Characterization of the serotonin transporter knockout rat: A selective change in the functioning of the serotonergic system

doi:10.1016/j.neuroscience.2007.03.030

Neuroscience

Volume 146, Issue 4, 8 June 2007, Pages 1662-1676

https://doi.org/10.1016/j.neuroscience.2007.03.030 Get rights and content

Abstract

Serotonergic signaling is involved in many neurobiological processes and disturbed 5-HT homeostasis is implicated in a variety of psychiatric and addictive disorders. Here, we describe the functional characterization of the serotonin transporter (SERT) knockout rat model, that is generated by N-ethyl-N-nitrosurea (ENU)-driven target-selected mutagenesis. Biochemical characterization revealed that SERT mRNA and functional protein are completely absent in homozygous knockout (SERT^−/−) rats, and that there is a gene dose-dependent reduction in the expression and function of the SERT in heterozygous knockout rats. As a result, 5-HT homeostasis was found to be severely affected in SERT^−/− rats: 5-HT tissue levels and depolarization-induced 5-HT release were significantly reduced, and basal extracellular 5-HT levels in the hippocampus were ninefold increased. Interestingly, we found no compensatory changes in in vitro activity of tryptophan hydroxylase and monoamine oxidase, the primary enzymes involved in 5-HT synthesis and degradation, respectively. Similarly, no major adaptations in non-serotonergic systems were found, as determined by dopamine and noradrenaline transporter binding, monoamine tissue levels, and depolarization-induced release of dopamine, noradrenaline, glutamate and GABA. In conclusion, neurochemical changes in the SERT knockout rat are primarily limited to the serotonergic system, making this novel rat model potentially very useful for studying the behavioral and neurobiological consequences of disturbed 5-HT homeostasis.

Section snippets

Animals

All experiments were approved by the Animal Care Committee of the Royal Dutch Academy of Science, the Free University of Amsterdam, the Radboud University Nijmegen, and the University of Groningen according to the Dutch legal ethical guidelines. Experiments were designed to minimize the number of required animals and their suffering.

The SERT knockout rat (Slc6a4^1Hubr) was generated by target-selected ENU-induced mutagenesis (for detailed description, see Smits et al., 2006). Briefly,

SERT knockout rat characteristics

High-throughput resequencing of target genes in a library of ENU-mutagenized rats (Smits et al., 2006) revealed a C to A transversion at position 3924 (relative to the start codon in ENSRNOG0000003476) in the SERT gene, resulting in a premature stop codon (TGC>TGA) in the third exon encoding the second extracellular loop (Fig. 1A), and thereby most likely resulting in a non-functional protein product. Northern blot analysis (Fig. 1B and C) revealed that the premature stop codon results in

Discussion

Using the recently established gene knockout technology (Zan et al 2003, Smits et al 2004, Smits et al 2006) we obtained a rat knockout for the SERT gene, and here we present the first comprehensive characterization of this novel model. We show that the ENU-induced premature stop codon results into a complete lack of SERT, and interestingly, that SERT^+/− rats show a gene-dosage effect with respect to SERT expression and function. The SERT^+/− rat may therefore be a novel model system for

Acknowledgments

We thank J. Vermond for his help with enzymatic measurements, F. Hogenboom and G. Wardeh for technical support with the superfusion experiments, and N. Leguit for HPLC analysis. This work was supported by the Dutch Ministry of Economic Affairs through the Innovation Oriented Research Program on Genomics (IGE1017) and the award “Exploiting natural and induced genetic variation in the laboratory rat” to EC from the European Heads of Research Councils and European Science Foundation EURYI

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¹: Present address: Evotec Pharmaceutical Division, Neuropharmacology, Hamburg, Germany (B. A. Ellenbroek); McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI, USA (B. M. G. Smits).

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Molecular neuroscienceCharacterization of the serotonin transporter knockout rat: A selective change in the functioning of the serotonergic system

Abstract

Section snippets

Animals

SERT knockout rat characteristics

Discussion

Acknowledgments

Behav Brain Res

Curr Opin Cell Biol

Brain Res Brain Res Rev

Neuropsychopharmacology

Neuropharmacology

Biol Psychiatry

J Neurosci Methods

Brain Res Dev Brain Res

Biol Psychiatry

Life Sci

Eur J Pharmacol

Genomics

J Neurosci Methods

Brain Res

Association of the serotonin transporter promoter regulatory region polymorphism and obsessive-compulsive disorder

Mol Psychiatry

Altered brain serotonin homeostasis and locomotor insensitivity to 3,4-methylenedioxymethamphetamine (‘ecstasy’) in serotonin transporter-deficient mice

Mol Pharmacol

Effects of chronic antidepressant treatments on serotonin transporter function, density and mRNA level

J Neurosci

Influence of life stress on depression: moderation by a polymorphism in the 5-HTT gene

Science

Inactivation of 5-HT(2C) receptors potentiates consequences of serotonin reuptake blockade

Neuropsychopharmacology

In vivo chronoamperometric measures of extracellular serotonin clearance in rat dorsal hippocampus: contribution of serotonin and norepinephrine transporters

J Pharmacol Exp Ther

Altered expression and functions of serotonin 5-HT1A and 5-HT1B receptors in knock-out mice lacking the 5-HT transporter

Eur J Neurosci

Association between low-activity serotonin transporter genotype and heroin dependence: behavioral and personality correlates

Am J Med Genet B Neuropsychiatr Genet

Modifications of the serotonergic system in mice lacking serotonin transporters: an in vivo electrophysiological study

J Pharmacol Exp Ther

Genetic variation in the serotonin transporter promotor region affects serotonin uptake in human blood platelets

Am J Med Genet

Molecular neuroscience
Characterization of the serotonin transporter knockout rat: A selective change in the functioning of the serotonergic system