Group I metabotropic glutamate receptors in the thalamus

Preprint of paper to be published in Neuropharmacology

Evaluation of agonists and antagonists acting at Group I metabotropic glutamate receptors in the thalamus in vivo.

T E Salt*, J P Turner & A E Kingston

Institute of Ophthalmology, University College London, 11-43 Bath Street,

LONDON EC1V 9EL, United Kingdom

and

Lilly Research Centre, Erl Wood Manor

Windlesham, Surrey GU20 6PH

KEYWORDS

thalamus; mglu1; mglu5; metabotropic glutamate receptor antagonist; LY367366; LY367385; LY393053; AIDA

SUMMARY

Recordings were made from single neurones in the ventrobasal thalamus of anaesthetised rats in order to evaluate the properties of several agonists and antagonists of Group I mGlu receptors. The selective mGlu1 receptor antagonist LY367385 was found to reduce excitatory responses to iontophoretically applied ACPD and DHPG whereas the mGlu5 agonist CHPG was resistant to antagonism. The antagonists LY367366 and LY393053 reduced responses to all three agonists, but without reducing responses to NMDA or AMPA. Although AIDA was also found to reduce mGlu agonist-evoked responses, this antagonist also produced significant reductions in responses to NMDA and AMPA. These data suggest that there are functional mGlu1 and mGlu5 receptors in the thalamus. Furthermore, LY367385 is a useful tool for investigating mGlu1 functions whereas LY367366 and LY393053 have a broader spectrum of action. The usefulness of AIDA as an antagonist in physiological experiments would appear to be limited by its effects against NMDA and AMPA.

INTRODUCTION

There is now compelling evidence to suggest that metabotropic glutamate (mGlu) receptors are involved in synaptic processes in various regions of the brain (Conn and Pin 1997). In particular, the Group I (mGlu1 and mGlu5) receptors appear to mediate postsynaptic metabotropic responses to glutamate, probably via coupling to inositol phosphate hydrolysis (Nakanishi 1992; Conn and Pin 1997). Until now, the lack of specific agonists and antagonists which are selective between mGlu1 and mGlu5 receptors has prevented the pharmacological and functional segregation of these receptors in postsynaptic processes. Recently, the mGlu5 receptor-selective agonist 2-chloro-5-hydroxyphenylglycine (CHPG) has been developed (Doherty et al. 1997), as have several newer antagonists such as 1-aminoindan-1,5-dicarboxylic acid (AIDA) (Moroni et al. 1997) and LY367385 Clark et al. 1997) which are reported to be mGlu1 receptor-selective. This opens up the possibility of revealing a functional distinction between mGlu1 and mGlu5 receptors in neuronal responses.

Previous work from this laboratory using a range of phenylglycine antagonists which are active at Group I mGlu receptors has indicated that such receptors mediate postsynaptic responses of ventrobasal thalamus neurones to the mGlu agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD), and that these receptors are involved in nociceptive responses of thalamic neurones (Eaton et al. 1993; Salt and Eaton 1994). However, although there is some suggestion of mGlu1 involvement (Salt and Turner 1998), these data do not allow conclusive determination of the degree of mGlu1 and mGlu5 mediation of responses. Indeed, although there is a large degree of mGlu1 mRNA expression in the thalamus (Masu et al. 1991; Shigemoto, Nakanishi, and Mizuno1992), there is also some expression for mGlu5 mRNA (Abe et al. 1992; Romano, Van den Pol, and O'Malley 1996), and immunohistochemical studies have revealed receptor protein for both of these receptor types in the thalamus (Martin et al. 1992; Romano, Van den Pol, and O'Malley 1996; Romano et al. 1995). The aim of the present study was to investigate a number of novel mGlu antagonists on the responses of single thalamic neurones to the mGlu agonists ACPD, (S)-3,5-dihydroxyphenylglycine (DHPG) and CHPG in an attempt to provide pharmacological evidence for the existence of functional mGlu5 and mGlu1 receptors in the thalamus, and to evaluate antagonists as a prelude to their use in studies of sensory responses. These antagonists are the mGlu1 receptor-selective AIDA (Moroni et al. 1997), LY367385 (Clark et al. 1997), the mGlu1/5 receptor-selective LY367366 (Bruno et al. 1999), and the broader spectrum LY393053 (Baker et al. 1998) (Figure 1).

METHODS

All experiments were carried out in adult Wistar rats, anaesthetised with urethane (1.2g/kg, Intra Peritoneal) and prepared for recording as previously described (Salt1987). All animal experiments were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines. Extracellular single-neurone recordings were made through the centre barrel (containing 4M NaCl) of seven-barrel iontophoretic electrodes. Outer electrode barrels 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- ethyl-4-isoxazolepropionate] 50mM, pH8; AIDA [(R,S)-1-aminoindan-1,5-dicarboxylic acid] 50mM, pH8; LY367385 [(+)-2-methyl-4-carboxyphenylglycine] 50mM, pH8; LY367366 [(±)-a-thioxanthylmethyl- -carboxyphenylglycine] 25mM in 75mM NaCl, pH8; LY393053 [(±)-a-thioxanthylmethyl-3- arboxycyclobutylglycine] 25mM in 75mM NaCl, pH8. In addition, one barrel contained 1M NaCl which was used for automatic balancing of iontophoresis currents in all experiments. 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 purchased from Tocris, apart from LY367385, LY367366, and LY393053 (from Lilly Research).

Thalamic neurones were identified on the basis of their stereotaxic location and responses to somatosensory stimuli, as described previously (Salt 1987; Salt and Eaton 1994). Regular repeated cycles (3-5 minute duration) containing two or (in most cases) three agonist ejections (10-20s durations, 40-95s intervals) were set up and initiated by a computer system. Agonist ejection parameters were adjusted so as to produce sub-maximal responses. Extracellular action potentials were gated and timed using the computer system, which could produce peristimulus-histograms of single-neurone activity both online and offline. 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 either ACPD or DHPG responses compared to responses to other agonists wherever possible, and the rows in the results tables (Tables 1 and 2) represent data obtained from such comparisons. Responses to agonists were quantified as the number of action potentials evoked by agonist ejection. The effects of antagonists on these responses were assessed by calculating the agonist response during antagonist application as a percentage of agonist response under control conditions. Baseline activity of neurones was not taken into account in these calculations as this was typically a low contribution of the overall count of action potentials.

RESULTS

Recordings were made from 65 neurones responsive to ejection of mGlu and ionotropic glutamate receptor agonists. In all experiments at least one mGlu agonist and one ionotropic glutamate receptor agonist was included in the study. All of the mGlu agonists were able to evoke excitatory responses which were similar in time course to those we have described previously for ACPD (Salt and Eaton 1991). It was a common finding that higher iontophoretic currents of CHPG (150-450nA) were required to produce responses which were similar or slightly smaller in magnitude to responses to either ACPD (40-100nA) or DHPG (50-100nA).

The antagonist AIDA was tested on 11 neurones with iontophoretic currents of 40-120nA. This compound reduced responses to ACPD in all cases, but was also found to reduce responses to AMPA and /or NMDA to a significant extent (Figure 2). These data are summarised in Table 1. In view of these data, AIDA was not investigated with other mGlu agonists. In contrast, LY367385 (5-40nA), LY367366 (5-60nA) and LY393053 (2-20nA) were all able to reduce responses to ACPD or DHPG with little effect on responses to either NMDA or AMPA (Figure 3), as summarised in Table 1. However, it is noteworthy that LY367385 was able to produce a slight, but significant, enhancement of responses to NMDA. (Tables 1 and 2). Recovery from the effects of LY367385 and LY367366 were typically seen within 1-15 minutes after termination of the iontophoretic ejection, whereas recovery from the effects of LY393053 had a more protracted time course (up to 25 minutes).

When tested in experiments (11 neurones) where CHPG was included, LY367385 was found to reduce selectively either ACPD or DHPG compared to CHPG (Figure 4), as summarised in Table 2. In contrast, LY367366 was found to reduce responses to both ACPD and CHPG (Figure 5) on the neurones tested (Table 2).

DISCUSSION

The finding that CHPG was able to evoke excitatory responses when applied to thalamic neurones indicates that there are functional mGlu5 receptors present on these neurones. Furthermore, the finding that it is possible to antagonise responses to ACPD and DHPG with less effect on responses to CHPG with LY367385 indicates the presence of a further population of receptors, presumably mGlu1 receptors. mRNA expression studies suggest that both mGlu1 and mGlu5 receptors are present, the former predominating (Masu et al. 1991; Abe et al. 1992). This dominance of mGlu1 receptors could perhaps explain the apparent weaker potency of CHPG compared to ACPD and DHPG based on iontophoretic currents seen in this study, although it must be remembered that it is not possible to quantify potency in iontophoretic experiments. Alternatively, as it is known that CHPG is a weak agonist in vitro (Doherty et al. 1997) it is quite conceivable that the lack of potency seen here is due to this intrinsic lack of potency. This matter could be resolved when more potent and selective mGlu1 and mGlu5 receptor agonists become available.

It is intriguing that both ACPD and DHPG could be antagonised to a large extent by LY367385, which is known to be selective for mGlu1 receptors (Clark et al. 1997). As both of these agonists are known to act at both mGlu1 and mGlu5 receptors, this finding suggests that most of the excitatory response seen upon iontophoretic application of these mixed agonists is mediated via mGlu1 receptors. This could be due to the preponderance of mGlu1 receptors compared to mGlu5 receptors in this brain area (see above). AIDA has also been described as an mGlu1-receptor-selective antagonist (Moroni et al. 1997). However, although this compound did reduce neuronal responses to ACPD, which is consistent with it being an mGlu receptor antagonist, it also reduced NMDA and AMPA responses to a significant extent. An effect of AIDA on NMDA receptors expressed in oocytes has been described recently (Contractor et al. 1998). Furthermore, an effect of AIDA on NMDA responses has also been found in an in vitro spinal cord study (Krieger, Grillner, and ElManira 1998), and another study found complex actions of this compound in the spinal cord (Pinkney et al. 1998). This would appear to limit its usefulness as a tool in physiological studies, and its use should be approached with caution unless suitable control experiments are carried out. In contrast, LY367366 and LY393053 appear to be more useful in that they antagonised postsynaptic responses to the mGlu agonists used in this study whilst having little effect on responses to NMDA or AMPA. This is consistent with previous findings with these compounds (Thomas et al. 1998; Baker et al. 1998; Bruno et al. 1999).

In conclusion, the data presented here indicate that in the ventrobasal thalamus there are functional mGlu1 and mGlu5 receptors which mediate excitatory neuronal responses, and that these mGlu5 receptors can be selectively activated by CHPG. Furthermore, LY367385 appears to be useful to antagonise mGlu1 receptors selectively in the thalamus, whereas LY367366 and LY393053 are more useful as broader-spectrum mGlu receptor antagonists.

REFERENCES

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Baker SR, Clark BP, Harris JR, Griffey KI, Kingston AE, and Tizzano JP. (1998). LY393675, an a-substituted-cyclobutylglycine, is a potent Group I metabotropic glutamate receptor antagonist. Society for Neuroscience Abstracts 28: 229.16.

Bruno V, Battaglia G, Kingston AE, O'Neill MJ, Catania MV, Di Grezia R, and Nicoletti F. (1999). Neuroprotective activity of the potent and selective mGlu1a metabotropic glutamate receptor antagonist (+)-2-methyl-4-carboxyphenylglycine (LY367385): Comparison with LY367366, an antagonist of mGlu1a and mGlu5 receptors. Neuropharmacology 38: 119-207.

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Doherty AJ, Palmer MJ, Henley JM, Collingridge GL, and Jane DE. (1997). (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) activates mGlu5 but not mGlu1 receptors expressed in CHO cells and potentiates NMDA responses in the hippocampus. Neuropharmacology 36: 265-267.

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TABLE 1

Effects of Antagonists on Amino Acid Responses

Antagonist	ACPD (% control)	DHPG (% control)	NMDA (% control)	AMPA (% control)	Current (nA)
A AIDA	20±3.0***	-	52±11.7*	55±14.5 *	87±6.9
	n=11		n=11	n=7	n=11
B	13±1.9***	-	128±8.3***	116±10.5	22±2.2
	n=27		n=27	n=18	n=27
C LY367385	-	13±3.5*	124±9.6	122±12.8	17±1.9
		n=6	n=6	n=4	n=6
D LY367366	20±4.4***	-	106±7.7	108±7.4	27±5.9
	n=13		n=13	n=7	n=13
E LY393053	10±3.0**	-	96±12.4	94±17.2	10±2.3
	n=10		n=10	n=9	n=10

Values are means (± standard error) of agonist responses in the presence of antagonist expressed as percentages of control values, and the mean (± standard error) iontophoretic current of antagonist used to block agonist responses. n values represent numbers of neurones in each group. A - effects of AIDA when tested against ACPD, compared to AMPA and/or NMDA; B- effects of LY367385 when tested against ACPD, compared to AMPA and/or NMDA; C - effects of LY367385 when tested against DHPG, compared to AMPA and/or NMDA; D,E - effects of LY367366 and LY393053, respectively in similar experiments. Values are significantly different from controls (Wilcoxon Signed Rank Test) as indicated: * P < 0.05; ** P < 0.01; *** P < 0.005.

TABLE 2

Comparison of Antagonists on CHPG and ACPD Responses

Antagonist	CHPG (% control)	DHPG (% control)	ACPD (% control)	NMDA (% control)	Current (nA)
A LY367385	78±9.1*	-	11±2.4**	128±10.3*	11±1.8
	n=9		n=9	n=9	n=9
B LY367385	74±6.7	25±0.7	-	111±10.6	10
	n=2	n=2		n=2	n=2
C LY367366	38±3.0*	-	31±6.7*	89±7.0	15±4.1
	n=6		n=6	n=6	n=6

Similar data to that in Table 1, but from the subset of neurones where the effects of antagonists on CHPG were directly compared with the effects on either ACPD or DHPG.

Figure 1

Structures of antagonists used in this study.

Figure 2

Peristimulus time histograms (PSTHs) of action potentials (spikes) counted into successive 1000ms epochs showing excitatory responses to iontophoretic ejections of ACPD, AMPA and NMDA at times indicated by the marker bars. The upper record is a control, below which is shown the same set of agonist applications during the co-application of AIDA. The lower record shows recovery from AIDA 15 minutes after the end of the iontophoretic ejection. Note that AIDA reduced responses to all three agonists.