AMPA receptor

Excitatory post-synaptic potentials (e.p.s.p.s) evoked by stimulation of the medial perforant path and depolarizations induced by excitatory amino acids were recorded from granule cells in the preparation of the hippocampal slice from the rat. The effects of (+/-)-2-amino-5-phosphonovalerate (APV), gamma-D-glutamylglycine (gamma DGG) and cis-2,3-piperidinedicarboxylate (PDA), antagonists of excitatory amino acids on these phenomena were compared. Gamma-DGG was the most effective antagonist of the e.p.s.p. Its action was reversible and not associated with any change in the passive membrane properties of the granule cells or in the apparent reversal potential of the e.p.s.p. Quantal analysis showed that the reduction in the e.p.s.p. paralleled the decrease in quantal size rather than quantal content, confirming a post-synaptic site of the action of Gamma-DGG. The potency of Gamma-DGG against the exogenous agonists was N-methyl-D-aspartate greater than kainate greater than or equal to quisqualate. APV had very little effect on the e.p.s.p. but was a selective antagonist of N-methyl-D-aspartate-induced depolarizations. PDA depolarized granule cells and increased their membrane input resistance. Although gamma DGG was a potent antagonist of both glutamate- and aspartate-induced depolarizations, no clear pattern of specificity could be found. The action of glutamate was unaffected by APV. These results indicate that the receptor for the transmitter at the synapses formed by the fibres of the perforant path with the granule cells is of the quisqualate and/or kainate type. The present data are consistent with the biochemical evidence that glutamate may be the endogenous transmitter at his synapse. [1]

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Our second approach to determine the postsynaptic contribution to quantal size variation was to apply γ-d-glutamylglycine (Gamma-DGG), a competitive AMPA receptor blocker that blocks less at higher glutamate concentration (Liu et al., 1999). At 200–400 μm, γ-DGG in the bath reduced the mEPSC amplitude by 26 ± 2% (n = 5 synapses), and shifted both the mEPSC amplitude distribution and the cumulative probability curve to the left (Fig. 6A,B). Similar to glutamate dialysis, the ratio (RDGG/Ctrl) between the mEPSC amplitude in control (ACtrl) and that during γ-DGG application was calculated. RDGG/Ctrl increased as ACtrl increased (Fig. 6C) (for a summary of five synapses, see Fig. 6F). Thus, γ-DGG reduced larger mEPSCs by a smaller fraction.
The results shown in Figure 6 strongly argue against the hypothesis that the size or the density of the postsynaptic receptor cluster is the only source of quantal size variation, because this hypothesis predicts no change of RDGG/Ctrl as ACtrl increases. Together, the experiments with glutamate dialysis (Fig. 4) and Gamma-DGG application (Fig. 6) suggest that larger mEPSCs, or at least a significant fraction of larger mEPSCs, were caused by higher glutamate concentrations in the synaptic cleft (for additional controls, see supplemental material 7, available at www.jneurosci.org). It should be pointed out that neither glutamate dialysis (Fig. 4) nor γ-DGG application (Fig. 6) could rule out the possibility that the postsynaptic receptor cluster size (or density) partially contributes to quantal size variation. [2]

To further assess whether the lack of LTD in ceftriaxone-treated rats depended on GLT-1 up-regulation, GLT-1 was blocked with the selective antagonist dihydrokainate (DHK; Arriza et al. 1994). DHK (15 μm) per se produced a modest but consistent decrease in fEPSP amplitude of 18 ± 2% and 14 ± 1% in controls (n= 6) and ceftriaxone-treated rats (n= 6), respectively (P= 0.26; Fig. 5C,D). The amplitude of fEPSPs rapidly regained control levels after washing out the drug. Interestingly, in ceftriaxone-treated rats LFS delivered in the presence of DHK was able to restore LTD. Forty minutes after LFS, the relative fEPSP magnitude was 0.70 ± 0.03 (n= 6; P= 0.002; Fig. 5E). fEPSP amplitude relative to DHK was 0.79 ± 0.03 (P= 0.01). In controls, the relative fEPSP amplitude obtained by delivering LFS to MF in the presence of DHK was 0.84 ± 0.05 (P= 0.05; n= 6; Fig. 5E). Although this value was lower in respect to CEF, it did not significantly differ from that obtained in the absence of the drug (P= 0.1). In addition, the effect of DHK on LTD was prevented by MCPG (500 μm), a broad-spectrum mGluR antagonist, in both controls (not shown) and ceftriaxone-treated rats (relative fEPSP magnitude: 0.96 ± 0.004, n= 5; P= 0.68; Fig. 5F), indicating that mGluR activation is essential for LTD induction at MF–CA3 synapses. To probe changes in synaptic glutamate transient concentrations, slices from both groups were exposed before and after LTD induction to Gamma-DGG/γ-DGG, a low affinity glutamate receptor antagonist (Liu et al. 1999). Because of the low affinity of γ-DGG, during synaptic glutamate transient a certain fraction of synaptic receptors will replace bound γ-DGG for glutamate, thus allowing assessment of changes in glutamate transient concentration in the synaptic cleft during basal synaptic transmission and LTD. Compared to controls, inhibition of fEPSP amplitude induced by γ-DGG (1 mm) was 0.38 ± 0.03 and 0.41 ± 0.03 in saline-treated (n= 5) and ceftriaxone-treated rats (n= 5), respectively. These values were not significantly different (P= 0.36). However, 15 min after LFS, the degree of γ-DGG-induced inhibition relative to controls was 0.59 ± 0.04 in untreated and 0.45 ± 0.03% in ceftriaxone-treated rats, respectively (Fig. 6). Mean values of fEPSPs inhibition obtained before and after LTD were significantly different in controls (P= 0.045) but not in ceftriaxone-treated rats (P= 0.74; see insets in Fig. 5). These results indicate that in controls (but not in ceftriaxone-treated rats), LTD was associated with a reduction in glutamate concentration in the cleft, further supporting its presynaptic locus of expression. [3]

Drugs were ejected ionophoretically from an independently mounted six-barrelled micropipette (6-9 ,m tip diameter) positioned along the dendritic tree of the impaled neurone in the molecular layer of the dentate gyrus at the same level of the stimulating electrode. After the cell had been impaled, the tip of the ionophoretic electrode was slowly advanced into the slice in 5-10 lam steps until a fast-rising depolarization of at least 5 mV could be observed in response to a brief (500-700 msec) application of glutamate. Ionophoretic barrels contained various combinations of the following drugs: L-glutamate (1 M; pH 8), L-aspartate (1 M; pH 8), N-methyl-D-aspartate (20 mm in 150 mM-NaCI; pH 8), quisqu*tte (20 mm in 150 mM-NaCl; pH 8), kainate (20 mM in 150 mM-NaCl; pH 8), APV (50 mm in 100 mM-NaCl; pH 8) yDGG/Gamma-DGG (200 mM; pH 8), PDA (200 mM; pH 8) and NaCl (1 M; pH 8). Retaining currents of 1-5 nA were applied to the individual barrels when necessary. At the end of each impalement, the effect of ionophoretic application of the drugs was re-tested to evaluate any electrical coupling between the ionophoretic pipette and the recording electrode. Results were stored on a Racal FM 4D tape recorder and later analysed with a PDP 11/23 computer.

Animals and hippocampal slice preparation [3]
Male Wistar rats (8–9 weeks old) and wild-type (WT) and GLT-1 KO mice (P15) received a daily intraperitoneal (i.p.) injection of saline or ceftriaxone (Rocefin, Roche; 200 mg kg−1 day−1 dissolved in saline) for 8 days. Twenty-four hours after the final injection, animals used for electrophysiological studies were anaesthetized with an i.p. injection of urethane (2 g kg−1) and killed.
Hippocampal slices from saline- or ceftriaxone-treated rats were prepared as described (Rosato-Siri et al. 2006). Briefly, brains were quickly removed from the skull and transverse hippocampal slices (400 μm thick) were obtained by cutting each hemisphere in ice-cold artificial cerebrospinal fluid (ACSF) containing (in mm): NaCl 126, KCl 3.5, NaH2PO4 1.2, NaHCO3 25, MgCl2 1.3, CaCl2 2.5, glucose 25, saturated with 95% O2–5% CO2 (pH 7.3–7.4). After a recovery period of at least 1 h, single slices were placed in the recording chamber and continuously superfused with oxygenated ACSF (2–3 ml min−1 at 33–34°C). Slices were firstly used for electrophysiological studies and then for immunocytochemical investigations.
Animals used for electron microscope studies (4 rats: 2 controls and 2 CEF-treated; and 8 mice: 2 untreated WT, 2 ceftriaxone-treated wild-type, 2 GLT-1 KO (Tanaka et al. 1997) and 2 GLT-1 KO treated with CEF) were anaesthetized with chloral hydrate (300 mg kg−1) 24 h after the final injection and perfused through the ascending aorta with a flush (∼1 min) of saline followed by 4% paraformaldehyde (PFA). Brains were removed and postfixed for 7 days before cutting; these sections were used for immunogold studies.

[1]. Blockade of amino acid-induced depolarizations and inhibition of excitatory post-synaptic potentials in rat dentate gyrus. J Physiol. 1983 Aug;341:627-40.

[2]. The origin of quantal size variation: vesicular glutamate concentration plays a significant role. J Neurosci. 2007 Mar 14;27(11):3046-56.

[3]. Up-regulation of GLT-1 severely impairs LTD at mossy fibre--CA3 synapses. J Physiol. 2009 Oct 1;587(Pt 19):4575-88.

D-gamma-Glu-Gly is a dipeptide formed from D-gamma-glutamyl and glycine residues.

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The primary conclusion of this investigation is that quisqualate/kainate receptors mediate excitation at the synapses formed by the perforant path fibres with the granule cells. This is based on (1) the higher potency of yDGG/Gamma-DGG compared with APV in antagonizing the e.p.s.p. evoked by stimulation of the medial perforant path; (2) the true post-synaptic site of action of yDGG and (3) the selectivity of yDGG and APVagainst N-methyl-D-aspartate-, quisqualate- and kainate-induced depolarization of granule cells. Synaptic antagonism yDGG was found to be more potent than APV in antagonizing the e.p.s.p. evoked in granule cells by stimulation of the medial perforant path. It is unlikely that the lack of effect of APV was due to inadequate accessibility, since both APV and yDGG were administered from adjacent barrels of the ionophoretic electrode at the same position along the dendritic tree of the impaled neurone and applications of APV smaller than those shown to have no effect on the e.p.s.p. blocked the N-methylD-aspartate response. Thus, N-methyl-D-aspartate receptors do not appear to be involved in the excitation at the synapses formed by the fibres of the perforant path with the granule cells. Instead an involvement of quisqualate and/or kainate receptors is very likely since synaptic antagonism by yDGG only occurred when the application was sufficient to block quisqualate and/or kainate responses. At the moment differentiation between these two receptor types seems difficult. Although other workers (Davies & Watkins, 1981; see Watkins & Evans, 1981) found that yDGG preferentially antagonized responses to kainate rather than quisqualate, we were unable to distinguish between the two types of receptors (in agreement with the findings of Salt & Hill, 1982 and Collingridge et al. 1983). Thus, the separation of quisqualate from kainate receptors must await the development of more specific antagonists. In a recent paper focussed on the lateral perforant path, Koerner & Cotman (1981) have reported l-2-amino-4-phosphonobutyrate as the most potent antagonist against the extracellularly recorded synaptic field potential in the middle molecular layer of the dentate gyrus and suggested the existence of 'a novel L-glutamate receptor not previously described'. However, any attempt to compare their results with the present data is hindered by methodological differences and the fact that these authors concentrated only on the action of the wo-phosphate series of excitatory amino acid antagonists. Furthermore the potency and selectivity of 1-2-amino-4-phosphonobutyrate as an excitatory amino acid antagonist has been strongly questioned at the spinal cord level (Evans et at. 1982) and more recently also on hippocampal CA1 pyramidal cells (Collingridge et al. 1983), where it not only enhanced the action of N-methyl-D-aspartate and quisqualate but also showed some excitatory action of its own. By the same reasoning, PDA was rejected as a useful antagonist in this study because of its excitatory action.[1]

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Mechanism of action of yDGG/Gamma-DGG The current view of e.p.s.p. generation at chemical synapses suggests that the e.p.s.p. amplitude is determined by the summation of a large number of ionic currents of similar amplitude generated by the opening of transmitter specific channels (Eccles, 1964; Ginsborg, 1967). One prediction of such a theory is that a continuous linear relationship exists between the amplitude of the synaptic potential and the associated change in conductance and that antagonists simply combine with specific receptors to reduce the number of channels. The synaptic antagonism by yDGG reported in this study closely followed these predictions. Thus, yDGG reduced the amplitude of the e.p.s.p. without any alteration in its apparent reversal level by limiting the change in membrane input resistance associated with the e.p.s.p. i.e. reducing the number of channels opened by the endogenous transmitter. The term 'apparent reversal level' has been used in this study since the value was determined by extrapolation. However, no marked deviation from linearity in the voltage-current relationship of granule cells was observed either in the hyperpolarizing direction (0 to -30 mV) (see Brown, Fricke & Perkel, 1981) or in the depolarizing direction (0 to + 15 mV). This linear relationship is in agreement with the results of Barnes & McNaughton (1980), who also reported an extrapolated reversal potential of the e.p.s.p.s of -18 mV. According to the quantal hypothesis of transmitter release the size of the miniature e.p.s.p. is determined by the sensitivity of the post-synaptic membrane to the endogenous transmitter (Katz, 1966; Martin, 1977; Takeuchi, 1977; Kuno, 1971). Thus the results of the quantal analysis performed in the presence of yDGG (i.e. a decrease in q with no change in m) provide further and independent evidence that yDGG acts directly on the granule cell membrane, decreasing its sensitivity to the endogenous transmitter. The validity of this analysis is supported by the similarity between the values of m and q reported in this study and those recently published by McNaughton et al. (1981, see Fig. 5, p. 960), who have calculated the quantal parameters of the e.p.s.p. evoked by stimulation of the medial perforant path using both the variance method and the method of failures and a different approach to correct the e.p.s.p. for non-linear summation.[1]

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The transmitter of the medial perforant path Glutamate and aspartate have long been suggested as the most likely candidates for a transmitter role at the synapses formed by the fibres of the perforant path with the granule cells (Nadler et al. 1977; Storm-Mathisen & Iversen, 1979; Wheal & Miller, 1980), with strong biochemical evidence favouring glutamate (White et al. 1977). In the spinal cord and higher centres it has recently been proposed that glutamate and aspartate function as mixed agonists since they are able to interact with all three types of excitatory amino acid receptor (cf. Watkins, 1980). Moreover, it has been shown that compounds which antagonize the N-methyl-D-aspartate receptor tend also to antagonize aspartate-induced excitations leaving glutamate relatively unaffected (Hicks, Hall & McLennan, 1978; Watkins & Evans, 1981). This observation has led to the suggestion that aspartate preferentially interacts with N-methylD-aspartate receptors while glutamate preferentially interacts with the quisqualate ones. In our system a reduction in the e.p.s.p. could occur in the absence of glutamate blockade (see Fig. 3) and Gamma-DGG/yDGG reduced the responses to glutamate and aspartate equally (in agreement with Salt & Hill, 1982), so it is not possible from these data to give preference to either glutamate or aspartate as being the endogenous transmitter of the perforant path. However, over-all the e.p.s.p. evoked by stimulation of the medial perforant path was always reduced by yDGG during blockade of quisqualate and/or kainate receptors and in those cells where yDGG was concomitantly tested against the e.p.s.p., aspartate and glutamate, the e.p.s.p. amplitude decreased by the same degree as the glutamate response. Thus, despite the fact that on the granule cells yDGG appears not to be as selective an inhibitor on aspartateinduced responses as in the spinal cord, we favour the view that the endogenous transmitter of the perforant path, and glutamate, both interact with the same receptor, and that the effect of both are blocked by yDGG. However, other amino acids or related acidic compounds cannot be excluded as the natural ligand for the synaptic receptors.[1]

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Fusion of a single vesicle induces a quantal response, which is critical in determining synaptic strength. Quantal size varies at most synapses. Its underlying mechanisms are not well understood. Here, we examined five sources of variation: vesicular glutamate concentration ([Glu]v), vesicle volume, ultrafast fusion pore closure, the postsynaptic receptor, and the location between release and the postsynaptic receptor cluster at glutamatergic, calyx of Held synapses. By averaging 2.66 million fusion events from 459 synapses, we resolved the capacitance jump evoked by single vesicle fusion. This capacitance jump, an indicator of vesicle volume, was independent of the amplitude of the miniature EPSC (mEPSC) recorded simultaneously at the same synapses. Thus, vesicle volume is not the main source of mEPSC variation. The capacitance jump was not followed by submillisecond endocytosis, excluding ultrafast endocytosis as a source of variation. Larger mEPSCs were increased to a lesser extent by presynaptic glutamate dialysis, and reduced to a lesser extent by Gamma-DGG (gamma-D-glutamylglycine), a competitive AMPA receptor blocker, suggesting that a higher glutamate concentration in the synaptic cleft contributes to the large size of mEPSCs. Larger mEPSCs were not accompanied by briefer rise times, inconsistent with the prediction by, and thus arguing against, the scenario that larger mEPSCs are caused by a shorter distance between the release site and the postsynaptic receptor cluster. In summary, the different amplitudes of mEPSCs were mainly attributable to release of vesicles having similar volumes, but different glutamate amounts, suggesting that [Glu]v is a main source of quantal size variation.[2]

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Furthermore, larger mEPSCs were reduced to a lesser extent by Gamma-DGG/γ-DGG, a competitive AMPA receptor blocker that blocks less at higher glutamate concentration (Fig. 6). These results suggest that larger mEPSCs, or at least a significant fraction of larger mEPSCs, are not attributable to a larger size or a higher density of postsynaptic receptor clusters, but rather result from higher glutamate concentrations experienced by postsynaptic receptors. Fourth, higher glutamate concentrations experienced by postsynaptic receptors were mainly not caused by a shorter distance between the release site and the postsynaptic receptor cluster, but by larger amounts of released transmitter (Fig. 7). In summary, different amplitudes of mEPSCs were mainly attributable to release of vesicles having similar volumes, but different glutamate amounts, suggesting that variation in [Glu]v is a main source of quantal size variation.[2]

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Glutamate transporters are responsible for clearing synaptically released glutamate from the extracellular space. By this action, they maintain low levels of ambient glutamate, thus preventing excitotoxic damage, and contribute to shaping synaptic currents. We show that up-regulation of the glutamate transporter GLT-1 by ceftriaxone severely impaired mGluR-dependent long-term depression (LTD), induced at rat mossy fibre (MF)-CA3 synapses by repetitive stimulation of afferent fibres. This effect involved GLT-1, since LTD was rescued by the selective GLT-1 antagonist dihydrokainate (DHK). DHK per se produced a modest decrease in fEPSP amplitude that rapidly regained control levels after DHK wash out. Moreover, the degree of fEPSP inhibition induced by the low-affinity glutamate receptor antagonist Gamma-DGG was similar during basal synaptic transmission but not during LTD, indicating that in ceftriaxone-treated rats LTD induction did not alter synaptic glutamate transient concentration. Furthermore, ceftriaxone-induced GLT-1 up-regulation significantly reduced the magnitude of LTP at MF-CA3 synapses but not at Schaffer collateral-CA1 synapses. Postembedding immunogold studies in rats showed an increased density of gold particles coding for GLT-1a in astrocytic processes and in mossy fibre terminals; in the latter, gold particles were located near and within the active zones. In both CEF-treated and untreated GLT-1 KO mice used for verifying the specificity of immunostaining, the density of gold particles in MF terminals was comparable to background levels. The enhanced expression of GLT-1 at release sites may prevent activation of presynaptic receptors, thus revealing a novel mechanism by which GLT-1 regulates synaptic plasticity in the hippocampus.

C9H13F3N2O7

318.20

71822-19-0

2935387-13-4; 71822-19-0; 6729-55-1

Typically exists as solids at room temperature

C(CC(=O)NCC(=O)O)[C@H](C(=O)O)N.C(=O)(C(F)(F)F)O

γDGG TFA; gamma-D-Glutamylglycine (trifluoroacetate salt); 71822-19-0; gamma-DGG (TFA); γ-D-Glutamylglycine TFA

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
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Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
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Oral Formulation 3: Dissolved in PEG400
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Oral Formulation 6: Mixing with food powders

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