Research reportA genetic screen for mouse mutations with defects in serotonin responsiveness
Introduction
Many studies have implicated disturbances of the serotonergic system in psychiatric disorders, such as alcoholism, aggression, schizophrenia and depression. For example, serotonin (5-HT) antagonists have proven useful in the treatment of schizophrenia, and selective serotonin reuptake inhibitors are among the most commonly prescribed anti-depressants. Thus, genetic factors required for a functional serotonergic system are attractive candidates in the search for genetic risk factors and for ideal drug targets in the treatment of human psychiatric disorders.
The incomplete penetrance and etiological heterogeneity of psychiatric disorders have made identification of the contributing genetic factors in human pedigrees challenging. For example, the serotonin receptor (5-HT) 2A has been implicated in some familial cases of schizophrenia, and is thought to contribute to the anti-psychotic activity of atypical anti-psychotics, such as clozapine [22], [27]. While polymorphisms in the 5-HT2A receptor gene have been identified, their functional consequences have been unclear. Similarly, polymorphisms in the serotonin transporter gene (SLC6A4) have been associated with several psychiatric disorders. Evidence of causality had remained elusive until recent imaging studies suggested that the level of activity observed in the amygdala was directly dependent on which allelic variant(s) was present in a given individual [24]. This general lack of functional correlates may reflect both the heterogeneity of affective disorders and the paucity of genes known to affect serotonergic neurotransmission directly or indirectly.
We have undertaken a small scale genetic screen to identify genes required for normal serotonergic responsiveness in the mouse. To date most ‘serotonergic’ mouse mutations have been generated by homologous recombination in embryonic stem cells. This process requires either pre-existing knowledge of a gene’s role in the serotonergic system or serendipitous observation of ‘serotonergic’ phenotypes, such as appetite deregulation or hyper-aggression. Furthermore, mutant mice generated by homologous recombination in embryonic stem cells are often on the 129Sv/J inbred background, which breeds poorly and is sub-optimal for many behavioral studies. Forward genetic screens using chemical mutagenesis with ethylnitrosourea (ENU) have strengths complementary to those of gene targeting approaches. Mutations are identified by their phenotype and are then used to identify novel genes required within the biologic pathway of interest. Although molecules that associate directly with a gene product can be identified by biochemical means, molecules that interact less directly or in a more complex or transitory manner often are best identified genetically. Such a forward genetic approach may be especially valuable in studying neuronal circuitry where intricate neuronal cross talk modulates behavioral responses. Such mutations and the identification of the affected genes would help dissect molecular mechanisms important in human neurological disorders, serve to develop more efficacious drugs, and possibly identify candidate genes for human neurological disorders. In conjunction with advances in the ability to map genes, identify transcriptional units, and directly clone point mutations, mutational analysis will play a pivotal role in understanding neural function in the coming decades.
In the mouse, mutagenesis with ENU is highly efficient. Both large scale as well as small focussed genetic screens have demonstrated the facility with which genetic screens in mice can be performed [1], [2], [10], [13], [19], [21]. After male mice have been treated with ENU, a single mutagenized male can produce 100–150 progeny (G1, ‘first generation’) each one of which represents one mutagenized gamete. Because ENU mutagenesis targets the spermatogonial stem cells of the mutagenized G0 male mouse, all G1 animals are non-mosaic [5]. On average the high efficiency of ENU mutagenesis results in identification of a new mutation in any single locus in one out of 500 to 1000 G1 animals. Thus, the mutational rate for ENU is 1.5×10−3/locus, and thus at least 300 times higher than the spontaneous mutational rate [19]. For saturation 2000–3000 G1 animals have to be screened in a recessive screen. Normally only dominant mutations can be identified in G1 progeny of mutagenized males. However, if the genetic background is ‘sensitized’ or the animals are ‘challenged’, mutations that are normally recessive or have less apparent phenotypes can be detected. Finally, ENU mutagenesis creates primarily point mutations, which may more closely replicate the allelic variants observed in humans.
Because neurotransmission is by its nature dosage sensitive, some mutations that affect neurotransmission directly or indirectly can be detected in the heterozygous state (in G1 animals). Such effects can be observed on the molecular level, as in, for example, mice carrying mutations in the 5-HT transporter [7]. The 5-HT transporter (5-HTT) regulates central 5-HT neurotransmission by taking up serotonin released in the extracellular space and causes adaptive changes in the levels of 5-HT1A and B receptor levels. In mice lacking 5-HTT, 5-HT1A receptor levels were decreased in the dorsal raphe nucleus (DRN) and increased in the hippocampus, while 5-HT1B receptor density was lowered in the substantia nigra. Intermediate changes were seen in 5-HTT+/− mice. In other cases gene dosage-sensitive behavioral effects have been observed. For example, mice lacking the dopamine transporter (DAT(−/−)) have high extracellular dopamine levels and are hyperactive [26]. This hyperlocomotion is decreased by acute administration of amphetamine-like psychostimulants. The decrease in hyperlocomotion is dependent on both drug and gene dosage. So, it should be possible to recover semi-dominant and dominant mutations that affect the serotonergic system.
Here we have used a pharmacologically induced behavioral assay to uncover strain-specific genetic variation and identify a mutation that affects serotonergic responsiveness. In the mouse a simple test for 5-HT responsiveness and, thereby, for 5-HT neuron function exists. Treatment of mice with 5-HT, 5-HT precursors, or 5-HT agonists, such as DOI (+-1-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane), results in a characteristic and unmistakable head twitch response, which can be scored visually [3], [4]. Pharmacologic experiments have shown that HTR is dosage dependent, can be reversed by antagonists in outbred ICR mice, and is dependent primarily on 5-HT2A receptor neurotransmission. Cross talk between serotonergic, dopaminergic, adrenergic, and GABAergic neurons has been detected in pharmacologic experiments using this assay [8], [20], [23], [25]. The serotonin agonist DOI, when administered intraperitoneally, elicits HTR in a dose-dependent manner, via the 5-HT2A receptors [6]. Previously, maximal HTR was observed in 28-day-old ICR mice, thereafter HTR, unlike other ‘serotonergic’ behaviors, decreased only very gradually [4]. Here we have reduced the actual scoring time to a 10 min interval to facilitate testing of mice for serotonergic defects. We have determined the genetic variability of DOI-induced head twitch response in five inbred mouse strains. Subsequently, we have used this pharmacologic behavioral assay to uncover animals with defects in serotonin responsiveness in a small scale mutagenesis screen.
Section snippets
Mice
Mice were housed in groups of two to five in vented racks on wood chip bedding with a standard 12 h light: 12 h dark cycle at 22±1 °C. Food (Purina mouse chow) and water were freely available. Mice from C57BL/6J, CAST/Ei, A/J, Balb/cJ, and C3HeB/FeJ inbred strains were obtained from the Jackson Laboratory at 3 weeks-of-age and allowed to acclimate for a week after shipping. Behavioral analyses were conducted on mice ranging in ages from 4 weeks to 9 months. For all strains, at least 10 animals
Variation of responsiveness to the serotonin agonist doi among inbred strains
We tested DOI induced HTR in five inbred mouse strains to examine the genetic variability of this trait and to test whether this protocol might uncover differences in serotonergic responsiveness among mice, which by simple observation appear behaviorally equivalent. We examined the HTR of C57BL/6J, CAST/Ei, C3HeB/FeJ, Balb/cJ, and A/J mice to 1 mg/kg DOI, because studies on ICR mice indicated that this dosage induced a substantial, but submaximal, HTR. C57BL/6J and CAST/Ei mice exhibited
Discussion
Of the five inbred strains examined, A/J mice showed the most dramatic difference in DOI-induced HTR. Although HTR had never been analyzed for A/J mice before, previously A/J mice have been categorized as a ‘non-anxiolytic’ strain by detailed behavioral analyses. Several chromosomal regions involved in the suppression of non-anxiolytic behaviors and in diazepam sensitivity have been identified by quantitative trait locus (QTL) mapping on recombinant inbred strains generated from C57BL/6J and
Acknowledgements
We are very grateful to Art Fredeen for help with statistical analyses. We would also like to thank Joe Culotti for helpful discussions and Dragana Vukasovic for invaluable secretarial assistance. This work was funded by an EJLB Foundation Scholar Award to S.P.C and by a generous gift from Mr Henry Bernick and Mrs Esther Bernick.
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Present address: The Graduate Program in Biological sciences, UCSF, CA, USA.