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.
Introduction
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.
Discussion
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).
