T E Salt and K E Binns

Institute of Ophthalmology, University College London, 11-43 Bath Street, LONDON EC1V 9EL
 

Keywords

metabotropic glutamate receptors; ventrobasal thalamus; pain; NMDA receptors; in vivo electrophysiology; somatosensory synapses
 
 

Previous work from this laboratory has shown that nociceptive responses of rat ventrobasal thalamus neurones can be reduced by N-methyl-D-aspartate (NMDA) antagonists and by selective metabotropic glutamate receptor mGlu1 antagonists. The recent development of the mGlu5-selective antagonist 6-methyl-2-(phenylethynyl)-pyridine (MPEP) now allows the direct probing of possible mGlu5 involvement in thalamic nociceptive responses. Extracellular recordings were made from single neurones in the ventrobasal thalamus and immediately overlying dorsal thalamic nuclei of adult urethane-anaesthetised rats using multi-barrel electrodes. Responses of neurones to iontophoretic applications of the mGlu5-selective agonist (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) were selectively reduced during continuous iontophoretic applications of MPEP. Similar applications of MPEP reduced neuronal responses to noxious thermal stimuli to 53±9.5 % of control responses. Co-application by iontophoresis of NMDA and metabotropic glutamate receptor agonists resulted in a mutual potentiation of excitatory responses. This effect could be reduced by either MPEP or the mGlu1 antagonist LY367385. These results, taken together with previous data, suggest that acute thalamic nociceptive responses are mediated by a combination of mGlu1, mGlu5 and NMDA receptor activation, and that co-activation of these receptors produces a synergistic excitatory effect. Thus blockade of any of these receptor types would have a profound effect on the overall nociceptive response.
 

It is known that metabotropic glutamate receptors can play a role in the signalling of nociceptive information both spinally and supra-spinally^{13,15,17,27,28,40,45}. The mGlu receptors can be divided into three groups, I-III, on the basis of their sequence homologies, their agonist and antagonist pharmacology, and their coupling to intracellular transduction mechanisms in expression systems^10,25. The Group I (mGlu1 and mGlu5) receptors, which are known to couple to postsynaptic inositol phosphate metabolism, have in particular been implicated in nociceptive responses in both the spinal cord and the thalamus^{8,13,15,17,28,40,45}. This is based on the use of antagonists with varying degrees of selectivity, and upon the use of antisense oligonucleotides. Some of these studies have suggested a role for mGlu1 receptors at both the level of the spinal cord⁴⁵ and the thalamus⁴⁰. However, the demonstration of a specific role for mGlu5 receptors in nociception has not been possible until recently due to the lack of specific antagonists for this receptor. The development of the selective mGlu5 antagonist MPEP has provided a suitable pharmacological tool for experimental purposes^18,37.
 

The ventrobasal (VB) thalamus is a pivotal relay and processing point for somatosensory information ascending from the spinal cord to the cerebral cortex, and as such it is clearly a prime potential site for the action of analgesic drugs^21,30. We have previously shown that mGlu1 receptors⁴⁰ and NMDA receptors¹⁴ contribute to nociceptive responses of rat thalamic neurones. The aim of the present study was to determine whether a role in acute nociceptive signalling could be identified for mGlu5 receptors in addition to mGlu1 receptors in the thalamus by the use of MPEP. Furthermore, in view of the known interactions between Group I mGlu receptors and NMDA receptors in several brain areas^{2,12,16,20,31}, and the involvement of NMDA receptors in thalamic nociceptive processing^4,14, it was of interest to determine whether functional interactions between NMDA responses and mGlu1 and mGlu5 responses could occur in the thalamus in vivo. We have attempted to investigate this using a number of agonists which show activity at Group I mGlu receptors and the selective mGlu1 and mGlu5 antagonists LY367385⁹ and MPEP¹⁸. Some of these results have been presented in abstract form.^35,36
 
 

Experiments were carried out in adult Wistar rats (300-500g) anaesthetised with urethane (1.2g/kg. I.P.), as previously described ³⁴. A tracheal cannulation was made and the rats were allowed to breathe spontaneously. In some animals an external jugular vein was cannulated to allow intravenous administration of drugs. The electrocardiogram waveform and rate was monitored throughout each experiment via limb surface electrodes. The electroencephalogram was recorded and monitored throughout the experiment via two screw electrodes fixed and cemented over the frontal-occipital cortex contralateral to the thalamus from which single neurone recordings were made. Anaesthesia was periodically supplemented during the experiment (50mg urethane, I.P. bolus) as necessary to maintain absence of withdrawal reflexes to hind paw pressure. All animal experiments were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines.
 

Extracellular recordings were made from single neurones in the VB thalamus and immediately dorsal thalamus using the central barrel of seven-barrel iontophoretic micropipettes. Action potential spikes were gated using a hardware spike-discriminator whose output pulses were timed and recorded by the CED1401 interface and computer system. The amplitude and shape of the gated action potentials were monitored throughout the recording session. Neurones were identified on the basis of their stereotaxic location and their responses to somatosensory (nociceptive and non-nociceptive) stimuli, as described previously ^19,30,34. Iontophoretic applications of glutamate receptor agonists and antagonists were made from the outer barrels of the micropipettes which contained one of the following aqueous solutions: ACPD [(1S,3R)-1-aminocyclopentane-1,3-dicarboxylate] 50mM, pH8; CHPG [(R,S)-2-Chloro-5-hydroxyphenylglycine] 100mM, pH8; DHPG [(S)-3,5-Dihydroxyphenylglycine] 50mM, pH5; NMDA [N-methyl-D-aspartate] 50mM, pH8; AMPA [(R,S)-a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate] 50mM, pH8; MPEP [6-methyl-2-(phenylethynyl)-pyridine] 2mM or 10mM in 150mM NaCl, pH5; LY367385 [(+)-2-methyl-4-carboxyphenylglycine] 50mM, pH8. In addition, one barrel contained 1M NaCl for automatic current balancing. All drugs were ejected iontophoretically as anions (with the exception of MPEP and DHPG), and prevented from diffusing out of the pipette by a retaining current (10-20nA) of opposite polarity to the ejection current. MPEP was provided by Novartis (Basel, Switzerland), LY367385 by Lilly Research Labs (Erl Wood Manor, UK), other drugs were purchased from Tocris (Bristol, UK) or Sigma.
 

Regular repeated cycles 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. Agonist ejection parameters were adjusted so as to produce sub-maximal responses. In the experiments where interactions between agonists were investigated, agonist ejections were adjusted to produce only minimal responses when ejected alone. The effects of antagonists were assessed by continuous iontophoretic application of antagonists during several cycles of agonist ejection. Antagonist ejection currents and durations were adjusted so as to achieve selective antagonism of appropriate agonist responses compared to responses to other agonists of the same neurones.
 

Nociceptive responses were evoked by immersion of part of either the contralateral hindpaw or the tail in water of 52C for 20-30sec. 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^13,14,29. When nociceptive responses were challenged with iontophoretic application of an antagonist, the duration and magnitude of the iontophoresis current was in all cases the same as that which had been shown to be effective and selective against the appropriate agonist on the same neurone.
 

Responses to agonists or noxious stimuli were quantified as the number of action potentials evoked by agonist ejection or stimulus. The effects of antagonists on these responses were assessed by calculating the agonist or stimulus response during antagonist application as a percentage of the response under control conditions. In agonist interaction experiments, the number of action potentials evoked by the co-application of agonists and by the application of the iGlu agonist alone and the mGlu agonist alone were counted and used to calculate an estimate of the degree of response potentiation as shown below:
 

The potentiation excess (Px) = [number of action potentials obtained ]

minus [number of action potentials expected]

Data from individual neurones were used to compute mean values of effects. Statistical comparisons of these values under control conditions and during antagonist applications were made using the Wilcoxon Signed Rank test. Results were deemed to be significant when P<0.05 .
 
 

Metabotropic Glutamate Receptors and nociceptive responses.
 

The effects of the mGlu5 antagonist MPEP applied iontophoretically on responses to the mGlu5-selective agonist CHPG¹², the broad-spectrum mGlu agonist ACPD¹⁰, and NMDA were investigated in 17 neurones. In all of these neurones responses to regular ejections of agonists were recorded over several 5-minute cycles prior to the ejection of MPEP. The antagonist was then continuously ejected for one or more agonist ejection cycles, and the MPEP ejection was terminated when a selective effect was seen or it was deemed that no effect was evident. Agonist ejection cycles were then continued until recovery from the effects of MPEP were seen (5-15 minutes). MPEP ejection currents and durations were adjusted so as to produce selective antagonism of CHPG responses whilst having little or no effect on NMDA responses. Under these conditions MPEP (20-120 nA) reduced responses to CHPG to32 % of control whilst having less effect on ACPD responses and no significant effect on NMDA responses (Table 1a). Six of these neurones were then studied further with noxious stimuli: the agonist ejection cycles were terminated and replaced with noxious stimuli applied at 5 minute intervals. When control responses to such stimuli had been established, MPEP was applied continously with the same ejection current and duration which had been found to be effective against CHPG on that neurone. Under these conditions MPEP was able to reduce the responses to noxious stimulation to an average of 53% of control levels (Table 1b, Figure 1). We have previously shown that the mGlu1-selective antagonist LY367385 ⁹ can reduce responses of thalamic neurones to ACPD compared to CHPG and reduce responses to noxious stimuli to 51% of control values ^40,41. These data are therefore included for comparison (Table 2).
 
 
 

Interactions between N-methyl-D-aspartate and Metabotropic Glutamate Receptor agonists.
 

In order to investigate the possibility for mutual enhancement of responses mediated by NMDA receptors and those mediated by Group I mGlu receptors, experiments were carried out where responses of thalamic neurones to NMDA and mGlu agonists alone were compared with those where the two types of agonist were applied together. In such experiments, responses to the two agonists applied together were greater than the arithmetic sum of the responses to the agonists when applied alone (Table 3). This was the case when NMDA was co-applied with any of CHPG, ACPD or DHPG (Figure 2). In some experiments, either MPEP or LY367385 was applied during this protocol. MPEP was able to reduce the effects of CHPG application, whereas LY367385 was able to reduce the effects of ACPD application (Figure 2 and Table 4). In order to investigate the selectivity of the interaction between NMDA and mGlu agonists, some experiments were also carried out with the ionotropic agonist AMPA and ACPD. It was found that there was a potentiation of responses when these agonists were co-applied, similar to that seen when NMDA and mGlu receptor agonists were co-applied (Table 3).
 
 

In the present study we have found that the mGlu5 antagonist MPEP reduces nociceptive responses of thalamic neurones. In addition, we have confirmed the previous findings by ourselves³⁷ and others¹⁸ that MPEP is a suitable antagonist to block mGlu5 receptor-mediated responses in the brain. It is known that mGlu5 receptors are present in the rat thalamus^1,26,32,43, and it is likely that these receptors are responsible for the excitatory responses seen upon iontophoretic application of CHPG, which are antagonised by iontophoretically-applied MPEP³⁷. Interestingly, responses to the mixed mGlu-receptor agonist ACPD were much less affected by MPEP, indicating that these responses are mediated predominantly by a receptor other than mGlu5. Given the comparatively high levels of mGlu1 receptors in the thalamus^23,26,42, it is probable that the major receptor contribution to the ACPD responses is via mGlu1. This is supported by our previous finding that thalamic responses to ACPD are very sensitive to the selective mGlu1 receptor antagonist LY367385⁴¹. It is also of interest that responses to CHPG were not completely blocked by MPEP (Table 1A) and that LY367385 in fact had a small effect on such responses (Table 2A). This suggests that CHPG may have some effect at mGlu1 receptors.
 

In this study, we have shown that application of either ACPD, DHPG or CHPG can potentiate responses to NMDA. Furthermore, these effects can be reduced by the selective antagonists LY367385 and MPEP. This suggests that activation of either mGlu1 or mGlu5 receptors results in potentiation of NMDA receptor-mediated responses. This is consistent with other reports in the literature which indicate that there is an interaction between NMDA and Group I mGlu receptors^{2,12,16,20,31}. However, it is also evident from our data that an interaction between AMPA and mGlu responses is possible in the thalamus, an effect also seen in other brain areas^3,6,7,11,22. It is known that activation of Group I mGlu receptors in the thalamus in vitro causes a postsynaptic depolarisation associated with a increase in input resistance, possibly due to a reduction in a membrane potassium conductance^24,44. Such an effect would result in the potentiation of other depolarising inputs mediated by an inward current, such as those due to NMDA or AMPA receptor activation. It is thus possible that the potentiation between NMDA/AMPA and mGlu responses seen in the present study is due to such a mechanism, rather than a specific interaction at the receptor level, or that the potentiation seen is a combination of these factors^3,22. This would require further investigation. Nevertheless, from a functional point of view, there is clearly a powerful facilitatory interaction between iGlu and mGlu1/mGlu5 receptor activation in the thalamus in vivo.
 

We have shown previously that nociceptive responses of thalamic neurones can be reduced by broad-spectrum mGlu antagonists^13,38 and by the mGlu1-selective antagonist LY367385⁴⁰, as well as by NMDA antagonists¹⁴. In this study we have now shown that thalamic nociceptive responses are sensitive to an mGlu5 antagonist. Interestingly, blockade of mGlu receptors does not result in a reduction of non-nociceptive (vibrissa/hair follicle afferent) responses in the thalamus¹³, although NMDA receptor blockade does reduce such responses³³. Taken together with the present findings, this indicates that there is a role for mGlu1, mGlu5 and NMDA receptors in the generation of thalamic nociceptive responses. It is intriguing that when broad-spectrum mGlu antagonists were used,¹³ a slightly greater reduction of nociceptive responses was seen than with the subtype-selective antagonists MPEP or LY367385. This further suggests that there is activation of both mGlu1 and mGlu5 and that these have distinct contributions to the nociceptive response. Furthermore, the potent effect of MPEP against nociceptive responses suggests that mGlu5 is an important contributor to supraspinal nociception even though mGlu1 is the predominant receptor in the thalamus. The current experiments indicate a role for Group I mGlu receptors in acute nociception. However, it is also evident that such receptors participate in chronic nociception and central sensitisation^5,27, a matter that will need to be addressed for thalamic responses in the future.
 

We have now shown that activation of mGlu1 and/or mGlu5 receptors can potentiate responses to NMDA in the thalamus and it is thus likely that these receptors interact synergistically to produce the nociceptive response in the thalamus. A consequence of this would be that blockade of any of these receptor types would have a profound effect on the overall nociceptive response. Furthermore, given the likely complexities of mGlu receptor interactions with presynaptic and postsynaptic, and excitatory and inhibitory synaptic elements in the thalamus ³⁹, it is probable that there are still further factors to be elucidated in this system.
 
 
 

Acknowledgements

We thank F Gasparini & R Kuhn for donations of MPEP and B Clark & A Kingston for donations of LY367385. This study was supported by Novartis and The Wellcome Trust.

A. Data for all neurones where MPEP was tested. B. Data from a sub-set of neurones where sensitivity of nociceptive responses to MPEP was also tested. Values in the table for each nociceptive or agonist response type are means of percentage of control + SEM for n neurones. Values marked with * or ** are significantly different from control values (P<0.05, P<0.01: Wilcoxon signed rank test).
 
 
 

Table 2. Effects of LY367385
 

LY367385	Nociceptive	CHPG	ACPD	NMDA	n
A		78±9.1*	11±2.4**	128±10.3*	9
B	51±5.8**		17±3.9**	110±8.6	10

 

A. Data for neurones where LY367385 was tested against ACPD and CHPG⁴¹. B. Data from neurones where sensitivity of nociceptive responses to LY367385 was tested⁴⁰. Values in the table for each nociceptive or agonist response type are means of percentage of control + SEM for n neurones. Values marked with * or ** are significantly different from control values (P<0.05, P<0.01: Wilcoxon signed rank test).
 

Table 3. Potentiation of the effects of NMDA and AMPA by co-application with Group I mGlu receptor agonists.
 

Co-applied agonists	response expected  (action potentials)	response obtained (action potentials)	Px	n
NMDA / ACPD	112 ± 23.7	419 ± 51.5**	307 ± 37.2	16
NMDA / CHPG	106 ± 12.9	325 ± 48.5**	218 ± 46.2	16
NMDA / DHPG	117 ± 42.4	269 ± 46.4*	152 ± 23.6	6
AMPA / ACPD	119 ± 45.2	513 ± 126.5**	393 ± 90.7	7

 

Comparisons of the response obtained with the response expected upon co-application of different iGlu and mGlu agonists, and the calculated Potentiation excess (Px) value. Values in the table are means + SEM for n neurones. Values marked with * or ** are significantly different from expected values (P<0.05, P<0.01: Wilcoxon signed rank test).
 
 
 
 
 
 
 

Table 4. Effects of antagonists on agonist potentiation.
 

Co-applied agonists/antagonist	response expected  (action potentials)	response obtained (action potentials)	Px	n
NMDA / ACPD	85 ± 21.5	428 ± 66.2	343 ± 57.0	6
NMDA / ACPD  with LY367385	70 ± 19.2	184 ± 42.9	114 ± 33.3*
NMDA / CHPG	104 ± 25.1	288 ± 65.4	184 ± 44.2	5
NMDA / CHPG  with MPEP	138 ± 55.4	79 ± 20.4	13 ± 35.6*

Similar values to those shown in Table 3, but taken from those experiments where either LY367385 or MPEP were applied. Note that the antagonists significantly reduced the Px values (* P<0.05: Wilcoxon signed rank test).

Responses of a single thalamic neurone to iontophoretic applications of ACPD, CHPG and NMDA (A) and noxious thermal stimulation (B). Records are peristimulus-histograms of action potentials ("spikes") collected in successive 1000ms epochs ("bins") under either control conditions, during MPEP iontophoresis, or recovery after the end of the MPEP ejection. Timing of agonist ejections and stimulation are shown by the marker bars. Note that MPEP selectively reduced responses to CHPG (A) and noxious stimulation (B), as indicated by the arrows.
 

Peristimulus time histograms showing potentiation between ACPD and NMDA (A) and CHPG and NMDA (B) taken from two different neurones. For details see figure 1.
 

A1 Responses to ACPD and NMDA alone and together under control conditions. Note the potentiation of responses when the agonists are co-applied. A2 Responses during the continuous application of LY367385, 15 minutes after the start of the antagonist ejection. A3 Recovery from the effects of LY367385.
 

B1-B3 Similar records showing potentiation between CHPG and NMDA, and the effect of MPEP, 10 minutes after the start of the antagonist ejection.
 

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

AMPA (R,S)-a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate

CHPG (R,S)-2-Chloro-5-hydroxyphenylglycine

DHPG (S)-3,5-Dihydroxyphenylglycine

iGlu ionotropic glutamate

LY367385 (+)-2-methyl-4-carboxyphenylglycine

MPEP 6-methyl-2-(phenylethynyl)-pyridine

mGlu metabotropic glutamate

NMDA N-methyl-D-aspartate

VB ventrobasal