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]

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γ-DGG (γ-DGG) is an AMPA receptor competitive blocker that blocks less glutamate at greater concentrations. Micro-EPSC (mEPSC) amplitude was decreased by 26±2% (n=5) at 200–400 μM due to the presence of γ-DGG in the bath. Additionally, the mEPSC amplitude distribution and cumulative probability curve were shifted to the left [2]. The strongest antagonist of excitatory postsynaptic potentials (EPSPS) is γ-DGG. Its effects are reversible and unrelated to alterations in the granule cells' passive membrane characteristics or in the EPSPs' apparent reversal potential. γ-DGG's postsynaptic site of action was confirmed when quantitative analysis revealed that the decline in EPSP followed a decline in quantal size rather than quantal content [1].

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-γ-Glu-Gly is a dipeptide composed of D-γ-glutamyl and glycine residues. The main conclusion of this study is that the quesquiate/algaeine receptor mediates the excitation of the synapse formed between the perforating fiber and granule cells. This conclusion is based on the following points: (1) γ-DGG/γ-DGG is more potent than APV in antagonizing excitatory postsynaptic potentials (EPSPs) induced by medial perforating fiber stimulation; (2) the true site of action of γ-DGG is located postsynaptic; and (3) γ-DGG and APV are selective for granule cell depolarization induced by N-methyl-D-aspartate, quesquiate, and algaeine. The study found that the synaptic antagonist yDGG is more effective than APV in antagonizing excitatory postsynaptic potentials (EPSPs) induced by medial perforating pathway stimulation of granule cells. The ineffectiveness of APV is unlikely to be due to insufficient dosage, as both APV and yDGG are administered via iontophoresis via adjacent electrode tubes at the same location on the dendrite of the punctured neuron, and APV can block the N-methyl-D-aspartate (NMDA) response with a dosage lower than that which has no effect on epsp. Therefore, NMDA receptors do not appear to be involved in the excitation of the synapse formed between the perforating pathway fibers and granule cells. Instead, quisquiate receptors and/or rutin receptors are likely involved, as the synaptic antagonism of yDGG only occurs when the dosage is sufficient to block the quisquiate and/or rutin response. Distinguishing between these two receptor types currently appears difficult. Although other researchers (Davies & Watkins, 1981; see Watkins & Evans, 1981) have found that γ-diacylglutamate (yDGG) preferentially antagonizes the rutin response rather than the quisquiate response, we cannot distinguish between these two receptors (consistent with the findings of Salt & Hill, 1982, and Collingridge et al., 1983). Therefore, the separation of quisquiate receptors from fumarate receptors awaits the development of more specific antagonists. In a recent paper focusing on the lateral perforation pathway, Koerner & Cotman (1981) reported that 1,2-amino-4-phosphonate butyrate is the most effective antagonist of extracellular synaptic field potentials recorded in the molecular layer of the dentate gyrus, and proposed the existence of "a novel L-glutamate receptor previously undescribed." However, due to methodological differences and the fact that these authors focused only on the effects of ω-phosphate series excitatory amino acid antagonists, any attempt to compare their results with current data is hampered. Furthermore, the potency and selectivity of 1,2-amino-4-phosphonate butyrate as an excitatory amino acid antagonist have been strongly questioned at the spinal cord level (Evans et al., 1982) and more recently in hippocampal CA1 pyramidal cells (Collingridge et al., 1983), where it not only enhances the effects of N-methyl-D-aspartate and quisquiate but also exhibits some excitatory activity itself. For the same reason, PDA was excluded from the list of effective antagonists in this study due to its excitatory effect. [1]

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Mechanism of action of γ-DGG/γ-DGG The current view on the generation of excitatory postsynaptic potentials (epsp) at chemical synapses is that the amplitude of epsp is determined by the superposition of a large number of ionic currents with similar amplitudes, which are generated by the opening of neurotransmitter-specific channels (Eccles, 1964; Ginsborg, 1967). One prediction of this theory is that there is a continuous linear relationship between the amplitude of the synaptic potential and the associated conductance changes, and the antagonist's role is simply to bind to specific receptors, thereby reducing the number of channels. The synaptic antagonistic effect of γ-DGG reported in this study is basically consistent with these predictions. Therefore, γ-DGG reduces the amplitude of epsp by limiting the membrane input resistance changes associated with excitatory postsynaptic potentials (epsp), thereby reducing the number of channels open by endogenous neurotransmitters, without changing its apparent reversal level. The term "apparent inversion level" is used in this study because the value was determined by extrapolation. However, no significant linear deviation was observed in the voltage-current relationship of granule cells in either the hyperpolarization direction (0 to -30 mV) (see Brown, Frike, and Perkel, 1981) or the depolarization direction (0 to +15 mV). This linearity is consistent with the findings of Barnes and McNaughton (1980), who also reported an extrapolated inversion potential of -18 mV for epsp. According to the quantization hypothesis of neurotransmitter release, the magnitude of the miniature excitatory postsynaptic potential (mEPSP) depends on the sensitivity of the postsynaptic membrane to endogenous neurotransmitters (Katz, 1966; Martin, 1977; Takeuchi, 1977; Kuno, 1971). Therefore, the quantization analysis results in the presence of γ-diacylglycerol (yDGG) (i.e., a decrease in q-value while the m-value remains unchanged) provide further independent evidence that yDGG acts directly on the granule cell membrane, reducing its sensitivity to endogenous neurotransmitters. The m-values and q-values reported in this study are similar to those recently published by McNaughton et al. (1981, see Figure 5, page 960), who used variance and failure methods to calculate the quantum parameters of EPSP induced by stimulation of the medial perforation pathway and employed different methods to correct for the nonlinear summation of EPSP. [1]

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The neurotransmitters glutamate and aspartate in the medial perforation pathway have long been considered the most likely neurotransmitter candidates for the synapses formed between perforation pathway fibers and granule cells (Nadler et al., 1977; Storm-Mathisen and Iversen, 1979; Wheal and Miller, 1980), and there is strong biochemical evidence to support glutamate (White et al., 1977). Recent studies have suggested that glutamate and aspartate act as mixed agonists in the spinal cord and higher nerve centers because they can interact with all three types of excitatory amino acid receptors (see Watkins, 1980). In addition, studies have shown that compounds that antagonize N-methyl-D-aspartate receptors tend to antagonize aspartate-induced excitatory effects as much as glutamate-induced excitatory effects (Hicks, Hall, and McLennan, 1978; Watkins and Evans, 1981). Based on this, it is hypothesized that aspartate preferentially interacts with N-methyl-D-aspartate receptors, while glutamate preferentially interacts with quisquiate receptors. In our system, even without glutamate receptor blockade, excitatory postsynaptic potentials (epsp) are reduced (see Figure 3). γ-DGG/γ-DGG have the same inhibitory effect on both glutamate and aspartate responses (consistent with the findings of Salt & Hill, 1982), therefore, based on these data, it is impossible to determine whether glutamate or aspartate is the dominant endogenous neurotransmitter in the perforation pathway. However, overall, γ-DGG consistently reduces excitatory postsynaptic potentials induced by medial perforation pathway stimulation when quisquiate and/or fumarate receptors are blocked. In cells where the effects of γ-DGG on excitatory postsynaptic potentials, aspartate, and glutamate responses were tested simultaneously, the reduction in the amplitude of excitatory postsynaptic potentials was similar to the reduction in the glutamate response. Therefore, although the selective inhibition of aspartate-induced responses by yDGG appears to be less pronounced in granulosa cells than in the spinal cord, we tend to think that both endogenous neurotransmitters and glutamate in the perforation pathway interact with the same receptor and that both are blocked by yDGG. However, the possibility that other amino acids or related acidic compounds may act as natural ligands for synaptic receptors cannot be ruled out. [1] Fusion of a single vesicle induces a quantization response, which is crucial for determining synaptic strength. The quantization size of most synapses varies. The underlying mechanisms are not fully understood. Here, we investigate five sources of variation: vesicle glutamate concentration ([Glu]v), vesicle volume, ultrafast fusion pore closure, postsynaptic receptors, and the location of the release point in a glutamatergic Held goblet synapse between the synaptic receptor cluster and the glutamatergic release point. By averaging 2.66 million fusion events from 459 synapses, we resolve the capacitive jump caused by single vesicle fusion. This capacitive jump is an indicator of vesicle volume and is independent of the amplitude of the miniature excitatory postsynaptic currents (mEPSCs) recorded simultaneously at the same synapse. Therefore, vesicle volume is not the primary source of mEPSC variation. No sub-millisecond endocytosis occurred after the capacitive jump, ruling out ultrafast endocytosis as a source of variation. Presynaptic glutamate dialysis slightly increased larger mEPSCs, while the competitive AMPA receptor blocker γ-DGG (γ-D-glutamylglycine) slightly decreased them, suggesting that higher glutamate concentrations in the synaptic cleft contribute to increased mEPSC size. Larger mEPSCs were not accompanied by shorter rise times, which is inconsistent with predictions, thus refuting the hypothesis that larger mEPSCs are caused by shorter distances between the release site and the postsynaptic receptor cluster. In summary, the differences in mEPSC amplitude are mainly attributed to similar vesicle volumes but different glutamate contents, indicating that [Glu]v is the primary source of quantum size variation. [2] Furthermore, γ-DGG/γ-DGG (a competitive AMPA receptor blocker with weaker blocking effect at high glutamate concentrations) showed little inhibitory effect on larger mEPSCs (Fig. 6). These results suggest that larger mEPSCs, or at least a large portion of larger mEPSCs, are not due to larger or denser postsynaptic receptor clusters, but rather to higher glutamate concentrations at the postsynaptic receptors. Fourth, the higher glutamate concentrations sensed by the postsynaptic receptors are not primarily due to a shorter distance between the release site and the postsynaptic receptor clusters, but rather to an increase in the released neurotransmitter mass (Fig. 7). In summary, the differences in mEPSC amplitudes are mainly due to similar vesicle volumes but different glutamate contents, suggesting that variations in [Glu]v are the primary source of quantum size variations. [2]

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Glutamate transporters are responsible for clearing glutamate released at the synapse. Through this function, they maintain lower extracellular glutamate levels, thereby preventing excitotoxic damage and contributing to the shaping of synaptic currents. We found that ceftriaxone upregulation of the glutamate transporter GLT-1 severely impaired mGluR-dependent long-term inhibition (LTD) induced by repetitive stimulation of afferent fibers at the rat lichen fiber (MF)-CA3 synapse. This effect was GLT-1-related because the selective GLT-1 antagonist dihydrolycanthine (DHK) could rescue the LTD. DHK itself slightly reduced the fEPSP amplitude, but the fEPSP amplitude rapidly recovered to control levels after DHK washout. Furthermore, the degree of fEPSP inhibition induced by the low-affinity glutamate receptor antagonist γ-DGG was similar during basal synaptic transmission but different during LTD, indicating that LTD induction did not alter the transient synaptic glutamate concentration in ceftriaxone-treated rats. In addition, ceftriaxone-induced GLT-1 upregulation significantly reduced the LTP amplitude at the MF-CA3 synapse but had no effect on the LTP amplitude at the Schaffer collateral-CA1 synapse. Immunogold labeling studies after embedding in rats showed increased density of gold particles encoding GLT-1α in astrocyte processes and moss-like fiber terminals; at the moss-like fiber terminals, gold particles were located near and within the active site. In CEF-treated and untreated GLT-1 KO mice used to verify immunostain specificity, the density of gold particles at the MF terminals was comparable to background levels. Enhanced GLT-1 expression at the release site may inhibit presynaptic receptor activation, thus revealing a novel mechanism by which GLT-1 regulates hippocampal synaptic plasticity.

C7H12N2O5

204.182

204.075

C, 41.18; H, 5.92; N, 13.72; O, 39.18

6729-55-1

6604701

White to off-white solid powder

586.6ºC at 760 mmHg

308.6ºC

-4.3

4

6

6

14

240

1

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

ACIJGUBIMXQCMF-SCSAIBSYSA-N

InChI=1S/C7H12N2O5/c8-4(7(13)14)1-2-5(10)9-3-6(11)12/h4H,1-3,8H2,(H,9,10)(H,11,12)(H,13,14)/t4-/m1/s1

(2R)-2-amino-5-(carboxymethylamino)-5-oxopentanoic acid

gamma-DGG; gamma DGG; 6729-55-1; gamma-D-Glutamylglycine; gamma-DGG; H-D-GLU(GLY-OH)-OH; gammaDGG; D-gamma-Glu-Gly; gamma-D-GLU-GLY; D-gamma-glutamylglycine; gammaDGG

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)

H2O : ≥ 150 mg/mL (~734.65 mM)

Solubility in Formulation 1: 25 mg/mL (122.44 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)