Moreover, in confirmation of a previous report, we find that disruption of the PSD-95 gene results in a greater magnitude of LTP. The lack of non-NMDA receptor-mediated fast component was obvious at P14, which coincides with the onset of ataxia in the stg mutant mouse (Qiao et al., 1996). Electrophysiological Imaging of Functional Architecture in the Cortical Middle Temporal Visual Area of, The Role of the Hyperpolarization-Activated Cationic Current, Developmental Increase in Vesicular Glutamate Content Does Not Cause Saturation of AMPA Receptors at the Calyx of Held Synapse, Visit Society for Neuroscience on Facebook, Follow Society for Neuroscience on Twitter, Follow Society for Neuroscience on LinkedIn, Visit Society for Neuroscience on Youtube, Impairment of AMPA Receptor Function in Cerebellar Granule Cells of Ataxic Mutant Mouse Stargazer. (A) Current traces showing NMDAR-mediated synaptic currents recorded in 10 μM glycine, 20 μM NBQX, and 0.1 mM Mg2+at −60 mV. These results suggest that the defect in MF to GC synaptic transmission is a major factor that causes the cerebellar dysfunction in the stg mutant mouse. These synapses are referred to as silent because NMDARs are nonfunctional at rest. Levy, Veeravan Mahadomrongkul, Tomoaki Shirao, Chiye Aoki , Patricio T. Huerta 3C; R2 = 0.04, n = 20). DOI: https://doi.org/10.1523/JNEUROSCI.19-14-06027.1999. This study was designed to investigate the expression and functional role of Ca2+-permeable AMPA receptors in developing chicken spinal motoneurons. As determined by 2P-uncaging on synapses from young (P7–P8) rat, we found that small, filopodia-like spines appeared to be largely devoid of AMPARs (Fig. 18), although they expressed NMDAR-mediated dependent current (Fig. We thus hypothesized that the silent synapses observed in KO mice would predominantly be located on smaller, less developed, filopodia-like spines. In these conditions, both AMPA and NMDARs are activated by synaptically released glutamate, and their respective contribution to the eEPSC was determined pharmacologically by administration of the NMDAR antagonist DL-AP5 (100 μM; Fig. To further examine at the translational level, we compared the size and amount of GluRα4 in the cerebellum. Heterozygous males (+/stg) and homozygous females (stg/stg) were mated to produce stg mutant mice. C, D,I–V relationships of MF–EPSCs from wild-type (C) and BDNF knockout (D) mice measured at the peak (○) and at 50 msec after the stimulus (●). Each trace is single-sweep record, and several traces are superimposed for each record. This developmentally regulated increase in mEPSC frequency was largely abolished in KO mice. Because we have not found any electrophysiological difference so far between +/+ and +/stg, nonmutant littermates including both genotypes were used in electrophysiological analyses as wild-type controls. AMPA receptors get 'pickled'. This is the second of five reports to emerge from the evaluation of the Markey Trust. An essential text, this is a fully updated second edition of a classic, now in two volumes. It provides rapid access to information on molecular pharmacology for research scientists, clinicians and advanced students. Lack of non-NMDA receptor-mediated fast component in MF–GC excitatory synaptic transmission in the stgmutant mice. Provides an authoritative summary of current knowledge of the biological basis of substance use behaviours, including their relationship with environmental factors. In parasagittal cerebellar slices prepared from wild-type and stg mutant mice, GCs were recorded in the whole-cell configuration (Edwards et al., 1989; Farrant et al., 1994; Ebradlidze et al., 1996; Takahashi et al., 1996). Regional diversity and developmental dynamics of the AMPA-receptor proteome in the . However, few studies to date have investigated the role of a key reactive oxygen species generator, NADPH oxidase, in mediating the signaling events leading to alterations in NMDA and AMPA receptor function following ischemia/reperfusion. The mutated subunit stargazin increases steady-state inactivation of the α1 Ca2+ channels in vitro, implying inappropriate Ca2+ entry through modified Ca2+ channels (Letts et al., 1998). BDNF and NT-4/5 enhance glutamatergic synaptic transmission in cultured hippocampal neurones. In addition, these silent synapses found in KO mice were observed on morphologically mature spines, suggesting that PSD-95 plays a distinct role in controlling glutamatergic synapse function and spine morphology. Thus, it is assumed that the defect of AMPA receptor function occurs in the post-translational process. The iGluRs are classified into four subfamilies distinguishable by function, sequence identity, and pharmacological properties: The α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors GluA1 to GluA4 are the main mediators of fast synaptic transmission, whereas the N-methyl-D-aspartate (NMDA) receptors (GluN1, GluN2A through . contributed new reagents/analytic tools; J.-C.B., D.-T.L., and M.-G.K. analyzed data; and J.-C.B. The blot was immunoreacted with anti-GluRα4 antibody at 1 μg/ml and visualized by chemiluminescence (ECL detection system, Amersham, Tokyo, Japan). (E) Traces depicting 2P-EPSCs at −70 mV and +40 mV, elicited from the spine shown, are shown during baseline, after bath administration of NBQX (20 μM) and after the subsequent administration of DL-APV (100 μM). Epub 2007/06/22. Long-lasting neurotrophin-induced enhancement of synaptic transmission in the adult hippocampus. In addition, the developmental profile of this defect is broadly consistent with the known expression profile of PSD-95, which begins to be highly expressed in the hippocampus at around P10 (14). Intriguingly, previous work on a PSD-95 KO mouse, reported no apparent changes in either AMPAR or NMDAR function (11). By contrast, we detected normal AMPA receptor-mediated responses at hippocampal synapses in thestg mutant mouse. function. Proper targeting was confirmed by Southern blot analysis of genomic DNA using the probe depicted in Fig. To isolate NMDA receptor-mediated components, MF–EPSCs were recorded at a holding potential of +40 mV in the presence of a non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX, 10 μm). We raised polyclonal rabbit IgG against the C terminus of the mouse GluRα4, which is low in homology with other mouse AMPA receptor subunits (Sakimura et al., 1990) (GenBank Accession No. We do not capture any email address. We observed no differences in the amplitude of mEPSCs between WT and KO mice at all ages tested (Fig. 4A). Synapse-specific regulation of AMPA receptor function by PSD-95 Jean-Claude Be'ique, Da-Ting Lin, Myoung-Goo Kang, Hiro Aizawa, Kogo Takamiya, and Richard L. Huganir* Department of Neuroscience, Johns Hopkins University School of Medicine and Howard Hughes Medical Institute, 725 North Wolfe Street, Baltimore, MD 21205 AMPAR subunits have large extracellular N-termini, three full transmembrane domains, and a cytoplasmic re-entrant loop, which forms the lining of the channel pore and, in GluA2, contains the RNA editing site that determines calcium permeability. In WT mice, we found that spine volume was not correlated with AMPA/NMDA ratios (Fig. These signals are conveyed through GCs and their axons, parallel fibers (PFs), to various neurons in the cerebellar cortex, including Purkinje cells (PCs), basket cells, stellate cells, and Golgi cells. C, D, Instantaneous I–Vrelationships of the NMDA (100 μm)-induced current evoked in GCs from wild-type (C) and stgmutant (D) mice. Those of the currents at 100 msec at +45 mV were 6.7 + 5.0 pA and 11.0 ± 11.8 pA (mean ± SD) for the wild-type (n = 20) and thestg mutant (n = 22) mice, respectively. Importantly, AMPA receptors are the principal glutamate receptors that mediate fast excitatory neurotransmission. Under deep pentobarbital anesthesia, adult mouse brains were excised from the skull and immediately frozen in powdered dry ice. Correspondence should be addressed to Masanobu Kano, Department of Physiology, Kanazawa University School of Medicine, 13-1 Takara-machi, Kanazawa 920-8640, Japan. A, B, Instantaneous I–Vrelationships of the AMPA (10 μm)-induced current evoked in GCs from wild-type (A) and stgmutant (B) mice. For more details, see Supporting Methods. Commun. In addition to these cerebellar phenotypes, the stg mutant mouse displays near-total reduction of brain-derived neurotrophic factor (BDNF) mRNA expression in the GC layer of the cerebellum, despite normal BDNF expression in other brain regions, including the hippocampus (Qiao et al., 1996). AB022913) was introduced into pGEX-4T-2 plasmid vector (Pharmacia Biotech AB, Uppsala, Sweden) to obtain glutathioneS-transferase (GST) fusion protein. They have modular structures, and each part has a specific task. PSD-95, a member of the membrane-associated guanylate kinase (MAGUK) superfamily of proteins, is a core component of the PSD and is thought to be important in the control of excitatory synapse function (2, 3). Based on the 35 Angstrom density map we published in 2005 and 2006 . Moreover, the amplitude ratio of EPSC(+45) to EPSC(−65) in the stg mutant mouse was not different from that in the wild-type mouse (Fig. We therefore systematically probed the function of individual synapses (i.e., determined whether they are functional or silent) by locally and transiently “uncaging” glutamate by laser photolysis of a glutamate analog, MNI-caged-l-glutamate (MNI-GLU). These results were obtained in age-matched littermates (P16–P20). Ionic currents were recorded with an Axopatch-1D patch-clamp amplifier (Axon Instruments). For the frequency of events: P8–P10, P = 0.3; n = 11 for WT and n = 4 for KO mice; P14–P20, P < 0.05; n = 10 for WT and n = 16 for KO mice; P21–P25, P < 0.05; n = 10 for WT and n = 8 for KO mice. The portion at the top recognizes and binds to glutamate (and similar neurotransmitters), and the portion at the bottom forms an ion channel through the membrane. It has been reported previously that the stgmutant mouse displays a selective and near-total reduction of BDNF mRNA expression in the cerebellar GC layer (Qiao et al., 1996) and that TrkB receptor-mediated tyrosine phosphorylation is downregulated (Qiao et al., 1998). 2F depicts two such spines from which we recorded 2P-EPSCs only when the cell was voltage-clamped at +40 mV. A, B, EPSCs elicited by MF stimulation in GCs from the wild-type (A) and stgmutant (B) mice at holding potentials of +40 mV (top panels) and −70 mV (bottom panels). and by Special Coordination Funds for Promoting Science and Technology from Science and Technology Agency (M.K., M.W.). Vollmar W, Gloger J, Berger E, Kortenbruck G, 2003; 40:763-74. L-glutamate is the major excitatory neurotransmitter in the mammalian central nervous system and acts on both ionotropic and metabotropic receptors. 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To distinguish between these possibilities, we carried out paired-pulse ratio analysis, a measure of presynaptic neurotransmitter release probability, and found no difference between WT and KO mice (Fig. X57497), 5′-GTGGCAAAGAATGCACAGAATATTAACCCATCTTCCTCGCAGAAT-3′ of the mouse GluRα2 subunit cDNA (nucleotide residues 2479–2523; GenBank Accession No. A reduction in the AMPA/NMDA ratio could reflect a reduction in the number (or function) of AMPARs, an increase in the number (or function) of NMDAR, or a combination of both. X57498), 5′-CTCACAAAGAACACCCAAAACTTTAAGCCTGCTCCTGCCACCAAC-3′ of the mouse GluRα3 subunit cDNA (nucleotide residues 2491–2535; GenBank Accession No. The MF–EPSC amplitude was much smaller at a holding potential of −70 mV than at +40 mV (Fig.1B). This broad range of AMPA/NMDA ratios for individual spines was also found in neurons from age-matched KO mice. In principle, the reduction in synaptic AMPAR function in KO mice could be caused by a decrease in synaptic expression of AMPARs across all synapses. Second, the distribution of AMPA/NMDA ratios obtained from 2P-uncaging onto individual spines revealed that a subset of spines in KO mice displayed equally high ratios to those obtained from spines in WT mice. PSD-95 is a major protein found in virtually all mature excitatory glutamatergic synapses in the brain. Studies in heterologous cells have shown that NMDA receptors containing NR2B-D subunits exhibit slower deactivation than those containing NR2A (19), raising the possibility that the longer decay kinetics observed in KO mice may reflect synaptic NMDAR of different subunit composition. The intensity profile of the spine is depicted by the blue trace. Regulation of synaptic responses to high-frequency stimulation and LTP by neurotrophins in the hippocampus. But uncontrolled environments could also potentially lead to faulty data and flawed conclusions. A) The membrane topology of an AMPA receptor subunit (AMPAR). However, we observed a small but significant increase in spine length in the KO mice (see Fig. Nat. Thus, it is clear that the loss of AMPA receptor function is not a common feature to excitatory synapses in thestg mutant brain but occurs preferentially at the MF–GC synapse. We recorded eEPSCs while clamping the cell at +40 mV. View all available purchase options and get full access to this article. Because the Mg2+ block of NMDA receptor channels is significant near the resting membrane potential (approximately −60 mV) (Mayer et al., 1984; Nowak et al., 1984;D’Angelo et al., 1994), usual excitatory synaptic transmission at low frequency is considered to be mediated mainly by AMPA receptors (D’Angelo et al., 1995). To examine whether the reduced non-NMDA receptor-mediated component of MF–EPSCs in the stg mutant mouse is caused by the defect in postsynaptic receptor function, we directly measured the whole-cell currents through AMPA receptors and NMDA receptors in GCs (Tempia et al., 1996). Thus, postsynaptic AMPA receptor function was selectively impaired instg mutant GCs, although the transcription of four AMPA receptor subunit genes in the stg GC was comparable to the wild-type GC. The current–voltage (I–V) relationship for the fast component was linear, whereas that for the slow component measured at 50 msec after the stimulus exhibited a characteristic outward rectification (Fig. Each I–V curve is an average of data from 9–12 different GCs. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Cerebellar circuits and synaptic mechanisms involved in classical eyeblink conditioning. The mean density of the band from the stg mutant cerebella (n = 5) was 93% of that from the wild-type cerebella (n = 5), showing no significant difference. From the evidence, we rather speculate that the lack of AMPA receptor function may lead to the reduced BDNF mRNA expression in GCs. We found that the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)subtype of glutamate receptor (AMPAR)-mediated synaptic transmission was reduced in these mice. This detailed volume explores key technologies that are used currently to investigate iGluR structure, function and physiology. Electrophysiology. and R.L.H. 4). Signals were filtered at 3 kHz and digitized at 20 kHz. Fresh frozen sections (20 μm in thickness) were prepared by cryostat and mounted on glass slides precoated with 3-aminopropyltriethoxisilane or poly-l-lysine (Sigma, St. Louis, MO). When the holding potential was changed to +45 mV, the slow EPSC component became larger (Fig.4A), and this was blocked by an NMDA-blocker,dl-2-amino-5-phosphonopentanoate (AP-5, 50 μm) (data not shown). In the hippocampal neurons and in the HEK293 cells transfected with different combinations of NMDA receptors, glycine preferentially acts on GIuN2A-containing NMDA receptors (GIuN2ARs), but not GIuN2B-containing NMDA receptors (GIuN2BRs), to . The author is at the Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA. Two-photon (2P) uncaging of MNI-glutamate onto individual spines suggested that the decrease in AMPAR function in the PSD-95 KO mouse stems from an increase in the proportion of “silent” synapses i.e., synapses containing N-methyl-d-aspartate (NMDA) receptors (NMDARs) but no AMPARs. Records were taken in the presence of tetrodotoxin (0.5 μm) and strychnine (1 μm). Bicuculline (10 μm) was always present in the saline to block spontaneous IPSCs. Impairment of AMPA Receptor Function in Cerebellar Granule Cells of Ataxic Mutant Mouse. In mediating fast synaptic communication in the brain, AMPA receptors require TARP auxiliary proteins. (1951). After biosynthesis, AMPA receptor subunits undergo phosphorylation and glycosylation (Kawamoto et al., 1995; Roche et al., 1996). Together, these results outline a general defect in glutamatergic transmission in mice lacking PSD-95. Although the results outlined above show that AMPA/NMDA ratios were not correlated to spine volume, it remains possible that there was a generalized reduction in spine volume in KO mice. Here, we have reexamined the role of PSD-95 in glutamatergic neurotransmission in the hippocampus by carrying out electrophysiological recordings in a mouse line carrying a complete PSD-95 gene deletion. 4A) that were significantly blocked by a non-NMDA antagonist, CNQX (10 μm) (data not shown).
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