A role for 5-ht6 receptors in retention of spatial learning in the Morris water maze
Introduction
Mammalian 5-HT receptors (14) have been cloned and sequenced (Barnes and Sharp, 1999). One of the most recent additions is the 5-ht6 receptor that was defined by molecular biology rather than pharmacological tools (Branchek and Blackburn, 2000). Both the rat and human (Ruat et al., 1993, Monsma et al., 1993, Kohen et al., 1996) 5-ht6 receptor have been cloned and hydropathic analysis demonstrates that it has a typical seven transmembrane spanning G-protein linked structure and is positively coupled to adenylyl cyclase. The 5-ht6 receptor mRNA is highly expressed in the limbic and cortical regions of the rat brain, including the olfactory tubercles, striatum, nucleus accumbens and hippocampus (Monsma et al., 1993, Ruat et al., 1993, Ward et al., 1995, Kohen et al., 1996, Gérard et al., 1996, Gérard et al., 1997). The general consensus is that the 5-ht6 receptor is not expressed in the periphery, although one group did report 5-ht6 mRNA in the stomach of the guinea pig (Ruat et al., 1993). By using polyclonal antibodies directed against the C-terminal region of the 5-ht6 receptor, Gérard et al. (1997) identified diffuse immunoreactivity in the neuropil of structures such as the olfactory tubercles, dendrites in the granular cell layer in the hippocampus and the medium spiny neurones in the caudate putamen of the striatum.
Prior to the recent development of selective, 5-ht6 receptor antagonists (Sleight et al., 1998, Bromidge et al., 1999, Isaac et al., 2000) tools for the in vivo characterisation of 5-ht6 receptor function were limited. Chronic intracerebroventricular (i.c.v.) injection of a 5-ht6 receptor specific antisense oligonucleotide (AO) directed against the initiation codon region of the mRNA has been successfully used by several groups to down-regulate 5-ht6 receptor expression in the CNS and determine concomitant behavioural alterations (Bourson et al., 1995, Yoshioka et al., 1998, Hamon et al., 1999). In the initial study (Bourson et al., 1995), chronic i.c.v. injection of a 5-ht6 receptor-directed AO was found to elicit a specific behavioural syndrome comprised of yawning, chewing and stretching which was accompanied by a 30% decrease in -LSD binding compared with that in controls, providing the first evidence that the 5-ht6 receptor was functionally expressed in the rat brain. With a similar AO protocol, Yoshioka et al. (1998) reported attenuation of conditioned fear stress-induced 5-HT release using cortical microdialysis probes in conscious rats, suggesting that the 5-ht6 receptor may be involved in certain anxiety disorders. In support of this suggestion, Hamon et al. (1999), recently reported that a similar AO treatment decreased social interaction and the percentage time spent on the open arms of an elevated plus maze.
Recently a number of selective 5-ht6 antagonists have been characterised (Sleight et al., 1998, Bromidge et al., 1999, Isaac et al., 2000) enabling the specificity of 5-ht6 receptor antisense-induced behavioural effects to be determined. The first centrally active 5-ht6 receptor antagonist to be developed, 4-amino-N-(2,6-bis-methylamino-pyramidin-4-yl)-benzene sulphonamide (Ro 04-6790), has a pKi of 7.35 and 7.26 for the rat and human 5-ht6 receptor, respectively, and over 100-fold selectivity for this compared with 24 other binding sites (Sleight et al., 1998). Initial behavioural studies showed that Ro 04-6790, produced a dose-related yawning and stretching syndrome analogous to that observed with 5-ht6 receptor AO treatment (Sleight et al., 1998). Furthermore, the stretching behaviour induced by both the 5-ht6 receptor AO and Ro 04-6790 were attenuated with non-selective muscarinic antagonists (atropine (both studies) and scopolamine (Ro 04-6790 only)), but not by the dopamine antagonist, haloperidol. This data suggested that there was a cholinergic, but no dopaminergic, component to this behaviour (Bourson et al., 1995, Bentley et al., 1999). Although a second selective 5-ht6 receptor antagonist, SB-271046 did not induce yawning when administered alone, it enhanced physostigmine-induced yawning (Routledge et al., 1999) in agreement with the role of the 5-ht6 receptor in this behaviour. The high level of expression of the 5-ht6 receptor in the hippocampus (Ruat et al., 1993, Bourson et al., 1995, Ward et al., 1995, Gérard et al., 1997) together with evidence for a link with cholinergic function suggests a role for this receptor in memory processes. Two preliminary studies have suggested that both the 5-ht6 receptor AO (Bentley et al., 1997) and the 5-ht6 receptor antagonist SB-271046 (Rogers et al., 1999) can enhance retention, but not acquisition, of a spatial learning task in the rat. This study provides the first detailed account of the role of the 5-ht6 receptor in a memory paradigm by comparing the effect of administration of the 5-ht6 receptor-directed AO with that of a selective 5-ht6 antagonist on performance in the Morris water maze.
Section snippets
Animals
Adult male, Lister hooded rats (Biomedical Services Unit, University of Nottingham derived from Charles River stock) weighing 280–310 g at the beginning of the study (group A, Fig. 1), were singly housed on a 12 h light–dark cycle (lights on at 07.00 hours) and given a weighed amount of food and water ad libitum. Room temperature (21±1°C) and humidity (55–65%) were maintained constant. A second group (group B, Fig. 1) of male, Lister hooded rats (Charles River, UK weighing between 250 and 260 g at
Antisense administration
All rats receiving ODNs or saline progressively learned to locate the hidden platform (p <0.05, by trial) during the 10 successive trials in the water maze (Fig. 2A) such that there was no significant difference irrespective of the treatment received. On the first day of training (trials 1–5), there was no drug-induced difference in the ability of the rats to locate the hidden platform (ANOVA, F(3,95) = 0.60; p = 0.62, by treatment). However, on the second day (trials 6–10) the overall average
Discussion
The results demonstrate that either chronic administration of the 5-ht6 receptor-directed AO or a selective 5-ht6 receptor antagonist during the acquisition period in a spatial learning paradigm does not affect the learning process per se, but may improve long-term memory retention of the task.
The ability to demonstrate a reduction in 5-ht6 receptor levels following treatment with AO is hampered by the lack of selective high affinity ligands for the receptor although the first of such ligands
Acknowledgements
M.L.W. is, and J.C.B. was, an M.R.C. case student supported by F. Hoffmann-La Roche, who also provided the opacifier and Ro 04-6790.
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Present address. Wolfson Centre for Age-Related Diseases, Kings College, London.