Reduction of sensory and metabotropic glutamate receptor responses in the thalamus by the novel mGluR1-selective antagonist S-2-methyl-4-carboxy-phenylglycine (LY367385).

T E Salt & J P Turner

11-43 Bath Street, LONDON EC1V 9EL.

Email: T.Salt@ucl.ac.uk

This document is a pre-print of a paper, the final version of which has been accepted for publication in 'Neuroscience'

Single-neurone recordings were made with extracellular multi-barrel iontophoretic electrodes in the ventrobasal thalamus and immediately overlying dorsal thalamic nuclei of adult male Wistar rats anaesthetised with urethane (1.2 g/kg, I.P.), as detailed previously²¹. The outer barrels were used for iontophoretic drug applications, and each contained one of the following substances, as Na⁺ salts: NMDA [N-methyl-D-aspartate], AMPA [(±)-a-amino-3-hydroxy -5-methyl-4-isoxazolepropionate], ACPD [1S,3R-aminocyclopentane-1,3-dicarboxylate], LY367385 [S-2-methyl-4-carboxy-phenylglycine] (all 50mM in water, pH 8.0-8.5), DHPG [S-3,5-dihydroxyphenylglycine] (50mM 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 (10-20nA) of opposite polarity to the ejection current. All drugs were obtained from Tocris, apart from LY367385 (gift from Lilly Research). Regular repeated cycles (5 minute 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-neurone activity.

Responses of neurones to iontophoretic applications of either ACPD (18 neurones) or DHPG (4 neurones) were significantly reduced during continuous iontophoretic application of LY367385 (10-40nA) whereas responses to either N-methyl-D-aspartate (NMDA) or (AMPA) were relatively unaffected (Table 1). The effects of the antagonist were seen from between 5 and 15 minutes after the start of the iontophoretic ejections, and lasted for between 5 and 20 minutes after the end of the ejections (Figure 1A). Given that LY367385 appeared to be a selective antagonist, we studied the effects this compound on the nociceptive responses of ten of these neurones. Nociceptive responses were evoked by immersion of part of either the contralateral hindpaw or the tail in water of 52^oC for 15-25sec. These stimuli were repeated at 5 minute intervals. Responses to such stimuli typically increased during the course of the stimulus and outlasted the stimulus by up to two minutes, 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 neurones was found to reduce the nociceptive responses of all of these neurones to 51±5.8% of their control responses (Figure 1B, Table 1). In addition, a further six of the 22 neurones recorded were studied to investigate the effects of LY367385 on non-nociceptive responses evoked by stimulation of the vibrissae with an air jet (2 second duration)²¹. The antagonist had little effect on these responses (101±8.1% of control responses), although responses of the same neurones to either ACPD or DHPG were reduced by LY367385.

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 mGluR1 over mGluR5⁴, 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 neurone dendrites^9,11,26, it seems highly likely that the ability of LY367385 to antagonise 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 neurones 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 localisation 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 utilise Group I mGluRs^6,14. This raises the intriguing possibility that the nociceptive response of thalamic neurones may be dependent upon a cortical input which may be mediated, at least partly, via mGluR1. It is of interest to note that we have previously found that thalamic nociceptive responses also show an involvement of NMDA receptors⁷. 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 Mg⁺⁺ 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 syndromes⁵.

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 neurones are partly mediated by mGluR1. This finding may be of importance in the development of novel analgesic therapies.

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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Table 1

Values in the table for each agonist or sensory response type are means of percentage of control + standard error of the mean for n neurones. 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).

Figure 1

Peristimulus time histograms from a single nociceptive thalamic neurone 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 1000ms 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 40nA prior to the start of the record), and lower records are recoveries commenced 5 minutes after the end of the antagonist ejection.

A: LY367385 antagonised the response to ACPD, but not the response to NMDA or AMPA.

B: LY367385 reduced the response to noxious thermal stimulation.

NMDA N-methyl-D-aspartate

AMPA (±)-a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate

ACPD 1S,3R-aminocyclopentane-1,3-dicarboxylate

LY367385 S-2-methyl-4-carboxy-phenylglycine

DHPG S-3,5-dihydroxyphenylglycine

mGluR metabotropic glutamate receptor

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