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14 avril 2005
British Journal of Pharmacology
1999; 126; 1537-1542
Investigation of stretching behaviour induced by the selective S-HT6 receptor antagonist, Ro 04-6790, in rats
Jane C. Bentley, Anne Bourson, Frank G. Boess, Kevin C. F. Fone, Charles A. Marsden, Nadine Petit & Andrew J. Sleight
Pharma Division, Preclinical Research, F. Hoffmann La Roche Ltd, 4070 Basel, Switzerland


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 1. The present study examined the effects of the selective 5-HT6 receptor antagonist 4-amino-N-(2, 6 bis-methylamino-pyrimidin-4-yl)-benzene sulphonamide (Ro 04-6790) on locomotor activity and unconditioned behaviour in male Sprague Dawley rats (230?300 g).

2. In non-quantified behavioural observations, animals treated with Ro 04-6790 (3, 10 or 30 mg kg-1, i.p) showed no overt behavioural signs except a dose-dependent reduction in locomotor activity and a behavioural syndrome of stretching, yawning and chewing. The latter behaviour was most pronounced between 30 and 90 min following the administration of Ro 04-6790.

3. Detailed analysis of the stretching and yawning behaviour showed that Ro 04-6790 (3, 10 or 30 mg kg-1, i.p.) dose-dependently induced stretching. The number of stretches observed following treatment with either Ro 04-6790 (10 mg kg-1 i.p.) or Ro-04-6790 (30 mg kg-1, i.p.) was significantly greater than that observed in saline-treated rats. The yawning behaviour, however, was not dose-dependent nor was the number of yawns in any of the drug treated groups significantly greater than in those treated with saline.

4. Pretreatment (30 min) with the non-selective muscarinic antagonists scopolamine (0.1, 0.3 or 1 mg kg-1, i.p.) and atropine (0.3, 1 or 3 mg kg-1, s.c.) but not methylatropine (1, 3 or 10 mg kg-1, s.c) significantly inhibited stretching induced by Ro 04-6790 (30 mg kg-1, i.p.).

5. The dopamine D2-like receptor antagonist, haloperidol (0.03, 0.1 or 0.3 mg kg-1, s.c.) given at the same time as Ro 04-6790 (30 mg kg-1, i.p.) had no effect on the stretching induced by the 5-HT6 antagonist.

6. These data suggest that systemic injection of the 5-HT6 antagonist, Ro 04-6790, produces a stretching behaviour that appears to be mediated by an increase in cholinergic neurotransmission in the CNS and which could be a useful functional correlate for 5-HT6 receptor blockade. There is no evidence for dopamine D2-like receptor involvement in this behaviour.
The 5-hydroxytryptamine6 (5-HT6) receptor is one of 14 receptors which mediate the effects of the neurotransmitter, 5-hydroxytryptamine (5-HT, Hoyer & Martin, 1997). The rat receptor was cloned by reverse transcription and polymerase chain reaction with degenerate primers derived from conserved regions of known G-protein coupled receptors (Monsma et al., 1993) or by low stringency screening with probes derived from the histamine H2 receptor (Ruat et al., 1993). Subsequently, the human receptor was identified (Kohen et al., 1994). 5-HT6 mRNA is present in olfactory tubercle, nucleus accumbens, striatum and hippocampus (Monsma et al., 1993; Ruat et al., 1993; Ward et al., 1995; Gérard et al., 1996). The localization of the 5-HT6 receptor protein has been studied with polyclonal antibodies raised to a synthetic peptide corresponding to part of the C terminal region (Leu393-Val415) of the 5-HT6 receptor protein. In addition to the regions expressing 5-HT6 mRNA, 5HT6-like immunoreactivity was found in the frontal and entorhinal cortex and the molecular layer of the cerebellum (Gérard et al., 1997). Electron microscopy showed that the immunoreactivity is localized on distal dendrites of pyramidal and granular cells in the hippocampus and on medium spiny neurones in the striatum (Gérard et al., 1997).
Although the 5-HT6 receptor has a distinct pharmacological profile, with a high affinity for clozapine-related compounds (Roth et al., 1994; Boess et al., 1997), in vivo investigation of receptor function has been hindered by the lack of selective agonists or antagonists. Chronic intracerebroventricular (i.c.v.) treatment with an antisense oligodeoxynucleotide (A.O.) produced a behavioural syndrome comprising of yawning, stretching and chewing (Bourson et al., 1995). This behavioural syndrome was not observed in either saline or scrambled oligodeoxynucleotide (S,O.) treated animals but was accompanied by a 30% reduction in the number of [3H]-Lysergic acid diethylamide ([3H]-LSD) binding sites (measured in the presence of 300 nM spiperone). Therefore, it was proposed that this behaviour is a result of a reduction in the expression of the 5-HT6 receptor in the CNS.
Recently, potent and selective 5-HT6 receptor antagonists, 4-amino-N-(2,6 bis-methylamino-pyrimidin-4-yl)-benzene sulphonamide (Ro 04-6790) and 4-amino-N-(2,6 bis-methylamino-pyridin-4-yl)-benzene sulphonamide (Ro 63-0563) have been characterized (Sleight et al., 1998). Both of these compounds are competitive antagonists at recombinant 5-HT6 receptors. The latter has been radiolabelled and 5-HT6 receptor binding sites have been identified in the striatum of both rats and pigs (Boess et al., 1998). Ro 04-6790 has an affinity (pki) of 7.3 for both the rat and human 5-HT6 receptor, has over two log units of selectivity with respect to 23 other receptor binding sites (including eight other 5-HT receptor subtypes and all five muscarinic receptor subtypes) and can be measured in the cerebro-spinal fluid of rats following systemic administration (Sleight et al., 1998). Interestingly, Ro 04-6790 produced a similar behavioural syndrome to that produced by 5-HT6 antisense oligonucleotide treatment in rats that had been habituated to the observation cages for 4 days prior to being administered with Ro 04-6790. In these animals, stretching behaviour could be dose-dependently produced by Ro 04-6790 although yawning failed to reach statistical significance when compared to saline treated animals (Sleight et al., 1998).
In the present report, we detail experiments undertaken to evaluate the consequences of 5-HT6 receptor antagonism by studying the effect of Ro 04-6790 on locomotor activity and unconditioned behaviour with particular emphasis on behaviour indicative of depressant, stimulant and autonomic properties (Irwin, 1968). In addition we also examined whether the stretching behaviour could be seen in animals that have not been habituated to the observation cages and the mechanisms that mediate this response.
The aim of the present study was to evaluate the effect of the selective 5-HT6 receptor antagonist, Ro 04-6790 on spontaneous rat behaviour in a novel environment and to determine whether any observed effect was centrally or peripherally mediated. These data suggest that systemic administration of Ro 04-6790 induces a stretching behaviour, which is centrally mediated. These data would also suggest that in vivo, the receptor is either constitutively active or is under tonic activation of the endogenous neurotransmitter, 5-HT, since the administration of 5-HT6 antagonists alone induces a behavioural syndrome. Although yawning was also observed following treatment with Ro 04-6790, it was not significantly greater in any of the drug treated groups than in the control groups. In addition, chewing was observed in animals treated with Ro 04-6790 but this behaviour was not quantified. In a recent publication we showed that Ro 04-6790 induces stretching behaviour in rats habituated to the observation cages (Sleight et al., 1998), however, the present data suggest that prolonged habituation is not necessary.
Pretreatment with the muscarinic antagonists, atropine and scopolamine which cross the blood-brain barrier, prevented the stretching induced by the 5-HT6 antagonist. Therefore, it is proposed that blockade of the 5-HT6 receptor facilitates cholinergic neurotransmission which, in turn gives rise to the stretching behaviour. Methylatropine which does not penetrate into the brain (Herz et al., 1965) had no effect on the Ro 04-6790-induced stretching, suggesting that the behaviour is centrally mediated. This is in agreement with previous reports showing that the 5-HT6 receptor is predominantly expressed in the CNS and not in the periphery (Monsma et al., 1993). In addition, although dopamine D2-like receptors have been implicated in this type of behaviour (Argiolas & Melis, 1998), haloperidol failed to attenuate the stretching induced by Ro 04-6790. This suggests that dopamine D2-like receptors are not involved in mediating the stretching response to 5-HT6 receptor blockade.
The present study is consistent with previous work by Bourson et al. (1995) using a 5-HT6 receptor-directed antisense oligonucleotides (AO). Chronic (4 days) i.c.v. treatment with AO produced a behavioural syndrome of stretching, yawning and chewing which was attenuated by atropine (0.3, 1.0, 3.0 mg kg, s.c.) but not by haloperidol (0.03 mg kg, s.c.) pretreatment. The studies with AO thus correctly predicted both that decreased 5-HT6 receptor function induces stretching and that this behaviour can be blocked by cholinergic but not by dopaminergic antagonists. These results show that AO can be a valuable tool for the evaluation of the function of novel receptors for which selective antagonists are not yet available, provided that the correct procedures and controls are used. This is one of the first examples of the successful use of AO to predict function that has subsequently been confirmed with selective antagonists. Most AO studies have examined receptors for which the effects of selective antagonists were already known or the observations with AO have not yet been confirmed pharmacologically (Wahlestedt et al., 1993a,b; Zhou et al., 1994). While the stretching behaviour observed after AO treatment was confirmed with the 5-HT, receptor antagonist Ro 04-6790, the yawning seen after Ro 04-6790 treatment was neither dose-dependent nor statistically significant compared with saline treated animals, while it was clearly present after AO treatment. There are a number of possible explanations for this difference between AO treatment and that of a 5-HT6 antagonist. For example, the distribution of the antisense given i.c.v. will be different from that of Ro 046790, and therefore the two treatments may be affecting different pools of receptor. Another explanation could be that the treatment of animals with both the AO and SO exhibited non-specific toxic symptoms (Bourson et al., 1995) and this may affect the expression of the yawning behaviour.
Alternatively, it is possible that either the AO or Ro 04-6790 treatment is not completely specific for the 5-HT6 receptor and may interfere with other receptors and proteins. Clearly although stretching seems to be a result of decreased 5-HT6 receptor function, it is important to study whether other selective 5-HT6 antagonists will produce yawning.
Stretching and yawning have been reported following central administration of adrenocorticotrophic hormone (ACTH) and a-melanocyte stimulating hormone (MSH) and related peptides (Gessa et al., 1967). Further investigation into the mechanism surrounding this particular behavioural syndrome has indicated the involvement of a variety of neurotransmitters (acetylcholine; Ferrari et al., 1963, dopamine; Ferrari et al., 1993), neuropeptides (morphine; Bertolili & Gessa 1981), and inorganic ions such as calcium (Argiolas et al., 1990) and nitric oxide (Poggioli et al., 1995). Few other studies, however, have examined stretching and yawning separately, but have scored both behaviours together making comparison with the current data difficult. Moreover, investigators observing solely a yawning note that occasionally there is a 'sudden stretching of the forelimbs' proceeding the yawning behaviour (Urba-Holmgren et al., 1977; Yamada & Furukawa, 1980). In the current study stretching and yawning were clearly dissociated and only stretching was dosedependent. Numerous factors may influence expression of such behaviours in particular the observation protocol, size of the observation box, extent of habituation to the environment and the age of the rats used. In addition, further investigation may identify other neurotransmitters that are involved in mediating 5-HT6 receptor antagonist-induced stretching.
In conclusion, we have demonstrated that a 5-HT6 antagonist, Ro 04-6790, induces a stretching behaviour which is centrally mediated and similar to that previously reported following AO treatment. The behavioural syndrome was reversed by muscarinic antagonists suggesting that 5-HT6 receptors modulate cholinergic neurotransmission. In agreement with this finding we have recently reported that 5-HT6 receptor-directed AO treatment enhances acquisition in the Morris Water Maze (Bentley et al., 1997), suggesting that this potentiation of acetylcholine neurotransmission may give rise to enhanced cognitive-responses. It is not known, however, whether these effects directly involve an increase in the release of acetylcholine from cholinergic neurones in the rat CNS. Nevertheless, the interaction between the 5-HT,, receptor and cholinergic neurotransmission is particularly interesting with respect to disease states such as, Alzheimer's disease, where there is clear evidence of a cholinergic deficit (Bartus et al., 1982).