Paul L. Ornstein

A hippocampal GluR5 kainate receptor regulating inhibitory synaptic transmission

The principal excitatory neurotransmitter in the vertebrate central nervous system, L-glutamate, acts on three classes of ionotripic glutamate receptors, named after the agonists AMPA (α-amino-3-hydroxy-5-methyl-4-isoxalole-4-propionic acid), NMDA ( N -methyl-D-aspartate) and kainate. The development of selective pharmacological agents has led to a detailed understanding ofthe physiological and pathological roles of AMPA and NMDA receptors. In contrast, the lack of selective kainate receptor ligands has greatly hindered progress in understanding the rolesof kainate receptors,. Here we describe the effects of a potent and selective agonist, ATPA (( RS)-2-amino-3-(3-hydroxy-5- tert -butylisoxazol-4-yl)propanoic acid) and a selective antagonist, LY294486 ((3SR, 4aRS, 6SR, 8aRS)-6-((((1H-tetrazol-5-yl) methyl)oxy)methyl)-1, 2, 3, 4, 4a, 5, 6, 7, 8, 8a-decahydroisoquinoline-3-carboxylic acid), of the GluR5 subtype of kainate receptor. We have used these agents to show that kainate receptors, comprised of or containing GluR5 subunits, regulate synaptic inhibition in the hippocampus, an action that could contribute to the epileptogenic effects of kainate.

LY341495 is a nanomolar potent and selective antagonist of group II metabotropic glutamate receptors

The in vitro pharmacology of a structurally novel compound, LY341495, was investigated at human recombinant metabotropic glutamate (mGlu) receptor subtypes expressed in non-neuronal (RGT, rat glutamate transporter) cells. LY341495 was a nanomolar potent antagonist of 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD)-induced inhibition of forskolin-stimulated cAMP formation at mGlu2 and mGlu3 receptors (respective IC50S of 0.021 and 0.014 microM). At group I mGlu receptor expressing cells, LY341495 was micromolar potent in antagonizing quisqualate-induced phosphoinositide (PI) hydrolysis, with IC50 values of 7.8 and 8.2 microM for mGlu1a and mGlu5a receptors, respectively. Among the human group III mGlu receptors, the most potent inhibition of L-2-amino-4-phosphonobutyric acid (L-AP4) responses was seen for LY341495 at mGlu8, with an IC50 of 0.17 microM. LY341495 was less potent at mGlu7 (IC50 = 0.99 microM) and least potent at mGlu4 (IC50 = 22 microM). Binding studies in rat brain membranes also demonstrated nanomolar potent group II mGlu receptor affinity for LY341495, with no appreciable displacement of ionotropic glutamate receptor ligand binding. Thus, LY341495 has a unique range of selectivity across the mGlu receptor subtypes with a potency order of mGlu3 > or = mGlu2 > mGlu8 > mGlu7 >> mGlu1a = mGlu5a > mGlu4. In particular, LY341495 is the most potent antagonist yet reported at mGlu2, 3 and 8 receptors. Thus, it represents a novel pharmacological agent for elucidating the function of mGlu receptors in experimental systems.

Kainate receptors are involved in synaptic plasticity

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 (α-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-decahydroisoquinoline-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.

A Critical Role of a Facilitatory Presynaptic Kainate Receptor in Mossy Fiber LTP

The mechanisms involved in mossy fiber LTP in the hippocampus are not well established. In the present study, we show that the kainate receptor antagonist LY382884 (10 microM) is selective for presynaptic kainate receptors in the CA3 region of the hippocampus. At a concentration at which it blocks mossy fiber LTP, LY382884 selectively blocks the synaptic activation of a presynaptic kainate receptor that facilitates AMPA receptor-mediated synaptic transmission. Following the induction of mossy fiber LTP, there is a complete loss of the presynaptic kainate receptor-mediated facilitation of synaptic transmission. These results identify a central role for the presynaptic kainate receptor in the induction of mossy fiber LTP. In addition, these results suggest that the pathway by which kainate receptors facilitate glutamate release is utilized for the expression of mossy fiber LTP.

Kainate GluR5 receptor subtype mediates the nociceptive response to formalin in the rat

In order to study the roles of the AMPA and kainate subtypes of non-NMDA glutamate receptors in the processing of persistent nociceptive information, compounds with varying activities at these receptors were examined for effects on the formalin-induced paw-licking behavior in rats. The selective AMPA antagonist, LY300164 and the mixed AMPA/kainate antagonist, NBQX, were compared for their effects on formalin-induced pain behavior. NBQX (3, 10, 20 mg/kg, i.p.), caused antinociception as well as ataxia whereas the selective AMPA antagonist, LY300164 (3,5,10 mg/kg, i.p.), did not cause antinociception at doses that did not produce ataxia. In view of the well documented distribution of kainate receptors on C fibres and of the kainate-preferring iGluR5 subtype on dorsal root ganglia (DRG), we tested a series of three decahydroisoquinolines with different profiles of activity between iGluR5 and AMPA receptors and all without activity on iGluR6, iGluR7 or KA2 subtypes. LY293558 (0.1, 1, 3, 5 mg/kg, i.p.), which had low micromolar affinity for both iGluR5 and 2 caused, like NBQX, both antinociceptive and ataxic effects. However, the selective iGluR5 antagonist LY382884 (5, 10, 30, 100 mg/kg, i.p.), exhibited antinociceptive actions without ataxia while the iGluR2 preferring antagonist LY302679 (5 mg/kg, i.p), caused ataxia but did not produce antinociceptive effects at that dose. These actions were stereoselective since the enantiomeric compounds, LY293559 and LY302680, were ineffective in these tests. The data strongly suggest an involvement of iGluR5 in the processing of nociceptive information.

The potent mGlu receptor antagonist LY341495 identifies roles for both cloned and novel mGlu receptors in hippocampal synaptic plasticity

Understanding the roles of metabotropic glutamate (mGlu) receptors has been severely hampered by the lack of potent antagonists. LY341495 (2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-y l)propanoic acid) has been shown to block group II mGlu receptors in low nanomolar concentrations (Kingston, A.E., Ornstein, P.L., Wright, R.A., Johnson, B.G., Mayne, N.G., Burnett, J.P., Belagaje, R., Wu, S., Schoepp, D.D., 1998. LY341495 is a nanomolar potent and selective antagonist at group II metabotropic glutamate receptors. Neuropharmacology 37, 1-12) but can be used in higher concentrations to block all hippocampal mGlu receptors, identified so far by molecular cloning (mGlu1-5,7,8). Here we have further characterised the mGlu receptor antagonist activity of LY341495 and have used this compound to investigate roles of mGlu receptors in hippocampal long-term potentiation (LTP) and long-term depression (LTD). LY341495 competitively antagonised DHPG-stimulated PI hydrolysis in AV12-664 cells expressing either human mGlu1 or mGlu5 receptors with Ki-values of 7.0 and 7.6 microM, respectively. When tested against 10 microM L-glutamate-stimulated Ca2+ mobilisation in rat mGlu5 expressing CHO cells, it produced substantial or complete block at a concentration of 100 microM. In rat hippocampal slices, LY341495 eliminated 30 microM DHPG-stimulated PI hydrolysis and 100 microM (1S,3R)-ACPD-inhibition of forskolin-stimulated cAMP formation at concentrations of 100 and 0.03 microM, respectively. In area CA1, it antagonised DHPG-mediated potentiation of NMDA-induced depolarisations and DHPG-induced long-lasting depression of AMPA receptor-mediated synaptic transmission. LY341495 also blocked NMDA receptor-independent depotentiation and setting of a molecular switch involved in the induction of LTP; effects which have previously been shown to be blocked by the mGlu receptor antagonist (S)-MCPG. These effects may therefore be due to activation of cloned mGlu receptors. In contrast, LY341495 did not affect NMDA receptor-dependent homosynaptic LTD; an effect which may therefore be independent of cloned mGlu receptors. Finally, LY341495 failed to antagonise NMDA receptor-dependent LTP and, in area CA3, NMDA receptor-independent, mossy fibre LTP. Since in the same inputs these forms of LTP were blocked by (S)-MCPG, a novel type of mGlu receptor may be involved in their induction.

A Role for Ca2+ Stores in Kainate Receptor-Dependent Synaptic Facilitation and LTP at Mossy Fiber Synapses in the Hippocampus

Compared with NMDA receptor-dependent LTP, much less is known about the mechanism of induction of NMDA receptor-independent LTP; the most extensively studied form of which is mossy fiber LTP in the hippocampus. In the present study we show that Ca2+-induced Ca2+ release from intracellular stores is involved in the induction of mossy fiber LTP. This release also contributes to the kainate receptor-dependent component of the pronounced synaptic facilitation that occurs during high-frequency stimulation. We also present evidence that the trigger for this Ca2+ release is Ca2+ permeation through kainate receptors. However, these novel synaptic mechanisms can be bypassed when the Ca2+ concentration is raised (from 2 to 4 mM), via a compensatory involvement of L-type Ca2+ channels. These findings suggest that presynaptic kainate receptors at mossy fiber synapses can initiate a cascade involving Ca2+ release from intracellular stores that is important in both short-term and long-term plasticity.

NMDA receptor antagonists suppress behaviors but not norepinephrine turnover or locus coeruleus unit activity induced by opiate withdrawal

Pretreatment with the non-competitive NMDA (N-methyl-D-aspartate) antagonist MK801 (0.5, 1.0 mg/kg, s.c.) suppressed the behavioral signs of withdrawal in morphine-dependent rats. However, the same doses of MK801 that suppressed morphine withdrawal also simultaneously produced phencyclidine (PCP)-like behaviors. Pretreatment with the competitive NMDA antagonist LY274614 (25, 50, 100 mg/kg i.p.) also suppressed the behavioral signs of withdrawal in morphine-dependent rats but did not produce PCP-like behavioral effects. Single unit recordings were made from noradrenergic neurons in the locus coeruleus (LC) and, at doses that suppressed morphine withdrawal behaviors, neither MK801 nor LY274614 blocked the withdrawal-induced activation of LC neurons. Biochemical analysis indicated that, at the same behaviorally relevant doses, neither MK801 nor LY274614 blocked the withdrawal-induced increase in norepinephrine turnover in the hippocampus, cerebral cortex, or hypothalamus. These results indicate that NMDA antagonists attenuate the behavioral signs of morphine withdrawal without blocking the withdrawal-induced increase in norepinephrine turnover or the withdrawal-induced increase in LC unit activity. In addition, non-competitive NMDA antagonists, like MK801, may not be useful to alleviate opiate withdrawal symptoms in man because of their PCP-like side effects. However, competitive NMDA antagonists, like LY274614, could be of great benefit for alleviating opiate withdrawal symptoms in man.

LY293558, a novel AMPA/GluR5 antagonist, is efficacious and well-tolerated in acute migraine.

Glutamatergic hyperactivity is implicated migraine pathogenesis. Also, LY293558, an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate (KA) receptor antagonist, is effective in preclinical models of migraine. We therefore tested LY293558 in acute migraine. We conducted a randomized, triple-blind, parallel-group, double-dummy, multicentre trial of 1.2 mg/kg intravenous (IV) LY293558, 6 mg subcutaneous (SC) sumatriptan, or placebo in the treatment of acute migraine. The primary efficacy variable was the headache response rate, i.e. headache score improvement from moderate/severe at baseline to mild/none at 2 h. Of 45 enrolled patients, 44 patients (20M:24F; mean age ± SD = 40 ± 9 years) completed the study. Response rates were 69% for LY293558 (P = 0.017 vs. placebo), 86% for sumatriptan (P < 0.01 vs. placebo) and 25% for placebo. LY293558 and sumatriptan were superior to placebo (P < 0.01 for all comparisons) on all other measures of improvement in pain and migraine associated symptoms. Fifteen percent of patients who took LY293558 reported adverse events (AEs) (n = 2; one mild, one severe). Fifty-three percent of patients who took sumatriptan (n = 8; seven mild, one moderate) and 31% of those who received placebo reported AEs (n = 5; four mild, one severe). The efficacy and safety results of LY293558 in this small migraine proof of concept trial, together with supportive preclinical data, provide evidence for a potential role of nonvasoactive AMPA/KA antagonists in treating migraine. Larger trials are needed to further test the hypothesis.

The GluR5 subtype of kainate receptor regulates excitatory synaptic transmission in areas CA1 and CA3 of the rat hippocampus

Activation of kainate receptors depresses excitatory synaptic transmission in the hippocampus. In the present study, we have utilised a GluR5 selective agonist, ATPA [(RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid], and a GluR5 selective antagonist, LY294486 [(3SR,4aRS,6SR,8aRS)-6-([[(1H-tetrazol-5-y l)methyl]oxy]methyl)-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3 -carboxylic acid], to determine whether GluR5 subunits are involved in this effect. ATPA mimicked the presynaptic depressant effects of kainate in the CA1 region of the hippocampus. It depressed reversibly AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor-mediated field excitatory postsynaptic potentials (field EPSPs) with an IC50 value of approximately 0.60 microM. The dual-component excitatory postsynaptic current (EPSC) and the pharmacologically isolated NMDA (N-methyl-D-aspartate) receptor-mediated EPSC were depressed to a similar extent by 2 microM ATPA (61 +/- 7% and 58 +/- 6%, respectively). Depressions were associated with an increase in the paired-pulse facilitation ratio suggesting a presynaptic locus of action. LY294486 (20 microM) blocked the effects of 2 microM ATPA on NMDA receptor-mediated EPSCs in a reversible manner. In area CA3, 1 microM ATPA depressed reversibly mossy fibre-evoked synaptic transmission (by 82 +/- 10%). The effects of ATPA were not accompanied by any changes in the passive properties of CA1 or CA3 neurones. However, in experiments where K+, rather than Cs+, containing electrodes were used, a small outward current was observed. These results show that GluR5 subunits comprise or contribute to a kainate receptor that regulates excitatory synaptic transmission in both the CA1 and CA3 regions of the hippocampus.