Vernon R. J. Clarke

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.

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.

Kainate receptors: subunits, synaptic localization and function.

Although it is well established that kainate receptors constitute an entirely separate group of proteins from AMPA receptors, their physiological functions remain unclear. The molecular cloning of subunits that form kainate receptors and the ability to study recombinant receptors is leading to an increased understanding of their functional properties. Furthermore, the development of kainate receptor-selective agonists and antagonists over the past few years is now allowing the physiological roles of these receptors and, in some cases, specific subunits to be investigated. As a consequence, the synaptic activation of postsynaptic kainate receptors and the presence of presynaptic kainate receptors that serve to regulate excitatory and inhibitory synaptic transmission have been described, and will be discussed in this article by Ramesh Chittajallu, Steven Braithwaite, Vernon Clarke and Jeremy Henley.

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.

Antagonists of GLUK5-containing kainate receptors prevent pilocarpine-induced limbic seizures

Developments in the molecular biology and pharmacology of GLUK5, a subtype of the kainate class of ionotropic glutamate receptors, have enabled insights into the roles of this subunit in synaptic transmission and plasticity. However, little is known about the possible functions of GLUK5-containing kainate receptors in pathological conditions. We report here that, in hippocampal slices, selective antagonists of GLUK5-containing kainate receptors prevented development of epileptiform activity—evoked by the muscarinic agonist, pilocarpine—and inhibited the activity when it was pre-established. In conscious rats, these GLUK5 antagonists prevented and interrupted limbic seizures induced by intra-hippocampal pilocarpine perfusion, and attenuated accompanying rises in extracellular L-glutamate and GABA. This anticonvulsant activity occurred without overt side effects. GLUK5 antagonism also prevented epileptiform activity induced by electrical stimulation, both in vitro and in vivo. Therefore, we propose that subtype-selective GLUK5 kainate receptor antagonists offer a potential new therapy for epilepsy.

Pharmacology of postsynaptic metabotropic glutamate receptors in rat hippocampal CA1 pyramidal neurones

1 Activation of metabotropic glutamate receptors (mGluRs) in hippocampal CA1 pyramidal neurones leads to a depolarization, an increase in input resistance and a reduction in spike frequency adaptation (or accommodation). At least eight subtypes of mGluR have been identified which have been divided into three groups based on their biochemical, structural and pharmacological properties. It is unclear to which group the mGluRs which mediate these excitatory effects in hippocampal CA1 pyramidal neurones belong. We have attempted to address this question by using intracellular recording to test the effects of a range of mGluR agonists and antagonists, that exhibit different profiles of subtype specificity, on the excitability of CA1 pyramidal neurones in rat hippocampal slices 2 (2S,1′S,2′S)‐2‐(2′‐carboxycyclopropyl)glycine (L‐CCG1) caused a reduction in spike frequency adaptation and a depolarization (1–10 mV) associated with an increase in input resistance (10–30%) at concentrations (≥50 μm) that have been shown to activate mGluRs in groups I, II and III. Similar effects were observed with concentrations (50–100 μm) of (1 3 Inhibition of the release of endogenous neurotransmitters through activation of GABAB receptors, by use of 200 μm (±)‐baclofen, did not alter the effects of (1S,3R)‐ACPD (50‐100 μm) (1S,3S)‐ACPD (100 μm) or L‐CCG1 (100 μm). This suggests that mGluR agonists directly activate CA1 pyramidal neurones. 4 Like these broad spectrum mGluR agonists, the racemic mixture ((SR)‐) or resolved (S)‐isomer of the selective group I mGluR agonist 3,5‐dihydroxyphenylglycine ((SR)‐DHPG (50‐100 μm) or (S)‐DHPG (20‐50μm)) caused a reduction in spike frequency adaptation concomitant with postsynaptic depolarization and an increase in input resistance. In contrast, 2S,1′R,2′R,3′R‐2‐(2′,3′‐dicarboxycyclo‐propyl)glycine (DCG‐IV; 100 μm) and (S)‐2‐amino‐4‐phosphonobutanoic acid (L‐AP4; 100‐500 μm), which selectively activate group II mGluRs and group III mGluRs, respectively, had no effect on the passive membrane properties or spike frequency adaptation of CA1 pyramidal neurones. 5 The mGluR antagonists (+)‐α‐methyl‐4‐carboxyphenylglycine ((+)‐MCPG; 1000 μm) and (S)‐4‐carboxyphenylglycine ((S)‐4CPG; 1000 μm), which block groups I and II mGluRs and group I mGluRs, respectively, had no effect on membrane potential, input resistance or spike frequency adaptation per se. Both of these antagonists inhibited the postsynaptic effects of (1S,3R)‐ACPD (50‐100 μm), (1S,3S)‐ACPD (30‐100 μm) and L‐CCG1 (50‐100 μm). (+)‐MCPG also reversed the effects of (SR)‐DHPG (75 μm). (The effect of (S)‐4CPG was not tested.) Their action was selective in that both antagonists did not reverse the reduction in spike frequency adaptation induced by carbachol (1 μm) or noradrenaline (10 μm) whereas atropine (10 μm) and propranolol (100 μm) did. 6 From these data it is concluded that the mGluRs in CA1 pyramidal neurones responsible for these excitatory effects are similar to the mGluRs expressed by non‐neuronal cells transfected with cDNA encoding group I mGluRs.

Characterisation of UBP296: A novel, potent and selective kainate receptor antagonist

Willardiine derivatives with an N3-benzyl substituent bearing an acidic group have been synthesized with the aim of producing selective antagonists for GLUK5-containing kainate receptors. UBP296 was found to be a potent and selective antagonist of native GLUK5-containing kainate receptors in the spinal cord, with activity residing in the S enantiomer (UBP302). In cells expressing human kainate receptor subunits, UBP296 selectively depressed glutamate-induced calcium influx in cells containing GLUK5 in homomeric or heteromeric forms. In radioligand displacement binding studies, the willardiine analogues displaced [3H]kainate binding with IC50 values >100 microM at rat GLUK6, GLUK2 or GLUK6/GLUK2. An explanation of the GLUK5 selectivity of UBP296 was obtained using homology models of the antagonist bound forms of GLUK5 and GLUK6. In rat hippocampal slices, UBP296 reversibly blocked ATPA-induced depressions of synaptic transmission at concentrations subthreshold for affecting AMPA receptor-mediated synaptic transmission directly. UBP296 also completely blocked the induction of mossy fibre LTP, in medium containing 2 mM (but not 4 mM) Ca2+. These data provide further evidence for a role for GLUK5-containing kainate receptors in mossy fibre LTP. In conclusion, UBP296 is the most potent and selective antagonist of GLUK5-containing kainate receptors so far described.

Synaptic activation of a presynaptic kainate receptor facilitates AMPA receptor-mediated synaptic transmission at hippocampal mossy fibre synapses.

The development of GluR5-selective kainate receptor ligands is helping to elucidate the functions of kainate receptors in the CNS. Here we have further characterised the actions of a GluR5 selective agonist, ATPA, and a GluR5 selective antagonist, LY382884, in the CA3 region of rat hippocampal slices. In addition, we have used LY382884 to study a novel synaptic mechanism. This antagonist substantially reduces frequency facilitation of mossy fibre synaptic transmission, monitored as either AMPA or NMDA receptor-mediated EPSCs. This suggests that GluR5-containing kainate receptors on mossy fibres function as autoreceptors to facilitate the synaptic release of L-glutamate, in a frequency-dependent manner.

Pharmacological evidence for an involvement of group II and group III mGluRs in the presynaptic regulation of excitatory synaptic responses in the CA1 region of rat hippocampal slices.

The actions of four mGluR antagonists, (+)-MCPG, MAP4, MCCG and (S)-4CPG, were evaluated against agonist-induced depressions of synaptic transmission at the Schaffer collateral-commissural pathway in rat hippocampal slices. (+)-MCPG (1 mM) reversed very effectively depressions of field EPSPs induced by (1S,3R)-ACPD and (1S,3S)-ACPD but had weak and variable effects on depressions induced by L-AP4. It had no effect on depressions induced by either (-)-baclofen or carbachol. In contrast, MAP4 (500 microM) reversed very effectively depressions induced by L-AP4 without affecting depressions induced by (1S,3S)-ACPD. MCCG (1 mM) had the opposite activity; it antagonized depressions induced by (1S,3S)-ACPD but not those induced by L-AP4. Finally, (S)-4CPG (1 mM) reversed small depressions of field EPSPs induced by high concentrations (50-100 microM) of (1S,3R)- and (1S,3S)-ACPD, but not L-AP4, whilst having no effect on large depressions induced by 10 microM (1S,3S)-ACPD in voltage-clamped cells. These results confirm and extend the effectiveness and selectivity of (+)-MCPG as an mGluR antagonist. The divergent effects of the group I antagonist, (S)-4CPG, can be explained by an indirect action on postsynaptic receptors which is manifest when high agonist concentrations are used in non-voltage-clamp experiments. The action of MCCG and MAP4 indicates that two pharmacologically-distinct mGluRs, belonging to classes II and III, can regulate synaptic transmission in the CA1 region via presynaptic mechanisms.

Characterisation of the effects of ATPA, a GLUK5 receptor selective agonist, on excitatory synaptic transmission in area CA1 of rat hippocampal slices

Kainate receptors are involved in a variety of synaptic functions in the CNS including the regulation of excitatory synaptic transmission. Previously we described the depressant action of the GLU(K5) selective agonist (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid (ATPA) on synaptic transmission in the Schaffer collateral-commissural pathway of rat hippocampal slices. In the present study we report several new features of the actions of ATPA at this synapse. Firstly, the effectiveness of ATPA is developmentally regulated. Secondly, the effects of ATPA decline during prolonged or repeated applications. Thirdly, the effects of ATPA are not mediated indirectly via activation of GABA(A), GABA(B), muscarinic or adenosine A(1) receptors. Fourthly, elevating extracellular Ca(2+) from 2 to 4 mM antagonises the effects of ATPA. Some differences between the actions of ATPA and kainate on synaptic transmission in the Schaffer collateral-commissural pathway are also noted.