Targeted gene deletion of the 5-HT3A receptor subunit produces an anxiolytic phenotype in mice
Introduction
Serotonin (5-HT) receptors are classified into seven groups (5-HT1–7), comprising a total of at least 14 structurally and pharmacologically distinct mammalian receptor subtypes (Hoyer et al., 1994). At the molecular level, 5-HT receptors are mostly seven transmembrane-spanning, G-protein-coupled metabotropic receptors (Barnes and Sharp, 1999). The 5-HT3 receptor is unique among this receptor family as the only member that is a ligand-gated ion channel Derkach et al., 1989, Maricq et al., 1991. Cellular studies indicate that the 5-HT3 receptor may form a cylindrical structure comprising five co-assembled subunits that surround a centrally gated channel (Boess et al., 1995). 5-HT binding occurs on the extracellular N-terminus of the 5-HT3A subunit (Eisele et al., 1993), but receptor activation is also enhanced by pharmacologically distinct allosteric sites (Lovinger and Zhou, 1993). Electrophysiologically, 5-HT mediates rapid excitatory responses through ionotropic 5-HT3 receptors (Derkach et al., 1989) and the activation of this channel results in rapid depolarization responses and subsequent desensitization Lambert et al., 1989, Yakel and Jackson, 1988.
The 5-HT3A receptor subtype was first cloned in 1991 (Maricq et al., 1991) and shown to be localized in the periphery and in limbic brain regions including the hippocampus, amygdala, and throughout the cortex (Tecott et al., 1993). Recently, a new class of 5-HT3 receptor subunit (e.g., 5-HT3B) was described and found to be co-expressed with the 5-HT3A subunit in human amygdala, caudate, and hippocampus (Davies et al., 1999). Heteromeric assemblies of human 5-HT3A and 5-HT3B subunits show channel conductance, calcium permeability, and current–voltage properties that closely resemble neuronal 5-HT3 channels (Davies et al., 1999). In rats, however, 5-HT3B subunit transcripts are restricted to peripheral neurons(Morales and Wang, 2002), which suggests that rodent neural 5-HT3 receptors might be 5-HT3A homomeric receptors or heteromeric receptors containing 5-HT3A subunits combined with subunits other than the 5-HT3B subunit. Indeed, a comparison of electrophysiological properties of rodent neural 5-HT3 receptors has shown the presence of receptors with both high and low conductances Hussy et al., 1994, Jones and Surprenant, 1994, Yang et al., 1992, which suggests the presence of at least two different 5-HT3 receptor channels (Fletcher and Barnes, 1998).
The antagonism of the 5-HT3 receptor produces a range of anxiolytic effects in various animal models of anxiety Costall and Naylor, 1991, Costall and Naylor, 1992a, Costall and Naylor, 1992b, Costall et al., 1990. These effects have been demonstrated in both rodents (Barnes et al., 1992a) and in primates (Barnes et al., 1990). In rodent conflict models of anxiety such as the light/dark box (Costall et al., 1989a) and the elevated plus maze Pellow and File, 1986, Pellow et al., 1985, selective 5-HT3 antagonists such as tropisetron, ondansetron, and zacopride can produce anxiolytic behavioral profiles in both rats and mice Barnes et al., 1992b, Cheng et al., 1994, Costall et al., 1989a, Jones et al., 1988 but some rodent studies have failed to find anxiolytic effects (Rodgers et al., 1997). Anxiolytic responses to 5-HT3 antagonists have also been reported in other rodent ethological anxiety models such as social interaction tests Cutler et al., 1997, Gao and Cutler, 1992a. Although the anxiolytic behavioral profile of 5-HT3 antagonists is similar to diazepam in these behavioral models, there appears to be no corresponding increase in sedation or reduction of locomotor behavior (Jones et al., 1988). There have been few clinical studies utilizing 5-HT3 antagonists to treat anxiety disorders; however, clinical trials have shown ondansetron to possibly be effective in treating generalized anxiety disorder (Freeman et al., 1997) and panic disorder (Schneier et al., 1996). In contrast to the pharmacological effects of 5-HT3 antagonists demonstrated in rodent models of anxiety, the administration of 5-HT3 agonists such as 2-methyl-5-HT has been demonstrated to produce anxiogenic effects when microinjected into either the dorsal raphe nucleus or the amygdala (Costall et al., 1989b).
Based on the evidence generated from the various 5-HT3 antagonists across a range of anxiety models in both primates and rodents and the concomitant changes observed under 5-HT3 agonists, it is reasonable to conclude that 5-HT3 receptors are involved in the modulation of anxiety-related behavior. Since neural 5-HT3 receptors might be homomeric or heteromeric receptors containing 5-HT3A subunits, and 5-HT3 antagonists show equal affinity for 5-HT3A and 5-HT3A/B receptors (Brady et al., 2001), the functional significance of specific 5-HT3 receptor subunits as they relate to anxiety has yet to be determined. To address this issue, we studied anxiety-related behavioral responses in mutant mice lacking the 5-HT3A receptor subtype.
Section snippets
Mice
5-HT3A receptor null mice were derived using homologous recombination as previously reported (Zeitz et al., 2002). F1 hybrid C57Bl/6J×129 heterozygous progeny were backcrossed with C57Bl/6J mice to produce F9 generation congenics.
Heterozygotes from the F9 generation were bred to generate wild-type and 5-HT3A null mutants used in the present study. Genotyping was conducted in Dr. David Julius' laboratory at the University of California at San Francisco as previously described (Zeitz et al., 2002)
Locomotor activity
To determine if any overt neurobiological changes exist due to the targeted mutation, we first tested 5-HT3A null mice and wild-type littermates in a novel open field environment for three consecutive days. The null mutation produced no effect on spontaneous locomotor activity (Fig. 1A). Moreover, locomotor activity by both genotypes decreased as a function of days in the test environment [F(2,255)=12.211, P<0.001] and time during each test session [F(5,255)=36.827, P<0.001], which indicates
Discussion
The primary finding of this study is that targeted gene deletion of the 5-HT3A receptor subunit produced an anxiolytic-like behavioral phenotype in mice. Initial screens of 5-HT3A null mice and wild-type littermate controls found no significant differences in motor ability, open field exploration, habituation to a novel environment, food or water intake, or body weight. This suggests that the null mutation did not alter the function of major homeostatic systems. However, the behavioral profile
Acknowledgements
This work, including mouse breeding and genotyping, was supported by funds provided by the State of California for medical research on alcohol and substance abuse through the University of California at San Francisco. We are grateful to David Julius, PhD, for initial contribution of the mice.
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- 1
Present address: Neurosciences Institute, Department of Pharmacology and Neuroscience, University of Dundee, Ninewells Hospital, Dundee DD1 9SY, Scotland, UK.
- 2
Present address: Pharmacology Department, Organon Laboratories Ltd., Newhouse, Lanarkshire, ML1 5SH, Scotland, UK.