Reduction of sensory and metabotropic glutamate receptor responses in the thalamus by the novel metabotropic glutamate receptor-1-selective antagonist S-2-methyl-4-carboxy-phenylglycine

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Neuroscience Vol. 85, No. 3, pp. 655–658, 1998 Copyright  1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(98)00048-7

Letter to Neuroscience REDUCTION OF SENSORY AND METABOTROPIC GLUTAMATE RECEPTOR RESPONSES IN THE THALAMUS BY THE NOVEL METABOTROPIC GLUTAMATE RECEPTOR-1-SELECTIVE ANTAGONIST S-2-METHYL-4-CARBOXY-PHENYLGLYCINE T. E. SALT* and J. P. TURNER Institute of Ophthalmology, University College London, 11–43 Bath Street, London EC1V 9EL, U.K. Key words: metabotropic glutamate receptor antagonist, mGluR1, LY367385, somatosensory, thalamus, nociception, iontophoresis.

Previous work has shown that responses of thalamic neurons in vivo to the metabotropic glutamate receptor agonists 1S,3R-aminocyclopentane-1,3-dicarboxylate and S-3,5-dihydroxyphenylglycine can be reduced by a variety of phenylglycine antagonists.5,22,23 Responses of thalamic neurons to noxious thermal somatosensory stimuli were reduced in parallel by these antagonists,5,22 indicating that these responses are mediated by Group I metabotropic glutamate receptors (i.e. metabotropic glutamate receptor-1 and/or metabotropic glutamate receptor-5), which are known to be linked to phosphoinositol phosphate hydrolysis.10,15,18 The recent development of S-2-methyl-4-carboxyphenylglycine as an antagonist which is highly selective for metabotropic glutamate receptor-1 compared to metabotropic glutamate receptor-5 on human receptors expressed in AV-12 cells,4 now offers the possibility of discriminating between these two receptor subtypes in order to distinguish which is involved in thalamic responses. We have made recordings from single somatosensory neurons in the thalamus of the rat, and find that S-2-methyl-4-carboxy-phenylglycine is able to reduce responses of neurons to 1S,3R-aminocyclopentane-1,3-dicarboxylate, S-3,5-dihydroxyphenylglycine, and noxious stimuli without significant effect on responses to either N-methylD-aspartate or ()-á-amino-3-hydroxy-5-methyl4-isoxazolepropionate. These results suggest that excitatory responses of thalamic neurons to 1S,3Raminocyclopentane-1,3-dicarboxylate and S-3,5*To whom correspondence should be addressed. Abbreviations: ACPD, 1S,3R-aminocyclopentane-1,3dicarboxylate; AMPA, ()-á-amino-3-hydroxy-5methyl-4-isoxazolepropionate; DHPG, S-3,5dihydroxyphenylglycine; LY367385, S-2-methyl-4carboxy-phenylglycine; mGluR, metabotropic glutamate receptor; NMDA, N-methyl--aspartate.

dihydroxyphenylglycine may be mediated by metabotropic glutamate receptor-1. Furthermore, the reduction of nociceptive responses by S-2-methyl4-carboxy-phenylglycine indicates that metabotropic glutamate receptor-1 is involved in thalamic nociceptive processing and that such antagonists may have analgesic properties.  1998 IBRO. Published by Elsevier Science Ltd. Single-neuron recordings were made with extracellular multi-barrel iontophoretic electrodes in the ventrobasal thalamus and immediately overlying dorsal thalamic nuclei of adult male Wistar rats (Harlan, U.K.) anaesthetized with urethane (1.2 g/kg, I.P.), as detailed previously.21 The outer barrels were used for iontophoretic drug applications, and each contained one of the following substances, as Na+ salts: N-methyl--aspartate (NMDA), ()á-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), 1S,3R-aminocyclopentane-1,3-dicarboxylate (ACPD), S-2-methyl-4-carboxy-phenylglycine (LY367385) (all 50 mM in water, pH 8.0–8.5), S-3,5dihydroxyphenylglycine (DHPG) (50 mM in water, pH 5.5), and 1M NaCl (for current balancing), and Pontamine Sky Blue dye (2.5% in 0.5M NaCl/0.5M Na acetate). All drugs were ejected iontophoretically as anions (with the exception of DHPG), and prevented from diffusing out of the pipette by a retaining current (10–20 nA) of opposite polarity to the ejection current. All drugs were obtained from Tocris, apart from LY367385 (gift from Lilly Research). Regular repeated cycles (5 min duration) of agonist ejections were set up and initiated by a computer system, and extracellular action potentials were gated and timed using the computer system, which could produce peristimulus histograms of single-neuron activity.

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T. E. Salt and J. P. Turner Table 1. Effects of S-2-methyl-4-carboxy-phenylglycine on amino acid and sensory responses

ACPD (% control) 142.6** n=18

DHPG (% control)

NMDA (% control)

AMPA (% control)

Nociceptive (% control)

Non-nociceptive (% control)

Current (nA)

81.6 n=4

1299.6 n=22

1178.9 n=22

515.8* n=10

1018.1 n=6

292.3

Values in the table for each agonist or sensory response type are means of percentage of control  S.E. of the mean for n neurons. The mean iontophoretic current for LY367385 is shown in the final column. Values marked with * or ** are significantly different from control values (P<0.05, P<0.01: Wilcoxon signed-rank test).

Fig. 1. Peristimulus time histograms from a single nociceptive thalamic neuron responding to iontophoretic applications of ACPD, NMDA and AMPA (A) or to noxious stimulation of the contralateral hindpaw (H/Paw, B). Histograms show action potential spikes counted into 1000 ms epochs (‘‘bins’’), and agonists/stimuli were presented as indicated by the markers above each record. In both A and B, upper records are controls, middle row of records are in the presence of LY367385 (ejected at 40 nA prior to the start of the record), and lower records are recoveries commenced 5 min after the end of the antagonist ejection. A: LY367385 antagonized the response to ACPD, but not the response to NMDA or AMPA. B: LY367385 reduced the response to noxious thermal stimulation.

Responses of neurons to iontophoretic applications of either ACPD (18 neurons) or DHPG (four neurons) were significantly reduced during continuous iontophoretic application of LY367385 (10–40 nA) whereas responses to either NMDA or AMPA were relatively unaffected (Table 1). The effects of the antagonist were seen from between five and 15 min after the start of the iontophoretic ejections, and lasted for between five and 20 min after the end of the ejections (Fig. 1A). Given that LY367385 appeared to be a selective antagonist, we studied the effects this compound on the nociceptive responses of 10 of these neurons. Nociceptive responses were evoked by immersion of part of either the contralateral hindpaw or the tail in water of 52C for 15–25 s. These stimuli were repeated at 5-min intervals. Responses to such

stimuli typically increased during the course of the stimulus and outlasted the stimulus by up to 2 min, as described previously.5,7,17 Application of LY367385 with the same iontophoretic currents and ejection durations which had produced selective antagonism on the same neurons was found to reduce the nociceptive responses of all of these neurons to 51  5.8% of their control responses (Fig. 1B, Table 1). In addition, a further six of the 22 neurons recorded were studied to investigate the effects of LY367385 on non-nociceptive responses evoked by stimulation of the vibrissae with an air jet (2-s duration).21 The antagonist had little effect on these responses (101  8.1% of control responses), although responses of the same neurons to either ACPD or DHPG were reduced by LY367385.

mGluR1 function in thalamus

The data obtained with LY367385 are consistent with our previous findings with a number of phenylglycine antagonists in the thalamus.5,22 Given the high selectivity of LY367385 for metabotropic glutamate receptor-1 (mGluR1) over mGluR5,4 the predominant expression of mRNA for mGluR1 compared to mGluR5 in the thalamus,1,12,20,24 and the immunohistochemical staining for mGluR1a rather than mGluR5 on the thalamic relay neuron dendrites,9,11,26 it seems highly likely that the ability of LY367385 to antagonize ACPD and DHPG responses in the present study is attributable to an action at mGluR1. Thus, this compound appears to be a useful tool to distinguish between mGluR1 and mGluR5 in studies of synaptic function. In this study, we have exploited this in order to test more directly whether sensory responses of thalamic neurons involve mGluR1: our finding that LY367385 reduces thalamic nociceptive responses now provides strong evidence that these responses are mediated in part by mGluR1. It is, of course, possible that other mGluRs may also contribute to this response, but more conclusive data cannot be obtained until appropriate selective antagonists (e.g., for mGluR5) are developed. The reduction of responses to nociceptive stimuli by LY367385 is the first direct pharmacological demonstration of a synaptic response mediated by mGluR1. The anatomical localization of mGluR1a in the thalamus suggests that it may be postsynaptic to cortical axon terminals.9,11,26 Furthermore, there is some electrophysiological evidence to suggest that the corticothalamic input may utilize Group I mGluRs.6,14 This raises the intriguing possibility that the nociceptive response of thalamic neurons may be dependent upon a cortical input which may be mediated, at least partly, via mGluR1. It is of interest

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to note that we have previously found that thalamic nociceptive responses also show an involvement of NMDA receptors.7 Thus it is likely that the complete physiological response to such stimuli is a summation of NMDA receptor and mGluR1 components (possibly with other components still to be identified). It is well known that NMDA receptor-mediated responses are voltage-dependent under physiological Mg2+ concentrations,13,16 and that postsynaptic Group I mGluR responses may be mediated via a decreased K+ conductance.3,14,25 Thus, an mGluR input would be in a good position to amplify the non-linear NMDA receptor-mediated response: this would make a combined NMDA receptor/mGluR1 input an effective signalling system. This could be further enhanced by the second-messenger-mediated facilitation of NMDA responses by Group I mGluRs which is known in several systems.2,8,19 This could also provide a mechanism for long-term changes in response to noxious inputs, which may be important in the development of central pain syndromes.5 CONCLUSION

In conclusion, we have shown that the novel antagonist LY367385 is a useful tool for studies of synaptic pharmacology aimed at identifying a role for mGluR1. We have used this compound to show that physiological responses to noxious stimuli of thalamic neurons are partly mediated by mGluR1. This finding may be of importance in the development of novel analgesic therapies. Acknowledgements—This work was supported by the Wellcome Trust. We are grateful to Dr A. E. Kingston, Lilly Research Centre, Windlesham, for the gift of LY367385 and helpful discussions.

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