Elsevier

Neuropharmacology

Volume 48, Issue 4, March 2005, Pages 492-502
Neuropharmacology

Genetic knockout and pharmacological blockade studies of the 5-HT7 receptor suggest therapeutic potential in depression

https://doi.org/10.1016/j.neuropharm.2004.11.015Get rights and content

Abstract

The affinity of several antidepressant and antipsychotic drugs for the 5-HT7 receptor and its CNS distribution suggest potential in the treatment of psychiatric diseases. However, there is little direct evidence of receptor function in vivo to support this. We therefore evaluated 5-HT7 receptors as a potential drug target by generating and assessing a 5-HT7 receptor knockout mouse. No difference in assays sensitive to potential psychotic or anxiety states was observed between the 5-HT7 receptor knockout mice and wild type controls. However, in the Porsolt swim test, 5-HT7 receptor knockout mice showed a significant decrease in immobility compared to controls, a phenotype similar to antidepressant treated mice. Intriguingly, treatment of wild types with SB-258719, a selective 5-HT7 receptor antagonist, did not produce a significant decrease in immobility unless animals were tested in the dark (or active) cycle, rather than the light, adding to the body of evidence suggesting a circadian influence on receptor function. Extracellular recordings from hypothalamic slices showed that circadian rhythm phase shifts to 8-OH-DPAT are attenuated in the 5-HT7 receptor KO mice also indicating a role for the receptor in the regulation of circadian rhythms. These pharmacological and genetic knockout studies provide the first direct evidence that 5-HT7 receptor antagonists should be investigated for efficacy in the treatment of depression.

Introduction

The 5-HT7 receptor is one of 14 distinct receptor subtypes thought to mediate the diverse range of CNS effects of the neurotransmitter, serotonin (reviewed in Barnes and Sharp, 2000). The 5-HT7 receptor is the most recent addition to the 5-HT receptor family having been cloned from a variety of species, including man, rat, mouse and guinea-pig (Plassat et al., 1993, Ruat et al., 1993, Shen et al., 1993, Tsou et al., 1994, Gelernter et al., 1995). The 5-HT7 receptor exhibits only low (40%) homology with other Gs coupled 5-HT receptors and is encoded by a single gene on human chromosome 10 (Krobert et al., 2001). Although it is found in four different isoforms (5-HT7a, 5-HT7b, 5-HT7c, and 5-HT7d), only two (5-HT7a and 5-HT7b) are present in both rat and human, and there appears to be no pharmacological differences between them (Gustafson et al., 1996). In situ hybridisation studies revealed CNS expression in amygdala, cortex, hippocampus, thalamus, septum, hypothalamus and suprachiasmatic nucleus (Gustafson et al., 1996). 5-HT7 receptors are defined pharmacologically by their high affinity for 5-CT, 5-HT, 5-MeOT and methiothepin, moderate affinity for 8-OH-DPAT and ritanserin and low affinity for pindolol, sumatriptan and buspirone (Sleight et al., 1995).

This CNS distribution, coupled with the receptor's relatively high affinity for several psychoactive drugs, including hallucinogenics (e.g. LSD), antipsychotics (e.g. clozapine) and antidepressants (e.g. amitriptyline), have implicated the 5-HT7 receptor as a potential therapeutic target in psychosis and depression. In support of this, chronic antidepressant treatment down regulates 5-HT7 receptor expression in rat hypothalamus (Mullins et al., 1999). Recent evidence also suggests that 5-HT7 receptors may be involved in the regulation of circadian rhythms (Lovenberg et al., 1993; Ehlen et al., 2001). Thus in rat SCN slices, 8-OH-DPAT-induced phase advances in spontaneous cell firing were attenuated by ritanserin, but not pindolol, suggestive of a 5-HT7 receptor pharmacology (Lovenberg et al., 1993, Thomas et al., 1998). However, until now little direct evidence of receptor function in vivo has been available to support any of these proposals.

In this study, we have examined the in vivo role of the 5-HT7 receptor in mice in which the receptor gene has been deleted by testing the mice in a range of behavioural assays sensitive to potential psychotic states. In addition, where a behavioural phenotype was observed, we also determined the effect of treating the wild type mice with the selective 5-HT7 receptor antagonist, SB-258719 (Forbes et al., 1998, Lovell et al., 2000). The data from both approaches provide the first, direct, preclinical validation of the 5-HT7 receptor as a therapeutic target for depression and add further support to its potential role in circadian/sleep disorders.

Section snippets

Compound synthesis

SB-258719 was synthesised and used for behavioural studies. (R)-[35S]-1-[2-(1-benzenesulfonyl-pyrrolidin-2-yl)-ethyl]-4-methyl-piperidine (termed compound A) was made following a reported generic synthetic route for such compounds (Collinson et al., 2002), and confirmed to be a high affinity 5-HT7 receptor antagonist suitable for use in autoradiography experiments. Desipramine HCl (DMI; 20 mg/kg; i.p.; Sigma–Aldrich, Poole, U.K.) was dissolved in 0.9% saline and administered in a dosing volume

Generation of 5-HT7 receptor knockout mice

5-HT7 receptor knockout mice were generated using a targeting vector to knockout exon 1 of the 5-HT7 gene (Fig. 1a). This vector was transfected into 129SvEv embryonic stem cells and six positive ES clones showing the targeted mutation were identified (Fig. 1b) and subsequently injected into C57Bl/6 blastocysts. A single independent line (line 3) was bred to generate homozygotic animals and all behavioural assessments have been carried out in male mice from the F3 or F4 generation of this line.

Characterisation of 5-HT7 receptor knockout mice and the selective 5-HT7 receptor antagonist, SB-258719

5-HT7 receptor knockout mice were generated by knocking out exon 1 of the 5-HT7 gene A single independent line (line 3) was bred to generate homozygotic animals and all behavioural assessments have been carried out in male mice from the F3 or F4 generation of this line. PCR analysis of tail DNA confirmed the successful generation of homozygous knockout mice at the genome level (Fig. 1c) while RT-PCR (data not shown) and autoradiographic studies (Fig. 1d) confirmed that functional 5-HT7 receptor

Acknowledgments

We would like to thank Dennis Dean and colleagues in the Labeled Compound Synthesis Group (LCS) at MRL Basic Research, Rahway for assistance in the generation of (R)-[35S]-1-[2-(1-benzenesulfonyl-pyrrolidin-2-yl)-ethyl]-4-methyl-piperidine.

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    MG and LJB contributed equally to this work.

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