GluR5 kainate receptor.

The ability of synapses to modify their synaptic strength in response to activity is a fundamental property of the nervous system and may be an essential component of learning and memory. There are three classes of ionotropic glutamate receptor, namely NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid) and kainate receptors; critical roles in synaptic plasticity have been identified for two of these. Thus, at many synapses in the brain, transient activation of NMDA receptors leads to a persistent modification in the strength of synaptic transmission mediated by AMPA receptors. Here, to determine whether kainate receptors are involved in synaptic plasticity, we have used a new antagonist, LY382884 ((3S, 4aR, 6S, 8aR)-6-((4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-decahydro isoquinoline-3-carboxylic acid), which antagonizes kainate receptors at concentrations that do not affect AMPA or NMDA receptors. We find that LY382884 is a selective antagonist at neuronal kainate receptors containing the GluR5 subunit. It has no effect on long-term potentiation (LTP) that is dependent on NMDA receptors but prevents the induction of mossy fibre LTP, which is independent of NMDA receptors. Thus, kainate receptors can act as the induction trigger for long-term changes in synaptic transmission. [1]

---

We therefore examined other members of this family of decahydroisoquinoline compounds using ligand-binding assays; one compound, LY382884, was identified as having considerably greater selectivity for GluR5 than for GluR2 (ref. 10). The ability of LY382884 to displace binding at a wide range of recombinant AMPA and kainate receptors is shown in Fig. 1a. Binding to GluR5 was displaced with an inhibition constant (Ki) of 4.0 ± 0.2 µM (n = 4), whereas binding to GluR1–4, GluR6, GluR7, KA2 and a heteromeric assembly of GluR6 and KA2 was displaced with Ki values in excess of 100 µM. The kainate receptor antagonist activity of LY382884 is shown for recombinant and native receptors in Fig. 1b–d. LY382884 antagonized kainate-induced currents in GluR5/GluR6 heteromers (Fig. 1b) but not in GluR6 homomers (Fig. 1c). The heteromer was made by co-expressing GluR6(Q), which alone generated strongly rectifying currents, with GluR5(R), which alone was non-functional; this co-expression yielded a more linear I–V relationship than GluR6 alone, in response to voltage ramps between -100 and +100 mV. In cells displaying this more linear rectification, LY382884 dose dependently reduced the current induced by 30 µM kainate and increased rectification, presumably because a greater proportion of GluR6 homomers contributed to the residual current (Fig. 1b). The absence of functional antagonism at recombinant GluR6 kainate receptors is shown in Fig. 1c, which compares LY382884 with the AMPA/ kainate receptor antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulphamoylbenzo(f)quinoxaline). LY382884 had no observable antagonistic effects at human GluR6 kainate receptors at concentrations of up to 100 µM. In rat dorsal root ganglion (DRG) neurons, which express GluR5 kainate receptors8,11,12, LY382884 inhibited currents evoked by kainate (30 µM) and a selective GluR5 kainate receptor ligand, ATPA ((RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid) (3 µM) in a concentration-dependent manner (half maximal concentration (IC50) 0.95 ± 0.16 µM (n = 6) and 1.19 ± 0.79 µM (n = 6), respectively. We also established the antagonistic activity of LY382884 at hippocampal AMPA and NMDA receptors (Fig. 1d). LY382884 had little or no effect on currents evoked by AMPA (30 µM) or NMDA (10 µM) at a concentration of 10 µM. [1]

---

To determine whether LY382884 can be used to antagonize neuronal kainate receptors selectively in an intact slice preparation, we performed experiments on the CA1 region of the hippocampus. We recorded excitatory postsynaptic potentials (EPSPs) mediated by AMPA receptors intracellularly and determined their sensitivity by sequentially applying increasing concentrations of the antagonist. LY382884 depressed the synaptic AMPA receptor-mediated response with an IC50 of 87 µM (n = 3; Fig. 2a). In these experiments, 10 µM was the maximum concentration that could be used before AMPA receptor-mediated synaptic transmission was affected. Neither monosynaptic γ-aminobutyric acid (GABA)A and GABAB receptor-mediated synaptic transmission13 nor passive membrane properties were affected by 10 µM LY382884 (n = 5; data not shown). Next, we determined the effectiveness of 10 µM LY382884 as a kainate receptor antagonist by testing its ability to inhibit the depression of AMPA receptor-mediated synaptic transmission induced by ATPA, using field potential recordings in slices obtained from juvenile rats14. ATPA depressed field EPSPs (fEPSPs), at a concentration of 1 µM (n = 5) or 3 µM (n = 6), respectively, by 57 ± 6 and 63 ± 6% under control conditions but by only 6 ± 2 and 18 ± 9% in the presence of LY382884 (data not shown). [1]

---

Given the high density of kainate receptors in area CA3 (ref. 15), we extended our analysis to this region. LY382884 similarly antagonized the depression of AMPA receptor-mediated EPSPs induced by ATPA. Thus, 1 µM ATPA depressed associational/commissural and mossy-fibre-evoked fEPSPs, respectively, by 34 ± 2 and 42 ± 11% under control conditions but by only 6 ± 4 and 10 ± 5% in the presence of 10 µM LY382884(n = 4; Fig. 2b). Synaptic transmission in area CA3 is also inhibited by activation of metabotropic glutamate (mGlu) receptors, raising the possibility that ATPA and LY382884 are acting through these receptors. However, the mGlu receptor antagonist (S)-α-methyl-4-carboxyphenylglycine (MCPG) had no effect on ATPA-induced depression (n =5), and LY382884 had no effect on depression induced by the mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD; n = 6; Fig. 2c). Furthermore, 10 µM LY382884 had no effect on depolarization of CA3 neurons induced by 30 µM (RS)-DHPG ((RS)-3,5-dihydroxyphenylglycine) (mean depolarizations before and after addition of LY382884, 10 ± 3 and 12 ± 2 mV, respectively; n = 8), precluding an effect of the antagonist on postsynaptic group I mGlu receptors. CA3 neurons possess postsynaptic kainate receptors that can be activated synaptically by brief, high frequency tetanus. LY382884 (10 µM) antagonized this kainate receptor-mediated excitatory postsynaptic current (EPSC), recorded under whole-cell voltage-clamp conditions at -70 mV, by 38 ± 4% (n = 7; Fig. 2d). [1]

---

Having established that LY382884 is a selective kainate receptor antagonist, we wished to determine whether kainate receptors are involved in the induction of LTP at mossy fibre synapses, given that LTP at these synapses is classically independent of NMDA receptors. Mossy fibre LTP was completely prevented, in a reversible manner, by LY382884. (The mean potentiation 60 min after tetanization (100 Hz, 1 s, test intensity) in the presence and after washout of LY382884 was 1 ± 4% and 48 ± 10%, respectively; n = 7; Fig. 3a, b.) The ability of LY382884 to block the induction of LTP was pathway specific, as NMDA receptor-dependent LTP in the CA3 region of the hippocampus, evoked by tetanic stimulation of associational/commissural fibres, was fully resistant to the actions of LY382884. The mean potentiation 60 min after tetanization in the presence of LY382884 was 45 ± 10 (n = 3), compared with 47 ± 6% in interleaved control experiments (n = 6; Fig. 3c). Furthermore, NMDA receptor-dependent LTP at CA1 synapses was also insensitive to LY382884 (n = 7; data not shown). [1]

---

It was possible that LY382884 blocked the induction of LTP by an action that was independent of its ability to antagonize kainate receptors. Mossy fibre LTP involves the activation of the cAMP–protein kinase A (PKA) pathway and can be induced by the stimulation of adenylyl cyclase by forskolin. Figure 4a shows input-specific, long-lasting (>4 h), PKA-dependent mossy fibre LTP induced by our standard tetanus protocol. LY382884 had no effect on forskolin-induced mossy fibre LTP (n = 6) when compared with interleaved controls (n = 6; Fig. 4b). Thus, its ability to inhibit LTP is unlikely to be due to an action on signal transduction or expression mechanisms involved in mossy fibre LTP. [1]

---

LTP was, however, fully blocked by 10 mM kynurenate (n = 5; Fig. 5b). We also found that 10 µM CNQX, a concentration that antagonizes kainate-induced currents mediated by GluR5 in CA3 neurons, blocked the induction of mossy fibre LTP (n = 4; Fig. 5c). In these two sets of experiments, we also examined the effects of LY382884 alone and of D(-)-2-amino-5-phosphonopentanoate (D-AP5) alone on mossy fibre LTP; in all cases, LY382884 blocked LTP whereas LTP was readily obtained in the presence of D-AP5 (the mean potentiation 60 min after tetanization in the presence of LY382884 alone was -2 ± 1%; 60 min after tetanization in the presence of D-AP5 alone it was 36 ± 11%; n = 9). CNQX (Ki for displacement of GluR5 binding, 2.9 ± 0.2 µM; n = 3) is about as effective as LY382884, whereas kynurenate is very weak as an antagonist at GluR5-expressing cells, with dose-dependent antagonism over the millimolar range. In three separate systems, kynurenate produced significantly (P < 0.05) more antagonism at 10 mM than at 3 mM: displacement of 100-µM kainate binding in GluR5-expressing HEK293 cells was 98 ± 1% at 10 mM kynurenate and 60 ± 1% at 3 mM (n = 3); inhibition of 100 µM kainate currents in DRG neurones was 89 ± 3 and 76 ± 1%, respectively (n = 3); depression of synaptic responses mediated by kainate receptors in CA3 neurons was 84 ± 7 and 42 ± 8%, respectively (n = 4). Thus, three structurally unrelated compounds antagonized both events mediated by kainate receptors containing GluR5 and mossy fibre LTP in the same rank order of potency: LY382884 = CNQX ≫ kynurenate. [1]

---

LY382884 shows selectivity between homomeric GluR5 and GluR2 receptors; it has been used in studies of global ischaemia and nociception. We have shown that LY382884 antagonizes responses mediated by kainate receptors at concentrations below those that affect synaptic processes mediated by AMPA or NMDA receptors. Like LY293558 (ref. 8) and LY294486 (ref. 9), LY382884 is highly selective for the GluR5 kainate receptor subunit. The earlier kainate receptor antagonists have been used to identify a role for GluR5 subunits in excitatory synaptic transmission in the hippocampus and amygdala. However, these compounds also antagonize AMPA receptors. Given its greater selectivity, LY382884 is a more useful antagonist with which to explore the functions of GluR5 in the brain.

Ligand-binding studies [1]
These experiments were performed using cell membranes prepared from frozen HEK293 cells expressing recombinant AMPA or kainate receptor subunits and using [3H]-AMPA and [3H]-kainate, respectively, as described.

Construction of the heteromer [1]
A GluR6(Q) stable HEK293 cell line was transfected with GluR52b(R) in the vector pCEP4, using Lipofectamine 2000. After three weeks of selection with 250 µg ml-1 hygromycin, the cells reached 30% confluency and were split for electrophysiology.

---

Electrophysiology using isolated cells [1]
Whole-cell voltage clamp recordings were made using extracellular solutions comprising (in mM) NaCl (138), CaCl2 (5), KCl (5), MgCl2 (1), HEPES (10) and glucose (10); pH 7.4. For HEK293 cells and hippocampal neurons, intracellular solutions comprised (in mM) CsCl (140), MgCl2 (1), diTris creatine phosphate (14), HEPES (10), BAPTA (15) and creatine phosphokinase (50 units ml-1); pH 7.15. For DRG neurons, intracellular solutions comprised (in mM) CsMeSO4 (125), CsCl (15), CsBAPTA (5), HEPES (10), CaCl2 (0.5) MgCl2 (3) and MgATP (2); pH 7.2. Experiments were performed at room temperature (20–22 °C). Drugs were applied by bath perfusion and exchange of solutions under these conditions took about 5 s. Voltage ramps were conducted between -100 and +100mV in 1 s. Experiments using recombinant receptors and DRG neurons were performed after incubation of cells with 250 µg ml-1 concanavalin A for 10 min to remove receptor desensitization. Hippocampal pyramidal neurons were cultured from E17 Sprague Dawley rat fetuses. AMPA receptor-mediated currents were obtained from cells (6–12 days in vitro) in the presence of tetrodotoxin (1 µM); NMDA receptor-mediated currents were recorded from cells (10–12 days in vitro) in the presence of glycine (10 µM) but without added magnesium. Curve fitting to the data points was based upon the equation y = 100(Dn/(Dn + ECn50)) using a slope fixed to a value of 1 and where D is the drug concentration. For antagonists, EC50= IC50. IC50 values were estimated from data obtained from at least four separate cells.

Electrophysiology in slice [1]
Experiments were performed on transverse rat hippocampal slices (400 µm) maintained in medium comprising (in mM) NaCl (124), KCl (3), NaHCO3 (26), NaH2PO4 (1.25), CaCl2 (2), MgSO4 (1) and D-glucose (10) (bubbled with O2/CO2 : 95/5%). Extracellular fEPSPs were recorded in areas CA1 and CA3 using glass microelectrodes (2–4 MΩ) containing 4 M NaCl, as described. Intracellular recordings were obtained using sharp glass microelectrodes (40–80 MΩ) filled with KMeS04 (2M). Whole-cell patch-clamp recordings were obtained blind using glass microelectrodes (5–7 MΩ; seal resistance ∼10 GΩ) filled with a solution comprising (in mM) CsMeSO3 (120), NaCl (1), MgCl2 (1), Mg-ATP (4), BAPTA (10), N-(2,6-dimethyl-phenylcarbamoylmethyl)-triethylammonium bromide (QX-314) (5) and HEPES (5), adjusted to pH 7.3, as described. Data are presented as mean ± s.e.m. throughout.

[1]. Kainate receptors are involved in synaptic plasticity. Nature. 1999 Nov 18;402(6759):297-301.

LY382884 is a member of benzoic acids.

C18H23NO4

317.37952542305

317.162

C, 68.12; H, 7.30; N, 4.41; O, 20.16

211566-75-5

656723

Typically exists as solid at room temperature

0.7

3

5

4

23

444

4

C1C[C@H]2CN[C@@H](C[C@H]2C[C@H]1CC3=CC=C(C=C3)C(=O)O)C(=O)O

YVMADKYPKNLVGU-BVUBDWEXSA-N

InChI=1S/C18H23NO4/c20-17(21)13-4-1-11(2-5-13)7-12-3-6-14-10-19-16(18(22)23)9-15(14)8-12/h1-2,4-5,12,14-16,19H,3,6-10H2,(H,20,21)(H,22,23)/t12-,14+,15-,16+/m1/s1

(3S,4aR,6S,8aR)-6-[(4-carboxyphenyl)methyl]-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid

LY382884; LY 382884; LY382,884; 211566-75-5; LY 382,884; 3-Isoquinolinecarboxylic acid, 6-[(4-carboxyphenyl)methyl]decahydro-, (3S,4aR,6S,8aR)-; CHEMBL274226; CHEBI:34808; (3S,4aR,6S,8aR)-6-[(4-carboxyphenyl)methyl]-decahydroisoquinoline-3-carboxylic acid; (3S,4aR,6S,8aR)-6-[(4-carboxyphenyl)methyl]-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid; LY-382884

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.

---

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)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

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)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
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)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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