A mutant mouse with a highly specific contextual fear-conditioning deficit found in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen

Abstract

Targeted mutagenesis in mice has shown that genes from a wide variety of gene families are involved in memory formation. The efficient identification of genes involved in learning and memory could be achieved by random mutagenesis combined with high-throughput phenotyping. Here, we provide the first report of a mutagenesis screen that has generated memory mutants in the mouse. We tested a group of N-ethyl-N-nitrosourea (ENU) mutagenized mice in the conditioned fear paradigm. We screened for both dominant and recessive mutations that caused impairments in contextual or tone fear conditioning. Heritability testing confirmed three fear conditioning mutants, i.e., Forgetful, Slowlearner, and Scatterbrain. All three have a learning or short-term memory deficit in contextual fear conditioning. Forgetful was further characterized and showed a highly specific phenotype. The contextual fear-conditioning deficit was apparent when Forgetful was trained with tone-shock pairings, but not when trained with shock alone. The deficit was not due to changes in shock sensitivity or anxiety. Forgetful was not impaired in two other memory tests (hidden platform version of Morris water maze and object recognition). Our data show that a mutagenesis screen can generate mutant mice with highly specific memory phenotypes that can supplement existing mice with targeted mutations. Mapping of Slowlearner found linkage to a region of chromosome 12 (LOD score of 6.5 close to D12Mit171), which suggests that ENU mutants should enable the positional cloning of genes involved in memory formation.Though memory is expressed at the behavioral level, it is dependent on molecular events. Knockout and transgenic mice have shown that a large number of genes are involved in memory formation (Mayford and Kandel 1999;Silva 2003;Tonegawa et al. 2003). Quantitative trait locus analysis in mice has identified many genomic regions that contribute to learning and memory (Caldarone et al. 1997;Wehner et al. 1997;Valentinuzzi et al. 1998;Steinberger et al. 2003). It has become apparent from these studies that memory formation requires the coordinated activity of a large number of genes encoding proteins involved in neurotransmission, signal transduction, regulation of gene expression, and the growth and differentiation of neuronal processes.Mutagenesis screens that combine random mutagenesis with high-throughput phenotyping provide an efficient method for genetically dissecting complex phenotypes. They can identify critical genetic components, and because of their unbiased character, can lead to the discovery of new mechanisms. Mutagenesis screens using Drosophila have revealed key insights into the genetics of learning and memory (Dubnau and Tully 1998). Complex behavioral phenotypes can also be dissected with mutagenesis screens that use model organisms more closely related to humans, as was demonstrated by the Clock mutant mouse (Vitaterna et al. 1994;King et al. 1997).Here, we provide the first report of a mutagenesis screen that has generated memory mutants ...

A Forward Genetics Screen in Mice Identifies Recessive Deafness Traits and Reveals That Pejvakin Is Essential for Outer Hair Cell Function

“…The ENU mutagenesis protocol and primary phenotypic screen has been described previously (Reijmers et al, 2006). All experiments were performed with littermates.…”

ENU Mutagenesis Screening for Dominant Behavioral Mutations Based on Normal Control Data Obtained in Home-Cage Activity, Open-Field, and Passive Avoidance Tests

“…DBF 1 mice showed significantly higher values than D2 for activity (0-5 min) of both sexes, total activity, and activity (0-10 min) of females. DBF 1 and D2 mice showed no significant differences for center % of both sexes, for activity (0-5 min) of both sexes, for activity (0-10) of males, and for activity (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) of both sexes. In summary, B6 mice were the most active, and DBF 1 mice showed values higher or similar to D2 in most of the activity parameters of both sexes, although male D2 mice exhibited a significantly higher value than DBF 1 for max for 1 min, and showed a significantly higher value than B6 or DBF 1 for max/total.…”

“…Many mutants exhibiting interesting behavioral traits have been generated by ENUmutagenesis, such as abnormal circadian rhythm [4,9,12,23], abnormal sensory-motor gating in the pre-pulse inhibition test [2,22], hyperactivity and hypoactivity in an open-field or novel cage [2,10,11], decreased anxiety in the light/dark test [2], learning deficit in contextual fear conditioning [20], and abnormal ethanol-induced locomotor activity [7]. Although most of the genes responsible for these ENU-induced behavioral mutants have not yet been identified, except those for circadian rhythm [9,12,23] and hyperactivity in novel cage [11], the genetic and phenotypic characterization of these mutants will provide new insights for elucidating the neural and molecular pathways of behavior.…”

Second-generation high-throughput forward genetic screen in mice to isolate subtle behavioral mutants

“…Although "emotionality" in rodents has been extensively studied as a model of anxiety disorder in humans, forward genetic screens in mice for behavior have not been as successful as in other phenotypic domains. There have been numerous large-scale screens carried out for neurobehavioral mutants; however, only a handful of mutants have been isolated using this approach (14,25,27,40,41), probably owing to the imprecise nature of many behavioral assays. We have also found that mutants isolated using a high threshold of detection have a high rate of false positives.…”

“…In addition, locomotor output assays are often used as endophenotypes for complex emotionality, and these outputs can be affected by systems independent of the behavioral pathway being investigated, leading to identification of animals with pleiotropic phenotypes (12,13). Moreover, mutations in genes known to regulate behaviors such as learning and memory, psychostimulant response, and open field behavior usually only produce a shift in the mean of 1 to 2 SDs (11,(13)(14)(15). Thus, when genetic screens are conducted to search for "strong" (greater than 3 SDs) mutants, the investigator is attempting to discover mutants that are more extreme than what would normally be found if a key gene, known to mediate the behavior, was deleted.…”