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Behavioral tests of animals in a controlled experimental setting provide a valuable tool to advance understanding of genotype-phenotype relations, and to study the effects of genetic and environmental manipulations. To optimally benefit from the increasing numbers of genetically engineered mice, reliable high-throughput methods for comprehensive behavioral phenotyping of mice lines have become a necessity. Here, we describe the development and validation of an anxiety test, the light spot test, that allows for unsupervised, automated, high-throughput testing of mice in a home-cage system. This automated behavioral test circumvents bias introduced by pretest handling, and enables recording both baseline behavior and the behavioral test response over a prolonged period of time. We demonstrate that the light spot test induces a behavioral response in C57BL/6J mice. This behavior reverts to baseline when the aversive stimulus is switched off, and is blunted by treatment with the anxiolytic drug Diazepam, demonstrating predictive validity of the assay, and indicating that the observed behavioral response has a significant anxiety component. Also, we investigated the effectiveness of the light spot test as part of sequential testing for different behavioral aspects in the home-cage. Two learning tests, administered prior to the light spot test, affected the light spot test parameters. The light spot test is a novel, automated assay for anxiety-related high-throughput testing of mice in an automated home-cage environment, allowing for both comprehensive behavioral phenotyping of mice, and rapid screening of pharmacological compounds. (C) 2015 Elsevier B.V. All rights reserved.

Genetic and environmental factors interact throughout life and give rise to individual differences, i.e., individuality. The diversifying effect of environmental factors is counteracted by genetic mechanisms to yield persistence of specific features (robustness). Here, we compared robustness between cohorts of isogenic mice of eight different commonly used strains by analyzing to what extent environmental variation contributed to individuality in each of the eight genotypes, using a previously published dataset. Behavior was assessed in the home-cage, providing control over environmental factors, to reveal within-strain variability in numerous spontaneous behaviors. Indeed, despite standardization and in line with previous studies, substantial variability among mice of the same inbred strain was observed. Strikingly, across a multidimensional set of 115 behavioral parameters, several strains consistently ranked high in within-strain variability (DBA/2J, 129S1/Sv A/J and NOD/LtJ), whereas other strains ranked low (C57BL/6J and BALB/c). Strain rankings of within-strain variability in behavior were confirmed in an independent, previously published behavioral dataset using conventional behavioral tests administered to different mice from the same breeding colonies. Together, these show that genetically inbred mouse strains consistently differ in phenotypic robustness against environmental variation, suggesting that genetic factors contribute to variation in robustness.

Both impulsivity in operant tasks and locomotor activity in a novel open field are known to predict the development of addiction-related behavior in rodents. In this study, we investigated to what extent impulsivity in the five-choice serial reaction time task and various measures of novelty exploration are controlled by shared genetic and environmental factors in 12 different inbred mouse strains. No genetic correlation was observed between the level of impulsivity and levels of activity, a low correlation was observed with traditional measures of anxiety-like behavior (impulsive strains tend to be less anxious) and a highly significant correlation was found between impulsivity and specific aspects of movement. Furthermore, we found that impulsivity and all measures of novelty exploration were under control of different environmental factors. Interestingly, in the dorsal medial prefrontal cortex, a brain region involved in impulsivity and activity in novelty exploration tests; these behavioral measures correlated with the expression of different genes (respectively, Frzb, Snx5, BC056474 and the previously identified Glo1). Taken together, our study shows that impulsivity and activity in novelty exploration tests are genetically and environmentally distinct, suggesting that mouse models of these behaviors provide complementary insights into the development of substance abuse disorder. © 2009 Blackwell Publishing Ltd/International Behavioural and Neural Genetics Society.

Individuals are able to change their behavior based on its consequences, a process involving instrumental learning. Studying instrumental learning in mice can provide new insights in this elementary aspect of cognition. Conventional appetitive operant learning tasks that facilitate the study of this form of learning in mice, as well as more complex operant paradigms, require labor-intensive handling and food deprivation to motivate the animals. Here, we describe a 1-night operant learning protocol that exploits the advantages of automated home-cage testing and circumvents the interfering effects of food restriction. The task builds on behavior that is part of the spontaneous exploratory tepertoire during the days before the task. We compared the behavior of C57BL/6J, BALB/cJ and DBA/2J mice and found various differences in behavior during this task, but no differences in learning curves. BALB/cJ mice showed the largest instrumental learning response, providing a superior dynamic range and statistical power to study instrumental learning by using this protocol. Insights gained with this home-cage-based learning protocol without food restriction will be valuable for the development of other, more complex, cognitive tasks in automated home-cages. (C) 2015 Elsevier B.V. All rights reserved.

Functional genetic analyses in mice rely on efficient and in-depth characterization of the behavioral spectrum. Automated home-cage observation can provide a systematic and efficient screening method to detect unexplored, novel behavioral phenotypes. Here, we analyzed high-throughput automated home-cage data using existing and novel concepts, to detect a plethora of genetic differences in spontaneous behavior in a panel of commonly used inbred strains (129S1/SvImJ, A/J, C3H/HeJ, C57BL/6J, BALB/cJ, DBA/2J, NOD/LtJ, FVB/NJ, WSB/EiJ, PWK/PhJ and CAST/EiJ). Continuous video-tracking observations of sheltering behavior and locomotor activity were segmented into distinguishable behavioral elements, and studied at different time scales, yielding a set of 115 behavioral parameters of which 105 showed highly significant strain differences. This set of 115 parameters was highly dimensional; principal component analysis identified 26 orthogonal components with eigenvalues above one. Especially novel parameters of sheltering behavior and parameters describing aspects of motion of the mouse in the home-cage showed high genetic effect sizes. Multi-day habituation curves and patterns of behavior surrounding dark/light phase transitions showed striking strain differences, albeit with lower genetic effect sizes. This spontaneous home-cage behavior study demonstrates high dimensionality, with a strong genetic contribution to specific sets of behavioral measures. Importantly, spontaneous home-cage behavior analysis detects genetic effects that cannot be studied in conventional behavioral tests, showing that the inclusion of a few days of undisturbed, labor extensive home-cage assessment may greatly aid gene function analyses and drug target discovery.

The objective of this study was to identify new candidate genes involved in experience-dependent plasticity. To this aim, we combined previously obtained data from recombinant inbred BXD strains on ocular dominance (OD) plasticity and gene expression levels in the neocortex. We validated our approach using a list of genes which alter OD plasticity when inactivated. The expression levels of one fifth of these genes correlated with the amount of OD plasticity. Moreover, the two genes with the highest relative inter-strain differences were among the correlated genes. This suggests that correlation between gene expression levels and OD plasticity is indeed likely to point to genes with a causal role in modulating or generating plasticity in the visual cortex. After this validation on known plasticity genes, we identified new candidate genes by a multi-step approach. First, a list was compiled of all genes of which the expression level in BXD strains correlate with the amount of OD plasticity. To narrow this list to the more promising candidates, we took its cross-section with a list of genes co-regulated with the sensitive period for OD plasticity and a list of genes associated with pathways implicated in OD plasticity. This analysis resulted in a list of 32 candidate genes. The list contained unproven, but not surprising, candidates, such as the genes for IGF-1, NCAM1, NOGO-A, the gamma2 subunit of the GABA(A) receptor, acetylcholine esterase and the catalytic subunit of cAMP-dependent protein kinase A. This was indicative of the viability of our approach, but more interesting were the novel candidate genes: Akap7, Akt1, Camk2d, Cckbr, Cd44, Crim1, Ctdsp2, Dnajc5, Gnai1, Itpka, Mapk8, Nbea, Nfatc3, Nlk, Npy5r, Phf21a, Phip, Ppm1l, Ppp1r1b, Rbbp4, Slc1a3, Slit2, Socs2, Spock3, St8sia1, Zfp207. The possible role of some of these candidates is discussed in the article.

Recognizing and avoiding aversive situations are central aspects of mammalian cognition. These abilities are essential for health and survival and are expected to have a prominent genetic basis. We modeled these abilities in eight common mouse inbred strains covering ∼75% of the species' natural variation and in gene-trap mice (>2000 mice), using an unsupervised, automated assay with an instrumented home cage (PhenoTyper) containing a shelter with two entrances. Mice visited this shelter for 20-1200 times/24 h and 71% of all mice developed a significant and often strong preference for one entrance. Subsequently, a mild aversive stimulus (shelter illumination) was automatically delivered when mice used their preferred entrance. Different genotypes developed different coping strategies. Firstly, the number of entries via the preferred entrance decreased in DBA/2J, C57BL/6J and 129S1/SvImJ, indicating that these genotypes associated one specific entrance with the aversive stimulus. Secondly, mice started sleeping outside (C57BL/6J, DBA/2J), indicating they associated the shelter, in general, with the aversive stimulus. Some mice showed no evidence for an association between the entrance and the aversive light, but did show markedly shorter shelter residence times in response to illumination, indicating they did perceive illumination as aversive. Finally, using this assay, we screened 43 different mutants, which yielded a novel gene, specc1/cytospinB. This mutant showed profound and specific delay in avoidance learning. Together, these data suggest that different genotypes express distinct learning and/or memory of associations between shelter entrance and aversive stimuli, and that specc1/cytospinB is involved in this aspect of cognition.

The DBA/2J inbred strain of mice has been used extensively in hearing research as it suffers from early-onset, progressive hearing loss. Initially, it mostly affects high frequencies, but already at 2-3 months hearing loss becomes broad. In search for hearing loss genes other than Cadherin 23 (otocadherin) and fascin-2, which make a large contribution to the high-frequency deficits, we used a large set of the genetic reference population of BXD recombinant inbred strains. For frequencies 4, 8, 16 and 32 kHz, auditory brainstem response hearing thresholds were longitudinally determined from 2-3 up to 12 weeks of age. Apart from a significant, broad quantitative trait locus (QTL) for high-frequency hearing loss on chromosome 11 containing the fascin-2 gene, we found a novel, small QTL for low-frequency hearing loss on chromosome 18, from hereon called ahl9. Real-time quantitative polymerase chain reaction of organs of Corti, isolated from a subset of strains, showed that a limited number of genes at the QTL were expressed in the organ of Corti. Of those genes, several showed significant expression differences based on the parental line contributing to the allele. Our results may aid in the future identification of genes involved in low-frequency, early-onset hearing loss.

A startle reflex in response to an intense acoustic stimulus is inhibited when a barely detectable pulse precedes the startle stimulus by 30-500 ms. It has been theorized that this phenomenon, named prepulse inhibition (PPI) of a startle response, is an automatic early-stage gating process contributing to the ability to focus attention. Deficits in PPI may therefore contribute to deficits in attentional processing. Both deficits are observed in schizophrenia spectrum disorders. Here, we investigated whether there is overlap in genetic control of PPI and attentional processing phenotypes in the panel of BXD recombinant inbred strains of mice. Using an individually titrated prepulse intensity to handle differences in perceived prepulse intensities among strains, we identified a significant quantitative trait locus (QTL) for PPI at the mid-distal end of chromosome 17. A measure of attentional processing in the five-choice serial reaction time task, response variability, mapped to a different locus on proximal-mid chromosome 16. In addition, the estimated genetic and environmental correlations between PPI and several attentional phenotypes were low and not significant. Taken together, the observation of separate genetic loci for PPI and attention and the absence of genetic and environmental correlations indicate that differences in sensorimotor gating do not contribute to differences in attentional performance. Therefore, it is worth pursuing the causative genes residing in both attention and PPI QTL, as these may contribute to separate molecular pathways implicated in neuropsychiatric diseases, such as schizophrenia. © 2011 The Authors. Genes, Brain and Behavior © 2011 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.

Extinction of instrumental responses is an essential skill for adaptive behavior such as foraging. So far, only few studies have focused on extinction following appetitive conditioning in mice. We studied extinction of appetitive operant lever-press behavior in six standard inbred mouse strains (A/J, C3H/HeJ, C57BL/6J, DBA/2J, BALB/cByJ and NOD/Ltj) and eight recombinant inbred mouse lines. From the response rates at the end of operant and extinction training we computed an extinction index, with higher values indicating better capability to omit behavioral responding in absence of reward. This index varied highly across the mouse lines tested, and the variability was partially due to a significant heritable component of 12.6%. To further characterize the relationship between operant learning and extinction, we calculated the slope of the time course of extinction across sessions. While many strains showed a considerable capacity to omit responding when lever pressing was no longer rewarded, we found a few lines showing an abnormally high perseveration in lever press behavior, showing no decay in response scores over extinction sessions. No correlation was found between operant and extinction response scores, suggesting that appetitive operant learning and extinction learning are dissociable, a finding in line with previous studies indicating that these forms of learning are dependent on different brain areas. These data shed light on the heritable basis of extinction learning and may help develop animal models of addictive habits and other perseverative disorders, such as compulsive food seeking and eating.

To study the heritability of different training stages of appetitive operant conditioning, we carried out behavioural screening of 5 standard inbred mouse strains, 28 recombinant-inbred (BxD) mouse lines and their progenitor strains C57BL/6J and DBA/2J. We also computed correlations between successive training stages to study whether learning deficits at an advanced stage of operant conditioning may be dissociated from normal performance in preceding phases of training.The training consisted of two phases: an operant nose poking phase, in which mice learned to collect a sucrose pellet from a food magazine by nose poking, and an operant lever press and nose poking phase, in which mice had to execute a sequence of these two actions to collect a food pellet. As a measure of magazine oriented exploration, we also studied the nose poke entries in the food magazine during the intertrial intervals at the beginning of the first session of the nose-poke training phase.We found significantly heritable components in initial magazine checking behaviour, operant nose-poking and lever press-nose poking. Performance levels in these phases were positively correlated, but several individual strains were identified that showed poor lever press-nose poking while performing well in preceding training stages. Quantitative trait loci mapping revealed suggestive likelihood ratio statistic peaks for initial magazine checking behaviour and lever press – nose poking. These findings indicate that consecutive stages towards more complex operant behavior show significant heritable components, as well as dissociability between stages in specific mouse strains. These heritable components may reside in different chromosomal areas.