesia. Probes were perfused for 5 h with artificial cerebrospinal fluid (CSF) containing 3 microM [14C]-L-arginine. Recovery of [14C]-L-citrulline in the effluent was used as a marker of NO production. In nine groups of rats, increases in [14C]-L-citrulline recovery were compared between right- and left-sided probes perfused with various combinations of the selective mGluR agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD); the mGluR antagonist, (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG); the NO synthase inhibitor, N-nitro-L-arginine (LNNA); the ryanodine sensitive calcium-release channel inhibitor dantrolene, the non-N-methyl-D-aspartate (NMDA); receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX); the NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine (MK-801); and the Na+ channel blocker, tetrodotoxin. Recovery of [14C]-L-citrulline during perfusion with artificial CSF progressively increased to 90 +/- 21 fmol/min (+/-SD) over 5 h. Perfusion in the contralateral hippocampus with 1 mM ACPD augmented [14C]-L-citrulline recovery to 250 +/- 81 fmol/min. Perfusion of 1 mM nitroarginine + ACPD inhibited [14C]-L-citrulline recovery compared to that with ACPD alone. Perfusion with 1 mM MCPG + ACPD attenuated ACPD enhanced [14C]-L-citrulline recovery. Perfusion of 1 mM dantrolene + ACPD inhibited the ACPD-evoked increase in [14C]-L-citrulline recovery. Perfusion of 1 mM MCPG or dantrolene without ACPD did not decrease [14C]-L-citrulline recovery as compared to CSF alone. ACPD-enhanced [14C]-L-citrulline recovery was not attenuated by CNQX, MK-801, or tetrodotoxin (TTX). Using an indirect method of assessing NO production in vivo, these data demonstrate that mGluR stimulation enhances NO production in rat hippocampus. Inhibition with dantrolene suggests that calcium-induced calcium release amplifies the inositol triphosphate-mediated calcium signal associated with mGluR stimulation, thereby resulting in augmented calcium-dependent NO production.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Benzoates; Biomarkers; Calcium Channels; Citrulline; Cycloleucine; Dantrolene; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glycine; Hippocampus; Male; Microdialysis; Muscle Proteins; N-Methylaspartate; Nerve Tissue Proteins; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Ryanodine Receptor Calcium Release Channel; Tetrodotoxin

The functional relevance of presynaptic glutamate receptors in controlling presynaptic Ca2+ influx and thereby transmitter release is unknown. To test if presynaptic Ca2+ entry in the hippocampus is controlled by glutamate autoreceptors, we created a hippocampal slice preparation for investigation of presynaptic Ca2+ signals with Ca(2+)-sensitive microelectrodes after lesioning of neurons by glucose deprivation or kainate. Stratum radiatum and alveus stimulation-induced postsynaptic field potential components were irreversibly abolished in areas CA1 and CA3 of lesioned slices, whereas stratum radiatum stimulation still evoked afferent volleys. Repetitive stimulation of the stratum radiatum still induced decreases in extracellular Ca2+ concentration. Repetitive stimulation of the alveus no longer induced decreases in extracellular Ca2+ concentration, suggesting complete damage of pyramidal cells. The stratum radiatum stimulation-induced decreases in extracellular Ca2+ concentration in lesioned slices were comparable to those elicited during application of the glutamate antagonists 6-cyano-7-nitroquinoxaline-2,3-dione and L-2-amino-5-phosphonovalerate. In lesioned slices the stimulus-induced presynaptic Ca2+ influx was reversibly reduced by kainate. RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), N-methyl-D-aspartate and glutamate without effects on afferent volleys. The kainate and N-methyl-D-aspartate effects on presynaptic Ca2+ signals were partly sensitive to 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(f)quinoxaline and L-2-amino-5-phosphonovalerate, respectively, while the AMPA effects were not significantly affected by 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(f)quinoxaline, suggesting involvement of a novel glutamate receptor subtype. The involvement of a novel glutamate receptor subtype was supported by our findings that ionotropic glutamate receptor agonists also reduce presynaptic Ca2+ influx under conditions of blocked synaptic transmission by 6-cyano-7-nitroquinoxaline-2,3-dione and L-2-amino-5-phosphonovalerate. 1-Aminocyclopentane-trans-1,3-dicarboxylic acid had no significant effect on presynaptic Ca2+ entry. Also, the presynaptic Ca2+ influx was not influenced by the glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione, 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(f)quinoxaline and L-2-amino-5-phosphonovalerate when applied alone. Low kainate concentrations (5 microM) reduced presynaptic Ca2+ signals in area CA3 but no

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Calcium; Cycloleucine; Electrophysiology; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; Glucose; Hippocampus; Kainic Acid; Male; N-Methylaspartate; Neuroprotective Agents; Organ Culture Techniques; Quinoxalines; Rats; Rats, Wistar; Receptors, Glutamate; Synaptic Transmission

1. Modulation of plateau properties in dorsal horn neurones was studied in a transverse slice preparation of the spinal cord of the turtle. In plateau-generating neurones high frequency stimulation of the ipsilateral dorsal root (10-20 Hz, 0.5-2 min) produced a slow depolarization (2.9 +/- 0.6 mV, mean +/- S.E.M.; n = 6) and enhanced the properties mediated by dihydropyridine-sensitive Ca2+ channels. The tetanic stimulus facilitated wind-up and after-discharges even when fast synaptic transmission was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10-20 microM), (+/-)-2-amino-5-phosphonopentanoic acid (AP5, 100 microM), bicuculline (10-20 microM) and strychnine (5-20 microM). 2. Application of cis-(+/-)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD, 10-50 microM) produced a slow depolarization (5.9 +/- 0.5 mV, n = 21) accompanied by an increase in input resistance (28.8 +/- 5.1%, n = 12). 3. ACPD increased the excitability by facilitating the plateau properties. In the presence of tetrodotoxin (TTX, 1 microM) a lower threshold and a slower decay of the plateau potential were observed. These effects resulted in facilitation of wind-up and prolonged after-discharges. 4. All ACPD-induced effects were blocked by alpha-methyl-4-carboxyphenylglycine (MCPG, 0.5-1 mM), a selective antagonist of metabotropic glutamate receptors. The selective agonist for the type I metabotropic glutamate receptor ((RS)-3,5-dihydrophenylglycine (DHPG, 50 microM)) reproduced all the effects of ACPD. 5. Application of a supposed neuromodulator, substance P (1-2 microM) produced a transient depolarization (4 +/- 0.6 mV) lasting 4-6 min during continued application of substance P. Variable effects on the input resistance were observed, a slight increase (12 +/- 2%) being the most frequent. In 61% of the cells, substance P induced a clear increase in excitability with no detectable change in input resistance or membrane potential. 6. The effects of substance P on plateau properties were indistinguishable from those produced by ACPD. Unlike the transient depolarization, the facilitation of the plateau properties persisted in the presence of the agonist. 7. The substance P-induced facilitation of the plateau potential was blocked by GR 82334 (5-10 microM), a selective NK-1 tachykinin-receptor antagonist, and was not affected by MEN 10376 (2 microM), a selective NK-2 antagonist. 8. The facilitation of plateau properties produced by dorsal root stimulation was also reduced by anta

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cycloleucine; Electrophysiology; Excitatory Amino Acid Antagonists; Neurokinin A; Neurons, Afferent; Neuroprotective Agents; Neurotoxins; Peptide Fragments; Receptors, Glutamate; Receptors, Metabotropic Glutamate; Receptors, Neurokinin-1; Spinal Cord; Substance P; Tetrodotoxin; Turtles

NF-kappaB and activator protein 1 (AP-1) are dimeric transcription factors involved in transcriptional regulation in many cells, including neurons. We have examined their activity during mouse cerebellum development, a postnatal process starting just after birth and completed by the fourth postnatal (PN) week. The activity of these factors was analyzed by binding of nuclear extracts to a synthetic oligonucleotide representing the kappaB site of human immunodeficiency virus or the AP-1 site of the urokinase promoter. NF-kappaB activity was observed from 7 PN, was restricted to the developing cerebellum, and was not observed in the early postnatal neocortex and hippocampus. On the other hand, AP-1 activity was not found in cerebellum but was present in both neocortex and hippocampus. Moreover, a kappaB-driven transgene was found to be increasingly expressed in the cerebellum from 5 PN to 10 PN but not in the adult. The regulation of NF-kappaB activation in mouse cerebellum was analyzed by intraperitoneal injection of glutamate receptor antagonists to 9 PN mice, which abolished NF-kappaB-binding activity, suggesting an endogenous loop of glutamate receptor activation. Glutamate receptor agonists, on the other hand, induced NF-kappaB nuclear translocation in the cerebellum of 5 PN mice, which is a stage in which NF-kappaB is not yet endogenously activated. This effect was specific for NF-kappaB and not observed for AP-1. In adult mice, NF-kappaB activity was absent in the cerebellum and was not induced by intraperitoneal injection of glutamate receptor agonists. These data show that NF-kappaB is specifically activated during cerebellum development and indicate an important role of glutamate receptors in this process.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Age Factors; Animals; Antibody Specificity; Cell Nucleus; Cerebellum; Cycloleucine; Cytoplasm; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Gene Expression Regulation, Developmental; Glutamic Acid; Injections, Intraperitoneal; Mice; Mice, Transgenic; N-Methylaspartate; Neuroprotective Agents; Neurotransmitter Agents; NF-kappa B; Transgenes

Synaptic depression of evoked EPSCs was quantified with stimulation frequencies ranging from 0.2 to 100 Hz at the single CNS synapse formed by the calyx of Held in the rat brainstem. Half-maximal depression occurred at approximately 1 Hz, with 10 and 100 Hz stimulation frequencies reducing EPSC amplitudes to approximately 30% and approximately 10% of their initial magnitude, respectively. The time constant of recovery from depression elicited by 10 Hz afferent fiber stimulation was 4.2 sec. AMPA and NMDA receptor-mediated EPSCs depressed in parallel at 1-5 Hz stimulation frequencies, suggesting that depression was induced by presynaptic mechanism(s) that reduced glutamate release. To determine the contribution of autoreceptors to depression, we studied the inhibitory effects of the metabotropic glutamate receptor (mGluR) agonists (1S, 3S)-ACPD and L-AP4 and found them to be reversed in a dose-dependent manner by (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG), a novel and potent competitive antagonist of mGluRs. At 300 microM, CPPG completely reversed the effects of L-AP4 and (1S, 3S)-ACPD, but reduced 5-10 Hz elicited depression by only approximately 6%. CPPG-sensitive mGluRs, presumably activated by glutamate spillover during physiological synaptic transmission, thus contribute on the order of only 10% to short-term synaptic depression. We therefore suggest that the main mechanism contributing to the robust depression elicited by 5-10 Hz afferent fiber stimulation of the calyx of Held synapse is synaptic vesicle pool depletion.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Afferent Pathways; Aminobutyrates; Animals; Brain Stem; Cycloleucine; Electric Stimulation; Glutamic Acid; Glycine; Neuronal Plasticity; Patch-Clamp Techniques; Rats; Rats, Wistar; Receptors, AMPA; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; Synapses

Stimulation of synaptoneurosome suspensions by the neurotransmitter glutamate gives rise to rapid loading of ribosomes onto mRNA and increased incorporation of amino acids into trichloroacetic acid-precipitable polypeptides. Metabotropic glutamate receptors (mGluRs) are responsible for this effect. Although simultaneous Ca2+ entry and mGluR stimulation do not change the response, entry of Ca2+ 30 s or 3 min before mGluR stimulation markedly depresses the polyribosomal loading. Either NMDA or ionophore (A23187) produces the depression. A calmodulin antagonist, W7, alleviates the effect, suggesting that inactivation of phospholipase A2 by calcium-calmodulin-dependent kinase II is partially responsible for the phenomenon. Thus, interaction between different classes of glutamate receptors affects the control of protein translation at the synapse. This effect may partially explain recent observations of negative interactions between receptor classes in induction of long-term potentiation.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calcimycin; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cerebral Cortex; Cycloleucine; Depression, Chemical; Gene Expression Regulation; Ionophores; Long-Term Potentiation; N-Methylaspartate; Nerve Tissue Proteins; Phorbol 12,13-Dibutyrate; Phospholipases A; Phospholipases A2; Protein Biosynthesis; Quisqualic Acid; Rats; Receptors, Metabotropic Glutamate; Ribosomes; RNA, Messenger; Sulfonamides; Synapses

The microinjection of L-glutamate (1-6 nmol/rat) and N-methyl-D-aspartate (NMDA 1-10 nmol/rat), ionotropic glutamate receptor (iGluR) agonists, into the nucleus raphe obscurus caused a concentration -dependent increase of arterial blood pressure. In contrast, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD, 14-42 nmol/rat), a metabotropic glutamate receptor (mGluRs) agonist, caused a concentration-dependent decrease in blood pressure. Pretreatment with D,L-2-amino-phosphono valeric acid (2-APV, 5 nmol/rat) a selective NMDA iGluR antagonist, and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b] cyclohepten-5,10-imine hydrogen maleate (MK801, 0.9 nmol/rat), a noncompetitive NMDA iGluR antagonist, blocked both the glutamate and NMDA pressor responses, while pretreatment with (+)-alpha-methyl-4-carboxyphenylglycine (MCPG, 0.05 nmol/rat), a mGluR1 antagonist, increased the glutamate-induced pressor effects and blocked the fall in blood pressure induced by t-ACPD. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX, 0.4 nmol/rat) a non-NMDA iGluR antagonist, did not affected the glutamate-induced hypertension. These observations indicate opposing roles for ionotropic and metabotropic receptors in the glutamate-induced blood pressure changes elicited from the nucleus raphe obscurus. Moreover, we suggest that the glutamate-induced hypertension may be due to the activation of NMDA ionotropic receptor subtypes and the metabotropic receptors may influence this activation through a reduction of excitability at level of synapses.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Analysis of Variance; Animals; Benzoates; Blood Pressure; Cycloleucine; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Glycine; Hypertension; Male; Microinjections; N-Methylaspartate; Neurotoxins; Raphe Nuclei; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Structure-Activity Relationship; Valine

L-Glutamate (Glu)-induced current responses were studied in 119 isolated OFF-type bipolar cells of the cat retina. Cells were recorded by the patch clamp technique in the whole-cell configuration. Glu induced a current carried by alkali metal ions and divalent cations with a permeability ratio of PNa:PK:PCs:PCa = 1:0.94:1.32:0.57. Sensitivity to Glu was highest in the dendritic region. Kainate and AMPA worked as potent agonists, but neither APB, L-aspartate, ACPD, nor NMDA (all at 100 microM) was effective. The Glu-induced response was antagonized by > 1 microM CNQX. We inferred that OFF-type bipolar cells have a non-NMDA receptor channel that is permeable to alkali metal ions with low selectivity, but not NMDA receptor or metabotropic Glu receptor.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Aminobutyrates; Animals; Aspartic Acid; Calcium; Cats; Chlorides; Cycloleucine; Dose-Response Relationship, Drug; Glutamic Acid; Glycine; In Vitro Techniques; Ion Transport; Kainic Acid; Membrane Potentials; N-Methylaspartate; Neurons; Patch-Clamp Techniques; Potassium; Retina; Retinal Cone Photoreceptor Cells; Sodium

Developmental changes in glutamate receptor agonist-produced enhancement of 4-beta-[3H]phorbol-12,13-dibutyrate binding ([3H]-PDBu binding), indicative of an intracellular translocation of protein kinase C (PKC), were investigated in cerebellar granule cells. Our observations demonstrate that the magnitude of glutamate-, NMDA-, and kainate-produced enhancement of PKC translocation was dramatically decreased between 2 and 12 DIV, whereas there was only a minor reduction in the corresponding response caused by the non-NMDA receptor agonist, AMPA. The maximally enhanced stimulation of PKC translocation caused by glutamate and NMDA was significantly reduced already at 4 DIV, whereas a significant reduction of the kainate-induced enhancement of [3H]PDBu binding was not observed until 8 DIV. Glutamate- and NMDA-induced responses were effectively blocked by the specific NMDA receptor antagonists MK-801 (1 microM) and APV (100 microM) as well as by the addition of Mg2+ into assay media. In contrast, the non-NMDA receptor antagonist, CNQX (10 microM), effectively blocked the kainate-induced enhancement of [3H]PDBu binding, but had no effect on the NMDA- and glutamate-induced stimulation of PKC translocation. The metabotropic glutamate receptor agonist, ACPD (up to 250 microM), had no effect on the translocation of PKC. Taken together, our data support the working hypothesis that the rapidly occurring changes in the glutamate receptor agonist-produced translocation of PKC are most likely due to a differential maturation of glutamate ionotropic receptor subtypes and/or to development-dependent alterations in mechanisms responsible for the coupling between the glutamate receptor subtypes and the activation of PKC translocation in cerebellar granule neurons.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Binding, Competitive; Biological Transport; Carcinogens; Cells, Cultured; Cerebellum; Cycloleucine; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Kainic Acid; Magnesium; N-Methylaspartate; Neurotoxins; Phorbol 12,13-Dibutyrate; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Tritium

Intracellular recordings were obtained from neocortical brain slices of adult rats maintained in vitro. The effect of metabotropic glutamate receptor activation on spike frequency adaptation in regular spiking layer II and III neurons was determined. Putative metabotropic glutamate receptor agonists and antagonists, as well as inhibitors of intracellular signaling systems, were tested. Activation of metabotropic glutamate receptors by bath applied (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD; 50-200 microM) reduced the first interspike interval and increased action potential frequency at all current intensities. This effect was not blocked by ionotropic glutamate receptor antagonists. Under these recording conditions, quisqualate (1-10 microM) similarly reduced spike frequency adaptation. Neither 1R,3S-ACPD, L-2-carboxycyclopropylglycine-I nor the putative presynaptic metabotropic glutamate receptor agonist, L-2-amino-4-phosphonobutyrate, mimicked the effects of 1S,3R-ACPD or quisqualate. Bath application of the putative metabotropic glutamate receptor antagonist, alpha-methyl-4-carboxyphenylglycine, competitively antagonized the excitatory actions of 1S,3R-ACPD. Another putative antagonist, L-2-amino-3-phosphonopropionate, failed to antagonize the reduction in spike frequency adaptation. Intracellular injection of guanosine-5'-O-(2-thiodiphosphate), a non-hydrolysable analog of GTP, inhibited the postsynaptic metabotropic glutamate receptor-mediated effects. However, the depression of synaptic transmission by 1S,3R-ACPD was not antagonized by this compound. The decrease in spike frequency adaptation by 1S,3R-ACPD was not prevented by prior exposure to the non-specific protein kinase inhibitors H-7 or H-8 (10 microM), the protein kinase A inhibitor H-89 (0.25 microM) or the protein kinase C inhibitor staurosporine (0.10 microM). These data suggest that the metabotropic glutamate receptor-mediated reduction in spike adaptation requires the activation of specific G-protein-coupled metabotropic glutamate receptor subtypes located on postsynaptic sites. The increase in neuronal excitability observed in the adult neocortex may be mediated either by an unidentified G-protein-coupled second messenger or via a membrane-delimited G-protein action.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Adult; Alanine; Amino Acids, Dicarboxylic; Animals; Benzoates; Cycloleucine; Enzyme Inhibitors; Frontal Lobe; Glycine; GTP-Binding Proteins; Guanosine Diphosphate; Humans; Isoquinolines; N-Methylaspartate; Nerve Tissue Proteins; Neurons; Protein Kinase Inhibitors; Protein Kinases; Quisqualic Acid; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Signal Transduction; Staurosporine; Sulfonamides; Thionucleotides

1. Whole-cell clamp experiments on Purkinje neurons in rat cerebellar slices were used to test whether glutamate transporters, detected immunocytochemically in the somata and dendrites of the cells, are functional in the cell surface membrane, and to investigate their role in terminating synaptic transmission. 2. A membrane current was detected with the pharmacology, voltage and ion dependence of a glutamate uptake current. Part of the current was generated by an anion conductance activated when uptake occurs. 3. With sodium and glutamate inside the cell, raising the external potassium concentration generated an outward current attributable to reversed operation of glutamate transporters. 4. The magnitude of the uptake current suggested that Purkinje cell transporters could help to terminate transmission at the climbing and parallel fibre to Purkinje cell synapses. Reducing postsynaptic glutamate uptake with intracellular D-aspartate prolonged the climbing fibre EPSC. 5. These data establish the existence of functional postsynaptic glutamate transporters, show that they contribute to terminating synaptic transmission, and suggest that they may play a role in the preferential death of Purkinje cells in ischaemia.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Aspartic Acid; Benzoates; Cycloleucine; Electrophysiology; Excitatory Amino Acid Antagonists; Glutamic Acid; Glycine; Ion Channel Gating; Kynurenic Acid; Membrane Potentials; Monosaccharide Transport Proteins; Neuroprotective Agents; Purkinje Cells; Rats; Receptors, Metabotropic Glutamate; Synaptic Transmission; Tetrodotoxin

1. Spontaneous inhibitory postsynaptic currents (IPSCs) and evoked IPSCs were recorded by a whole-cell patch-recording technique from cultured Purkinje cells of the rat. The size of spontaneous IPSCs, after a train of depolarizing pulses was applied to the Purkinje cells, increased to 163 +/- 6% (mean +/- S.E.M., n = 7 cells) of the control levels measured before the stimulus train. 2. The GABAergic postsynaptic currents were recorded under voltage clamp from the synapse formed between two Purkinje cells. These IPSCs increased to 218 +/- 31% (n = 4) of control levels after depolarizing stimulation was applied to the postsynaptic Purkinje cells. Size-increased IPSCs were observed as long as recording continued and the phenomena will be called potentiation in this paper. 3. Intracellular application of Ruthenium Red (20 microM) did not block the potentiation of spontaneous IPSCs induced by the depolarizing stimulus (165 +/- 9%, n =6), but heparin (2 mg ml-1) partially blocked the potentiation (123 +/- 10%, n = 6). Heparin applied together with Ruthenium Red (20 microM) blocked potentiation completely (96 +/- 5%, n = 8) at concentrations higher than 1 mg ml-1. 4. Intracellular free calcium concentrations ([Ca2+]i) was monitored as the ratio of fura-2 fluorescences excited at 340 and 380 nm. In control cells, [Ca2+]i was increased by each depolarizing pulse. When Purkinje cells were dialysed with heparin or heparin with Ruthenium Red, the rise in [Ca2+]i was suppressed. 5. Bath application of thapsigargin (1 microM) blocked the potentiation (99 +/- 2%, n = 4) and suppressed the rise in [Ca2+]i. 6. When 30 mM BAPTA was applied intracellularly, a train of depolarizing pulses failed to induce potentiation of IPSCs and failed to raise [Ca2+]i. The results from points 3-6 suggest that the increase in [Ca2+]i, most probably coupled with the release from intracellular stores especially from the inositol trisphosphate (IP3)-sensitive stores, is crucial for the potentiation of IPSCs. 7. Bath application of a metabotropic glutamate receptor activator (t-ACPD, 200 microM) increased both the amplitude and frequency of spontaneous IPSCs and increased the [Ca2+]i slightly in dendrites. The inward current induced by the puff-applied GABA (2 microM) was increased, after t-ACPD application, to 186 +/- 36% of the control level (n = 3). Bath application of quisqualate (2 microM) caused a rapid increase in [Ca2+]i in dendrites and in the cell body and increased both the amplitud

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calcium; Calcium Channels; Chelating Agents; Cycloleucine; Egtazic Acid; Enzyme Inhibitors; Estrenes; Evoked Potentials; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; Heparin; Neuroprotective Agents; Phosphodiesterase Inhibitors; Purkinje Cells; Pyrrolidinones; Rats; Receptors, Metabotropic Glutamate; Ruthenium Red; Synapses; Thapsigargin; Type C Phospholipases

Cone-driven ON-type bipolar cells were patch clamped in white perch retinal slices. Application of glutamate activated a current (IGlu) that was mediated by a conductance increase. The reversal potential for IGlu followed ECl closely when the intracellular chloride concentration was varied. IGlu was not blocked by 100 microM picrotoxin or 1 microM strychnine, indicating that it was not caused by inhibitory input. IGlu is not mediated by a typical ionotropic glutamate receptor since it was not activated by kainate, AMPA, or NMDA, or blocked by kynurenic acid, CNQX, DNQX, or AP-V. Further, IGlu is not mediated by a known metabotropic glutamate receptor since it was not activated by quisqualic acid, AP-4, ACPD, or ibotenate. IGlu required the presence of extracellular sodium and could be partially inhibited by the glutamate uptake inhibitors THA and tPDC. This is suggestive of sodium-dependent glutamate transport. However, when intracellular sodium was greatly increased, neither the magnitude nor reversal potential of IGlu was substantively affected. Thus, IGlu appears to involve a chloride channel activated by a glutamate receptor with transporter-like pharmacology. IGlu is localized to the dendrites of the bipolar cell, where bipolar cells receive an endogenous glutamatergic input from photoreceptors. Further, the reversal potential of the light response in these cells is the same as that of IGlu. Thus, it seems likely that IGlu is the current responsible for the cone component of the ON bipolar cell light response in the teleost retina.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Chloride Channels; Chlorides; Cycloleucine; Dendrites; Glutamic Acid; Ibotenic Acid; In Vitro Techniques; Kainic Acid; Kynurenic Acid; Membrane Potentials; N-Methylaspartate; Neurotoxins; Patch-Clamp Techniques; Perches; Picrotoxin; Quinoxalines; Quisqualic Acid; Receptors, Metabotropic Glutamate; Retina; Retinal Cone Photoreceptor Cells; Strychnine

The effect of excitatory amino acids (EAA) on phosphatidylinositol (PI) turnover in human cerebral cortical slices was investigated. Trans-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) increased inositol phosphate (IP) formation in the 1-1000 microM range. Quisqualic acid (QA) was maximally effective at 10-100 microM, showing an inverse correlation between concentration and effect in the 100-1000 microM range. The glutamate metabotropic receptor antagonist 2-amino-3-phosphonopropionic acid (AP3), the ionotropic non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the NMDA channel blocker dizolcipine (MK-801) failed to prevent the PI response to ACPD (1000 microM). However, CNQX (100 microM) modified the concentration-response curve of QA reducing the effect of QA 10 microM by approx. 50% and enhancing that of QA 1000 microM by 2-fold. In addition, CNQX (100 microM) together with MK-801 (100 microM) unmasked the ability of L-glutamate (L-GLU) 3000 microM to stimulate PI turnover. The effect of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) on the EAA-induced PI turnover was also studied. AMPA (0.1-1 microM) potentiated the response to submaximal (30 microM) ACPD and (1 microM) QA concentrations. However, higher AMPA concentrations (10 microM) failed to synergize with ACPD 30 microM and, in addition, inhibited the PI turnover maximally stimulated by QA 10 microM. These results further support the presence of the glutamate metabotropic receptor in the human neocortex. In addition, they show the occurrence of a concentration-related dual interaction between AMPA and glutamate metabotropic receptor activation in the IP formation in this brain area.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Adolescent; Adult; Aged; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Calcimycin; Cerebral Cortex; Child; Cycloleucine; Dizocilpine Maleate; Female; Glutamic Acid; Humans; Male; Middle Aged; N-Methylaspartate; Phosphatidylinositols; Quisqualic Acid; Receptors, AMPA; Receptors, Metabotropic Glutamate; Signal Transduction; Tetrodotoxin

This study examined the binding of (S)-[3H]AMPA, the radiolabelled active isomer of AMPA, to rat brain synaptic membranes. Under non-chaotropic conditions specific binding of 10 nM (S)-[3H]AMPA represented 33 +/- 2% of the total; this increased to 74 +/- 1% in the presence of 100 mM KSCN. (S)-[3H]AMPA binding was inhibited by non-NMDA receptor agonists and the antagonists NBQX and CNQX, with the following rank order of potency: NBQX > (S)-AMPA > or = quisqualate > CNQX > L-glutamate > domoate > or = kainate > (R)-AMPA. NMDA, and the metabotropic glutamate receptor agonist (1S,3R)-ACPD, up to 100 microM, did not inhibit (S)-[3H]AMPA binding. A number of willardiine analogues all effectively inhibited (S)-[3H]AMPA binding with the rank order of potency: (S)-5-fluorowillardiine > (S)-5-nitrowillardiine > (S)-5-trifluoromethylwillardiine > (S)-5-bromowillardiine approximately (S)-5-chlorowillardiine > (S)-5-cyanowillardiine > (S)-willardiine > (S)-5-iodowillardiine > (S)-6-methylwillardiine > (S)-5-methylwillardiine. This rank order closely reflects data from equilibrium measurements made, under voltage clamp, on cultured hippocampal neurons. In contrast the respective (R)-enantiomers and the racemate mixtures of (R,S)-3, 5 and 6-isowillardiine were relatively inactive. Similar IC50 values and thus rank orders of potency for the willardiines were observed in the presence of 100 mM KSCN.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Alanine; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Brain; Cycloleucine; Male; Pyrimidinones; Quinoxalines; Radioligand Assay; Rats; Rats, Wistar; Receptors, AMPA; Stereoisomerism; Synaptic Membranes; Tritium; Uracil

Single channel potassium currents activated indirectly via stimulation of metabotropic receptors were recorded in cell-attached patches on hippocampal neurones in slices from neonatal rats. When metabotropic receptors in the area outside of the patch are stimulated by glutamate, quisqualate and trans-ACPD, single channel currents appear with a predominant conductance level of about 20 pS. From the reversal potential and substitution of ionic species, we established that the currents are carried by potassium ions. Using selective blockers of ionotropic receptors in conjunction with specific agonists for the metabotropic receptor, it was ascertained that the channel observed is linked to metabotropic receptors. The currents display submaximal conductance states and fluctuate rapidly between evenly-spaced conductance sublevels.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cycloleucine; Glutamic Acid; Hippocampus; In Vitro Techniques; Neurons; Potassium Channels; Quisqualic Acid; Rats; Rats, Wistar; Receptors, Metabotropic Glutamate; Stimulation, Chemical; Valine

The mobilization of inositol triphosphate ip3 by N-methyl D-aspartate (NMDA) and kainate, two excitatory amino acid EAA receptor agonists, was studied in cultured chick retina cells as a function of culture differentiation. Kainate (EC50 = 30 microM) stimulated from 6 to 9-fold the production of [3H]ip3 between E8C3 (embryonic day 8 plus 3 days in vitro) and E8C13. The kainate response was blocked by CNQX (100 microM) by more than 80% until stage E8C9. MK-801, however, was totally ineffective in preventing the kainate induced ip3 generation. [3H]ip3 production evoked by NMDA was increased 4-fold above basal levels at E8C3. As cultures differentiated, [3H]ip3 production promoted by NMDA decreased to 2.5-fold at E8C6 to 1.6-fold the basal levels in cultures at later stages of differentiation. The removal of Mg2+ from the incubating medium at E8C3 increased the NMDA mediated [3H]ip3 production by 80%. However, at more differentiated stages of the cultures, when cells were not responsive to NMDA, removal of Mg2+ plus the addition of 1 mM glycine did not change the pattern of the response. Although NMDA mediated ip3 production is almost absent in more differentiated cultures, NMDA is able to induce [3H]GABA release in E8C3 and E8C13 cultures with characteristics that reflect typical NMDA receptor activation: it is highly potentiated by the absence of Mg2+ and by the presence of glycine. The NMDA induced production of [3H]ip3 at E8C3 was entirely blocked by MK-801 (100 microM) and APV (100 microM) but not by CNQX.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cells, Cultured; Chick Embryo; Cycloleucine; Dizocilpine Maleate; gamma-Aminobutyric Acid; Inositol 1,4,5-Trisphosphate; Kainic Acid; N-Methylaspartate; Neurotoxins; Receptors, N-Methyl-D-Aspartate; Retina; Time Factors

1. The effects of metabotropic glutamate receptor (mGluR) activation on synaptic inhibition were examined using whole-cell recordings of spontaneous and miniature inhibitory synaptic currents from CA3 pyramidal cells in rat hippocampal slices. 2. The mGluR agonist (1S,3R)trans-1-aminocyclopentane-1,3-dicarboxylic acid (tACPD) increased spontaneous IPSC (spIPSC) frequency by up to 5-fold. At doses above 5 microM the increase was transient (15-45 s) and was followed by a decline to control frequency. In these conditions, elevating external K+ from 2 to 8 mM could still increase spIPSC frequency. 3. Miniature IPSCs (mIPSCs) were recorded in the presence of 1 microM TTX, 5 mM Mg2+ and nominally zero Ca2+. At concentrations above 50 microM, tACPD induced a sustained, reversible reduction in mIPSC frequency by up to 43%. 4. Quisqualate, at doses as low as 50 nM, increased spIPSC frequency, but did not affect mIPSC frequency at concentrations up to 10 microM. 5. The specific mGluR2 and 3 agonist (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV, 3 microM) reduced mIPSC frequency by 40 +/- 4% but did not increase spIPSC frequency. 6. The putative mGluR antagonist L-2-amino-3-phosphonopropionate (L-AP3, 1 mM) blocked the effect of tACPD on mIPSC but not spIPSC frequency. The broad-spectrum antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG, 500 microM) blocked both responses. 7. mGluR activation also had dual effects on IPSCs evoked by focal extracellular stimulation. Application of 5 microM tACPD increased the mean amplitude of evoked IPSCs by 112 +/- 9%, largely by reducing the proportion of response failures. In contrast, IPSC amplitude was reduced to 44 +/- 1% of control values by 3 microM DCG-IV. 8. These results suggest hippocampal inhibitory cells express two distinct mGluR subtypes. One receptor (possibly mGluR1 or 5) is located on somato-dendritic membrane and enhances cell excitability. Another (mGluR2 or 3) is present at inhibitory terminals and reduces the probability of GABA release.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Cycloleucine; gamma-Aminobutyric Acid; Hippocampus; In Vitro Techniques; Kinetics; Neurotoxins; Pyramidal Cells; Quisqualic Acid; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Synapses; Synaptic Transmission; Tetrodotoxin

We have reported previously that transient application of a specific metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD) can induce a slow-onset form of long-term potentiation (LTP) of synaptic transmission in the CA1 region of rat hippocampal slices [Bortolotto Z. A. and Collingridge G. L. (1993) Neuropharmacology 32, 1-9]. Here we have investigated further the mechanisms involved in the induction and expression of ACPD-induced LTP. Unless otherwise stated, field excitatory postsynaptic potentials (EPSPs) were recorded in stratum radiatum in response to low frequency (0.033 Hz stimulation) of the Schaffer collateral-commissural pathway and 10 microM ACPD was added for 20 min to the perfusate. ACPD-induced LTP was still observed following blockade of GABAA receptor-mediated synaptic inhibition using picrotoxin (50 microM) and was not the result of a change in the presynaptic fibre volley. Intracellular recording from area CA1 revealed an increase in the size of the EPSP but no associated change in membrane potential or input resistance. However, ACPD-induced potentiation was never seen when intracellular electrodes contained the Ca(2+)-chelating agent 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; 0.5 M). In area CA3, ACPD elicited a slow-onset LTP of the intracellularly recorded EPSP, evoked by stimulation of associational fibres. In contrast to area CA1, 10 microM ACPD depolarized CA3 neurones. Unlike certain other forms of tetanus- and chemically-induced potentiation, ACPD-induced LTP was not affected by the L-type Ca2+ channel antagonist nimodipine (50 microM). It was, however, prevented by delivering low frequency stimulation (900 shocks at 1 Hz) immediately following termination of the application of ACPD; an effect which was inhibited by the specific N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonopentanoate (AP5; 50 microM). ACPD failed to induce LTP of pharmacologically-isolated NMDA receptor-mediated EPSPs. The induction of ACPD-induced LTP was blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), in a reversible manner. In slices in which area CA3 had been removed ACPD failed to induce LTP when applied alone or together with AMPA. However, a slow-onset form of LTP was induced, in slices lacking area CA3, when a tetanus (100 Hz, 1 sec) was delivered in the presence of ACPD a

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Calcium Channel Blockers; Cycloleucine; Evoked Potentials; Excitatory Amino Acid Agonists; Female; Hippocampus; In Vitro Techniques; Long-Term Potentiation; Nimodipine; Rats; Receptors, N-Methyl-D-Aspartate; Receptors, Presynaptic

Metabotropic glutamate receptors (mGluRs) form a receptor family that consists of diverse receptor subtypes; now, numbering 8--exclusive of splice variants. (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) has been suggested to be a selective agonist for the mGluRs. We have recently reported that, in rat dorsolateral septal nucleus (DLSN) neurones, a 1S,3R-ACPD-preferring inward current (ACPDi) persists in pertussis toxin-treated rats. We now report that this ACPDi-current: (1) persists in DLSN neurones dialyzed with a stable analog of GTP, namely, GTP gamma S; (2) exhibits a negative slope region with inward rectification in its I-V relationship; (3) persists in neurones superfused with tetrodotoxin or low calcium solutions; (4) is dependent upon both sodium and calcium ions; and (5) is independent of a reduction in temperature. Furthermore, pharmacological data suggest that this current may be activated by a unique type of excitatory amino acid (EAA) receptor, i.e. a receptor which prefers "metabotropic" EAA agonists and is insensitive to AP5 or CNQX. Activation by ACPD of inward currents associated with a conductance increase have also been reported at cultured mouse cerebellar Purkinje neurones; in slices of rat hippocampal CA1 neurones and slice cultures of hippocampal CA3 neurones. We suggest that this ACPDi current may play an important role within the CNS in the induction of long-term potentiation and other neurological processes; processes attributed previously to currents associated with NMDA receptor activation.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cycloleucine; Excitatory Amino Acid Antagonists; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); In Vitro Techniques; Male; Neurons; Patch-Clamp Techniques; Pertussis Toxin; Purkinje Cells; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Temperature; Virulence Factors, Bordetella

A grease-gap recording technique which allows the monitoring of presynaptic d. c. potentials without contamination of potentials from postsynaptic elements was used to examine presynaptic actions of glutamate agonists in the CA1 region of rat hippocampus. Presynaptic depolarizations through the activation of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)- and 2-amino-5-phosphonovaleric acid (APV)-sensitive receptors could be induced by applied agonists. In addition, the N-methyl-D-aspartate (NMDA)-induced depolarization was smaller in the presence of extracellular Mg2+ suggesting some similarity to postsynaptic NMDA receptors. The (1S,3R)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD)-induced depolarization was antagonized by L-2-amino-3-phosphonopropionic acid (L-AP3) but was also sensitive to APV+CNQX, creating ambiguity as to the type of receptors involved. These results suggest that the activation of glutamate autoreceptors leads to a presynaptic depolarization.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cycloleucine; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; In Vitro Techniques; Magnesium; Male; Membrane Potentials; N-Methylaspartate; Patch-Clamp Techniques; Potassium Channels; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Receptors, Presynaptic

The purposes of our study were to determine the type of respiratory changes that would occur when either an excitatory amino acid receptor agonist or an uptake inhibitor was administered into the caudal subretrofacial area. This was done by microinjecting either L-glutamate or L-pyrrolidine-2,4-dicarboxylate (L-trans-2,4-PDC) into the caudal subretrofacial area while monitoring tidal volume, respiratory rate, mean arterial blood pressure and heart rate. Bilateral microinjection of 2.5 nmol of L-glutamate into the caudal subretrofacial area produced apnea in eight of eight animals tested, and the duration of apnea was 27 +/- 2 s. To determine the type of L-glutamate receptor responsible for mediating the apneic response, antagonists of the N-methyl-D-aspartate (NMDA) and non-NMDA receptor, namely, 3-[(RS)-carboxypiperazin-4-yl]-propyl-phosphonic acid (CPP), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, were tested. Neither antagonist in doses that blocked NMDA (in the case of CPP) and amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) (in the case of CNQX) blocked apnea elicited by L-glutamate. In addition, kynurenic acid, an antagonist of NMDA and non-NMDA ionotropic receptors, failed to block the effect of L-glutamate. Microinjection of the metabotropic receptor agonist drug, trans-L-1-amino-1,3-cyclopentone-dicarboxylic acid (L-trans-ACPD), into the caudal subretrofacial area failed to have any effect on respiratory activity. Because of the inability to block the effect of L-glutamate in the caudal subretrofacial area, and the lack of effect of L-trans-ACPD, the data suggest that the apneic response produced by L-glutamate is mediated by an as yet undefined receptor. Microinjection of the L-glutamate uptake inhibitor, L-trans-2,4-PDC, was found to produce apnea. Using the dose of 0.5 nmol of L-trans-2,4-PDC, we examined the type of excitatory amino acid receptor that mediated the response. Neither pretreatment with the NMDA receptor antagonist, CPP, nor the non-NMDA receptor antagonist, CNQX, affected L-trans-2,4-PDC-induced apnea. However, combined use of these two antagonists prevented L-trans-2,4-PDC-induced apnea. These data suggest that the effect of synaptically released exitatory amino acid at the caudal subretrofacial area on breathing is apnea, and that this effect is mediated by simultaneous activation of both NMDA and non-NMDA ionotropic receptors.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Apnea; Blood Pressure; Cats; Cycloleucine; Dicarboxylic Acids; Excitatory Amino Acid Antagonists; Female; Glutamic Acid; Heart Rate; Kynurenic Acid; Male; Neurotoxins; Neurotransmitter Uptake Inhibitors; Piperazines; Pyrrolidines; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Respiration; Respiratory Function Tests; Tidal Volume; Trigeminal Caudal Nucleus

The aim of the described experiments was to use immunohistochemistry to visualize the release of GABA from specific retinal amacrine cells following ischaemia and to establish the involvement of defined glutamatergic receptors. In initial experiments, rabbit retinas were exposed in vitro to excitatory amino acid agonists alone or in combination with a putative antagonist, or in physiological solution lacking oxygen and glucose, or in solution containing potassium cyanide for 45 min at 37 degrees C. The nature of the GABA immunoreactivity was then examined by immunohistochemistry. In other in vitro experiments, retinas were first allowed to accumulate exogenous serotonin before exposing the tissues to the combinations as described. These tissues were then processed immunohistochemically for the localization of serotonin. In yet other experiments, the intraocular pressure of a rabbit's eye was raised to about 110 mmHg for 60 min and a reperfusion time of 45 min allowed before dissecting the retina and processing for the localization of GABA immunoreactivity. The other eye served as a control. Of the excitatory amino acid agonists tested, only N-methyl-D-aspartate, kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid caused a change in the GABA immunoreactivity. The N-methyl-D-aspartate effect was specifically antagonized by dizocilpine maleate, dextromethorphan and memantine, and was characterized by a reduction in the number of GABA-immunoreactive perikarya. The GABA "staining" in the inner plexiform layer also appeared as four clear bands. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- and kainate-induced effects were both antagonized by 6-cyano-2,3-dihydroxy-7-nitroquinoxaline-2,3-dione and partially by kynurenic acid at the concentrations used. Here, the amount of GABA-positive perikarya was greatly reduced and three immunoreactive bands appeared in the inner plexiform layer. However, for low concentrations of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid four GABA-immunoreactive bands could be identified in the inner plexiform layer. The normal GABA immunoreactivity of the inner plexiform layer also appeared to be in defined bands in retinas which received an ischaemic insult either by reducing the availability of glucose and oxygen, exposing the tissue to potassium cyanide or raising the intraocular pressure of an eye. In these cases the number of GABA-positive perikarya was also reduced. Only alpha-amino-3-hy

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Aminobutyrates; Animals; Cycloleucine; Dextromethorphan; Eye Proteins; gamma-Aminobutyric Acid; Glucose; Glutamates; Glutamic Acid; Intraocular Pressure; Ischemia; Kainic Acid; Kynurenic Acid; Memantine; N-Methylaspartate; Oxygen; Potassium Cyanide; Quinoxalines; Rabbits; Receptors, AMPA; Receptors, Glutamate; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Retina; Retinal Vessels; Serotonin

To investigate the electrophysiological effects of the stimulation of the metabotropic excitatory amino acid receptors, we applied trans-1-amino-cyclopentane-1,3-dicarboxylate, an agonist of this type of receptors, on presumed rat dopamine cells intracellularly recorded in vitro. Trans-1-amino-cyclopentane-1,3-dicarboxylate (3-30 microM, t-ACPD) caused a sustained increase of the spontaneous firing rate and a depolarization. When the membrane potential was held at about the resting level (-50, -60 mV), by the single-electrode voltage-clamp technique, t-ACPD induced an inward current. In 57% of the tested cells the inward current was associated with a decrease of the apparent input conductance. In the remaining cells no obvious changes in membrane conductance were observed. The active form of t-ACPD, (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylate [3-50 microM, (1S,3R)-ACPD] also produced a reversible inward current on the dopaminergic cells and this was antagonized by (S)-4-carboxy-3-hydroxyphenylglycine (300 microM), a selective antagonist of the (1S,3R)-ACPD-induced depolarization on central neurons. The (1S,3R)-ACPD-induced inward current was not antagonized by L-2-amino-3-phosphonopropionic acid (100 microM), an antagonist of the t-ACPD-induced activation of inositide synthesis. 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM), an alfa-amino-3-hydroxy-5- methyl-isoxazole propionic acid/kainate antagonist, DL-amino-5-phosphonopentanoic acid (30 microM), an N-methyl-D-aspartate antagonist, and scopolamine (10 microM), a muscarinic antagonist, did not significantly affect the actions of t-ACPD. A block of synaptic transmission obtained by applying tetrodotoxin failed to prevent the action of t-ACPD.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Alanine; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Cycloleucine; Dopamine; Electrophysiology; Female; Glycine; Male; Neurons; Potassium; Quinoxalines; Rats; Rats, Wistar; Receptors, Metabotropic Glutamate; Stimulation, Chemical; Tetrodotoxin

1. The effects of agonists of on the evoked N-wave complex in slices of mouse have been studied: most experiments were carried out using slices perfused with Mg(2+)-free solution to which 10 microM of either 6,7-dinitroquinoxaline-2,3-dione or 6-cyano-7-nitroquinoxaline-2,3-dione was applied. 2. Following agonist washout, a slowly developing, long lasting potentiation of the complex occurred which was confined to the mediated component of the potential. The relative agonist potencies were 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD, 5-250 microM) = quisqualate (5-50 microM) > 1RS,3RS-cis-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD, 25-1000 microM) > L-glutamate (0.25-2.5 mM); NMDA, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and L-aspartate were inactive. 3. Potentiation of the NMDA receptor-mediated component by 1S,3R-ACPD (0.1 mM) was non-competitively antagonised by S-(+)- but not R-(-)-2-amino-3-phosphonopropionate (AP3, 0.125 mM), equally by D-(-) and L-(+)-2-amino-4-phosphonobutyrate (0.25 mM) and also by the protein kinase C inhibitors sphingosine, (25 microM), sangivamycin (25 microM) and 5-(isoquinolinylsulphonyl)-3-methylpiperazine (50 microM). 4. In a series of input-output experiments, 1S,3R-ACPD (0.1 mM) reversibly reduced the latency to peak of the NMDA receptor-mediated component at submaximal stimulus intensities, an effect blocked by S-(+)-AP3 (0.125 mM). On agonist washout, there was an increase in the area of the receptor-mediated component over all stimulus intensities, an effect blocked by the inhibitors of protein kinase C and by S-(+)-AP3 (0.125mM). 4-beta-Phorbol-12,13-diacetate (2.5 muM) also potentiated the component, an action inhibited by protein kinase C inhibitors but not by S-(+)-AP3. IS,3R-ACPD (0.1mM) had no significant effect on postsynaptic responses evoked by NMDA, AMPA and kainate, but significantly reversed a partial antagonism of NMDA responses produced by 7-chlorokynurenate (2.5 muM). The K+evoked release of glycine was selectively and significantly increased in the presence 0.1mM 1S,3R-ACPD(antagonized by 0.125 mM S-(+)-AP#) whereas following agonist washout, release of glycine fell to control levels but there was a significant increase in release of aspartate(antagonized by 25 muM sangivamycin and 0.125 muM S-(+)-AP3). It is concluded that mediate (i) a reduction in the latency of the mediated component of potentials by a mechanism that is independent of protein kinase C but wh

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Amino Acids; Animals; Cycloleucine; Ibotenic Acid; In Vitro Techniques; Male; Mice; Neurotransmitter Agents; Olfactory Bulb; Potassium; Quinoxalines; Receptors, Glutamate; Synapses; Synaptic Transmission

1. Inhibitory cell activity and inhibitory postsynaptic potentials impinging spontaneously on pyramidal cells were recorded in the CA3 region of hippocampal slices from guinea-pig. We compared the effects on synaptic inhibition, of tetanic stimuli in the presence of antagonists of ionotropic excitatory amino acid receptors, and of application of agonists of metabotropic glutamate receptors. 2. Tetanic stimulation of afferent fibres caused an increase, of duration 0.5-2.5 min, in the frequency of spontaneous Cl(-)-mediated IPSPs. Inhibitory cell firing increased due to a depolarization and a reduction of after-hyperpolarizing potentials. 3. Tetanic stimulation induced, in some experiments, rhythmic bursts of IPSPs and transformed the firing pattern of some inhibitory cells from a discharge of single action potentials to rhythmic bursts of three to five action potentials. 4. Application of the metabotropic glutamate receptor agonist, trans-1-amino-cyclopentane-1,3-dicarboxylic acid (tACPD), at concentrations from 3-10 microM increased the frequency of spontaneous IPSPs. In some slices tACPD caused IPSPs to occur rhythmically. IPSP frequency did not continue to increase with concentrations of tACPD above 20 microM. 5. tACPD depolarized inhibitory cells and reduced after-hyperpolarizing potentials. High concentrations (50-100 microM) of tACPD excited inhibitory cells to potentials at which they no longer discharged fast action potentials. 6. Both tetanic stimulation and tACPD led to the appearance in pyramidal cell pairs of simultaneous IPSPs which were not previously observed, suggesting that the same group of inhibitory cells was excited in both cases. 7. Low concentrations of tACPD (3-10 microM) enhanced IPSP responses to tetanic stimuli, while the effects of tetanic stimuli were occluded in the presence of high concentrations (20-30 microM) of tACPD. 8. We suggest that activation of metabotropic glutamate receptors during tetanic stimulation leads to a post-tetanic excitation of inhibitory cells that mediate Cl(-)-dependent IPSPs.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Cycloleucine; Electric Stimulation; Guinea Pigs; Hippocampus; In Vitro Techniques; Membrane Potentials; Neurons, Afferent; Picrotoxin; Potassium Chloride; Pyramidal Cells; Quinoxalines; Receptors, Metabotropic Glutamate; Synapses

The effects of a novel antagonist of metabotropic glutamate receptors were investigated in CA3 pyramidal cells in hippocampal slice cultures of the rat. Earlier experiments showed that selective activation of metabotropic glutamate receptors with low concentrations of an agonist, 1S, 3R-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD), induced an inward current associated with a decrease in membrane conductance and inhibition of the slow calcium-dependent potassium current. These responses were strongly and reversibly reduced by the antagonist, (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG, 0.5-1 mM). In the presence of antagonists of ionotropic glutamate receptors, stimulation of the afferent mossy fibres evoked postsynaptic responses in CA3 pyramidal cells which paralleled those observed with exogenously applied metabotropic glutamate receptor agonists, i.e. a slow inward current and a reduction of calcium-dependent potassium current. Both responses were greatly reduced by bath-applied MCPG (1 mM). These results show that MCPG acts as an effective antagonist at metabotropic glutamate receptors coupled to potassium conductances in the hippocampus. Furthermore, they confirm that glutamate release from presynaptic terminals can modulate postsynaptic properties by activation of metabotropic glutamate receptors.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Benzoates; Bicuculline; Cycloleucine; Evoked Potentials; Glycine; Hippocampus; In Vitro Techniques; Nerve Fibers; Neurotoxins; Pyramidal Cells; Quinoxalines; Rats; Receptors, Glutamate; Synapses

The role of quisqualate (QUIS) metabotropic receptors in the synaptic transmission in the striatum was investigated using the cortico-striatal slice preparation. Low concentrations (1-30 microM) of trans-1-amino-cyclopentyl-1,3- dicarboxylic acid (t-ACPD), a selective agonist of QUIS metabotropic receptors, decreased glutamate-mediated synaptic potentials (EPSPs) evoked in the striatum by the stimulation of cortico-striatal fibers. This agonist decreased also GABA-mediated depolarizing synaptic potentials evoked by intrastriatal stimulation in the presence of 6-cyano-7-nitro-quinoxaline-2,3- dione (CNQX); this effect was less potent than the action of t-ACPD on glutamate-mediated potentials. Low concentrations of t-ACPD did not affect the intrinsic membrane properties of striatal neurons and their postsynaptic responses to exogenous glutamate and GABA. Higher concentrations (50-100 microM) to t-ACPD caused membrane depolarizations and inward currents in several neurons. Our data suggest that low concentrations of t-ACPD selectively reduce synaptic transmission while higher concentrations of this agonist may cause a direct excitatory action on striatal neurons.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Corpus Striatum; Cycloleucine; gamma-Aminobutyric Acid; Glutamates; Glutamic Acid; In Vitro Techniques; Neurotoxins; Quinoxalines; Rats; Receptors, AMPA; Receptors, Neurotransmitter; Synapses

We have previously reported that, in the rat dorsolateral septal nucleus (DLSN), metabotropic glutamate receptor (met-GluR) agonists evoked a slow depolarization accompanied by an increase in membrane conductance and burst firing. We have speculated that the burst firing elicited by met-GluR agonists may be due to activation or enhancement of a non-specific cation current, which exists in some DLSN neurons. Now we report that a slow afterdepolarization (sADP) mediated by a non-specific cation current was potentiated by both 1S,3R-ACPD and quisqualate. In addition, met-GluR agonists unmask a sADP in DLSN neurons which did not show a sADP under control conditions. Our data suggest that a non-specific cation current can be potentiated by activation of the met-GluR.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cycloleucine; Evoked Potentials; Glutamates; Ibotenic Acid; In Vitro Techniques; Male; Membrane Potentials; Neurons; Prosencephalon; Quinoxalines; Quisqualic Acid; Rats; Rats, Wistar; Receptors, Glutamate

The expression of a receptor subtype for one type of excitatory amino acid agonist, t-ACPD, was examined in developing Purkinje cells of cerebellar slices. The t-ACPD-induced responses were compared with those induced by QA in current response, single cell Ca2+ imaging and changes in the miniature currents in the same preparation. It was found that t-ACPD induced a single component of inward current, and an increase in the frequency of miniature currents associated with the presence of external Ca2+, but was ineffective at mobilizing intracellular Ca2+ even in the presence of external Ca2+. The present study suggests the expression of at least two types of metabotropic receptors in the Purkinje cell region, one of which, expressed in the Purkinje cell dendrites, is highly sensitive to QA, but relatively insensitive to t-ACPD, and the other of which is a t-ACPD-sensitive receptor expressed on the presynaptic terminals of the neurons making synapses onto Purkinje cells.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calcium; Cerebellum; Cycloleucine; Dendrites; Evoked Potentials; Fluorescent Dyes; Heterocyclic Compounds, 3-Ring; In Vitro Techniques; Membrane Potentials; Neurotoxins; Purkinje Cells; Quinoxalines; Quisqualic Acid; Rats; Receptors, Metabotropic Glutamate; Receptors, Neurotransmitter; Synapses

An amino acid, 3,5-dihydroxyphenylglycine (DHPG) induced current responses in Xenopus oocytes expressing a metabotropic glutamate receptor clone mGluR1. Apparent EC50 of DHPG for mGluR1 was slightly lower than that of (+-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD). DHPG responses were partially inhibited by 2-amino-3-phosphonopropionic acid (AP-3). DHPG had no effect on ionotropic glutamate receptors whose expression was induced in the oocytes following injection of poly(A)+ mRNA of rat brains. In hippocampal slices, DHPG produced slow excitation of pyramidal cells, resulting from a depression of Ca(2+)-dependent K+ current and a voltage-dependent K+ current. These results indicate that DHPG is a specific and potent agonist of mGluRs.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Alanine; Animals; Cycloleucine; Egtazic Acid; Glycine; Hippocampus; In Vitro Techniques; Male; Oocytes; Potassium Channels; Quinoxalines; Rats; Rats, Wistar; Receptors, Glutamate; Resorcinols; RNA, Messenger; Xenopus

Exposure of hippocampal and cortical slices to quisqualate induces a 30- to 100-fold decrease in the half-maximal concentration of L-2-amino-4-phosphonobutanoic acid (L-AP4) required to depolarize neurons. This sensitization persists for hours and has previously been shown to be induced only by quisqualate, via the interaction of quisqualate with a 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)-insensitive site. Here we tested the hypothesis that quisqualate may act on phosphoinositide (PI) metabolism to enhance the response to L-AP4, and found that sensitization to L-AP4 was induced by trans-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD), an agonist selective for PI-coupled excitatory amino acid receptors, and by carbachol and norepinephrine, agonists at other PI-coupled receptors. However, these compounds produced only a 2- to 5-fold sensitization to L-AP4, that was of shorter duration than that induced by quisqualate. These results suggest that sensitization to L-AP4 may be induced, at least in part, via PI-coupled receptors, and that the sensitivity of neurons to an excitatory amino acid agonist may be modified by heteroreceptor activation of the PI second messenger system.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Aminobutyrates; Animals; Carbachol; Cerebral Cortex; Cycloleucine; Hippocampus; Ibotenic Acid; In Vitro Techniques; Male; Neuromuscular Depolarizing Agents; Neurons; Norepinephrine; Quinoxalines; Quisqualic Acid; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear

Stimulation of the quisqualate (QA) subtype of glutamate receptor increased the expression of phosphate activated glutaminase (needed for neurotransmitter glutamate synthesis) and the ability to release neurotransmitter glutamate in cultures of glutamatergic cerebellar granule neurons. In contrast, QA had no significant effects on the lactate dehydrogenase activity and amount of protein. The QA-mediated elevation in glutaminase activity was blocked by the ionotropic QA receptor antagonist CNQX and mimicked by the ionotropic QA receptor agonist AMPA, but not by the metatropic QA receptor agonist t-ACPD. The increase in Ca2+ influx essentially through activation of L-type channels, and not the mobilization of internal Ca2+ stores, was responsible for these QA receptor-mediated long-term changes in cerebellar granule neurons.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Calcium; Calcium Channels; Cerebellar Cortex; Cycloleucine; Diltiazem; Glutaminase; Ibotenic Acid; Ion Channel Gating; L-Lactate Dehydrogenase; Neuronal Plasticity; Neurons; Nifedipine; Quinoxalines; Rats; Rats, Inbred Strains; Receptors, AMPA; Receptors, Neurotransmitter

beta-N-methylamino-L-alanine (L-BMAA) is an excitotoxin whose neurodegenerative effects are associated with its agonist properties at the N-methyl-D-aspartate (NMDA) receptor. We measured the effects of L-BMAA on inositol phosphate (InsP) formation in primary cultured striatal neurons. This culture is almost devoid of glial cells and the pharmacology of glutamate receptors is well-defined. This allowed us to show that L-BMAA induced InsP formation via a direct action at the glutamate metabotropic (Qp) receptors coupled to InsP formation. We demonstrated that L-BMAA is a full-agonist of the Qp receptor, but with a low potency. Therefore, the neurotoxic properties of L-BMAA might implicate the activation of the Qp receptor in association with the NMDA receptor.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Amino Acids, Diamino; Animals; Carbachol; Cells, Cultured; Corpus Striatum; Cyanobacteria Toxins; Cycloleucine; Dizocilpine Maleate; Embryo, Mammalian; Glutamates; Inositol; Inositol Phosphates; Kinetics; Mice; Neurons; Neurotoxins; Phorbol 12,13-Dibutyrate; Quinoxalines; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter

Activation of metabotropic quisqualate receptors by trans-ACPD (trans-1-aminocyclopentane-1,3-dicarboxylic acid) caused a reduction in the amplitude of the synaptic response elicited by stimulation of the Schaffer collateral projection and recorded intracellularly from area CA1 in slices of rat hippocampus. Pharmacological agents were used to isolate components of the response mediated by N-methyl-D-aspartate (NMDA) receptors, non-NMDA receptors, and gamma-aminobutyric acid (GABA) receptors. Each of these components was reduced during the trans-ACPD application. These results indicate that one subtype of glutamate receptor may be able to decrease the synaptic efficacy of other subtypes and may provide an important means for balancing the synaptic enhancement processes often studied in the hippocampus.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cycloleucine; Electrophysiology; Hippocampus; In Vitro Techniques; Male; Quinoxalines; Rats; Receptors, Amino Acid; Receptors, Cell Surface; Synapses; Synaptic Transmission

The complex modulation of cytoplasmic free calcium concentration ([Ca2+]c) in primary cultures of cerebellar granule cells in response to glutamate receptor agonists has been the subject of several contradictory reports. We here show that 3 components of the [Ca2+]c response can be distinguished: (1) Ca2+ entry through voltage-dependent Ca2+ channels, following KCl- or receptor-evoked depolarization, (2) Ca2+ entry through NMDA receptor channels, and (3) liberation of internal Ca2+ via a metabolotropic receptor. Depolarization with KCl induced a transient [Ca2+]c response (subject to voltage inactivation) decaying to a sustained plateau (largely inhibited by nifedipine). The NMDA response was potentiated by glycine, totally inhibited by (+)5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801), and blocked by Mg2+ in a voltage-sensitive manner. Polarized cells displayed small responses to quisqualate (QA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA). Depolarization enhanced a transient response to QA, but not to AMPA. Trans-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD), a selective agonist for the metabolotropic glutamate receptor, caused a transient elevation of [Ca2+]c, which was blocked by prior exposure to QA but not AMPA. The prolonged [Ca2+]c response to kainate (KA) can be resolved into 2 major components: an indirect NMDA receptor-mediated response due to released glutamate and a nifedipine-sensitive component consistent with depolarization-mediated entry via Ca2+ channels. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), QA at greater than 10 microM, and AMPA (but not trans-ACPD) reversed the KA response, consistent with an inactivation of the KA receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Calcium; Calcium Channels; Cell Membrane; Cells, Cultured; Cerebellum; Cycloleucine; Dizocilpine Maleate; Ibotenic Acid; Kainic Acid; Magnesium; Membrane Potentials; Neurons; Quinoxalines; Quisqualic Acid; Rats; Rats, Inbred Strains; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Cycloleucine; Electric Stimulation; Electrophysiology; Hippocampus; Ibotenic Acid; In Vitro Techniques; Male; Neurons; Oxadiazoles; Quinoxalines; Quisqualic Acid; Rats; Receptors, Glutamate; Receptors, Muscarinic; Receptors, Neurotransmitter