Genes, Brain and Behavior (2009) © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd/International Behavioural and Neural Genetics Society Activity and impulsive action are controlled by different genetic and environmental factors M. Loos†, S. van der Sluis‡, Z. Bochdanovits§, † ¶ ∗∗ I. J. van Zutphen , T. Pattij ,O.Stiedl , Impulsivity and novelty seeking are core aspects of human Neuro-BSIK Mouse Phenomics consortium††, personality (Bouchard & Mcgue 2003) that received a great ∗ A. B. Smit† and S. Spijker ,† deal of attention, not the least because they have been associated with substance abuse disorder (Kreek et al. †Departments of Molecular and Cellular Neurobiology , 2005; Wills et al. 1994). In rats, individual differences in ‡Functional Genomics , §Medical Genomics , ¶Anatomy and impulsivity and the locomotor response to a novel open Neurosciences, and **Behavioral and Cognitive Neuroscience field (OF), a putative measure of novelty seeking, are Group, Center for Neurogenomics and Cognitive Research, predictive of aspects of drug self-administration behavior Neuroscience campus Amsterdam, VU University, Amsterdam, (Belin et al. 2008; Diergaarde et al. 2008; Ellenbroek & Cools The Netherlands, and ††Collaborators: Brussaard AB, Borst JG, 2002; Piazza et al. 1989). These individual differences can Elgersma Y, Galjart N, van der Horst GT, Levelt CN, Pennartz arise from genetic differences among individual rats and/or CM, Smit AB, Spruijt BM, Verhage M, de Zeeuw CI from environmental factors. Given the relevance of animal *Corresponding author: S. Spijker, PhD, Department of Molec- models of predictive factors of addiction-related behavior, we ular and Cellular Neurobiology, VU University, De Boele- examined whether shared, or alternatively, distinct genetic laan 1085, 1081 HV Amsterdam, The Netherlands. E-mail: and/or environmental factors control impulsivity and various [email protected] measures of novelty exploration. Inbred strains of mice provide the opportunity to separate Both impulsivity in operant tasks and locomotor activity genetic and environmental factors. When kept under highly in a novel open field are known to predict the controlled conditions, differences between inbred mouse development of addiction-related behavior in rodents. strains result from additive genetic effects and gene-by- In this study, we investigated to what extent impulsivity environment interactions (Crabbe et al. 1999). Thus, high in the five-choice serial reaction time task and various correlation between measures of impulsivity and novelty measures of novelty exploration are controlled by exploration across strain means would indicate that these shared genetic and environmental factors in 12 different measures are controlled by common genetic or gene-by- inbred mouse strains. No genetic correlation was environment interaction effects (genetic correlation), albeit observed between the level of impulsivity and levels of such correlations contain some residual environmental activity, a low correlation was observed with traditional influence because of estimation of strain means (Crusio measures of anxiety-like behavior (impulsive strains 2006). In contrast, behavioral differences among isogenic tend to be less anxious) and a highly significant mice of the same strain result from environmental correlation was found between impulsivity and specific effects idiosyncratic to each individual, and hence high aspects of movement. Furthermore, we found that within-strain correlations of impulsivity and measures impulsivity and all measures of novelty exploration of novelty exploration would indicate that these are were under control of different environmental factors. controlled by common environmental factors (environmental Interestingly, in the dorsal medial prefrontal cortex, correlation). a brain region involved in impulsivity and activity in Several operant tasks of impulsivity exist (Evenden 1999), novelty exploration tests; these behavioral measures broadly categorized into those that measure impulsive action correlated with the expression of different genes or impulsive choice (Winstanley et al. 2006). In this study, (respectively, Frzb, Snx5, BC056474 and the previously the murine version of the five-choice serial reaction time identified Glo1). Taken together, our study shows task (5-CSRTT; Pattij et al. 2007; Robbins 2002) was used, that impulsivity and activity in novelty exploration in which premature responses to a food predictive stimulus tests are genetically and environmentally distinct, are a measure of impulsive action, previously shown to be suggesting that mouse models of these behaviors predictive of increased motivation to self-administer addictive provide complementary insights into the development drugs in rats (Belin et al. 2008; Diergaarde et al. 2008). of substance abuse disorder. The locomotor response in a novel OF, predictive of Keywords: 5-CSRTT, BXD, dark/light box, discriminant amphetamine self-administration (Piazza et al. 1989), is analysis, elevated plus maze, hypophagia, novel object, open the net result of several interacting behavioral constructs field, principal component analysis, recombinant inbred (most notably anxiety, novelty seeking and general activity). Therefore, in this study novelty exploration was also Received 7 April 2009, revised 13 July 2009, accepted for measured in tests that differ in the anxiogenic nature of publication 24 July 2009 novel stimuli, the motivational salience of novel stimuli and doi: 10.1111/j.1601-183X.2009.00528.x 1 Loos et al. the required activity to cover the novel stimulus. In addition Novel cage to total activity, novelty exploration was also measured in On days 4 and 11, mice were transferred to a novel clean cage with terms of the locomotor strategy and location of activity. fresh bedding containing the metal cup with the familiar snack. Both the latency until the first touch of the cup and latency to start eating Finally, because the dorsal part of the rodent medial the snack were recorded manually. The mean of both days was prefrontal cortex (mPFC) is involved in both impulsivity in the used for analysis. If a subject did not eat within 720 seconds, the 5-CSRTT (Muir et al. 1996) and activity in a novel OF (Deacon maximum time was assigned. After testing, both nesting material et al. 2003; Holmes & Wellman 2009), we subsequently and tube were transferred to the novel home cage. analyzed gene expression data of this brain region (Hovatta et al. 2005) to identify genes whose expression was Open field specifically correlating with either impulsivity or activity in On day 14, mice were introduced into a corner of the white square a novel OF. OF (50 × 50 cm, walls 35 cm high) illuminated with a single white fluorescent light bulb from above (130 lx), and exploration was tracked for 10 min (12.5 frames/seconds; EthoVision 3.0, Noldus Information Technology). Time spent in, and number of entries Materials and methods into the center square area (20 × 20 cm), was measured using EthoVision. The Strategy for the Exploration of Exploration software Subjects (SEE; Kafkafi et al. 2005) was used to smoothen path shape to calculate the total distance moved. Furthermore, SEE uses the Six-week-old male mice (n = 12 per strain) were obtained from distribution of speed peaks to parse the locomotor data into slow Jackson Laboratory (A/J, C3H/HeJ, C57BL/6J, DBA/2J, FVB/NJ), local movements (lingering episodes) and progression segments, from our own breeding colony (BXD2, BXD6, BXD11, BXD21, BXD32 which together constitute all distance traveled. In addition to the and BXD40) or Taconic Farms (129S6/SvEvTac). Mice arrived in traditional measures in the OF, describing the animal tendency to the facility in six different batches in a period spanning 18 weeks. engage in exploratory behavior, SEE was used to calculate the Batches 1–5 consisted of BXD mice of different strains, batch 6 number of progression segments and the median duration of a entirely consisted of the remaining six strains. Mice were housed lingering episode. SEE also enables the calculation of measures individually in Macrolon cages on sawdust bedding, which were, that describe the strategy of movement once exploration has been for the purpose of animal welfare, enriched with cardboard nesting initiated: the median distance traveled per progression segment, the material and a curved PVC tube. A metal food cup was placed in median duration of a progression segment, the number of stops per the home cage (for the purpose of habituation; used in subsequent distance and the median acceleration during a progression. hypophagia test). Food (Harlan Teklad) and water were provided ad libitum, except food supply during the weeks of operant testing. All mice were habituated to the facility for 14 days before testing started. During habituation, mice were familiarized four times, on Dark/light box separate days, to a highly palatable snack (crumbles of cream cracker) On day 15, mice were introduced into the dark compartment (<10 × × × × placed in a metal food cup. Housing rooms were controlled for lx; 25 30 30 cm, length width height) of a dark/light box temperature, humidity and light/dark cycle (0700 h lights on, 0700 h (DLB; TSE-Systems; Baarendse et al. 2008), 15 seconds later, the lights off). All experimental procedures were approved by the local motorized black door to the identically sized brightly lit compartment animal research committee and complied with the European Council (320 lx) was opened and